Mechanism of conjugation and recombination in bacteria

Summary Development of resistance to ²³P-decay of donor genetic determinants after their transfer into the female cell is dependent on unabated DNA synthesis. A similar dependence upon DNA synthesis was found in recombinational events. Both processes show a similar time-course. The DNA synthesis, involved, seems distinct from physiological replication of the chromosome. The formation of the structure resistant to ³²P-decay is going on concomitantly with recombinational process and is completed within 45 to 55 minutes after transfer, before beginning of the replication of recombinant structure. The bearing of these facts on the molecular mechanism of genetic recombination is discussed.     

Mechanism of conjugation and recombination in bacteria

Summary For inhibition of DNA synthesis an antibiotic, edeine, acting specifically on DNA replication, was used. The inhibition of DNA synthesis in F^– cells caused only small decrease (three to four-fold) in recombination frequency. On the other hand a full inhibition of DNA synthesis in Hfr cells affected the recombination to the high extent, lowering its frequency 20–40 fold, at the same time lowering to the similar degree chromosome transfer (measured by zygotic induction frequency). However, the partial inhibition of DNA synthesis in Hfr cells, amounting to about 10 per cent of the control, permitted normal chromosome transfer and normal level of recombination. The results do not agree with Jacob and Brenner's model of chromosome transfer, yet they do not unequivocally confirm Bouck and Adelberg's model. It is possible that the limited DNA synthesis is necessary for other processes, and not for completing of the replication round. The results do not exclude also, that some residual DNA synthesis in female cells is of importance in mating.     

Mechanism of conjugation and recombination in bacteria XVI

Summary Protein synthesis by ribosomes from several cryptopleurine-resistant yeast mutants is also resistant to emetine and tubulosine. These mutants can be classified into two different types: Class I mutants which display high levels of resistance to emetine and tubulosine and Class II mutants that are only weakly resistant to tubulosine and are slightly more sensitive to emetine than those of Class I. Apparently all mutants have similar levels of resistance to cryptopleurine. The distinct phenotypes of Class I and Class II strains are expressed through their 40S ribosomal subunit. Genetic analysis has shown that the mutations to cryptopleurine resistance are allelic and that in a particular case (strain CRY6) the pleiotropic phenotype is a result of the expression of the cryl locus. It is suggested that Class I and Class II mutants arise from two independent mutational events within the cryl allele. in heterozygous (+/cryl) diploids both the sensitive and the resistant genes are expressed as shown by studies of the action of cryptopleurine on polyphenylalanine-synthesizing system derived from each parental sensitive and resistant haploid strain and heterozygous diploid strains. The apparent dominance of sensitivity over resistance which may be observed in vivo in heterozygous (+/cryl) diploids has been explained in terms of the mode of action of the inhibitors.     

Mechanism of conjugation and recombination in bacteria. XXI. Role of F factor genes in post-conjugational recombination in Escherichia coli K-12

Temperature sensitive dnaA recipient crossed at the restrictive temperature with HfrH, free from contaminating F+ cells, forms recombinants almost as proficiently as at the permissive temperature. The merozygotes are able to synthesize DNA at 42 degrees C, although the recipient and donor cells do not incorporate 3H-thymine. A substantial fraction of Lac+ recombinants, irrespective of the mating temperature, is temperature resistant (42 C-R); 15% from among those mated at 35 C and 30% from those mated at 42 C. The presence of dnaA mutation in these Lac+ 42 C-R recombinants was ascertained by co-transduction with ilv. Cell division at 42 C is inhibited in the Lac 42 C-R recombinants by acridine orange. The presence of F factor DNA in these recombinants was demonstrated directly by DNA: DNA hybridization. Suppression of dnaA mutation in Lac+ 42 degrees C-R recombinants and their sensitivity to acridine orange at 42 degrees C suggests that at least part of the F factor is integrated into the recombinant chromosome. A large fraction of the Lac+ 42 degrees C-R recombinants (up to 80%) is sensitive to male phage R17 and fertile. In crosses with HfrC there is a marked decrease of recombination frequency at 42 degrees C in the dnaA recipient. The fraction of Lac+ 42 degrees C-R recombinants is low (up to 10%) and the 42 degrees C-R recombinants are neither sensitive to male phage nor fertile. The results are discussed on the basis of the previously proposed model of post-conjugational recombination.     

Mechanism of conjugation and recombination in bacteria. XVII. Further evidence for single-stranded regions in recipient DNA during conjugation in Escherichia coli K-12.

The secondary structure of recipient DNA mated with Hfr strain was investigated by CsCl density gradient fractionation. After 45 min of HfrH64 X 3h-f-ab1157 mating one-fourth of the radioactive recipient DNA was recovered as a single-strand but only after shearing of cell lysates prior to centrifugation. This heavier than native DNA fraction of radioactive material (obtained after the first centrifugation) was degraded by single-strand specific nuclease S from Aspergillus oryzae. These findings thus confirm the authors' earlier results suggesting that in the course of mating are generated local single-stranded regions in recipient DNA.     

Mechanism of conjugation and recombination in bacteria. XX, Recombination DNA synthesis during mating in Escherichia coli K-12.

In crosses of dnaA recipients with HfrH at restrictive temperature a functional recombinant structure and viable recombinants were formed at a nearly normal rate, whereas recombination in crosses with HfrC was markedly inhibited. Recombinational DNA synthesis, as determined by 3H-thymine incorporation, was found in dnaA merozygotes from HfrH crosses. The recombinational synthesis was completed shortly after the end of mating. The replication of recombinant chromosomes in dnaA Lac+ recombinants started 120 minutes after interruption of mating, and at the restrictive temperature was sensitive to acridine orange, thus pointing to the presence of integrated F factor in the recombinant chromosome.     

Timing of initiation of chromosome replication in individual Escherichia coli cells.

The synchrony of initiation of chromosome replication at multiple origins within individual Escherichia coli cells was studied by a novel method. Initiation of replication was inhibited with rifampicin or chloramphenicol and after completion of ongoing rounds of replication the numbers of fully replicated chromosomes in individual cells were measured by flow cytometry. In rapidly growing cultures, with parallel replication of several chromosomes, cells will end up with 2n (n = 1, 2, 3) chromosomes if initiation occurs simultaneously at all origins. A culture with asynchronous initiation may in addition contain cells with irregular numbers (not equal to 2n) of chromosomes. The frequency of cells with irregular numbers of chromosomes is a measure of the degree of asynchrony of initiation. After inhibition of initiation and run-out of replication in rapidly growing B/r A and K-12 cultures, a small fraction of the cells (2-7%) contained 3, 5, 6 or 7 chromosomes. From these measurements it was calculated that initiation at four origins in a single cell occurred within a small fraction, 0.1, of the doubling time (tau). A dnaA(Ts) mutant strain grown at permissive temperature exhibited a very large fraction of cells with irregular numbers of chromosomes after drug treatment demonstrating virtually random timing of initiation. A similar pattern of chromosome number per cell was found after treatment of a recA strain.     

Mechanism of conjugation and recombination in bacteria. XVIII. Polarity of donor DNA strands transferred to the recipient as determined by DNA-RNA hybridization.

Polarity of donor DNA strand transferred into recipient during conjugation in Escherichia coli K-12 was determined by DNA-3H-RNA hybridization. Lambda prophage was used as a marker. The defective lysogen Hfr H (lambdat11) as a donor and thermosensitive F- CR34 dnaB strain as recipient were used. Two sets of hybridization experiments, with 1-strand specific lambda mRNA and lambda mRNA specific for both phage strands but with large excess of r-strand specific mRNA, were carried out. Strand 1 of lambda DNA was detected preferentially in recipient cells mated at restrictive temperature, when Hfr transferred its genophore in the order gal-lambda-bio. Thus the genophore is transferred with 5'OH at its origin.