MCB 140 09-19-08 1

“Bacterial” genetics

MCB 140 09-19-08 2

“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 MedicineNobel Lecture, June 4, 1934

3MCB 140, 2/25/05

DNA RNA protein

central dogma of molecular biology

MCB 140 09-19-08 47.20

MCB 140 09-19-08 5

Max Delbrück (1906 -1981) & Salvador Luria (1912 - 1991), Cold Spring Harbor Laboratory, Long Island NY, Summer 1941

MCB 140 09-19-08 6

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

MCB 140 09-19-08 7

The idea smacks of the pre-Mendelian, pre-Darwinian notion of the inheritance of acquired characteristics; Luria damned bacteriology as the “last stronghold of Lamarckism.”

Judson p. 55

MCB 140 09-19-08 8

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

MCB 140 09-19-08 9

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

MCB 140 09-19-08 107.4

MCB 140 09-19-08 11

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 phage-resistant colonies per culture are found. Ask, if there is a

difference between these two sample sets.

MCB 140 09-19-08 12

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

MCB 140 09-19-08 13

Brock p. 59

MCB 140 09-19-08 14

MCB 140 09-19-08 15

MCB 140 09-19-08 16

George Beadle (left) and Edward Tatum (right) receiving their Nobel Prizes

MCB 140 09-19-08 17Nature Reviews Genetics 3, 397-403

MCB 140 09-19-08 18

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-of-function 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

MCB 140 09-19-08 19

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

MCB 140 09-19-08 20

Thirty points on the midterm, so wake up

Nature Reviews Genetics 5, 949-954

MCB 140 09-19-08 21

Ka-BOOOMIt 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

MCB 140 09-19-08 227.23

MCB 140 09-19-08 23

Joshua Lederberg

MCB 140 09-19-08 24

“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.

MCB 140 09-19-08 2515.11

J. Lederberg, E. Tatum (1946)Novel genotypes in mixed cultures of biochemical

mutants of bacteria. Cold Spring Harbor Symp. Quant. Biol. 11: 113-114.

MCB 140 09-19-08 26

Paramecium

MCB 140 09-19-08 27

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

MCB 140 09-19-08 28

William Hayes

MCB 140 09-19-08 29

Selman Waksman

streptomycin

MCB 140 09-19-08 30

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 minimal-medium plate containing streptomycin.

Wait and see.

MCB 140 09-19-08 31

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!!

MCB 140 09-19-08 32

“'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

MCB 140 09-19-08 33

MCB 140 09-19-08 34

Fig. 15.12

MCB 140 09-19-08 35

15.12

MCB 140 09-19-08 3615.13

MCB 140 09-19-08 3715.14

MCB 140 09-19-08 38

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.

Fig. 15.15

MCB 140 09-19-08 39

MCB 140 09-19-08 40

MCB 140 09-19-08 41

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).

Cours de Physiologie et de génétique bactériennes, 1957Illustration 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

MCB 140 09-19-08 42Fig. 15.21

The life cycle of a temperate phage

MCB 140 09-19-08 43

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

MCB 140 09-19-08 44

Annual budget: $3,780.00

MCB 140 09-19-08 45

MCB 140 09-19-08 46

MCB 140 09-19-08 47

MCB 140 09-19-08 48

MCB 140 09-19-08 49

MCB 140 09-19-08 50

Bronfenbrenner

MCB 140 09-19-08 5115.19

MCB 140 09-19-08 52

MCB 140 09-19-08 53

Further reading

Horace Judson

The Eighth Day of Creation

Thomas Brock

The Emergence of Bacterial Genetics