Download Lecture 8: Gene mapping in bacteria Escherichia coli

Lecture 8: Gene mapping in bacteria
Escherichia coli
Theodor von Escherich,
photo around 1900
1. Bacterial cultures
2. Bacterial conjugation
3. Mapping with Hfr strains
4. Recombination in bacterial crosses
Bacterial cultures
Bacteria can be grown in liquid media and on solid (agar) media
Minimal media are those that contain the minimum
nutrients possible for colony growth, generally without
the presence of amino acids, and are often used to
grow "wild type" microorganisms
Minimal media contain carbon source (e.g. glucose), salts and water
Wild type bacteria are usually prototrophic: they can synthesize all
necessary compound “from scratch”. Prototrophs can grow on minimal
media. Prototrophy can be lost by mutations leading to auxotrophy.
Auxotrophy is the inability of an organism to synthesize a particular
compound required for its growth. Auxotrophs cannot grow on minimal
media.
Both prototrophic and auxotrophic bacteria
can grow on enriched media that contain the
nutrients required to support the growth of a
wide variety of organisms, including some of
the more fastidious ones
http://en.wikipedia.org/wiki/Growth_medium
bacterial cells do
have phenotypes
Joshua Lederberg
Edward L. Tatum
A general approach to
detect recombination in
bacteria
The Nobel Prize in Physiology or Medicine 1958
"for their discovery that genes act by regulating definite
chemical events"
George Wells Beadle
1/4 of the prize
California Institute of
Technology (Caltech)
Edward Lawrie Tatum
1/4 of the prize
Rockefeller Institutute for
Medical Research, NY
"for his discoveries
concerning genetic
recombination and the
organization of the genetic
material of bacteria"
Joshua Lederberg
1/2 of the prize
University of Wisconsin
Madison, WI
Lederberg and Tatum’s experiment (1946)
The parental strains were
auxotrophic: they could
not grow on minimal
medium
Prototrophic recombinant
progeny were formed with
freq. 10-7
Bacterial conjugation
Bacterial conjugation
(“F-factor”)
happens with frequency of 30%
François Jacob and Elie Wollman
(both at Inst. Pasteur, France)
From left to right: François Jacob,
Jacques Monod and André Lwoff,
who were awarded the Nobel
Prize for Physiology or Medicine
in 1965 (for regulation of lacoperon, which will be considered
later in this course).
Interrupted-conjugation experiment of Jacob and Wollman (1957)
Hfr: strs azir tonr lac+ gal+ x F-: strr azis tons lac- galConjugation
Interruption
Plating
exconjugants
(all strr)
azir tonr etc.
Interrupted-conjugation experiment of Jacob and Wollman (1957)
Hfr: strs azir tonr lac+ gal+ x F-: strr azis tons lac- gal-
Formation of an Hfr cell: frequency 10-3 per each F+ cell
Transfer of the chromosome by Hfr during conjugation
freq. 10-4 per each
Hfr cell
gal+ lac+ tonr azir
gal+ lac+ tonr azir
gal+ lac+tonr azir
Genetic map of the genes in bacteria
100
25
gal
20
15
lac
10
ton azi
5
0 / Origin
Recombination during bacterial conjugation
donor DNA
meroploid
cells
(partial
diploid)
recipient’s
chromosome
Single crossover would make the
chromosome linear and the cell would die
Insertion of F factor into bacterial chromosome
O – ‘origin’ of transfer
The order of gene transfer is determined by polarity
and site of integration of F-factor in the chromosome
• in each Hfr cell line the polarity and the site are different
• hence the exact gene transfer order are not the same
• but the relative gene order is the same – as in the original chromosome
Genetic (old) and sequencing-based (1997) maps of E. coli
100 min
Conjugation
summary
10-7
10-3
10-4
~30%
Conjugation
summary
10-7
~30%
Important:
10-3
10-4
F+ cells DO NOT transfer their genes directly:
F+ convert into Hfr (10-3) and those Hfr cells
would then perform the transfer (10-4),
thereby creating an APPEARANCE of F+
(10-4 X 10-3 =10-7)
transfering genes at 10-7
Production of an F’ factor
a gene (e.g. lac+) carried by F’
is transferred with 30% freq.,
but all others still at 10-7
F’ factor carrying lac gene