The Kinetics of the Mating Process in Escherichia coli

Abstract

SUMMARY: When broth cultures of donor (HfrH) and recipient (F-) strains of Escherichia coli K-12 are mixed, zygotes are formed by the transfer of part of the donor chromosome to the recipient cell. The donor parent thus becomes dispensable as soon as transfer is accomplished. The kinetics of zygote formation can therefore be studied by treating samples, removed at intervals from a parental mixture, with virulent bacteriophage to which only the donor parent is susceptible. Only zygotes already formed at the time of treatment can segregate a recombinant ceil. A lag of 8-10 min. precedes a linear rise in the number of zygotes when selection is made for inheritance of the donor nutritional markers T + L + only. The formation of zygotes inheriting the marker Lac+ as well as T + L + shows a lag of about 18 min. These lag periods represent the times required for the genes T + L + and Lac +, respectively, to enter the F- cells and confirm the finding of Wollman & Jacob (1955) that chromosome transfer is an oriented process and that the donor genes penetrate the F- cell in the same order as their arrangement on the chromosome. The process of zygote formation in the equivalent F + × F- cross has also been studied by the phage method. Although the yield of T + L + recombinants is c. 2 × 10⁴ times less than in the Hfr × F- cross under the same conditions, the times of entry of the donor genes T + L + and Lac + are the same in both crosses. In the Hfr × F- cross, significant zygote formation does not occur in unsupplemented buffer but requires the presence of both glucose and sodium aspartate. Zygote formation is a temperature-dependent process which occurs in the absence of multiplication of either parent and is unaffected by the presence of deoxyribonuclease. The number of zygotes (and therefore of recombinants) formed in a given time is a function of two independently variable factors: (i) the frequency and intimacy of chance concacts; (ii) the speed of chromosome transfer which is related to energy production. Decrease of temperature from 37° to 32° about doubles the time required for any given Hfr gene to be transferred to an F- cell. Alteration of the parental population density, or the pH values of the medium (Fisher, 1957b), does not affect the times of entry of Hfr genes into the F- cells but does modify the rate of effective contact formation. Segregation of haploid recombinant cells from Hfr × F- zygotes, at 37°, takes place in nutrient broth at about 140 min., and on minimal agar at about 160 min., after mixing the parental suspensions. The phenotypic expression of resistance to sodium azide, inherited from the Hfr parent, commences shortly after the zygotes are formed and becomes complete just before segregation; resistance to phage T₁, however, is not expressed at all until the time of segregation, and requires four generations of the recombinant segregants for completion.