Overview of Conjugation

F+ x FF+, F+ products

Generation and transfer of Hfr chromosome

Insertion of the F factor into the E. coli chromosome Multiple positions Two orientations The order of gene transfer depends the position and the orientation of the inserted F facter in the Hfr chromosome

Transfer of the integrated F factor results in transfer of genomic markers

Hfr a+ x F- a-

Hfr a+ + F- a+

Two types of conjugation.

F+ x F- crosses 107 F+ cells x 107 F- cells 104 different Hfrs

All markers transferred equally at about 1/107 frequency Hfr x F- crosses 107 Hfr cells x 107 F- cells all 107 same Hfr

Markers transferred in specific order at about 1/104-1/103 frequency

Hfr Strain1 = MZXWC 2 = LANCW 3 = ALBRU 4 = ZMURB

M U R B L A

MZXWC WCNAL ALBRU BRUMZ

Z X W C N

The Genetic Map of Ecoli

minutes

How to do fine scale mapping.

Interrupted mating experiments cannot distinguish the relative position of genes within about 2 minutes of each other, and thus give only a rough idea of gene location. Do 1. Three point crosses. 2. Reciprocal crosses. Both use recombination frequencies to determine gene order. Lets look more closely at what happens in the Fcell (the exconjugant).

Recombination (crossover) in F- cell.

incomplete genome from the donor Hfr chromosome. complete genome from F- cells

Interrupted mating says: mal arg/met

10 minutes 6 minutes

Use a 3 point cross to figure out order of arg and met.

Hfr strsarg+met+mal+ X F- strRarg-met-mal-

How to do the experiment????

StrS
(book says mal+,met+)

StrR

1. Select for mal+, the last marker, ensures that all genes transfer. That way you only look at recombination. 2. Replica plate onto both arginine or methionine minus plates..

met arg

mal

Crossovers? 2 Most frequent 2 Pretty frequent 2 Less frequent, smaller interval 4

least frequent

Reciprocal Crosses
Another way for ordering markers that are close to one another, also takes advantage of the lower frequency of quadruple crossovers. a
or

bc cb

Cross I Hfr strs a+ b+ c- x F- strr a- b- c+

select strr a+ b+ c+
Cross II Hfr strs a+ b- c+ x F- strr a- b+ c-

select strr a+ b+ c+

If the order is abc, then will get many more a+b+c+ exconjugants from cross I than cross II. If the order is acb, then will get many more a+b+c+ exconjugants from cross II than cross I.

Cross 1
a+ If abc a- b- c+ F- a- b+ c- Hfr a+

Cross 2
b- c+

Hfr F-

b+ c-

a+ If acb a-

c- b+ Hfr c+ b- F-

a+

c+ b- Hfr

a-

c- b+

F-

Therefore If abc cross I > cross II,

If acb cross II > cross I.

F elements (F prime elements)

F factors which carry a part of the chromosome. Allows very high frequency transfer of F markers. Allows one to make partial diploids. Can use for complementation tests.

Bacteria tend to cluster genes of similar function.

Bacterial chromosome

F+

metA metB metC

Hfr

F plasmids have every thing they need to transfer.

Bacterial chromosome

metA metB metC

Bacterial chromosome Bacterial chromosome F

metA metB metC

metA metB metC

F male

F- female

Bacterial chromosome

metA metB metC

metA metB metC

F partially diploid male

Complementation test, an example.

Bacterial chromosome

metA- metB+ metC+

Bacterial chromosome

metA metB met??- metC

F met- m1 male Your met- mutant

F- met- m2 female Your friends met- mutant

metA- metB+ metC+

Bacterial chromosome

metA metB met??- metC

F partial diploid Test for complementation by growing on met- plates. If it grows on met- plates = If no growth = Different genes = complement. Same gene = fail to complement = metA-.

Lederberg and Zinder, 1951

Question: Is there conjugation in Salmonella (closely related bacteria)? phe+trp+met-his- x phe-trp-met+his+ Found prototrophs Experiment: U-tube to separate two auxotrophic strains, then check for growth on minimal media

U- tube experiment

(phe+trp+met-his-)

(phe-trp-met+his+)

minimal media no prototrophs

<< the size of bacteria ~ the size of P22 Phage

minimal media Prototrophs!!

Bacterial Phage
Conclusion: Instead of conjugation, they discovered phagemediated transfer of bacterial genes, or

TRANSDUCTION.