Microbiology 260- test two
example of quorum sensing
( first discovered) gram- postive bacterium called streptococcus pneumoniae vibrio fishcheri - marine luminescent bacterium lives within the light organ of certain fish and squids
nutrient availability and growth
- During log phase, cells grow as quickly as they can for the conditions available -nutrient concentrations may be limiting, limiting growth -abundance of nutrients allows transport mechanisms to be saturated, so growth increase is minimal for adding in even more nutrients
Isolation of Pure Cultures
-Population of cells arising from a single cell developed by Robert Koch -Allows for the study of single type of microorganism in mixed culture -Spread plate, streak plate, and pour plate are techniques used to isolate pure cultures
Quorum Sensing Systems
-Processes regulated by quorum sensing involve host-microbe interactions -symbiosis - Vibrio fischeri and bioluminescence in squid -pathogenicity and increased virulence factor production -DNA uptake for antibiotic resistance genes
the growth curve
-observed when microorganism are cultivated in batch culture -log of cell number vs time have five distinct phases: - lag phase - log phase - stationary phase - death phase - long- term stationary phase
Thermophiles would be expected to grow
55 to 85 degrees C
catabolic/amphibolic pathways
Enzyme catalyzed reactions whereby the product of one reaction serves as the substrate for the next - pathways also provide material for biosynthesis
proteins synthesis inhibitors- aminogylcoside antibiotics
Large group, all with a cyclohexane ring, amino sugars Bind to 30S ribosomal subunit, interfere with protein synthesis by directly inhibiting the process and by causing misreading of the messenger RNA Resistance and toxicity effect mostly gram-negative pathogens Aminoglycoside-induced mutation suppression (stop codon readthrough) as a therapeutic strategy for Duchenne muscular dystrophy
electron carriers
Located in plasma membranes of chemoorganotrophs in bacteria and archaeal cells Located in internal mitochondrial membranes in eukaryotic cells Examples of electron carriers include NAD, NADP, and others
Cytokinesis( steps)
steps: selection of site for septum formation assembly of Z ring (composed of protein FtsZ assembly of cell wall synthesizing machinery constriction of cell and septum formation
defined v.s synthetic media examples
table 7.5
Biofilm Microorganisms
the EPS and change in attached organisms' physiology protects microbes from harmful agents UV light, antibiotics, antimicrobials when formed on medical devices, such as implants, often lead to illness sloughing off of organisms can result in contamination of water phase above the biofilm such as in a drinking water system
Anabolism
the synthesis of complex organic molecules from simpler ones requires energy from fueling reactions
accessory pigments
transfer light energy to chlorophylls e.g., carotenoids and phycobiliproteins accessory pigments absorb different wavelengths of light than chlorophylls
what are the energy units of joule?
units of work capable of being done by a unit of energy 1 cal of heat is equivalent to 4.1840 J of work J
ultra radiation
wavelength most effectively absorbed by DNA is 260 nm (lethal) mutations to death cases formation of thymine dimer in DNA requires direct exposure on microbial surface DNA damage can be repaired by several repair mechanisms
protein and amino acid catabolism
*Protease: hydrolyzes protein to amino acids *Deamination: removal of amino group from amino acid -resulting organic acids converted to pyruvate, acetyl-CoA, or TCA cycle intermediate >can be oxidized via TCA cycle >can be used for biosynthesis >can occur through transamination
Catabolism of Other Carbohydrates
- Many different carbohydrates can serve as energy source - Carbohydrates can be supplied externally or internally (from internal reserves)
what are the two energy resources for bacterial and archaeal species
- much less energy is available from oxidation of inorganic molecules than glucose due to more positive redo potentials
counting chambers
-Easy, inexpensive, and quick -Useful for counting both eukaryotes and prokaryotes -Cannot distinguish living from dead cells
factors of influencing antimicrobial drugs
-ability of drug to reach site of infection -susceptibility of pathogen to drug -ability of drug to reach concentrations in body that exceed MIC of pathogen
exponential phase
-also called log phase -rate of growth is constant -population is most uniform in terms of chemical and physical properties during this phase
membrane filter technique
-bacteria from aquatic samples are trapped on membranes -membrane soaked in culture media -colonies grow on membrane -colony count determines # of bacteria in sample
factors that affect ATP yield
-bacterial ETCs are shorter and have lower P/O ratios -ATP production may vary with environmental conditions -PMF in bacteria and archaea is used for other purposes than ATP production (flagella rotation) -precursor metabolite may be used for biosynthesis
Measurement of Microbial Growth
-can measure changes in number of cells in a population -can measure changes in mass of population
temperature and microbes
-cannot regulate internal temp -enzymes have optimal temp for functionality (high temps may inhibit them & be lethal) Cardinal Growth Temperatures: +minimal +maximal +optimal
lag phase
-cell synthesizing new components -varies in length
Direct counts on membrane filters
-cells filtered through special membrane that provides dark background for observing cells -cells are stained with fluorescent dyes -useful for counting bacteria -with certain dyes, can distinguish living from dead cells
disk diffusion test
-disks impregnated with specific drugs are placed on agar plates inoculated with test microbe -drug diffuses from disk into agar, establishing concentration gradient -observe clear zones (no growth) around disks
Measurement of Cell Mass
-dry weight -quantity of a particular cell constituent( chlorophyll, protein, DNA, ATP) -turbidometric measures (light scattering)
Flow cytometry
-microbial suspension forced through small orifice with a laser light beam -movement of microbe through orifice impacts electric current that flows through orifice -instances of disruption of current are counted -specific antibodies can be used to determine size and internal complexity
Turbidostat
-regulates the flow rate of media through vessel to maintain a predetermined turbidity or cell density -dilution rate varies -no limiting nutrient -turbidostat operates best at high dilution rates - constant nutrients for the microbial environment and circulation to remove waste - continuous culture system - photo cell to measure the tobidity ( amount of light scattered)
Psychrophiles would be expected to grow
0 to 20 degrees C
Psychrotrophs would be expected to grow
0 to 35 degree C
bacterial cell cycle consist of three stages?
1. a period of growth after the cell Is born 2. chromosome replication and partitioning period 3. cytokinesis during which septum and daughter cell forms.
turbidostat differs from chemostat by
1. dilution rate varies rather than being constant - tubidostat culture media contains all nutrients- no limit 2. high dilutions rates rather than low dilutions
Mesophiles would be expected to grow
20 -40 degrees C
Hyperthermophiles would be expected to grow
85 to 113 degrees C
non-cyclic electron flow
A route of electron flow during the light reactions of photosynthesis that involves both photosystems and produces ATP, NADPH, and oxygen. The net electron flow is from water to NADP+.
cyclic electron flow
A route of electron flow during the light reactions of photosynthesis that involves only photosystem I and that produces ATP but not NADPH or oxygen.
the mechanism of enzyme reactions
A typical exergonic rxn: A+B --> AB++ -->C+D Transition-state complex - resembles both the substrates and the products Activation energy - energy required to form transition-state complex Enzyme speeds up reaction by lowering Ea
Metabolic antagonist
Act as antimetabolites antagonize or block functioning of metabolic pathways by competitively inhibiting the use of metabolites by key enzymes Are structural analogs molecules that are structurally similar to, and compete with, naturally occurring metabolic intermediates block normal cellular metabolism
antiviral drugs for herpes virus
Acyclovir inhibits herpes virus DNA polymerase thymidine kinase Adenine arabinoside (vidarabine) inhibits herpes virus enzymes involved in DNA and RNA synthesis and function Valacyclovir prodrug form of acyclovir Ganciclovir and others anti-herpesvirus drugs Foscarnet inhibits herpes virus DNA polymerase
chemoorganotrophic fueling process- what are they?
Also called chemoheterotrophs Processes: aerobic respiration anaerobic respiration fermentation
feedback inhibition
Also called end-product inhibition Inhibition of one or more critical enzymes in a pathway regulates entire pathway pacemaker enzyme -catalyzes the slowest or rate-limiting reaction in the pathway Each end product regulates its own branch of the pathway Each end product regulates the initial pacemaker enzyme Isoenzymes -different enzymes that catalyze same reaction
Drug resitance
An increasing problem -once resistance originates in a population it can be transmitted to other bacteria -a particular type of resistance mechanism is not confirmed to a single class of drugs Microbes in abscesses or biofilms may be growing slowly and not be susceptible Resistance mutants arise spontaneously and are then selected
antiviral drugs
Azidothymidine (AZT) or zidovudine- nucleotides reverse transcriptase inhibitor ritonavir- viral protease inhibitor Forcarnet- viral fusion inhibitor Cidofovir(HPMPC)- inhibitors of via DNA polymerase.inhibits viral DNA polymerase papovaviruses, adenoviruses, herpesviruses iridoviruses, and poxviruses amantiadine- blocks viral penetration and uncoating oseltamivir- inhibits neuraminidase
noncompetitive inhibitor
Binds to enzyme at a site other than active site Causes shape change that makes enzyme unable to bind substrate
Solutes and Water Activity
Changes in osmotic concentrations in the environment may affect microbial cells hypotonic solution (lower osmotic concentration) water enters the cell cell swells and may burst hypertonic (higher osmotic concentration) water leaves the cell membrane shrinkage from cell wall (plasmolysis) may occur
stationary phase
Closed system population growth eventually ceases, total number of viable cells remains constant active cells stop reproducing or reproductive rate is balanced by death rate possible reason for this phase: nutrient limitation, limited O2, toxic waste accumulation, critical population density reached
The ability of a drug to reach the site of the infection depends on what?
Depends in part on mode of administration oral some drugs destroyed by stomach acid topical parenteral routes nonoral routes of administration Drug can be excluded by blood clots or necrotic tissue
fueling reactions: what are the basic needs of a organism
Despite diversity of energy, electron, and carbon sources used by organisms, they all have the same basic needs: 1. ATP 2. reducing power to supply electrons for chemical reactions 3. precursor metabolites ( small molecules that provide carbon skeleton needed for biosynthesis
example of anaerobic respiration
Dissimilatory nitrate reduction -use of nitrate as terminal electron acceptor, making it unavailable to cell for assimilation or uptake Denitrification reduction of nitrate to nitrogen gas in soil, causes loss of soil fertility
antiviral drugs
Drug development has been slow because it is difficult to specifically target viral replication Drugs currently used inhibit virus-specific enzymes and life cycle processes
Electron transport chain _ redox pairs
Each carrier is reduced and then reoxidized Carriers are constantly recycled The difference in reduction potentials electron carriers, NADH and O2 is large, resulting in release of great deal of energy - in gram negative bacteria
Effects of pH and Temperature
Each enzyme has specific pH and temperature optima Denaturation loss of enzyme's structure and activity when temperature and pH rise too much above optima
Electron Transport Chain (ETC)
Electron carriers organized into ETC -first electron carrier having the most negative E'o -the potential energy stored in first redox couple is released and used to form ATP -first carrier is reduced and electrons moved to the next carrier and so on
Frist Law of Thermodynamics
Energy can be neither created nor destroyed total energy in universe remains constant however, energy may be redistributed either within a system or between the system and its surroundings
chemical equilibrium
Equilibrium consider the chemical reaction A + B ⇌ C + D reaction is at equilibrium when rate of forward reaction = rate of reverse reaction Equilibrium constant (Keq) expresses the equilibrium concentrations of products and reactants to one another
Role of ATP in metabolism
Exergonic breakdown of high energy ATP is coupled with endergonic reactions to make them more favorable Guanosine 5'-triphosphate, cytosine 5'-triphosphate and uridine 5'-triphosphate also supply some energy ATP has the ability to transfer phosphoric group to another molecule suited energy united because it a high energy molecule that can hydrolyzes into ADP
standard free energy change
Free energy change defined at standard conditions of concentration, pressure, temperature, and pH ΔGo´ standard free energy change at pH 7 directly related to Keq ΔGo´ = -2.303RT•logKeq
E.coli divisomes proteins and their functions
Fts Z- forms Z ring FtsA,ZipA- anchors Z ring to plasma membrane FtsK- chromosomes segregation and separation of chromosomes dimers FtsQLB- may provide a scaffolded for assembly of proteins involved in peptidoglycan synthesis Ftsl1- peptidoglycan synthesis FtsN- thought to trigger construction imitation
Overcoming Drug Resistance
Give drug in appropriate concentrations to destroy susceptible Give two or more drugs at same time Use drugs only when necessary Possible future solutions -continued development of new drugs -use of bacteriophages to treat bacterial disease
oxygen concentration
Growth in presence of different oxygen concentrations depends on a microbe's metabolic processes, electron transport chains (ETC), terminal electron acceptor used aerobe: grows in presence of atmospheric oxygen (O2) which is 20% O2 obligate aerobe - requires O2 anaerobe: grows in the absence of O2 obligate anaerobe: usually killed in presence of O2 microaerophile: requires 2-10% O2 facultative anaerobes: do not require O2 but grow better in its presence aerotolerant anaerobes: grow with or without O2
the light reaction in an oxygenic photosynthesis
H2O not used as an electron source; therefore O2 is not produced Only one photosystem involved Uses bacteriochlorophylls and mechanisms to generate reducing power Carried out by phototrophic green bacteria, phototrophic purple bacteria, and heliobacteria
dilution susceptibility test
Involves inoculating media containing different concentrations of drug broth or agar with lowest concentration showing no growth is MIC if broth used, tubes showing no growth can be subcultured into drug-free medium broth from which microbe can't be recovered is MLC - MIC is determined through if the lowest of antibiotic results in no growth in 20 hours after incubation - MLCis determined if tubes showing no growth
main electrons carriers
Located in plasma membranes of chemoorganotrophs in bacteria and archaeal cells Located in internal mitochondrial membranes in eukaryotic cells Examples of electron carriers include NAD, NADP, and others NAD nicotinamide adenine dinucleotide NADP nicotinamide adenine dinucleotide phosphate FAD flavin adenine dinucleotide FMN flavin mononucleotide riboflavin phosphate Coenzyme Q (CoQ) a quinone also called ubiquinone Cytochromes use iron to transfer electrons iron is part of a heme group Nonheme iron-sulfur proteins e.g. ferrodoxin use iron to transport electrons iron is not part of a heme group
Protein synthesis inhibitors
Many antibiotics bind specifically to the bacterial ribosome binding can be to 30S (small) or 50S (large) ribosomal subunit Other antibiotics inhibit a step in protein synthesis -aminoacyl-tRNA binding -peptide bond formation -mRNA reading translocation
energy sources
Many different energy sources are funneled into common degradative pathways Most pathways generate glucose or intermediates of the pathways used in glucose metabolism Few pathways greatly increase metabolic efficiency
oxidation-reduction (redox) reactions
Many metabolic processes involve oxidation-reduction reactions (electron transfers) Electron carriers are often used to transfer electrons from an electron donor to an electron acceptor Transfer of electrons from a donor to an acceptor -can result in energy release, which can be conserved and used to form ATP -the more electrons a molecule has, the more energy rich it is
pH
Measure of the relative acidity of a solution negative logarithm of the hydrogen ion concentration
Microbial Metabolism and Its Importance
Metabolism is the total of all chemical reactions in the cell and is divided into two parts
Carbohydrates
Monosaccharides converted to other sugars that enter glycolytic pathway Disaccharides and polysaccharides cleaved by hydrolases or phosphorylases
pH tolerance mechanism of microbes
Most microbes maintain an internal pH near neutrality the plasma membrane is impermeable to proton exchange potassium for protons Acidic tolerance response pump protons out of the cell some synthesize acid and heat shock proteins that protect proteins Many microorganisms change the pH of their habitat by producing acidic or basic waste products
The Influence of Environmental Factors on Growth
Most organisms grow in fairly moderate environmental conditions Extremophiles grow under harsh conditions that would kill most other organisms ( table 7.2)
the autoinducer is called
N- acylhomoserine lactone (AHL) - found in many gram negative bacteria - diffuse across plasma membrane - once inside the cell, induces expression of target gene regulating a variety of functions
proteins synthesis inhibitors- Chloramphenicol
Now is chemically synthesized Binds to 23s rRNA on 50S ribosomal subunit and inhibits peptidyl transferase reaction Toxic with numerous side effects so only used in life-threatening situations
Redox: Two Half Reactions
One is electron donating (oxidizing reaction). One is electron accepting reaction (reducing reaction). Acceptor and donor are conjugate redox pair: acceptor + e- donor each reaction consist of two half reactions
fermentation
Oxidation of NADH produced by glycolysis Pyruvate or derivative used as endogenous electron acceptor Substrate only partially oxidized Oxygen not needed Oxidative phosphorylation does not occur ATP formed by substrate-level phosphorylation
the development of chemotherapy
Paul enrich(1904) developed concept of selective toxicity identified dyes that effectively treated African sleeping sickness Sahachiro Hato (1910) working with Ehrlich, identified arsenic compounds that effectively treated syphilis Gerhard Domagk, and Jacques and Therese Trefouel (1935) discovered sulfonamides and sulfa drugs
second law of thermodynamics
Physical and chemical processes proceed in such a way that the disorder of the universe increases to the maximum possible entropy = amount of disorder in a system
aerobic respiration
Process that can completely catabolize an organic energy source to CO2 using -glycolytic pathways (glycolysis) -TCA cycle -electron transport chain with oxygen as the final electron acceptor - produces ATP ( most of it indirectly via the activity of the election transport chain), and high energy electron carriers
Inhibitors of cell wall synthesis- vancomycin and
Produced by streptomycin oreinatlis Glycopeptide antibiotics Inhibit cell wall synthesis Vancomycin - important for treatment of antibiotic-resistant staphylococcal and enterococcal infections - previously considered "drug of last resort" so rise in resistance to vancomycin is of great concern tagets gram-positive bacteria of the genus staphylococcus and some members of the genus Clostridium( gangrene and sever diarrhea), Bacillus( food poisoning), streptococcus( strep throat), and Enterococcus ( UTI) "drug of the last resort"
Z Ring Formation - Role in Septation
Protein FtsZ tubulin homologue, found in most bacteria and archaea polymerization forms Z ring, filaments of meshwork In E.coli, MinCDE system limits Z ring to cell center MinC, MinD, MinE oscillate from one side of cell to other high concentration of MinC at poles prevents formation of Z ring at those locations
Enzymes are
Protein catalysts -high specificity for the reaction catalyzed and the molecules acted on -substance that increases the rate of a reaction without being permanently altered Substrates = reacting molecules Products = substances formed by reaction Some enzymes are composed solely of one or more polypeptides Some enzymes are composed of one or more polypeptides and nonprotein components
Thermophiles and Hyperthermophiles
Protein structure stabilized by a variety of means e.g. more H bonds e.g. more proline e.g. chaperones Histone-like proteins stabilize DNA Membrane stabilized by variety of means e.g. more saturated, more branched and higher molecular weight lipids e.g. ether linkages (archaeal membranes)
Chemostat
Rate of incoming medium = rate of removal of medium from vessel An essential nutrient is in limiting quantities - constant nutrients for the microbial environment and circulation to remove waste - continuous culture system
what are toxic O2 derivatives called? examples?
Reactive oxygen species (ROS) superoxide radical hydrogen peroxide hydroxyl radical
how do microbes adapt to changes in osmotic concentration
Reduce osmotic concentration of cytoplasm in hypotonic solutions mechanosensitive (MS) channels in plasma membrane allow solutes to leave Increase internal solute concentration with compatible solutes to increase their internal osmotic concentration in hypertonic solutions solutes compatible with metabolism and growth
an example of chromosome partitioning
Replisome pushes, or leads to condensation of, daughter chromosomes to opposite ends ParA/ParB proteins in C. crescentus ParA polymerizes to form filaments ParB binds DNA at parS site near origin of replication; technically, ParB binds 2 copies of parS site since DNA has been replicated ParA interaction with 1 of the 2 ParB/parS complexes causes it to depolymerize, pulling one copy of the DNA away system in almost 70% of 400 sequenced bacterial genomes more work remains to understand partitioning in bacteria
Interdomain communication
Rhizium interactions with leguminous planet for nitrogen fixation - "speak" by secretions - plant released molecules called flavonoids, which take up by the bacterium and binds bacterium to bacterial protein called NodD - activates the production chemicals called nod D factors
antimicrobial agents are classified as
Side effects - undesirable effects of drugs on host cells Narrow-spectrum drugs - attack only a few different pathogens Broad-spectrum drugs - attack many different pathogens Cidal agent - kills microbes Static agent - inhibits growth of microbes
how do chromosomes replicate?
Single origin of replication - site at which replication begins Terminus - site at which replication is terminated, located opposite of the origin Replisome - group of proteins needed for DNA synthesis DNA replication proceeds in both directions from the origin Origins move to opposite ends of the cell "STRO"
Inhibitors of cell wall synthesis Cephalosporins
Structurally and functionally similar to penicillins Broad-spectrum antibiotics that can be used by most patients that are allergic to penicillin Four categories based on their spectrum of activity
Metabolic antagonist- Sulfonamides or sulfa drugs
Structurally related to sulfanilamide, a para aminobenzoic acid (PABA) analog PABA used for the synthesis of folic acid and is made by many pathogens sulfa drugs are selectively toxic due to competitive inhibition of folic acid synthesis enzymes
Metabolic antagonist- Trimethrprim
Synthetic antibiotic that also inferred with folic acid production it binds to dihydrofolate reductase (DHFR), the enzyme responsible for converting dihydrofolic acid to tertradrofolic acid, competing against the dihydrofolic acid substrate Broad spectrum Can be combined with sulfa drugs to increase efficacy of treatment combination blocks two steps in folic acid pathway used to treat respiratory and middle ear infections, UTI, and traveler's diarrhea
antiviral for influenza
Tamiflu -anti-influenza agent -a neuraminidase inhibitor - though not a cure for influenza, has been shown to shorten course of illness Amantidine -used to prevent influenza infections blocks. -penetration and uncoating of influenza virus
electron movement and reduction potentials
The greater the difference between the E0 of the donor and the E0 of the acceptor ⇓ the more negative the ΔGo´
Anti-protozoan drugs
The mechanism of drug action for many antiprotozoal drugs is not precisely known Examples of available drugs some antibiotics that inhibit bacterial protein synthesis are used against protozoa chloroquine and mefloquine - malaria metronidazole - Entamoeba infections atovaquone - Pneumocystis carinii and Toxoplasma gondii
chemiosmotic hypothesis
The most widely accepted hypothesis to explain oxidative phosphorylation -protons move outward from the mitochondrial matrix as e- are transported down the chain -proton expulsion during e- transport results in the formation of a concentration gradient of protons and a charge gradient -the combined chemical and electrical potential difference make up the proton motive force (PMF)
reverse polymers
Used as energy sources in absence of external nutrients e.g. glycogen and starch cleaved by phosphorylases (glucose)n + Pi → (glucose)n-1 + glucose-1-P glucose-1-P enters glycolytic pathway e.g. poly-hydroxybutyrate PHB → → → acetyl-CoA acetyl-CoA enters TCA cycle
Heterogeneity in Biofilms
a mature biofilm is a complex, dynamic community of microorganisms heterogeneity is differences in metabolic activity and locations of microbes interactions occur among the attached organisms - exchanges take place metabolically, DNA uptake and communication
Law of Thermodynamics
a science that analyzes energy changes in a collection of matter called a system (e.g., a cell) all other matter in the universe is called the surroundings
E-test MIC
a test by testing the antimicrobial sensitivity place a E-strip directly on the agar each strip contains a gradient of an concentration of antibiotic and is labeled with the MIC scale values
Barotolerant
adversely affected by increased pressure, but not as severely as nontolerant organisms
strict aerobic microbes
all strict anaerobic microorganisms lack or have very low qualities of superoxide and catalase these microbes tolerate O2 anaerobes must be grown without O2
post- translational regulation regulation of enzymes activity
allosteric regulation covalent modification
the tricarboxylic acid cycle
also called the citrus acid and Kerb's cycle common in aerobic bacteria, free-living protozoa, and most algae, and fungi major role is as a source of Carbon skeletons for use in biosynthesis For each acetyl-CoA molecule oxidized, TCA cycle generates: 2 molecules of CO2 3 molecules of NADH one FADH2 one GTP
factors influencing ability of drug to reach concentration exceeding MIC
amount administered route of administration speed of uptake Tate of clearance ( emliniatisnf rom body)
Structure of enzymes
apoenzyme- protein component of an enzyme cofactor- nonprotein component of an enzyme - prosthetic group - firmly attached -coenzyme - loosely attached, can act as carriers/shuttles holoenzyme= apoenzyme +cofactor
The reproductive strategies of eukaryotic microbes
asexual and sexual, haploid or diploid Bacteria and Archaea haploid only, asexual - binary fission, budding, filamentous all must replicate and segregate the genome prior to division
visible light
at high intensities generates singlet oxygen (1^O2). - powerful oxidizing agents carotenoid pigments - protect many light-exposed microorganisms from photooxidation
quorum sensing
bacterial cells in biofilms communicate in a density-dependent manner produce small proteins that increase in level as microbes to competent state - DNA uptake occurs, bacteriocins are released
persister cells
bacterial cells that survive killing by antibiotics but remain sensitive to that antibiotic upon being regrown
mechanism of action of selective and differential media ( table 7.7)
blood agar- enriches and differential Eosin methylene blue agar (EMB)- selective and differential MacConkey agar (MAC)- slsective and differential Mannitol salt agar- selective and differential
how do wall less microbes protect themselves
by reducing the osmotic concentration
covalent modification
can stitch on and off usually occurs through the addition and removal of particular chemical group. most studied is Escherichia coli Advantages of this method -respond to more stimuli in varied/sophisticated ways -regulation of enzymes that catalyze covalent modification adds second level of control
Chemolithotrophy
carried out by chemolithotrophs electrons released from energy source which is an inorganic molecule -transferred to terminal electron acceptor (usually O2) by ETC ATP synthesized by oxidative phosphorylation Much less energy is produced - the reduction potentials of inorganic molecules are much more positive
bacterial cell cycle
cell cycle is sequence of events from formation of new cell through the next cell division most bacteria divide by binary fission two pathways function during cycle DNA replication and partition cytokinesis
mechanical work
cell motility and movement of structures within cells energy required such as portioning chromosomes during cell division
microbial growth on a solid surface
colony characteristics that develop when microorganisms are gown on agar surfaces aid in identification microbial growth in biofilms is similar differences in growth rate from ages to center is due to oxygen, nutrients an toxic products, cell may be dead in some areas
what is the most common metabolic channeling
compartmentation Differential localization of enzymes and metabolites differential distribution of enzymes and metabolites among separate cell structures or organelles can generate marked variations in metabolite concentrations
What does RpoS do?
component of RNA polymerase holoenzyme responsible for binding to DNA and innate transcription helps encode enzymes that help bacteria survive starvation and proteins
proteins synthesis inhibitors- Macrolides
contain12- to 22- carbon lactone rings linked to one or more sugars Erythromycin binds to 50S ribosomal subunit to inhibit bacterial protein synthesis - broad spectrum antibiotics against gram- negative bacteria used with patients that have an allergy to penicillin and treatment of whooping cough, diphtheria , diarrhea
Cellular Growth and Determination of Cell Shape 9 (steps)
determined by peptidoglycan synthesis in bacteria penicillin binding proteins (PBPs) - link peptidoglycan strands and catalyze controlled degradation for new growth autolysins - PBP enzymes that degrade peptidoglycan at site where new units are added Cocci and Rods Cocci divisome - new peptidoglycan forms only at septum FtsZ determines site of cell wall growth FtsZ may recruit PBPs for synthesis of septum Rods are similar but elongate prior to septation MreB determines cell diameter/elongation as Z ring forms
systemic mycoses
difficult to control and can be fatal three common drugs amphotericin B - binds sterols in membranes 5-flucytosine - disrupts RNA function fluconazole -low side effects, used prophylactically
PMF drives ATP synthesis
diffusion of protons back across membrane (down gradient) drives formation of ATP - ATP synthase - uses PMF down gradient to catalyze ATP synthesis - function like rotary engine ( conformational changes)
Kirby-Bauer Test
disk diffusion test essential for groups of bacteria commonly showing resistance. (drug susceptibility) Standardized method for disk diffusion test Sensitivity/resistance determined using tables relating zone diameter with microbial resistance Table values plotted, used to determine if effective concentration of drug in body can be reached
Dilution rate and microbial growth
duration rate- rate at which medium flows though vessel relative to vessel size note- cell density maintained at wide range of dilution rates - chemostat operates best at low dilution rate
Superficial mycoses
e.g. Candida topical and oral disrupt membrane permeability and inhibit sterol synthesis disrupts mitotic spindle; may inhibit protein and DNA synthesis
Photosynthesis
energy from light trapped and converted to chemical energy a two-part process light reactions: light energy is trapped and converted to chemical energy dark reactions: energy produced in the light reactions is used to reduce CO2 and synthesize cell constituents
Deinococcus radiodurans
extremely resistant to DNA damage
Antifungal drugs
fewer effective agents because of similarity of eukaryotic fungal cells and human cells -many have low therapeutic index and are toxic easier to treat superficial mycoses than systemic infections -combinations of drugs may be used
Septation
formation of cross walls between daughter cells
Catabolism
fueling reactions energy-conserving reactions provide ready source or reducing power (electrons) generate precursors for biosynthesis
amphibolic pathways
function both as catabolic and anabolic pathways important ones -Embden-Meyerhof pathway -pentose phosphate pathway -tricarboxylic acid (TCA) cycle
method of streak plate
grab loop of colony go to plate and streak sterilized loop turn plate a quarter and start where you streaked the first time... coming into clean agar where no strike happened sterilized loop turn plate again, and go into the second streak area.... pulled into a third new agar area and finish by a swirling tail
Acidophiles pH
grow in acidic environments growth optimum between pH 0-5.5
Halophiles
grow optimally in the presence of NaCl or other salts at a concentration above about 0.2M
The continuous culture of microorganisms
growth in an open system continual provision of nutrients continual removal of wastes maintains cells in log phase at a constant biomass concentration for extended periods achieved using a continuous culture system
antennas
highly organized arrays of chlorophylls and accessory pigments captured light transferred to special reaction-center chlorophyll -directly involved in photosynthetic electron transport Photosystems antenna and its associated reaction-center chlorophyll Electron flow → PMF → ATP
Regulation of Metabolism
important for conservation of energy and materials maintenance of metabolic balance despite changes in environment
Growth
increase in cellular constituents that may result in: increase in cell number increase in cell size groth refers to populations growth rather than growth of individuals cells
How enzymes lower activation energy
increasing concentration of substrate at active site of enzyme By orienting substrates properly with respect to each other in order to form the transition-state complex Induced fit model for enzyme-substrate interaction process: 1. App and glucose bind to the active site of enzyme ( hexokinase) 2. enzyme undergoes conformational change that strains the substrates and brings them close together ( induced fit) 3. substrates are converted to products 4. products are realeased
identify target for anti-HIV drugs
infection begins with HIV - fusion inhibitors block this step once inside a host cell, HIV unchaste and its reverse transcrptase (RT) forces the host to make DNA from the viral RNA - RT inhibitors block this step the viral DNA is transcribed and translated into polyproteins that are cut to release viral proteins - protease inhibitors block this step viral DNA is added to the host DNA by the action of viral integrase inhibitors block this step
chemoorganic fueling process - respiration
involved use of electron transport chain 2 types: - aerobic respiration= final electron acceptor of oxygen - aerobic respiration= final electron acceptor is different exenous acceptor such as NO-3,SO4^2,CO2,Fe3+,or SeO4^2- As electrons pass through the electron transport chain to the final electron acceptor, a proton motive force (PMF) is generated and used to synthesize ATP
the streak plate
involves technique of spreading a mixture of cells on an agar surface so that individual cells are well separated from each other involves use of bacteriological loop each cell can reproduce to form a separate colony (visible growth or cluster of microorganisms)
allosteric regulation
is altered to small enzyme known as allosteric effector the effector binds reversibly by a non-covalent to a regulatory site separate from a catalytic site and cases to change shape of the enzyme. postive effector activity increase activity negative effecter activity decrease activity
common features aspects of metabolism for all organisms
life obeys the law of thermodynamics the energy cells obtain from their environment is most often conserved as molecule called ATP oxidation- reduction (redox) reactions play crital roel in energy conservation the chemical reactions that occur in cels are organized into pathways each reaction of pathway is catalyzed by an exam or ribosome the functioning of biochemical pathways id regulated
minimal inhibitory concentration (MIC)
lowest concentration of drug that inhibits growth of pathogen
minimal lethal concentration (MLC)
lowest concentration of drug that kills pathogen
Chlorophyll
major light-absorbing pigments different chlorophylls have different absorption peaks
what are the nutritional types of mircroorganisms
majority of the pathogens are known for: -photolithoautottroph (photoautotrophs)- uses light and CO2 as their carbon resource -chemoorganoheterotrophs (chemoheterotrophs)- uses inorganic ( iron, sulfur) to derive both energy and electrons for biosynthesis ecological importance photoorganoheterotrophs- common inhabitants of polluted lakes and streams chemolithoautotrophs-used to reduce organic compounds made by autotrophs chemolithotrophs- reduces inorganic molecules as their energy and electron source that derive on carbon from organic sources table 11.2
effects of different oxygen levels on microbe growth
many microbes have protect themselves from toxic O2 products.
complex media
media that contain some ingredients of unknown chemical composition examples: peptone (gelatin hydrolysate), beef extract, and final pH 6.8
three major mechanism in regulating a metabolism
metabolic channeling regulation fo the synthesis of a particular enzyme ( transcriptional and translational) direct stimulation or inhibition of the activity of a critical enzyme - post- translational
Biofilms formation
microbes reversibly attached conditioned surface and release polysaccharide, proteins, and DNA to form extracellular polymeric substance (EPS) additional polymers are proceeded as microbes reproduce and biofilms mature
pressure
microbes that live on land and water surface live in 1 atmosphere (atm) some Bacteria and Archaea live in deep ea wit very high hydrostatic pressure
the embden- Meyerhof pathway
most common pathway for glucose degradation to pyruvate in ages of aerobic respiration occurs in eh cytoplasmic matrix of most organisms, planet sea animals ( cytosol) Oxidation step - generates NADH, high-energy molecules used to synthesize ATP by substrate-level phosphorylation function with the presence of O2 two phases: - six carbon phase= ATP is used to phosphorylate glucose twice, resulting in fructose. This primes the pump by adding phosphate to each end of the sugar. organism makes the ATP and more is being made int he pathway. NADH and ATP is produced - three carbon phase= begins with fructose 1,6-biophosphate is cleaved into two halves , resulting dihyroxyhasetone phosphate glceraldeyde 3-phosphate and formed by cleaved molecules. catalyzed in 6 steps
Biofilms
most microbes grow attached to surfaces( sessile) rather than free floating ( planktonic) these attache microbes are members of complex, slim enclosed communities called ---- ubiquitous innate in water can be formed on any conditioned surface
inhibitors of the cell wall synthesis- penicillin
most penicillin have 6-aminopeniclianic acid and differ from one another according to the next amino acid group. contains a beta- lactam ring, essential for molecule. bacteria resistors produce an enzyme called penicilinases ( beta-lactamases). this inactivates the antibiotic by hydrolyzying the beta- lactam ring bond Mode of action -blocks the enzyme that catalyzes transpeptidation (formation of cross-links in peptidoglycan) -prevents the synthesis of complete cell walls leading to lysis of cell -acts only on growing bacteria that are synthesizing new peptidoglycan
Inhibitors of cell wall synthesis
most selective antibiotics penicillin, cephalosporins, vancomycin, and bacitracin have a high therapeutic index because they target structures and functions not found in eukaryotic cell
anti- HIV drugs five classes
nucleotide reverse transcriptase inhibitors( e.g. azidothymidine) nucleotide reverse transcriptase inhibitors - prevents HIV DNA synthesis by binding to and inhibiting the reverse transcriptase enzyme protease inhibitors- block the activity of the HIV protease that needed for the production of all viral proteins integrate inhibitors - that prevent the incorporation of the HIV genome into the host's chromosomes fusion inbitiors- relatively new category of drug. precent HIV entry into cells - most successful drug is a cocktail to curtail resistance
Oligotrophic
nutrient poor environment
Eutrophic
nutrient rich environment
method of spread plate
on agar, a small amount of the sample is pipetted to the center of a solidified medium the glass spreader is sterilized by dipping it into ethanol and briefly flaming it when cooled then used to spread the sample evenly over the surface of the medium
Types of Penicillins
penicillin G - high activity against most gram- positive bacteria penicillin V- same spectrum more acid resistant than G ampicllin- active against gram-negative and positive bacterium; acid stable. carbencillin- active again gram- negative bacteria such as Psuedomonas and Proteus; acid stable, not well absorbed by small intestines piperacillin- extended spectrum, active abasing gram-negative bacteria. i.e E.coli, Pseudomonasm, Enterobacter, Proteus, and Klebsiella spp. ticarillin- similar to carbenicillin but more against Pseudomonas
light reactions in oxygenic photosynthesis
photosynthetic eukaryotes and cyanobacteria oxygen is generated and released into the environment most important pigments are chlorophylls
two major classes of anti fungal drugs?
polyenes azoles - both block fungal cell membrane synthesis
Inhibitors of cell wall synthesis-teicoplanin
produced by Actinoplanes teichomyceticus similar to Cancomcyin and has fewer side effects ative against staphylococci, enterococci, streptococco, clostridia, listeria spp. and many other gram-positive pathogens.
proteins synthesis inhibitors- Lincosamines
produced by streptomycin bacteria Broad spectrum of antibiotic activity against anaerobic microbes; less activity against aerobes Used sparingly because they can support (indirectly) the growth of C. difficile, which can result in other disease states Clindamycin is an example, and is used to treat infections caused by Bacteroides fragilis, as well as some staphylococcal and streptococcal infections
Barophilic (peizophilic) organisms
require or grow more rapidly in the presence of increased pressure change membrane fatty acids to adapt to high pressures
extreme halophiles
require salt concentrations of 2M and 6.2M extremely high concentrations of potassium cell wall, proteins, and plasma membrane require high salt to maintain stability and activity
pour plate
sample is serially diluted diluted samples are mixed with liquid agar mixture of cells and agar are poured into sterile culture dishes both may be used to determine the number of viable microorganisms in an original sample
characteristics of antimicrobial drugs
selective toxicity - ability of drug to kill or inhibit pathogen while damaging host as little as possible therapeutic dose - drug level required for clinical treatment toxic dose - drug level at which drug becomes too toil for patient therapeutic index - ration of toxic dose to therapeutic dose
method of a pour plate
setup an original sample to make a serial dilution to make each tube have less bacteria as you go down the series take a sample of each tube and make agar plates
spread plate
small volume of diluted mixture containing approximately 30-300 cells is transferred spread evenly over surface with a sterile bent rod
examples of self-splicing ribozymes
splicing of pre-RNA= tetrahymena spp. splicing of mitochondria rRNA and mRNA = numerous fungi splicing of chloroplast tRNA, rRNA,and mRNA= plants and algae splicing of viral mRNA= virus ( hepatitis delta virus)
viable counting methods- spread/ pour plates
spread and pour plate techniques - diluted of bacteria spread over solid agar surface or mixed with agar and poured into Petri plate - after incubation the numbers of organism are determined by counting the number of colonies multiplied by the dilution factor - results expressed as in units of colony forming units(CFU)
proteins called made in response to starvation ?
starvation proteins
Strict Anaerobic Microbes
strict anaerobic microbes lack / have a very low qualities of superoxide dimutase and catalase these mocriboes cannot tolerate O2 aerobes much be grown without O2
environmental effects on enzyme activity are
substrate concentration pH temperature
what are fungal infections subdivided in?
superficial mycoses subcutaneous mycoses systemic mycoses
aerobes and facilitate aerobes contains protective enzymes that protect themselves against toxic O2... they are?
superoxide dismutase (SOD) catalase peroxidase
functional types of media
supportive media- tryptic soy broth and tryptic soy agar enriched media- blood that is added to supportive media to enable faster growth selective media- allow growth of a particular microorganism, while inhibiting the growth of others. selective gram-negative bacteria. ( MacConkey agar) differential media- that distinguish among different of microbes and even permit tentative in deification of microorganisms based on biological characteristics ( blood agar, MacConkey agar)
chemical work
synthesis of complex biological molecules energy is needed
nucleic acid synthesis inhibition- Quinolones
synthetic drug that contain the 4- quinolone ring used to treat wide variety o infections types: ciprofloxacin, norfloxacin, and ofloxacin Nalidixic acid first synthesized quinolone (1962) Act by inhibiting bacterial DNA-gyrase and topoisomerase II broad spectrum antibiotic ( both kills gram positive and negative)
transport work
take up of nutrients, elimination of wastes, and maintenance of ion balances requires energy
culture media can be classified by
the chemical constituents from which they are made their physical nature their function
electron transport chain
the mitochondrial electron chain transport chain (ETC) = a series of e- carriers, operating together to transfer e- from NADH and FADH2 to a terminal e- acceptor, O2 E- flow from carriers with more negative reduction potentials (E0) to carriers with more positive E0
death phase
two alternative hypotheses - cell are viable but not cultural (VBNC) - programmed cell death prolonged decline in growth - bacterial population continually evolves - process marked by successive waves of genetically - natural selection occurs
Autotroph
use carbon dioxide as their sole or principal carbon source must obtain energy from other sources
Hetrotroph
use organic molecules as carbon sources which often also serve as energy source can use a variety of carbon sources
anaerobic respiration
uses electron carriers the than O2 generally yields less energy because of activation energy of electron acceptor is less than activation energy of O2
chemoorganic fueling processes- fermentation
uses endogenous electron acceptor -usually an intermediate of the pathway used to oxidize the organic energy source e.g., pyruvate Does not involve the use of an electron transport chain nor the generation of a proton motive force ATP synthesized only by substrate-level phosphorylation
nucleic acid synthesis inhibition
variety of mechanisms block DNA replication -inhibition of DNA polymerase inhibition of DNA helicase -block transcriptionA inhibition of RNA polymerase Drugs not as selectively toxic as other antibiotics because bacteria and eukaryotes do not differ greatly in the way they synthesize nucleic acids
ionizing radiation
x-rays and gamma rays mutations to death ( sterilization) disrupts chemical structure of many molecules, including DNA - damage may be repaired by DNA repair mechanisms if small dose
Exergonic reactions are
ΔGo´ is negative (reaction proceeds spontaneously)
Endergonic reactions are
ΔGo´ is positive (reaction will not proceed spontaneously)
Theoretical vs. Actual Yield of ATP
• amount of ATP produced during aerobic respiration varies depending on growth conditions and nature of ETC • under anaerobic conditions, glycolysis only yields 2 ATP molecules
Mathematics of Growth
• generation (doubling) time - time required for the population to double in size - varies depending on species of microorganism and environmental conditions - range is from 10 minutes for some bacteria to several days for some eukaryotic microorganisms
ATP yield during aerobic respiration
• maximum ATP yield can be calculated - includes P/O ratios of NADH and FADH2 - ATP produced by substrate level phosphorylation • the theoretical maximum total yield of ATP during aerobic respiration is 38 - the actual number closer to 30 than 38
culture media
• need to grow, transport, and store microorganisms in the laboratory • culture media is solid or liquid preparation • must contain all the nutrients required by the organism for growth • classification - chemical constituents from which they are made - physical nature - function
electron transport and oxidative phosphorylation
• only 4 ATP molecules synthesized directly from oxidation of glucose to CO2 • most ATP made when NADH and FADH2 (formed as glucose degraded) are oxidized in electron transport chain (ETC)
Some Media Components
• peptones - protein hydrolysates prepared by partial digestion of various protein sources • extracts - aqueous extracts, usually of beef or yeast • agar - sulfated polysaccharide used to solidify liquid media; most microorganisms cannot degrade it
the breakdown of glucose pyruvate
• three common routes - Embden-Meyerhof pathway - pentose phosphate pathway - Entner-Duodoroff pathway
effects of ( substrate)
Rate increases as [substrate] increases No further increase occurs after all enzyme molecules are saturated with substrate
Ribozymes
Thomas Cech and Sidney Altman discovered that some RNA molecules also can catalyze reactions example: catalyze peptide bond formation self-splicing involved in self-replication
lipid catabolism
Triglycerides common energy sources hydrolyzed to glycerol and fatty acids by lipases -glycerol degraded via glycolytic pathway -fatty acids often oxidized via β-oxidation pathway
Vibrio Cell Wall Growth and Cell Shape Determination
Vibrio (comma-shaped bacteria) FtsZ - forms Z ring MreB - helical polymerization throughout cell crescentin - intermediate filament homologue localizes to short, curved side of cell asymmetric cell wall synthesis forms curve
Viable counting: Alive or dead?
Whether or not a cell is alive or dead isn't always clear cut in microbiology - cells can exist in a variety of states between 'fully viable' and 'actually dead'
what are the energy units of calorie?
amount of heat energy needed to raise 1 gram of water from 14.5 to 15.5°C cal
common anti fungal drugs
amphotericin B and Nystantin- drugs that bind to sterols resulting membrane damage. ketoconazole and miconazole- drugs that inhibit sterol synthesis resulting in altered membrane permeability griseofulvin- drug that inhibits acid synthesis protein synthesis, and cell division 5-flyctosine- drugs that disrupts RNS function
who discovered streptomycin? and what is it?
an antibiotic active against T.B - discovered by Selman Waskman (1944) - Nobel price was awarded by 1953, chloramphenicol, terramycin, neomycin, and tetracycline isolated
what regulates luminescence by producing small, diffusible molecules called
autoinducer
what are the energy sources for microbial life
bases on sources : -phototrophs - pH -chemotrophs - oxidation of organic or inorganic compounds based on electron sources: -lithographs- reduced inorganic molecules -organotrophs - organic molecules
types of media culture
chemical physical - can be defined/ synthetic or complex
what are the three types of cell's work?
chemical work transport work mechanical work
chemotherapeutic agents
chemicals used to treat disease destroy pathogenic microbes or inhibits their growth within the host most are antibiotics - microbial products or their derivatives that kill susceptible microbes or inhibit their growth( i.e penicillin)
two continuous culture system
chemostat turbidostat
direct measurements of cell numbers
direct cell count - counting chambers - electronic counters- flow cyotmetry - on membrane filters
microbial population size can indirect and direct....
direct( hard to see if cell are alive or dead) -counting chamber -flowcytometer -electronic counters -fluorescent microscopy indirect -viable counting techniques ( standard- spread plate, i.e pour plate, or the membrane filter) - dry weight - turbidity - amount of cell component
competitive inhibitor
directly competes with binding of substrate to active site
Cytokinesis
division of the cytoplasm to form two separate daughter cells
standard reduction potential
equilibrium constant for an oxidation-reduction reaction A measure of the tendency of the reducing agent to lose electrons more negative E0 ⇒ better electron donor more positive E0 ⇒ better electron acceptor
proteins synthesis inhibitors- Tetracylines
family of antibiotics with a four ring structure in which a variety of side chains are attached. these antibiotics are similar t the amino-glycosides int hat they can combine with the 30S unit of the ribosomes, inhibiting proteins synthesis. broad spectrum antibiotics that are against most bacteria, including the intracellular pathogens ( rickettsias, chalmudiae, and mycoplasmas ) - sometimes used to treat acne
Penicillium
first discovered by Ernest Duchesne(1896), but discovery was lost accidentally discovered by Alexander Fleming (1928) - observed pencilian activity on contaminated plate - did not think could be develop further effectiveness demonstrated by Florey, chain and heatley Fleming, Florey, and chain received Nobel prize in 1945 for discovery and production of penicillin
Neutrophiles pH
growth optimum between pH 5.5-7
Alkaliphiles pH
growth optimum between pH 8.5-11.5
what drugs are used to treat superficial fungal infection
mincoazole ketoconazole clontrimazole
patterns and colonies bacteria depend on what factors
nutrient difffusion and availability bacterial chemotaxis presence of liquid on the surface hardness of the agar
Osmophiles
organisms that live in environments high in sugar as solute
enzymes --- the reaction
speed up
