Transcript “Bacterial” genetics I - Department of Molecular & Cell Biology

“Bacterial” genetics
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“What are the genes? What is the nature of the
elements of heredity that Mendel postulated as
purely theoretical units? … Frankly, these are
questions with which the working geneticist has
not much concern himself…
If the gene is a material unit, it is a piece of a
chromosome; if it is a fictitious unit, it must be
referred to a definite location in a chromosome.
… Therefore, it makes no difference in the actual
work in genetics which point of view is taken.”
T.H. Morgan
The Relation of Genetics
to Physiology and Medicine
Nobel Lecture, June 4, 1934
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DNA  RNA  protein
central dogma of molecular
biology
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3
7.20
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Max Delbrück (1906 -1981) & Salvador Luria (1912 - 1991),
Cold Spring Harbor Laboratory, Long Island NY, Summer 1941
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Today even the layman thinks of resistant
bacteria as originating from mutation …
but when Luria and Delbrück first got
together, conventional bacteriologists were
by no means clear that microorganisms
could be tought about genetically… Many
believed that resistance was some kind of
adaptation induced, in a few of the
bacteria in a culture, by the exposure to
the antibacterial agent.
Judson p. 55
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The idea smacks of the preMendelian, pre-Darwinian
notion of the inheritance of
acquired characteristics;
Luria damned bacteriology
as the “last stronghold of
Lamarckism.”
Judson p. 55
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Let’s all do science in Nevada
One Saturday evening … Luria went to
a faculty dance… There, watching the
fluctuating returns obtained by
colleagues gambling on a slot
machine, he thought of the experiment
that would distinguish between
resistance induced in bacteria and
resistance resulting from previous
spontaneous mutation upon which
selection acts.
Judson p. 55
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What Luria perceived was that previous
spontaneous mutation would pay out
jackpots of resistant bacteria that would
fluctuate much more widely in size than
those paid out by induction. He tried the
first experiment on the following morning
and wrote off to Delbrueck; Delbrueck
promptly replied that Luria really ought to
go to church …
Judson p. 55
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7.4
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What Luria actually did
Sample set A:
1. Inoculate bacteria
into individual
cultures (1
bacterium per
culture).
2. Let it grow up to a
large number.
Sample set B:
1. Take an aliquot of
bacteria, and start a
culture (which will
therefore not be
clonal).
2. Let them grow up to
a large number
Expose both to phage, and count, how many phageresistant colonies per culture are found. Ask, if there is a
difference between these two sample sets.
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S. Luria, M. Delbrück (1943)
Mutations of bacteria from virus sensitivity to
virus resistance. Genetics 28: 491-511.
“If the production of resistance began only at the
moment of exposure to phage, then it wouldn’t
matter whether the bacteria came from many
individual cultures or one bulk culture. … When
Luria performed the experiment, though, the
twenty separate cultures showed much wider
fluctuations from the average number of resistant
colonies, indicating that a few of the individual
tubes contained resistant bacteria from near the
beginning of the overnight growth period.”
Judson p. 56
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Brock p. 59
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George Beadle (left) and Edward Tatum (right) receiving their Nobel Prizes
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Nature Reviews Genetics 3, 397-403
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Beadle and Tatum:
the rationale
In 1940, while teaching at Stanford, Tatum reviewed the nutritional-growth-factor
requirements that distinguish related bacterial species. This raised the question of
whether mutations can lead to nutritional requirements in species that do not already
require a growth factor. As Beadle was already familiar with Neurospora from his
contact with Dodge and Lindegren, he recognized it as an ideal organism with which
to pursue this question. First, Neurospora could be grown on a simple minimal
medium that contained inorganic salts, sucrose and a single vitamin. The idea of
imposing further nutritional requirements by mutation was plausible. Moreover,
Neurospora cultures were haploid, and therefore recognition of recessive, loss-offunction mutations should be straightforward. And most importantly, Neurospora had
orthodox Mendelian genetics, an attribute that would be vital in a continuing dispute
about the role of genes — the idea still persisted among embryologists that the
fundamental information regarding body plan, organ systems and the 'epigenetic'
features of development lay in the cytoplasm. Beadle and Tatum did their
experiments in part to convince many sceptical biologists that genes control the
fundamental processes of life, and not just the final touches of development, such as
wing shape or eye pigment. To show this, it was important to use a eukaryote that
was simpler than Drosophila and to focus on metabolic functions that could not
possibly be considered as final touches.
Nature Reviews Genetics 3, 397-403
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Beadle and Tatum:
the beginnings
"Observing him [Tatum] writing sequences of reactions on
the blackboard, I suddenly realized how stupid we had
been all these years. Here were all those enzymatic
reactions already worked out by competent biochemists.
If our gene–enzyme concepts were correct, then we
ought to be able to identify the genes immediately
responsible for specifically known enzyme-catalysed
reactions. So why not reverse the approach? Instead of
looking for reactions by enzymes controlled by known
genes, why not look for genes that control already known
chemical reactions? We might then expect to find
mutations ... characterized by an inability to synthesize
essential diffusible substances such as vitamins, amino
acids and other building blocks of the cell's protoplasm."
Nature Reviews Genetics 5, 949-954
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Thirty points on the midterm,
so wake up
Nature Reviews Genetics 5, 949-954
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Ka-BOOOM
It took Beadle and Tatum only 5 months to find the first 3 nutritional
mutants in their irradiated cultures; one required pyridoxin, another
needed p-aminobenzoic acid and the third required thiamin.
The first public announcement of their accomplishment was at a
Caltech seminar where Beadle went to recruit people for his
research group. Not surprisingly, his news was a bombshell.
Norman H. Horowitz, one of the people he recruited, recalls it
clearly: The talk lasted only half an hour, and when it was suddenly
over, the room was silent. The silence was a form of tribute. The
audience was thinking nobody with such a discovery could stop
speaking after just 30 minutes — there must be more.
Superimposed on this thought was the realization that something
historic had happened. Each one of us, I suspect, was surveying, as
best he could, the consequences of the revolution that had just
taken place."
Nature Reviews Genetics 5, 949-954
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7.23
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Joshua Lederberg
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“Conjugation” in bacteria
Take strain of E. coli that is auxotrophic for
two distinct nutrients (thiamine and
leucine).
Take different strain of E. coli that is also
auxotrophic for two distinct nutrients, but
different ones (biotine and cysteine).
Mix the two.
Ask, if ANY NOVEL PHENOTYPES
APPEAR.
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J. Lederberg, E. Tatum (1946)
Novel genotypes in mixed cultures of biochemical
mutants of bacteria. Cold Spring Harbor Symp.
Quant. Biol. 11: 113-114.
15.11
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Paramecium
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B.H. and A.H.
In the pre-Hayes period, mating in bacteria
was envisioned as a conventional sex
process, perhaps modified by aspects of
“relative sexuality,” but nevertheless a
standard haploid/diploid/meiosis
mechanism. After Hayes, it was known
that bacteria were not just small cells, but
constituted a completely different kind of
cell … The terms prokaryote and
eukaryote were not introduced until 1962.
Brock p. 87
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William Hayes
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Selman Waksman
streptomycin
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Hayes expt:
Take strain A, which is
streptomycin-resistant, and
auxotrophic for biotin and
methionine.
Take strain B, which is
streptomycin-sensitive, and
auxotrophic for threonine and
leucine.
Mix the two on a minimalmedium plate containing
streptomycin.
Wait and see.
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Rich husband, poor wife
is not the same as poor husband, rich wife
Cross #1:
Strain A (StrR, B-, M-)  Strain B (StrS, L-, T-)
Result: streptomycin completely inhibits prototroph
formation (i.e., appearance of B+,M+,L+,T+
bacteria) if added before conjugation is
complete.
Cross #2:
Strain A (StrS, B-, M-)  Strain B (StrR, L-, T-)
Result: streptomycin has no effect whatsoever.
You can add it after the onset of conjugation, yet
prototrophs will still form!!
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“'I discussed these results with Denny Mitchison
and I think it was he who first suggested that one
of the parents, A, might be acting as a gene
donor and the other, B, as a recipient'.
It was from this experiment that the concept of
asymmetry in bacterial sexuality arose. Parent B
was the recipient or 'female', the continued
viability of which was essential for the whole
process of recombination and segregation, while
the A donor or 'male' cell was dispensable once
genetic transfer had been effected.”
W. Hayes (1952) Recombination in Bact. coli K 12;
unidirectional transfer of genetic material. Nature
169: 118.
Brock p. 89
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Fig. 15.12
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15.12
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15.13
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15.14
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Fig. 15.15
E. Wollman, F. Jacob (1955) Sur le mecanisme du transfert de materiel genetique
au cours de la recombinaison chez E. coli K12. CR Academie Sciences 240: 2449.
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Cours de Physiologie et de génétique bactériennes, 1957
Illustration humoristique des cours par une série de dessins de F. Lavallé
Légendes de Georges Cohen
http://www.pasteur.fr/infosci/archives/mon/im_laval.html
Interruption de la pénétration de l'ADN mâle dans le cytoplasme de la bactérie
femelle à l'aide d'un warring blender (simple mixer ménager).
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The life cycle of a temperate phage
Fig. 15.21
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Three aspects of phage biology
with long-term impact
1. Transduction (phage carrying additional
genetic information from cell to cell)
 oncoretroviruses
2. Lysogeny (phage resident in bacterial
genome)
 latent viruses in eukaryotic genomes
3. Recombination between phage
 the fine structure of a gene
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Annual budget: $3,780.00
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Bronfenbrenner
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15.19
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Further reading
Horace Judson
The Eighth Day of Creation
Thomas Brock
The Emergence of Bacterial Genetics
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