Microbiology Exam 1

What are the two types of *Single-stranded RNA virus* genomes? *(Start of PID 25-27: Virus replication and classification Cards 476-496)*

(+) sense RNA genome: carry a mRNA type positive RNA genome (-) sense RNA genome: carry a RNA genome that does not have an open reading frame (ORF) in the RNA and carries a *RdRp* ssRNA

Assays which require live virus:

1. Foci formation 2. Plaque formation/assay 3. Syncytium formation 4. Virus neutralization tests (NT): Antibodies directed against the whole virus particle often inactivates the virus particle such that virus infection is inhibited

What are toxic effects of antimicrobial agents?

1. Hypersensitivity-allergic reactions 2. Hematological reactions 3. Gastrointestinal problems

Steps of transcription

1. Initiation 2. Sigma release 3. Elongation 4. Termination

Translation initiation steps

1. Initiation Factors 1 and 3 bind to the 30S subunit preventing the 50S subunit from binding 2. The shine-dalgarno sequence or start codon sequence binds to the subunit (IF-3 leaves--not shown in picture) 3. The initiator tRNA (f-met tRNA) is carried by IF2-GTP to the subunit and binds to the P-site 4. Initiation factors leave and the 50S subunit binds forming the 70S ribosome

*Aminoglycosides*

* Streptomycin*, kanamycin, tobramycin, gentamicin, netilmicin, amikacin, and neomycin The aminoglycosides are highly polar, positively-charged compounds which do not readily cross membranes. Therefore, they are not absorbed from the gut and cannot be administered orally (except for sterilization of the gut prior to surgery). Also transport across bacterial membranes is slow. They are ineffective against intracellular infections. Anaerobiosis retards entry into cells by reducing the membrane potential, hence *they are not effective against anaerobic organisms.* *30S ribosome subunit inhibitor*

*Negative control* of the lac operon

*A specific control of the lac operon* (one of two controls of the lac operon) A repressor binds to the operator site and prevents transcription.

What is the function of teichoic Acids?

*Activate alternative pathway of complement* Adhesion of bacteria to host cells Involved in *antigenicity* of cell wall (S. aureus polysaccharide A is a teichoic acid) Lipoteichoic acid anchors wall to cell membrane and functions as an epithelial cell *adhesin*

*Azotobacter* undergoes what type of fermentation?

*Alcoholic (bacterial) fermentation* producing ethanol and CO2

*Pseudomonas* undergoes what type of fermentation?

*Alcoholic (bacterial) fermentation* producing ethanol and CO2

What are the different types of bacterial fermentation? (7)

*Alcoholic Fermentation*- produces 2 ATPs, ethanol, and CO2 *Homolactic Fermentation*- produces 2 ATPs and lactate *Butyric Acid Fermentation*- produces 2 ATPs, butyrate, butanol, acetone, and CO2 *Mixed Acid Fermentation*- produces 3 ATPs, lactate, acetate formate, ethanol, and CO2 *Heterolactic Fermentation*- produces 1 ATP and lactate *Alcoholic (Bacterial) Fermentation*- produces 1 ATP, ethanol, and CO2 *Butanediol Fermentation*- produces 1 ATP, acetone, butanediolh, and CO2

*Yeast* undergoes what type of fermentation?

*Alcoholic fermentation* producing ethanol and CO2

*All the antivirals are directed against viral enzymes or proteins.*

*All the antivirals are directed against viral enzymes or proteins.*

How do viruses become resistant to drugs?

*All the antivirals are directed against viral enzymes or proteins.* These drugs are used by viral enzymes resulting in inhibition of virus growth. Often during drug therapy, viral variants which fail to utilize the drug arise. These drug-resistant viruses carry proteins which do not recognize the drug. The mutant proteins carry altered amino acids in them. Thus viral genes coding for these mutant proteins carry corresponding nucleotide changes.

What is the toxicity of polyene compounds?

*Amphotericin B* - Renal damage, anemia, hypokalemia (low potassium) *Nystatin* - exhibits similar but greater toxicity than amphotericin B and is used only in topical applications

What is the clinical use of polyene compounds?

*Amphotericin B* is used for the treatment of systemic fungal infections (aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis and mucormycosis). *Nystatin* is a second-line drug for the treatment of *candidiasis* when resistance develops to azole compounds.

*Sulfonamides*

*Antimetabolites of p-aminobenzoic acid*- one of the substrates in the first enzymic reaction in the pathway leading to the synthesis of tetrahydrofolic acid. Competitive inhibitors of dihydrofolic acid synthesis Mechanism of Action: Sulfonamides inhibits dihydropteroic acid synthesis in the pathway leading to the synthesis of tetrahydrofolic acid by *inhibiting the enzyme, dihydropteroate synthetase.* Tetrahydrofolic acid is essential for one-carbon transfer reactions necessary for the synthesis of DNA, RNA, lipids, vitamins and amino acids. Mammalian cells do not synthesize folic acid but obtain folic acid by dietary uptake. Spectrum of Activity: Equally against susceptible gram-positive and gram-negative bacteria Clinical Use: Treatment usually in combination with trimethoprim of urinary tract infections Treatment of rheumatic fever caused by Group A streptococci in patients allergic to penicillin Toxicities: Renal blockage, hypersensitivity, GI problems

What is the product of the trpR gene?

*Aporepressor* Cannot bind to the operator unless *tryptophan* is present as a *co-repressor* Since the promoter and operator sequences overlap, the bound repressor *prevents RNA polymerase from binding to the trp promoter* (How is this different than the lac operon?--the bound lac repressor on the operator prevents the movement of RNA polymerase from promoter to the structural genes.) Mutations in the trpR gene or operator locus causes constitutive expression of trp mRNA

*Clostridium* undergoes what type of fermentation?

*Butyric Acid fermentation* producing butyrate, butanol, acetone, and CO2

*Second Generation Cephalosporins*

*Cefaclor, cefoxitin, cefuroxime, and cefamandole* Spectrum of Activity: Improved activity against gram-negative bacilli such as members of the family Enterobacteriaceae, but less active against gram-positive organisms. More resistant to cephalosporinases Clinical Use: As second line drugs in seriously ill patients As an alternative to aminoglycosides in patients with renal impairment For preoperative prophylaxis

OriC

*Chromosome replication starts at a precise site known as the OriC site* Replication is *bidirectional,* so two replication forks proceed in opposite directions around the chromosome and meet near the top locus.

*Lincosamides*

*Clindamycin and lincomycin* Mechanism of Action: Clindamycin and lincomycin bind to the 50S ribosomal subunit and interfere with peptide bond formation Spectrum of Activity: • Clindamycin - similar spectrum as erythromycin and lincomycin with *greater activity against anaerobic bacteria* especially, Bacteroides fragilis • Lincomycin - similar spectrum as erythromycin Clinical Use: Treatment of infections by Bacteroides fragilis or other anaerobes in patients allergic to penicillin Toxicities: Pseudomembranous colitis (inflammation of the digestive tract) due to inhibition of most of the anaerobic flora of the bowel and overgrowth by clindamycin-resistant Clostridium difficile *50S ribosome subunit inhibitor*

Escherichia coli Plasmid-encoded virulence traits?

*Co1 plasmids* cause production of *colicins* that kill sensitive normal flora and thus facilitate the *colonization* of the intestine. *K88 plasmids* specify *capsular K antigens* that permit *adherence to intestinal mucosa* and thus further facilitate colonization of the host. Plasmids specifies the production of two *enterotoxins, a heat-labile enterotoxin (LT) and a heat stable enterotoxin (ST),* that are similar to but not quite as potent cholera enterotoxin thus *causing a diarrheal syndrome* similar to but not as severe as caused by Vibrio cholera. *Epithelial penetration factor plasmid* permits E. coli to penetrate cells in the intestinal mucosa to cause *ulceration of the intestine* and thus causing a diarrheal syndrome similar to but not as severe as caused by Shigella.

Conjugative transposition

*Conjugative transposons* are characterized by their ability to move between bacteria cells by a process that requires cell-to-cell contact *in the absence of a plasmid.* Conjugative transposition was first described for plasmid-free strains of Enterococcus faecalis that contained a tetracycline-resistance determinant on a transposon called Tn917.

Nucleocapsid

*Contained by all viruses* Composed of a protein shell (cage) and genetic material. *A complex of viral genome and a protective shell of protein aggregates known as a capsid* Some nucleocapsids carry glycoproteins at their surface The nucleocapsid can exist in two geometric symmetries: helical or icosahedral/cubical

What are common characteristics of cell components shared between Gram-negative and gram-positive cells?

*Cytoplasmic Membrane* *Peptidoglycan cell wall* (what is the difference in thickness and cross-links?) *Glycocalyx (and Capsule)* *Pili (frimbrae)* *Flagella* *Cytoplasmic inclusions* *Ribosomes* *Periplasm*

How long does DNA replication take in bacterial cells?

*DNA replication requires 40 minutes* in *slow growing cells* undergoing one round of DNA synthesis/cycle. In *fast growing cells*, DNA replication is still *40 minutes*, but more copies of DNA is created. Replication will initiate again on a strand before its own replication has completed. So new rounds of replication must start before previous ones are terminated. Chromosome replication is regulated by *frequency of initiation*.

Stringent Response

*Decrease in RNA synthesis associated with amino acid depletion or starvation* *due to the relA locus* If a bacterial culture is transferred from a growth medium in which growth is rapid to one in which growth is slow, this is referred to as *downshift*. The ribosome content of each cell decreases from a higher value to a lower value. This is expected since otherwise the cells would have more ribosomes than needed. The decrease is accomplished by allowing DNA synthesis to proceed without synthesis of rRNA. This inhibition of rRNA synthesis is associated with amino acid depletion or starvation and is called stringent response.

*Bordetella pertussis* Example

*Disease/condition:* whooping cough *Sensor protein:* BvgS *Regulator protein:* BvgA *Signal:* Mg+2, SO4-2, temperature *Regulated virulence properties:* Pertussis toxin, adenylase cyclase, toxin/hemolysin, filamentous hemagglutinin

R plasmids

*Drug resistance plasmids* Harbor drug resistant genes or determinants Can be isolated in a number of different bacteria genera (Shigella, E. coli, Salmonella, Vibrio, Bacillus, Pseudomonas and Staphylococcus.) Usually conjugative and self-transmissible Their presence in a host cell confers resistance on their host cell to a variety of antibiotics Most R plasmids have two contiguous segments of DNA. One is called *RTF (resistance transfer factor)* which carries genes for conjugative functions. The other segment, the *r-determinant or resistance determinant*, carries genes for antibiotic resistances. Resistance to penicillin, ampicillin, chloramphenicol, streptomycin, kanamycin and sulfonamides singly or in combinations appear commonly.

Drug synergy

*Drug synergy or synergism* is produced when a mixture of drugs exert a rate of killing at minimal inhibitory concentrations which reflects a greater than additive effect of the drugs acting alone. Drug synergy can be observed with bacteriostatic as well as bactericidal drugs. Typical combinations of drugs which complement each other are drugs which exhibit different mechanisms of action, inhibit different pathways or inhibit different enzyme reactions in the same pathway.

Artificial Transformation

*Electroporation* the action or process of introducing DNA or chromosomes into bacteria or other cells using a pulse of electricity to briefly open the pores in the cell membranes.

*Macrolides*

*Erythromycin*, azithromycin, clarithromycin, and dirithromycin Mechanism of Action: Erythromycin binds to the 50S ribosomal subunit and blocks the translocation reaction of protein synthesis by specifically interfering with the release of the charged tRNA bound to the donor site (P site) of the ribosome after peptide bond formation Spectrum of Activity: Gram-positive bacteria, penicillinase-producing Staphylococcus aureus, Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia trachomatis Clinical Use: Treatment of Mycoplasma pneumoniae and Legionella pneumophila (Legionnaires' disease) infections, diphtheria, pertussis Treatment of patients allergic to penicillin who have infections caused by Group A streptococci or pneumococci Toxicities: Gastrointestinal upset *50S ribosome subunit inhibitor*

*Gas Vapor Sterilants*

*Ethylene oxide* is toxic and potentially explosive *Plasma gas* is hydrogen peroxide vaporized (highly ionized)-- Reacts with membranes, proteins and nucleic acids. Used for *sterilization* of medical instruments *Chlorine dioxide gas* denatures protein by oxidation-- Lack of toxicity used for industrial cooling systems *Formaldehyde gas* is limited use/carcinogenic

Staphylococcus aureus Plasmid-encoded virulence traits?

*Exfoliatin toxin causes scaled skin syndrome in young babies* an affliction of the skin of newborns called scalded skin syndrome is caused by a plasmid-associated toxin

Clostridium perfringens Plasmid-encoded virulence traits?

*Exotoxin causes diarrheal disease in domestic animals* One type of diarrheal disease in domestic animals is caused by C. perfringens. The disease is associated with a plasmid-encoded toxin.

Periplasmic Space

*Found in gram-negative bacteria* (small space in gram-positive cells) Space between outer & cytoplasmic membrane Periplasm is fluid that contains: Degradative enzymes (nucleases, phosphatases, penicillinases, etc)

Spores

*Found in gram-positive bacteria* Example: Clostridium sp. and Bacillus anthracis Are dormant and dehydrated Form in response to nutritional deprivation Resistant to heat, chemicals, drying, freezing Contains chromosome, proteins, ribosomes High concentrations of *calcium dipicolinic acid*

Operon

*Genes and their regulatory regions* A genetic regulatory system found in bacteria and their viruses in which genes coding for functionally related proteins are clustered along the DNA. This feature allows protein synthesis to be controlled coordinately in response to the needs of the cell. The picture is the lac operon

What is the clinical use of aminoglycosides?

*Gentamicin, tobramycin, and amikacin are the most useful.* They are active against many gram-negative bacilli, including pseudomonads. *Gram-positive bacteria are generally resistant to these three drugs, with the exception of Staphylococcus aureus.* The other aminoglycosides are limited to specific infectious niches, due to toxicity and emergence of resistance. For example, streptomycin is the drug of choice for Francisella tularensis and Yersinia pestis infections. In combination with penicillin or vancomycin, streptomycin is used in the treatment of endocarditis caused by Enterococcus faecalis or viridans streptococci, and in combination with other drugs for the treatment of tuberculosis.

Sterilization method: *Heat kills by denaturation of macromolecules*

*Heat kills by denaturation of macromolecules* *Autoclaving:* steam under pressure is the preferred method of sterilization-- *121 degrees Celsius, 15 lbs. of pressure for 20 minutes* *Dry heat* sterilizes at *160 degrees Celsius for 2 hours* *Boiling water does not kill spores*, but does kill vegetative cells Spore test strips - used to evaluate effectiveness of sterilization. (Bacillus stearothermophilus)

Heme acquisition systems

*Hemophores* secreted by bacteria remove heme from hemo proteins (hemoglobin)

Subunit or recombinant vaccine

*Hepatitis B vaccine* - envelope glycoprotein (HBsAg) - only one protein - genetically engineered yeast as source *Human Papilloma Vaccine* contains a recombinant capsid protein Example: Gardasil and Cervarix

*Leuconostoc* undergoes what type of fermentation?

*Heterolactic fermentation* producing lactate

*Lactobacillus* undergoes what type of fermentation?

*Homolactic fermentation* producing lactate

*Streptococcus* undergoes what type of fermentation?

*Homolactic fermentation* producing lactate

Methods of viral entry

*Horizontal spread*: person-to-person *Vertical spread*: mother to offspring, fetal infection Insects: Arboviruses Animal: Zoonoses, Rabies Iatrogenic inoculation: Medical practice transfusion- blood transfusion (HIV, CMV, HBV, HCV) Prion disease in humans: Corneal transplant, neurological surgery Growth hormone from pituitary of dead cadavers, Creutzfeldt-Jacob disease

Hypersensitivity-allergic reactions

*Hypersensitivity-allergic reactions* can be caused by any antimicrobial agent. Penicillins can produce either immediate, IgE-mediated, or delayed hypersensitivity reactions. *Cutaneous reactions* have been reported with every class of antimicrobial.

Types of viral infections

*Inapparent or subclinical infections*: Active virus replication, but *no symptoms* are experienced 50-90% (virus is there, but no symptoms) *Acute infections*: Active replication and *severe symptoms* Example: Flu, mumps, measles (virus and disease) *Persistent infections*: Active infection and variable disease for months to years Long term infection with or without overt disease *Latent infections*: Tiny amount of genome and an inactive virus hiding in select tissues An inactive virus hiding in the body A type of persistent infection, with no virus replication (hiding virus) *Congenital infections*: Infections that can affect a fetus Example: Rubella, CMV, HIV etc.

Explain the formation of *HFr cells*

*Integration of the F plasmid into the bacteria chromosome* occurs normally, however rarely, in cultures of F+ cells. The chromosome remains a single, circular DNA molecule and F behaves as if it were part of the bacterial chromosome. When a culture of *Hfr cells is mixed with an F- culture, conjugation occurs.* In this instance, *DNA replication begins in the Hfr cell and a copy is transferred to the F- cell.* The direction of replication is such that a small part of F is transferred first and the major part is transferred last. Because the bacteria are very small and in constant motion and because it would take over 100 minutes to transfer the entire chromosome, the mating pair usually breaks apart before transfer of the entire chromosome can occur. *Therefore, the female cell receives a large fragment of the male chromosome and a small functionless fragment of F.* Because of this the female recipient remains a female and the result of this cross is as follows: *Hfr x F- → F-*

How do interferons protect against viral infections?

*Interferons inhibit protein synthesis in infected cells by inducing the activation of RNAse L and Protein Kinase P1 (R)* Interferons induce the transcription of 2'5' oligoadenylate synthetase (OAS) gene. The OAS enzyme directly activates Ribonuclease L.

What is the toxicity of imidazoles?

*Ketoconazole, fluconazole and itraconazole* are less toxic than the other azole compounds and can be taken orally for the treatment of systemic fungal infections. *Clotrimazole and miconazole* are toxic when taken orally and are used mostly for the topical treatment of skin fungal infections.

Oxazolidonones

*Linezolid* (ZyvoxTM) Mechanism of Action: Prevents formation of functional 70S initiation complex, which is essential for bacterial translation process Spectrum of Activity: Bacteriostatic against enterococci and staphylococci and bactericidal against most strains of streptococci

Chemotherapeutic agent

*Man-made compounds* which exhibit *selective toxicity* for select groups of microorganisms

Sterilization method: *Heavy metals*

*Mercury, Silver, and Arsenic* form *mercaptides* with sulfhydryl groups of cysteine *Silver nitrate* is bactericidal for gonococcus; used routinely for prophylaxis of *ophthalmia neonatoru and burn patients* *Phenylmercury salts* control of pseudomonads in cosmetics

*Penicillinase-resistant penicillins*

*Methicillin* Nafcillin Isoxazolyl penicillins (cloxacillin, dicloxacillin, oxacillin) Spectrum of Activity - Similar to penicillin G (Gram-positive bacteria & Neisseria) but less active Clinical Use: Staphylococcal infections

*Escheichia* undergoes what type of fermentation?

*Mixed Acid fermentation* producing lactate, acetate formate, ethanol, and CO2

*Salmonella* undergoes what type of fermentation?

*Mixed Acid fermentation* producing lactate, acetate formate, ethanol, and CO2

*Shigella* undergoes what type of fermentation?

*Mixed Acid fermentation* producing lactate, acetate formate, ethanol, and CO2

Acid-Fast Bacteria

*Mycobacteria, Nocardia sp.* Most mycobacteria are not pathogenic, and are readily isolated from the environment. These bacteria are gram-positive, aerobic, non-sporulating, non-motile, and often pleomorphic. They are typically smaller than other bacteria. Mycobacteria are acid-fast because of lipid-rich cell envelope. Stain red with *carbolfuchsin* Decolorization with acid-alcohol (Ziehl-Neelsen) *Mycolic acid* - basis for acid-fast staining (In the picture-- A are non-acid fast and B are- acid fast)

Which form of inorganic nitrogen can be directly assembled into amino acids? a. NH4+ b. NH3 c. NO3 d. N2

*NH3* is the only form of inorganic nitrogen that can be directly assembled into amino acids.

There are two types of *transcription regulation* in prokaryotes:

*Negative Regulation*-An inhibitor keeps transcription turned off and an anti-inhibitor (*inducer*) is needed to turn the system on-- Example: Lac Operon *Positive Regulation*-An effector molecule (protein, small molecule, or molecular complex) activates a promoter-- Example: Trp Operon

Neisseria gonorrhoeae

*Neisseria gonorrhoeae* are *naturally competent* meaning they're always competent, but they will only take up specific DNA sequences from their own species.

Polyene compounds

*Nystatin and Amphotericin B* (anti-fungal agents) *Spectrum of Activity: Fungi* Clinical Use: *Amphotericin B* is used for the treatment of systemic fungal infections (aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis and mucormycosis). *Nystatin* is a second-line drug for the treatment of *candidiasis* when resistance develops to azole compounds. Toxicities: *Amphotericin B* - Renal damage, anemia, hypokalemia (low potassium) *Nystatin* - exhibits similar but greater toxicity than amphotericin B and is used only in topical applications

How do β-Lactam antibiotics work?

*Penicillins inhibit the transpeptidation reaction of cell wall synthesis* by irreversible binding to the transpeptidase enzyme. This binding inhibits cross-bridge formation during peptidoglycan synthesis.

What are characteristics of cell envelope components specific to Gram-positive cells?

*Peptidoglycan cell wall*- 40+ layers; 90% cross-links between monomers *Teichoic Acids (and lipoteichoic acids)* *Spores* *Acid-Fast bacteria*

What are characteristics of cell envelope components specific to Gram-negative cells?

*Peptidoglycan cell wall*- one to two layers; less cross-links between monomers *Outermembrane*-- *LPS (Lipid A, O antigen, and core polysaccharide [KDO])* *Porins* (some are in gram positive bacteria) *Mycoplasma bacteria* (lack cell wall) *Lipooligosaccharide (LOS)*

Disinfection method: *Phenolics*-*(Specifically asked on the study guide)*

*Phenolics* cause membrane damage and protein denaturation Lysol Hexachlorophene (not used anymore) Chlorhexidine - replaced hexachlorophene as skin disinfectant Triclosan - deodorant, soaps

The mechanism of antigenic variation in Pili

*PilS* genes are incomplete silent genes with no promoter regions, so proteins cannot be transcribed *PilE* genes are intact genes that are expressed In recombination of PilS and PileE genes, variance of proteins can arise By changing gene expression, new proteins are produced. The host's antibodies will not recognize the new protein and evade the immune system. This why there is no cure for gonorrhea

Lipooligosaccharide (LOS)

*Present in Neisseria gonorrhoeae, N. meninigitidis* *Lacks the O antigen portion of LPS* Readily shed from the bacteria Resemble glycosphingolipids of mammalian cell membranes Important virulence factor-- Role in evasion of host immune response

Define repression and depression in terms of transcription

*Repression*- a decrease in synthesis of an enzyme or series of enzymes mediated by a specific molecule; the decrease observed in the synthesis of a metabolite when the intracellular level of the metabolite reaches a certain concentration. *Derepression*- removal of repression; the increase in the level of a metabolite when the intracellular level of the metabolite drops to a certain concentration. Example: trp operon

Lipid A

*Responsible for toxicity of endotoxin, pyrogenic* Consist of a chain of *phosphorylated D-glucosamine disaccharide units with fatty acids attached* Most commonly observed fatty acid is *b-hydroxymyristic acid*

Another way to describe peptidoglycan synthesis:

*Step 1* Inside the cell, glucosamine (NAG) is converted to MurNAc (NAM) and then reacts with UTP to form UDP-MurNAc (UDP-NAM) Next, a pentapeptide is attached to UDP-MurNAc in series of reactions. *Step 2* UDP-MurNAc pentapeptide (UDP-NAM-pentapeptide) is attached to bactoprenol located in the membrane. UMP is released. GlcNAc (NAG) is added to make the disaccharide building block (bactoprenol-P-P-NAG-NAM-pentapeptide) *Step 3* Bactoprenol translocates the disaccharide-pentapeptide precursor to the outside of the cell. The pyrophosphate link between itself and the bactoprenol is the energy used to drive the reaction by transglycosylases. *Step 4* Outside the cell, peptide chains are cross-linked to each other by peptide bond exchange (transpeptidation) Cross-linking is catalyzed by membrane-bound transpeptidases Transpeptidases are also called penicillin binding proteins (PBPs)

There are new drugs that inhibit 50S ribosomal function of Gram-positive cocci

*Streptogramins* - a combination of quinopristin and dalfopristin (30%/70%) sold under the trade name of Synercid. Synercid is the first antibiotic approved for *treatment of patients with serious or life-threatening infections associated with vancomycin-resistant Enterococcus faecium.* The drug is also approved for complicated skin and skin structure infections caused by Staphylococcus aureus (methicillin-susceptible) or Streptococcus pyogenes. *Ketolides* - fourth generation macrolides. These drugs are highly potent against most gram-positive pathogens. The exceptions to the spectrum of activity of ketolides are methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. *Ketolides are highly active against drug-resistant Streptococcus pneumoniae.*

Disinfection method: *Surfactants*-*(Specifically asked on the study guide)*

*Surfactants* cause membrane damage and lysis Anionic detergents - soaps have little direct antibacterial effect cationic detergents - quaternary ammonium compounds that are highly bactericidal, but inactive against spores and most viruses

Ketolides

*Telithromycin* Mechanism of Action: Blocks protein synthesis by binding to 50S ribosomal subunit Spectrum of Activity: Drug-resistant Streptococcus pneumoniae Clinical Use: Respiratory tract infections

Measurement of antimicrobial susceptibility- *Define MIC and MBC*

*Terms used to express relative potency of antimicrobial toward an organism* *Minimum inhibitory concentration (MIC)*: Lowest concentration of agent required to inhibit the growth of an organism *Minimum Bactericidal Concentration (MBC)*: Lowest concentration of drug that kills 99.9 % of the population of organisms

*Tetracycline* mechanism of action

*Tetracycline binds transiently to the 30S ribosomal subunit and blocks attachment of amino-acylated tRNA to the mRNA-ribosome complex (A site)* *30S ribosome subunit inhibitor*

What is the main difference between Cephalosporin generations?

*The cephalosporins are broad-spectrum agents. As a rule, gram-positive activity diminishes while gram-negative activity improves as one progresses from first to third generation agents.*

Transduction

*Transduction* involves the transfer of genetic information from one cell to another cell mediated by bacteriophage. There are two types of transduction called *generalized and specialized transduction.*

Transformation frequency equation

*Transformation frequency* is defined as the ratio of the number of cells that were transformed and the total number of cells that were used for the transformation experiment. Example: StrS recipient cells - (10^7) Transforming DNA - (1 mg) Calcium chloride - (10 mM) Incubate - 37oC - 30 min. Add DNAse to terminate rx. Plate on nutrient agar plates containing streptomycin *Transformation frequency =# of Transformants x 100/ total cells* 105/107 x 100 =1.0%

Sterilization method: *Physical agents* (3)

*Ultraviolet Light* - 240 to 280 nm leads to pyrimidine dimer formation in microbial DNA *(Specifically asked on the study guide)* Poor ability to penetrate Minimizes airborne cross-infection in labs/hospitals *Ionizing Radiation* - gamma rays, x-rays lead to single-strand breaks and double strand breaks in microbial DNA *Filtration* sterilization of heat-labile materials HEPA filters for air; nitrocellulose filters for solutions

*Fourth Generation Cephalosporins*

*cefepime and cefpirome* Spectrum of Activity: Active against most Enterobacteriaceae and Pseudomonas aeruginosa Increased stability to beta-lactamases Clinical Use: As second line drugs in seriously ill patients infected with susceptible aerobic gram-negative bacilli As an alternative to aminoglycosides in patients with renal impairment For preoperative prophylaxis

*Third Generation Cephalosporins*

*cefotaxime, cefsulodin, cefixime, and ceftriaxone) Spectrum of Activity: Less active against gram-positive bacteria, but much more active against the Enterobacteriaceae Clinical Use: As second line drugs in seriously ill patients infected with susceptible aerobic gram-negative bacilli As an alternative to aminoglycosides in patients with renal impairment For preoperative prophylaxis

*First Generation Cephalosporins*

*cephalothin, cephalexin, and cefazolin* Spectrum of Activity: Most gram-positive cocci, many common gram-negative bacilli *except Enterobacter species, indole-positive Proteus species and Pseudomonas species* Clinical Use: As second line drugs in seriously ill patients As an alternative to aminoglycosides in patients with renal impairment For preoperative prophylaxis

Imidazoles (Azoles)

*ketoconazole, miconazole,clotrimazole, fluconazole, and itraconazole* (anti-fungal agents) *These compounds are not antibiotics. These drugs are synthetic azole derivatives.* Clinical Use: Azole compounds are used for the treatment of superficial and systemic fungal infections. Several of these compounds exhibit toxicities less than that of amphotericin B and flucytosine. Toxicities: *Ketoconazole, fluconazole and itraconazole* are less toxic than the other azole compounds and can be taken orally for the treatment of systemic fungal infections. *Clotrimazole and miconazole* are toxic when taken orally and are used mostly for the topical treatment of skin fungal infections.

Number of cells present at a given is time *b* equations

*one original cell:* b= 1 x 2^n n= number of divisions occurring during a given time period *More than one original cell:* b= a x 2^n where "a" is the number of organisms present in the original inoculum

How many strands of mRNA is produced by bacterial transcription?

*only one strand is transcribed* The *Coding strand or "+" strand* is not transcribed. 5'---->3' It has the same sequence as mRNA. (replace Ts with Us) The strand that is copied is called the *transcribed strand or the (- strand).* 3'-->5' The sequence of a gene is usually written as the sequence of coding strand. 5' ATGCTAT 3' --coding strand 3' TACGATA 5' --template/noncoding strand 5' AUGCUAU 3' --mRNA transcript

*Rho independent* Transcription Termination

- series of U's in the transcript - inverted repeat (form stem-loop structures)

What is the temperature range a *Psychrophile* can survive in?

-5 to 30 degrees celsius

Major steps in virus life cycle in cultured cells:

-virus attachment, entry, uncoating -gene expression from viral genomes -viral nucleocapsid assembly -virus exit

How many ATP does *Azotobacter* yield from Alcoholic (bacterial) fermentation?

1

How many ATP does *Enterobacter* yield from Butanediol fermentation?

1

How many ATP does *Klebsiella* yield from Butanediol fermentation?

1

How many ATP does *Leuconostoc* yield from heterolactic fermentation?

1

How many ATP does *Pseudomonas* yield from Alcoholic (bacterial) fermentation?

1

What factors have contributed to the widespread prevalence of drug-resistant plasmids in bacteria?

1. *Plasmid transfer in nature*- Conjugation has been demonstrated in vivo in fish, pigs, chickens, calves, sheep, mice, rats, guinea pigs, man, root nodules on leguminous plants and soil. The presence of antibiotics in feed for cattle, poultry and fish not only applies selective pressure but also tends to reduce the lipid content of the intestinal contents and this increases the frequency of conjugation. 2. *Selection of plasmids specifying drug resistance by widespread use of antibiotics.* Antibiotics are used in animal feed and fish farms and subsequently can pass from these sources by storm water runoff to rivers, lakes and municipal water supplies. The use of antibiotics in hospitals may also apply selective pressure acting to enhance the prevalence of bacteria with drug resistance plasmids, which increases the difficulty in controlling nosocomial infections. 3. *Occurrence of conjugation*- Conjugation occurs in a variety of genera including Escherichia, Shigella, Salmonella, Yersinia, Vibrio, Nocardia, Streptomyces, Enterobacter, Streptococcus, Agrobacterium, Rhizobium, Erwinia, Citrobacter, Hafnia, Chromobacterium Rhodospirillum, Neisseria, Aeromonas, and Bordetella. Each year new conjugation systems are reported. Although intrageneric conjugal gene transfer is more prevalent than intergeneric transfer, it should be emphasized that E. coli can donate and/or receive genetic material by conjugation to and/or from members of all of the above genera except Streptococcus, Neisseria, Mycobacterium, Nocardia and Streptomyces.

Retrovirus Replication

1. A (+) diploid RNA Retrovirus enters a host cell 2. Synthesis of a (-) sense DNA strand from one of the (+) sense RNA strands occurs in the cytoplasm by a reverse transcriptase brought by the virus 3. Digestion of the original (+) sense RNA strand by RNase H then occurs in the cytoplasm 4. : Synthesis of (+) DNA complimentary strand from the (-) DNA strand is completed in the cytoplasm 5. Transport of dsDNA is then taken into the nucleus 6. Integration of the dsDNA into the host chromosomes (now referred to as a provirus) 7. Normal synthesis of mRNA by DNA dependent RNA pol II occurs 8. mRNA splicing occurs and the viral mRNA is transported to the cytoplasm 9. protein synthesis, capsid assembly, budding (numbers don't aline with the picture)

Positive ssRNA replication

1. A (+) ssRNA genome enters a host cell 2. *Translation* of the *original* viral *positive sense RNA* genome by *host ribosomes* is completed to produce a *viral RdRp*, and other proteins. 3. The Viral RdRp binds to the original viral genome to make a *(-) sense antigenome* that is complementary to the viral genome. (no ORFs!)-- makes a complementary RNA strand 4. Viral RdRp binds to (-) sense viral complementary RNA and makes abundant (+) sense viral genomes 5. Translation of (+) sense genomes occurs to make massive amounts of viral proteins for assembly

Lytic Life Cycle

1. Attachment or adsorption of phage to specific receptors on bacterial surface 2. Passage of nucleic acid genome from phage through bacterial cell wall into cytoplasm of host cell (injection of genome into cytoplasm of bacterium) 3. Conversion of bacterium to a *phage-producing cell.* Following infection by most phages, the host bacterium loses the ability to replicate or to transcribe its DNA. This shutdown of host DNA and RNA synthesis is accomplished in a variety of ways depending on the phage. 4. The phage genome then directs the synthesis of phage nucleic acid. Many bacterial replication proteins are used during this process. *RNA phages, however, encode their own replication enzymes because bacteria do not have enzymes that replicate RNA.* 5. Assembly of phage particle in a process is called *morphogenesis.* Structural proteins are brought together and assembled into a phage particle using catalytic proteins. Assembly essentially involves encapsidation of the phage nucleic acid into the protein coat. 6. Release of new phage and lysis of bacterium-- In general, a phage encoded degradative enzyme such as *lysozyme* attacks the cell wall of the host bacterium and causes cell lysis and release of progeny phage. *The Cro protein promotes lytic life cycles-- mutations in the cro gene result in phage that cannot enter the lytic life cycle.*

*Elongation* phase of chromosome replication

1. The DNA is unwound ahead of the replication fork by DnaB (helicase) which is accompanied by *DNA gyrase*. 2. Two DNA polymerase III complexes are present. Both polymerases can only *polymerize nucleotides 5'---> 3'*. Thus, both strands must be synthesized in the 5'--->3' direction. 3. The copy of the parental 3'--->5' strand is synthesized continuously. It is the *leading strand*. As the helix unwinds, the 5'---->3' parental strand is copied in a discontinuous fashion through synthesis of *Okazaki fragments. This is the lagging strand.* 4. *DNA polymerase I* ligases the okazaki fragments

Stages in viral pathogenesis:

1. entry into host 2. primary replication at the site of entry spread in host 3. cell and tissue tropism 4. host immune response 5. cell injury 6. viral clearance or persistence

Negative ssRNA replication

1.A (-) ssRNA genome enters a host cell, along with its own RdRp enzyme 2. Transcription of incoming *original (-) sense genome* by *virus associated RdRp* to produce *(+) sense mRNAs* 3. . Translation of mRNAs to produce proteins including RdRp. 4. Transcription from the original negative sense genome RNA by RdRp also generates a *(+) sense genome size intermediate* which is used as a template to generate a (-) sense genomic RNA 5. Repetition of this RNA synthesis then occurs

What is the temperature range a *Mesophile* can survive in?

10 to 45 degrees celsius

Intercalating agents (ICR 191, acridine dyes)

1CR 191 and acridine dyes (acridine orange, proflavine, and acriflavin) Interact with DNA secondary structure creating distortions which promote replication errors *Causes addition and deletion of bases*

1CR 191

1CR 191 is a planar, acridine-type compound not usually mutagenic until alkylating function is introduced. This compound intercalates between base pairs causing additions and deletions of bases and consequent frameshifts.

How many ATP does *Clostridium* yield from homolactic fermentation?

2

How many ATP does *Lactobacillus* yield from homolactic fermentation?

2

How many ATP does *Streptococcus* yield from homolactic fermentation?

2

How many ATP does *Yeast* yield from alcoholic fermentation?

2

What is the temperature range a *Thermophile* can survive in?

25 to 80 degrees celsius

P/O ratio=2, how many ATPs are produced?

26

How many ATP does *Escherichia* yield from mixed acid fermentation?

3

How many ATP does *Salmonella* yield from mixed acid fermentation?

3

How many ATP does *Shigella* yield from mixed acid fermentation?

3

*All (+) sense RNA viruses have cubical nucleocapsids, undergo translation first when they enter cells, and maintain a cytoplasmic life cycle. 3 families of enveloped viruses 3 families of naked viruses*

3 families of enveloped viruses-- Toga, Flavi, Corona 3 families of naked viruses-- Picorna, Calici, Astro (Don't have to memorize these for exam 1)

P/O ratio=3, how many ATPs are produced?

38

5-bromouracil (BU)

5-bromouracil (BU) is an analog of thymine and primarily pairs with adenine. Thymine can sometimes assume an enol form that can pair with guanine. This conversion can give rise to mutations during replication. 5-BU that is in the enol form exists for a greater fraction of the time than the keto form of thymine which pairs with adenine. Therefore, the potential for causing mutation by 5-BU is greater than that of the enol form of thymine. If BU replaces a thymine, in subsequent rounds of replication, 5-BU can base pair with guanine, which in turn pairs with cytosine resulting in formation of a GC base pair. 5-BU can induce GC to AT as well as from AT to GC transitions.

Base analogs

5-bromouracil (adenine in the *imino* configuration) *Compounds with structures similar to bases that can be incorporated into DNA* Causes GC to AT and AT to GC transitions

What environmental elements make up bacteria?

50 % Carbon 20% Oxygen 14% Nitrogen 8% Hydrogen 3% Phosphorus 1% Sulfur, potassium, and sodium 0.5% Calcium, chloride, and magnesium 0.2% Iron

What is the macromolecular composition of bacteria?

55% Protein 20% RNA 9% Lipid 3.4% LPS 3% DNA

Methicillin-resistant Staphylococcus aureus (MRSA)

90% of all S. aureus strains are resistant to penicillin MRSA infections occur most frequently among persons in hospitals and healthcare facilities (nursing homes, dialysis centers etc) who have weaken immune systems Usually resistant beta-lactam agents Often MRSA are multiply resistant to other commonly used antimicrobials (erythromycin, clindamycin and tetracyline) *Vancomycin drug of choice for severe MRSA infections*

Attenuator

A *cis-active regulatory region* A region within which the activity of RNA polymerase can be stopped, depending on the *secondary structure* of the region, prior to entering the structural genes. The attenuator is a transcription termination site within an operon and is located before the structural genes of the operon. In the trp operon, the attenuator is located within the leader sequence (trpL) after the trpO, but before trpE (refer to the picture in "Trp Operon" flash card)

Gene *(Start of PID 10: Microbial Variation and Mutations)*

A *gene* is a unit of genetic material and can be either functional or nonfunctional. Genes are passed from parent to offspring and has the ability to mutate to alternative states, to recombine with other similar units and to function within an organism resulting in some particular observable characteristic (phenotype). The symbols "+" or "-" after the gene denote the particular state and tells you whether the gene is wild type or mutated, respectively. The term *allele* indicates alternative forms of a particular gene.

*Catabolite Repression* of the lac operon

A *general control of the lac operon* (one of two controls of the lac operon) Studies indicate that some catabolic breakdown product of glucose (no exact identity is yet known) prevents activation of the lac operon by lactose, so the effect is called catabolite repression. The effect of the glucose catabolite is exerted on an important cellular constituent called cyclic adenosine monophosphate (cAMP).

Glycolysis

A 10 step metabolic pathway that converts *one glucose* into *two molecules of pyruvate.* *Two net ATP molecules* are generated by substrate level phosphorylation. (4 gross ATP) *Two NADH molecules* are formed as well

Acyclovir (Zovirax)

A Purine nucleoside analog (cyclic Ribose replaced by linear alcohol) that is considered a chain terminator The virus' thymidine kinase (TK) phosphorylates the analog to Acyclovir monophosphate Cellular kinases make diphosphate and triphosphate The *Acyclo-GTP* competes for GTP and inhibits *viral DNA polymerase* Used against Herpes Simplex Virus Type 1 and 2 (Herpes: HSV-1 & 2) Not effective against Cytomegalovirus (CMV-A common virus that infects people of all ages) Drug resistance may be due to viral TK and/or viral DNA polymerase mutations Side effect: Encephalopathy

Bacteriophage

A bacteriophage also known as a *phage* is an obligate intracellular parasite of bacteria Bacteriophage cannot persist independently Bacteriophage must reside within a host bacterium The bacteriophage genome possesses the genes to encode a variety of proteins. Most of these proteins are synthesized to facilitate production of more phage particles within in the host bacterium. *All phages use the protein synthetic machinery and the energy generating systems of the host to propagate and survive.* For continued survival, phages must deliver its genomic nucleic acid inside of a bacterium and must also protect itself from environmental agents. The life cycle of bacteriophages is dedicated to transforming the host bacterium into a phage-producing factory yielding large numbers of progeny phages.

Lysogen

A bacterium containing a complete set of phage genes (prophage) An infected cell with a bacteriophage in the lysogenic life cycle A lysogen cannot be *reinfected or superinfected* by a phage of the type that *first lysogenized the bacterium*, i.e., a lysogen is immune to superinfection and this trait is called *immunity*. A lysogen can initiate a lytic cycle by a process called *induction.* For example, ultraviolet light will cause excision from the bacterial chromosome of a lambda prophage and induce phage production.

Spheroplasts

A bacterium or plant cell bound by its plasma membrane, the cell wall being deficient or lacking and the whole having a spherical form.

Duplication

A base sequence repeats itself and causes identical sequences to appear in tandem. AGGT*CCGTA*GCAAC Wild Type AGGT*CCGTACCGTA*GCAAC Tandem Duplication

Mutant

A cell carrying a mutation

Viral Genome Reassortment

A characteristic of *all segmented genome RNA viruses* RNA segments are exchanged between related viruses Examples: Orthomyxo, Bunya, Arena, and Reo families responsible for influenza pandemics

Antibiotic *(Start of PID 14-16: Antimicrobial Agents Cards)*

A chemical *produced by microorganisms* which exhibits *selective toxicity* for other select groups of microorganisms.

Mutagen

A chemical or physical agent that interacts with DNA and causes a mutation.

Regulon

A common regulatory element that controls genes that are involved in a related function, but are dispersed on the bacterial chromosomes So in other words, a regulatory element that can control genes not continuous with it. Example: arginine regulon

Viable count

A culture of bacteria is diluted in a broth and then plated, each plate increases in dilution. Then bacteria are incubated and colonies are counted. Each colony came from one cell Colonies have millions of cells Best plate count is typically between 30 to 300 colonies Best plate count x Dilution factor= Colony-forming units Example Picture: 65 x 10^3= 6.5 x 10^4 cells per milliliter of original sample

Determining Bacterial Number Example

A flask is inoculated to a density of 5 x 10^4 cells/ml. How many cells are there at the end of 120 minutes if the generation time is 30 min.? *Step 1* Tg=t/n so n=t/tg n= 120/30 = *4* *Step 2* b=a x 2^n b= (5 x 10^4) x 2^4 b= (5 x 10^4) x 16 b= 80 x 10^4 = *8.0 x 10^5 cells*

Inversion

A gene rearrangement in which a segment of DNA is cut out and reinserted with a reverse orientation. AGGT*CCGTA*GCAAC Wild Type ACGT*ATGCC*GCAAC Inversion

Mutation

A lesion in a gene; An alteration in the primary sequence of DNA; Can be induced by mutagens

Kinetics of bacterial killing

A linear line indicates on bacterial target, but a bent line indicates more than one bacterial target. Plots of log # survivors vs. time are linear Exponential with time Not all organisms die at once Rate of killing increases with increasing temperature and increasing concentration

Live-attenuated virus

A live virus that multiplies in the human body, but the disease causing ability is lost (attenuation) Examples: oral polio (Sabin) mumps, measles, rubella (MMR) yellow fever (17D) varicella-zoster (VZV) Rotavirus Nasal flu Adenovirus (military purposes only)

Oxidation-Reduction Potential (Eh)

A measure of the tendency of a chemical species to acquire electrons and thereby be reduced. Measured in volts (V), or millivolts (mV). Each species has its own intrinsic reduction potential; the more positive the potential, the greater the species' affinity for electrons and tendency to be reduced. Positive Eh indicates the oxidative state (lost electrons) Negative Eh indicates the reduced state (gained electrons) *Eh for Aerobes: ~+0.3 volt* *Eh for Anaerobes: -0.2 volt*

Missense mutation

A missense mutation occurs when the result of a mutation is that the amino acid sequence is affected such that *one amino acid is replaced by another amino acid.* Missense mutations may result in total inactivation of an enzyme or just subtle changes in catalytic activity. Gross changes do not occur in the topology of the enzyme even when activity is altered. These altered enzymes are detectable with antibodies prepared against the native enzyme. Because of their serologic cross-reactivity these enzymatically inactive proteins are called *"cross-reactive material" (CRM).* *Point Mutation-- can be transition or transversion*

Define Mutagen

A mutagen is a chemical agent or a physical agent that causes mutations to occur. Their modes of action may differ but they alter the nucleotide sequence in DNA. They exert their effect by promoting errors in replication or in the repair of DNA.

Forward mutation

A mutation in which the mutant phenotype is initially achieved. For example, construction of an Arg+ → Arg- mutant strain.

Nonsense mutation

A mutation results in a *termination codon is generated*, the mutation is considered nonsense. Three kinds of nonsense mutations, each representing one of three particular base sequences, are produced that do not correspond to any amino acid. a. UAG b. UAA c. UGA Nonsense mutations result in truncated proteins where the length of the polypeptide and the effect on biological activity of the protein depend on where in the coding sequence the termination codon is generated. *Point Mutation-- can be transition or transversion*

Leaky mutation

A mutation that results in a *gene product being semi-functional.* For example, a bacterium carrying a mutation in a gene encoding an enzyme involved in adenine biosynthesis grows very slowly in the absence of adenine. A true mutant will not grow at all in the absence of adenine.

Obligate Parasite *(Start of PID 12: Phage Biology and transduction Cards 571-593)*

A parasite that entirely depends upon a host for its nourishment, reproduction, habitat, and survival

Prophage

A phage genome integrated in the bacterial chromosome A prophage can be induced by various agents that damage the lysogenic bacterium to initiate the lytic life cycle (induction)

Foscarnet

A phosphate analog known as Trisodium phosphonoformate that *inhibits viral DNA polymerase* Licensed for CMV retinitis (Cytomegalovirus retinitis, also known as CMV retinitis, is an inflammation of the retina of the eye that can lead to blindness.) Also blocks HIV Reverse Transcriptase

Stem and loop structure by plasmid containing inverted repeats

A plasmid is denatured Rapid cooling allows self-annealing Annealing forms a stem and loop structure due to inverted repeats

Mobilizable plasmid

A plasmid that can prepare their DNA for transfer but cannot transfer its own DNA.

Self-transmissible plasmid

A plasmid that is both mobilizable and conjugative, A plasmid that can prepare its DNA for transfer to another bacterial cell and then mediate its own transfer via conjugation.

What is a prion?

A protein particle that is believed to be the cause of brain diseases such as BSE, scrapie, and Creutzfeldt-Jakob disease. Prions are not visible microscopically, contain no nucleic acid, and are highly resistant to destruction.

Ribavirin

A purine nucleoside analog that inhibits both DNA and RNA viruses by RdRp A nasal spray against Respiratory Syncytial virus (RSV) bronchiolotis and pneumonia Recently used against Hantaan and arena viruses

Transversion Mutation

A pyrimidine may also be replaced by a purine or vice versa to cause a transversion.

Explain a Two-component regulatory system

A signal activates the *sensor protein* to become an active protein kinase that phosphorylates itself and donates the phosphoryl group to a response regulator. The phosphorylated *response regulator* acts on DNA to either allow or prevent the expression of a specific set of genes.

*Streptomycin resistance*

A single amino acid replacement in S12 protein of the 30S ribosomal subunit allows certain bacteria to be resistant to streptomycin

Amantidine and Rimantadine

A tricyclic amine that blocks the entry/uncoating of Flu A only. Binds to matrix proteins of influenza virus A Inhibits Flu-A, Flu B or C entry and uncoating can still occur

Hemagglutination Inhibition (HI)

A virus specific antibody (in test serum) blocks hemagglutination caused by a virus. *Many viruses carry hemagglutinins on surface*

Killed or inactivated vaccine

A whole virus particle that were heated or chemically inactivated Do not multiply in the human body Examples: killed polio (Salk) influenza A&B (every year) rabies hepatitis A

Two-component regulatory system

A widely used mechanisms in bacteria to control virulence factor expression and involves coordinate regulation of multiple genes in response to an environmental signal (signal transduction). In this system, two gene products interact to control the transcription of other genes whose expression is thought to be required in a specific host environment. The two proteins that make up the system are a *sensor protein* with kinase activity (autokinase or sensor kinase) and a *response regulator* that activates transcription after being phosphorylated by the sensor protein. *The sensor/transmitter is activated by a signal to become an active protein kinase that phosphorylates itself and donates the phosphoryl group to a response regulator. The phosphorylated form of the response regulator acts on DNA to either turn on or turn off transcription of a specific set of genes.* A single bacterial species may have a number of two-component systems that allow the bacterium to adapt to a variety of environmental conditions.

Turbidity

A word describing how light passes through a sample of liquid as a measure of how many particles are suspended in that liquid. Microbiologists use turbidity as a measure of cell density within a culture sample. Microbiologists use machines called photometers and spectrophotometers that shine different types of light through culture samples to determine turbidity. The general assumption is that the higher the turbidity, the higher number of cells within the culture.

Abortive transduction

Abortive transduction occurs when the DNA transferred to the recipient cell does not become integrated into the genome of the recipient cell and is not replicated. Since this DNA is not replicated, it is not stably inherited and will be loss during subsequent cell divisions.

Asepsis

Absence of microorganisms in living tissue

Absolute defective mutants

Absolute defective mutants display mutant phenotype under all conditions of growth, temperature, etc.

Acridine dyes

Acridine dyes (proflavin, acridine orange intercalate between stacked bases causing additions and deletions resulting in a shift in the reading frame.

Inhibitors of Chromosome Replication

Actinomycin D (intercalate between base pairs) Proflavine (intercalate between base pairs) Ethidium bromide (intercalate between base pairs) Mitomycins (cause cross-linking) Metronidazole (cause DNA strands to break) Nitrofurans (cause DNA strands to break) DNA gyrase inhibitors- Quinolones

Ion Dependent Transport (IDT)

Active Transport Used for the accumulation of *ions and amino acids* Uses a hydrogen ion (H+)- pmf (or another cation to drive the transport process) Example: lactose permease of E. coli Also referred to as *Shock-insesnsitive*

Binding Protein Dependent Transport (BPDT)

Active Transport Used to transport *sugars and amino acids* Uses binding proteins in the periplasm that binds the solute to be transported, admitting it into a membrane channel to be transported across the membrane Example: histidine, maltose, galactose Also referred to as *Shock-Sensitive*

*Fourth Generation Cephalosporins* affect what type of bacteria?

Active against most Enterobacteriaceae and Pseudomonas aeruginosa Increased stability to beta-lactamases

Vidarabine

Adenine arabinoside is phosphorylated by *cellular enzymes* creating a chain terminator for viral proteins Used against HSV encephalitis and Zoster (VZV)--Herpes (don't know if we need to know for exam)

2- aminopurine

Adenine in the *imino configuration* is a mutagen in that it pairs with cytosine (2- aminopurine). The pairing with cytosine is weak because it forms only a single hydrogen bond. When this occurs, there is a transition from an AT pair to a GC pair after several rounds of replication.

*There are 3 families of DNA viruses that are non-enveloped/naked. All are one-segmented and cubical 1/3 families is single stranded 1/3 families is circular, but others are linear 1/3 families is bullet shaped, but the others are small*

Adeno- linear and bullet Papova-circular and small Parvo- ssDNA, linear, and small (Don't have to memorize these for exam 1)

What occurs if you give more than one antibiotic to a resistant bacteria?

Administration of several drugs at once can lead to counteracting drug resistance Example: Resistance to drug A is 10-6 Resistance to drug B is also 10-6 Given in combination - frequency of a bacterium becoming resistant to A and B = 10-12

M. Tuberculosis and Bacillus Subtilis are examples of what type of bacteria? a. aerobe b. anaerobe c. facultative anaerobe d. aerotolerant e. microaerophilic

Aerobic Bacteria

What are the types of energy metabolism bacteria can undergo? *(Start of PID 4-5: Bacterial Physiology and Nutrition Cards 55-147)*

Aerobic Respiration Anaerobic Respiration Fermentation Catobolism of glucose can go through three different pathways: Glycolysis, TCA cycle, and the hexose monophosphate shunt (Pentose Phosphate Pathway)

S. pneumonia and S. pyogenes are examples of what type of bacteria? a. aerobe b. anaerobe c. facultative anaerobe d. aerotolerant e. microaerophilic

Aerotolerant anaerobic bacteria

DNA virus replication

After entry into the cell, DNA is transported to the nucleus where DNA replication (by host polymerase), mRNA synthesis (by host RNA polymerase II), and splicing occurs. Then mRNA is transported out of the nucleus to undergo protein synthesis All DNA viruses undergo this except *poxviruses*

Agar Diffusion Techniques

Agar Diffusion (Kirby-Bauer) Techniques Used to determine whether an infectious agent is susceptible to one or more antimicrobials Infectious agent is spread on agar plate and discs or strips impregnated with antimicrobials are applied to the agar surface and the plates are incubated for 18-24 hrs Zones of inhibition are monitored and compared with known values to determine susceptibility for a particular infectious agent

Bactericide

Agent that kills

Bacteriostatic

Agent that prevents growth temporarily

Chloramphenicol Resistance summary

Agent: Chloramphenicol Plasmid-borne: yes Mechanism of resistance: Acetylation of OH groups

Ciprofloxacin resistance summary

Agent: Ciprofloxacin Plasmid-borne: no Mechanism of resistance: Mutated DNA gyrase

Erythromycin resistance summary

Agent: Erythromycin Plasmid-borne: yes Mechanism of resistance: Methylation of 23S RNA

Methicillin resistance summary

Agent: Methicillin Plasmid-borne: no Mechanism of resistance: altered penicillin binding protein

Nalidixic acid resistance summary

Agent: Nalidixic acid Plasmid-borne: no Mechanism of resistance: Mutated DNA gyrase

Penicillin resistance summary

Agent: Penicillins/cephalosporins Plasmid-borne: yes Mechanism of resistance: β -lactam ring hydrolysis by β -lactamases (penicillinases* and *cephalosporinases*) Altered penicillin binding protein

Rifampin resistance summary

Agent: Rifampin Plasmid-borne: no Mechanism of resistance: Altered beta subunit of RNA polymerase

Aminoglycoside resistance summary

Agent: Streptomycin Plasmid-borne: yes Mechanism of resistance: Inactivating enzymes

Sulfonamide resistance summary

Agent: Sulfonamide Plasmid-borne: yes Mechanism of resistance: Resistant dihydropteroate synthetase

Tetracycline resistance summary

Agent: Tetracycline Plasmid-borne: yes Mechanism of resistance: Excretion

Trimethoprim resistance summary

Agent: Trimethoprim Plasmid-borne: yes Mechanism of resistance: Resistant dihydrofolate reductase

Vancomycin resistance summary

Agent: Vancomycin Plasmid-borne: yes Mechanism of resistance: Altered peptidoglycan binding site

Disinfection method: *Alcohols*-*(Specifically asked on the study guide)*

Alcohols - disorganize lipids and denature proteins Used in the range of 70-95% (70% alcohols are the best because they have a higher water composition) Not effective against bacterial spores and many viruses

Sterilization method: *Aldehyde sterilization*

Aldehydes are high level disinfectants/sterilants Target functional groups on proteins *Formaldehyde* - alkylating agent that replaces H atom in COOH, SH or OH groups on proteins with hydroxymethyl group *Formalin* - 37% formaldehyde; used in preserving tissues *Glutaraldehyde* - alkylating agent 2% solution is called *Cidex* 10x more effective than formaldehyde as sporidical agent and not as toxic.

Genetic Transformations occur in the following genera except: Haemophilus Neisseria Bacillus Staphylococcus Streptococcus

All of these organisms are *competent* meaning they have the ability to take up transforming DNA and be genetically changed by it. *Neisseria gonorrhoeae* are *naturally competent* meaning they're always competent, but they will only take up specific DNA sequences from their own species. *S. pneumonia and H. influenzae* are *transiently competent* meaning they are induced or artificial competent.--- Bacillus subtitles and S. pneumoniae do not require specific DNA uptake sequences and will take up any DNA, however, only *homologous DNA* will recombine into their chromosome, A *competence activator protein* is needed in Streptococcus.

Common virus characteristics

All viral genomes undergo transcription and replication (multiplication) All viruses code for at least a few virus specific proteins Only inside a host cell can viruses express their genes and replicate their genomes All viruses make use of host translational apparatus

Virus composition

All viruses contain a structure known as *nucleocapsid.* Some viruses enclose their nucleocapsid within a *lipid envelope.* If a virus particle contains an envelope, it is known as enveloped. If a virus particle contains only nucleocapsid, it is known as naked or non enveloped.

What characteristics do all viruses lack?

All viruses require a host cell to multiply because they *lack machinery to translate mRNA.* They also lack the ability to generate energy, they do not have a cytoplasm or electron transport (no action potential), and they lack cellular structure.

Viral Genome Recombination

Almost all RNA and DNA viruses can undergo this process Cleavage and Ligation of DNA genomes Template switching by polymerase on RNA genomes-- meaning polymerase jumps from one genome to another

Gastrointestinal problems caused by antimicrobial agents

Almost all antibiotics, by *altering the gastrointestinal flora*, can cause an *overgrowth of Clostridium difficile*, which produces a toxin that causes serious colon reactions. Alteration of microbial flora by antibiotics also can result in overgrowth of Candida in the mouth, vagina, or gastrointestinal tract.

Pili

Also known as *Fimbriae* hairlike structures on surface of bacteria smaller in diameter than flagella Expressed by the *PilE gene* Two types: *F pili* are sex pili and are involved in conjugation *Common Pili* are used for cell adherence and attachment-- without Pili, bacteria cannot attach to the host epithelium and invade the host. important virulence factors - E. coli, N. gonorrhoeae

Viral Phenotypic mixing

Also known as phenotypic masking

Viral Phenotypic masking

Also known as phenotypic mixing When two or more similar viruses infect same cell, the genome of one virus can be packaged into the capsid of the other virus (masking) or by a mixture of capsid proteins (mixing). *But, the genome of the progeny virus dictates the identity of the subsequent population.*

Rifamycins

Also referred as *Rifampin* Mechanism of Action: Inhibits DNA-dependent RNA polymerase which blocks RNA transcription and ultimately protein synthesis Spectrum of Activity: Broad, including mycobacteria Clinical Use: Important in the treatment of *tuberculosis, leprosy* and in the prophylaxis of meningococcal meningitis

Transposons

Also referred to as *mobile genetic elements* or *hopping genes* Segments of double stranded DNA that can move from place to place within a genome or between genomes. Transposons can move from the chromosome to a plasmid, from a plasmid to the chromosome, from one plasmid to another, or from one site on the same molecule to another The insertion of a transposon into a new site in DNA is called *transposition.* Can encode conjugation functions (referred to as conjugated transposons) Types: insertion elements (IS) and composite structures containing them Tn3 class

*Sulfones*

Also referred to as Dapsone Mechanism of Action: Similar to that of sulfonamides Spectrum of Activity: Gram-positive and gram-negative bacteria, acid-fast bacteria (Mycobacterium leprae) Clinical Use: *Treatment of leprosy.* Dapsone is too toxic for use against routine bacteria infections. Toxicities: (highly toxic) polyneuritis, psychosis

Nitroimidazoles

Also referred to as Metronidazole Mechanism of Action: The reduction of the nitro group of the compound is believed to result in the *production of free radicals that damage DNA and inhibit replication.* Spectrum of Activity: anaerobic bacteria and certain parasites Clinical Use: Treatment of infections caused by anaerobic gram-negative bacilli (Bacteroides fragilis) and parasites (Trichomonas vaginalis)

Peptidoglycan- Cell wall

Also referred to as murein or mucopeptide A rigid mesh that provides cell shape and provides osmotic protection (prevents cell lysis) Linear polysaccharide chains form the backbone of cell wall *~ 40 layers thick in gram-positive bacteria and ~ 1-2 layers thick in gram-negative bacteria*

Nitrofurantoin

Also referred to as nitrofurans Mechanism of Action: The drug must be *reduced* in the organism to become active and create highly reactive electrophilic intermediates that can-- The precise mechanism of action is not known, but probably is associated with *causing damage (breakage of strands) to DNA & react with bacterial ribosomal proteins non-specifically causing complete inhibition of protein synthesis* Spectrum of Activity - gram-positive and gram-negative bacteria Clinical Use - Urinary tract infections; prevention of bacteriuria after prostatectomy *Treatment of bladder bacteriuria caused by Escherichia coli, Klebsiella, Enterobacter, indole-negative Proteus mirabilis and enterococci*

Chemical mutagen

Alters a base already incorporated in DNA and changes its bonding specificity Chemically alters a base so that a new base pair appears in daughter cells in a later generation Examples include: • Nitrous acid • Hydroxylamine •Alkylating agents (EMS, MMS, nitrosoguanidine) • Base analogs (5-bromouracil) • Intercalating agents (ICR 191, acridine dyes) • Ultraviolet light

*Extended spectrum penicillins*

Aminopenicillins (*ampicillin, amoxicillin*) Carboxypenicillins (carbenicillin, ticarcillin) *Amdinocillin* Spectrum of Activity- Similar to penicillin G (Gram-positive bacteria & Neisseria) as well as Enterococcus faecalis, Haemophilus influenzae and many coliforms (amdinocillin) Clinical Use: Urinary and respiratory infections Enteric fever In combination with other drugs in severe systemic infections

Semisynthetic antibiotic

An *antibiotic* whose structure has been *altered by chemical reactions.*

*Spectinomycin* State the mechanism of action, spectrum of activity, clinical use, and toxicity

An aminocyclitol antibiotic- contains aminocyclitol ring but lacks amino sugar residue Mechanism of Action: Interferes with mRNA-ribosome interaction *but causes no misreading* Spectrum of Activity: Neisseria gonorrhoeae (penicillinase-producing and chromosomally mediated penicillin-resistant strains) Clinical Use: Clinical use restricted to treatment of uncomplicated gonorrhea in patients allergic to penicillin Toxicities: neuromuscular paralysis ototoxicity nephrotoxicity *30S ribosome subunit inhibitor*

Gancyclovir

An extra hydroxymethyl group in Acyclovir is added Phosphorylated initially by both viral and cellular enzymes Diphosphate and triphosphate formation by cellular enzymes occurs *Selectively inhibits viral DNA polymerases* Effective against CMV and other Herpes viruses Resistant mutants are due to TK and DNA polymerase mutations

Phenotype

An observable property of an organism Measurable property produced by the genotype in cooperation with environment For example, a cell that can synthesize the amino acid leucine is denoted Leu+, if it cannot, it is denoted Leu-.

What are heterotrophs?

An organism that is able to form its nutritional requirements from complex organic substances, like *glucose.*

What are autotrophs?

An organism that is able to form nutritional organic substances from simple inorganic substances such as *carbon dioxide.*

C. Botulinum and Bacteroides are examples of what type of bacteria? a. aerobe b. anaerobe c. facultative anaerobe d. aerotolerant e. microaerophilic

Anaerobic Bacteria

Direct ELISA

Antibody coated well

Antigenic Variation

Antigenic variation results in changes in the composition or structure of molecules residing on the surface of organisms that allows the organism to avoid recognition by specific antibodies occurring during the course of infection. The changes are produced by genetic rearrangements that occur at a high frequency within the population. Chronic and repeated infection is characteristic of diseases caused by infectious agents that undergo antigenic variation. Antigenic variation occurs in African trypanosomes (causative agent of African sleeping sickness), Borrelia species (which causes relapsing fever) and Neisseria gonorrhoeae. Antigenic variation which occurs in these organisms results from the reassortment and recombination of duplicated gene segments.

How can antimicrobial agents cause toxicity?

Antimicrobial agents can cause toxicity: (1) directly, (2) they can interact with other drugs to influence the toxicity of another agent, or (3) they can alter microbial flora

Antimicrobial agents function by which general mechanisms?

Antimicrobial agents function by the following general mechanisms: 1) inhibition of bacterial cell wall synthesis 2) inhibition of cytoplasmic membrane function 3) inhibition of nucleic acid synthesis 4) inhibition of protein synthesis 5) inhibition of metabolic pathways

What are the clinical uses for *Second Generation Cephalosporins*?

As second line drugs in seriously ill patients As an alternative to aminoglycosides in patients with renal impairment For preoperative prophylaxis

What are the clinical uses for *First Generation Cephalosporins*?

As second line drugs in seriously ill patients As an alternative to aminoglycosides in patients with renal impairment For preoperative prophylaxis

What are the clinical uses for *Fourth Generation Cephalosporins*?

As second line drugs in seriously ill patients infected with *susceptible aerobic gram-negative bacilli* As an alternative to aminoglycosides in patients with renal impairment For preoperative prophylaxis

What are the clinical uses for *Third Generation Cephalosporins*?

As second line drugs in seriously ill patients infected with *susceptible aerobic gram-negative bacilli* As an alternative to aminoglycosides in patients with renal impairment For preoperative prophylaxis

State the steps in virus life cycle

Attachment to cells/entry Genome replication, RNA synthesis Accumulation of capsid proteins Nucelocapsid assembly Release of virus particles

Attenuation

Attenuation is a mode of regulation prevalent in amino acid biosynthetic systems that occurs by translational control of transcription termination. *This process senses the level of specific aminoacylated tRNA in a cell rather than the level of free amino acid.* Attenuation effects a reduction of the transcription of distal proteins of an operon and can reduce transcription 8-10 fold.

What is the clinical use of imidazoles?

Azole compounds are used for the treatment of superficial and systemic fungal infections. Several of these compounds exhibit toxicities less than that of amphotericin B and flucytosine.

What is the mechanism of action of imidazoles?

Azole compounds exert fungistatic activity by *inhibiting synthesis of ergosterol*, an integral component of the cell membrane, and rendering the cytoplasmic membrane more permeable. The result is leakage of intracellular contents.

Bacillus subtilis

Bacillus subtilis- a rod-shaped, Gram-positive bacteria that is found in soil and the gut of humans and some types of animals. Does not require specific DNA uptake sequences and *will take up any DNA* *Only homologous DNA* will recombine into the recipient chromosome

What is the mechanism of action of *bacitracin*?

Bacitracin inhibits the dephosphorylation of the pyrophosphate form of the phospholipid carrier (Bactoprenol-P) which prevents the phospholipid carrier from reacting with the peptidoglycan precursor, UDP-N-acetylmuramyl- pentapeptide. Also referred to as the polypeptide antibiotic

*Semisynthetic penicillins*

Bacteria became resistant to the original penicillin so the structure of the original penicillin was altered to change the spectrum of activity and the acidic and enzymatic susceptibility to degradation. The chemical modification of penicillin gave rise to the *semisynthetic penicillins* which can be grouped into *extended spectrum*, *penicillinase-resistant* and *anti- pseudomonas* penicillins.

How can you classify bacteria based on oxygen utilization?

Bacteria can be classifies as aerobes, aerotolerant, microaerophilic, facultative anaerobes, and anaerobes

Nutrients that support the growth of bacteria are called media (medium).

Bacteria can grow in *minimal medium* (inorganic salts, glucose, etc) or *nutrient medium* (everything they need, also referred to as Lysogeny (LB) broth)

Aerotolerant Anaerobe Bacteria

Bacteria that *always ferment glucose* but are insensitive to the presence of oxygen. They live by fermentation alone whether or not O2 is present in their environment. (tolerate oxygen) Growth response to *aerobic and anaerobic conditions* Enzymes: *Superoxide Dimutase (SOD)* (contain other enzymes like peroxidase) Examples: *S. pneumonia and S. pyogenes*

Protoplast

Bacterial cell whose cell wall has been removed

Central Dogma

Bacterial cells undergo DNA replication Transcription Translation

What factors influence bacterial growth?

Bacterial growth rate depends on several factors: Species of bacterium Chemical composition of the medium Temperature

Bacterial promoters

Bacterial promoters are *binding sites* for Holoenzyme RNA polymerase Consist of a *-10* sequence called *Pribnow box* that has a sequence of bases similar to TATAAT (consensus sequence) And another sequence called a *-35 region.*

Base analog mutagens

Base analog mutagens are substances other than standard nucleic acid bases which can be incorporated into a DNA molecule by the normal polymerization process. 5-bromouracil (BU) and 2- aminopurine

Transition Mutation

Base substitutions involve the replacement of a pyrimidine with a different pyrimidine or a purine with a different purine. These are called transitions.

What is the clinical use of polymyxins?

Because of toxicity, polymyxins should only be used as second-line drugs in the treatment of gram-negative infections. Polymyxin B has been prepared for topical and ophthalmic use, usually in combination with other compounds such as neomycin and bacitracin.

Explain the role of endonuclease in bacterial conjugation

Before transfer of the F plasmid to the recipient cell occurs, *an endonuclease* nicks one strand of the F plasmid at the end of the origin of replication of the plasmid. The 5' end of the single strand is transferred and is converted to a duplex molecule followed by circularization in the recipient cell. Since the female cell acquires the F plasmid, the female cell is converted to a male cell. Therefore, a cross between an F+ cell and an F- cell results in the following: F+ x F- → F+

*Natural Penicillins*

Benzyl penicillin (penicillin G) Phenoxymethyl penicillin (penicillin V) Spectrum of Activity-Gram-positive bacteria, Neisseria Clinical Use: Streptococcal and pneumococcal infections Sensitive staphylococcal infections (penicillinase-negative) Clostridial infections Meningitis, gonorrhea, syphilis, anthrax, actinomycosis

*Beta-lactamase inhibitors*

Beta-lactams, such as *clavulanic acid and sulbactam*, have little or no antimicrobic activity, can bind irreversibly to β-lactamase enzymes making them inactive. These compounds are used in combination with a β-lactam antibiotic. Examples are combinations of amoxicillin and clavulanic acid (augmentin), ticarcillin and clavulanic acid and ampicillin and sulbactam. Spectrum of Activity: β-lactamase-producing coliforms, staphylococci, Bacteroides Clinical Use: Urinary and respiratory infections Skin and soft tissue infections caused by amoxicillin-resistant bacteria

What is another name for shock-sensitive transport?

Binding Protein Dependent Transport (BPDT) Transport is mediated by binding proteins in the periplasm

Antibody dependent complement mediated cell lysis

Binding of IgG or IgM antibody to virus infected cell causes activation of complement Membrane attack complex thus destroys virus-infected cells C3b bound antibody-coated target cells can also be phagocytosed This type of antibody and complement mediated target cell destruction is also a Type II Hypersensitivity reaction

Dimeric DNA polymerase

Both strands are synthesized in concert by a *dimeric DNA polymerase* situated at the replication fork. Thus the 5'--->3' parental strand (lagging) must wrap around in trombone fashion so that the unit of the dimeric DNA polymerase replicating it can move along it in the 3'---->5' direction.

Broth Dilution Method

Broth or Agar Dilution Method Serial dilutions of an antimicrobial agent are made in broth or agar Then a standardized concentration of organisms is added to broth or spread on the agar followed by incubation for 16-18 hrs. The lowest concentration of drug required to inhibit growth is considered the MIC

Correlation Of Mutagenesis And Carcinogenesis Using Ames Test

Bruce Ames and collaborators utilized a series of histidine auxotrophs of Salmonella typhimurium to develop a test that can be used to determine by measurement of the reversion from histidine auxotrophy (His-) to prototrophy (His+) if carcinogenic compounds are capable of increasing the mutation frequency. Tests are done by spreading an auxotrophic strain on minimal glucose plates in the presence of the carcinogen or mutagen. An increase in the reversion rate is assessed by counting (His+) colonies and comparing the counts with counts of control plates. An added dimension of the test involves including in the medium a sterile microsomal preparation to act as a metabolic activator. If the compound displays carcinogenic activity only in presence of liver microsomes, this suggests that the compound has to be converted to an active form to have biological activity.

What is the clinical use of tetracyline?

Brucellosis Cholera Infections of Chlamydia Rickettsia and Mycoplasma pneumoniae Urinary tract infections caused by sensitive organisms Usually don't use in small children

*Enterobacter* undergo what type of fermentation?

Butanediol fermentation producing acetonin, butanediol, and CO2

*Klebsiella* undergoes what type of fermentation?

Butanediol fermentation producing acetonin, butanediol, and CO2

Catabolite activator protein (CAP)

CAP is a dimeric protein and consists of identical 22,500 dalton subunits. The protein has binding sites for cyclic AMP and DNA Coded for by the crp gene *cAMP can bind to CAP forming a complex, this complex then can bind to a short nucleotide sequence that lies before the lac promoter sequence*

Receptor-Ligand interactions during viral attachment:

CD4 and chemokine receptors-HIV,gp120 Sialic acid-Influenza, HA ICAM-1- Rhinovirus CR2 (C3d receptor)- EBV Acetyl Choline receptor- Rabies

Antibodies bind to the __________ of naked viruses.

Capsid Antibodies bind to the capsid of naked viruses.

*Anti-Pseudomonas penicillins*

Carbenicillin Ticarcillin Ureidopenicillins (*azlocillin, mezlocillin, piperacillin*) Spectrum of Activity -Similar to extended spectrum penicillins, in addition, Pseudomonas aeruginosa, Klebsiella and most Proteus species Clinical Use: Urinary and respiratory infections Infections from burns In combination with other drugs in severe infections

Binding Protein Dependent Transport in Bacteria Summary

Carrier mediated? Yes Concentration? Goes against the conc. gradient Specific? Yes Energy Expended? ATP Solute modified during transport? no

Ion Dependent Transport in Bacteria Summary

Carrier mediated? Yes Concentration? Goes against the conc. gradient Specific? Yes Energy Expended? Pmf Solute modified during transport? no

Facilitated Diffusion in Bacteria Summary

Carrier mediated? Yes Concentration? Goes with the conc. gradient Specific? Yes Energy Expended? no Solute modified during transport? no

Group Translocation System in Bacteria Summary

Carrier mediated? Yes Concentration? N/A Specific? Yes Energy Expended? PEP Solute modified during transport? Yes (phosphate added)

*Positive sense RNA viruses* denoted (+) sense ssRNA

Carry *infectious RNA* No RdRp is present in the virus particle, but is later translated in a host cell from RNA The introduction of pure RNA into cells leads to infection and cell death! Viral genome and its mRNA (when made) are of *same polarity* or sense

Colicin E1

Causes impairment of ATP formation which causes cessation in all macromolecular synthesis.

How can Transposable Elements lead to mutagenesis?

Causes mutations by "hopping" into genes and interrupting the genes Many bacteria contain long DNA segments that are mobile, i.e. capable of movement from site to site on DNA, called transposable elements. Included in the transposable elements are insertion sequences (IS elements) and transposons. When insertion sequences and transposons replicate, one copy of the element remains at the original insertion site and the other copy is inserted in another region of a replicon such as a plasmid or bacteria chromosome. This process is called transposition. When transposition occurs, the element frequently inserts itself into a gene, thereby mutating that gene. E. coli can be infected by a transposable bacteriophage called Mu which inserts its DNA during its replication cycle into the bacteria chromosome at random sites, and thereby generates mutations.

Nitrous acid

Causes oxidative deamination of *cytosine to uracil*, *adenine to hypoxanthine* and *guanine to xanthine.* Causes transition mutations

Ultraviolet Light

Causes pyrimidine dimer formation Mainly between thymines

Colicin E2

Causes single strain nicks in DNA and therefore interferes with DNA synthesis. Also referred to as DNAse

Syncytium formation

Cell-cell fusion to form giant cells Syncytia is formed by fusion of an infected cells with neighboring cells leading to the formation of multi-nucleate enlarged cells. Examples of viruses that cause this formation: HIV Paramyxo Herpes

Lac operon: *Lactose being the sole carbon source in the medium*

Cells growing in a medium containing lactose as sole source of carbon will have *high levels of beta-galactosidase* which is needed to split lactose into glucose and galactose. The binding of the *inducer (lactose)* to the repressor results in a conformational change of the repressor which prevents the binding of the repressor to the operator region.

Cell-Mediated Immunity

Cells infected with viruses express two types of antigenic determinants at their cell surface 1. intact glycoproteins as part of protein trafficking- specific to enveloped viruses 2. endogenously processed viral peptides bound to MHC class I proteins- all viruses

Diauxie Growth Curve

Cells will metabolize glucose first. A growth lag follows depletion of glucose that represents the amount of time it takes to turn on transcription of the lactose operon.

*Cephalosporins*

Cephalosporins are derivatives of *7-aminocephalosporanic acid*, the basic structure of which is a beta-lactam ring fused with the *six-member dihydrothiazine ring* (instead of the five-member thiazolidine ring characteristic of the penicillins) *There are four generations of cephalosporins* *R1 and R2* represent side chains that determine most of the antibacterial and pharmacological properties of the cephalosporin) *B* represents the β-lactam ring *A* represents the dihydrothiazine ring *β-lactamases* act on the C-N bond in β-lactam rings *Amidases* act on the O=C-N bond between the β-lactam ring and acyl side chain

How is drug resistance achieved in bacterial cells? *(Start of PID 17: Mechanisms of drug resistance Cards 381-410)*

Chromosomal mutations Genetic Exchange (R-factors and plasmids) Transposons Biochemical mechanisms

IFN-gamma is used to treat what conditions in the United States?

Chronic Granulomatosis

Why is plasmid-mediated resistance more important than chromosomal-mediated resistance?

Clinical Significance Plasmids have high rate of transfer Transfer mediated by Conjugation Occurs in many species

What are two sporulating genera that have medical significance?

Clostridium sp. and Bacillus anthracis

Why are antimicrobial drugs used in combinations?

Combinations of drugs may be used: • To achieve a synergistic killing effect • To prevent the emergence of resistant organisms during therapy • To broaden the antimicrobial spectrum for empirical therapy • Treatment of polymicrobial infections

Nonsense Codons

Commaless Code Universal

Azidothymidine (AZT)

Commonly referred to as AZT or Azidothymidine (Zydovudine) A *thymidine analog (3' OH replaced by N3)* is phosphorylated by cellular enzymes and used as a *competitive Inhibitor of Reverse Transcriptase.* The chain terminator for DNA synthesis has a higher affinity for RT than host DNA polymerase Used against HIV Resistant viruses carry mutations in reverse transcriptase

Sterilization *(Start of PID 18: Sterilization and disinfection Cards 411-436)*

Complete killing or removal of all living organisms from a particular location or material, *including the killing of spores.*

Cell Envelope

Composed of the: Cytoplasmic Membrane Cell Wall (peptidoglycan) Capsule Outer Membrane Other adherent materials

What sugars are found in peptidoglycan?

Composed of two alternating amino sugars: N-acetylglucosamine (NAG) N-acetylmuramic acid (NAM)

Conditional mutants

Conditional mutants do not always show mutant phenotype. Expression of the mutant phenotype depends on physical conditions. For example, a temperature-sensitive (Ts) mutant strain behaves normally at 30°C and as a mutated strain at 42°C. The product of the gene is inactive at the higher non-permissive temperature not the gene. Activity is regained by returning to the lower permissive temperature.

What are the types of genetic transformations observed in bacteria?

Conjugation - cell to cell contact Transduction - mediated by virus Transformation - uptake of naked DNA

Conjugation in gram-positive bacteria cells

Conjugation also occurs in gram-positive bacteria. It appears that a diversity of conjugation systems exists among gram-positive bacteria but none of these systems appear to use pili to mediate cell-to-cell contact. Cell-to-cell contact in Enterococcus faecalis is mediated by *oligopeptides called sex pheromones* that are produced by certain cells that are devoid of plasmids. In the presence of pheromones, plasmid-containing cells produce an *aggregation substance* that mediates their aggregation with pheromone-producing cells. The phenomenon of conjugative transposition is observed primarily in gram-positive bacteria (not exclusively) and involves a conjugative transposon.

Lipopolysaccharide (LPS)

Considered an *endotoxin* Has 3 regions: Lipid A core polysaccharide O antigen

Core polysaccharide

Consists of outer and inner core The *outer core* has structural variability The *innner core* shows low structural variability Has unique *octose, 2-keto-3-deoxyoctulosonic acid (KDO)* Associated with serologic specificity Branched polysaccharide of 9-12 sugars

What determines the lytic/lysogeny decision?

Controlled by the relative amounts of cI and Cro proteins *cI and Cro proteins are repressor proteins* *Cro protein* is a repressor of synthesis of cI protein and *cI* is a repressor of the synthesis of Cro protein In lysogeny, cI protein is produced preferentially over the Cro protein During lytic phage growth, Cro protein is produced and cI repressor is not produced

Spore Germination

Conversion of spores to vegetative cells Stimulated by disruption of outer coat-- stress, pH, heat Requires water, triggering nutrient (alanine) Outgrowth

Cotransduction

Cotransduction is the simultaneous transfer of two or more markers during the same transduction event. Cotransduction allows one to map closely linked genes relative to each other. The closer two genes are to each other the higher the probability that they will be cotransduced, i.e., packaged in the same phage head.

Capsule

Covers cell surface and is composed of polysaccharide polymers (*Exception - poly-D-glutamic acid of Bacillus subtilis*) Function: *Antiphagocytic*; hinders opsonization (blocks attachment of bacteria to phagocytes so antibodies have to be made against capsules to help with opsonization) Mediates adhesion of bacteria to human tissues Helps in laboratory identification-- *Quellung reaction*

Gram Stain

Crystal violet (primary stain) Gram's iodine (mordant) Decolorization - ethyl alcohol Safranin - counterstain

Viruses can be identified by their effect on cells in culture.

Cytopathic effects (CPE) include: Necrosis- cell death or lysis Inclusion formation: deposition of viral antigens Cytomegaly- increase in cell size Syncytium formation- cell to cell fusion causing multinucleated cells cytoplasmic vacuolization Transformation: uncontrolled growth (*Foci* are spots of uncontrolled growth of cells upon infection with tumor viruses)

What is required for DNA replication in a medium?

DNA parental template strand (dNTPs-deoxribonucleotide triphosphates) Primer template (NTPs-ribonucleotide triphosphates) Magnesium Free 3' OH group Proper enzymes

DNA Genome viruses

DNA sequences can be: Double-stranded DNA (most DNA viruses) Single-Stranded DNA (less than 1% of DNA viruses-- ex: *parvovirus*) Circular DNA genome viruses are *DNAse sensitive* *Except for poxviruses*, ALL DNA VIRUSES REPLICATE THEIR DNA IN THE *NUCLEUS* DNA genome viruses undergo: DNA replication---> DNA transcription---> mRNA translation---> protein Example: Herpes Virus and Hepatitis B virus

Operator

DNA sequences which modulate and control the expression of structural genes. This is achieved by binding of repressor proteins to the operator loci. Operators are usually located between the promoter region and the structural genes whose synthesis they control. Repressor-operator interactions are referred to as *negative regulatory systems* because the binding of a repressor protein to the operator prevents transcription of structural genes.

How does DNA virus replication occur in poxviruses?

DNA stays in cytoplasm The virus encodes and carries all enzymes for genome replication and RNA synthesis (picture is just an example, don't memorize)

There are two types of virus genomes

DNA viruses contain DNA RNA viruses contain RNA

Daptomycin

Daptomycin (CubicinTM) - cyclic lipopeptide Mechanism of Action: Exact mode of action unclear Thought to cause rapid depolarization of the cell membrane due to potassium efflux which leads to disruption of DNA, RNA and protein synthesis Spectrum of Activity: - Staphylococcus aureus (MSSA, MRSA, VRSA) - Ampicillin and vancomycin-resistant enterococci - Penicillin-resistant Streptococcus pneumoniae

Sporogenesis

Depletion of specific nutrients Spore mRNA transcribed Duplication of the chromosome Formation of core Formation of cortex (unusual peptidoglycan) Formation of protein coat (keratin) Formation of exosporium (protein lipid membrane)

Southern Blot

Detection of viral DNA by nucleic acid hybridization carried out on membrane supports Isolate virus-specific DNA Fragment viral DNA by restriction digestion Separate the fragments through agarose or *acrylamide gel* by electrophoresis Transfer separated DNA fragments to membrane support Hybridize immobilized DNA using labeled nucleic acid probes.

Western Blot

Detection of viral proteins or antibody by antigen-antibody reaction carried out on membrane supports. Viral antigens are separated by electrophoresis through acrylamide gels, transferred to membrane supports and probed with antigen specific antibodies, similar to ELISA method. Virus-infected tissue or samples analyzed for their protein profile. First total proteins in the clinical sample is solublized & complexed with SDS. Using *polyacrylamide gel electrophoresis* the proteins are separated according to their size. The resolved proteins are transferred to synthetic membranes as replica. The viral proteins are probed with specific antisera or test sera.

Northern Blot

Detection of virus-specific RNA by nucleic acid hybridization carried out on membrane supports Isolate viral RNA and separate them on denaturing agarose or acrylamide gels Transfer separated RNA onto membrane supports Hybridize immobilized RNA using labeled nucleic acid probes.

Dideoxy Inosine (ddI) and Didoxy Cytidine (ddC)

Dideoxy Inosine (ddI) is also known as Didanosine Didoxy Cytidine (ddC) is also known as Zalcitabine Chain terminators during viral DNA Synthesis by inhibiting reverse transcriptase

Passive Immunity

Direct administration of immune globulins Given: -immunocompromised people -recent exposure to virus (rabies, etc) -children born to infected mothers - corticosteroid- transplant patients Examples: rabies vaicella-zoster hepatitis B measles

Antiseptics

Disinfectants that can be used on the body

Selective toxicity

Drugs must be toxic to some function necessary for the survival of microorganisms while exerting little or no adverse action on the host. This important property of antimicrobials is called selective toxicity.

Dry heat sterilization

Dry heat sterilization technique requires longer exposure time (1.5 to 3 hours) and higher temperatures than moist heat sterilization. Various available methods of dry heat sterilization are; hot air oven, incineration, flaming (wire loop) etc. Dry heat does most of the damage by oxidizing molecules. The essential cell constituents are destroyed and the organism dies. The temperature is maintained for almost an hour to kill the most difficult of the resistant spores. *Dry heat* sterilizes at *160 degrees Celsius for 2 hours*

What is ppGpp?

During amino acid starvation, an unusual nucleotide is produced, *3'-diphosphate 5'-diphosphate guanosine (ppGpp) or guanosine tetraphosphate.* *This compound is the intracellular signal for the stringent response.* *Referred to as stringent factor* The relA locus is primarily responsible for the stringent response. The product of the relA gene is an enzyme known as *ribosome-associated stringent factor.* It is an enzyme that is responsible for the synthesis of ppGpp. *Stringent factor (ppGpp) is located exclusively on 50S ribosomes.*

Translation Elongation

E

Explain the GT system in E. Coli

E. Coli uses a group translocation process to transport glucose The carrier in the membrane is a protein channel specific for glucose. Glucose enters the channel and is phosphorylated by the PTS system. The system derives energy from PEP (phosphoenolpyruvate) which is hydrolyzed to pyruvate. Glucose is phosphorylated to form *Glucose-6- phosphate*

ELISA

ELISA- Enzyme linked immunosorbent Assay Detection using antigen-antibody reaction amplified through enzyme-substrate reaction and color development.

Alkylating agents

EMS-Ethyl methane sulfonate (EMS), Methyl methansulfonate (MMS) and nitrosoguanidine *Reacts with guanine so that it ionizes and base pairs with thymine* Causes primarily GC to AT transitions, but can also cause transversions

E-Test

ETEST is a well-established method for antimicrobial susceptibility testing in microbiology laboratories around the world. ETEST consists of a predefined gradient of antibiotic concentrations on a plastic strip and is used to determine the Minimum Inhibitory Concentration (MIC) of antibiotics, antifungal agents and antimycobacterial agents.

Other types of genetic transformation

Eclipse- Single-stranded form of transforming DNA has no transforming ability. Transformation by Plasmids - Calcium chloride Organisms not naturally transformable (E. coli) made transformable using calcium or *electroporation* Transfection- Transformation of cells with bacteriophage DNA

Electron Transport Chain

Electrons flow downhill, energetically Energy released in this process is used to generate the Proton Motive Force (pmf) which is used by the cell to make ATP

Elongation Factors

Elongation Factor TU and G are used EF-Tu-GTP EF-G-GTP

What is another name for glycolysis in bacteria?

Embden Myerhof-Parnas

What type of bacteria are *tetracycline resistant*?

Enterobacteriaceae

Bacterial Taxonomy

Enterobacteriaceae Family has Escherichia coli and Salmonella Typhimurium members Escherichia is the genus and Coli is the species Salmonella is the genus and Typhimurium is the species

What bacteria are vancomycin resistant?

Enterococci found in the intestines, female genital tract, skin that can cause urinary tract infections, wound infections, endocarditis These are the leading cause of nosocomial infections that occur in hospitals. The are typically resistant to many other antibiotics.

Things you should know about each virus (not exam 1 though) *(Start of PID 23-24: General properties of viruses-PID Lecture 8 overview of viruses is included in here, they are bolded Cards 437-475)*

Enveloped or non-enveloped Genetic material Nucleocapsid symmetry Route of entry Subcellular site of multiplication Organs affected and diseases caused Major clinical features of the disease Known distinguishing features between related viral diseases Vaccines or antiviral Unique aspects if any

Inhibitors of metabolic pathways

Enzymatic activity or other essential reactions in microorganisms may be inhibited by compounds that are similar in structure to the natural substrate. These compounds are called *antimetabolites*. Bacteria contain many enzyme systems that are not found in mammalian cells or the enzymes in bacteria may have a higher affinity for an analogue than the comparable enzyme in mammalian cells. *Therefore, antimetabolites represent an excellent means of selectively inhibiting microbial functions.* Antimetabolites---> Metabolite inhibitors---> Metabolite analogues Sulfonamides p-Aminosalicyclic acid Sulfones Trimethoprim Isoniazid

Biochemical mechanisms of drug resistance include:

Enzymatic inactivation of the drug Inhibition of uptake Drug excretion Modification of target site Synthesis of resistant pathways

Episomes

Episomes are genetic elements that are capable of replicating either autonomously or as an integral part of the chromosome of the host bacterium. Examples: The F-plasmid (F factor) and bacteriophage lambda

Which drug may be causing this patients symptoms? a. Dapsone b. Isoniazid c. Ethambutol d. PAS

Ethambutol

Sterilization method: *Ethylene oxide sterilization*

Ethylene oxide is a common gas used for low temperature sterilization. Alkylating agent Conditions - 10% ethylene oxide in CO2 (50-60 degrees Celsius) Heat-labile materials Targets protein (SH, COOH,OH groups)' Alkylates nucleic acids

Ethylmethane sulfonate (EMS) and nitrosoguanidine

Ethylmethane sulfonate (EMS) and nitrosoguanidine are alkylating agents that react primarily with guanine and to some extent with adenine and cause G C to A T transitions. The addition of an alkyl group at position N-7 of the purine ring results in formation of a quaternary nitrogen at that position. This stimulates ionization of the ring. *In the ionized form the alkylated guanine can pair with thymine instead of cytosine*, which results in a transition from a G C to an A T base pair. The alkylated purine also has a labile N-glucoside bond which hydrolyzes easily to yield a depurinated state and when replication occurs, any base can be inserted at that site.

What is the toxicity of cycloserine?

Exhibits central nervous system toxicity

Plasmids *(Start of PID 13- Plasmids, Transposons, conjugation)*

Extrachromosomal genetic elements that are capable of autonomous replication. Closed circular DNA Capable of changing the phenotype of the bacterial host cell Plasmids can be conjugative (can mediate their own transfer to another bacterial cell by conjugation), non-conjugative (cannot undergo conjugation), or cryptic (specify no known function)

Which type of membrane transport is *least* used in bacteria?

Facilitated Diffusion Transport of *Glycerol* into bacteria is the only well known facilitated diffusion system in bacteria

True or false: The rate of killing by disinfectants/sterilants increases with increasing temperature and decreasing concentration

False The rate of killing by disinfectants/sterilants increases with increasing temperature and increasing concentration

What are other *anti-fungal agents*?

Flucytosine (5-fluorocytosine) Griseofulvin

What is the clinical use of flucytosine?

Flucytosine is used in the treatment of cryptococcosis, invasive forms of candidiasis and chromomycosis. Flucytosine is used frequently in combination with amphotericin B for treatment of severe cases of cryptococcal meningitis.

Virus Envelope

Found in some viruses Composed of a lipid bilayer Derived from host cells-- thus all enveloped viruses enter the host cell, multiply and come out with an envelope Some viruses derive their envelope from cytoplasmic membranes, some from nuclear membranes and some from microsomal membranes Although some viruses carry cell membranes with them, no action potential exists across the membrane The envelope of viruses is important for virus entry and infectivity Envelopes of viruses almost always carry one or more virus specific transmembrane glycoproteins. Can be destroyed by organic solvents (but does not affect naked viruses)

Frederick Griffith's Bacterial transformation Experiment

Frederick Griffith conducted a series of experiments using Streptococcus pneumoniae bacteria and mice. In his experiments, Griffith used two related strains of bacteria, known as R and S. R strain- When grown in a petri dish, the R bacteria formed colonies, or clumps of related bacteria, that had well-defined edges and a rough appearance (hence the abbreviation "R"). The R bacteria were nonvirulent, meaning that they did not cause sickness when injected into a mouse. S strain- S bacteria formed colonies that were rounded and smooth (hence the abbreviation "S"). The smooth appearance was due to a polysaccharide, or sugar-based, coat produced by the bacteria. This coat protected the S bacteria from the mouse immune system, making them virulent (capable of causing disease). Mice injected with live S bacteria developed pneumonia and died. As part of his experiments, Griffith tried injecting mice with heat-killed S bacteria (that is, S bacteria that had been heated to high temperatures, causing the cells to die). Unsurprisingly, the heat-killed S bacteria did not cause disease in mice. The experiments took an unexpected turn, however, when harmless R bacteria were combined with harmless heat-killed S bacteria and injected into a mouse. Not only did the mouse develop pnenumonia and die, but when Griffith took a blood sample from the dead mouse, he found that it contained living S bacteria! Griffith concluded that the R-strain bacteria must have taken up what he called a "transforming principle" from the heat-killed S bacteria, which allowed them to "transform" into smooth-coated bacteria and become virulent.

Other methods of killing: *Freezing*

Freezing can be lethal, but generally bacteriostatic Not reliable for sterilization Valuable method for preserving bacteria agents such as glycerol, dimethyl sulfoxide protect cells against damage by freezing liquid nitrogen at 180 degrees Celsius and refrigerated at -70 degrees Celsius.

Frequency of Cell Division

Frequency of Cell Division is determined by: Species Environmental conditions Growth medium

Viropexis

Fusion between endosomal membrane, and viral envelope in cytoplasm, at acidic pH Many enveloped viruses

Conjugation

Gene transfer that requires cell to cell contact and a conjugative plasmid *Male cells* possess a circular piece of extrachromosomal DNA denoted by *F (fertility factor)* and called a sex plasmid. *Donor male cells are designated F+.* *Female cells which lack this factor are designated F-.* When a culture of males is mixed with a culture of female cells, male-female pairs are formed. *Contact of male cells with female cells is by way of sex pili or F pili.*

Generalized transduction

Generalized transduction is the transfer of any portion of the host or donor cell genome by a bacteriophage. During normal nucleic acid packaging, the packaging apparatus occasionally packages chromosomal DNA rather than phage DNA. When a transducing particle that contains the chromosomal DNA binds to a bacterial cell, the DNA can be injected into the bacterium by the phage and become integrated into the genome of the new cell by generalized recombination. Therefore in a series of these events, many different genetic loci from a host cell can be transferred to a recipient cell population. In short-- When the extrachromosomally located phage begins its replicative cycle, the bacterial DNA is hydrolyzed into small fragments which, during assembly, can be accidentally incorporated into a phage head. Subsequent infection of a bacterium with such a phage releases functional bacterial DNA into the newly infected cell.

What is genetic transformation? *(Start of PID 11: Genetic Transformation Cards 204-219)*

Genetic Transformation is the process by which foreign DNA is introduced into a cell. Important: generates *genetic diversity*, helps with *DNA repair*, can be used to *map genes* in molecular genetics, and can be used to *reintroduce DNA8 into cells after DNA has been manipulated There three mechanisms of genetic transformation in bacterial cells: conjugation, transduction, and transformation.

What are the five amino acids bacteria incorporate ammonia directly into?

Glutamate Glutamine Aspartate Asparagine Alanine Bacteria incorporate ammonia into these five amino acids. All other amino acids are derived from these by transamination reactions.

Embden Myerhof-Parnas

Glycolysis in bacteria

Antibodies bind to the __________ of enveloped viruses.

Glycoproteins found on the envelope Antibodies bind to the glycoproteins of enveloped viruses.

What bacteria contain β -lactamases?

Gram-Positive Bacteria contain extracellular enzymes that can be induced by the beta-lactam to become β -lactamases. Gram-Negative Bacteria contain enzymes in the periplasm that are constitutive β -lactamases.

What bacteria are affected by tetracycline?

Gram-positive and gram-negative bacteria (aerobic and anaerobic) cell wall-deficient organisms (Mycoplasma and L-forms) some obligate intracellular bacteria (Rickettsia and Chlamydia)

*Natural penicillins* affect what type of bacteria?

Gram-positive bacteria Neisseria

*Extended Spectrum penicillins* affect what type of bacteria?

Gram-positive bacteria Neisseria Enterococcus faecalis Haemophilus influenzae coliforms

*Anti-Pseudomonas penicillins* affect what type of bacteria?

Gram-positive bacteria Neisseria Enterococcus faecalis Haemophilus influenzae coliforms Pseudomonas aeruginosa Klebsiella most Proteus species (we don't have to memorize this according to professor)

What bacteria are affected by bacitracin?

Gram-positive bacteria Neisseria gonorrhoeae

What transport system does *E. Coli* use to transport *glucose*?

Group Translocation- PTS System

What transport system does *S. Aureus* use to transport *lactose*?

Group Translocation- PTS System

What transport system does *Streptococcus mutans* use to transport *glucose, mannose, and fructose*?

Group Translocation- PTS System

Sepsis

Growth of harmful microorganisms in living tissue

Facultative Anaerobe Bacteria

Growth response to *aerobic and anaerobic conditions* Enzymes: *Catalase and Superoxide Dimutase (SOD)* Examples: *E. Coli and S. aureus*

Microaerophilic Bacteria

Growth response to *aerobic and anaerobic conditions* Enzymes: Small amounts of *Catalase and Superoxide Dimutase (SOD)* Examples: *Campylobacter and Jejuni* Use oxygen when carrying out aerobic respiration. They live in an environment where oxygen level is low. Some of the microaerophiles can also perform anaerobic respiration.

Aerobe Bacteria

Growth response to *aerobic conditions* Enzymes: *Catalase and Superoxide Dimutase (SOD)* Examples: *M. Tuberculosis and Bacillus Subtilis*

Anaerobe Bacteria

Growth response to *anaerobic conditions* Enzymes: *DO NOT* have Catalase and Superoxide Dimutase (SOD) Examples: *C. Botulinum and Bacteroides* Oxygen is a toxic substance, which either kills or inhibits their growth Also referred to obligate anaerobes

What nucleotides are found in the leader regions of trpL?

Guanine and cytosine Can assume many different secondary structures depending on the availability of tryptophan

Haemophilus influenzae

Haemophilus influenzae-- a type of bacteria that can cause many different kinds of infections. These infections range from mild ear infections to severe diseases, like bloodstream infections. Doctors consider some of these infections "invasive." Invasive disease happens when the bacteria invade parts of the body that are normally free from germs. *Transiently Competent* These bacteria transformasomes react with conserved sequences meaning they only recognize specific uptake sequences on DNA. Their transformasomes mediate the uptake of transforming DNA. DNA receptor proteins also recognize the specific DNA sequences Only take up DNA from their own species DNA binding and uptake is very specific Donor DNA taken into competent cells intact but transforming DNA is incorporated as single strand

Disinfection method: *Halogens*

Halogens are oxidizing agents *Chlorine* - oxidizing agent; kills all vegetative cells water disinfectant *(Specifically asked on the study guide)* *Iodine* - oxidizing agent; used as a tincture of 2% iodine causes hypersensitivity reactions and stains (sporicidal) *Iodophors* - consist of detergents and iodine (cause less hypersensitivity and staining). *Hydrogen peroxide* - concentrations in the range of 3-6% effectively kill most bacteria At concentrations of 10-25%, it can effectively kill spores.

Reverse Transcriptase

Has three different functions: A RNA dependent *DNA polymerase* that produces a complimentary (-) sense DNA strand Ribonuclease H (hybrid) that digests the original RNA strand A *DNA dependent* DNA polymerase that produces a complimentary (+) sense DNA strand

Flagellar arrangement

Have different arrangements peritrichous- flagella all over polar- single flagellum at one pole (monotrichous) Produces the swarming phenomena observed in Proteus and Bacillus

What is the main function of flagella?

Helps cells move during *bacterial chemotaxis* -- the process of moving in specific directions in response to environmental stimuli Bacteria swim in counterclockwise direction toward chemoattractants. They swim away from repellants. Chemotaxis depends on a signaling pathway that terminates at the flagellar motor. Signaling starts with the binding of chemoattractants or repellants to *chemoreceptors* in the membrane. This leads to either methylation or demethylation of methyl-accepting chemotaxis proteins that in turn eventually trigger either a counterclockwise or clockwise rotation of the flagellum and tumbling.

Reverse Transcriptase PCR

Helps determine RNA genome estimation Observed in real time RT-PCR used to diagnose viruses

Hematological reactions

Hematological reactions can range from the life-threatening events that occur in 1 in 60,000 individuals who receive chloramphenicol to the development of hemolytic anemia in individuals who take sulfonamides and have a deficiency of the enzyme glucose-6-phosphate dehydrogenase. *Many antimicrobials depress blood platelet activity.*

Hepatitis B is an example of a *pararetro-virus* and a DNA genome virus

Hepatitis B DNA enters the nucleus and is replicated, as well as transcribed by RNA polymerase II in the nucleus. The Hepatitis B mRNA is then reverse transcribed into DNA again in the cytoplasm by DNA polymerase (a virus Reverse Transcriptase (RT).

*Herpes* is an example of a DNA genome virus

Herpes DNA enters the nucleus and is replicated by DNA polymerase. The viral DNA is then transcribed by RNA polymerase II in the nucleus. The herpes mRNA is later translated into a protein by the host ribosome.

*There are 3 families of DNA viruses that are enveloped. They are all cubical and have only one segment 1/3 families is circular and small 2/3 families are linear and large*

Herpes- large Hepadna- circular, small Pox-large/brick (Don't have to memorize these for exam 1)

Hfr plasmid

Hfr designates a high frequency of recombination male cell and the F plasmid exists as a part of the bacteria chromosome *F factor is integrated into donor (male) cell*

Lac operon: *Glucose and lactose are both carbon sources in the medium* High Glucose Low Lactose

High Glucose levels Low Lactose Low cAMP levels CAP does not bind to DNA Repressor does not bind to operon because lactose is present-- (inducer-repressor) *Lac mRNA produced in small amounts* *Little β-galactosidase and other proteins are expressed* (second diagram on picture)

Lac operon: *Glucose being the sole carbon source in the medium* High Glucose No Lactose

High Glucose levels No Lactose Low cAMP levels CAP does not bind to DNA Repressor binds to operon (no inducer present) *No Lac mRNA produced* *No β-galactosidase and other proteins produced*

Levels of disinfection

High-level disinfection - (chlorine compounds, glutaraldehyde) Intermediate-level disinfection - (alcohols, phenolic compounds) Low-level disinfection -(quaternary ammonium compounds) i.e. benzalkonium chloride

Naked virus characteristics

Highly stable in sewage water, soil, and heat *All intestinal viruses are naked viruses* Stable to intestinal proteases and bile salts

Bacterial Transcription Initiation

Holoenzyme RNA polymerase binding to the promoter site is key to obtain proper translation initiation RNA polymerase binding is enough to unwind the DNA and form a transcription bubble

RNA Polymerase

Holoenzyme RNA polymerase is composed of 4 polypeptides: alpha - 37 kDa (2 subunits) beta-150 kDa beta'- 160 kDa sigma - 70 kDa The Core Enzyme is composed of *a2bb'*

Bacteriophage structures

Icosahedral tailless (PM2) Icosahedral head with tail (lambda)--The tailed phages do not always have a collar and can have from 0-6 tail fibers, the number depending on the phage type. Filamentous (M13)

What is the fate of *beta-galactosidase* when lactose and glucose are present in a medium?

If both lactose and glucose are present, synthesis of β-galactosidase is not induced until all the glucose has been utilized.

Bacteriostatic

If the action of a drug *inhibits the growth of a cell* and *can be reversed by the removal of the drug* from the system which allows the cell to survive and resume replication, the action of the drug is *bacteriostatic.* Inhibits growth but action of agent can be reversed by removal of agent

What occurs if there is a *high amount of tryptophan tRNA* detected in a cell?

If there is a high amount of trp tRNA in a bacteria cell, then *trp operon ribosomes* do not stall at *strand one* of the trpL sequence. If the translating ribosome does not stall at the tandem codons, then the ribosome is in contact with strand two so strand two and strand three cannot base pair By the time strand four is synthesized, it cannot pair with strand two, so strand four pairs with strand three, forming the *termination structure 3:4* When all translation is blocked and there are no translating ribosomes on the transcript, the combination of stem-loop structures 1:2 and 3:4 forms instead of stem-loop 2:3. This causes transcription of the tryptophan operon to terminate, since there is no need to produce this mRNA when all protein synthesis is blocked.

What occurs if there is a *low amount of tryptophan tRNA* detected in a cell?

If there is a low amount of trp tRNA in a bacteria cell, then *trp operon ribosomes* stall at *strand one* of the trpL sequence. This stalling prevents strand one and strand two of the leader sequence to base pair and form a loop. Instead, strand two pairs with strand three By the time strand four is synthesized, it can no longer pair with strand three, preventing the formation of the *termination structure 3:4* RNA polymerase will not stop at the attenuator and structural genes of the operon will be translated

Co-Transferred genes

If two genes are observed to be co-transferred, one can conclude that they are close together on the chromosome. Frequencies of co- transfer increase as distance between two genes decreases. (Meaning the frequency is higher the closer the genes are together) Thus, cotransfer can be used to order a sequence of genes on the chromosome. Co-transferred genes & Co-transformation Frequency (%) X and Y --- 50% X and Z --- 5% Y and Z --- 25% gene sequence -- xyz

*Second Generation Cephalosporins* affect what type of bacteria?

Improved activity against gram-negative bacilli such as members of the family Enterobacteriaceae, but less active against gram-positive organisms. More resistant to cephalosporinases

Outermembrane

In *Gram-negative Bacteria* the cell wall is composed of a single layer of peptidoglycan surrounded by a membranous structure called the *outer membrane* Outer membrane is covalently attached to the peptidoglycan cell wall through a lipoprotein layer Contains *lipopolysaccharide (LPS)* The Outer Membrane is held together by divalent cation (Mg++ and Ca++) linkages between phosphates on LPS and hydrophobic interactions between LPS and proteins. The asymmetric lipid bilayer consists of external layer composed of LPS and inner layer composed of phospholipids Reinforces cell shape Provides protection Excludes hydrophobic molecules Antibiotics penetrate Outer Membrane slowly Disruption of Outer Membrane results in penetration of large hydrophobic molecules --Outer Membrane is disrupted by polymyxin (cationic basic peptide) that acts like detergent and EDTA (ethylene diamine tetracetic acid)

Gene Mutations

In general there are two types of alterations can occur in the nucleotide sequence of DNA: The *substitution* of one base pair for another or *deletion or insertion (addition)* of a base pair or DNA segment. Base substitutions involve the replacement of a *pyrimidine with a different pyrimidine* or a *purine with a different purine.* These are called *transitions.* A *pyrimidine may also be replaced by a purine* or vice versa to cause a *transversion.* The addition or deletion of bases from DNA shifts the translation "reading frame" of the coded message and causes *frameshift mutations.* Mutations involving single base changes are referred to as point mutations.

Indirect ELISA

In the indirect ELISA test, the sample antibody is sandwiched between the antigen coated on the plate and an enzyme-labeled, anti-species globulin conjugate. The addition of an enzyme substrate-chromogen reagent causes color to develop. This color is directly proportional to the amount of bound sample antibody.

Tricarboxylic Acid Cycle (TCA)

In the presence of oxygen, pyruvate undergoes this cycle. The TCA cycle allows organisms to generate more energy per mole of glucose than is possible from glycolysis (and fermentation) alone. *Products per pyruvate*: 1 GTP, 2 CO2, 3 NADH, 1 FADH2- carried to the electron transport chain (ETC) where NADH can produce 3 moles of ATP and FADH2 can produce 2 moles of ATP.

Colicin E3

Inactivates 30S ribosomes by removing 50 terminal nucleotides from 16S RNA.

*β -lactam resistance*

Inactivation of β-lactam antibiotics can be completed by *β -lactamases*, which include *penicillinases* and *cephalosporinases*

Incompatibility Groups

Incompatibility is used to classify plasmids into groups. *Plasmids in the same incompatibility group cannot reside in the same cell while plasmids in different incompatibility groups can reside in the same cell.* Certain closely related plasmids when introduced into the same cell are mutually exclusive, i.e. only one plasmid will be stably inherited in subsequent generations.

Mutator genes

Increases mutation frequencies There are genes that in a mutant state cause the spontaneous mutation frequencies of other genes to be increased. These genes are called mutator (mut) genes. Example: E. coli has about six different genetic loci within which alterations increase mutation frequencies. MutT causes AT to CG transversions. The mutator function of bacteriophage T4 is associated with the 3' - 5' exonuclease activity of its DNA polymerase, which provides a proofreading function.Impairment of this proofreading function causes GC to TA transversions. In a dam- mutant there is no methylation of DNA. The dam locus methylates cellular DNA and protects its DNA from degradation by its own restriction endonucleases.

What is Infectious RNA?

Infectious RNA is pure RNA that have the ability to cause infection No viral proteins are needed from outside of the cell, but are translated from the RNA RdRp is generated in cytoplasm Replication of RNA in the cell leads to infection and virus release *positive sense viruses*

Inhibitors of Protein synthesis

Inhibitors of the 30S ribosomal subunit: aminoglycosides, tetracyclines, and spectinomycin Inhibitors of the 50S ribosomal subunit: chloramphenicol, marcolides, and lincosamides

Fosfomycin

Inhibits step one of peptidoglycan synthesis NAG is created from a glucose precursor and then converted into UDP-NAG. UDP-NAG is then converted into *UDP-NAM* by a phosphoenolpyruvate (also known as enol pyruvate transferase-- inhibited by *fosfomycin*).

What are the three phases of DNA replication in bacterial cells?

Initiation Elongation Termination

70S Initiation Complex

Initiation Factor 1= IF1 Initiation Factor 2= IF2 Initiation Factor 3= IF3

Sulfonamide structure

Instead of the SO2NHR on the sulfonamide, p-amino benzoic acid contains a COOH seen the second structure in the picture

Protein Kinase P (R1) system

Interferon induces augmented synthesis of PKR (autophosphorylation of PKR) PKR adds phosphate group to eIF2 (elongation factor 2) *eIF2 phosphorylation is inactivated leading to inhibition of protein synthesis* (GTP step in the picture cannot occur)

2'5' oligoadenylate synthetase (OAS) System

Interferon induces the transcription of 2'5' oligoadenylate synthetase gene (OAS also activated by viral RNA genome sequence) OAS activates Ribonuclease L *RNase L degrades all RNAs in virus infected cells which inhibits protein synthesis*

Interferons

Interferons are secreted mammalian cell proteins Interferons are host specific, but not virus specific extremely potent (need only very small amounts) IFN alpha and IFN beta are produced by *all* immune cells IFN-gamma is produced by T cells IFN-alpha is considered antiviral and anti-tumor and is induced by RNA virus infections, dsRNA, and ds synthetic polyribonucleotides (Poly IC). IFN-gamma is involved with Immunomodulation (immune response inducer) and is induced by mitogens, antigens, and IL-2.

*Termination* phase of chromosome replication

Involves the termination locus and the *tus protein.* *T1* is involved in the termination of *clockwise forks* and *T2* is involved in the termination of *counterclockwise forks*. *Tus protein* binds to the locus and prevents DNA from unwinding. *It inhibits helicase.*

Idoxuridine

Iodo-uridine resembles thymidine and is phosphorylated within cells Used against HSV keratitis (Herpes keratitis is a viral infection of the eye caused by the herpes simplex virus (HSV).)-- Herpes (don't know if we need to know for exam)

What is another name for shock-insensitive transport?

Ion Dependent Transport (IDT) Uses a cation to drive the transport process Example: Lactose permeate of E. Coli Lactose is transported against its conc. gradient simultaneously as H+ is allowed to move down its concentration gradient. Generates a pmf that drives the interaction

What transport system does *E. Coli* use to transport *lactose*?

Ion Dependent Transport (IDT) also referred to as Proton symport

Antimetabolites Effective Against Mycobacterial Infections

Isoniazid Sulfones p-Aminosalicylic acid (PAS) Ethambutol

*Isoniazid*

Isonicotinic acid hydrazide; INH Mechanism of Action: Not known, but structural similarity of INH to that of nicotinamide and pyridoxal suggests the drug might act as a antimetabolite against either of these vitamins *The most widely accepted mechanism is that INH activity is due to an inhibition of the synthesis of mycolic acid* Spectrum of Activity: *Mycobacterium tuberculosis* Clinical Use: Infections of Mycobacterium tuberculosis; usually used in combination with other drugs (isoniazid plus rifampin or isoniazid plus rifampin and pyrazinamide) Toxicities: Symptoms of vitamin B6 deficiency such as peripheral neuritis and seizures, hepatic toxicity

Phases of Bacterial Growth

Lag phase- variable in length and no cell division Exponential or logarithmic phase- cells are dividing at a constant rate Stationary phase- decreased growth rate, depletion of nutrients and the beginning of cell death Death phase- cell death *Bacteria reproduce by binary fission*

Vir mutants

Lambda phage contains a repressor-operator system. The repressor gene is called *cI*. The repressor protein binds to two *operators, OL and OR*, which are adjacent to two *promoters, PL and PR.* (L and R = leftward and rightward, respectively, and refer to direction of synthesis of two early mRNA molecules). *In a lysogen, the cI repressor is synthesized continuously and in excess.* Therefore, cI repressor is always bound to the operators in a lysogen and pL and pR are unavailable for binding by RNA polymerase which prevents transcription from the two early promoters. This binding of cI repressor keeps the prophage in an "off" state. If the lysogen is infected by another lambda phage, excess repressor molecules already present will bind to the operators of the incoming infecting DNA molecule which will prevent transcription of the incoming phage DNA. Mutations in the cI gene, which cause cI repressor to be nonfunctional, will result in mutant phage that can only engage in the lytic life cycle. Mutations in OL and OR that cause these loci to lose the ability to interact with repressor generate what are called *vir mutants* which cannot establish repression. Additionally, vir mutants can superinfect and grow in a lysogen because these phages are insensitive to repressor.

What are R-factors?

Large, self-transmissible plasmids Widely distributed in gram-negatives Replicate independently of the chromosome Many Copies Transfer to same or different species

*There is only one type of virus that has Single-stranded RNA that is diploid-- Retroviruses. There are two types of families*

Lentiviruses- HIV 1 and HIV 2 Oncogenic- HTLV 1 and HTLV 2

*All retroviruses are ssRNA diploid, one-segmented, linear, cubical, enveloped, and bullet shaped--Two families*

Lentiviruses- HIV 1 and HIV 2 Oncogenic- HTLV 1 and HTLV 2 (Don't have to memorize these for exam 1)

*Third Generation Cephalosporins* affect what type of bacteria?

Less active against gram-positive bacteria, but much more active against the Enterobacteriaceae

Cytoplasmic Membrane

Lipid bilayer Semi-permeable Composed of 70% protein and 30% phospholipids No sterols (formed in eukaryotic cells)-- *Exception - Mycoplasma sp*

Give an example of *Cis-active regulatory sequences*.

Loci linked together (conjoined) to the genes whose activity they control or modulate Example: promoter and operator genes

Give an example of *Trans-active regulatory sequences*.

Loci not conjoined to the genes whose activity they control Example: repressor gene

Characteristics of life vaccines

Long lasting IgG and IgA Cell-mediated immunity Infectious contaminants Immunosupression is not used Storage difficulty Reversion to wild type may occur Herd immunity

Siderophore Transport

Low molecular weight iron binding proteins secreted from bacteria that bind *iron* in the environment. Iron-loaded siderophores are bound to receptors on surface of bacteria and internalized. Released iron is released as a nutrient source. (blue circle in the picture)

Lysogenic conversion

Lysogenic conversion is an event dependent on the establishment of lysogeny between a bacteriophage and the host bacterium. *A change in the virulence phenotype of a bacterium due to lysogeny establishment* Several medically important phenotypic traits associated with bacteriophage have been found to be the result of lysogenic conversion. *The production of toxin by the diphtheria bacillus, Corynebacterium diphtheriae*, is dependent on lysogenization of the bacteria with β phage to make the bacteria tox+. The production of erythrogenic toxin by members of the Group A streptococci has also been shown to be the result of lysogenization by bacteriophage. Toxin production by Clostridium botulinum, types C and D, requires the participation of bacteriophages. Lysogenic conversion is also involved in altering the antigenic structure of Salmonella species. The outer membrane polysaccharides comprising the O antigens, which serve as the basis for serological classification of Salmonella, can be altered by the presence of a bacteriophage.

Effects of lysozyme or penicillin on LPS

Lysozymes hydrolyze the bond between N-acetyl glucosamine and N-acetylmuramic acid Both lysozyme and penicillin treatment of gram- positive bacteria produce *protoplasts in hypertonic medium.* Similar treatment of gram-negative bacteria produces *spheroplasts*.

Bacteriophage Life cycles

Lytic Life Cycle-- results in lysis of host bacterium Lysogenic Life Cycle-- transcription of phage genome is repressed There are two distinct phage life cycles, the lytic and lysogenic life cycles. In the lytic life cycle, the phage converts an infected bacterium into a phage factory, many progeny phage are produced and released after lysis of the host cell. A phage capable of carrying out only lytic growth is called *virulent.* *The lysogenic life cycle is observed only with phages containing double-stranded DNA.* Usually the phage genome becomes integrated into the chromosome of the host bacterium. The phage genome remains in the chromosome until it is induced to carry out the lytic life cycle. A phage that can carry out both the lytic and lysogenic life cycle is called a *temperate phage.* The lysis-lysogeny decision of temperate phage lambda is governed by the products of the cI and cro genes. In lysogeny, cI protein is produced preferentially over the Cro protein, which is a repressor of cI synthesis and is necessary for several features of the lytic life cycle. During vegetative phage growth, Cro protein is produced and cI repressor is not produced. Mutations in the cI gene generate phage that cannot enter the lysogenic life cycle while mutations in the cro gene result in phage that cannot enter the lytic life cycle.

*Marcolide resistance*

Macrolide antibiotics - erythromycin, lincomycin, clindamycin Targets are modified by methylation of the 23S ribosomal RNA in susceptible gram-positive bacteria Methylase - plasmid encoded

Peptidoglycan Synthesis

Made of N-acetyl Muramic Acid (NAM) alternating with N-acetyl glucosamine (NAG) which are cross linked with chains of four amino acids. *Stage one (occurs in the cytoplasm):* 1. NAG is created from a glucose precursor and then converted into UDP-NAG. UDP-NAG is then converted into *UDP-NAM* by a phosphoenolpyruvate (also known as enol pyruvate transferase-- inhibited by *fosfomycin*). 2. 5 amino acids are then added to UDP-NAM. 1st-- L-alanine (L-ala) 2nd-- D-glutamic acid (D-glu) 3rd-- Meso-diaminopimelic acid (m-DAP) in *gram negative* or Lys-(gly)5 in *gram positive* 4th and 5th-- D-alanyl-d-alanine (D-ala-D-ala) Creating UDP-NAM-L-ala-D-glu-m-DAP- D-ala-D-ala which is referred to as *UDP-NAM-Pentapeptide* *Stage two (inner side of the membrane)* 3. UDP-NAM-Pentapeptide is transferred from UDP to *bactoprenol-phosphate (P)*. This lipid protein is also referred to as *Undecaprenol-P* 4. UDP-NAG is then transferred to the bactoprenol-P-P-NAM-pentapeptide creating the *final peptidoglycan precursor* 5. The final NAM-NAG Precursor is transported across the cell membrane by the bactoprenol lipid to the outer-membrane with the help of *peptidoglycan synthase*. *Stage three (outer-membrane)* 6. NAM-NAG precursors are then added to the pre-existing peptidoglycan strand by *transglycosylases*. 7. Transpeptidation reaction occurs, releasing d-ala and forming a cross-link bond between D-ala (carboxyl group) and m-DAP or Lys-(Gly)5 (amino group). This is completed by *transpeptidase.* *Carboxypeptidases* cleave the terminal D-alanine. Good Video: https://www.youtube.com/watch?v=JRHOjgwXBrI

Prototrophs

Make all their nutrients from basic components

How is bacterial cell growth measured?

Measuring turbidity (Spectrophotometer) Viable count Determine dry weight and measure amount of DNA, RNA, protein

*Ethambutol*

Mechanism of Action: *Inhibits the polymerization of arabinoglycan*(critical constituent of mycobacteria cell wall) Spectrum of Activity: Mycobacterium tuberculosis Clinical Use: *Mycobacterium tuberculosis infections* (usually used in combination with rifampin, isoniazid, and pyrazinamide) Toxicities: Optic neuritis (visual acuity should be monitored during therapy)

*Chloramphenicol* State the mechanism of action, spectrum of activity, clinical use, and toxicity

Mechanism of Action: Chloramphenicol binds to the 50S ribosomal subunit and inhibits peptide bond formation, by inhibiting the peptidyl transferase reaction of protein synthesis Spectrum of Activity: gram-positive and gram-negative bacteria, rickettsiae, chlamydiae Clinical Use: Haemophilus influenzae meningitis, anaerobic infections, typhoid fever Toxicities: Inhibition of blood-forming cells in bone marrow (marrow hypoplasia) Progressive bone marrow aplasia with aplastic anemia and agranulocytosis (occasionally) Gray syndrome in neonates with abdominal, circulatory and respiratory dysfunction *50S ribosome subunit inhibitor*

Griseofulvin

Mechanism of Action: Griseofulvin is an oral fungistatic agent specific for fungi whose cell walls contain *chitin*. This drug interferes with protein assembly. *This drug inhibits mitotis in the metaphase by interfering with the assembly process of tubulin into microtubules by binding to proteins involved in tubulin assembly.* Clinical Use: Griseofulvin is standard therapy in the management of *dermatophytic infections* of the skin and nails *especially in the treatment of tinea capitis (ringworm of the scalp) which is a common childhood fungal infection.*

*p-Aminosalicylic acid (PAS)*

Mechanism of Action: Similar to that of sulfonamides Spectrum of Activity: Primarily Mycobacterium tuberculosis Clinical Use: PAS is used as a second-line drug against Mycobacterium tuberculosis infections and is reserved for use in the treatment of multidrug-resistant tuberculosis. Toxicities: Nausea, vomiting, hypersensitivity

Flucytosine (5-fluorocytosine) mechanism of action

Mechanism of Action: The fungistatic effect of flucytosine, a fluorine analog of cytosine, results from its antimetabolite properties. Flucytosine, after uptake by the fungus, is converted by deamination to fluorouracil, which is incorporated into RNA and inhibits protein synthesis. Flucytosine is also converted into fluorodeoxyuridine monophosphate which interferes with DNA synthesis by inhibiting thymidylate synthetase.

Humoral Immunity *(Start of PID 31-32: Diagnostics, interferons, and vaccines Cards 511-570)*

Mediated by B cells and immunoglobulins Antibodies are generated to recognize protein antigens and glycoprotein antigens on enveloped viruses. IgM, IgG, and IgA all can be directed towards virus antigens Binding of antibodies to virus particles leads to abolition of infectivity a process known as *virus neutralization* Antibody binding to virus particle sterically interferes with virus binding to host cells Neutralizing antibodies effectively inactivate circulating free virus particles

Sulfonamide resistance

Mediated by R plasmids Encode sulfonamide-resistant dihydropteroate synthetase

Trimethoprim resistance

Mediated by R plasmids Encode trimethoprim-resistant dihydrofolate reductase

Suppression of missense mutations

Mediated by altering the anticodon of a tRNA molecule so that the termination codon is read Suppression can also occur for missense mutations but this kind of suppression is inefficient. Suppression of missense mutations can involve 1) an altered anti-codon on tRNA that does not affect charging of the tRNA by it cognate tRNA synthetase 2) mischarging of mutant tRNA by tRNA synthetase 3) a mutant tRNA synthetase that misacylates a mutant tRNA.

*Chloramphenicol Resistance*

Mediated by two mechanisms: Chloramphenicol acetyltransferase - *acetylates* antibiotic to an acetyl or diacetyl ester Responsible for widespread resistance in gm+/gm- Anaerobic bacteria can reduce a *p-nitro group* on the drug plasmid-encoded

What is the most important transposon, medically?

Medically the most important class of transposon is the *composite type* containing antibiotic resistance genes Can be inverted or direct repeats

Merozygotes

Merozygotes arise when the genetic material from one bacterial cell is only partially transferred into another cell during conjugation, transduction, or transformation. Merozygotes in E. Coli were used to determine what occurs if the lac operon is mutated. Also referred to as *partial diploids and merodiploids*

Semi-conservative replication experiment

Meselson-Stahl Experiment Meselson and Stahl conducted their famous experiments on DNA replication using E. coli bacteria as a model system. They began by growing E. coli in medium, or nutrient broth, containing a "heavy" isotope of nitrogen, 15-N (An isotope is just a version of an element that differs from other versions by the number of neutrons in its nucleus.) *When grown on medium containing heavy 15-N the bacteria took up the nitrogen and used it to synthesize new biological molecules, including DNA.* After many generations growing in the 15-N medium, the nitrogenous bases of the bacteria's DNA were all labeled with heavy 15-N. Then, the bacteria were switched to medium containing a "light" 14-N isotope and allowed to grow for several generations. *DNA made after the switch would have to be made up of 14-N, as this would have been the only nitrogen available for DNA synthesis.* Meselson and Stahl knew how often E. coli cells divided, so they were able to collect small samples in each generation and extract and purify the DNA. They then *measured the density of the DNA (and, indirectly, its 15-N and 14-N content)* using density gradient centrifugation. This method separates molecules such as DNA into bands by spinning them at high speeds in the presence of another molecule, such as cesium chloride, that forms a density gradient from the top to the bottom of the spinning tube. *Density gradient centrifugation* allows very small differences—like those between 15-N and 14-N-labeled DNA to be detected. After reviewing each generation the experiment done by Meselson and Stahl demonstrated that DNA replicated semi-conservatively, meaning that each strand in a DNA molecule serves as a template for synthesis of a new, complementary strand.

Hydroxylamine

Modifies cytosine so that it base pairs with adenine Causes GC to AT transitions Hydroxylamine reacts specifically with cytosine and converts it to a modified base that pairs only with adenine so that a G C pair ultimately becomes an A T pair.

Autoclaving mechanism

Moist heat sterilization using autoclave is commonly used for the sterilization of biohazardous trash, heat and moisture resistant materials such as aqueous preparation (culture media). This method is also used for the sterilization of surgical dressings and medical devices. *Moist heat destroys microorganisms by the irreversible denaturation of enzymes and structural proteins.* The temperature at which denaturation occurs varies inversely with the amount of water present. Sterilization in saturated steam thus requires precise control of time, temperature, and pressure. Pressure serves as a means to obtain the high temperatures necessary to quickly kill microorganisms. steam under pressure is the preferred method of sterilization *121 degrees Celsius, 15 lbs. of pressure for 20 minutes*

Protease Inhibitors

Molecular studies have indicated that viral proteases play a critical role in the life cycle of many viruses by effecting the cleavage of high-molecular-weight viral polyprotein precursors to yield functional products or by catalyzing the processing of the structural proteins necessary for assembly and morphogenesis of virus particles. Inhibition of viral protease leads to accumulation of non-functional viral polyproteins *Inhibitios block formation of functional proteins from polyprotein precursors, and therefore infectivity of virus is lost* Inhibitors can be used against HIV, herpesvirus, RSV (respiratory syncytial virus), and Flu A Example: Amantidine, Rimantadine

Anaerobic Respiration

More efficient than fermentation, but not as efficient as aerobic respiration Bacteria use compounds other than oxygen as terminal electron acceptors (NO3-, SO4-2. CO2)

*First Generation Cephalosporins* affect what type of bacteria?

Most gram-positive cocci Many common gram-negative bacilli *except Enterobacter species, indole-positive Proteus species and Pseudomonas species*

IFN-beta is used to treat what conditions in the United States?

Multiple Sclerosis

Mutagenesis

Mutagenesis is the process of producing mutations by changing the primary sequence of DNA Changes are generally reflected in a change in a particular property of the cell. Mutations may be spontaneous (occurs in nature without the addition of a mutagen) or induced (caused by a mutagen).

Auxotrophs

Mutants that have a nutritional requirement that they cannot produce Examples: Trp+ bacteria becomes a Trp- bacteria and cannot code tryptophan Lac+ bacteria becomes a Lac- and cannot breakdown Lactose Bacteria that are resistant to drugs go from EmS to EmR

What occurs if there is a relA mutation?

Mutations in the relA locus give rise to cells called *relaxed* that continue to synthesize stable RNA during amino acid starvation

Chromosomal-mediated resistance

Mutations occur in drug targets or transport systems Spontaneous mutations between 10 -7 to 10 -9 Less important than plasmid-mediated resistance

Mutations in which gene of the lac operon results in the lack of β-galactosidase?

Mutations responsible for the lack of beta-galactosidase were shown to lie *in the lacZ gene.* Genotype: *I+O+Z+* --- lac region is haploid --- Synthesis of beta-galactosidase is inducible BUT when the Genotype:*I+O+Z-* --- lac region is haploid --- Synthesis of beta-galactosidase is *negative*

Suppression mutations

Mutations that mask or correct the effects of a prior mutation Mutations that do not correct the original mutation but restore the original phenotype are called suppressor mutations. Reversal of the mutant phenotype by another mutation distinct from original mutation is called *suppression*.

Mycoplasma

Mycoplasma is a genus of bacteria that lack a cell wall around their cell membrane. Without a cell wall, they are unaffected by many common antibiotics such as penicillin or other beta-lactam antibiotics that target cell wall synthesis. No cell wall Variable morphology Sterols in cytoplasmic membrane

What type of bacteria do have sterols ?

Mycoplasma sp

What is the peptidoglycan monomer?

NAM-NAG attached to a tetrapeptide Tetrapeptide composed of alanine, glutamic acid, meso diaminopimelic acid, alanine The tetrapeptide has both D & L amino acids Another alanine is added to form a pentapeptide, but is removed during synthesis

Transglycosylases

NAM-NAG precursors are added to the pre-existing peptidoglycan strand by *transglycosylases*.

Nalidixic Acid and Fluoroquinolones

Nalidixic Acid and Fluoroquinolones (*ciprofloxacin, norfloxacin*) Mechanism of Action: Nalidixic acid and the fluoroquinolones inhibit DNA replication of susceptible bacteria. *They inhibit the A subunit of DNA gyrase or topoisomerase II (nicking- closing enzyme).* Spectrum of Activity: *Nalidixic acid*- aerobic gram-negative bacilli (not pseudomonads) *Fluoroquinolones* - broader than nalidixic acid, including enterococci, staphylococci and Pseudomonas aeruginosa Clinical Use: Urinary tract infections (cystitis) caused by gram-negative and gram-positive bacteria.

What are the two types of penicillins?

Natural Penicillins Semisynthetic penicillins

Supercoiled DNA

Negative Coil- underwound DNA that is twisted in the opposite direction of the helix Positive Coil- Overwound DNA twisted in the direction of the helix

What is the toxicity of polymyxins?

Nephrotoxicity is commonly observed with polymyxin B and, and less frequently with colistin (polymyxin E). Polymyxins are topically applied due to their severe toxicity

Neuraminidase, hemagglutinin

Neuraminidase (NA): a glycoprotein (enzyme) found on the surface of virally infected host cells, that catalyzes the breakdown of glycosides containing neuraminic acid, i.e. sialic acid. Hemagglutinin (HA): a viral glycoprotein found on the surface of virally infected cells. Can cause hemagglutination and allow virus cells to attach to target cells. These glycoproteins contain *sialic acid.* Sialic acid: Sticky monosaccharides

Neuraminidase Inhibitors (Oseltamivir and Zanamivir)

Neuraminidase inhibitors are analogues of sialic acid. Their proposed mechanism of action is to block the active site of neuraminidase and leave *uncleaved sialic acid residues* on the surfaces of host cells and influenza viral envelopes. Viral hemagglutinin binds to the uncleaved sialic acid residues The result is viral aggregation at the host cell surface and a reduction in the amount of virus that is released and can infect other cells *Oseltamivir and Zanamivir both affect Flu A and B by blocking virus release and spread*

Neurologic toxicity caused by antimicrobial agents

Neurologic toxicity is fairly uncommon, but the aminoglycosides can damage the auditory or vestibular apparatus if the clinician does not pay close attention to the amount given to the patient.

Virus Evolution *(Start of PID 28-320: Viral genetics and pathogenesis Cards 497-510)*

New RNA viruses arise faster than DNA viruses RdRp is error-prone (no proof-reading function) *Spontaneous random mutations* which may allow mutant genome accumulation due to some advantage over wild type. ex: drug resistance, altered cell tropism Large amounts of viral variants arise during RNA virus replication Some of these variants might carry altered tissue and host specificity as well as drug sensitivity

Explain nitrous acid involvement with mutagenesis

Nitrous acid converts amino groups to keto groups by oxidative deamination. Cytosine, adenine and guanine are converted to uracil, hypoxanthine and xanthine, respectively. These bases can form the base pairs UA,HC andXC. Therefore,the changes are GC to AT and A T to G C transitions.

Lac operon: *Lactose is the sole carbon source in the medium* No Glucose Normal Lactose

No Glucose Normal levels of Lactose High cAMP levels AMP-CAP binds to DNA Repressor does not bind to operon because lactose is present-- (inducer-repressor) *Lac mRNA produced in abundance* *β-galactosidase and other proteins are expressed*

Trifluridine

Nucleoside analog used against HSV-1 keratitis Herpes keratitis is a viral infection of the eye caused by the herpes simplex virus (HSV). (don't know if we need to know for exam)

HIV Inhibitors

Nucleoside analogs (NRTI): Chain Termination-- AZT and ddI Non-nucleoside analogs (NNRTI): Reverse Transcriptase activity is dead-- Nevaripine and Efavirenz

Nucleoside Analogs Mechanism of Action

Nucleoside analogs are phosphorylated in the infected cell A reverse transcriptase incorporates them into the DNA Chain termination occurs blocking cDNA synthesis ( RNA to DNA) Many of the antiviral nucleoside analogues are blocked at the 3' hydroxyl group of the deoxyribonucleic acid, which results in failure of elongation of the nascent DNA molecule. Drug resistance can be due to mutations in RT

One-step growth curve:

Observes infection in a tissue cell culture Intracellular line describes the virus inside of the cell Extracellular line describes the virus once released from the cell *Eclipse period*: no viral gene expression yet No infectious virus found inside cells Virus is in an enclosed compartment *Latent period*: no extracellular virus produced Virus replication occurs High level of virus inside cells no virus released from cells

*(-) sense RNA viruses can be multi-segmented. One RNA segment: 3 families Two RNA segments: 1 family Three RNA segments: 1 family Eight RNA segments: 1 family*

One RNA segment: Rhabdoviridae, Paramyxoviridae, Filoviridae Two RNA segments: Arenaviridae Three RNA segments: Bunyaviridae Eight RNA segments: Orthomyxoviridae (Don't have to memorize these for exam 1)

Transposon Tn3

One member of a large family of related elements that encode resistance to a variety of antibiotics. Tn3 itself contains genes for β-lactamase.

Flagella

Organelles used for locomotion Composed of 3 parts (basal body, hook, filament) Composed of flagellin - helically coiled protein subunits Contains *H antigens* which are used in identification of Salmonella/Enterobacteriaceae Undergo phase variation Propel cells by rotation motion Proton motive force provides energy for rotation

Organic Solvents

Organic solvents destroy lipid bilayers that hold *peplomer spikes* that attach viruses to cells Since all envelopes are made up of lipid bilayer, they are sensitive to organic solvents (ether, chloroform) and non-ionic detergents (NP40, Triton X100). Thus all enveloped viruses are inactivated by organic solvents, whereas non-enveloped viruses are not. Causes infectivity to be lost in enveloped viruses, but not naked viruses

Butyric Acid Fermentation

Organism: *Clostridium* Pathway: *Glycolysis* Products: *Butyrate, Butanol, acetone and CO2* ATP Yield: *2* (number 6 in the picture)

Butanediol (Acetoin and mixed acid) Fermentation

Organism: *Enterobacter and Klebsiella* Pathway: *Glycolysis* Products: *Acetonin, Butanediol, and CO2* ATP Yield: *1*

Mixed Acid Fermentation

Organism: *Escherichia, Salmonella, Shigella* Pathway: *Glycolysis* Products: *Lactate, acetate formate, ethanol, and CO2* ATP Yield: *3* Occurs when bacteria utilize two or more different pathways in the terminal steps of fermentation.

Heterolactic Fermentation

Organism: *Leuconostoc* Pathway: *Phosphoketolase* (The pentose phosphoketolase pathway (PPK) is a branch of the pentose phosphate pathway) Products: *Lactate* ATP Yield: *1*

Alcoholic (bacterial) Fermentation

Organism: *Pseudomonas and Azotobacter* Pathway: *Entner-Doudoroff* (An alternative pathway to glycolysis) Products: *Ethanol and CO2* ATP Yield: *1*

Homolactic Fermentation

Organism: *Streptococcus and Lactobacillus* Pathway: *Glycolysis* Products: *lactate* ATP Yield: *2*

Alcoholic Fermentation

Organism: *Yeast* Pathway: *Glycolysis* Products: *Ethanol and CO2* ATP Yield: *2*

What is the toxicity of aminoglycosides?

Ototoxicity (vestibular and auditory branches of the eighth cranial nerve) nephrotoxicity (kidney toxicity)

Majority of viral infections are subclinical. (70-98%)

Pathogenicity of a virus is due to the affect it has on cells and how it is recognized by the immune system: alteration in the cellular synthetic processes altered cellular transport cell-cell fusion transformation immunological attack on infected cells immune complex disease (viral antigens) autoimmune responses

What inhibits transpeptidation?

Penicillin inhibits cross-linking by binding irreversibly to transpeptidases

What are the targets for β-Lactam antibiotics?

Penicillin-binding proteins

Inhibitors of cell wall synthesis

Penicillins (β-lactam) Cephalosporins (β-lactam) Cycloserine Vancomycin Bacitracin

Penicillin Structure

Penicillins are derivatives of 6-aminopenicillanic acid The basic structure is a β-lactam ring linked to thiazolidine ring *R* represents *side chains* (determines most of the antibacterial and pharmacological properties of the penicillin) *β-lactamases* act on the C-N bond in β-lactam rings *Amidases* act on the C-N bond between the β-lactam ring and acyl side chain

True or False: Penicillins are the least toxic of all of the antibiotics.

Penicillins are the least toxic of all of the antibiotics. Five to ten per cent of individuals treated will become sensitized to the drug About 1% of the sensitized individuals can undergo anaphylaxis.

Phase variation

Phase variation is another type of altered gene expression caused by DNA rearrangements. *In phase variation, the expression of a particular gene product is turned on or off at high frequency.* Oscillation between on and off states occurs by two mechanisms: 1. One mechanism involves *site- specific inversion of a DNA segment* which contains a transcriptional promoter. Examples of this mechanism of phase variation are type 1 fimbriae expression in E. coli and expression of flagellar types in Salmonella. In the "on" state, the promoter element is oriented so that transcription of the gene product can take place. Inversion of the element orients the promoter in a direction that does not allow for transcription to occur (the "off" state). 2. The second mechanism of phase variation is associated with the *occurrence of short nucleotide repeats at the 5' ends of genes.* Such repeats can be gained or lost at high frequency as a result of strand misalignment during normal DNA replication and repair. Events of this type disrupt the integrity of a gene's translational reading frame. Phase variation of virulence-associated Opa proteins N. gonorrhoeae is associated with this mechanism. A common feature of this type of phase variation is that the number of nucleotides of the repeat element is not three, nor is it divisible by three.

What is the toxicity of vancomycin?

Phlebitis (vein inflammation) Ototoxicity (ear poisoning) Nephrotoxicity (poisoned kidney)

PO Ratio

Phosphate/Oxygen ratio In bacteria cells, the electron transport chain is shorter, containing only 2 sites of enzymatic action. Therefore, the PO ratio is typically lower (ratio-2), generating ~26 ATP. Bacteria with a ratio of 3 can produce ~38 ATP.

*There are 3 families of (+) sense RNA viruses that are non-enveloped/naked. They are all linear, cubical, and small*

Picorna Calici Astro (Don't have to memorize these for exam 1)

Pili in Neisseria

Pili genes undergo: *Phase variation* (switch on/off) *Antigenic variation* (variable C-terminus) Tips of pili (fimbriae) may have proteins (lectins) that bind to specific sugars, i.e. mannose. N. gonorrhoeae evades host immune system by changing surface proteins, i.e. pilin, outer membrane proteins by gene conversion

Plasmid mobilization

Plasmid mobilization is the presence of one plasmid in a cell facilitating the transfer of another plasmid in the same cell. For example, the F-plasmid can provide missing conjugative functions to the non-conjugative plasmid, ColE1 and ColE1 can be transferred to another bacterial cell.

Vancomycin resistance

Plasmids encode a gene known as Van A Alteration in drug target-- bacteria do not contain D-ala-Dala, but D-alanyl-lactate instead Altered cell wall peptidoglycan precursor Patients that have vancomycin resistant infection are difficult to treat Quinupristin/dalfopristin (streptogramins) tradename = Synercid Daptomycin, linezolid

Bacteriocinogenic plasmids

Plasmids which encode the production of *bacteriocins.* *Bacteriocins* are low molecular weight and heat stable proteins that are *bactericidal* (kills bacteria) for certain bacteria. Different bacteriocins produce different effects. The cessation (stopping) of bacterial DNA, RNA, or protein synthesis is the most common effect of bacteriocins. *Colicins are bacteriocins produced by E. coli.-- Colicin E1, E2, and E3*

What is the mechanism of action of polyene compounds?

Polyene compounds bind steroids in the membrane which disrupts the structural integrity of the membrane and allows important components to leak from cells. Polyenes preferentially bind ergosterol which is found in the membranes of fungi over cholesterol which is found in the membranes of mammalian cells.

PCR

Polymerase chain reaction Extremely sensitive procedure used to detect very low amounts of viral nucleic acids Viral nucleic acid is amplified often million fold The amplification requires a thermostable enzyme (*Taq polymerase*) Two complementary nucleic acid primers are extended on the template. Multiple cycles of extension ultimately results in large amounts of nucleic acids

Inhibitors of cytoplasmic membrane function

Polymyxins (polymyxin B and polymyxin E-colistin) Polyene compounds (anti-fungal agents- nystatin and amphotericin B) Imidazoles (non-antibiotic anti-fungal agents- ketoconazole, miconazole, clotrimazole)

What is the mechanism of action of polymyxins?

Polymyxins are cationic detergents and attach to anionic binding sites in the cell membrane. The lipophobic groups of the polymyxins interact with phospholipids which disrupt the membranes and change the permeability of the membranes. Polymyxins have been found to block the biologic effect of endotoxin lipopolysaccharide (LPS) of gram-negative bacteria presumably by their binding to phospholipids.

What bacteria are affected by polymyxins?

Polymyxins are most effective against gram-negative bacteria. Polymyxins are usually reserved for serious Pseudomonas aeroginosa infections which do not respond to other drugs.

What are other ways β -lactam antibiotics can be resisted?

Poor penetration of drugs Altered penicillin binding proteins

Pathophysiologic Effects of LPS

Powerful stimulator of innate immune responses Activates B cells and induces macrophages to release cytokines (interleukin 1, 6, TNF) Complement and coagulation cascades are activated Fever and shock Shwartzman reaction (DIC) follows Death from massive organ dysfunction Peptidoglycan can also cause vascular changes leading to shock, but less potent.

Peroxidase

Present in aerotolerant bacteria

Superoxide Dismutase

Present in: Aerobes Facultative anaerobes Aerotolerant anaerobes Microaerophiles

Catalase

Present in: Aerobes Facultative anaerobes Microaerophiles

Bacitracin

Prevents the regeneration of bactoprenol P

Ribosomes

Primary function is *protein synthesis* 70S = 50S + 30S Composed of 60% RNA and 40% protein *30S subunit* -16S RNA and 21 proteins *50S subunit*- 23S RNA; 5S RNA and 34 proteins

Ionizing Radiation

Produces free radical which causes single- stranded breaks in DNA and also deletions exhibits greater penetrance than ultraviolet radiation

Polycistronic mRNA

Prokaryotic mRNAs are polycistronic (polygenic) and usually carry information for the synthesis of several polypeptides from a single mRNA. Synthesis of each polypeptide chain in a polycistronic mRNA is determined by an AUG initiation codon and one or more stop codons that release the finished polypeptide from the ribosome. Several enzymes that act in sequence in a single metabolic pathway are regulated in a unit due to polycistonic mRNA.

Prophage Induction

Prophage induction occurs when a lysogenic bacterium becomes damaged. Inducing agents such as ultraviolet radiation causes DNA damage that results in the activation of the SOS repair pathway and the generation of single-stranded regions of DNA. In prophage induction by ultraviolet light, an unknown chain of events leads to the cleavage of cI repressor by the protease activity of the *RecA* protein, a major element in SOS repair and genetic recombination. Since induction cannot occur in a recA- mutant, this destruction of the repressor appears to be the cause of prophage induction.

What is the proposed mechanism of prion disease induction?

Protein induced conformational disorder PCD, prion disease Also referred to *Kuru* in humans

Prion

Proteinaceous infectious agent *Prion protein (Prp)* is coded by cellular gene (*PrPc*) Prp is normally expressed in neurons Prp is membrane anchored glycoprotein Phenol-- urea sensitive The normal PrP is sensitive to protease digestion, but can become *nuclease insensitive*-- example: Scrapie prion protein (PrPsc) is resistant to toprotease digestion Altered Prp protein may possesses amino acid changes - proposed mechanism of disease induction:

*Negative sense RNA viruses* denoted (-) sense ssRNA

Pure RNA that is *not infectious* Require a virion associated RdRp to cause infection The viral mRNA is *complementary* to viral genomic RNA 3'------UAC----------AUU---5' [(-) sense] 5'------AUG----------TAA---3' [(+) antigenome] Picture titles include: Virus type, viral protein synthesis, viral genome replication

Fermentation

Pyruvic acid (pyruvate) is converted in to energy molecules- CO2 & H20 (aerobic conditions) and organic molecules (anaerobic conditions) *Occurs mainly in the absence of oxygen* There are many types- alcoholic, homolactic, butyric acid, mixed acid, heterolactic, etc. *Used in bacteria*

Inhibitors of Nucleic Acid Function

Quinolones Nitrofurantoin Nitroimidazoles Rifamycins

RNA Genome viruses

RNA sequences can be: Single-stranded, non-segmented (*monopartite*), positive sense or negative sense 5' ------------------------> 3' Single-stranded, segmented (*multipartite*) ------------------------> -----------------> --------> Single-stranded, diploid ------------------------> ------------------------> Double-stranded 5' ------------------------> 3' 3' <------------------------ 5' RNA genome viruses are RNAse and alkali sensitive RNA genome viruses undergo: RNA replication---> RNA transcription---> mRNA translation---> protein Example: MMR virus and HIV

What is RdRp?

RNA-dependent RNA polymerase

Porins

Referred to as outer membrane proteins (OMPs) Trimeric proteins Each subunit has a pore (1 nm diameter) through which chemicals and nutrients traverse outer membrane Allow small hydrophilic solutes to traverse the membrane (less than 700 daltons) Barrier for large or hydrophobic antibiotics and proteins such as lysozyme

Wild Type

Refers to an organism or a gene most often found in the natural setting (example: E. Coli are Lac+) Genes of bacteria isolated from nature are considered wild type. In terms of nutritional requirements, some bacteria can synthesize all of their nutritional needs and do not require nutrient supplements. These organisms are referred to as *prototrophs.* Derivatives of wild type genes are termed *mutant genes*. Mutant strains that are defective in synthesizing a particular nutrient are referred to as *auxotrophs.*

Genotype

Refers to the actual genetic composition of an organism. The Leu- cell must have a defective gene preventing it from synthesizing leucine. Therefore, it is referred to as leu- or leu without minus sign. In written text, genotypes are written in lower case letters and in italics. There are several genes involved in the synthesis of leucine. To denote a mutant strain of a particular gene A, one would write leuA.

O antigen

Repeating carbohydrate units (50-100) Occur in a variety of combinations Attached to the core and extends away from the cell Distinguishes serotypes (strains) of a species Accounts for antigenic diversity among serotypes Mutants that lack O antigens are sensitive to bile salts and antibiotics to which wild type cells may be resistant

*Initiation* phase of chromosome replication

Replication proceeds bidirectionally from OriC (245 bp). 1. *DnaA protein* binds to 9bp repeats in the OriC. 2. *DnaB (helicase)* then binds and further unwinds DNA. 3. *Single-strand binding proteins (SSB)* bind to SS regions to hold the strands open. 4. *Primase* then binds to protein complex forming a *primosome*. A RNA primer is synthesized at each replication fork by primase. 5. Two *DNA polymerase III holoenzyme complexes* bind at each fork- one synthesizing leading strand and the other synthesizing lagging strand.

RecA protein

Required for *homologous recombination* in which homologous sequences are merely exchanged Nonhomologous recombination events require very little sequence homology - does not require RecA Examples: site-specific (integration of lambda bacteriophage) into E. coli chromosome Transposition (insertion of transposons into E. coli chromosome)

*Tetracycline resistance*

Resistant organisms cannot accumulate tetracycline within the cell because of excretion/efflux

Can Methicillin-resistant Staphylococcus aureus (MRSA) be resistant to vancomycin?

Resistant to other antibiotics Resistant to most known penicillins and cephalosporins Only treated with Vancomycin Produces new penicillin binding protein-pbp2' (mecA) Vancomycin resistance has been reported (VRSA) Mechanism involves VanA resistance gene in some isolates and in others a thickening of the peptidoglycan and decreased cross-linking seem to be involved.

Routes of viral entry:

Respiratory routes: Droplet infections through mucosa (IgA, NK cells, macrophage defenses) Gastrointestinal route: oral-fecal routes lead to naked viruses in feces (acidity of stomach leads to Rhinovirus inactivation; *bile salts inactivate all enveloped viruses*) Venereal (sexual intercourse route): occur mostly in adults, but observed in neonates (HIV, Herpes, CMV, Papilloma) Virus spread to the nervous tissue or distant organs under multiple routes: - neural or axonal route - blood, blood brain barrier (hematogenous route)

Reverse mutation

Reversion (back mutation or reverse mutation) The process in which the wild-type phenotype is regained. For example, if 104 Arg- bacteria are plated on agar lacking arginine, no colonies will form. If 107 bacteria are plated, a few colonies will appear. These colonies can make their own arginine. These are called revertants and the cells exhibit an Arg+ phenotype. They can grow in the absence of arginine.

*There are 6 families of (-) sense RNA viruses that are enveloped. They are all linear, helical, and bullet shaped These families range from 1 segment, 2 segments, 3 segments, and 8 segments*

Rhabdo (one-segment) Paramyxo (one-segment) Filo (one segment) Arena (2 segments) Bunya (3 segments) Orthomyxo (8 segments)

*Rho dependent* Transcription Termination

Rho protein is involved in termination because it has helicase activity *most RNA synthesis is terminated by Rho proteins*

Shine-Dalgarno Sequence

Ribosome binding site consensus sequence is 5'AGGAGGU3' Important for the binding of mRNA to the 30S complex This sequence base pairs to a region at the 3' end of the 16S rRNA This positions the *AUG codon* so that it binds to an initiator tRNA anticodon

Teichoic Acids

Rich in glycerol phosphate/ribitol phosphate *Attached via phosphate groups to muramic acid (NAM) in the peptidoglycan* *Found in gram-positive bacteria* Absent in gram-negative bacteria Lipoteichoic acids (membrane associated) Nature of modifications can define serotype (sugars, amino acids)

Peptidoglycan

Rigid Gives bacterial cells shape Protects bacteria from *osmotic lysis* Has unique components Target of many antibacterial agents

Semi-conservative replication

Semi-conservative replication. In this model, the two strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand. This results in two DNA molecules with one original strand and one new strand.

What is the clinical use of vancomycin?

Serious infections caused by multi-resistant staphylococci or streptococci or if patient is hypersensitive to penicillin Clostridium difficile infections of the bowel Used as a last resort

Streptococcus species Plasmid-encoded virulence traits?

Several species of Streptococcus produce *hemolysins (lyse red blood cells)* that are encoded on plasmid DNA.

What is the toxicity of bacitracin?

Severe nephrotoxicity restricts clinical usefulness to topical administration only

Characteristics of killed vaccines

Short lived IgG No Cell-mediated immunity No Infectious contaminants Immunosupression is used No storage difficulty Reversion to wild type does not occur No Herd immunity

Viral entry/uncoating

Simple engulfment by cells-- Viral envelope fuses with cell membrane at neutral pH The envelope is left at the surface and the *capsid enters the cell* Syncytium formation (cell-cell fusion that occurs with giant cells after a viral infection) Fusion at the endosomal membrane (acidic pH) is called *viropexis*

Liver or kidney damage caused by antimicrobial agents

Since a number of antibiotics are metabolized in the liver, *hepatic toxicity* can occur. This has been of particular concern when isoniazid is used to treat tuberculosis. *Renal toxicity* can follow the use of aminoglycosides while other drugs may damage renal tubular cells.

Characteristics of a bacterial chromosome

Single, circular, covalently closed strand of DNA referred to as a *nucleoid* Double stranded, *negatively supercoiled* 2% of cellular dry weight Molecular weight of ~ 3 x 109 daltons No histone proteins 1300 mm in length (~ 1 mm)

Small drug-resistance plasmids

Small drug-resistance plasmids are not usually called R plasmids. These plasmids lack the ability to facilitate their own transfer but in some instances are mobilizable. These plasmids usually carry one or two antibiotic resistance genes and are commonly used in gene cloning experiments. Plasmid pBR322 is an example.

Transferrin and lactoferrin receptors

Some bacteria are capable of direct recognition of transferrin and lactoferrin through receptors present on bacterial cell surface. Transferrin/lactoferrin receptors are present in pathogenic *Neisseria*

What occurs after a virus enters a host cell?

Some stay in the cytoplasm Some go to the nucleus *All of them translate their mRNA in the cytoplasm*

Specialized transduction

Specialized transduction is mediated by a bacteriophage that has a *high specificity for integrating into a certain site* on the bacterial chromosome. *This specificity restricts transfer of genetic information to those genes that are in the immediate vicinity of the integration site.* For example, the chromosome integration site for E. coli bacteriophage lambda is in the region of the chromosome that encodes genes for galactose utilization. Invariably, lambda transfers this region to recipient cells.

Viral Plaques

Spots of localized cell death on cultured cells forming holes in a lawn of cells. Only caused by a virus that causes cell death

Focus formation

Spots where cells form a mountain due to over growth Transformed cells grow on top of one another, eventually forming foci Tumor viruses cause foci on cells in culture

Tetracycline toxicity

Staining and impairment of bone and teeth structure (contraindicated (withhold treatment) during pregnancy and in children up to 8 years of age) Gastrointestinal disturbances due to alteration of the normal flora Development of oral or vaginal opportunistic yeast infections

What bacteria are affected by vancomycin?

Staphylococci Streptococci Clostridia Many gram-positive bacteria including anaerobes

Cytoplasmic Inclusions

Storage granules (metachromatic) Act as storage mechanism when nutrients are low. Identifying feature in *Corynebacterium diphtheriae* Different types: Polymetaphosphates - volutin granules (Babes- Ernst) Glycogen-like polymers Poly b-hydroxybutyric acid

What are the clinical uses for *natural penicillins*?

Streptococcal and pneumococcal infections Sensitive staphylococcal infections (penicillinase-negative) Clostridial infections Meningitis, gonorrhea, syphilis, anthrax, actinomycosis

Streptococcus pneumoniae

Streptococcus pneumoniae (S. pneumoniae) are lancet-shaped, gram-positive, facultative anaerobic bacteria with over 90 known serotypes. Most S. pneumoniae serotypes can cause disease, but only a minority of serotypes produce the majority of pneumococcal infections. Pneumococci are common inhabitants of the respiratory tract and may be isolated from the nasopharynx of 5-90% of healthy persons, depending on the population and setting. *Transiently competent* Competent state is induced by a *competence activator* which triggers the synthesis of new proteins. Cells then develop capacity to bind DNA. Does not require specific DNA uptake sequences and *will take up any DNA* once competence is induced. *Only homologous DNA* will recombine into the recipient chromosome. One strand is degraded and the remaining strand is transported into the cell. Transforming DNA integrates as single strand in homologous region (new strand replaces old) and a *heteroduplex* is created.

There are newer drugs to treat infections by positive gram-positive cocci (MRSA)

Streptogramins Ketolides Oxazolidonones Daptomycin

Streptogramins

Streptogramins - *quinupristin/dalfopristin* Cyclic peptides - SynercidTM Mechanism of Action: *Dalfopristin* binds to 50S ribosomal subunit and induces a conformational change that facilitates binding of quinupristin; also prevents peptide chain elongation *Quinupristin* causes premature release of peptide chains from ribosomes Spectrum of Activity: Primarily restricted to healthcare-associated infections caused by vancomycin-resistant Enterococcus faecium (but not E. faecalis) Methicillin-susceptible Staphylococcus aureus or Streptococcus pyogenes

When is Stringent factor synthesized?

Stringent factor is inactive during normal protein synthesis and no ppGpp is made. *Stringent factor is synthesized when a ribosome engaged in translation is occupied by an uncharged tRNA* Activation of stringent factor occurs when the 50S subunit is intact in a 70S ribosome particle and is bound to mRNA, engaged in translation. Additionally, a site on the ribosome must be occupied by an uncharged tRNA molecule rather than a charged tRNA. During amino acid starvation, the tRNA species that normally carries that amino acid will be uncharged. This leads to production of ppGpp which signals that protein synthesis has stopped and that additional rRNA should not be made.

Inhibitors of Tetrahydrofolate Synthesis

Sulfonamides Trimethoprim

Bacterial swarming

Surface spreading phenomena of motile bacteria Example: Proteus and Bacillus

Helical Nucleocapsid

Symmetry of nucleocapsid: Helical Can have two-fold rotation axis perpendicular to helix

Icosahedral Nucleocapsid

Symmetry of nucleocapsid: Icosahedral also referred to as cubical Contain 20 triangular faces, identical elements on the surface Can be related by 2, 3 and 5-fold rotation axes

What is one common rule about most regulatory mechanisms in bacterial cells? *(Start of PID 9: Regulation of gene expression Cards 148- 203)*

Synthesis of a gene product is turned on when the gene product is needed Synthesis of a gene product is turned off when the gene product is not needed

Match the test tube letters with the type of bacteria Aerobes Facultative anaerobes Anaerobes Aerotolerant anaerobes Microaerophiles

Test Tube 1- Aerobes Test Tube 2- Anaerobes Test Tube 3- Facultative anaerobes Test Tube 4- Aerotolerant anaerobes Test Tube 5- Microaerophiles

Translation Inhibitors

Tetracycline: Prevents binding of aminoacyl-tRNA to the A site on 30S ribosomal subunit Aminoglycosides (streptomycin): Misreading of the mRNA Macrolides (erythromycin): Interact with 50S ribosomal subunit and inhibits translocation Chloramphenicol: Binds 50S subunit and inhibits peptidyl transferase *Fusidic acid*: Inhibits translocation of peptidyl-tRNA from the acceptor to donor site

What is the leader region in the trp operon?

The *leader region* is the sequence that precede the enzyme-encoding portion of the transcript. The leader region is rich in codons for the amino acid it controls. This leader transcript encodes a leader peptide which is rich in those amino acids. If the amino acid that is being controlled is in short supply, translation is stalled at the multiple codons of the transcript. In the case of the trp operon, trpL is the leader region and tryptophan is the amino acid controlled.

Other examples of positive genetic regulatory protein

The *malT gene*, which encodes a protein that is required to activate the genes of the maltose operon used for maltose utilization. The product of the *araC gene* functions in unliganded form as a repressor and in the arabinose-bound form as an activator of transcription therefore acting as both a positive and negative regulator.

Antimicrobial Agent Spectrum

The *spectrum* of an antimicrobial is related to the *range of activity that an antimicrobial exhibits.* An antimicrobial may possess a *narrow spectrum* with antimicrobial activity on only a restricted group of organisms, genera of organisms or a species of organism or may have a *broad spectrum* and have activity against a wide range of organisms.

How is the AMP-CAP complex a *positive regulatory system*?

The AMP-CAP complex binds to DNA and enhances transcription by rendering the promoter site more accessible to RNA polymerase

Bacterial Cell Cycle *(Start of PID 7-8: DNA replication Cards 594-639)*

The Bacterial cell cycle resembles eucaryotic cell cycle in slow growing bacteria. The *c period* takes *40 minutes* and DNA replication occurs. The *D period* takes *20 minutes* and cell division occurs.

tRNA

The CCA sequence is conserved at the 3' end of all mature tRNA molecules to function as the site of amino acid attachment. This sequence is acquired and maintained by stepwise nucleotide addition by the ubiquitous CCA enzyme, which is an unusual RNA polymerase that does not use a nucleic acid template for nucleotide addition.

F+ plasmid

The F plasmid exists as a autonomously replicating episome *F factor present and free*-- donor (male) cell

F' plasmid

The F plasmid exists as an autonomously replicating episome containing small segments of DNA derived from the bacterial host chromosome and is designated F prime. Merogeonote (diploid for a gene)

F- Factor

The Fertility factor allows genes to be transferred from one bacterium carrying the factor to another bacterium lacking the factor by conjugation.

HIV is an example of a *retro-virus* and a RNA genome virus

The HIV retrovirus RNA enters the cytoplasm of the host CD4 T cell and is replicated by the virus' RNA polymerase- *virus-Reverse Transcriptase (RT)* into a DNA molecule. The viral DNA is then transcribed by the host cell's RNA polymerase II in the nucleus. The HIV mRNA is then translated into a protein by the host ribosome.

Explain why the LacI gene is considered *trans dominant* in merozygotes.

The LacI gene controls inducibility of the lac operon by encoding the lac repressor protein. Genotype: *I-O+Z+/I+O+Z-* (LacI gene is mutated on the original bacterial chromosome and the LacZ gene is mutated on the plasmid) The lac region is diploid and synthesis of beta-galactosidase is *inducible*... why? Well... Although I- on the original bacteria chromosome is mutated, the plasmid's I+ can still express a repressor protein. The plasmid's Lac repressor can influence the original bacteria's chromosome. So since one LacI gene (on the plasmid's chromosome) works and one LacZ gene (on the original bacteria's chromosome) works, beta-galactosidase can still be induced under the right conditions. I+ is on the opposite chromosome of the Z+ gene, so it is considered *trans.* In other words: The plasmid's LacI can produce a repressor protein that influences both chromosomes and therefore still induces beta-galactosidase production. (Trans-acting: Affects genes located on a different DNA molecule)

Select the correct term: The TCA cycle is apart of (anaerobic or aerobic) respiration.

The TCA cycle is apart of aerobic respiration because oxygen is used as the final electron acceptor of the ETC. Helps generate 19 times more energy than fermentation (~38 ATP)

Viral complementation

The ability of a viral gene or protein to rectify the deficiency of related gene or protein of another virus. Complementation can occur when either one or both of two viruses that infect a cell have a mutation that results in a nonfunctional protein. The non- mutated virus "complements" the mutated one by making a functional protein that serves for both viruses. One clinically important example of complementation is hepatitis B virus providing its surface antigen to hepatitis delta virus, which is defective in its ability to produce its own outer protein.

Bactericidal

The action of a drug is *bactericidal* if the action of the drug *causes irreversible damage to the cell* and *results in the death of the cell.* Agent exerts a killing action that is irreversible

Frameshift mutation

The addition or deletion of bases result in a change in the reading frame. The base sequence is read in groups of three bases when it is being translated into an amino acid. The addition or a deletion of a base changes the reading frame. A base addition causes all downstream codons to be read incorrectly and the entire amino acid sequence following the mutation is altered. Frameshift mutations can result in gross structural changes of polypeptides

What is the mechanism of action for *cycloserine*?

The bactericidal activity of cycloserine lies in its structural similarity to alanine. *Cycloserine is a competitive inhibitor* of two sequential reactions in the synthesis of peptidoglycan in which *D-alanine is incorporated.* These reactions are catalyzed by alanine racemase and D-alanyl-D-alanine synthetase.

Flagellar phase variation

The basis for the control of flagellar phase variation in Salmonella typhimurium is dependent on an invertible DNA sequence. In Salmonella, flagella antigens may be of two types, H1 and H2. These antigens are alternatively expressed. This is called phase variation. H2 flagellin gene is adjacent to a 970 base pair sequence that is capable of existing in either orientation with respect to H2. In one orientation, a promoter at one end of the sequence directs transcription of the H2 gene and a contiguous gene which encodes a repressor of the H1 gene. This orientation allows repression of H1 flagellar phenotype. The opposite orientation of the 970 base pair sequence permits no transcription of H2 and the repressor gene. Therefore, the cell expresses H1 flagellar antigen The hin gene product is a site-specific recombinase that controls inversion of the invertible sequence and has significant protein sequence similarity with the tnpR gene product of Tn3, a transposon. The recombinase recognizes inverted repeats found at either end of the controlling sequence. The phase variation system may represent the acquisition by the bacterial cell of a portion of a transposable element.

Capsid Shell

The capsid shell is made up of hundreds of identical protein units called capsid proteins (protomers, capsomeres) Viral genomes can be covered with single or two layers of protein shells (capsids) In naked viruses (non-enveloped viruses), the capsid shell mediates entry Function: protects genetic material Contains antigenic determinants which promote antibody production and cell-mediated immunity Also referred to as a protein shell (cage)

What is the significance of antigenic variation in bacterial surface components, such as the antigenic variation of pili?

The characteristics allows bacteria to invade the host immune system by changing surface components

What accounts for the color differentiation between gram positive and gram negative bacteria observed in step 3 of the Gram Stain?

The color differentiation observed is due to the *thickness* of the cell walls. Gram negative bacteria cell walls are very thin composed of one or two layers of peptidoglycan, on the other hand gram positive cells have multiple layers of peptidoglycan When alcohol is added to the bacteria, the gram positive pores close retaining the color. The gram negative cells do the same, but their wall is thinner so color is not retained.

Disinfection

The destruction of bacteria by processes that fail to meet the criteria for sterilization.

What is the fate of beta-galactosidase when the operator gene is mutated in merozygotes?

The fate of beta-galactosidase depends on the mutation of the operator gene (lacO) and where the gene is positioned. The expression of the lacZ gene is affected only when it was conjoined with lacO on the same stretch of DNA. Example 1- Genotype: *I+O+Z-/I+OcZ+* (LacZ gene is not functioning on the original bacteria's chromosome, the LacO is mutated to be constitutive (none stop so it will work despite the repressor being present or not) on the plasmid's chromosome) The lac region is diploid and synthesis of beta-galactosidase is *constitutive*... why? Oc is on the plasmid is *cis* to the only functioning LacZ gene in the cell, which is also on the plasmid. So the constitutive influence is going to affect beta-galactosidase production Example 2- Genotype: *I+OcZ-/I+O+Z+* (LacZ gene is not functioning on the original bacteria's chromosome and the LacO is mutated to be constitutive (none stop so it will work despite the repressor being present or not) on the original bacteria's chromosome) The lac region is diploid and synthesis of beta-galactosidase is *inducible*... why? Oc is on the original bacteria's chromosome and is *trans* to the only functioning LacZ gene in the cell which is on the plasmid. So the constitutive influence is not going to affect beta-galactosidase production because it cannot influence the other chromosome. (Cis-acting: Affects only genes on the same DNA molecule. Trans-acting: Affects genes located on a different DNA molecule)

Fluctuation test

The fluctuation test of Luria and Delbruck Used resistance to phage infection as a selectable marker Addresses the question of whether exposure to a selective agent induces mutations at the time of exposure or merely select for cells possessing pre-existing mutations. Proved that mutations in organisms occur as a spontaneous mutational event that can be selected for using the appropriate selective conditions Showed that mutations do not arise as a result of some direct genetic change brought about by an environmental agent *Spontaneous mutagenesis produces mutant cells that arise spontaneously in nature and in the laboratory.*

Virulence genes

The genes that give pathogenic bacteria the ability to cause disease Regulated in a way that allows pathogens to express the products of these genes when environmental conditions are optimal for their effectiveness. Typically regulated by a singly regulatory protein that controls a number of operons that are said to be members of the same regulon. Most pathogens only express virulence genes inside of the eukaryotic host that indicates that the environment inside the host turns on expression of these genes. Virulence genes are genes that when inactivated render the bacterium nonpathogenic but does not affect its ability to grow outside of the host.

Genetic Code

The genetic code is considered *degenerate and non-overlapping* Triplet code 64 possible amino acids may be encoded More than one triplet code can be used to code for the same amino acid (degenerate) The code is also non-overlapping-- example: UUU and UUC both code for phenylalanine, but cannot code for another amino acid

Bacteriophage genomes

The genome of bacteriophages consists of a *single nucleic acid molecule* either DNA or RNA; never both. The nucleic acid may be either: double-stranded or single-stranded and linear or circular DNA OR single- stranded or double-stranded linear RNA An *exception is the φ6 phage of Pseudomonas phaseolica*, whose genome is composed of three linear double-stranded RNA molecules (segmented genome). The bases of bacteriophage DNA are often glycosylated and/or methylated. The genomes of bacteriophage can also contain unusual or substituted bases.

Disinfectant potency

The increase in *concentration* of the disinfectant, the more potent The increase in *time/exposure* of the disinfectant, the more affective the killing *pH* influences the degree of ionization and enzyme function in bacterial cells An increase in *temperature* leads to an increase in killing Nature of Organism Extraneous material

N-formylmethionine

The initiating amino acid in bacteria

insertion sequence IS10

The insertion sequence IS10 encodes only an IS10-specific transposase (tnp).

Antibody-dependent cell mediated cytotoxicity (ADCC)

The intact viral glycoproteins on the *host cell surface* can be recognized by antibodies. The antibody bound infected host cell can be recognized by many nonsensitized immunocytes-- Natural killer cells, monocytes, neutrophils that express Fc receptors for IgG These immunocytes recognize IgG coated infected host cells, and lyse them Host cell lysis proceeds without phagocytosis Host cell lysis by this mechanism involve no complement fixation ADCC of virus-infected cells is classified under type II hypersensitivity reaction

Lac Operon (in E. Coli)

The lactose operon is composed of a promoter, an operator and three structural genes for three enzymes- *beta-galactosidase, galactoside permease, and galactoside transacetylase*, which allows E. coli to use lactose as a carbon source. The regulatory gene (*lacI*) coding for a repressor protein is located near the operator but is not continuous with the operator. The DNA for the structural genes is transcribed as a single polycistronic mRNA.

cI and cro genes

The lysis-lysogeny decision of temperate phage lambda is governed by the products of the cI and cro genes. In lysogeny, cI protein is produced preferentially over the Cro protein, which is a repressor of cI synthesis and is necessary for several features of the lytic life cycle. During vegetative phage growth, Cro protein is produced and cI repressor is not produced. Mutations in the cI gene generate phage that cannot enter the lysogenic life cycle while mutations in the cro gene result in phage that cannot enter the lytic life cycle. (In lysogeny, cI protein is produced preferentially and inhibits the lytic life cycle; The Cro protein promotes lytic life cycles )

Explain why spores are heat resistant

The mechanisms that account for heat resistance include the impermeability of the endospore coat, the dehydration of the cytoplasm and the production of special proteins that protect the spore's DNA.

What is the mechanism of action of Aminoglycosides?

The mode of action of *streptomycin* is the best studied of those of the aminoglycosides. *Aminoglycosides attach to the 30S ribosomal subunit and cause misreading of mRNA and inhibits protein synthesis.* This is a two-stage process: (1) a small amount of drug gets into the cell and causes misreading of mRNA, leading to the synthesis of abnormal membrane proteins which allow a greater influx of drug. This higher concentration of drug is enough to cause inhibition of protein synthesis. When a cell wall synthesis inhibitor such as penicillin is used in combination with streptomycin, inhibition of cell wall synthesis by penicillin leads to damage to the membrane and allows more streptomycin (or other aminoglycoside) to traverse the membrane. *This is a classical example of drug synergism.*

Nucleoside Analogs

The nucleoside analogues are an important class of antiviral agents now commonly used in the therapy of human immunodeficiency virus (HIV) infection, hepatitis B virus (HBV), cytomegalovirus (CMV) and herpes simplex virus (HSV) infection. *The nucleoside analogues resemble naturally occurring nucleosides and act by causing termination of the nascent DNA chain.* These agents are generally safe and well tolerated as they are used by the viral, but not human polymerases in DNA replication. Nucleoside analogues are a large class of agents that include drugs for cancer (cytarabine, gemcitabine, mercaptopurine), rheumatologic diseases (azathioprine, allpurinol) and even bacterial infections (trimethoprim).

Polymyxins

The polymyxins are a group of polypeptide antibiotics. Polymyxin A, B, C, D, and E have been isolated. Of the five polymyxins, only two are currently in clinical use, *polymyxin B and E (colistin).*

Lysogeny

The process of forming a lysogen by infecting a bacterial culture with a temperate phage persistence of a prophage without phage replication and destruction of bacterial cell

bacteriophage one step multiplication curve

The production of progeny phage as a function of the time after infection is described by the one step multiplication curve. 1. A dense suspension of bacteria is infected with phages and incubated for a few minutes to allow for phage attachment or adsorption to a specific receptor on the surface of the host bacterium. 2. The culture is diluted and incubated further and samples are then plated on sensitive bacteria to produce plaques on a lawn of bacteria. The plaque assay is used to determine the number of progeny phage that are produced after infection. 3. Within the first few minutes after infection, the original parental phage are no longer infective. The stage of intracellular phage growth during which the infected host cell contains no material capable of initiating a plaque is called the *eclipse phase.* During the eclipse phase, the parental phage particle is reduced to a non-infective form probably because of damage to its physical integrity during entry inside of the host cell. The eclipse phase ends when mature intact infective progeny have been constructed under the direction of non-infective parental phage. 4. The plaque-forming units of the culture remain constant for the first 20 minutes or so after infection. This is the *latent phase.* The latent phase represents the time that elapses between the time of infection with phage and the appearance of progeny phage. (in the picture, time from bullet 1 to bullet 3) 5. After hours, plaque-forming units rise rapidly until a plateau is reached after minutes. This is the *rise phase.* (bullet 3 to 4 in the picture) The ratio of the final titer of the plateau to the initial titer of phage-infected bacteria is the burst size. *Burst size* corresponds to the average number of progeny phage particles produced per bacterium.

Complete the sentence: The rate of synthesis of tRNA and rRNA in bacteria is coordinated with the growth rate of the ______________.

The rate of synthesis of tRNA and rRNA in bacteria is coordinated with the growth rate of the cell. The concentration of these molecules is greatest in rapidly growing cells.

What is enzyme induction?

The regulatory gene, *lacI*, codes for a repressor protein of the lac operon and is located near the operator The lac repressor when bound to operator *prevents the movement of the polymerase from the promoter into the structural genes* which causes transcription of lac mRNA to be halted. Inducer drives the repressor from the operator allowing transcription to occur. This is called enzyme induction.

Deletions

The removal of a number of bases from the coding sequence. Deletion mutations can result in gross genetic changes.

Transcription

The synthesis of RNA using DNA as a template *Requires a DNA-dependent RNA polymerase* Types of RNA include: mRNA tRNA rRNA *occurs in the 5' to 3' direction*

What is the toxicity of flucytosine?

The toxicity of flucytosine is directed against the gastrointestinal tract (abdominal pain, diarrhea, nausea, vomiting), liver (hepatitis) and bone marrow (leukopenia, thrombocytopenia).

Cell wall synthesis

The uridine nucleotide precursor, *UDP-N-acetyl muramic (NAM) acid*, bearing its pentapeptide is synthesized UDP-NAM-peptide is attached to the carrier molecule, *bactoprenyl phosphate*, and *N-acetyl glucosamine (NAG) is added* The NAM-NAG disaccharide with attached peptide is added to the growing glycan chain of the cell wall and cross-links are established by transpeptidation.

*Measles, Mumps, and Rubella (MMR) Virus* is an example of a RNA genome virus

The viral RNA enters the cytoplasm of a host cell and is replicated by the virus' RNA polymerase- *RdRp*. The viral RNA is then transcribed by RdRp in the cytoplasm. The MMR mRNA is later translated into a protein by the host ribosome.

Characteristics of the DNA polymerases involved in DNA replication in *E. Coli*

There are 5 DNA polymerases in E. coli. (I, II, III, IV, V) All use 4 dNTPs as substrates, eliminating ppi All require a primer-template *DNA polymerase III* is the major polymerizing enzyme during chromosome replication - All synthesize DNA 5'---->3' - Proofreading carried out by 3'--->5' exonuclease activity *DNA polymerase I* is involved in gap-filling and primer removal and DNA repair *DNA polymerase II* is also involved in repair of DNA. The enzyme edits errors made by Polymerase III on the *lagging strand.* *Polymerases IV & V* are error prone polymerases

Lysogenic Life Cycle *(This is a way bacteria can obtain virulence genes example-corynebacterium diphtheria)*

There are two types of lysogenic cycles. The most common is exemplified by E. coli *phage lambda.* 1. The DNA of lambda is injected into the bacterium. 2. Transcription of lambda mRNA is turned off by a repressor after a brief period of transcription to produce proteins need for integration of lambda genome into bacterial chromosome. 3. After integration, the phage genome replicates as a part of the bacterial chromosome. *A second type is shown by E. coli phage P1* where there is no integration of the phage genome into the chromosome of the bacterium. Instead, the phage DNA becomes *a plasmid-like element* and independently replicates itself in the cytoplasm of the bacterium.

*Double-stranded RNA viruses* denoted dsRNA

They all carry a vision associated RNA polymerase,*RdRp*, in the virus particle *Pure RNA not infectious* Transcription of mRNAs from the dsRNA template is completed by RdRp. Then translation occurs. For replication, the positive sense strand is used as a template for the (-) sense strand. Then the two strands anneal and assemble. *Only example is reovirus families*

*Retrovirus*

They carry *diploid RNA* that are *two identical (+) sense RNA* The RNAs are capped and polyadenylated A reverse transcriptase is present in the viral particle A DNA intermediate is generated in replication Both cytoplasmic and nuclear life cycle Provirus integration into chromosomes--meaning their DNA genome can integrate into host chromosome

*Ambisense RNA viruses*

They carry a RNA polymerase *RdRp* in the virus particle Half of the genome is (+) sense and the remaining half is (-) sense, but for practical purposes they can be considered (-) RNA viruses by the way they replicate All Arena viruses and some Bunya viruses

Envelope glycoproteins

They exist as transmembrane These glycoprotein monomers form multimeric complexes on the surface of Found on virus specific envelope and found on infected cell membranes Attach to receptors on cell surfaces Obtained through membrane fusion Also referred to as peplomer spikes

R plasmid fact

Through the years the rate of microbial evolution has been such that today conjugative plasmids and drug resistance plasmids have greatly increased in prevalence and the tendency has been toward the evolution of R plasmids with multiple drug resistance.

*There are 3 families of (+) sense RNA viruses that are enveloped. They are all linear and cubical. One out of three families is large*

Toga-small Flavi-small Corona- large (Don't have to memorize these for exam 1)

DNA replication

Topoisomerase= gyrase

Inducible Transcription

Transcription of genes is turned on in response to the presence of an inducer Also called induction- an increase in synthesis of an enzyme or series of enzymes mediated by a specific molecule (inducer) ex: lac operon

Constitutive Transcription

Transcription of genes occurs in the absence of an inducer Example: Transcription occurs all the time

What is the first step after a (-) ssRNA virus enters a cell?

Transcription of genome to mRNAs by already present RdRp

What is the first step after a (+) ssRNA virus enters a cell?

Translation of genome into proteins including RdRp

Transpeptidation

Transpeptidation reaction occurs, releasing d-ala and forming a cross-link bond between D-ala (carboxyl group) and m-DAP or L-lys-(gly)5 (amino group). This is completed by *transpeptidase.* Cross-linking occurs about 90% in gram-positive and 20-30% in gram-negative Transpeptidases are also called penicillin binding proteins (PBPs)

What is the function of the cytoplasmic membrane?

Transport Site of attachment for enzymes DNA replication Electron transport (energy generation) Oxidative phosphorylation Cell wall biosynthesis Flagella movement

Transposon Tn10

Transposon Tn10 is composed of two (nearly identical) copies of IS10 bracketing a gene for tetracycline resistance (tetR). Both IS10 L (left) and IS10 R (right) contain the IS10 transposase gene; however, the protein specified by IS10L has reduced activity due to several mutations.

*Trimethoprim*

Trimethoprim is an anti-folic acid agent which is a synthetic structural analog of the pteridine portion of folic acid. *This drug is frequently used in combination with one of the sulfonamides.* A typical combination would be sulfamethoxazole and trimethoprim. This is an example of drug synergy. Mechanism of Action: Trimethoprim *competitively inhibits dihydrofolate reductase*, the last enzyme in the pathway leading to the synthesis of tetrahydrofolic acid. Spectrum of Activity: Gram-positive and gram-negative bacteria, Pneumocystis carinii Clinical Use: Urinary tract infections, Pneumocystis carinii infections Toxicities: Hypersensitivity, folate deficiency (potentially)

TrpL

TrpL is a *162 base pair leader region* located in the attenuator of the tryptophan operon and has two tandem trp codons. During translation, ribosomes may stall at either of the two tandem trp codons. The stalling is long enough for the rest of the transcript to form a secondary structure. The position of the ribosome on the transcript determines which one of the alternative RNA secondary structures will form.

True or False: Surface-exposed iron receptors could be viable vaccine candidates for prevention of infection.

True

True or False: The TCA cycle supplies key intermediates (alpha-ketoglutarate, pyruvate, oxoloacetate, etc) for synthesis of amino acids, lipids, purines, and pyrimidines.

True

True or False: The ability of some microorganisms to cause disease is associated with special virulence determinants carried on plasmids.

True It should be noted that virulence plasmids are not contained to any specific genera but are widespread.

True or False: LacI and lacO mutations affect the rates of synthesis of all enzymes in Lac Operon.

True The expression of all enzymes in the lac operon is coordinate. The rate of synthesis of the permease and the transacetylase changes by the same factor as beta-galactosidase when presented with inducer. LacI and lacO mutations affect the rates of synthesis of all enzymes coordinately. The coordinate synthesis of these enzymes is explained by the synthesis of a polycistronic mRNA containing the information for all three enzymes.

True or False: Eukaryotic cells have multiple origin sites for replication, but prokaryotic cells only have one.

True The only start site for replication is the OriC site Consists of 245+ base pairs

Trp Operon

Tryptophan is synthesized in five steps, each requiring a particular enzyme. The enzymes are encoded by genes trpA, B, C, D and E. The trpE gene is the first one translated The genes encoding the enzymes of the trp biosynthetic pathway are adjacent to one another and in the same order as their use in the biosynthetic pathway. The genes are transcribed as a polycistronic trp mRNA. There is a promoter-operator region and an *attenuator* region.

tRNA synthetase

Tryptophanyl tRNA synthetase Produces tRNA

Viral Genotyping Mixing

Two or more related genomes are packaged in one particle Example: polio 1 and 2 genome with a polio 2 capsid

Respiration

Two types: Aerobic and Anaerobic Oxygen is used in some form as the final electron receptors of energy generation pathways Aerobes use oxygen Anerobes use compounds other than oxygen like NO3- Both are more efficient in producing ATP than fermentation

*Aminoglycosides resistance*

Two ways: Alteration of transport protein - anaerobic bacteria Inactivating enzymes (most common mechanism)-- enzymes are plasmid-encoded or transposons Mechanisms of inactivating enzymes include: acetylation of amino groups phosphorylation of OH groups adenylation of OH groups

DNA Gyrase

Type II Topoisomerase Relieves supercoils ahead of the replication fork To introduce negative supercoils in DNA requires ATP To relax negative supercoils in DNA does not require ATP Inhibitors: *quinolones* (ciprofloxacin, norfloxacin, novobiocin, coumermycin, nalidixic acid)

Other methods of killing: *Ultrasonic Vibrations*

Ultrasonic Vibrations do not have practical value in sterilization

Ultraviolet light

Ultraviolet light causes pyrimidine dimers by linking 5,6 unsaturated bonds of adjacent pyrimidines to form a cyclobutane ring. Three types dimers produced are thymine-thymine, thymine-cytosine and cytosine-cytosine with thymine-thymine dimers produced most frequently. The mutations that result from UV exposure are not directly caused by dimers but rather result from an error prone process of post replication repair called "SOS repair". This type of repair is induced by the presence of dimers.

Quellung reaction

Under microscopes, capsules cannot be seen unless they are stained or undergo the quelling reaction. This process consists of swelling the capsule with antiserum and then allowing antibodies to bind to the chemical antigen that caused swelling.

Enveloped virus characteristics

Unstable in the environment Require *aerosols* for stability and transmission-- coughing, sneezing, and body fluids Can be transferred through droplet infections Inactivated by *bile salts* in the GI tract

Calcium dipicolinate

Up to 20% of the dry weight of the endospore consists of calcium dipicolinate within the core, which is thought to stabilize the DNA.

What are the clinical uses for *extended spectrum penicillins*?

Urinary and respiratory infections Enteric fever In combination with other drugs in severe systemic infections

What are the clinical uses for *Anti-Pseudomonas penicillins*?

Urinary and respiratory infections Infections from burns In combination with other drugs in severe infections

What is the clinical use of cycloserine?

Used in the treatment of drug-resistant cases of *tuberculosis* in combination with several other drugs (usually in cases of reinfection) Spectrum of activity: broad

Group Translocation- PTS (phosphotransferase) System

Used to transport *sugars* Composed of several distinct components GT systems specific for one sugar may share some of their components with other GT systems Sugar is brought into the cell and simultaneously phosphorylated by phosphoenolpyruvate (PEP). The system involves participation of at least 4 proteins that act as phosphate carriers of the high energy phosphate group from PEP to the incoming sugar. Saves metabolic energy *Found in mostly obligate and facultative anaerobes*

Active Immmunization

Vaccines given to activate immunoglobulin production Vaccines can be: live-attenuated virus killed or inactivated virus subunit or recombinant vaccine

What is the mechanism of action for *vancomycin*?

Vancomycin *inhibits translocation of the peptidoglycan precursor* to the outside of the membrane where it is linked to pre-existing peptidoglycan. The inhibition occurs because *vancomycin binds irreversibly to the acyl-D-alanyl-D-alanine terminus* of the membrane-bound peptidoglycan precursor (the pentapeptide linked to N-acetyl muramic acid). Also referred to as glycopeptide antimicrobic

What inhibits transglycosylase activity?

Vancomycin binds to the terminal ends of D-alanine residues

What are the functions of surface glycoproteins of viruses?

Viral attachment to host cells Virus budding Neutralizing antibodies are directed against these glycoproteins - may function as fusion proteins Hemagglutinins (agglutinate erythrocytes) Neuraminidases (digests sialic acid residues on glycoproteins)

Explain why capsules are considered virulence factors

Virulence factors help bacteria invade the host, evade host defenses, and cause disease. Capsules contain *K antigens* which help determine *serological types* (this is how it is known that S. pnueminiae has 84 different serological types ) Capsular polysaccharides used as antigens in certain vaccines Examples: Streptococcus pneumoniae and Neisseria meningitidis

CD8+ cytotoxic T cell mediated host cell lysis

Virus infected host cells express MHC Class I complexed viral peptides Sensitized CD8+ T cells recognize these viral peptides on infected host cells and lyse them No humoral antibodies are involved in this cytotoxicity reaction This is a virus specific cell mediated immune response Thus CD8+ T cell mediated host cell lysis is classified under Type IV hypersensitivity reaction

What characteristics are common to *all viruses*?

Viruses are *obligate intracellular parasites* that: attach to enter cells multiply their genetic material make mRNAs translate proteins from the *host ribosome* create a nucleocapsid then leave the cell. (Obligate means capable of functioning or surviving only in a particular condition or by assuming a particular behavior)

Relative sizes of Viruses

Viruses are between 20-300 nm in size Animal cells are larger than bacteria which are bigger than viruses--- one bacteria is approximately ~100 viruses and one human cell is ~100 bacteria Electron microscopes and x-ray crystallography are used to see viruses, not light microscopes

Plaque Assay

Viruses causing cell death can be counted by this procedure. A series of virus dilutions are made, and these diluted virus stocks are allowed to infect cells in culture. An agar overlay prevents the virus from spreading during infection. Thus each virus particle gives rise to localized cell death as a result of confined replication. A series of holes or plaques are observed on tissue culture plates after 2-3 days of infection with a given virus. By counting the number of plaques, the number of particles can be ascertained. Plaque assay is possible only if a virus can cause cell death.

Viral Life Cycle

Viruses enter host cell, use many of the cellular functions, multiply and form thousands of new progeny virus particles, and come out the host cell.

Viruses are filterable agents, bacteria can be retained by specific filters.

Viruses pass through pores of membrane filters because they are small, but bacteria are retained on membranes.

Antivirals

Viruses utilize host cell machineries for multiplication Most of the enzymatic activities needed for virus replication, are also needed for host cell function Hence antivirals that inhibit virus life cycle also affect host cell metabolism and cause serious side effects on extended use. *Most of them are nucleoside analogs* 11 compounds are licensed for use in U.S.A *Azidothymidine (AZT)* *Dideoxy Inosine (ddI) and Didoxy Cytidine (ddC)* *Acyclovir (Zovirax)* *Gancyclovir* Vidarabine Idoxuridine Trifluridine *Foscarnet* *Ribavirin* *Amantidine and Rimantadine* *Oseltamivir and Zanamivir*

Glycocalyx

Viscous material extending from bacterial cell Adhesive polymer Secreted by bacteria When loosely attached it is considered a *slime layer (S)*, but when tightly bound to the cell wall it is referred to as a *capsule*

What is the fate of β-galactosidase when the LacI gene is mutated?

When LacI is mutated, β-galactosidase is produced constitutively (none stop) Genotype:*I-O+Z+* --- lac region is haploid --- Synthesis of beta-galactosidase is *constitutive*

Suppression of frameshift mutations

When a frameshift mutation occurs by base addition all codons downstream are read incorrectly. To correct a frameshift mutation, the original correct reading frame must be established and can be achieved by: a. removal of the added base b. intragenic second-site suppression in which a nearby base is removed or two extra bases are added c. extragenic suppression by a mutant tRNA molecule having a four-letter anticodon. For example, a mutant tRNAGly has an extra C in the anticodon loop to read CCCC. The extra C allows pairing with a sequence of four G's or the added base could serve as a spacer that forces the next codon read to be displaced by one base.

Explain the formation of *F' cells*

When an F plasmid excises from the bacterial chromosome imprecisely, chromosomal genes adjacent to the site of insertion of F are found associated with the plasmid. *A plasmid containing both F genes and chromosomal genes is called an F' plasmid.* It is usual to describe an F' plasmid by the chromosomal genes it is known to possess. For example, F'lac contains the genes for lactose utilization. If an F'lac plasmid is transferred into a host strain with chromosomal lac genes, the strain will become a merodiploid which is diploid the lac locus. F' plasmids can be transferred from an F' male to a female cell. This occurs usually with transfer of the entire F' plasmid. Therefore, the female recipient is converted to an F' male. Therefore, a cross of this type results in the following: *F'x F- → F'*

Silent mutation

When an amino acid substitution has no detectable effect on the phenotype of a cell or when there is a base change without an amino acid alteration because of the redundancy of genetic code.

Stringent/Relaxed Response Summary *(specifically asked on study guide)*

When bacteria are moved from a medium of high nutrients to low nutrients, they may undergo amino acid starvation. If there are not a lot of amino acids present, then there is no need for lot of ribosomes If there is no need for ribosomes, then the need of rRNA production is not necessary and needs to stop Steps: 1. An uncharged tRNA binds to the 50S subunit of the ribosome in the A site. 2. *RelA gene* is triggered to be transcribed, producing *ribosome-associated stringent factor.* 3. *ribosome-associated stringent factor* will catalyze the production of *ppGpp (stringent factor)*. 4. *ppGpp will the bind to RNA polymerase*, preventing the transcription of rRNA No rRNA will be produced, so no ribosomes will be made *Relaxed* cells have a Real mutation and still produce rRNA despite amino acid starvation

Lac Operon Genetics

When discussing lac operon genetics, genotypes express the genes that make up the lac operon: *I+* means LacI gene works correctly and the *lac repressor* functions correctly, i.e. works when lactose is not present and does work when lactose is present *O+* means the operator gene functions correctly and is inhibited when the repressor is bound *Z+* means the LacZ gene functions correctly and β-galactosidase is synthesized Partial diploid cells contain two chromosomes instead of one, so there genes are expressed side by side with a slash. (example: I+O+Z+/I+O+Z+) Genotypes are written in order from repressor (i) to operator (o) to β-galactosidase gene (z).

Temperate

a phage capable of entering either the lytic or lysogenic life cycle

extragenic suppression

extragenic suppression involves two distinct mutations at different loci on the chromosome with one of the mutations canceling the other mutation.

E. Coli and S. aureus are examples of what type of bacteria? a. aerobe b. anaerobe c. facultative anaerobe d. aerotolerant e. microaerophilic

facultative anaerobic bacteria

What bacteria are affected by aminoglycosides?

gram-positive and gram-negative bacteria, including tubercle bacillus Anaerobiosis retards entry into cells by reducing the membrane potential, hence *they are not effective against anaerobic organisms.*

Pasteurization

inactivation of important pathogens in milk, etc.

intragenic suppression

intragenic suppression requires two distinct mutations within a single gene and the effect of one mutation cancels the other mutation.

IFN-alpha is used to treat what conditions in the United States?

kaposi Sarcoma (AIDS) Hairy Cell Leukemia (HTLV-II) Condyloma acuminata ( HPV) Hepatitis B hepatitis C

transformasomes

membranous extension responsible for binding and uptake of dNA, found on the surface of transformation competent haemophilus influenzae bacteria.

Campylobacter and Jejuni are examples of what type of bacteria? a. aerobe b. anaerobe c. facultative anaerobe d. aerotolerant e. microaerophilic

microaerophilic bacteria

What clinical specimens for viral diagnosis are taken?

nasopharyngeal washings sputum conjunctival swab serum blood CSF feces tissue biopsy vesicles

Bacteriophage Protein coat

nucleic acid is surrounded by a *protein coat or capsid.* The protein capsid is made of usually identical subunits packed to form a prismatic structure. *The protein coat protects the phage nucleic acid from damaging nucleases and other harmful substances.* Some phages have a tail that serves as an attachment organ and as a tube through which phage DNA can pass during the infection process of the phage. Additionally, some bacteriophages contain lipid material but this is not a common feature of phages.

How does hydrogen concentration influence bacterial growth?

pH concentration can inhibit the growth of bacteria because hydrogen concentration affects enzyme function Example: E. Coli survive in neutral pH and Lactobacilli are found in the mouth and require a pH of 5.5 to 5.8

How are viruses typically diagnosed?

proteins: ELISA and Western blot antibodies: ELISA and Western blot nucleic acid: PCR, southern, northern *PCR is the most sensitive detection method*

Generation time (tg)

tg= t/n t= time elapsed n=number of generations log2b=log2 a + n----> n=log10b-log10a/0.301 (log102)

*β-lactam Antibiotics* mechanism of action

β-Lactam antibiotics (penicillins and cephalosporins) act by binding to target cytoplasmic membrane proteins called *penicillin-binding proteins (PBPs).* Most penicillin-binding proteins are enzymes involved in the terminal stages of peptidoglycan synthesis. *Penicillins inhibit the transpeptidation reaction of cell wall synthesis* by irreversible binding to the transpeptidase enzyme, a penicillin-binding protein. This binding inhibits cross-bridge formation during peptidoglycan synthesis. *β-Lactam antibiotics are only active against replicating bacteria*, i.e., cells actively synthesizing cell wall material.

Transcription Inhibitors

• Rifampin •Streptovaricin •Streptolydigin •Actinomycin D