LECTURE 8: GENETICS OF BACTERIA & THEIR VIRUSES I

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

BACTERIAL CONJUGATION • 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

BACTERIAL CONJUGATION • experiment ... contact ? • selective filter prevents cell contact • no growth (prototrophs) on minimal medium •  contact required for recombination

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

BACTERIAL CONJUGATION • 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

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

BACTERIAL CONJUGATION • 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

BACTERIAL CONJUGATION • Hfr x F– recombination of donor genes in host

BACTERIAL CONJUGATION • Hfr x F– recombination of donor genes in host • terms: exogenote and endogenote

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

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

BACTERIAL CONJUGATION • interrupted-mating • selective markers • donor is strs • recipient is strr • origin of replication transferred first • 1st transferred markers most frequent in exconjugants

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

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

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

BACTERIAL CONJUGATION • 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)

BACTERIAL CONJUGATION • so far, genetic transfer only • recombination of Hfr exogenote & F– endogenote ...

BACTERIAL CONJUGATION • exogenote enters cell ... merozygote = partial diploid • single recombination event (3x, 5x, ...) nonviable • double recombination event (4x, 6x, ...) viable

leu+ arg+ met+ leu+ arg+ met+ leu+ arg+ met+ MORE LIKELY  LESS LIKELY BACTERIAL CONJUGATION • 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+ BACTERIAL CONJUGATION • determination of gene order by gradient of transfer Hfr leu+ arg+ met+ strs x F–leu–arg– met – strr  • of those markers transferred... Hfr F– leu– arg – met –

leu+ arg+ met+ BACTERIAL CONJUGATION • determination of gene order by gradient of transfer Hfr leu+ arg+ met+ strs x F–leu–arg– met – strr  • of those markers transferred... which also recombine? Hfr F– leu– arg – met –

leu+ arg+ met+ BACTERIAL CONJUGATION • determination of gene order by gradient of transfer Hfr leu+ arg+ met+ strs x F–leu–arg– met – strr  met+ = 100% arg+ = 60% leu+ = 10% Hfr F– leu– arg – met –

BACTERIAL CONJUGATION • bias in recovery of markers • gradient of transfer used fordetermination of gene orderonly •  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

Schaum’s Outlines – Genetics 3rd Ed., CH12, pp. 321 – 325 4th Ed., CH10, pp. 349 – 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