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 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

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

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

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+ MORE LIKELY  LESS LIKELY

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... leu+ arg+ met+ Hfr F– 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? leu+ arg+ met+ Hfr F– 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% leu+ arg+ met+ Hfr F– leu– arg – met –

BACTERIAL CONJUGATION 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

BACTERIAL CONJUGATION high-resolution mapping by recombinant frequency

BACTERIAL CONJUGATION high-resolution mapping by recombinant frequency

BACTERIAL CONJUGATION high-resolution mapping by recombinant frequency

Schaum’s Outlines – Genetics 3rd Ed., CH12, pp. 321 – 325 4th Ed., CH10, pp. 349 – 355

Schaum’s Outlines – Genetics 3rd Ed., CH12, pp. 321 – 325 4th Ed., CH10, pp. 349 – 355

Schaum’s Outlines – Genetics 3rd Ed., CH12, pp. 321 – 325 4th Ed., CH10, pp. 349 – 355

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