4_Bacteriology IV

Question 1

what characteristics of bacteria help it make many mutations passed along during division?

Answer 1

• Bacteria are haploid –> so any mutations are passed to daughter cells upon division
• Single copy of chromosome inherited
• Effects of genetic change are not masked

Question 2

describe how DNA replication errors occur

Answer 2

• DNA polymerase can misincorporate nucleotides
• DNA polymerase normally has proofreading activity and can repair the mismatch
• occasionally mistakes are not repaired and SNP single nucleotide polymorphisms arise

Question 3

what are the types of DNA damaging agents that cause lesions in DNA?

Answer 3

• Base analogs
  
  • 5-bromouracil (Bu)
  • 2-aminopurine
• Intercalating agents
  
  • acriflavine
  • ethidium bromide
  • acrydine orange
• Ultra-violet (sunlight)
  
  • pyrimidine dimers
• Chemicals that react with DNA
• X-rays (–> free radicals –> damage to DNA)

Question 4

what induces DNA repair mechanisms?

Answer 4

• the SOS (a global response to DNA damage in which the cell cycle is arrested and DNA repair and mutagenesis is induced. It is an error-prone repair system that contributes significantly to DNA changes observed in a wide range of species.)
• these are similar to those in humans, adn repair the DNA damage

Question 5

what induces error-prone polymerases?

Answer 5

under conditions of starvation or extreme stress (lots of DNA damage)

Question 6

what polymerases read past lesions;

what are the results of this?

Answer 6

• Error prone polymerases such as PolV and PolIV read past the lesions and do not proofread
• This causes misincorporation of nucleotides.
  
  • These polymerases do not have proofreading capabilities so the mutations stay.
• responsible for increased mutation rates under stress
  
  • oxidation causes DNA damage and
  • starvation causes stalls in DNA replication

Question 7

how do the Pol V and Pol IV polumerases differ?

Answer 7

• Pol V replicates past pyrimidine dimers caused by UV light
• Pol IV replicates past various other lesions in DNA

Question 8

bacteria exist as populations:

define microbiota and disease

Answer 8

• microbiota: mixed species populations
• disease: single species populations; exist as pathogens
  
  • error prone polymerases allow cells in the population to vary in hopes that the mutations may be beneficial

Question 9

error prone polymerases help give rise to antibiotic resistant mutants.

how can we try to prevent pathogens from becoming completely resistant to antibiotics?

Answer 9

• The use of multiple antibiotics to treat infections by pathogens that use error prone polymerases, reduces the chance that these pathogens will become completely resistant to antibiotics.
• For example if 1/10^-7 bacteria become resistant to a single antibiotic the risk of resistant infection is 10⁷.
  
  • When antibiotics are combined the chance that a bacterium will become resistant to both antibiotics becomes 10¹⁴.

Question 10

horizontal gene transfer (HGT):

define, and what are the major mechanisms

Answer 10

• horizontal gene transfer of resistance genes is an efficient way of generating antibiotic resistant bacteria.
• major mechanisms of HGT:
  
  • transformation
  • conjucation
  • transduction

Question 11

what are the major mechanisms of horizontal gene transfer,

and define each

Answer 11

• transformation: trasnfer of free DNA
• conjugation: plasmid transfer
• transduction: transfer of viral delivery

Question 12

which types of bacteria can take up DNA?

Answer 12

• Only competent bacteria can take up naked DNA.
  
  • Transformation is DNase sensitive because the DNA is free outside the cell and can be degraded by the DNase enzyme.
• Gram-positive bacteria take up any DNA
• Gram-negative bacteria can only take up OWN DNA

Question 13

how is DNA integrated in transformation?

Answer 13

homologous recombination

Question 14

what is needed for an incoming gene to be integrated into the chromosome by homologous recombination?

Answer 14

• requires a stretch of homology between the incoming DNA and the chromosomal DNA
• RecA protein
• heteroduplex formation
• replication to resolve the heteroduplex

Question 15

how can antibiotic resistance genes be integrated into the chromosome?

Answer 15

• directly if there is enough homology or
• be introduced into the DNA if flanking on both sides by genes containing homology

Question 16

what is a mechanism that allows for development of supragenome?

define the supragenome?

Answer 16

• Competence is one mechanism that allows for the development of a pan genome (supragenome)
• Bacteria with a supragenome have:
  • conserved core of genes present in all strains
  • genes from a pool of distributed genes that differ between strains

Question 17

bacterial plasmids:

characteristics

Answer 17

• Covalently closed, double stranded circular DNA
• Usually not essential for bacterial growth
• Replicate separately from the chromosome

Question 18

three types of bacterial plasmids

Answer 18

• Narrow host range (present in limited number of bacterial species)
• Broad host range plasmids replicate in many species
• Conjugative plasmids can transfer

Question 19

describe the transfer of plasmids by conjucation prototype F-plasmid E.Coli

Answer 19

1. The F+ cell is the donor and contains the F plasmid.
2. The F- cell is the recipient cell and does not contain the plasmid.
3. The F+ donor synthesizes a pilus, which is a Type IV secretion system.
4. One strand of the DNA is transferred to recipient.
5. The strands in both cells are replicated so both the donor and recipient now have the plasmid.
6. The recipient has been converted to a donor, so the process is infectious (epidemic) and the plasmid is spread. through the bacterial population

Question 20

can an F-plasmid integrate into the chromosome?

what does it form?

Answer 20

• F-plasmid can integrate into the chromosome
• Forms an Hfr

Question 21

what are the 3 outcomes of an Hfr?

Answer 21

1. F+ :The F plasmid excises precisely and nothing happens to the plasmid or chromosome.
2. F’ :The F plasmid excises and carries of a small piece of chromosomal DNA with it. The F’ plasmid is now infectious and the chromosomal fragment basically becomes part of the plasmid.
3. Hfr mating : The pilus and Type IV secretion system are induced and a transfer event is initiated from the integrated plasmid so a large piece of chromosome is introduced into the recipient cells. The plasmid does not recircularize and is lost (NOT infectious) but genes from the donor chromosome can enter the recipient by homologous cross over events.

Question 22

how can plasmids be detected on agarose gels?

Answer 22

• Plasmids can be visualized from a DNA preparation as distinct bands on an agarose gel.
• Plasmids can be larger or smaller than the chromosomal fragments present on the gel (the chromosome shears during DNA isolation).

Question 23

between what does gene transfer events occur?

Answer 23

can occur between bacteria of different species and genera

Question 24

bacteriophages:

define, and characteristics

Answer 24

• bacterial viruses
• can switch between lytic and lysogenic phases

Question 25

lytic cycle of bacteriophages

Answer 25

• In the lytic cycle, bacterial chromosomal DNA is often degraded into small pieces.
• The viruses replicate their nucleic acid, package the DNA and lyse the bacteria

Question 26

lysogenic cycle of bacteriophage

Answer 26

• In the lysogenic cycle, the phage integrate into the chromosome by non-homologous self-directed recombination.
• The viral genetic material replicates as part of the chromosome.

Question 27

how can a bacteriophage in the lytic cycle mediate HGT?

Answer 27

• by generalized transduction:
  
  • when the bacteriophage packages a fragment of chromosomal DNA instead of viral DNA.
  • the fragment is injected into a new bacterial cell.
  • the chromosomal fragment can be incorporated into the bacterial chromosome by homologous recombination.
  • the phage does not contain viral DNA, so the bacterial cells is not infected by the virus

Question 28

how can a bacteriophage in the lysogenic cycle mediate HGT?

Answer 28

• by specialized transduction
  
  • when a lysogenic phages converts to the lytic cycle it can occasional take a piece of chromosomal DNA adjacent to the integration site with it.
  • the piece of chromosomal DNA is carried with the phage DNA and is introduced into the bacterial chromosome when the phage reintegrates upon entering the lysogenic cycle
  • the integration is mediate by the phage and is non-homologous recombination

Question 29

transposon:

define, and how is it mediated?

Answer 29

• A transposon mobile element transposes (moves) from one piece of DNA to another.
• The movement is mediated by the action of a transposase enzyme that inserts the transposon into the DNA via binding to the inverted repeats that flank the transposon (insertion sequences; IS).

Question 30

non-homologous recombination:

define

Answer 30

• Since the insertion sequence and transposase direct the insertion a transposon can insert anywhere in the chromosome and does NOT require RecA or homology
• Insertion and excision are usually precise, but excision can occasionally cause deletions.

Question 31

why are transposons not considered replicons?

Answer 31

• Transposons become part of the DNA, and therefore are not replicons
• Transposons are never free like phages or plasmids

Question 32

transposons:

functions

Answer 32

• Transposons can carry multiple different genes between the insertion sequences.

Question 33

conjugative transposons:

define and functions

Answer 33

• Conjugative transposons are also known as ICE (integrative and conjugative elements)
• Fxn: encode genes encoding their conjugative transfer to other bacteria.

Question 34

MRSA SCCmec:

define

Answer 34

• is a large chromosomal cassette containing the methicillin resistance genes and can contain many other resistance and virulence factors
• this is a novel type of transposon-like mobile element whose mechanism of movement is unknown.
  • the different cassettes are variable in genetic content.
  • the MRSA SCCmec are often flanked by insertion sequences, transposons and integrated plasmids in them

Question 35

pathogenicity islands:

define

Answer 35

• large mobile genetic elements encoding virulence factors
• present in pathogenic strains and absent in commensal strains of the same species
• related to transposons sometimes, but not always, have Insertion Sequence elements and integrase
• tend to be found in tRNA genes

Question 36

what can also result from antibiotic resistance?

Answer 36

• often causes a loss in fitness like slower growth rate or reduced survival in stationary phase
• In the absence of antibiotic selection, antibiotic resistant strains are often outcompeted and lost from the bacterial population

Question 37

where is antibiotic resistance more common?

Answer 37

• Antibiotic resistant strains are more common in hospitals where there is more antibiotic selection and less common in the community where there is less antibiotic selection.