Academic journal article Genetics

NinR- and Red-Mediated Phage-Prophage Marker Rescue Recombination in Escherichia Coli: Recovery of a Nonhomologous Imm[lamda] DNA Segment by Infecting [Lamda]imm434 Phages

Academic journal article Genetics

NinR- and Red-Mediated Phage-Prophage Marker Rescue Recombination in Escherichia Coli: Recovery of a Nonhomologous Imm[lamda] DNA Segment by Infecting [Lamda]imm434 Phages

Article excerpt

ABSTRACT

We examined the requirement of λ recombination functions for marker rescue of cryptic prophage genes within the Escherichia coli chromosome. We infected lysogenic host cells with λimm434 phages and selected for recombinant immλ phages that had exchanged the imm434 region of the infecting phage for the heterologous 2.6-kb immλ region from the prophage. Phage-encoded activity, provided by either Red or NinR functions, was required for the substitution. Red^sup -^ phages with ΔNinR, internal NinR deletions of rap-ninH, or orf-ninC were 117-, 12-, and 5-fold reduced for immλ rescue in a Rec^sup +^ host, suggesting the participation of several NinR activities. RecA was essential for NinR-dependent immλ rescue, but had slight influence on Red-dependent rescue. The host recombination activities RecBCD, RecJ, and RecQ participated in NinR-dependent recombination while they served to inhibit Red-mediated immλ rescue. The opposite effects of several host functions toward NinR- and Red-dependent immλ rescue explains why the independent pathways were not additive in a Rec^sup +^ host and why the NinR-dependent pathway appeared dominant. We measured the influence of the host recombination functions and DnaB on the appearance of oriλ-dependent replication initiation and whether oriλ replication initiation was required for immλ marker rescue.

MARKER rescue recombination to produce gene sub-LV-L stitutions involves exchanges within regions of homology straddling a marker of interest. Strong modern evidence for the shuffling of phage gene modules in nature is provided by the stx phages and prophages of Escherichia coli, which share the genome organization of bacteriophage ë (BRUSSOW et al. 2004). Early ë workers identified phage-prophage marker rescue, where an infecting ë was capable of rescuing a gene present on a homologous cryptic prophage in a lysogenic cell. SIGNER and WEIL (1968) used a spot test involving the rescue of an h (unspecified host range) marker from rec+ cells with a cryptic ë prophage (deleted for a large portion of prophage, including the imm region) that was infected by ë/*\ and \h recombinants were selected on host cells that were resistant to infection by \hx but sensitive to Xh. Using this assay, SIGNER and WEIL (1968) were able to screen hydroxylamine-treated infecting phage for deficiency in marker rescue. Several mutants with reduced ability to rescue prophage markers were subsequently mapped as recombination-defective red mutants. ECHOLS and GINGERY (1968) recovered \sus+ recombinants that were formed by marker rescue between an infecting Xsus phage and a defective prophage in a lysogen. Both studies concluded that Red functions of ë were required for phage-prophage marker rescue in E. coli hosts defective for the host recA function. The ë Red-dependent recombination activity (reviewed by STAHL 1998; KUZMINOV 1999; COURT et al 2002) depends upon the expression of ë genes exo and bet (or Reda, Redp; combined, Red) along with gam. Red-dependent recombination is initiated by double-strand breaks, and when marked Red+ ë phages infect cells blocked for DNA replication, the ë ? ë exchanges are focused near the cos ends, the only site of an initiating doublestrand break (TARKOWSKI et al. 2002). Murphy and coworkers (MURPHY 1998; MURPHY et al. 2000) constructed an E. coli strain in which the cellular recBCD genes (SMITH 2001) were replaced with exo-bet and placed under /acpromoter control. Theyfound that the ë activities supported recombination between the cellular chromosome and linear DNA fragments at an elevated level. Recombination in these ArecBCD cells, lacking Gam, depended upon Exo and Beta, was greatly reduced in recA mutants, and required host recombination genes recQ, recO, recR, recF, and ruvC, but not recjor recG (MuRPHY 1998; POTEETE et al 1999; MURPHY et al 2000; POTEETE and FENTON 2000). The ë Red functions can facilitate chromosomal engineering, i.e., substituting or disrupting genes in an E. …

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