MCB 3020 FINAL EXAM

Be familiar with the use of pUC19 cloning vector in delivering foreign DNA to a bacterial cell including how to screen for transformants (Fig. 12.10).

-. the pUC19 plasmid is a standard for cloning because it contains multiple cloning sites (MCS) which are artificial DNA cut sites for many different restriction enzymes, which help to the insertion of a gene often in the lacZ gene, which is useful in the blue-white color-screening system to select from recombinants. -The pUC19 plasmid includes an ampicillin resistance gene that is helpful for further selection.

Which of the following are benefits to bacteria that form biofilms?

-Ability to evade the immune system of a host -Ability to remain in a favorable niche -Ability to live in close association with other microbial cells

CRISPR defense system against viruses and foreign DNA.

(a) Immunization. (b) Interference

· How are the following symbioses classified (mutualism, commensalism, parasitism): Lichen, Legume-Root Nodule, Crown Gall, Coral - Symbiodiniaceae, Termite - gut microbes, bobtail squid - A. fischeri

- Lichen: Mutualistic symbiosis, Mutualistic symbiosis between one or two species of fungi and an alga/cyanobacterium. - Legume-Root Nodule: Mutualistic symbiosis, Mutualistic symbiosis between legume roots (plant) and rhizobia (nitrogen-fixing bacteria). The nodule is a consequence of the infection of the bacteria to the plan. - Crown Gall: Parasitic symbiosis, 1) Parasitic symbiosis of Agrobacterium tumefaciens (Rhizobium relative) with plants. The crown gall is the tumor caused by the infection of this bacterium to a plant. -Coral - Symbiodiniaceae: Mutualistic symbiosis, Mutualistic symbiosis between stony corals (staghorn coral) and the dinoflagellate Symbiodinium. -Termite - gut microbes: Mutualistic symbiosis, Mutualistic symbiosis between termites and microbial symbionts found in their internal digestive organs (gut). -Bobtail squid - A. fischeri: Mutualistic symbiosis, Mutualistic symbiosis between the Hawaiian bobtail squid and the bioluminescent A. fischeri bacteria.

·How does an oxygen microenvironment impact the spatial distribution of bacteria (Fig. 20.3)?

- Microenvironments are micrometer-scale spaces surrounding a microbial cell or group of cells, and the presence of oxygen in one of these environments can play a significant role on the spatial distribution of different bacterial species. - Obligated aerobic bacteria will be present in the outermost layer of the particle, consuming most of the oxygen, with microaerophiles close to the outer layer as they require just very low levels of oxygen. - Anaerobic organisms could thrive near the center, while facultative aerobes could be distributed throughout the particle.

What is the difference between a prebiotic, probiotic, and synbiotic?

- Prebiotic: - These are food additives that promote the growth and activity of beneficial microorganisms, usually in the gastrointestinal tract. - Probiotic: : These are live microorganisms which, when administered in adequate amounts, confers a health benefit on the host. - Synbiotic: - These are mixtures of prebiotics and probiotics that beneficially affects the host by altering the microbiome.

What is the endosymbiotic theory and how is it related to mitochondria and chloroplasts?

- The endosymbiotic theory states that the mitochondria and the chloroplasts were descendants of respiratory and phototrophic bacterial cells, respectively. - According to the hypothesis, the non-phototrophic hosts associated with these symbiotic bacteria, gaining new forms of energy metabolism while bacterial cells received a stable and supportive growth environment inside the host cell. Over time, the originally free-living symbionts became an intimate part of the eukaryotic cell.

What are endotoxins, what type of bacteria produce them and what type of assay is used to detect them?

- The endotoxins are the toxic lipopolysaccharides (LPS) found in the cell walls of most gram-negative bacteria. These are cell-bound structures, and they are only released in toxic amounts when the cell lyse. -The main type of assay used for the detection of endotoxins in a mixed solution is the Limulus Amoebocyte Lysate (LAL) assay, which uses the mobile cells in the fluids of the horseshoe crab Limulus polyphemus, as endotoxins specifically causes lysis of the amoebocytes. A gel is formed with precipitation and a change in turbidity if endotoxins are present and has been used on clinical samples (serum and CSF) to detect presumptive evidence of gram-negative bacteria infection.

How does catabolite repression affect the expression of the lac operon and how can it lead to diauxic growth?*

- The lac operon is only expressed when lactose is present as allolactose must bind to the repressor to prevent the repressor from binding to the operator for the genes to be transcribed. - Catabolite repression is a type of global control and can affect a range of processes related to carbon use. A consequence of this control might be diauxic growth, which is a phenomenon where 2 exponential growth phases are present. Glucose is a better source of energy than lactose, therefore the cell grows quicker when glucose is present. However, if depleted, the cell growth will stop forming a lag period, resuming once the other energy source is found (in this case, lactose).

Be familiar with the major sources of global methane emissions

- There are many natural sources of methane, as well as other several linked to human activities, which include wetlands (including melting permafrost), ruminants, and rice cultivation, while fossil fuel, biomass burning, and increasing number of domesticated ruminants are the main contributions from the human side. - The methane emissions from the human side, just fossil fuels use along with biomass burning release up nearly 1/4 of the total emissions on Earth.

Which of the following are mechanisms of horizontal gene transfer (choose all that apply)

- Transduction -Transformation -Conjugation

In the arg operon, arginine servers as the effector molecule and is specifically a corepressor. Which of the following is true?

- Under normal circumstances, transcription of the arg operon is turned on - When media is supplemented with arginine, the arg operon is repressed - Transcription of the arg operon is under negative regulatory control

You are cloning using the pUC19 vector. After transforming E.coli cells and plating them in the presence of ampicillin, you observe only white colonies on your plate? Which of the following is a possible explanation for this observation?

- You forgot add X-gal to your plate -All the E.coli cells took up plasmids containing the DNA insert

Which of the following is true of the process of gene insertion using CRISPR genome editing?

- it requires a single sgRNA -it requires homologous recombination -it requires the Cas9 protein

All of the following are true of polymerase chain (PCR) ?

- it requires a thermostable DNA polymerase such as Taq -it requires DNA primers -it is used to make many copies of a specific region of DNA

What methods were used to determine microbial composition of the human microbiome?

- the nucleic acid sequencing methods, as the vast majority of microorganisms cannot yet be readily cultured or enumerated using growth-dependent approaches. -. Metagenomic sequencing and the other omics tools for microbial ecology have been extremely useful for the human microbiome investigation.

Which of the following is true of Type, III, IV, and VI secretion systems in gram negative becateria

- they are considered one-step translocases - they secrete molecules outside of the bacterial cell and intro a host cell

Why are prokaryotes able to carry out coupled transcription and translation while eukaryotes cannot?

-Prokaryotic cells are able to rapidly carry out simultaneous transcription and translation, whereas eukaryotic cells cannot because eukaryotic cells have a nucleus. In eukaryotic cells, transcription takes place in the nucleus and then the transcripts need to be moved out of the nucleus before translation can occur. - Prokaryotic cells often have plasmids, but this does not explain their ability to carry out simultaneous transcription and translation. They require both mRNA and rRNA, as do eukaryotic cells.

What molecules and bacterial structures play a role in adherence?

-Receptor molecules coating the surfaces of both the pathogen and cells of its host are often critical for the adherence step. Adhesins are receptors on the pathogen surface composed of glycoprotein or lipoprotein that enable the pathogen to bind with the outer layer of the host cells. -There are other bacteria that can form a capsule composed with either polysaccharides or polypeptides which surface contain specific receptors for adherence facilitation, as well as assisting to the overall attachment process due to its sticky nature. -Fimbriae (usually fimbriae type I as they are distributed uniformly on the surface of the bacterium) and pili (longer and less numerous than fimbriae) can function by specifically binding to host cell surface glycoproteins. Flagella may also facilitate on adherence. As summary, these three structures

What are the dominant bacterial groups found in each of the following areas of the human microbiome (skin, saliva, urogenital tract, gastrointestinal tract)?

-Skin: Propionibacterium with >50%, -Saliva: Streptococcus with >25%, -Urogenital tract: Lactobacillus with >80%, -Gastrointestinal tract: Bacteroidetes with >50%,

· How can CRISPR be used to edit genomes (Fig 12.42)?

-The CRISPR/Cas9 system provides the most powerful and precise tool yet for altering eukaryotic genomes in living cells (genome editing) and has been used successfully to edit the genomes of plants, animal embryos, and human cell lines. -Cas proteins that function as endonucleases (cut nucleic acids) when guided by a specific RNA (crRNA in bacteria), which can be synthesized (synthetic guide RNA, sgRNA) to both recruit the S. pyogenes Cas9 protein and binds to the desired DNA sequence to cut in the genome of virtually any cell. A protospacer adjacent motif (PAM) must occur on target DNA for Cas9 to cut the DNA sequence with its two endonuclease domains. You can use CRIPSR for insertion or deletion as follows:

How can you determine the virulence of different pathogens?

-Virulence is the net outcome of host-pathogen dynamic relationship interactions. It is a quantifiable entity, especially if a pathogen is lethal, by the LD50 ("Lethal Dose") which is the number of cells of a pathogen (or virions) that kills 50% of the population of the host organism in a test group. Highly virulent pathogens frequently show little difference in the number of cells required to kill 100% of a test group as compared with the LD50.

Bright-field

-contrast results from cells absorbing or scattering light differently from their surroundings. Pigmented organisms are easier to see and bacterial cells can be stained to increase their contrast. -A very common differential stain is the Gram stain, dividing two categories in either Gram-positive (purple/violet) or Gram-negative (pink/red) bacteria. However, the tradeoff is that usually staining kills the cells and can distort their features.

Which of the following are associated with genome editing using CRISPR?

-sgRNA -Cas9protien -PAM

Which of the following are ways in which bacteria can be beneficial to humans?

.-Production of fermented foods -Gut bacteria aiding in digestion -Bioremediation of contaminated environments - Production of biofuels

What information does a phylogenetic tree contain?

A phylogenetic tree is a diagram that depicts the evolutionary history of the organisms on the tree and bears some resemblance to a family tree. It can be built by using molecular sequences that provide a record of past evolutionary events.

You have discovered a new species of bacteria and are interested in determining what genes are present in its genome. Which biological molecule and 'omic techniques would be the most appropriate for determining the organism's genetic potential?

DNA, genomics

What 3 major processes does the central dogma encompass?

DNA> RNA > Protein

What technique is used to differentiate between gram - and gram + bacteria in the laboratory?

Differential Staining is used to look for Gram negative or positive bacteria. The stains react differently with different kinds of bacteria, and it was developed by Hans Christian Gram on 1848

What organism produces each of these? What disease are they associated with? What is their mode of action (activity)? Refer to Table 25.2 Diptheria toxin

Diphtheria Exotoxin: This toxin is produced by the gram-positive bacterium Corynebacterium diphtheriae, causing the disease known as Diphtheria, which activity inhibits the eukaryotic protein synthesis

Scanning electron microscope

It is used for optimal 3D imaging of the cell. The specimen is coated with a thin film of heavy metal and even large specimens can be observed, and the depth of field is extremely good. It has a wide range of magnifications but only the surface of an object is typically visualized. SEM also provides black and white images only.

Fluorescent Microscopy

It visualizes specimens that fluoresce (emit light) by using filters, making the cells appear to glow in a black background. DAPI is a widely used fluorescent dye that stains the cells bright blue because it complexes with the cell's DNA. It is widely used in clinical diagnostic microscopy, and in microbial ecology for enumeration of bacteria in a natural environment or cell suspicion.

cAMP

Key molecule in metabolic control systems. Because it is derived from a nucleic acid precursor (RNA adenosine), it is a regulatory nucleotide. Catabolite repression is an indirect result of the presence of a better energy source (glucose). The direct cause of catabolite repression is a low level of cyclic AMP: a. Glucose enters the cell b. Cyclic AMP level is lowered c. CRP cannot bind DNA d. RNA polymerase fails to bind to the promoters of operons subject to catabolite repression

What types of staining are available to microbial ecologists and how are they used?

Non-specific 1. DAPI (4',6-diamindino-2-phenylindole) 2. Acridine Orange 3. SYBR Green I:

· How are microbes harmful?

They can be harmful by forming biofilms, it is when they grow on surfaces and can foul pipes and pipelines.

Which are used to visualize and count, target phylogeny, distinguish living from dead, target transcription, or target translation?

Viability Staining: living from dead GFP: transcription or target translation Fluorescence in situ hybridization: Target phylogeny

What is the typical structure of a virion in a naked virus or an enveloped virus?

Virions are the extracellular form of a virus, and it allows the virus to travel from one host cell to another. Naked virus: Nucleic acid, capsid (composed of capsomeres), nucleocapsid Enveloped virus: Nucleic acid, capsid, Glycoprotein and enveloped

Electron microscopy

uses electrons instead of visible light (photons) to image cells and structures. -transmission electron microscopy -scanning electron microscopy

Light microscopy

uses photons to look at bacteria that aren't present to the eye (staining can help). - Bright-field -Phase-contrast -Dark-Field Microscopy -Fluorescent -Differential interference contract (DIC) microscopy -Confocal Scanning Laser Microscopy (CSLM)

What protein secretion system types are present in gram-negative bacteria? Know characteristics of each. One Step system:

▪ Type I, Type III, Type IV and Type VI secretion systems are one-step secretion systems. They move molecules through a channel both the cytoplasmic and outer membrane and do not require Sec or Tat. ▪ a) Type I systems are ABC transporters that move molecules through a channel (e.g., E. coli bacteriocins kill neighbor bacteria). b) Type III secretion systems release molecules, often toxins, to the environment or to other cells. Commonly used by pathogenic bacteria. (e.g., effector molecules on Chlamydia and Salmonella). c) Type IV secretion systems release molecules to the environment or into other cells and are important in moving DNA during conjugation. (e.g., Agrobacterium transfer of Ti plasmid to host plant cells). d) Type VI system secretions are used by bacteria as weapons to compete with other bacterial or to attack eukaryotic cells. (e.g., V. cholerae way to deliver toxins to human intestines, and P. aeruginosa way to inject enzymes to competing bacteria).

Be able to compare Fermentation, Aerobic Respiration, and Anaerobic Respiration with regard to ATP generation and generation of reducing power?

- 1) Fermentation: used when respiration is not an option. Total net ATP production of 2, as glycolysis is the only reaction used for ATP synthesis. This process regenerates NAD+ by donating electrons from NADH to pyruvate in order to achieve redox balance. 2) Aerobic Respiration: in this process, oxygen (O2) is used as an external/terminal electron acceptor. The total net production of ATP molecules is approximately 38, with 2 produced during glycolysis, 2 more on the Tricarboxylic Acid (TCA/Citric acid/Krebs) Cycle, and a theoretical maximum of 34 ATP molecules by Oxidative Phosphorylation. The main reducing power of this process is found on the Electron Transport Chain (ETC) that includes NADH, FADH2, flavins, cytochromes and quinones which oxidation and reduction helps for the formation of a H+ gradient with a proton motive force. 3) Anaerobic respiration: This kind of respiration is very similar to aerobic respiration, with the difference that the terminal/external electron acceptor is a molecule other than O2, usually nitrate (NO3-), nitrite (NO2-), or sulfate (SO42-). The total net ATP production varies depending on the conditions, but it is safe to state that this process yields less ATP molecules than aerobic respiration, but more than fermentation. The reducing factors are also the molecules associated with the ETC.

What is a fecal transplant and what type of infection has it been used to treat with ~90% effectiveness?

- A fecal transplant is the transfer of microbiota from the colon of one individual into the colon of another, and the goal is to reintroduce a normal microbiota into the gut of someone with an intestinal disease of bacteria origin. -This method has been proven to be extremely successful against the opportunistic infection of the toxigenic Clostridioides difficile, which is an antibiotic resistant pathogen. The fecal transplant has proven an efficacy of curing C. difficile infection without relapse of nearly 90% versus only 25% of efficacy with standard antibiotic therapy.

How does a virulent (lytic) phage differ from a temperate phage?

- A virulent phage (ss ds DNA or RNA) always proceeds to kill its host cell through lysis once infection begins, while some viruses called temperate phages that have double stranded DNA, although capable of a virulent cycle, can infect their host and establish a symbiosis. - The temperate phage enters a stage called Lysogeny, which is a state where the viral genome is replicated in step with the genome of the host. The cell will now be called a lysogen (carrier of the prophage) when the prophage (viral DNA now part of the host cell genome) forms.

What is attenuation and how is it valuable in clinical medicine?

-Attenuation is the decrease or loss of virulence of a pathogen, which can happen when pathogens are kept in laboratory cultures rather than isolated from diseased animals. -Attenuated strains of various pathogens are valuable to clinical medicine as they are often used for the production of vaccines, especially viral vaccines. This process helped on the development of the first rabies vaccine, and have been employed on vaccines for measles, mumps, rubella, chicken pox/shingles, and yellow fever.

What is coral bleaching and how does it relate to the symbiosis between the coral animal and Symbiodiniaceae?

-Coral bleaching is the loss of color from host tissue caused by the lysis or expulsion of Symbiodiniaceae, which are pigmented symbionts, revealing the underlying white limestone skeleton. -. Usually, healthy corals will harbor millions of cells of Symbiodinium per cm2 of tissue. This phenomenon has been recorded and noticed on environments with elevation of temperatures, usually attributed to elevation on levels of atmospheric CO2, as elevated temperatures and high irradiance impair the photosynthetic apparatus of the dinoflagellates, resulting in the production of reactive oxygen that cause damage to both symbiotic partners. For this reason, it is thought that bleaching is a protective immune response of the coral host that destroys the compromised symbiont.

· How does prokaryotic diversity compare among Coastal Marine Environment

1) Coastal Marine Environment: The molecular analysis revealed that the clearly dominant prokaryote in the ocean is Proteobacteria, with more than half of the prokaryotes sampled being a Proteobacteria class. Following Proteobacteria, we have Cyanobacteria, Bacteroidetes, and to a lesser extent Marinimicrobia and Actinobacteria; Firmicutes are only minor components.

What is the steps of polymerase chain reaction (PCR)?

1) Denaturation: The template DNA is heated to denature it (separate the double strand), and 2 DNA oligonucleotide complementary primers to target sequence are added in excess to ensure that most of template strand anneal to a primer when the mixture cools. 2) Annealing: DNA polymerase extends the primers using original DNA as the template. 3) Elongation/Extension: The mixture is heated again after an incubation period to separate the strands, but now the target gene is present in twice the original amount.The mixture is cooled to allow the primers to hybridize with complementary regions of newly formed DNA.

How does prokaryotic diversity compare among freshwater

1) Freshwater Environment: 1) Proteobacteria also compromises the greater part of diversity, due to its metabolic diversity. Actinobacteria and Bacteroidetes species are significantly diverse, but in lesser abundance compared to Proteobacteria, while Acidobacteria is only found on a tiny fraction.

How does the process of gene deletion compare to gene insertion?

1) Insertion: Only one cleavage site and corresponding sgRNA are needed, which will activate the cell's own DNA repair machinery, by adding a piece of DNA containing sequences with homology to the cut site that will activate homologous recombination, yielding genomic insertion. 2) Deletion: Two target cleavage sites flanking the DNA sequence to be deleted must be identified and corresponding sgRNAs designed. The nonhomologous double-strand DNA break repair pathway will be employed to ligate following the deletion event. No additional piece of DNA is needed because one nucleotide is either added or removed during the re-ligation of the two strands.

What three major techniques are used to measure microbial growth? What are the benefits and drawbacks of each?

1) Microscopic Cell Count: it is a quick and easy way of estimating microbial cell numbers. However, without special staining techniques, dead cells cannot be distinguished from live cells, and precision is difficult to achieve. 2) Viable Cell Count: spread-plate method and pour-plate method is performed by spreading the microbes on solid media and counting colonies, hence is often called a plate count. It is important that the number of colonies on the medium not be too many or too few, as some colonies may fuse leading to erroneous measurements. Turbidity: It is the measurement with a spectrophotometer, which make it a quick and easy to perform techniquethat can be made without destroying or significantly disturbing the sample. However, the measurements can occasionally be problematic, as some organisms do not grow evenly on liquid medium, clumping and biofilm have to be minimized and some bacteria are just naturally planktonic, staying suspended in liquid

Which 'omics provide information about potential activity, gene expression, or activity of communities and single cells (Fig 19.21)?

1) Potential Activity: Metagenomics and genomics 2) Gene Expression: Metatranscriptomics and Metaproteomics. 3) Activity of Communities and Single Cells: Metabolomics and single-cell genomics.

How does prokaryotic diversity compare among soil

1) Soil Environment: the molecular data revealed the dominance of Proteobacteria presence on several soils samples, making up nearly 1/2 of the total phylotypes, followed by Acidobacteria (acid-tolerant, mostly aerobic and chemoorganotrophs), Actinobacteria and Firmicutes. Bacteroidetes is very diverse but not very abundant in most soils.

How is transcription of the lac, mal, and arg operons controlled? What transcription factors and effectors are involved? Are the operons inducible or repressible? What metabolic processes are associated with each?

1) The arg operon is repressible. When the cell is on an arginine rich environment, it doesn't need to make more on its own. For this reason, the arginine becomes a corepressor that binds to a specific repressor protein (ArgR) present in the cell. This causes the transcription to be physically blocked as RNA Pol can't either bind or proceed.

Next Generation (Next-Gen) Sequencing

1) These are a wide variety of methods used for reading and generating long sequences of DNA for genome assembly, which are widely used by molecular microbiologists today. One popular method is MinION sequencing, as it does not require a cloning step. PCR products can be used directly for sequencing as DNA fragments are separated and amplified by the device. Useful for environmental metagenomic studies with the goal of reconstructing genomes from members of the microbial community, finding minor phylotypes possibly missed by a clone library, and determining different phylotypes of a single gene in a mixture of different cells, which neither cloning nor DGGE are capable to do so.

Be familiar with the partners in each symbiosis listed above and know their contribution to the symbiotic relationship: Bobtail squid - A. fischeri Partners

A. fischeri Partners: The partners in this mutualism are the bioluminescent bacterium Aliivibrio fischeri that benefits from the nutrients found in the guts of the squid, and the Hawaiian bobtail squid (Euprymna scolopes) that benefits from the bioluminescence of the bacteria as a means of camouflage at night, increasing its survival rate.

How is quorum sensing utilized by A. fischeri?

Aliivibrio fischeri is able to emit light by the enzyme called luciferase, which is encoded by the lux operons under the control of activator protein LuxR. The lux operon is induced when a specific AHL of A. fischeri becomes high enough, causing the binding of LuxR and activate transcription of luciferase.

What type of viruses are found in greatest abundance in the human virome and how might they play a role in host-independent immunity?

Anellovirus, Papillomavirus, Polyomavirus, Adenovirus, Circovirus, Pepper mild mottle virus. These are bacteriophages, play a protective role and can server as a first line of defense with mucosal linings and surfaces and produces a symbiotic relationship, that provides a form of host-independent immunity.

What factors can affect the development and maintenance of the gut microbiome?

Antibiotics, medicine, -Diet -Delivery Method -Immune system -Metabolism

At what life stage does the human microbiome begin to develop?

At birth

What are biofilms?

Biofilms are assemblages of bacterial cells attached to a surface and enclosed in an adhesive matrix that is the product of excretion by calls and cell death. These structures trap nutrients and help to prevent the detachment of cells on dynamic surfaces. There are at least 3 proposed reasons for biofilm formation: 1) Microbial self-defense mechanism 2) Remaining in a favorable niche 3) Close association between bacterial cells

How do phototrophs, chemoorganotrophs, and chemolithotrophs differ?

By the way they make ATP, as explained above. 1) Phototrophs: These organisms obtain energy for metabolism from light. They usually contain chlorophylls and other pigments that convert light energy into ATP, and thus do not require chemicals as a source of energy. They can use either oxygenic photosynthesis (plants, cyanobacteria and algae) or anoxygenic photosynthesis (bacteria). 2) Chemoorganotrophs: Organisms that obtain its carbon and reducing power from organic molecules and chemical reactions. Most microorganisms in laboratory culture, and most pathogenic bacteria, use carbohydrates or proteins as their energy source and are thus chemoorganotrophs. 3) Chemolithotrophs: These organisms can oxidize inorganic compounds (e.g., H2, Fe2+, S0, or NH4+) as energy sources (electron donors). These are important in many biogeochemical cycles. Chemolithotrophic groups like "sulfur" bacteria, "iron" bacteria, or "nitrifying" bacteria can be found, as usually they form related groups based on similar inorganic compounds.

What are the major carbon reservoirs on earth and how is carbon cycled between them?

Carbon travels as CO2 through all of the Earth's major C reservoirs: the atmosphere, the land, the oceans, freshwaters, sediments and rocks, and biomass. The carbon and oxygen cycles are intimately linked. 1) The largest carbon reservoir on Earth by far is the sediments and rocks of Earth's crust. 2) A large amount of carbon is found in land plants (major sites of phototrophic CO2 fixation). 3) There is a larger amount of carbon present in dead organic material than in living organisms. This dead material is called humus. 4) The atmospheric carbon possesses the most rapid means of transfer, as CO2 is removed from the atmosphere, primarily via photosynthesis of land plants and marine microorganism. The most important contribution of CO2 to the atmosphere is by microbial decomposition of dead organic material.

What role do CRP and cyclic AMP play?

Catabolite repression relies on the activator protein cyclic AMP receptor protein (CRP). CRP is an allosteric protein and binds to DNA only if it has first bound a cAMP -If cyclic adenosine monophosphate (cAMP) levels are higher than glucose, then the lac operon will be expressed if lactose is present regardless of whether glucose is present. This is because the cyclic AMP receptor protein (CRP) will create a positive control (CRP will bind to CRP-binding site on the DNA promoter region), and in the presence of lactose (or any suitable inducer present) the LacI protein will not block transcription. - Of note, if cAMP levels are artificially increased, then this could overcome the effects of glucose. This is because the direct cause of catabolite repression is a low level of cAMP, while the indirect cause is the presence of glucose as a better energy source.

DAPI (4',6-diamindino-2-phenylindole):

Cells stained with DAPI will fluoresce blue. All microbial cells are readily visible and easy to enumerate..

SYBR Green I:

Cells stained with it will fluoresce a very bright green to all organisms, including viruses. Provides excellent enumeration of aquatic virus populations and other microorganisms.

Acridine Orange:

Cells stained with it will fluoresce orange or greenish orange. All microbial cells are readily visible and easy to enumerate.

What the central dogma

Central dogma is the process by which the instructions in DNA are converted into a functional product: DNA replication, transcription (DNA to RNA), and translation (RNA to protein).

What organism produces each of these? What disease are they associated with? What is their mode of action (activity)? Refer to Table 25.2 Chlorela entertoxin

Cholera Enterotoxin: This exotoxin is produced by Vibrio cholerae, causing the disease known as Cholera. It induces fluid loss from intestines.

Confocal Scanning Laser Microscopy (CSLM):

Computer-controlled microscope that couples a laser to a fluorescent microscope, which generates high contrast 3D images to allow the viewer to access several planes of focus in the specimen. The cells can be stained with fluorescent dye to be more distinct. It is useful for the analysis of thick layers of specimen, as the laser is adjusted to go layer by layer.

Which of the following are characteristics of the gram + bacterial cell envelope?

Contains teichoic acid in the peptidoglycan layer

What is the significance of endospore formation in a bacterium?

Endospores are highly differentiated, dormant, resistant to heat, radiation, chemical exposure, drying, and lack of nutrients. Development survival structures to endure unfavorable growth conditions. Ideal for dispersal via wind, water, or animal gut. Present only in some gram-positive bacteria (e.g., Bacillales and Clostridiales).

The lac operon is said to be under both positive and negative control. Which of the following best describes the circumstances under which the lac operon is induced (transcription is turned on)

In the presence of lactose and the absence of glucose

Which of the following methods separates DNA based on size and charge

Gel electrophoresis

Are you able to differentiate between the cell envelopes of gram - and gram + bacteria (composition and function)?

Gram-Positive Cell Wall: Thick layer of peptidoglycan, stabilized by peptide cross-links often containing peptide inter-bridges. It is susceptible to penicillin (blocks formation of peptide cross-links). It also contains Teichoic & lipoteichoic acids. The peptidoglycan is also susceptible to lysozyme. cytoplasmic membrane Gram-Negative Cell Wall: Thin layer of peptidoglycan, cytoplasmic membrane, outer membrane, and proteins

What organism can be found in the stomach (pH 2) that has been associated with gastric ulcers and cancer?

Helicobacter: a highly acid-tolerant bacterium that colonized the stomachs of about 50% of all humans can cause upper GI tract disease, including ulcers and developing stomach cancer.

What are the basic steps involved in generating a phylogenetic tree?

In order to build a phylogenetic tree, we will need to sequence the 16S rRNA gene. After the 16S rRNA sequence is obtained, there are 3 basics steps that must be taken into account: 1. Sequence Alignment 2. Distance Matrix Calculation 3. Adding Nodes and Branches

How to use pUC19?

In order to use pUC19, a suitable restriction enzyme (cut sites within MCS in the lacZ gene encoding for beta-galactosidase) must be chosen to cut both the foreign DNA restriction sites and vector, which will be introduced on E. coli cells after ligation. These cells will be plated on media containing both ampicillin (antibiotic) and X-gal (lactose analog). Cells without the plasmid will not be present as they will be killed by the antibiotic. Cells with recombinant DNA will remain, but not all the colonies will posses the desired cloned DNA. X-gal is a colorless substance that when is cleaved by beta-galactosidase generates a blue product. As the MCS of the cloned DNA were inserted on lacZ, beta-galactosidase should not be expressed. Therefore, white colonies on the plated media would be recombinant cells with your cloned DNA, and blue colonies would be recombinant cells without your cloned DNA.

What are the major human impacts on the nitrogen cycles?

In terms of the Nitrogen Cycle, human activity doesn't have the same extent as with the C cycle, yet there are still significant effects found in it, as the two systems are closely coupled: 1. Haber-Bosch synthesis of N fertilizers 2.Agricultural Run-Offs: 3.Unknown Effects on Nitrogen Cycle:

What major steps in pathogenicity lead to infection and development of disease (Fig. 25.1)?

In the process of infection, which is the growth of microorganisms on or in the host, the steps are as follows: 1) Adherence: This is the first step of infection that is required to initiate the disease process, but it is not sufficient to initiate disease on its own because the host has many innate defenses that can thwart infection. 2) Colonization/Invasion: This is the process of entry to the host epithelial cells after adherence in order to initiate disease and continue the infection process. 3) Multiplication: This is the step of microbial growth inside the host cells and the induction of virulence factors and toxins production. Of note, exposure to pathogens is NOT part of the infection process, but it is essential for infection to occur. After infection, the development of disease could be trigger, noting that disease is the term reserved for actual tissue damage or injury that impairs host function caused by the pathogen. The following steps are part of the disease development: 1) Toxicity and Invasiveness: The toxins released during the multiplication steps can have local or systemic effects, and the virulence factors can provide further growth at original and distant sites of the host's body. 1) Tissue or Systemic Damage.

Differential Interference Contrast (DIC) Microscopy

It employs a polarizer in the condenser to produce polarized light, creating an optical effect of 3D perspective, which enhances subtle differences in cell structure. Different cellular structures or various types of inclusions present in some bacteria, appear more 3D than other forms. DICM is usually used on unstained cells that are invisible on bright-field microscopy without the need for staining.

Phase-Contrast Microscopy

It is commonly used for the observation of living preparations, as the cells do not often have to undergo staining to be observed. Based on the principle that cells differ in refractive index from their surroundings. It results on a dark image on a light background (phase ring).

What is the significance of leghemoglobin and nod factors in Legume-Root Nodule symbiosis?

Leghemoglobin is important in fixing nitrogen for bacteria, it binds , protecting nitrogenase. -The protein allows the bacteria to obtain sufficient oxygen for respiration, while keeping the unbound O2 low in the nodule environment -The leghemoglobin bound O2 to free O2 ratio in a functional root nodule is 10,000:1. The production of this protein is induced by interaction between symbiotic partners (rhizobia and plants). -Leghemoglobin is an O2-binding iron-containing protein found in root nodules that functions as an "oxygen buffer," as the rhizobia need a microaerophilic condition (low oxygen) -Nod Factors are lipochitin oligosaccharides encoded on the nodABC gene (universal, encoding for "Nod backbone") that function as primary rhizobial signaling molecules that triggers in legumes to develop root nodules that host the bacteria as nitrogen-fixing bacteroids. The nod backbone can be modified by the positive regulatory NodD protein (promoter) encoded on the nodD gene, which can be induced or inhibited by flavonoids (organic molecules) produced by the plant, providing a mechanism that can allow the symbiosis with a wider range of different legume species.

· Be familiar with the partners in each symbiosis listed above and know their contribution to the symbiotic relationship: Lichen Partners:

Lichen Partners: The partners in question are a chemotrophic fungus (usually ascomycete) that provides a firm anchor, inorganic nutrients through lichen acid reactions and water sequestration, and either an alga or cyanobacterium (phototroph) that provides organic nutrients to the fungus.

When cloning using the pUC19 vector, how can you confirm that a bacterial cell has taken up the plasmid without DNA instead?

Look for blue colonies growing in the presence of ampicillin

How are microbes beneficial?

Microorganisms represent a major fraction of Earths' biomass, and their activities are essential to sustaining life: - Oxygen (O2) is a significant result of microbial activities. - Evolution of animals and plants are heavily influenced by microbial activities, microbial symbioses, and pathogens (minority of microbes). - Humanity and microbes also are interconnected; from infectious diseases, foods (fermentation and agriculture), water, fertility of soils (agriculture), health of our animals, to the fuel of machines and technology.

How do the lytic and lysogenic phases of a temperate phage differ?

Lytic cycle: phage components are synthesized and assembled in the host cell. Lysis occurs to release new virions. Lysogenic: viral DNA is integrated into host DNA. Viral DNA is replicated with host DNA at cell division.

How is transcription of the lac, mal, and arg operons controlled? What transcription factors and effectors are involved? Are the operons inducible or repressible? What metabolic processes are associated with each?

Mal regulon controlled:1) The mal operon is inducible. The mal operon encodes for the enzymes necessary to catabolize maltose. An activator protein called MalT is available in mentioned operon and cannot bind to the DNA unless maltose is present as an effector molecule. Because the MalT have multiple activator-binding sites in multiple mal operons, it is said that the enzymes for maltose are encoded on the maltose regulon (series of operons controlled as a unit).

Viruses are not considered living organisms because they are acellular and require the machinery of a host cell to replicate. As a result, we refer them as

Obligate intracellular parasites

What genes are typically targeted for microbial community diversity analysis?

On microbial community diversity analysis, the typically targeted genes are the ones encoding for the subunit ribosomal (SSU) rRNAs, or in other words, the 16S rRNA gene sequence.

How is quorum sensing utilized by pathogenic organisms like E. coli ?

Pathogenic E. coli (Shiga toxin-producing) produces an AHL called AI-3 that induces virulence genes, while the host intestinal cells produce epinephrine and norepinephrine. These 3 signal molecules result in phosphorylation and activation of 2 transcriptional factors that activate genes encoding for motility and toxin secretion, making the infection more effective.

You are given a mixed culture containing two different species of bacteria. You are told that one species has an outer membrane containing LPS in its cell envelope while the other species lacks an outer membrane in its cell envelope but contains a thick layer of peptidoglycan. What first steps would you take in the lab to obtain pure cultures of each organism?

Perform the streak plate method to obtain individual colonies, followed by gram staining of individual colonies

· How are plasmids similar to and different from the bacterial chromosome?

Plasmid: Relatively short circular or linear, extrachromosomal Bacterial chromosome:Extremely long, usually circular Similarity: Both have double-stranded DNA. Plasmids are circular or linear double stranded DNA molecules that replicate separately from the chromosome and are typically much smaller than chromosomes. Bacterial chromosomes and plasmids may host a transposable element, which are sequences of DNA that are inserted into other DNA molecules but can move from one site on the DNA molecule to another.

What organism produces each of these? What disease are they associated with? What is their mode of action (activity)? Refer to Table 25.2 Botulinum Toxins

Produced by Clostridium botulinum, which causes the disease known as Botulism, causing flaccid paralysis with the activity of these toxins.

You have discovered a new bacterium and are growing it up in culture in the lab. To maintain the growing culture over time and to keep cell density low, you have been taking a small sample and placing it in fresh media each evening before you head home for the day. In a rush to meet your parents for dinner, you forget to do this. The next morning when you get to the lab, you notice that your culture appears to be glowing. What regulatory system is most likely driving this?

Quorum Sensing

Transcription is catalyzed by which of the following enzymes?

RNA polymerase

Which of the following correctly describe RT-PCR

RT-PCR generates cDNA from an mRNA template using reverse transcriptase

How does RT-PCR differ from traditional PCR?

Reverse transcription PCR (RT-PCR) is an extension of the standard PCR that synthesizes DNA from an mRNA template. This method uses reverse transcriptase, a retroviral enzyme, in order to convert RNA into complementary DNA (cDNA). In order to do this conversion, a primer must be attached to the 3' end of the target RNA, or to the Poly(A) tail of target eukaryote mRNA. Then, RNase H hydrolyzes the RNA that leaves the single-stranded cDNA as template to be followed by a standard PCR procedure.

What are the major virulence factors in Salmonella (Fig. 25.11)?

Salmonella species infect humans and lead to various GI illnesses. They encode a large number of virulence factors, including: 1) Type I fimbriae: facilitates attachment to the host cells 2) Several different classes of exotoxins: (a) Enterotoxin causing diarrhea, (b) cytotoxin inhibits host cell protein synthesis as well as helping on adherence. 3) Antiphagocytic proteins: these block engulfment of bacterial cells by host phagocytes 4) Siderophores: these are organic molecules that allow bacteria to outcompete the host cell on iron sequestration. 5) Endotoxin: This causes fevers in the host, present on the LPS layer. With the exception of the endotoxins, the rest of the virulence factors are encoded on genes present on mobile DNA (Pathogenicity Islands) rather than on the chromosome

What are the major human impacts on the carbon cycles?

Since the Industrial Revolution, the human activities are having major effects on the Carbon and Nitrogen Cycles. Carbon Cycle, humans have the greatest impact on the release of CO2 trough burning of fossil fuels and extensive ongoing deforestation 1.Effects in the Atmosphere -Radiative Forcing (units are W∙m-2): -Annual Greenhouse Gas Index (AGGI): 2.Acidification of the Oceans 3.Expansion of Oxygen Minimum Zones (OMZs

Diversity of microbial species in a community are expressed by richness and abundance. What is the difference between species richness and species abundance?

Species richness is the expression of the total number of different species present, while species abundance, by contrast, is the proportion of each species in the community

Phylogenetic Stains:

Specific fluorescent methods used to identify how many species, phyla or domains are present in a sample. a) Phylogenetic Fluorescent In-Situ Hybridization (FISH): I.) This method utilizes rRNA probes that are fluorescing oligonucleotides complementary to sequences encoding for SSU rRNA (16S or 23S rRNA in Bacteria and Archaea, or 18S or 28S in eukaryotes). The stains penetrate the cells without lysing them and hybridize with rRNA directly in the chromosome. Hybridization can be manipulated as specific as the researcher requires it. Limited with slow-growing organism due to small number of ribosomes present in them. b) Catalyzed reporter deposition (CARD)-FISH: Modified FISH method using the enzyme peroxidase instead of a fluorescent dye and a fluorescently labeled substrate called tyramide. These are hybridized on a specific mRNA. Even low abundance mRNA can be visualized by fluorescent microscopy. It also can be used on phylogenetic studies with microbes that grow very slowly (e.g., the ones found in the open ocean). b) Bioorthogonal noncanonical amino acid tagging (BONCAT)-FISH: Works as a direct measure of translational activity. The cells are incubated with a modified methionine containing a reactive alkyne group and then treated with a fluorescent reporter containing azide groups that bonds with the reactive groups. The methionine analog with the reactive alkyl group used on this method is called noncanonical (nonstandard) amino acid.

What are the three major types of symbioses and how are they distinguished from one another?

Symbioses are intimate relationships between organisms, often developed through prolonged association and coevolution. The 3 major types of symbioses: 1) Mutualism: both symbiotic partners benefit from the relationship. 2) Parasitism: one symbiotic partner benefits while the other is harmed. 3) Commensalism: one symbiotic partner benefits while the other is unaffected (does not benefit nor is harmed).

What is the role of CRISPR in bacteria?

The main function of CRISPR (Clustered Regular Interspaced Short Palindromic Repeats) is to seek out and destroy foreign nucleic acids. In bacteria, it functions to maintain the cell stability and the integrity of its genome, either by protecting it by destroying viral DNA (and also viral RNA) or destroying certain plasmids and other genes obtained from horizontal transfers.

What organism produces each of these? What disease are they associated with? What is their mode of action (activity)? Refer to Table 25.2 Tetanus toxin

Tetanus Toxin/Tetanospasmin: The microorganism Clostridium tetani produces this toxin that causes the disease known as Tetanus, which activity causes spastic paralysis.

What are the 3 major mechanisms by which ATP is generated and how do they differ from one another?

The ATP is generated through 1 of 3 mechanisms. (a) Substrate-level phosphorylation: energy-rich substrate bond hydrolyzed directly to drive ATP formation (e.g., hydrolysis of phosphoenolpyruvate). (b) Oxidative phosphorylation: Movement of electrons generates proton motive force (electrochemical gradient) used to synthesize ATP. (c) Photophosphorylation: light used to generate proton motive force that powers ATP synthesis.

What is the general structure of a cytoplasmic membrane and how does it function?

The Cytoplasmic Membrane is Selectively Permeable, composed of a Phospholipid bilayer of hydrophobic tails and hydrophilic heads. The main functions of the plasma membrane are: (a) permeability barrier (b) protein anchor (c) energy conservation.

What advantages does high throughput next-gen sequencing of microbial communities have over sequencing of a single gene?

The Next-Generation Sequencing long reads and tremendous volume of data are particularly useful for environmental metagenomics where the goal is to reconstruct genomes from members of the microbial community. They can detect low abundance phylotypes along with their more abundant neighbors through deep sequence analyses, identifying rare and potentially important components of a microbial community.

What are the key processes and microbes involved in the Nitrogen Cycle?

The Nitrogen Cycle is an essential cycle for microbial life that is composed of specific redox reactions that serve as its key processes. These reactions are: 1. Nitrogen gas (N2) Fixation 2. Denitrification 3.Ammonification 4.Nitrification 5.Anammox:

What is the significance of the Ti plasmid in Crown Gall?

The Ti (Tumor inducing) plasmid is a large plasmid found on the parasitic bacterium Agrobacterium tumefaciens (a.k.a. Rhizobium radiobacter) that contains the genes encoding for tumor formation (onc gene) and synthesis of modified amino acids called opines (ops genes). The section of the Ti plasmid containing both the oncogenes and the opine synthesis genes will be called transferred DNA (T-DNA) during infection, as this structure will be the only one entering the plant cell to induce tumorigenesis, not the whole plasmid. The T-DNA will enter the cell with the help of the vir genes: virA (protein kinases, activates virG), virG (activator proteins, activates other vir genes), virD (endonucleases for T-DNA isolation), and virB (11 different proteins for type IV secretion system). The process resembles bacterial conjugation.

What are the three categories of exotoxins?

The exotoxins are toxic proteins secreted by the pathogen as it grows, which can travel from a site of infection and cause damage at distant sites. There are 3 categories of exotoxins in terms of their mechanism: 1) AB-Type Exotoxins: These toxins consist of two subunits, A and B. The B component binds to a host cell surface molecule, facilitating the transfer of the A subunit across the cytoplasmic membrane, where it damages the cell. These can be potent toxins and can be found expressed on diphtheria, tetanus, botulism, and cholera. 1) Cytolytic Exotoxins, or Cytotoxins: These are soluble proteins that damage the host cytoplasmic membrane, causing cell lysis and death. Cytotoxins are also referred as hemolysins due to be more easily observed in assays that use RBCs. These toxins are usually phospholipases (phospholipid bilayer destroyers), but there are other types. An example of this is the α-toxin, a pore-forming cytotoxin produced by growing Staphylococcus aureus cells, which kills the cells due to efflux of cytoplasmic content through the pores, without nicking the phospholipid bilayer. 1) Superantigen Exotoxins: These exotoxins usually trigger an immune response called superantigen poisoning, which can be triggered by certain types of food poisoning, by toxic shock syndrome, or by pyrogenic fever. Toxic Shock Syndrome is the classic systemic effect of a toxic superantigen resulting from exposure of certain exotoxins secreted during infection by certain strains of S. aureus and S. pyogenes. The TSS can be fatal, much more for immunocompromised individuals (HIV patients, cancer patients ongoing treatment, and elders).

Dark-Field Microscopy

The light is directed from the sides of the specimen and only light that is scattered when it hits the specimen reaches the lens. Particularly good for observation of microbial motility, as bundles of flagella are often resolvable with this technique.

Can you recognize the major bacterial morphologies and arrangements?

The main bacterial morphologies are: - Coccus/Cocci (round) - Bacillus/Bacilli (rods) - Spirillium/Spirilla (rod-like) - Vibrio (coma-shaped) - Spirochete (helical shape) - Budding and Appendaged - Filamentous. Their arrangements can be paired (Diplo), Tetrads (4), cuboidal-manner (Sarcinae), grape-like clusters (Staphylo), or chains (Strepto).

· How does prokaryotic diversity compare among soil (Fig. 20.14), freshwater (Fig. 20.20), and coastal marine (Fig. 20.31) environments?

The molecular method used to generate the prokaryotic diversity snapshot for each environment was 16S rRNA gene sequencing. The results were as follows:

Be familiar with the partners in each symbiosis listed above and know their contribution to the symbiotic relationship: Legume-Root Nodule Partners

The partners are nitrogen-fixing bacteria (rhizobia), usually Alpha- or Betaproteobacteria, that provides organic nitrogen to the plant through the conversion of N2 to NH3 (nitrogen fixation) after infection, and legumes (plant) that provide the bacteria with O2 necessary for bacterial respiration in its root nodules through the protein leghemoglobin.

Be familiar with the partners in each symbiosis listed above and know their contribution to the symbiotic relationship: Coral - Symbiodiniaceae Partners

The partners here are the phototrophic dinoflagellate Symbiodinium that provides organic nutrients (fixed carbon through sugars, glycerol and amino acids) to the corals, and the stony corals (phylum Cnidaria) which provide shelter/protection (through structures in their cells called symbiosomes), more efficiency on light gathering through its structure (one of the most efficient solar radiation collectors in nature), and inorganic nutrients (nitrogen, phosphorus and inorganic carbon) to the dinoflagellate.

Be familiar with the partners in each symbiosis listed above and know their contribution to the symbiotic relationship: Termite - gut microbes Partners

The partners in this mutualistic relationship are the wood-eating termites (higher termites Nasutitermes & Microcerotermes, and lower termite Reticulitermes) which provide protection and a nutrient-rich environment to a wide diversity of bacteria, and the anaerobic acetogen bacteria (Spirochetes of the genus Treponema, and Bacteroidetes in certain higher termites that digest wood) which provide acetate and other organic acids (primary carbon and energy sources) produced during microbial fermentation to the termite, as well as providing glycosyl hydrolases that assists with breaking down the cell wall (cellulolytic activity) of plant and/or fungal material (hydrolyzes cellulose and hemicellulose).

Be familiar with the partners in each symbiosis listed above and know their contribution to the symbiotic relationship: Crown Gall Partners

The partners in this relationship are the bacterium A. tumefaciens (or Rhizobium rhizogenes) as the parasite that causes a tumorlike growths on diverse plants (hosts). The callus (tumor) is formed with an uncontrolled growth (similar to an animal malignant tumor) by the Ti plasmid injected by the bacterium, which also encodes for opines (modified amino acids). The parasite inhabits the callus structure which provides the bacterium with nutrients (Carbon, Nitrogen and Phosphate) taken from the plant through the opines.

How is quorum sensing utilized by pathogenic organisms like S. Aureus:?

The pathogenic Staphylococcus aureus uses a small peptide called Autoinducing peptide (AIP) as the autoinducer, encoded by the argD gene. ArgC sensor kinase binds with AIP and results in autophosphorylation, which increases transcription of specific genes that code for virulent proteins.

What is the purpose of polymerase chain reaction (PCR)

The purpose of Polymerase Chain Reaction (PCR) is to isolate and amplify segments (genes) of the target DNA in their pure form. PCR is essentially DNA replication in vitro, doubling the amount of DNA with each round of amplification, leading to an exponential increase in the target DNA. There are 3 basic steps involved in this process, which are:

What is the purpose of molecular cloning and what are the basic steps involved?

The purpose of molecular cloning is to move desired genes from the original source to a small and manipulable genetic element (Vector), resulting in recombinant DNA, that can be placed on an appropriate host where the desired cloned DNA will be replicated. The 2 basic steps for this procedure are: 1) Inserting the DNA into a cloning vector: Restriction enzymes cut the phosphodiester bond that result in double stranded breaks. These enzymes are used on cloning vectors (independently replicating elements that can both carry and replicate cloned DNA segments). 2) Inserting the vector into a host: The recombinant DNA molecules are introduced into a suitable host organism where they can replicate. It often yields a mixture of recombinant constructs.

Transmission electron microscopy

The resolving power is much greater than light microscopy, allowing to view structures at the molecular level. Objects as small as individual proteins and nucleic acid molecules can be visualized by TEM. It is a black and white image.

What cell structure is the target of cytolytic exotoxins?

The target of cytolytic exotoxins (cytotoxins) is the cytoplasmic membrane of the host cell. This attack can be either by destroying the cytoplasmic membrane through lecithinases (phospholipid lecithin used as a substrate), phospholipases, or by opening pores through the membrane as done by Staphylococcal α-toxin.

· Be familiar with the different types of environmental effects on growth. How are organisms classified based on their ability to cope with different environmental conditions (temp., pH, O2 availability, etc.)?

There are different environmental factors that can have an effect on the growth of microorganisms. Based on these factors, the following classifications can be made: 1) Temperature: The bacteria can be classified as (a) Psychrophiles (Cold-loving, optimal temp between 1-5 ºC), (b) Psychrotroph (optimal temp between 20-30 ºC, associated with food spoilage), (c) Mesophiles (moderate-temperature, optimal temp between 30-40 ºC), (d) Thermophiles (Heat-loving, optimal temp between 60-70 ºC), and (e) Hyperthermophiles (optimal temp between 90-100 ºC, only Archaea cells). 2) Oxygen (O2) Availability: The bacteria can be (a) Obligate Aerobes (grow only when O2 is available), (b) Facultative Anaerobes (grows best with O2, but also grows without it), (c) Obligate Anaerobe (Cannot grow when O2 is present), and (d) Aerotolerant Anaerobe (grows equally well with or without O2). 3) pH/Acidity & Alkalinity: bacteria can be classified as (a) Acidophiles (pH < 5, few bacteria), (b) Neutrophiles (pH = 6-8, most bacteria), or (c) Alkaliphiles (pH ~ 8.5, few bacteria, including Nitrobacter). 4) Water (H2O) Availability/Osmotic Pressure: In this case, the bacteria will be classified in accordance to the NaCl (salt/dissolved solute) concentrations in aquatic environment, with (a) Non-halotolerant (no growth in the presence of NaCl concentrations), (b) Halotolerant (does not required NaCl for growth, but can growth in the presence of it), (c) Halophile (organisms grow in environments with 5-10% concentration of NaCl), and (d) Extreme Halophile (>10% NaCl, near saturation, for growth). There are other classifications associated with water availability, such as osmophiles (grows best in the presence of high levels of sugar) and xerophiles (grow best in very dry environments).

Viability Staining:

This method uses 2 dyes, one green and one red, it allows instant assessment of both abundance and viability (live or dead organisms) on laboratory cultures. However, not suitable for use in direct microscopic examination.

· How do we measure the amount of virus in a sample in the lab?

To measure the amount of a bacteriophage virus in a sample, a typically done laboratory technique is the plaque assay in which the titer (estimate # of infectious virions present per volume fluid, expressed in PFU [Plaque Forming Units]) is quantified. When a lytic virus infects a host cell, a zone of cell lysis called plaque forms and appears as a clear area in the cell culture. When plaques are present, it represents that successful viral infection has occurred.

What protein secretion system types are present in gram-negative bacteria? Know characteristics of each. Two Steps system:

Type II and Type V secretion systems are common in gram-negative bacteria and are two-step secretion systems that use the Sec and Tat secretion systems. a) Type II systems transport completed folded proteins from the periplasm to the extracellular environment. (e.g., Vibrio cholerae toxins causative of cholera). b) Type V systems is the simplest structurally and are also called autotransporters. The protein to be secreted are transported by the Sec system and secreting them extracellularly. Requires chaperon proteins for proper folding. (e.g., adhesion proteins on E. coli and H. influenzae to attach to host cells).

You have performed a PCR to amplify a 300bp fragment of the 16S rRNA gene. Which of the following would be the best way to confirm that your PCR product is the correct size?

Use gel electrophoresis to run the product on a gel alongside a ladder containing a 300bp fragment

What are virulence factors?

Virulence factors are toxic or destructive substances produced by the pathogen that directly or indirectly enhance invasiveness and host damage by facilitating and promoting infection. Many of these factors are enzymes.

What is quorum sensing

a regulatory system that monitors the population levels and controls gene expression based on cell density, and it is employed by the production of a specific signal molecule called autoinducer, which usually diffuses freely across the cell envelope in either direction. The first class of autoinducers to be classified are the ones called acyl homoserine lactones (AHLs).

Biofilms medically relevant examples

a) Antibiotic and human immune cells resistance to the bacterial cells inside the biofilm. b) Tenacious biofilms caused by the bacterium Pseudomonas aeruginosa (also Burkholderia cepacia), which forms in the lungs of humans suffering from cystic fibrosis. c) 10 million people a year in the US experience biofilm infections from implants or intrusive medical procedures. d) Dental plaque is a typical biofilm that contains acid-producing bacteria responsible for dental caries (importance of a routine oral hygiene). e) Biofilms that develop in water distribution pipes might compromised the safety of drinking water, leading to outbreaks due to periodic release of pathogenic cells. f) Biofilms have been associated with several medical and dental conditions that include, but not limited to, periodontal disease, chronic wounds, kidney stones, tuberculosis, Legionnaires' disease, and Staphylococcus infections.

Reverse transcription PCR (RT-PCR)

is an extension of the standard PCR that synthesizes DNA from an mRNA template. This method uses reverse transcriptase, a retroviral enzyme, in order to convert RNA into complementary DNA (cDNA). To do this conversion, a primer must be attached to the 3' end of the target RNA, or to the Poly(A) tail of target eukaryote mRNA. Then, RNase H hydrolyzes the RNA that leaves the single-stranded cDNA as template to be followed by a standard PCR procedure.

How is transcription of the lac, mal, and arg operons controlled? What transcription factors and effectors are involved? Are the operons inducible or repressible? What metabolic processes are associated with each?

lac operon controlled: 1) : The lac operon is both inducible and repressible. The lac operon encodes for enzymes that use lactose. A common enzyme is the β-galactosidase, which cleaves lactose into glucose and galactose. If lactose is available in the environment, it will be used as an inducer in the form of allolactose, which binds the lactose repressor LacI in the operator region and causes allosteric changes in the protein, allowing RNA Pol to transcribe the lac operon.

· What are the major steps involved in homologous recombination?

▪In homologous recombination, the RecA protein is essential. The steps are as follows: 1) Endonuclease (enzyme that cuts DNA) begins the process by nicking one strand of the donor DNA molecule. This strand is separated by proteins with helicase activity. 2) The single-stranded segment binds single-strand binding (SSB) protein. 3) RecA then binds to the single strand, forming a complex that promotes base pairing in the recipient DNA, which displaces the other strand of the recipient DNA. This step is called strand invasion. 4) Recombination intermediates (cross-strand exchange) form heteroduplex regions, which is a DNA helix composed of single strands from 2 different DNA molecules. 5) In the resolution process, the linked molecules are then resolved (separated) by enzymes that cut and rejoins the strands of both original DNA molecules. 6) Two products possible: splices or patches. This depends on where strands are cut during the resolution process.