Transcript GENETICS – BIO 300

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
a) exogenote enters cell
... merozygote =
partial diploid
b) single recombination
event (3x, 5x, ...)
nonviable
c) double recombination
event (4x, 6x, ...)
viable
BACTERIAL CONJUGATION
a) exogenote enters cell
... merozygote =
partial diploid
b) single recombination
event (3x, 5x, ...)
nonviable
c) double recombination
event (4x, 6x, ...)
viable
BACTERIAL CONJUGATION
a) exogenote enters cell
... merozygote =
partial diploid
b) single recombination
event (3x, 5x, ...)
nonviable
c) 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

MORE
LIKELY

LESS
LIKELY
leu+
arg+
met+
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

Hfr
leu+
arg+
met+
leu–
arg –
met –
F–
of those markers transferred...
BACTERIAL CONJUGATION
determination of gene order by gradient of transfer
Hfr leu+ arg+ met+ strs x F– leu– arg– met – strr

Hfr
leu+
arg+
met+
leu–
arg –
met –
F–
of those markers transferred... which also recombine?
BACTERIAL CONJUGATION
determination of gene order by gradient of transfer
Hfr leu+ arg+ met+ strs x F– leu– arg– met – strr

Hfr
leu+
arg+
met+
leu–
arg –
met –
F–
met+ = 100%
arg+ = 60%
leu+ = 10%
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