Microbiology Exam 2

Name at least four different levels for regulating gene/protein activity.

Transcription, translation, and post-translational control.  Transcription and translation can be regulated at the level of initiation, elongation, and termination.  Post-translational control may involve covalent modification of the protein (e.g. phosphorylation/de-phosphorylation) or non-covalent modification (such as feedback inhibition for allosteric enzymes).

Describe the important sequence regions of a bacterial promotor.  What function do these sequences perform? 

The -35 and -10 regions of promoter DNA allow recognition and binding of the sigma factor portion of RNA polymerase holoenzyme.

What is the sigma factor cycle in bacterial transcription?

The sigma factor is a subunit of RNA polymerase that must associate with the RNA polymerase core enzyme in order to specifically bind to promotor sequences and allow transcription initiation to occur.  After initiation, the sigma factor is released from the core enzyme, which continues to transcribe the gene into an mRNA.

How are sugar catabolic operons induced and yet amino acid biosynthetic operons are repressed by a similar operator-repressor mechanism (i.e. explain the apparent contradiction here)? 

The apparent contradiction has to do with the activity state of the repressor: active in the absence of inducer in the case of the catabolic operons, and inactive in the absence of the co-repressor (e.g. cognant amino acid) in the case of the biosynthetic operons.  Thus addition of inducer inactivates the repressor for the catabolic operons, while it activates the repressor when the co-repressor is present to form an active holorepressor (i.e. the small molecule effectors perform opposite functions on the repressor proteins).

Compare and contrast positive versus negative regulation in the case of operons controlled by simple a DNA-binding protein whose binding site is adjacent to the promotor.

The difference between positive and negative regulation in this case has to do with the promity of the binding site of the regulator protein relative to the promoter.  In the case of repressors, the repressor-binding site overlaps with the promoter or lies downstream of it, thus preventing binding of RNA polymerase or blocking its progress.  In the case of activators, the activator-binding site lies immediately adjacent and upstream of the promoter so that it is able to enhance the binding and stability of RNA polymerase at the promoter site by making appropriate protein-protein interactions with RNA polymerase.

 What are the two components in a typical two-component regulatory system?  What third activity is needed for the system to operate correctly?  Why? 

The two components are the senor kinase and the response regulator.  A phosphatase is needed in order to reset the system back to the ground state.

Briefly explain how Quorum Sensing works?  When do microorganisms employ this type of regulation? 

A particular microorganism makes a small molecule autoinducer, which can be taken up by similar species.  Once the autoinducer reaches a critical concentration, it can bind to and inactivate a repressor protein inside the cell, thus turning on the relevant regulatory system.  Thus autoinducer concentration is a function of cell density, and responses only occur once sufficient cell density is achieved.  This insures that sufficient microorganisms are present in order to orchestrate a particular response.

What is diauxy?  What key regulatory molecules/macromolecules comprise this system, and where/how do they function?

Diauxy refers to the preferential use of a particular carbon source until it is exhausted, followed by utilization of the non-preferred carbon source during the second phase of growth.  The key regulator is catabolite activator (CAP) protein, a positive activator/transcription factor, along with adenyl cyclase that makes cAMP.  In the presence of a preferred sugar such as glucose, adenyl cyclase is inhibited.  However following glucose depletion and exhaustion, adenyl cyclase is activated and produces cAMP, which binds to and activates CAP.  The CAP-cAMP complex can now bind adjacent to promoter sequences such as the lac promoter, in order to activate the lac operon if lactose is also present.

Name at least two regulatory systems that are controlled by alternative sigma factors.  When is this type of regulation particularly useful? 

A variety of global regulator responses are controlled by alternative sigma factors: nitrogen assimilation, iron transport, heat shock, unfolded periplasmic protein response, etc.  This system is good for activating groups of functionally related genes.

What is the structure of a factor-independent terminator? How does this structure compare to a rho-dependent terminatior. 

A factor-independent terminator contains a GC-rich sequence that produces a stem-loop structure in the RNA followed by a poly U sequence (e.g. UUUUUU).  The former sequence is an RNA polymerase pause site, while the latter sequence allows dissociation of the RNA from the DNA during transcription, since A-U base pairs exhibit the weakest hydrogen bonding of any natural base pair.  A rho terminatior also has the RNA polymerase pause site, but lacks the poly U sequence.  However, it has a rho entry site on the RNA immediately upstream of the pause site.  This allows rho protein to travel up the nascent mRNA and effect transcription termination.

What enzymatic activities does Rho possess and how does this explain how transcriptional termination occurs through Rho action? 

Rho protein has both a nucleic acid specific ATPase activity and a DNA-RNA-duplex-dependent helicase activity.  Thus it can utilize its ATPase activity to travel up a nascent mRNA where it reaches the DNA-RNA duplex region.  It helicase activity can then upwind the DNA-RNA duplex, thus causing transcriptional termination.

What is a Shine-Dalgarno sequence?  What is its function? 

It is a sequence (5’-GGAGGU-3’ consensus) that is typically immediately upstream of the AUG start site on mRNA.  It is complementary to the 3’ end of 16S RNA and allows correct positioning of the mRNA relative to the 30S ribosomal subunit during translation initiation.

Antisense RNA was originally discovered in bacterial gene regulation.  How would this form of gene regulation work? 

Antisense RNA can bind at or near the translational initiation region (e.g. SD or AUG) to block translation initiation.

What is a Riboswitch? How does it control gene expression? 

Riboswitches are a form of translational regulation where a small molecule (e.g. metabolite) can regulate one of two different mRNA conformations.  Metabolite binding stabilizes an mRNA conformation where the structure is incapable of translation initiation (e.g. where the SD or AUG is blocked in a hydrogen-bonded secondary structure), while the unbound state adopts an mRNA conformation that is competent for translation initiation.

RNA-binding proteins are often found to possess a second activity: autologous regulation.  Explain this statement using the regulation of the ribosomal protein L11 L1 operon of E. coli as an example. 

In this latter case L1 protein normally binds to 23S rRNA during assembly of the 50S ribosomal subunit.  However when L1 protein is produced in excess, it has a lower affinity, RNA-binding site upstream of the L11 L1 coding region on its mRNA.  Binding at this site, blocks translation initiation of L11 and L1 proteins.

What is translational coupling? 

Translational coupling refers to the situation where a downstream coding region on a polycistronic mRNA is dependent on the coding region immediately upstream of it in order to be translated.  Such downstream coding regions lack their own SD sequences.  In such cases, the ribosomes never fully release during termination of the upstream gene, but rather, they slide over to the next gene and reinitiate at the next AUG codon.

Name the three different genetic exchange mechanisms found in microorganisms. 

Transformation, transduction, and conjugation.

What kinds of factors are induced in order to make a microorganism competent for DNA update during transformation? 

These include: cell surface DNA-binding proteins, a DNA transporter within the plasma membrane, and single-stranded DNA-binding proteins within the cytoplasm.  In addition, if duplex DNA is taken in via a single stranded form, then a membrane-bound nuclease is required.  Proteins like RecA are constitutive and not induced.

Outline the steps needed for generalized transduction. 

The bacteriophage must infect a donor cell, replicate, and accidentally package host DNA into a new viral particle, which must be released upon cell lysis.  The transducing phage must infect a bacterial recipient, injecting the donor DNA into the new host.  This piece of donor DNA can be recombined into the recipient’s genome via RecA-promoted recombination to give rise to a transductant.

Outline the steps needed for specialized transduction.

The bacterial lysogenic virus must infect a donor cell, where its DNA inserts into the host genome as a provirus. 

During induction, the proviral genome needs to undergo a defective excision, leading to the production of a specialized transducing phage that has both viral and host genes physically linked to one another. 

This aberrant DNA molecule is packaged into a viral particle, which must be released by cell lysis. 

The specialized transducing phage can then infect a bacterial recipient, injecting its DNA into the new host. 

The DNA containing both viral and host genes can be recombined into the recipient’s genome—typically at the proviral attachment site to form a transductant.

What is a provirus?  What is a lysogen? 

A provirus is the state of a lysogenic virus where its genome is stably integrated into the host bacterial genome.  A lysogen is a bacterial strain that carries a provirus stably integrated into its genome.

What core elements must all plasmids possess?

They must have an origin of replication and appropriate replication proteins needed for initiation of DNA replication at that origin.  In addition, if the plasmid is linear, it must possess telomers at its ends and a telomerase.

How does a conjugative plasmid differ from a non-conjugative plasmid?

A conjugative plasmid must possess a transfer origin, DNA replication proteins capable of initiating DNA replication at this origin, and genes encoding a pilus and pilus assembly genes that promote the conjugal structure needed for DNA transfer. 

Non-conjugative plasmids lack all of these elements.

What features allow a conjugative plasmid to associate with the bacterial chromosome to form an Hfr?

Integration of the conjugative plasmid typically occurs by homologous recombination (RecA-promoted) utilizing IS sequences and other homologous transposons on both the plasmid and chromosome.

If an Hfr E. coli strain is mated with an F-^- E. coli recipient, can the recipient strain become an Hfr?  If so, would this be a frequent or rare event?  Justify your answer.

Yes the recipient can become an Hfr, but rarely, since the entire chromosome must be transferred (the end of the F factor is the last piece of DNA to be transferred in this process).   This occurs rarely because fluid motion generally breaks up mating pairs prior to this late event that takes 100 minutes of mating pair formation.

What is an R factor?

They are conjugative plasmids that have multiple genes for resistance to antibiotics or other growth inhibitors used as chemotherapeutics.

Draw the structures of simple versus composite transposons.  Label the relevant parts.

Appropriate diagrams can be found in your text (e.g. Figure 11-30 in the 12^th edition).  One would want to label the inverted or direct repeats found at the ends of the transposon, the gene for the transposase enzyme found within the middle of the transposon, and in the case of a composite transposon, the captured drug resistant genes that lie within the middle of that structure and are flanked by the two simple transposons.

• Transposons: DNA elements that catalyze their own movement from one DNA molecule to another

• Encode an enzyme (transposase) that acts on ends of transposon to catalyze transposition

• Capable of gene capture to form composite transposons

• Composite transposons facilitate the spread of antibiotic resistance

• Transposition into plasmids facilitates spread of antibiotic resistance between different bacterial species (esp. for broad host-range plasmids).

Figure legend: The arrows above the maps show the direction of transcription of any genes on the elements. The gene encoding the transposase is tnp. (a) IS2 is an insertion sequence of 1327 bp with inverted repeats of 41 bp at its ends. (b) Tn5 is a composite transposon of 5.7 kbp containing the insertion sequences IS50L and IS50R at its left and right ends, re-spectively. IS50L is not capable of independent transposition because there is a nonsense mutation, marked by a blue cross, in its transposase gene. The genes kan, str, and bleo confer resistance to the antibiotics kanamycin (and neomycin), streptomycin, and bleomycin.

Briefly explain the mechanism of gene capture to form a composite transposon.

During transposition, a second transposon inserts itself immediately downstream of a homologous transposon.  In this case, transposition utilizing the 5’ repeat of the upstream transposon and the 3’ repeat of the downstream transposon creates a composite transposon that captures the DNA region and any associated genes that lies between the two transposons to create a composite transposon.

Distinguish between Type I and Type II restriction enzymes.

Type I enzymes are complex consisting of R, M, and S subunits.  These restriction enzymes recognize a particular sequence, but cut it at a variable distance, leaving a variety of different DNA ends (thus not useful for cloning).  By contrast, Type II enzymes are simple single subunit enzymes (having either restriction or modification functions).  The restriction enzyme both recognizes a particular sequence and cleaves it precisely (thus useful for cloning).

What are the advantages and disadvantages of using plasmids as vectors of recombinant DNA (rDNA) cloning?

Advantages: small size, thus easily isolated and manipulated. 

Disadvantages: limited DNA capacity (generally under 20 kb), high copy number can cause instability of cloned DNA.

Why would one use a virus as a cloning vector?

Allows larger DNA inserts; generally good stability of cloned DNA (only requires virus replication not host viability), genomic libraries easily manipulated (as simple phage stock). used for toxic genes

What is a shuttle vector and why is it employed in rDNA work?

It is a plasmid that can be maintained in two different types of hosts (and thus must have different origins of replication and selective markers that work in each host).  It is utilized for transferring cloned genes between two distinct hosts.

What features are needed in order to construct a shuttle vector?

It must have different origins of replication and selective markers that work in each host.

When would one utilize an expression vector? 2 reasons

When one wants to (1) carefully regulate gene expression under a particular set of conditions or

(2) overproduce dramatically a particular gene product.

Give two reasons as to why one would want to utilize a gene fusion vector.

(1) To study the activity of a particular promoter under different conditions where the natural gene product was difficult to detect (e.g. during development). 

(2) To obtain localization information of a particular gene product within the subcellular compartments of the cell (GFP tagging).

What is the difference between a genomic and cDNA library?

A genomic library contains DNA fragments of an organisms genome (i.e. in the nature intron-exon state) cloned into a particular vector system within a particular host.  By contrast, a cDNA library is made by reverse transcription of mRNAs, and such libraries lack introns and are good for bacterial expression.

Provide at least two different kinds of strategies for isolating your favorite gene from a cDNA library starting with its purified protein product.

1. One method involves obtaining N-terminal amino acid sequence of the relevant protein, designing an appropriate oligonucleotide probe(s) for this sequence utilizing the genetic code, and then screening the cDNA library with this labeled oligonucleotide probe to find the particular clone.

2. By contrast, if sufficient protein can be purified, then an antibody can be raised to this protein, and then the labeled antibody can be utilized to screen the cDNA library to find the particular clone.

In the context of rDNA technology, what is a BAC and why is it utilized?

BAC is a bacterial artificial chromosome utilized for cloning large rDNA fragments.  The F factor has been engineered to be a BAC, since its copy number is similar to the chromosome and it can accept very large pieces of rDNA (>300 kb).

In the context of rDNA technology, what is a YAC and why is it utilized?

YAC is a yeast artificial chromosome utilized for cloning large rDNA fragments.  YACs are mini-chromosomes that have a yeast origin of replication (ARS), a centromer, telomers, a selectable marker (e.g. URA3), and which can hold very large pieces of rDNA (up to 800 kb).

What is trophophase? What is idiophase

Trophophase: It is the growth phase of a microorganism, when nutrients are turned into cell mass.

Idiophase: It is the stationary phase of a microorganism, when nutrients are converted into secondary metabolites, unique to specific microorganisms.

Distinguish between primary metabolites and secondary metabolites and give an example of each.

Primary metabolites are utilized or excreted during the growth of an organism and are intimately related to catabolic and anabolic reactions needed for cell growth.  Fermentation end products like lactate and ethanol are good examples. 

Secondary metabolites are idiosyncratic compounds produced during stationary phase and are unrelated to growth: compounds such as penicillin, tetracycline, and other antibiotics are good examples of secondary metabolites.

List 4 characteristics of secondary metabolism/metabolites.

Non-essential for cell viability

Relatively specific for a given microorganism

Often produced as a family of related compounds

Production is highly dependent on growth media

Dramatic overproduction is often possible

What are some of the difficulties in finding novel antibiotics and in bringing them to market?

Discovery problems include:

(1) Many isolates turn out to be previously discovered antibiotics. 

(2)Novel antibiotics may lack a broad spectrum,

(3) have poor stability in vivo, or

(4) have toxicities. 

Market problems include: (1) long and (2) expensive development times—particularly multiple clinical trials.  Often this results in only short periods of patent protection before generics come on the market and (3) cut revenue returns.

What is semi-synthetic penicillin? Provide an example and indicate any advantages to this new form of penicillin.

It is when penicillin, produced microbially, is chemically altered by organic chemists.  An example is ampicillin, which is a modification of the N-acyl group on the penicillin nucleus.  It is a broad spectrum penicillin that works against both Gram negative as well as Gram positive bacteria.

What is meant by the term “broad spectrum antibiotic”?

A broad spectrum antibiotic is one that works against divergent bacterial species, especially, but not exclusively, both Gram negative as well as Gram positive microorganisms.

List 4 targets to antibiotics in a typical bacterial cell.

DNA gyrase, RNA polymerase, specific targets within the 50S and 30S ribosomal subunits, peptidoglycan synthesizing enzymes, lipid biosynthetic enzymes, enzymes involved in folate metabolism.

List 4 distinct mechanisms that promote antibiotic resistance in bacteria.

Tighter cell membrane due to mutation

Production of enzymes that inactivate or destroy antibiotics

Loss of affinity of antibiotic targets due to mutations

Efflux pumps that pump out antibiotic

Development of resistant biochemical pathway due to new, uninhibited enzyme

What are some of the practices that help to promote the spread of antibiotic resistance?

Use of antibiotics for prophylaxis in animal feeds.   

Use of antibiotics to treat an ailment without diagnosing their true need (e.g. viral infections).

Failure to take antibiotics when needed (e.g. the poor and homeless).

Discontinuing antibiotic usage prematurely.

Lack of effective national health care system (e.g. people that act as carriers or chronically sick to spread infections).

What is meant by “rational drug design”?  Provide an example.

It is when the X-ray structure of a drug target is available so that small molecule drug inhibitors can be designed in silico utilizing this X-ray structure, organically synthesized, and then tested for efficacy.  An example includes inhibitors of the HIV protease: squinavir and indinavir.

What is the definition of a virus?

An element composed in part of nucleic acid that can alternate between an inert extracellular state that is infectious (i.e. virion or viral particle) and an intracellular metabolically-active replicative state.

How are viruses categorized according to the Baltimore scheme? Provide the basic logic for each viral classification type. 

The Baltimore scheme utilizes the centrality of messenger RNA (taken as positive) to classify viruses into dsDNA, ssDNA, + ssRNA, - ssRNA, dsRNA, and retroviruses.  This system immediately allows assessment of which enzymes, if any, must be carried by the virion in order to replicate itself.

What are the two basic shapes for non-enveloped viruses?

Helical and icosahedral (a 20-sided shell)

How do enveloped viruses acquire their envelope?  What key proteins assist in this process?

They acquire their envelope by budding through the cytoplasmic membrane.  Viral glycoproteins that promote capsid association with the membrane as well as membrane distortion and fusion are needed in this process.

Briefly define the principal 5 stages of a typical lytic viral life cycle.

These include (1) attachment to the susceptible host cell, (2) penetration of the nucleic acid into the cell cytoplasm, (3) replication of the viral genome and production of viral proteins, (4) assembly and packaging of new infectious viral particles, and (5) release of infectious virus into the surrounding area.

What is a virus plaque, and how can it be used to quantify a virus stock?  What type(s) of viruses do not form plaques?

A virus plaque is a region in a plaque assay plate where a virus has repeated infected and lysed susceptible cells to produce a zone of clearing.  By serially diluting a viral stock and performing a plaque assay, one is able to quantify the infectious virus within the stock.  Viruses that do not kill their hosts and cause latent infections, persistent infections, or oncogenic transformation would not form plaques.

What is a focus, and how can it be used to quantify an oncogenic virus stock?

A focus is a region of a focus assay plate or tissue culture flask substrate where the oncogenic virus has infected and transformed a cell, and that transformed cell has lost contact inhibition such that it has grown up into a visible mound of cells called a focus (as opposed to the surrounding uninfected cells that are contact inhibited and form a monolayer).  By serially diluting a viral stock and performing a focus assay, one is able to quantify the oncogenic viral titer in the stock.

Explain the terms ID₅₀ and LD₅₀ when it comes to quantification of viral or microbial virulence.

ID₅₀ is the dilution of the viral or bacterial stock required to infect 50% of test animals and produce a characteristic disease.  LD₅₀ is the dilution of the viral or bacterial stock required to lethally infect 50% of test animals with a potentially lethal disease.  Both parameters depend on the route of transmission in the assay as well as the particular test animal utilized.

List 4 different outcomes that are possible after cellular infection with an animal virus? 

These include an acute infection with high production of virus often accompanied by cell lysis, a persistent infection with lower virus production with continued cell viability, a latent infection with no virus production and normal cell viability, and oncogenic transformation where a viral oncogene transforms the cell into a cancer cell.

What virus families possess oncogenes?  

1. Some prominent DNA viruses include Polyomaviruses (tumors in mice and humans), Human papilloma virus (oropharyngeal cancers, cervical carcinomas), Herpes viruses (Epstein Barr virus/Burkitt’s lymphoma; HHV8/Kaposi’s sarcoma), Hepatitis B virus (Liver cancers).  Retroviruses such as Human T cell Leukemia Virus 1 (HTLV-1) also possess an oncogene that induces leukemias.

What is a viroid and how does it differ from a virus? 

Viroids are infectious RNA molecules that can self propagate themselves and can produce specific diseases in plants.

What is a prion?  How is a protein able to propagate disease?

Prions are infectious proteins (PrPsc) that are able to self-propagate themselves and produce disease in animals (e.g. mad cow disease, scrapie in sheep) and humans (kuru, Creutzfeldt-Jacob Disease).  They do this by ingestion of diseased nerve tissue (e.g. brain) from the dead animal.  Upon entering nerve cells and interacting with a normal cellular homologue (PrPc), the PrPsc prion acts as a chaperone to facilitate misfolding of the PrPc protein to the PrPsc state, which facilitates PrPsc protein aggregation, which is responsible for the neuropathological properties.  As nerve cells die, the brain exhibits the characteristic spongiform encephalopathy.

List 2 strategies that viruses utilize in order to shut down host metabolic activity and propagate themselves.

1. .  A bacterial virus like T7 inhibits host RNA polymerase, thus shutting down host transcription; it makes its own RNA polymerase that uniquely recognizes its own promoter sequences.  Polio virus degrades the host cap-binding protein that is needed for host protein synthesis; its RNA genome has an internal ribosome entry site (IRES) that does not need this host protein, thus allowing for viral protein synthesis to continue.

Briefly outline the replication strategy of poliovirus, starting with virion RNA. 

+ Strand virion RNA enters the cytoplasm and can be utilized directly as mRNA to make a single viral polyprotein, which self cleaves itself into a number of viral proteins.  One of these proteins is the viral replicase that copies the + strand RNA into – strand RNA that is utilized as the template for making additional + strand RNA copies.  These new RNAs can either be utilized as mRNA to make the capsid components or as genomic RNA that is packaged into assembled virions.  See Figure 21.13 for additional details.

Briefly outline the replication strategy of influenza virus, starting with virion RNA. 

The – strand virion RNA enters the cell with its bound RNA replicase, which synthesizes a + strand RNA using the – strand RNA as the template.  The newly synthesized + strand RNA can either be utilized as a template to make additional – strand virion RNA copies, or alternatively, it can be utilized as mRNA to make viral proteins.  The viral proteins are assembled into a nucleocapid that packages the eight – strand RNA segments with their associated RNA replicase.  The RNA-containing nucleocapsids then associate with the membrane via membrane-bound viral glycoproteins which facilitate budding of the virus from the cell, and the creation of the viral envelope around the nucleocapsid.  See Figure 21.16 for additional details.

Briefly outline the replication strategy of a retrovirus, starting with virion RNA. 

The viral RNA enters the cell with its bound reverse transcriptase and integrase proteins.  The reverse transcriptase copies the viral RNA into the first single-stranded DNA copy, and then the enzyme uses its RNase H activity to degrade and remove the RNA strand from this DNA-RNA duplex.  The reverse transcriptase then synthesizes the second strand of DNA to form the DNA duplex copy of the viral genome, which then integrates into the host genome utilizing the viral integrase.  Once in the host genome, the host RNA polymerase copies the integrated proviral DNA, making full length viral RNA genomes that can be packed into virons, along with viral mRNAs that can be translated into viral proteins that form the structural proteins of the virion.   See the appropriate figure in your book for additional details.

What feature of influenza virus biology is responsible for antigenic drift?

Point mutations within the viral hemaglutinin and neuraminidase genes lead to diminished binding of neutralizing and opsinizing antibody, respectively, to the corresponding proteins within the influenza virion particle.

What feature of influenza virus biology is responsible for antigenic shifts and pandemics? 

Since influenza virus has a segmented genome of 8 RNA segments, a mixed infection of different viral strains within a host cell can lead to reassortment of these RNA segments, giving rise to a brand new recombinant viruses that are not recognized by existing protective immunity (i.e. memory B cells) within a given human population.