Integration of bacteriophage Mu at host chromosomal replication forks during lytic development.

Abstract

The target site for bacteriophage Mu integration in a lytic cycle of infection was investigated. DNA synthesis in five Hfr strains of Escherichia coli K-12 (3,9,10). Mu can also promote the integration of plasmids and direct the transposition of chromosomal markers (11-13). As is the case with some insertion elements and transposons, Mu prophages in stable lysogens are flanked by a direct repeat of five base pairs of host DNA (14,15). No form of Mu that is free of host DNA has yet been observed.One possible explanation for the ability of this phage to integrate randomly utilizes host chromosomal replication forks as target sites for Mu insertion. In a population of cells replicating asynchronously, the chromosomal replication forks must be distributed over all parts of the bacterial genome; integration of Mu at host replication forks would therefore result in an apparent lack of site specificity for insertion. Integration at host replication forks has been associated with Mu insertion events leading to the formation of stable lysogens (16,17).In this study we asked if the integration events that occur in a lytic cycle of infection involve host chromosomal replication forks. We constructed a system consisting of five Escherichia coli K-12 Fig. 1. Nonsuppressing (Su-) derivatives of the Hfr stocks were isolated by using the procedure of Templin et al. (22). The his-4 allele from strain AB1157 (23) was introduced into each Hfr strain by phage P1 transduction. Strain AT3757 was constructed by phage P1 cotransduction of the pdxAl::Mu -and ara + alleles from AT796 into a spontaneous arabinose-negative mutant of strain AT3753.Bacteriophages. Mucts62SamlO04 was obtained by heat induction of strain MH1671. Mucts62AamlO93 and Mucts62Bam7234 were obtained by heat induction of strains MH219 and MH2808, respectively. A phage strains JC1929 (XN7N53cI26) and JC1930 (h80iXN7N53cI26), used for the construction of Su-strains, were the gift of A. J. Clark.Media. Bacteria were cultured in L broth or in M9 minimal medium (24). Dilutions and washings were done in 0.85% NaCI, except where otherwise noted. Agar plates contained 1% agar (Difco) in L broth plus 5 mM MgSO4 and nalidixic acid (Sigma) at 30 ,ug/ml; agar overlays contained 0.3% agar in L broth plus nalidixic acid at 15 ,ug/ml. Amino Acid Starvation and Nalidixic Acid Treatment.Overnight cultures of Hfr strains in M9 minimal medium supplemented with histidine at 40 ug/ml were diluted 1:100 in 40 ml of the same medium and grown at 37°C for 2 hr with vigorous shaking. After this period of incubation, the cells were collected by filtration (Amicon, 0.45 ,m), washed, resuspended in 40 ml of M9 without histidine, and incubated at 37°C for 200 min to allow existing rounds of replication to terminate (25)(26)(27). We have previously determined by thymidine incorporation assays that DNA synthesis ceases in these his-4 strains after 180 min of starvation (data not shown). Starved cells were collected by filtration and resuspended in 10 ml of L broth Abbreviations: cfu, ...

Replication forks of Escherichia coli are not the preferred sites for lysogenic integration of bacteriophage Mu

“…The hypothesis of preference for replication forks for lysogenic integrations was supported by the frequencies of gene inactivation in nalidixic acid-treated cells (22) and by sequential inactivation of genes in synchronized cultures infected at intervals (21). Comparative kinetics of zygotic induction of synchronized, Mu-infected Hfr strains indicated that in a lytic pathway as well, at least the earlier transpositions occur preferentially at replication forks (9).These results were satisfactory because they were in keeping with the prevailing ideas that Mu exploits nicks or gaps as well as special proteins found in the "replisome" (9) and that replication of Mu DNA is an obvious step for its integration (9). However, it is now generally accepted that each transposon participates in determining the specificity of the staggered cut made at the target (13), thus eliminating the need for existing nicks.…”

“…These results were satisfactory because they were in keeping with the prevailing ideas that Mu exploits nicks or gaps as well as special proteins found in the "replisome" (9) and that replication of Mu DNA is an obvious step for its integration (9). However, it is now generally accepted that each transposon participates in determining the specificity of the staggered cut made at the target (13), thus eliminating the need for existing nicks.…”

“…The hypothesis of preference for replication forks for lysogenic integrations was supported by the frequencies of gene inactivation in nalidixic acid-treated cells (22) and by sequential inactivation of genes in synchronized cultures infected at intervals (21). Comparative kinetics of zygotic induction of synchronized, Mu-infected Hfr strains indicated that in a lytic pathway as well, at least the earlier transpositions occur preferentially at replication forks (9).…”

“…Secondary transpositions to many different sites also occur, as is reflected by the variability of host sequences at both ends of the viral DNA (3,4). The ability of Mu DNA to integrate into many sites led to the hypothesis that it is inserted preferentially at chromosome replication forks (9,21,22). The hypothesis of preference for replication forks for lysogenic integrations was supported by the frequencies of gene inactivation in nalidixic acid-treated cells (22) and by sequential inactivation of genes in synchronized cultures infected at intervals (21).…”

Recombination involving transposable elements: role of target molecule replication in Tn1 delta Ap-mediated replicon fusion.

“…Along with earlier observations on transposition from nonreplicating bacteriophage genomes (5), these experiments indicated that replication of donor replicons was not essential for transposable element DNA rearrangements. On the other hand, studies of the specificity of bacteriophage Mu insertion into the E. coli chromosome suggested a particular affinity for the replication fork and, consequently, a possible recombinational role of the target molecule's replication mechanism (6,7). In order to study this possibility further, we examined the effect on Tnl -specific replicative recombination of blocking target replicon duplication.…”