chapter 8: genetic analysis + mapping in bacteria and bacteriophages

importance to study bacteria and viruses

-disease causing agents, so important study subjects

-useful genetic models

special features in Prokaryotes

-haploid DNA (no homologous chromosome)

-no mitosis or meiosis

-chromosome DNA is circular

-plasmids

-extrachromosomal DNA (circular)

-genetic exchanges still occurs in prokaryotes

genetic exchange in prokaryotes involves

-a DNA molecule from an external source

-replacement of chromosomal information

how closely linked two genes are influences

-likelihood one molecule that includes both genes will be transferred to host simultaneously

-likelihood one recombination event will include both genes

genetic information can be

transferred from one bacterium to another, resulting in an altered genotype

-vertical or horizontal gene transfer

Conjugation: transfer of DNA between cells

1. Conjugation:

2. Transformation

3. Transduction

conjugation

transfer of DNA between cells

transformation

introduction of DNA into cells

transduction

introduction of viral DNA into cells

growth of bacteria and isolation of mutants

-grow in liquid or solid media

-minimal medium

-complete medium

minimal medium

salts + NH4+ + glucose

complete medium

minimal medium + yeast extract / amino acids

bacterial growth phenotypes

-prototrophs

-auxotrophs

-resistance mutants

prototrophs

wild type, strains that will grow on minimal medium

auxotrophs

mutant strains that can only grow on minimal medium if it is supplemented with some biochemical

-trp+, trp-, gal-

trp+

strain can make tryptophan

trp-

strain requires addition of tryptophan to medium

gal-

strain cannot grow on galactose as sole carbon source

reisstance mutants

will grow on media containing an antibiotic

-strR, strS

strR

resistant to streptomycin

strS

sensitive (wild type)

bacterial mutants typically identified by

growth phenotypes

spontaneous mutations are source of

variation in bacterial mutants

bacterial mutants are

haploid so phenotypes are directly affected

conjugation is a

DNA transfer between cells

evidence of conjugation in bacteria

-only some strains can be donors, named F+

-cells that receive, F-

-Lederberg and Tatum, 1946

conjugation requires

physical contact

-U tube experiment

in U tube experiment the medium

passes back and forth across filter and cells do not

conjugation is

unidirectional

F factor is a

plasmid

plasmids are composed of a

double-stranded closed circle of DNA

plasmids exist in

multiple copies in the cytoplasm

plasmids may contain

one or more egenes

plasmids use the same

replication enzymes as host

plasmids are distributed to

daughter cells

plasmids replicated independently of the

bacterial chromosome

F factor plasmids confer

fertility

F factor plasmids contain genes for

sex pilus formation on which genetic recombination depends

conjugation of F plasmid

-F+ plasmid is copied into the F- cells

-F- cells become F+

-F= to F- conjugation occurs in 1x10^-7 cells

steps in F plasmid conjugation

1. conjugation occurs between F+ and F- cell

2. one strand of F factor is nicked by endonuclease and moves across conjugation tube

3. DNA complement is synthesized on both single strands

4. movement across conjugation tube is complete + DNA synthesis is complete

5. ligase closes circles + conjugates separate

Hfr strains are

special types of DNA donors

Hfr

high frequency recombination (1 x 10-4)

in Hfr F+ plasmid is integrated into the

chromosome

in Hfr genes on the bacterial chromosome are

transferred to F- cells

in Hfr F+ plasmid is

not transferred

in Hfr F- cells do not become

F+

conjugation between Hfr and F- strains leads to

transfer of genes on the chromosome

recombination between the incoming DNA and the chromosome can

place the new DNA into the chromosome

-basis for mapping in prokaryotes

once the molecule is present in the cell

crossing over can occur between these two molecules of DNA

frequency at which two genes are included in the same recombination event is dependent on

their physical distance from one another on the molecule

interrupted mating technique is used to

map genes in E. coli

in interrupted mating technique

-conjugation is interrupted, resulting in recipients with various number of bacterial genes

-number of transferred genes increases with amount of time of conjugation

-genes close + the F+ factor is first transferred

-more time = more genes transferred

-transferred genes replace the genes in the chromosome by homologous recombination

gene order is determined by

transfer time

genes closer to the F+ are

transferred and recombined sooner than others

an ordered linear transfer of genes is correlated with the

length of time conjugation proceeded

with a time map gene order and distance between genes could be

predicted

time maps are the basis for

first genetic map in bacteria

strains with F+ plasmid inserted at different locations of time map can be used for

mapping

conjugation rarely goes on for long enough for

F factor itself to be transferred

order of gene transfer in different Hfr strains shows

the E. coli chromosome is circular

through recombination the F DNA can

shuttle in and out of the bacterial chromosome

-when it comes out it may take a piece of the chromosome with it

merozygotes are the only time there is

diploidy in bacteria

there are different types of plasmids which include

different genes

R plasmids consist of two components

1. resistance transfer factor (RTF)

2. one or more r- determinants

RTF encodes genetic information essential to

transferring the plasmid between bacteria

R-determinants confer resistance to

antibiotics

col plasmids encode

colicins that can kill neighboring bacteria

genetic recombination in bacteria is by

transformation

-uptake of "free DNA"

genes that are close enough to each other to be

cotransformed are linked

after transformation, DNA recombines with the

chromosome, creating a heteroduplex

transduction in bacteria

-genetic recombination in bacteria via bacteriophages

-transfer DNA between bacteria by Viruses

phage

bacterial virus

phage can

recombine with one another

phage can also recombine with

bacterial DNA (=transduction)

viral transduction does not require

cell to cell contact

when phage lyse host

they leave plaques

counting plaques is how we

assay phage phenotypes

sometimes we have to dilute to

get an accurate count

not all phage

lyse host cells

viruses that lyse cells are called

virulent

lysogeny occurs when

-the phage DNA integrates into the bacterial chromosome

-it is replicated along with the chromosome

-it is passed to daughter cells

some viruses (temperate viruses) can integrate their DNA into the bacterial chromosome and remain dormant

DNA into the bacterial chromosome and remain dormant

-lysogeny

lysogenic bacterium can be induced to

enter a lytic virus replication cycle by environmental conditions

life cycle of bacteriophages (lytic cycle) of T4 phage of e.coli

phage DNA enters bacterium

host DNA is degraded

-virus takes over host cell

machinery and replicates

-viral particles are assembled and release

T4 phage of E. coli has _____ genes

150

bacterial DNA can be packed into

virus

like transformation, generalized transduction can be used in

linkage and chromosomal mapping of the bacterial chromosome

two closely aligned (linked) genes can be simultaneously

transduced (cotransduction)

the closer linked genes are to each other

the greater the frequency of transduction

the precise order of genes can be

determined in transduction

plaque morphology associated with mutations are

frequently used phenotypes for mapping genes on phage chromosome

two and three point mapping in phage

# of recombination events between two genes is proportional to the relative distance between the genes

results of a cross involving the h and r genes in phage T2 (hr+ x h+r)

mixed infection with two phage types

rec proteins are essential to

bacterial recombination

genetic recombination is a

regulated process in bacteria, like eukaryotes

bacterial recombination requires

functional gene products from RecA, and RecBCD

bacterial mutants deficient in any of these components (functional gene products) don't

undergo recombination