Ch 3: Bacterial Genetics

Question 1

Competent

Answer 1

the ability to bind free DNA to the cellular membrane and import it into the cell

Question 2

Transformation

Answer 2

• uptake of naked DNA from the environment by a competent cell
• captured DNA is incorporated by homologous recombination.
• some bacteria are capable of natural transformation:
• H. Influenza, Strep pneumoniae, bacillus species and neisseria
• the penicillin-binding protein mutations of strep pneumoniae occurs via transformation

Question 3

Transduction

Answer 3

• transfer of bacterial DNA by a phage vector
• phage picks up the bacterial DNA through an error in phage production
• doesn’t require cell-cell contact
• 2 types: generalized and specialized
• high host cell specificity of bacteriophage limits transduction to a transfer mechanism btw members of the same bacterial species.
• the staph aureus resistance to methicillin is chromosome mediated and transferred by transduction
• in pseudomonas, imipenem resistance is transferred from one member of the species to another during transduction by wild-type bacteriophage.

Question 4

plasmid

Answer 4

• extrachromosomal genetic elements
• circular, DS DNA
• protected from DNAase
• replicate autonomously in bacterial cells
• carry a variety of genes that are NONESSENTIAL for bacterial life
• including: fertility genes (tra operon) antibiotic resistance, bacterial exotoxins

Question 5

bacteriophage

Answer 5

• a virus that infects and replicates within a bacterium

- phage = bacteriophage = bacterial virus

Question 6

homologous recombination

Answer 6

• mechanism to incorporate short, linear pieces of DNA into chromosome
• must be some sequence homology
• recombinase A (recA) is required
• 1:1 exchange of DNA (aka DNA swap)

Question 7

site-specific recombination

Answer 7

• mechanism used to combine circular pieces of DNA (plasmids, temperate phage, transposons)
• doesn’t require homology
• no DNA lost; DNA added to existing circular genome
• requires restriction endonucleases
• integration of temperate phage DNA into a bacterial chromosome creates a prophage
• Both lysogeny and the formation of the Hfr chromosome require site-specific recombination of a small circle of extrachromosomal DNA to be incorporated into the chromosome of the cell.
• In lysogeny, the small circle of DNA is a viral genome; and in the Hfr chromosome, the small circle of DNA is a conjugative plasmid. Of the two, only the Hfr chromosome is used during conjugation. Homologous recombination is required following conjugation with an Hfr cell, but is not involved in the formation of the Hfr chromosome.

Question 8

prophage

Answer 8

• integration of temperate phage DNA into a bacterial chromosome creates a prophage
• temperate phage aka prophage

Question 9

temperate phage

Answer 9

• a phage that has both options (lytic replication or lysogeny)
• temperate refers to the ability of some bacteriophages to display a lysogenic (can integrate into chromosome) life cycle.
• Many (but not all) temperate phages can integrate their genomes into their host bacterium’s chromosome,
• together becoming a lysogen as the phage genome becomes a prophage.
• A temperate phage is also able to undergo a productive, typically lytic life cycle, where the prophage is expressed, replicates the phage genome, and produces phage progeny, which then leave the bacterium.

Question 10

conjugation

Answer 10

• gene transfer from one bacteria to another involving direct cell-cell contact
• fertility factors (in plasmid or episome) control conjugation
• a single strand of the double helix DNA is transferred from donor (male, bacteria that has fertility factor and f-pilus) to the recipient (female cell)
• chromosomal genes transferred in by conjugation have to be stabilized by homologous recombination (in a Hfr x F- cross)
• plasmid genes transferred by conjugation circularize and are stable without combination (in an F+ x F- cross) because an entire strand of the double-stranded DNA is transferred, the complementary strand is synthesized in the recipient, and the result is the acquisition of a perfectly stable circle of double-stranded plasmid DNA.
• conjugation w/ recombination may produce new genetic combos

Question 11

lysogenic conversion

Answer 11

• when bacteriophage DNA is inserted into the bacterial chromosome
• this enhanced virulence = lysogenic conversion
• When a bacterium acquires new pathogenicity via the incorporation of the genome of a temperate phage into its chromosome, it is called lysogenic conversion. In this way, normal flora diphtheroids in the oropharynx can be converted to the pathogen Corynebacterium diphtheriae, and the disease diphtheria can result.

Question 12

exogenotes

Answer 12

• short linear pieces of DNA

- can be taken up by cels in homologous recombination

Question 13

F factors

Answer 13

• fertility plasmids
• have a series of important plasmid “fertility” genes called the transfer or tra region; which codes for:
  1. sex pili
  2. genes whose products stabilize mating pairs
  3. genes that direct conjugal DNA transfer
• have OriT
• have insertion sequences where the plasmid can be inserted into the bacterial chromosome combining to make one larger molecule of DNA

Question 14

OriT

Answer 14

• origin of transfer
• region on fertility plasmid where a single strand break in the DNA will be made and where transfer of the single strand begins.
• the insertion sequence (where the plasmid can be inserted into the bacterial chromosome combining to make one larger molecule of DNA) is opposite of the origin, so that when the DNA is straightened out, the IS is in the middle.
• thus OriT will be transferred first and then the rest of the plasmid genes
• the last genes to be transferred = tra region

Question 15

F+ cell

Answer 15

donor cells where the fertility plasmid is in its free state

Question 16

F- cell

Answer 16

• recipient cell; lacks fertility factor

Question 17

Hfr

Answer 17

• donor cell where the fertility factor has been inserted into the bacterial chromosome
• this integrated plasmid = an episome

Question 18

Conjugal Cross

Answer 18

• in the F+ x F- cross, one strand of the entire plasmid is transferred and the F- cell becomes F+
• no bacterial genes are transferred

Question 19

Generalized Transduction

Answer 19

• error of lytic virus life cycle –> any gene can be transferred
• a generalized transducing phage is produced when the phage with a lytic (virulent) life cycle puts a piece of bacterial DNA into its head.
• All bacterial genes have an equal chance of being transduced
• transferred genes must be stabilized by homologous recombination

Question 20

specialized transduction

Answer 20

• error of temperate virus life cycle –> specific genes get transferred
• requires an error in excision
• only genes near the virus insertion site can be transferred
• any transferred genes must be stabilized by homologous recombination
• the resulting phage (virus) isn’t a functional virus because it doesn’t have its whole genome. It can still inject its DNA into a new bacterial cell, but it can’t replicate and make new viruses.
• specialized: occurs when an error is made in the life cycle of a temperate (lysogenic) phage.
• temperate phage introduce their genomic DNA into the bacterial chromosome at a specific site and then excise it later to complete their life cycle
• if errors are made during the excision process, then bacterial chromosomal DNA cab be carried along into the next generation of viruses

Question 21

virulent phage

Answer 21

infect bacterial cells, always making more virus and lysing the cells = lytic replication

Question 22

temperate phage

Answer 22

• often infect without lysing the cells because they have the ability to repress active phage replication and to stably integrate their DNA into the bacterial chromosome
• in the absence of functional repressor proteins, they may also replicate lytically

Question 23

lytic infection

Answer 23

• either by phage or viruses
• leads to production of viruses and their release by cell lysis
• virulent viruses can only go into lytic life cycles and can accidentally carry out generalized transduction

Question 24

lytic life cycle

Answer 24

1. phage adsorbs to specific bacterial cellular receptors and injects its DNA into bacterial cell
2. early transcription/translation of phage DNA to convert cell to a phage factory and to replicate phage DNA
3. following replication of DNA phage structural proteins are replicated at a higher rate
4. assembly of phage (morphogenesis) and release of phage by cell lysis (these phages are free to go on and each repeat this cell process)
   - if, during assembly, a phage incorporates a piece of bacterial DNA by mistake = a transducing phage.
   - called a generalized transducing phage bc every gene had an equal chance of being incorporated.
   - when this phage infects a new cell (#2), it will inject this DNA from cell #1 creating the opportunity for new combinations of genes (if homologous recombination produces allelic exchange).

Question 25

lysogeny

Answer 25

• lysogeny = a state of a bacterial cell with a stable phage DNA (generally integrated into the bacterial DNA), to undergoing lytic replication either because it is repressed or defective
• when the cell DNA replicates, the phage DNA also replicates, and as long as the repressor protein is not damaged, the lysogenic state continues ad infinitum
• defective phage (or defective viruses in the human equivalent) can’t go into active replication unless a helper virus is present

Question 26

phage mediated pathogenic factors

Answer 26

• COBEDS
• C: cholera toxin
• O: presence of specific prophage in salmonella can affect “O” antigens
• B: phage CE β or DE β cause clostridium botulinum to produce Botulinum toxin
• E: Exotoxins A-C (erythrogenic or pyogenic) or strep pyogenes
• D: prophage β causes corynebacterium diphtheria to make Diphtheria toxin
• S: Shiga toxin
• cobeds: when 2 people share a bed, someone gets pregnant (with phage)
• lysogenic conversion

Question 27

generalized vs specialized transduction mechanism

Answer 27

• Generalized transduction = error in assembly during lytic life cycle
• Specialized transduction = error of excision; requires previous stable insertion of prophage DNA into bacterial genome (lysogeny)

Question 28

bacteria that lack mycelia acids are intrinsically resistant to

Answer 28

isoniazid

Question 29

bacteria that lack peptidoglycan (i.e. mycoplasma) are intrinsically resistant to

Answer 29

penicillin

Question 30

chromosome mediated antibiotic resistance

Answer 30

• genes that determine resistance are located on the bacterial chromosome
• MC these genes modify the RECEPTOR for a drug, so the drug can no longer bind ex) mutation in a gene for a penicillin binding protein
• in general, causes low-level drug resistance rather than high, but even low-level resistance can be significant (i.e. strep pneumo in meningitis)

Question 31

plasmid mediated drug resistance

Answer 31

• genes often code for enzymes that modify the drug
• transposition has produced multiple drug-resistance plasmids
• R factors are conjugative plasmids carrying genes for drug resistance
• one section of the DNA (containing OriT and tra gene region) mediates conjugation
• other section (R determinant) carries genes for drug resistance. Multiple genes seem to have been inserted through transpositional insertion into a hot spot.

Question 32

transposable elements

Answer 32

• transposons are mobile and can move themselves or a copy from one molecule of DNA to another (jumping genes)
• found in eukaryotic, bacterial and virus cells
• have at least one gene for transposase (enzyme involved in the movement)
• create additional mutations w/ their insertion into another totally unrelated gene
• have indirect repeats on each end
• create direct repeats where they land (flank indirect repeat with direct repeats)
• multiple transposons can insert into the same piece of DNA and create multiple drug-resistant plasmids

Question 33

physical linkage of there fertility factor to the bacterial chromosome promotes transfer of bacterial genes in a linear order. w/ _____________ transferred w/ highest frequency

Answer 33

• bacterial genes closest to the insertion site are transferred with the highest frequency
• frequency of transfer is proportional to distance from the insertion site

Question 34

R factor

Answer 34

• R factors are multiple drug-resistance plasmids that are capable of mediating conjugation.
• When they are transferred, the bacterium receiving the plasmid receives multiple drug resistances in a single conjugative event.

Question 35

What is the most rapid and effective means of transfer of drug resistance in gram-negative bacteria?

Answer 35

• Answer = Conjugation using an R factor (multiple drug-resistance plasmids that are capable of mediating conjugation)
• In the Hfr conjugal transfer, a few chromosomal genes may be transferred to the recipient.
• In generalized and specialized transduction, a few chromosomal genes may be transferred using a virus vector.
• In transformation, chromosomal genes from dead cells may be acquired following homologous recombination.

Question 36

F+ + F- mating

Answer 36

• In the F+ to F– conjugal cross, the F+ donor cell donates one strand of the entire plasmid across the conjugal bridge to the F– partner.
• The second strand of plasmid DNA in each partner is then synthesized, so at the end of the process, two F+ cells exist with identical fertility factor plasmids.
• Doesn’t need to undergo stabilization through homologous recombination because an entire strand of the double-stranded DNA is transferred, the complementary strand is synthesized in the recipient, and the result is the acquisition of a perfectly stable circle of double-stranded plasmid DNA.

Question 37

In which DNA transfer mechanisms can the statistical probability of gene transfer and stabilization be used as a measure of genetic distance?

Answer 37

• In Hfr conjugation and specialized transduction, the success with which a particular genetic trait is transferred and stabilized in the recipient cell is directly proportional to the genetic distance between that gene and a particular locus on the chromosome.
• In the case of the Hfr transfer, the proximity to oriT is the determining factor.
• In the case of specialized transduction, it is the proximity to the insertion point of the viral genome that determines the likelihood of transfer.

Question 38

Accumulation of drug resistance genes (DRGs) into multiple drug resistance plasmids can be detected by

Answer 38

• identification of characteristic flanking sequences (direct and indirect)

Question 39

Drug resistance in neisseria gonorrhoeae

Answer 39

• 2 plasmids are required to transfer drug resistance
• drug resistance genes are located on non conjugative plasmids (plasmids that have lost their tra operon but retained oriT)
• these plasmids may be transferred by conjugation as long as there is another fertility factor in the same cell w/ a functional tra operon.
• this process is called mobilization

Question 40

drug resistance via transduction

Answer 40

• high host cell specificity of bacteriophage limits transduction to a transfer mechanism btw members of the same bacterial species.
• the staph aureus resistance to methicillin is chromosome mediated and transferred by transduction (most of the other antibiotic resistance in staph aureus is transferred by plasmids)
• in pseudomonas, imipenem resistance is transferred from one member of the species to another during transduction by wild-type bacteriophage.

Question 41

drug resistance via transformation

Answer 41

the spread of penicillin-binding protein mutations in strep pneumoniae occurs via transformation

Question 42

drug resistance in Staph A.

Answer 42

• resistance to methicillin is chromosome mediated and transferred by transduction (via bacteriophage)
• most of the other antibiotic resistance in staph aureus is transferred by plasmids
• acquired vancomycin resistance (vanA and vanB) from E. faecalis via transposon on a multi-drug resistant conjugative plasmid.
• in s. aureus, the transposon moved from the e. faecalis plasmid to a multi-drug resistant plasmid in s. aureus.
• The new s. aureus super multi-drug resistant plasmid now contains resistance genes to methicillin, β-lactams, vancomycin, aminoglycosides, trimethoprim and some disinfectants and can be transferred to other strains via conjugation

Question 43

What treatment would be expected to destroy bacterial spores?

Answer 43

• Autoclave at 121 C, 15 lb pressure for 20 minutes
• Autoclaving at the parameters listed is a sterilizing technique. All viable living organisms are killed by such a treatment.
• dry heat: 2h 180C
• UV radiation (formation of thymine-thymine pairs on adjacent DNA bases)

Question 44

sterilization vs disinfection

Answer 44

• sterilization = complete removal or killing of all viable organisms
• disinfection = removal or killing of disease-causing organisms. Compounds for use on skin = antiseptics

Question 45

minimal inhibitory concentration = MIC

Answer 45

lowest concentration of drug showing no visible growth

Question 46

minimal bactericidal concentration = MBC

Answer 46

• lowest antibiotic concentration showing no growth on subculture to media without the antibiotic
• MBC will be = to or > than the MIC
• use when you have an immunocompromised patient

Question 47

Hfr x F- cross

Answer 47

• chromosomal genes closest to OriT are transferred
• the last genes to be transferred would be the tra region
• transferred genes must be stabilized by homologous recombination
• the bridge does not remain long enough to transfer the tra operon (complete transfer takes ~2h); recipient remains F-

Question 48

if something has a complex carbohydrate cell wall it is a

Answer 48

• fungus
• bacteria have peptidoglycan cell walls
• parasites don’t have cell walls
• prions are infectious protein
• viruses are acellular

Question 49

if something causes a granuloma, it’s most likely causal agent is

Answer 49

• the fact that is intracellular

Question 50

what do gram - bacteria have that gram + bacteria lack?

Answer 50

outer membrane

Question 51

what genetic material is found in pathogenic corynebacterium diphtheria but not in nonpathogenic normal flora diphtheroids?

Answer 51

an integrated temperate phage

Question 52

how is a prophage created?

Answer 52

through site-specific recombination of a temperate phage and bacterial DNA

Question 53

Assuming the following cells have no plasmids other than those mentioned, which cell type would contain 2 molecules of DNA: F+, F-, Hfr

Answer 53

• F+ would contain the bacterial chromosome and fertility factor

Question 54

the process by which bacterial or plasmid DNA may be mistakenly incorporated (during assembly) into one phage being produced by the lytic life cycle and then that DNA-transferred to another bacterial cell which may acquire some new genetic traits is called?

Answer 54

• generalized transduction
• lytic viruses are only capable of generalized transduction
• for specialized transduction only episomal plasmid DNA (incorporated into the bacterial chromosome near an attachment site) or chromosomal DNA could be picked up

Question 55

lysogenic conversion

Answer 55

• is a change in pathogenicity due to the presence of a prophage
• usually this means increased pathogenicity

Question 56

lysogeny refers to

Answer 56

the incorporation of a prophage into a chromosome