archea- a part monolipid part bilayer

(no phospolipid bilayer, no cytoplasmic membrane)

diversity by the 16/18s Ribosomal Rna and their sequence- found in all free living forms of life (minus the Viruses) 

Old way (using metabolism) 

We use the 16s (bacterial) and 18s Eukaryote Ribosomal DNA

we use the variable regions to compare how diffrent they are 

Prokaryotes- appeared 3.8 billion years ago

Eukarya

 Eukarya- 2.8 billion years ago

Cyanobacteria- 2.5 billion years ago

Plants and animals .5 billion years ago 

Are all bacteria small?

Are all viruses Small? 

No- there are always size exeptions

Peptidoglican Synthesis- Especially crosslinking reactions

penacilin family

Vancomicin

Protien Synthesis

tetracycline- macroslides

natural

Synthetic

Semi-synthetic 

Cure infections (some that we thought we could never cure)

treatment of Surgical Patients

Cancer treatment 

kills or inhibits bacterial growth at low concentrations

Broad spectrum

Minimal side effects on body and good microbiota

Pharmocoknetic

must get to site of infection in a high concentration 

Good-

Reduced need to diagnose a dangrous infectious disease

Bad-

Effects on normal microbiota as well as effects on environmenta microbiota 

1st we grow bacteria on Agar or Broth media

it depends on what bacteria we are growing, no universal media.

2nd-  make sure we get a pure culture-

the gram stain is often nonconclusive becuase bacteria look similar.

Gram stain doesnt work well with non-clinical bacteria 

Kirby Bauer Test- Qualitative test of inhibition

MIC- Quantitative test of inhibiton

MBD- tests killing not just inhibition 

1st step Microbial DNA is cut with a restriction enzyme

2nd step- large fragments seperated onto a gel by a pulsating electric field

3rd step-Gel stained profiles compared

Long incubation time with endonuclease, easy to contaminate DNA with nuclease

If the restriction cuts partially we see a diffrent pattern 

Sequence will determine where primers will hybridize

size of primer not critical (except it must be long enough to be specific)

number of reactions determines amount of product

temperature must be just right (not to high not to low) 

allows directional movement in bacteria that have flagella

sensing of compounds- like a radar 

multiple proteins.-

the shaft is composed of one protein while the receptor binder is usually composed of multiple proteins. 

Assembled on surface of cell sticking outwards

binding specific

also very thin pili are called fimbrae.

Pathogens love PILI (like Ecoli UPEC) 

The top portion is the most active layer of biofilm while the lower (surface portion that sticks to the surface) is the most metabolically inert level of the biofilm.

A biofilm does not need to be composed of only one type of species. 

The environmental biofilms- form on pipes, trees, etc

the medical biofilms form on implants

They protect the bacteria

Are usaully composed of polysacharides but some are composed of proteins

even polysacharide capsuls have some protein components

during infection the polysacharide prevent the phagocyte from killing the bacteria

in the external environment the capsul may protect the bacteria from drying out. 

No, they can not

both meningitis and Pnuemonie (have capsuls) 

The peptidoglycan monomers are synthesized in the cytosol of the bacterium where they attach to a membrane carrier molecule called bactoprenol.

Bacterial enzymes called autolysins break both the glycosidic bonds at the point of growth along the existing peptidoglycan, as well as the peptide cross-bridges that link the rows of sugars together.

Bactoprenol and transglycosidase enzymes then insert the new peptidoglycan monomers into the breaks in the peptidoglycan.

Transglycosidase enzymes catalize the formation of glycosidic bondsbetween the NAM and NAG of the peptidoglycan momomers and the NAG and NAM of the existing peptidoglycan.

Finally, transpeptidase enzymes reform the peptide cross-links between the rows and layers of peptidoglycan to make the wall strong.

Beta- lactamase is excreted by gram positive cells

in gram negetive it is secreted to peraplasmic space in gram negetive bacteria

Clavulanic Acid

- No antibiotic action

-Keeps antibiotics from being destroyed

LPS- Lypopolysacharide, (neutral sugars, Lipid A)

Lipid A embedded in the outer membrane of Gram Negetive cells

LTA- (sugar phosphates lipid,)

Lipid embedded in the cytoplasmic membrane of GM positive.

TA

Teichoic Acid- binds to Peptidoglycan. 

Inflammatory Compoud

Lipid portion activates cytokyinse

Lps and LTA are released when the Bacteria lysis

Stimulates redness and swelling from a wound, stimulates Leukocytes to migrate to the area.

In bloodstream they can cuase leakage of blood fluids drop in blood pressure and death 

Transport proteins (to transport things in and out of the cell)(most but not all require energy to function)

Penicillin binding proteins

Electron transport system

Protein secretion system

Sensor protein 

Take up nutrients against a gradient

Energy can come from direct hydrolysis of ATP , PEP

or symport or antiport of ions- harness energy by the electron transport chain.

the smallest bacteria come from Parasitic Bacterial genomes

the largest ones come from soil bacteria 

True-

bacterial genomes are from .6 to 7mb

while the smallest Eukaryotic genome is 12 mb

at least 5 bacterial species have 5-8 thousand genes 

True and False they usually have on chromosome but during rapid division they might have more than one in the cell at a particular time.

They also have a lot of Plasmids. 

the chromosome replicates

the chromosome seperates

a septum begins to form- a ring around the cell.

the septum begins to shrink and cuts the cell into two cells 

Lag phase- bacteria introduced, new genes are expressed

Exponential phase- bacteria reproduce like crazy

stationary phase- reproduction halted due to nutrient limitations

Death phase- resources run out. Bacteria lose viability.

Form a spore, sporulation

not terminal 

How do spores differ from Regenerative bacteria?

Spores are metabollically inert- non dividing

More resistant to heat/ drying/radiation

contains all components necessary for regenerative growth  and vegetative growth

have a tough protein coat 

After the chromosome replicates-

the cytoplasmic membrane invaginates

and the septum begins to form

once the septum is complete

the cytoplasmic membrane of the bacterium engulfs the newly formed spore

Peptidoglycan is deposited around the cell of the spore- the original dna begins to degrade

A protein coat forms around the spore

Bacterium lysis and the spore is released.

Mutations in target proteins lead to resistance to what?

Antibiotic is pumped out as fast as it is pumped in.

Resistance to tetracycline 

two ways

by two component regulatory systems

which control expression of many bacterial genes

and by Quorum sensing-

Replication of Bacteria to a certain quantity 

Where are autoinducers seen and how do they differ between gram negetavie and gram positive cells?

they are seen in Quorum sensing.

in gram positive they are Peptides

In gram negetive they are homoroseine lactones. 

We are going to take a promoter from a certain bacteria and a gene that codes for a certain protein. Like ecoli's beta galactose

we are going to fuse them and then introduce them back to the original population of Bacteria

Measure the amount of Beta galactose in the population and we know how active this expression is. 

Primers Amplify a small region

of the gene producing a labeled product

Machine monitors the appearance of labeled amplicon

Amount of RNA dictates when the threshold is passed

Quantitative becuase measurement is taken during early linear phase of amplification.

No information about final protein concentration.

Physiological impact or change

Depends on accuracy of annotation of genes.

most scientists- recomment RT-PCR supplements. 

Horizontal Gene Transfer

occurs between 

A Narrow range, between species or a closely related set of species.

Can happen by DNA Uptake

Generalized Phage transduction

Or by a lysogenic phage 

can cross genus and Phylus lines.

What are they

 Conjugation and self transmissible plasmids

In natural transformation only only a single strand of RNA comes into the cell.

only certain bacteria have the apparatus to take it up (the pores/ proteins)

(some cases it can only occur in a narrow amount of time)

In artificial transformation-

Bacteria is forced to take up a double stranded segment by the use of heat/salt shock or electrical shock.

both need HOMOLOGOUS RECOMBINATION 

Requires Homologous recombination

Phage acidentially packages host DNA instead of Phage DNA 

1st by positive or negetive strand

2nd by nonsegmented genome or by Diploid genome

3rd By naked or enveloped DNA

A naked virus has its capsul created and becomes part of the cell.

A enveloped virus comes in and carries everything.

the + strand codes for the MRNA when translated it gives the code for RNA replicase, Capsids, and Envelope Proteins.

the + strand is replicated by the replicase into the - strand and 

then Replicase makes it into the minus strand again.

Capsid and envelop proteins surround it and we have a copy of another Virus ready to go out and infect again.

the plus strand is replicated into a SSDNA copy. Afterwards the SSDNA is made into a DSDNA and then the DSDNA intergrates into the host chromosome.

The DSDNA uses host machinery to make a +RNA progeny and a +Rna mRNA. The +RNA Mrna makes Reverse Transcriptase, Envelop Proteins, and Capsid that are used to package the Virus and send it out to infect other Cells.

Viral DNA is transcribed-

 into a viral Polymera, Capsid proteins, and envelope proteins

the host or Viral polymerase transcribes the Viran DNA into viral Progeny.

Capsids and envelop proteins envelop the virus and it goes on to replicate in other cells.

THE CELL DIES

THEIR is a Latent Infection-which will become a Persistant infection- the cell keeps making these Virons

A tumor forms due to the Virus

The Virus Genome is lost.

Viruses can cuase some cancers.

HPV (DNA virus) can cuase cervical warts and cervical cancer.

Prevention involves Cauterizing Warts

The Vaccine covers the HPV types that cuase cancer

Hepititus B (DNA virus) cuases Liver Cancer as well as Liver diease

their is a vaccine

Cell entry, fusion inhibitors

Uncoating inhibitors

Mrna and Genome synthesis inhibitors

Translation inhibitors

they elicit antibodies against the virus

which bind proteins and prevent attachment by steric hinderance . They attach to host cells and direct T cells to kill host cells

Types of Vaccines

live attenuated viruses

Killed Virus

Isolated Surface proteins

it is a Enveloped virus with a Helical Shape

Single stranded RNA Genome (negetive strand) segmented into 8 segments

mammalian cell receptors Sialic Acid (C-9 sugar)

Types A B C

(A is the most dangerous Kind)

Viral attachment protein (Hemaglutin) H

as well as Neuramanidase (involved in budding from the cell)

HIGH RATE OF MUTATION (UNUSUAL FOR A VIRUS)

Through genetic drift and genetic shift.

the  virus infects the same kind of life form (reinfects human or reinfects Animal)

Reasortment occurs through homologous recombination and creates a virus with diffrent surface proteins

the immune response for the virus becomes ineffective. THis is much worse than Genetic Drift.

through antigenic types of H and N

the most recent type of influenza is H1N1

the Avian Flu type is H5N1

The release of the budding influenza virus from the cell.

Mutations in neuraminidase can inhibit the Tamiflu from working.

Virus cell interactions.

RT-PCR

PCR

ELISA

Western Blot

for PCR and RT PCR you must use diffrent primers for a diffrent virus

When it comes to microbes, why does size matter to someone who is trying to identify them?

Obviously, size determines the kind of microscopy you would use to view the microbe of interest.  Size is also an issue if you are using filtration to purify a fluid or to separate one type of microbe from another.  For example, a filtration unit designed to remove diarrhea-causing protozoa from stream water would probably not remove bacteria and would certainly not remove viruses.  During the anthrax attacks, microbiologists were often asked if the masks you can buy in hardware stores would protect a person from inhalation anthrax.  The answer is no, except insofar as the bacterial spores have attached themselves to a much larger particle of dust or soil.  Special filters (called HEPA filters) had to be used to filter out bacteria.  These filters are expensive and make breathing more difficult.

Identification of bacteria and archaea rely heavily on biochemical tests or DNA tests, whereas identification of eukaryotic microbes relies more on morphology (appearance).  Why is this so?

As is evident even from the simple diagrams shown in slides 2 and 3, bacteria and archaea tend to show much less morphological diversity than eukaryotic microbes such as fungi, protozoa and algae.  However, bacteria and archaea show a much greater metabolic diversity than eukaryotic microbes. For example, some bacteria can “breathe” sulfur or arsenic, using respiration of such substances as a source of energy.  Eukaryotes cannot do this unless, as has happened in some cases, they have “enslaved” bacteria or archaea (known as endosymbionts) that do the energy-yielding respiration reactions for them.  Even such widespread eukaryotic organelles such as mitochondria and chloroplasts were once bacteria.

How do bacteria and archaea differ from each other?  Are these differences consistent with the rRNA gene-based tree of life?

From the rRNA-based tree of life, it is clear that archaea are as distinct phylogenetically from bacteria as they are from bacteria.  Under the microscope, most bacteria and archaea look pretty much alike.  But archaea do not have a phospholipid bilayer membrane like bacteria and other forms of life do.  Instead they have a phospholipid membrane with a somewhat different structure.  The RNA transcription machinery of archaea is more like that of eukaryotes than that of bacteria.  Some other differences are included in your handout.

How do bacteria differ from protozoans and from algae?  Make an educated guess as to why antibacterial compounds usually do not work on eukaryotic microbes.

See the handout.   The main differences are size and the presence of a nuclear membrane in the eukaryotes.   One practical consequence of these differences is that antibacterial compounds generally do not kill or inhibit the growth of eukaryotes.  Either the bacterial target of the antibiotic is absent in eukaryotic cells (e.g., the bacterial cell wall) or is different enough from the eukaryotic equivalent (e.g. the ribosome) that there is little or no effect of the antibiotic on the eukaryote. 

How are the conserved and variable regions of an rRNA gene used to assess the relatedness of microbes to each other? 

The conserved regions are used to align the DNA sequences of rRNA genes from different microbes.  The variable regions are the regions that contain information about the differences (e.g. species, genus).  This will become very important when we cover the use of PCR amplification of rRNA genes and subsequent sequence analysis to identify microbes.

Is the rRNA gene-based tree of life consistent with the time line shown on the last slide?  Can you even compare the two?

Technically, the two are not strictly comparable because the rRNA gene tree is a measure of relatedness rather than a direct measure of evolutionary time (e.g., the fossil record).  Also note, that the rRNA-based tree is derived from modern organisms.  However, there is a relationship between diversity and evolutionary time because the genetic divergence of two organisms from each other takes time.  Also, there is a rough agreement between the time line and the tree of life.  That is, bacteria appear in the tree of life to have appeared before the eukaryotes and certainly long before plants and animals.  The cyanobacteria, for which we do have a fossil record in the form of the oxygen-generated banded iron formations and stromatolytes (fossilized masses of these bacteria), appear on the tree to be later than some of the deep branching anaerobic bacteria.    Finally, there are bacteria (more than first thought) that are surprisingly resistant to radiation compared to other types of microbes, another trait that would have helped land dwelling bacteria to survive before the ozone layer formed.

What is an antibiotic?  Why aren’t all antibiotics bactericidal?  Why would a bacteriostatic antibiotic work to eliminate bacteria from the body?

You might think that a bacteriostatic antibiotic would be useless, but such antibiotics can be very effective in an immunocompetent person.  The reason is that the antibiotic keeps bacterial numbers from increasing so that the defenses of the human body (especially neutrophils) can kill them.  People with impaired host defense systems, however, need bactericidal antibiotics.

What factors are important for cultivating a bacterium?

The composition of the medium (source of carbon, nitrogen, etc.), temperature, pH, and atmosphere are all important.  Different bacteria have different requirements, so there is no universal medium.

            You need a pure culture of metabolic tests and for antibiotic susceptibility tests such as the Kirby Bauer, MIC and MBC tests.  The reason is that if you have a mixture of bacteria, one may have the activity or antibiotic resistance being tested and obscure the activities of other bacteria in the mixture.  Even if the contaminating bacteria is initially present in very low numbers in the sample, it may overgrow the organism of interest on something like a Kirby Bauer or MIC test if it is a faster grower than the initially predominant bacterium. 

In the meningitis example used in class, why was the lab technician doing a Gram stain on spinal fluid before getting a pure culture?

What does the MIC test tell you that the Kirby Bauer test doesn’t?  What about the MBC test?

The MIC test is more quantitative and gives the concentration of antibiotic that is sufficient to inhibit the growth of the bacterium being tested.  Both measure the concentration of antibiotic that inhibits growth.  The MBC determines the concentration that kills the bacterium.  If an antibiotic is bacteriostatic, there may not be an MBC.  In any case, the MBC is usually higher than the MIC.  Why go to the trouble of doing an MBC test?  Usually MBC tests are not done unless the patient has an immune system that is impaired, so that it is important to have an antibiotic that kills the bacteria.

You accidentally mix two strains, one of which has an MIC of 4 μg/ml and one of which has an MIC of 16 μg/ml, but you think you have a pure culture so you go ahead and do an MIC test.  What is the MIC most likely to be?

            Probably 16μg/ml, unless the organism with an MIC of 16 μg/ml is unable to grow in the MIC medium.

How does 16SrRNA gene sequence differ from the cultivation approach?

Does the PCR-based molecular census give a quantitative representation of the bacterial population?  Why might some members be missed altogether?

How does the PCR “census” of a bacterial population differ from PCR identification of a bacterium you have isolated as a pure culture?

How does PFGE differ from the PCR “census” approach?

            In the application described in class, PFGE is being used to differentiate different strains of the same species.  PFGE has a much finer degree of resolution than sequences of rRNA genes.  Sequences of rRNA genes are used to distinguish bacteria at the species level rather than the strain level because the genes are so highly conserved.  Also, you can use PCR-sequencing to determine what organisms are present in a specimen.  The way PFGE was used in this application, you would start out knowing the species of the microbe in advance.

Could you use PFGE to characterize a bacterial population?

            The PFGE profile from a single bacterium is already pretty complex.  The profile from a population would be VERY complex, too complex to see the profile components clearly.  Also, different strains would look different so even a population that contained only a single species could have a very complex pattern.

In the rRNA gene census approach, why is there a cloning step?

Why is morphology (shape) more useful for identification of viruses and eukaryotes than for identification of bacteria?

            [Something to remember about viruses.  Viruses are not free-living organisms and thus cannot be plated on agar medium.  They need to be plated on monolayers of the specific cells they infect.]

Why is morphological analysis of viruses usually a method of last resort?

Why is there no single primer set for PCR amplification of viruses?

Which of the DNA-based methods described for bacteria could be used on eukaryotic microbes?

Making and using flagella takes a lot of energy.  Why would a bacterium need to move at all?

Why would different types of bacteria use different types of motility?

How could a nonmotile (i.e., nonflagellated) bacterium like Shigella move from place to place?

Will all antibodies that bind flagella bind to the surface of the bacteria?

            If the flagella are exposed, the antibodies can bind to the flagella of an intact bacterium, but in the case of spirochetes (flagella between the cytoplasmic membrane and outer membrane) the antibodies could not get to the flagella.

How do pili resemble/differ from capsules and flagella?

If you added a protease to a bacterial culture, what surface features is the bacterium most likely to lose?

How would the aforementioned protease be likely to affect the capabilities of the bacterium?

Why might a medical biofilm be a serious threat to a patient’s life?   What is the physician’s likely response?

How could you prevent the formation of a biofilm in the first place?

Most biofilms have a structure that includes channels that run through the biofilm?   What is the advantage to the bacterium of this type of biofilm structure?

Why are gram-negative bacteria generally resistant to lysozyme, an enzyme that attacks the PG backbone?  More resistant to many antibiotics than gram-positive bacteria?

How do LTA and LPS resemble each other?  How are they different from each other, pili and flagella?

How does the function of the cytoplasmic membrane differ from that of PG?

Why does PG synthesis start inside the cell cytoplasm and then move to the outside of the cytoplasmic membrane?

How does the mechanism of penicillin differ from that of vancomycin?

How does a bacterium become resistant to penicillin?  To vancomycin?  Why can’t the same mechanism work for both antibiotics – or is there one that does?

What is clavulanic acid and how does it work?  Is there a type of resistance that clavulanic is powerless against?

1.  Bacteria tend to have smaller genomes than eukaryotes.  Does this mean that they necessarily have fewer genes?  Why do bacteria need so many genes if they are such simple creatures?

.  Bacteria tend to pack their genomes with genes, with little noncoding DNA.  This is not true for many eukaryotes.  This is the reason a bacterium that has a smaller genome than a eukaryote can actually in some cases have more genes.  Bacteria are noted for their adaptability.  The genetic basis for this adaptability is having genes that are useful in different environmental conditions. This is also the reason that regulation is so important for bacteria.

.  The first bacteria that were studied had single circular chromosome.  Some had plasmids too, but the plasmids carried genes that were not essential for existence (e.g., resistance genes rather than rRNA genes).  As more bacteria were studied, it became apparent that some of them have multiple chromosomes and some have linear chromosomes.  [Do not memorize the organism names in slides 2 and 3.  Just get the big picture.]

3.  Suppose you took bacteria grown in medium A and inoculated them into medium B and found that the lag phase was 2 hr long.  If you take bacteria from late exponential phase in medium B and reinoculate them into medium B, what would you expect to happen to the lag phase?  What if you took bacteria that were well into stationary phase and did the same experiment?

.  Lag phase is the period during which bacteria change gene expression patterns to adapt to the new medium.  If the “new” medium is the same as the one the bacterium just came from, the lag phase should be shorter.  This is true if actively growing bacteria are transferred.  Bacteria deep into stationary phase may be changing their patterns of gene expression to allow them to survive starvation.  Thus, even if the same number of viable bacteria are transferred, the lag period may be lengthened by the need to adapt to the availability of nutrients.

3.  Suppose you took bacteria grown in medium A and inoculated them into medium B and found that the lag phase was 2 hr long.  If you take bacteria from late exponential phase in medium B and reinoculate them into medium B, what would you expect to happen to the lag phase?  What if you took bacteria that were well into stationary phase and did the same experiment?

.  Obviously on average, bacteria can divide only every 12 hours. If they divided as rapidly as they can in the laboratory, we would not be here to talk about microbiology.  The point of this question is that although the growth curve seen in slide 6 is the one usually used, bacterial growth in nature is usually not like the growth of a single strain in medium with an excess of nutrients.

5.  How does sporulation differ from vegetative growth (binary fission). How does a spore differ from a vegetative bacterium.

.  Review the steps in sporulation.  Sporulation involves asymmetric division and the result is one spore, not two daughter cells.  See slide 9 for characteristics of a spore.  Spores are impervious to many conditions, including heat and antibiotics.  In the case of inhalation anthrax, which is caused by inhalation of spores, the spores have to germinate before antibiotics are effective. 

6.  An extract taken from a bacterium contains a membrane protein that is labeled when incubated with labelled ATP. If the protein is a regulatory protein, what type of regulation is it probably mediating?

.  The protein is most likely to be a member of a two component regulatory system, either the sensor or the response regulator.  Note that phosphorylation is not involved in quorum sensing or other types of gene regulation you have seen in previous courses.

7.  How do the signals differ in the case of a two component system and a quorum sensing system?  How are the two types of process turned off when the conditions they are sensing change?

The signal sensed by a two components system sensor protein is an external signal.  The signal sensed by the quorum sensing system is generated by the bacterium itself.  The reason for this difference is that a two component system is sensing changes in the external environment, whereas a quorum sensing system is sensing the concentration of the bacteria themselves.  A two component system is turned off by the phosphatase that removes the activating phosphate group from the DNA binding protein.  In the case of a quorum sensing system, dilution of the bacterial culture will also dilute the signal. 

9.  Normally transcription of lacZ is up-regulated by the presence of lactose and the absence of glucose.  How would transcription of lacZ be affected if it was fused to a promoter that normally controls a gene whose synthesis is influenced by nitrogen?

A gene fusion is created by cloning and what is measured is the activity of LacZ (beta-galactosidase).  This is an indirect measure of lacZ mRNA levels.  In RT-PCR, there is no cloning step.  Primers amplify the mRNA from the gene of interest itself (not a reporter gene).  So mRNA is measured directly.  Measurements made with a gene fusion are quantitative (amount of beta-galactosidase activity), whereas RT-PCR is qualitative.  The reason it is qualitative is that after a certain number of cycles of amplification, the number of amplicons levels off.  If you do enough cycles, amplicons representing a lower abundance mRNA can “catch up” with amplicons from a higher abundance mRNA.  In real time or quantititative RT-PCR, the amplification process is monitored and stopped long before it reaches the saturation point. 

.  An open reading frame is defined by a start codon and a stop codon.  Sometimes, there can be multiple start codons in the same region, making it difficult to known which one is the correct one.  The function of the protein encoded by the open reading frame can be deduced by looking for proteins of known function in the protein databases that resemble it in amino acid sequence. 

A microarray is basically a fancy version of the dot or spot blot, with a DNA “spot” representing each gene being sampled by the array.  What is measured is hybridization of labeled mRNA from an organism to the DNA spots on the microarray.  This can be made quantitative and is thus considered a quantitative method.  As with the other forms of gene expression measures, it is necessary to compare at least two separate treatments to determine whether a message is produced at a higher, lower or similar rate under different conditions.  RT-PCR also measures the level of mRNA directly, but is usually done only on one or a few genes at a time.  Real time RT-PCR is generally done on one gene at a time.  

13.  Scientists often leave rRNA genes off of a microarray chip.  Why? [Hint:  What are the relatively amounts of rRNA and mRNA?]

.  In the case of eukaryotic cells, it is possible to selectively isolate mRNA because eukaryotic mRNA has a polyA “tail”.  Some bacterial mRNAs have a similar tail but in practice, an RNA sample isolated from a bacterium consists mainly of rRNA, with any single mRNA being in the minority of molecules.  Thus, the rRNA signal will dominate if there is rDNA on the chip.  tRNAs are not quite as much of a problem but can still dominate mRNA signals.

14.  Microarrays can cost as much $400 apiece. If microarrays are all you do, you need at least 8 replicates for each treatment to establish statistical significance in the changes you are detected.  You are a cheapskate and decide to buy 2 microarrays only, then take another route to establishing the significance of a change in gene expression you find.  How would you proceed?

.  Some scientists who are tired of being ripped off by the microarray manufacturers will do two chips, one for each condition they are interested in.  This tentatively identifies the mRNAs of interest.  Then, they use quantitative RT-PCR to measure levels of the mRNAs from the genes whose expression appears interesting.  The point of this question is to get you to think about the differences and similarities between microarrays and RT-PCR.

15.  How do the examples of antibiotics given in this section resemble/differ from penicillin and vancomycin in their mechanism of action?  How does resistance to them resemble/differ from resistance to penicillin or vancomycin?

.  The antibiotics used as examples here have in common with penicillin and vancomycin that they bind to a bacterial target (protein in the case of the DNA and RNA synthesis inhibitors, protein/rRNA in the case of the protein synthesis inhibitors) and thereby inhibit the activity of that target.  However, whereas penicillin binds covalently to its target (penicillin binding proteins), the antibiotics used as examples in this section bind noncovalently.  Some of the same types of resistance mechanisms seen for penicillin or vancomycin resistance , e.g., mutation of a target molecule, are also seen in the case of these antibiotics.  The efflux pump mechanism is different because these antibiotics have to enter the cell.  Thus, preventing transport (which for some reason bacteria seem not to do) or expelling the antibiotic from the cell become options for resistance.  [FYI.  There are also enzymes that covalently modify and inactivate some protein synthesis inhibitors, but these were omitted for simplicity.]  The main resistance point is that the fact an antibiotic has to cross the CM make it an option for the cell to employ a resistance mechanism (efflux) that would not be useful in the case of antibiotics such as penicillin and vancomycin that do not have to enter the cell.

Why are phage-mediated transfer processes considered to be narrow host range processes?  How would this change (or would it?) if the piece of DNA contained a transposon?

In both generalized phage transduction and lysogeny, there needs to be a receptor on the surface of the recipient cell.  Usually, such receptors are found only in a single species or closely related species.  In the case of generalized transduction, there has to be enough identity between the segment packaged by the phage and the chromosome of the recipient for homologous recombination to integrate the incoming DNA.  The entire DNA segment does not have to be identical to the chromosomal sequences, but you need to have near identity on either side of a foreign gene or a mutation if you want it to integrate by a double cross-over recombination event.

.  In natural transformation, only a single strand of the DNA is taken up.  Also, there is a special protein complex that mediates binding and eventual uptake of the single stranded DNA.   In the case of artificial transformation, double stranded DNA segments are taken up and no specialized receptor complex is involved.  DNA uptake is forced by exposing the recipient bacterium to chemical/heat stress or to an electric field.

.  How do generalized transduction and artificial transformation resemble each other?  How does generalized transduction differ from lysogeny?

.  Conjugation appears to be independent of a receptor and to be capable of occurring between very distantly related species.  The plasmid or the conjugative transposon has to be able to survive in the recipient cell.  A single strand of DNA is transferred, a feature that may explain why restriction enzymes are not as effective in eliminating incoming DNA as they are in eliminating DNA transferred by bacteriophages (which is double-stranded).  Restriction enzymes cleave double-stranded DNA.

If a plasmid can transfer from A to B but does not replicate in B, is the plasmid necessarily lost in B?

.  If the plasmid contains DNA that has identity to DNA on the recipient’s chromosome, the plasmid can integrate by homologous recombination.  If the plasmid carries a transposon, the transposon may be saved by transposing to the chromosome, even though the plasmid itself is lost due to inability to replicate.  (Note that the transposition process is independent of homologous recombination, so even if a strain lacks a functional homologous recombination system, the transposon can still be retained in the recipient.)

The self-transmissible transposon carries the genes that encode the proteins that form the bridge between the cytoplasm of the donor and the cytoplasm of the recipient, as well as the protein(s) that nick the plasmid and initiate transfer.  A mobilizable plasmid only provides the nicking protein; it uses the transfer proteins provided by the self-transmissible element.  Since conjugative transposons also contain genes that encode the mating bridge, these elements can also mobilize plasmids.

How can a transposon affect an open reading frame?  In the case of transposon mutagenesis, which type of event are you most likely to see? 

.  If the transposon integrates near an open reading frame, but not in the promoter region, there will be no effect.  If the transposon integrates into the promoter region, it can change the way the gene is regulated because it replaces the promoter with one of its own.  If the transposon integrates within an open reading frame, transcription is disrupted and the protein encoded by the gene is no longer made.  This is the type of event you are most likely to detect if you are screening for loss of a trait, although sometimes integration of a transposon into the promoter reduces gene expression sufficiently to make gene expression too low for the trait to be seen.

.  How can you determine which gene your transposon interrupted?  If your transposon carries a promoterless lacZ gene, what is the probability that the lacZ gene would be expressed if it interrupted an open reading frame?  (Assume the lacZ gene has a ribosome binding site.)

Transposons used for transposon mutagenesis have some sort of selectable marker, such as an antibiotic resistance gene.  Using a restriction enzyme that cuts only once within the transposon or cuts only outside of the transposon, you can clone a mixture of transposon and adjacent chromosomal DNA into a plasmid and select for the resistance marker.  Then, you can sequence the DNA outside the transposon ends.  The transposon that integrates into an open reading frame may integrate in either orientation.  If the lacZ gene is transcribed in a direction opposite to the direction of transcription of the open reading frame, there will be no LacZ production.  Thus, in theory, you have a 50% chance that the transposon carrying the promoterless lacZ will be expressed. 

.  How can a transposon insertion or a gene disruption affect expression of genes other than the one in which they insert?

.  If the gene interrupted is part of an operon, the transposon will affect genes downstream of the disrupted gene because transposons usually contain terminators that terminate transcription of the mRNA.  This is also true for a single crossover gene disruption.

What are the properties of a “suicide” plasmid?  If a suicide plasmid does not contain a cloned region, what happens to it in the nonpermissive strain?

“Suicide” plasmids need to be able to replicate in some strain so that DNA can be introduced into it by cloning, but the plasmid must not be able to replicate in the recipient strain.  Otherwise, all you would detect in the recipient is the replicating plasmid.

How do proteins that mediate adherence differ between naked and enveloped viruses?

Why do some viruses only infect certain mammalian cells?

What type of target (receptor) on a mammalian cell would allow a virus to infect most or all cells?

A receptor like sialic acid that is found on many cell types would have this effect.

How might an antiviral strategy (vaccine or treatment) inhibit the attachment step?

Vaccines such as the flu vaccine contain envelope proteins and elicit antibodies that bind the viral surface protein so that it cannot make specific contact with the mammalian cell receptor due to steric hindrance.  Antiviral vaccines often target surface proteins to prevent attachment (adherence), the initial step in infection.

You could also use a free form of the mammalian cell receptor to coat the virus and prevent binding to the receptor attached to the mammalian cell surface.  This has been attempted with influenza virus by making a nasal spray that contained sialic acid.  So far this has not worked, but it could be (in the future) a useful way to prevent infection, a way that does not require the weeks-long induction period required by vaccines.

How might an antiviral compound inhibit the uncoating step?  The budding step?

Influenza virus is a good example of both.  Influenza virus is taken up by endocytosis and ends up in a vesicle that starts to acidify.  This acidification process causes a change in the conformation of the matrix proteins that attach the envelope to the capsid, thus starting the uncoating process.  Presumably a change in conformation of the capsid proteins completes the process by removing the capsid.  Amantadine stabilizes the matrix proteins so that they do not change conformation and thus prevents the early stage of the uncoating process.  So any compound that prevented the conformational changes in matrix or capsid proteins that occur in acidified vesicles could prevent uncoating of viruses that enter the cell in this way.  Of course, this would not work for viruses that fuse directly with the mammalian cell membrane and do not enter

Newer drugs like Tamiflu bind to the influenza envelope protein neuraminidase and inhibit its enzymatic activity.  This activity is essential for the step in which the budding virus is released from the cell.  If the viral particle is not released from the mammalian cell it invaded, it cannot infect other cells.

via endocytic vesicles.

What types of proteins are found in a virus’ nucleoprotein core?

Structural proteins that stabilize the genome and enzymes like replicases or reverse transcriptase that are needed in the early stage of viral genome replication.

Why do some viruses have to have preformed enzymes to replicate their genome? Which type(s) of virus are most likely to need a preformed replicase in their nucleoprotein core?

Why do some viruses carry preformed replication proteins even if they would seem not to need them?

Probably the availability of the preformed replicase aids the virus in taking over the mammalian cell biosynthetic machinery more efficiently.

What are some examples of antiviral compounds that inhibit viral replication?

            Note that both of these examples are HIV specific and would not work with most other viruses.  They target viral activities that are different from mammalian cell activities and thus reduce toxicity to mammalian cells.  [FYI:   In the case of other viruses, for example herpes viruses, there are compounds that interfere with replication of the viral genome.  It is also a nucleotide analog but is different from AZT.]

How does reassortment differ from mutation in the case of influenza virus?  Why do you need a segmented genome to do this (reassortment)?

See figure in handouts.  Be sure to understand the reassortment process.

How could reassortment create a “killer” influenza virus?

Given that HIV is an RNA virus, what would you predict about its genetic variability?

RNA viruses are likely to be mutable than DNA viruses, although not all RNA viruses mutate as rapidly as influenza virus and HIV.

Why is a combination of compounds used to treat people with HIV infection?

Would a protease inhibitor work against influenza viruses?

. 

What is the significance of the mutant co-receptors for HIV that some people possess?

How does the ELISA detection system differ from the Western blot test for HIV or the RT-PCR (of PCR, for a DNA virus) type test?

Contains a copy of the bacterial genome

Contains contents fromt he cytoplasm of the mother cell

Is surrounded by a multilayer peptidoglican protein wall

Is more resistant to heat and drying than the mother cell