36. Bacterial Genetics

Question 1

key genetic differences making bacteria unique?

Answer 1

haploid

no nucleus

no introns

30S (INCL 16S rRNA) + 50S ribosomes

Question 2

the presence of 16S rRNA sequences are used for what?

Answer 2

to identify and quantify individual bacterial species from a complex mixture of human cells and microbial cells (ie from stool sample/microbiome)

Question 3

conserved regions vs hypervariable regions of 16s rRNA?

Answer 3

conserved: used to design universal PCR primers for amplification of the 16s gene across all bacterial species
hypervariable: used for species specific identification w/in 16S rRNA genome

Question 4

which can replicate independently from the chromosome: plasmids or transposons?

Answer 4

plasmids - have own replication genes but are sometimes integrated into chromosome

transposons cannot replicate independently (must be inserted into another replicating element like the chromosome, plasmid, or bacteriophage)

Question 5

the bacteriophage and pathogenicity island of bacteria are on the chromosome or plasmid?

Answer 5

chromosome

Question 6

what enzyme lets transposons jump b/w plasmids and genes?

Answer 6

transposase

Question 7

transposons (Tn) are simple elements that contain genes that allow them to transpose…along with what other elements?

Answer 7

one or more genes encoding a phenotype - mostly antibiotic resistance like B-lactamase

Question 8

besides conferring a new phenotype like abx resistance, insertion of a Tn (transposon) can do what?

Answer 8

inactivate genes or can mediate other rearrangements (duplication, deletion, inversion)

Question 9

Tns are flanked on either side by what?

Answer 9

inverted repeats ( what causes the hairpin loop often seen on EM)

Question 10

what are insertion sequences (IS)?

Answer 10

essentially transposons without an obvious phenotype like abx resistance - can mediate rearrangements or inactivate target genes. Have transposase (tnp) genes plus inverted DNA sequences at ends

Question 11

IS elements can form part of more complex transposons called what?

Answer 11

composite transposons (can lock phenotypic gene like abx resistance or toxin in the middle)

Question 12

what are bacteriophages?

Answer 12

viruses that infect bacteria, not mammalian cells - there are 2 lifestyles for bacteriophages:

1. lytic phages can lyse and kill cells (virulent phage)
2. lysogenic phage (temperate phage or prophage) do not immediately lyse cells but can insert their DNA into the chromosome and replicate along with the cell. Many toxins can be encoded on phage genome and so if the phage DNA is incorporated into the chromosome, the bacteria can express the toxin

Question 13

lytic phages attach to the outside of the cell and inject their genome into the bacterial cell. New copies of the phage genome and hte phage proteins are produced inside the bacterial cell and assemble into complete phage particles. The cell is then lysed and the phage can now infect other cells….why is this clinically relevant?

Answer 13

therapies to kill abx-resistant bacteria

Question 14

life cycle of a temperate bacteriophage?

Answer 14

phage DNA is integrated into the bacterial chromosome and the lytic cycle is suppressed. Bacterial cell can grow and express the genes encoded on the phage (sometimes toxins) and the viral DNA is replicated along with the chromosomal DNA. At some point, a signal (eg UV light, heat) can induce the lysogenic phage to become lytic and the lytic cycle results

Question 15

what are pathogenicity islands?

Answer 15

large blocks of DNA containing multiple virulence genes (located on the region of house-keeping genes present in non-pathogens (house-keeping genes encode normal growth and metab of non-pathogenic and pathogenic bacteria)

often have different DNA composition from the rest of the genome, suggesting that the entire block was via horizontal gene transfer

eg. LEE pathogenicity island of E.coli O157:H7 can lead to HUS

Question 16

what are the 3 major ways in which bacteria can exchange genetic info?

Answer 16

transformation, conjugation, and transduction

Question 17

transformation?

Answer 17

the process whereby naked DNA from one cell is taken up by another cell through the cell wall without ant participation of other bacteria or virus….

the Griffith experiment: pathogenic strep pneumoniae (pneumococcus) have a smooth polysacch. capsule on their surface, which acts as an antiphagocytic factor

1. life smooth pneumococci injected into mice causes death and smooth pneumococci are recovered from dead mice
2. head killed smooth pneumococci don’t kill mice
3. rough pneumococci do not kill mice
4. live rough (do not kill mice on their own) and killed smooth (do not kill mice on their own) are injected together into mice, death results and smooth, live cells are recovered

Question 18

conjugation?

Answer 18

occurs via cell-to-cell contact (sex)

mediated by a pilus encoded on a plasmid

explanation of discovery and how it works:
model conjugation system was orig described in E.coli and is encoded in a plasmid called “F” (fertility factor). Strain with the F plasmid = F+ and can serve as a donor. A strain w/o the plasmid =F- and can serve as the recipient.

ssDNA from the plasmid enters into the recipient cell where the complementary strand is synth to form a complete ds circle.

complementary strand in the donor cells is also synth so that both cells have complete copies of the plasmid

Question 19

the F plasmid is a special kind of plasmid called what? (ie how does it integrate into the chromosome)

Answer 19

an episome that can integrate into the chromosome (via IS elements on teh plasmid that recombine w/identical IS elements on the chromosome)

Question 20

when the F plasmid is integrated into the chromosome, it is called what?

Answer 20

an HFR (high frequency recombination) because it can transfer adjacent chromosomal genes into a recipient strain, not just plasmid sequences

Question 21

transduction?

Answer 21

gene transfer involving phage, which can pick up genes from the host cell and incorporate them into the progeny phage.

the phage w/foreign DNA can attach to a recipient cell and inject the phage DNA along with the foreign DNA into the recipient cell.

Question 22

why do bacteria have to change the expression of their own genes?

Answer 22

to rapidly adapt to new environments

Question 23

bacteria have multiple genes physically linked that are co-regulated in what?

Answer 23

an operon

Question 24

multiple operons can be linked together in a regulatory network (not necessarily physically linked) in a _____.

Answer 24

regulon

Question 25

the most important means of bacterial gene regulation occurs is by what?

Answer 25

the initiation of txn, w/activator or repressor proteins increasing or decreasing the levels of txn

Question 26

describe the two-component regulatory system?

Answer 26

utilizes a membrane protein called a sensor and a cytoplasmic regulatory protein.

sensor proteins sense changes in the env’t (salt, pH, ions, etc) and transmits a signal to the regulator protein which binds DNA and modulates txn

mechanism of signal transduction is PHOSPHORYLATION

eg global regulation of multiple virulence factors in bordetella pertussis (pertussis toxin) by a positive effector (Bvg) whose activity is regulated in response to environmental conditions

Question 27

quorum sensing?

Answer 27

a mechanism of cell-cell communication whereby small molecs produced b bacterial cells can be sensed by other bacteria in a population resulting in modulation of gene expression. Bacteria produce small molecs (autoinducers). As the density of a bacterial population increases, the density of the autoinducer increases. These small molecs can be taken up by other cells and activate gene expression.

So bacteria express a different set of genes at low density than at high density. Allows a unicellular org to act as multicellular. Many of the genes regulated in this way are virulence factors.

eg pseudomonas aeruginosa has genes encoding toxins and biofilm formation that are regulated by quorum sensing. At low initial inoculum, these genes are not expressed (saving energy and preventing early immune response to these factors). As the cell density increases, these genes are expressed so factors inportant in later stages of infection are expressed

Question 28

clinical implications of quorum sensing?

Answer 28

antimicrobial therapy - prevent quorum sensing signaling to prevent or decrease virulence gene expression - don’t need to kill org w/antibiotic, but decrease # and virulence until the body’s immune system can clear it

Question 29

E.coli and quorum sensing?

Answer 29

only a few cells are required for infection, so the pathogen is not talking to its siblings, but it can detect autoinducer molecs produced by commensal (resident) intestinal E.coli flora, which then serves as signal to activate gene txn of virulence factors in the O157 strain

Question 30

Diptheria toxin regulation by diptheria tox repressor (DtxR)?

Answer 30

host tissues are low iron env’t thanks to transferrin or lactoferrin that bind iron and make it unavailable to bacteria. A repressor of diptheria toxin (DtxR) is functional in high iron conditios (ie non-host conditions), thereby repressing expression of toxin. In low iron (host) conditions, the repressor becomes inactive (apo-DtxR), thereby allowing expression of toxin

Question 31

phase variation vs antigenic variation?

Answer 31

phase: reversible switch b/w off and on expression phases (gene inversion, slipped strand mispairing)
antigenic: expression of antigenically distinct but functionally conserved moieties within a population (homologous recombination from silent gene copies or programmed rearrangement)

phase variation can lead to antigenic variation in some cases

Question 32

Inversion?

Answer 32

can lead to phase variation - switching back and forth b/w “off” and “on” expression.

Salmonella has two different flagellar antigens and only one is expressed at a time. THe transition takes place at a regular frequency. Switch b/w one phase and the other is controlled by an invertible sequence of DNA. Eg: In on phase the promoter promotes one phase and on the same gene is a repressor for the other phase. In the other phase, the segment is inverted so that the promoter reads in the opposite direction, so that the first phase is not transcribed (and neither is the repressor)

Question 33

slipped strand mispairing (SSM)?

Answer 33

mechanism of phase variation that acts at the level of translation, not txn.

Tandemly repeated nucleotides, 1-2 or 4-5 nucleotides in length (not a multiple of 3) that can be deleted during DNA replication, resulting in frame shifts and premature stop codons.

addition or deletion of one base can lead to a premature stop codon and non-production of the Byg regulatory element in pertussus.

Question 34

SSM in Neisseria Opa proteins?

Answer 34

the Opa genes have multiple repeats of a 5-nucleotide sequence of CTCTT. Deletion or amplification of the number of repeats can lead to premature stop codon (because not a multiple of 3)

Question 35

what is the most important mechanism of antigenic variation?

Answer 35

involves movement of genes from an inactive expression site (eg a site lacking a promoter) into an active expression site. Expression of these previously silent genes leads to production of novel proteins/antigens = gene conversion or programmed rearrangement

eg Neisseria gonorrhoeae pili - the ability to change the surface antigen allows the bug to escape the immune system - also acts via HOMOLOGOUS RECOMBINATION

Question 36

what 2 things contribute to the antigenic variation of Neisseria Opa proteins?

Answer 36

phase variation and gene conversion (homologous recombination)

Question 37

antigenic variation in Borrelia?

Answer 37

multiple silent copies of a major surface protein (VMP) are stored on plasmids. Recombination of these silent genes into the expression site in the plasmid gives rise to antigenic variation - causes relapsing fever in humans

Question 38

some abx resistance can arise by single chromosomal nucleotide change s- like changes in the DNA gyrase gene leading to nalidixic acid resistance. But, the majority of resistances arise from what?

Answer 38

acquisition of new genes

Question 39

what is an integron?

Answer 39

genetic element that incorporates resistance genes that lack a promoter next to an integrase gene with a strong outwardly reading promoter that transcribes the resistance gene.

Question 40

mechanisms of abx resistance?

Answer 40

1. inactivate antibiotic: eg PCN “destroy it”
2. block antibiotic transfer “avoid it” - altered permability or activate efflux like tetracycline
3. substitute pathway - eg vancomycin resistance changes to peptidoglycan from D-ala-D-ala to D-ala-D-lac
4. alter target site - eg erythromycin resistance modifies ribosome; rifampin resistnace selects for an altered RNA polymerase (“live w/it”)

Question 41

in GNs, the cheif means of transfer b/w bacteria is what?

Answer 41

conjugative plasmids

Question 42

analysis of plasmid profiles, restriction fragment length polymorphisms (RFLP), or sequence analysis of indiv genes, can show what?

Answer 42

if bacterial isolates are related and linked to a common source