bacterial genetics I and II Flashcards
how many copies of genes exist in the bacterial genome?
only one - bacterial genomes are haploid
what is the structure of the bacterial genome?
single, double-stranded circular DNA molecule
what does progeny DNA consist of?
semiconservative replication = progeny molecules are a parental strand and a nascent strand
what is the directionality of bacterial DNA replication? where does it start?
starts at origin of replication
bidirectional from there
what is required for initiation of DNA synthesis?
bacterial origin recognition protein that binds to the origin
what is the genotype?
exact nucleotide sequence of the genome
what is a mutation?
change in genotype - any change in the base sequence of DNA
what does it mean for something to be wild type?
designation of the genotype of an organisms found in the wild
what is phenotype?
observable characteristics of an organism
what is a silent mutation?
mutation that does not give rise to a change in phenotype
why are some mutations silent?
1: genetic code is redundant (ie several 3-letter combinations can code for the same AA)
2: conservative changes in AA may not affect function (so if you change, for example, a valine to an alanine, there might not be a change in function since both are nonpolar)
what are point mutations? what are the types of point mutation (list)?
single base changes
1: transitions
2: transversions
3: missense
4: nonsense
what is a transition?
type of point mutation
purine replaced by a pruine (A to G) or pyrimidine replaced by pyrimidine (C to T)
what is a transversion?
type of point mutation
purine replaced by pyrimidine or vice versa
what is a missense mutation?
type of point mutation
when the mutation results in a change of the AA encoded by a codon
what is a nonsense mutation?
type of point mutation
when the mutation changes the codon to a stop codon => shortened protein
what is a deletion (in terms of mutation)?
removal of one or more nucleotides
what is an insertion (in terms of mutation)?
addition of one or more nucleotides
what is a frameshift?
shift in reading frame caused by the insertion or deletion of nucleotides
what is a revertant mutation? what is the difference between a true revertant and a suppressor?
one that restores a wild type phenotype
true revertants are a reversal of the original mutation, whereas suppressors are mutations that occur at a second site and restore wild type phenotype
how could you directly select for mutants?
grow bacteria on solid agar containing ampicillin
bacteria should die, but if mutated for resistance to ampicillin, will survive
what is auxotrophy?
inability to synthesize an essential metabolite such as an AA
what is an auxotroph? what is a prototroph?
auxotrophs = mutants that lack the ability to synthesize an essential metabolite prototroph = wild type of that
how would you select for auxotrophs?
an auxotroph will only grow on a medium that contains the molecule that they canât make whereas the prototroph will be able to grow on minimal medium (just a nitrogen and phosphate source)
what are the basic mechanisms by which antibiotic resistant strains arise?
1: mutation to antibiotic resistance
2: transfer of antibiotic resistance genes
what are conditional mutations?
mutations that exhibit a mutant phenotype only under certain conditions - eg temp sensitive
what is a temperature sensitive mutation? why does this occur?
type of conditional mutation
some are silent at one temperature (the permissive temperature) but not at the non-permissive temperature
because mutation in protein allows it to assume a normal conformation at the permissive temp but forces it to assume an abnormal conformation at the nonpermissive temp
what is genetic recombination?
process by which two genetic elements combine to form one
what are the three types of genetic recombination? (list)
1: general (homologous) recombination
2: site-specific
3: illegitimate recombination
what is general recombination?
type of genetic recombination
requires extensive DNA homology
what is site-specific recombination?
type of genetic recombination
requires a small region of homology
what is illegitimate recombination?
type of genetic recombination
requires no homology
occurs at a very low freqency
what is small subunit (SSU) rRNA analysis used for?
to quantify bacteria in mixed populations
because SSU rRNA is highly conserved but has enough variability to have a unique sequence for most species
what is the benefit of whole genome sequencing?
reveals pathogenesis related genes and metabolic tendencies and potential weaknesses
permits parallel study and deeper understanding of each
only way to identify obligate intracellular and difficult to culture pathogens
what is a bacteriophage? what do they consist of?
bacterial virus
nucleic acid genome (DNA or RNA) surrounded by a protein shell/capsid
what are bacteriophages used for?
as model systems for research on molecular biology and genetics of virus reproduction
what is the nucleocapsid structure of a bacteriophage?
some with either icosahedral or helical symmetry
others have complex structure = head, tail and tail fibers
what determines the species of bacteria that a bacteriophage infects?
viral coat of proteins of the phage
how would you grow bacteriophages?
infect an actively growing bacterial culture
reproduce and usually cause host cell lysis - release progeny
centrifuge culture to remove lysed cells
bacteriophage in supernatant
how would you quantify bacteriophages?
by plaque assay
phage placed on lawn of sensitive bacteria
measure the amount of death they create
what will a plaque assay tell you about bacteriophages?
quantification
species have different morphology
each plaque has several million phage
what is the lytic pathway?
involves phage multiplication and release of newly formed phage following host cell lysis
what life cycle do virulent phage follow?
the lytic pathway only
what is the lysogenic pathway?
does not result in production of progeny phage or bacterial killing
what life cycle do temperate phage follow?
either the lytic or lysogenic pathway
what are the steps in the life cycle of a virulent DNA phage?
1: adsorption
2: introduction of DNA - nucleic acid goes in, protein coat stays out
3: transcription of phage DNA and inhibition of host transcription
4: replication of phage DNA
5: synthesis of phage capsid proteins
6: morphogenesis and packaging of the phage genomes
7: lysis and release
what occurs during the adsorption step in phage life cycle?
binding of phage coat protein to cell membrane receptors
what is the one-step growth curve? what does it tell us about the phage reproductive cycle?
kinetic analysis of phage reproduction
indicates that phage do not reproduce by binary fission
phage nucleic acid enters cell => phage factory
viral components synthesized and then assembled into mature phage particles late in infection
what are the characteristic of virulent phage?
- plaques they make - infect cells? - affect on host?
produce clear plaques
infect cells, lyse them, and then infect more cells
what are the characteristics of temperate phage?
- plaques - types of infection
turbid plaques
can pick either virulent or lysogenic infection
what is a virulent infection?
lytic infection
occurs in most cell populations
virulent phage always do this
temperate phage can
what is a lysogenic infection?
one that does not result in the production of progeny phage and bacterial lysis
instead, phage DNA becomes integrated into host chromosome
phage genome then propagated as a prophage when the bacteria divide
what is bacterophage lambda?
well-characterized temperate phage
once injected, makes choice to undergo either lytic or lysogenic infection
what happens if a phage lambda undergoes lysogenic infection?
chromosome circularizes and integrates at specific sites in the bacterial chromosome
what is site specific recombination?
phage chromosome has similar sites and recombination between bacterial and phage chromosomes
leads to integration (lysosomy)
what is a prophage?
integrated phagic DNA
what does the prophage do to ensure its continuation?
expresses a repressor of expression of all the genes required for lytic infection so that the prophage is happily carried along as a passenger in the bacterial chromosome
what happens if the host bacterium infected by a phage is stressed?
repressor protein is inactivated
prophage excises and undergoes a lytic cycle
what is complementation?
rescue of phenotype of a mutant incapable of making a certain protein by supplying the protein to the mutant organism
putting mutants into groups based on the mutated gene
genetic test that can be used to determine the number of independent genetic elements if one has isolated a series of mutants with the same phenotype
what are restriction enzymes? what do they allow bacteria to do?
recognize specific DNA sequences
allow bacteria to eliminate the negative consequences of bacteriophage infection by degrading the infecting foreign DNA enzymatically
how do bacteria prevent their own DNA from being degraded by restriction enzymes?
have other enzymes that chemically alter their DNA on specific sequences so itâs not a target for the restriction enzymes
what types of sequences do restriction and modifying enzymes recognize?
certain palindrome sequences
how can restriction enzymes be used in genetic research?
allow reproducible cutting of DNA at certain locations
allow for specific cloning of pieces of DNA, isolation in a gel, and then insertion into a plasmid
what are transposons?
mobile genetic elements that are integrated into bacterial chromosomes or plasmids and are capable of jumping from one location in DNA to another
what are insertion sequences (IS)?
special sequences that transposons have
at ends
responsible for transposonâs ability to integrate at random locations
what type of recombination do transposons do?
nonhomologous (illegitimate)
what do the simplest transposons consist of?
just IS
what enzyme is required for transposition? where is it encoded?
requires transposase enzyme - facilitates illegitimate recombination
encoded by IS element
what can transposons carry?
some have gene encoding antibiotic resistance
some have toxin genes
these have medical relevance
how can antibiotic resistance be transmitted to another bacteria?
some transposons duplicate themselves during transposition so that theyâre retained at their original site
can result in gene moving from the bacterial chromosome to a conjugal plasmid
this plasmid can move to another bacteria
what happens if transposons insert into bacterial genes?
can disrupt them and cause mutations
usually null mutations - genes with transposon insertions get completely disrupted
what are the three mechanisms for bacterial gene transfer? (list)
1: transformation
2: transduction
3: conjugation
what is transformation?
mechanism of bacterial gene transfer
transfer of genetic information to a bacterium following the uptake of naked DNA from outside the cell
what is transduction?
mechanism of bacterial gene transfer
transfer of a gene from one bacterium to another by a phage that has mistakenly replaced part or all of its genome with some of its hostâs DNA
what is conjugation?
transfer of genetic info that requires direct cell-to-cell contact
eg transfer of conjugal plasmids and transfer of portions of the bacterial chromosome by Hfr strains
exchange of genetic info between the two parents is unequal
what are the steps of transformation? what if the DNA is chromosomal versus plasmid?
DNA binds to the bacterial membrane
enters the cell
if chromosomal: undergoes homologous recombination with host chromosome
if plasmid: replicates autonomously, independent of chromosome
how is transformation used in research?
used for linkage mapping
plus (more importantly) recombinant DNA plasmids can be easily introduced into bacterial strains by transformation
what is competence?
the ability of bacteria to be transformed some bacteria (such as e coli) which are not normally competent can be made competent (in this case by treatment with CaCl2 or electroporation)
what are the two types of transduction? (list) whatâs the difference?
generalized transduction - transfer of a random region of DNA
specialized transduction - transfer of a specific region of DNA
what causes generalized transduction?
a unique group of bacteriophage that produce some phage particles that contain only host DNA
due to mistaken encapsidation of host chromosomal DNA fragments into phage particles - host DNA can be from any part of the bacterial genome
what is generalized transduction used for in research?
mapping studies
phage preparation grown on donor strain of bacteria can be used to infect recipient strain
this strain will have genetic markers from the donor strain
how does specialized transduction occur (mechanism)?
lysogenic prophage inserts into specific area of DNA
if converts to lytic, can accidentally leave some of its genome behind and take some of host DNA with it
will have DNA from both host bacterium and the phage
which genes can be transduced in specialized transduction?
only those near the site of integration in the bacterial chromosome
how do phage transfer toxin genes?
thought to be via specialized transduction
what are plasmids?
extrachromosomal, circular DNA molecules that are capable of autonomous replication
what is the F factor/plasmid?
first type of plasmid discovered
able to transfer genes from a donor strain bearing an F factor to a recipient F- strain
what do plasmids usually carry?
optional genes that confer additional phenotypic properties
eg: ability to donate DNA by conjugation, resistance to antibiotics, metals, UV, bacteriophage
production of proteases, toxins, antigens, hemolysis
what are R factors?
most medically important class of plasmids encode resistance to various antibiotics conjugative (R is a particular form of F)
what are bacteriocidins?
class of plasmids that encode narrow spectrum antibiotics called bacteriocins - bacteria use against one another
what is the genome structure of plasmids?
circular, supercoiled, doublestranded DNA
how would you separate plasmids from chromosomal DNA?
via density centrifugation or agarose gel electrophoresis
what makes plasmids capable of autonomous replication?
they have their own origin of replication
what is copy number? what controls it?
number of plasmids per cell
number of copies per chromosome
controlled by plasmid encoded repressor of replication that binds at the site of replication initiation
how are plasmids classified?
in incompatibility groups - inability to coexist in the same cell
what makes plasmids incompatible?
similar plasmids have the same repressor mechanisms controlling copy number
if have same repressor mechanism, usually share extensive DNA homology and form an incompatibility gene
what is the role of IS elements in plasmids?
give plasmids ability to insert themselves into chromosome and can result in genomic rearrangements like insertions and deletions => plasmid evolution
what is the role of IS elements in F plasmids?
allow integration into the host chromosome to form an Hfr strain
what is the role of IS elements in R plasmids?
responsible for generation of multiply resistant R factors - permit single resistance genes to move onto plasmids that already carry other resistance genes
what does the F plasmid encode?
encodes proteins required for conjugal transfer - conjugation bridge (sex pilus)
tra genes (required for transfer)
transfer origin that is nicked to initiate conjugation
what is the sex pilus?
aka conjugation bridge
made of pilin
encoded by F plasmid
allows transfer of genetic material from one bacterium to another during conjugation
what is an Hfr cell?
one that has an integrated F+ plasmid - occurs via homologous recombination between IS elements or by IS-mediated transposition of the entire F plasmid
what is an Fâ plasmid?
occurs when F+ plasmid is excised from the chromosome and the excision is imprecise => chromosomal genes getting carried on the F
what happens if an Hfr mates with an F- cell?
can transfer donor chromosomal genes to the F- cell
what is the ames assay?
test for chemical mutagens
measures the ability of a chemical to revert a his- strain of S. typhimurium to wild type in the presence of extract of rat liver microsomes, provided to metabolize procarcinogens
what are crossfeeding assays?
way to determine the order in which several genes act in a pathway for the biosynthesis of an AA
what is degeneracy?
property that each AA is specified by more than one codon - one of the reasons why mutations are sometimes silent
what is an episome?
extrachromosomal DNA element that is capable of autonomous DNA replication, but can also integrate into the chromosome
eg F
what is a genetic map?
order of genes in a chromosome relative to each other or to some other fixed point on the chromosome
what is illegitimate recombination?
genetic recombination between two DNA molecules having no homology - method by which transposons integrate into a chromosome
