lecture 8: genetics of bacteria & their viruses i fch5 key concepts fworking with microorganisms...

LECTURE 8: GENETICS OF BACTERIA & THEIR VIRUSES I

CH5 key concepts

working with microorganisms

bacterial conjugation

CHAPTER 5: KEY CONCEPTS fertility factor (F) permits bacterial cells to transfer DNA to other

bacteria cells through conjugation F can be integrated or cytoplasmic when integrated, F can transfer host chromosome markers

through conjugation bacteriophages can transfer DNA from one bacterial cell to

another in two ways ... generalized transduction is the transfer of randomly incorporated

bacterial chromosome fragments specialized transduction is the transfer of specific genes near

phage integration sites these methods of gene transfer facilitate construction of detailed

maps of bacterial genomes

WORKING WITH MICROORGANISMS

so far... recombination & mapping in eukaryotes

now... prokaryotes & viruses resolution

3 ways to incorporate & recombine DNA in bacteria:

1. conjugation – plasmid-mediated transfer

2. transformation – absorb from environment

3. transduction – bacteriophage-mediated transfer

WORKING WITH MICROORGANISMS

WORKING WITH MICROORGANISMS binary fission known rate ~ °C liquid medium plating ...

serial dilutions ... 1 cell ~ 107 cells

visible colonies or undiluted ... lawn

use both methods

WORKING WITH MICROORGANISMS strains prototrophs =

wild type grow on minimal

medium auxotrophs =

mutants do not grow on

minimal medium nutrition carbon source

resistant mutants

BACTERIAL CONJUGATION

do bacteria have genetic exchange & recombination ?

Lederberg & Tatum, 1946

Escherichia coli (E. coli)

single circular “chromosome”

haploid


experiment ... contact requirement ?

2 strains, > 1 mutation no colonies on A or B ... no spontaneous back

or reversion mutations BUT… some colonies

(10-7) on mixed ... prototrophs from

recombination


experiment ... contact ? selective filter prevents

cell contact no growth (prototrophs)

on minimal medium contact required for

recombination


Hayes, 1953 genetic transfer in bacterial “crosses” unidirectional donor & recipient strains ... not really sex ( & ) as strains donate unequally


fertility factor – F plasmid F+ donor & F– recipient strains F+ x F– both F+ unidirectional rolling circle plasmid replication F DNA transferred through a pore in the pilus


the F plasmid can integrate into the host chromosome generates a high frequency recombinant strain ... Hfr


Hfr transfers part of the host genome during conjugation

Hfr x F– F– rarely converted to Hfr or F+

isolate & purify Hfr from F+ for mapping


Hfr x F– recombination of donor genes in host


Hfr x F– recombination of donor genes in host terms: exogenote and endogenote


Wollman & Jacob, 1957 – gradient of transfer selective marker – donor is strs & recipient is strr origin of replication is transferred first


mapping in E. coli by interrupted-mating donor genes recombined into host genome


interrupted-mating selective markers

donor is strs

recipient is strr origin of replication

transferred first 1st transferred markers

most frequent in exconjugants


mapping in E. coli by interrupted-mating distance measured in time (min)


bacterial chromosome is circular integration of F factor is pseudo-random integration in either orientation


F factor integrates by recombination pairing regions of homology (hatched) episome = plasmid with free & integrated states


F plasmid = episome

F+ & Hfr replicate during transfer

F+a+ x F–a– F+a–

~10–3 F+a+ Hfr a+

Hfr a+ x F–a– F–a– (exogenote lost) or F–a+ (exogenote incorporated)

so far, genetic transfer only

recombination of Hfr exogenote & F– endogenote ...



a) exogenote enters cell ... merozygote = partial diploid

b) single recombination event (3x, 5x, ...) nonviable

c) double recombination event (4x, 6x, ...) viable


gradient of transfer bridge spontaneously breaks

early marker transfer more likely than late

Hfr leu+ arg+ met+ strs x F– leu– arg– met – strr

leu+ arg+ met+

leu+ arg+ met+

leu+ arg+ met+

MORE LIKELY

LESS LIKELY


determination of gene order by gradient of transfer


of those markers transferred...

leu+ arg+ met+

leu– arg – met –

Hfr

F–




of those markers transferred... which also recombine?

leu+ arg+ met+


Hfr

F–




met+ = 100%arg+ = 60%leu+ = 10%

leu+ arg+ met+


Hfr

F–


bias in recovery of markers

gradient of transfer used for determination of gene order only

to determine map distances, select late marker to ensure transfer of all relevant genes ... high resolution mapping

BACTERIAL CONJUGATION high-resolution mapping by recombinant frequency

Scha

um’s

Out

lines

– G

enet

ics

3rd E

d., C

H12

, pp.

321

– 3

254th

Ed.

, CH

10, p

p. 3

49 –

355

BACTERIAL CONJUGATION marker transfer by

episomes ... F'

a) integrated F Hfr

b) imprecise excission of F

c) incorporation of genes

d) transfer to F––

BACTERIAL CONJUGATION AND RECOMBINATION MAPPING: PROBLEMS

in Griffiths chapter 5, beginning on page 179, try questions #1-3, 5-10, 12, 13, 15, 22, 23, 25-27

begin with the solved problems on page 177 if you are having difficulty

look at the way Schaum’s Outline discusses conjugation (pp. 338-341) and mapping (pp. 349-355)

try Schaum’s Outline questions 10.19 and 10.20 on page 361

lecture 8: genetics of bacteria & their viruses i fch5 key concepts fworking with microorganisms...

Documents

conjugation fhfr x f

host slide

recombination slide

bacterial conjugation

bacterial conjugation

bacterial conjugation

hfr slide

f prototrophs