Salmonella recD mutations increase recombination in a short sequence transduction assay

Abstract

We have identified recD mutants of Salmonella typhimurium by their ability to support growth of phage P22 abc (anti-RecBCD) mutants, whose growth is prevented by normal host RecBCD function. As in Escherichia coli, the recD gene ofS. typhimurium lies between the recB and argA genes at min 61 of the genetic map. Plasmids carrying the Salmonella recBCD+ genes restore ATP-dependent exonuclease V activity to an E. coli recBCD deletion mutant. The new Salmonella recD mutations (placed on this plasmid) eliminate the exonuclease activity and enable the plasmid-bearing E. coli deletion mutant to support growth of phage T4 gene 2 mutants. The Salmonella recD mutations caused a 3-to 61-fold increase in the ability of a recipient strain to inherit (by transduction) a large inserted element (MudA prophage; 38 kb). In this cross, recombination events must occur in the short (3-kb) sequences that flank the element in the 44-kb transduced fragment. The effect of the recD mutation depends on the nature of the flanking sequences and is likely to be greatest when those sequences lack a Chi site. The recD mutation appears to minimize fragment degradation and/or cause RecBC-dependent recombination events to occur closer to the ends of the transduced fragment. The effect of a recipient recD mutation was eliminated if the donor P22 phage expressed its Abc (anti-RecBC) function. We hypothesize that in standard (high multiplicity of infection) P22-mediated transduction crosses, recombination is stimulated both by Chi sequences (when present in the transduced fragment) and by the phage-encoded Abc protein which inhibits the host RecBCD exonuclease.The recB, recC, and recD genes of Escherichia coli and Salmonella typhimurium encode subunits of exonuclease V and provide a major bacterial recombination function (1,4,23,25,26,33,39,61). This enzyme also contributes to maintenance of cell viability and DNA repair. The RecBCD enzyme has several in vitro activities: a highly processive ATP-dependent double-stranded exonuclease and helicase, an ATP-dependent single-stranded exonuclease, and an ATP-stimulated endonuclease. The enzyme recognizes a specific DNA base sequence (Chi) and responds by mediating exchanges nearby (32, 58; reviewed in references 29, 49, and 53).E. coli strains that carry a recB or recC null mutation are deficient for all of the identified RecBCD enzymatic activities and display multiple phenotypic defects (30,31,33,57). Because of their deficiency in DNA damage repair, recBC mutants are sensitive to mitomycin and UV light; they also show reduced cell viability and reduced transductional and conjugational recombination (12,23). E. coli strains that carry recD mutations lack all exonuclease activity but retain the ATP-dependent helicase (1, 9, 29, 41, 43). These mutants show only a slight elevation of recombination ability (9, 34). Although recD mutants are recombination proficient, the recombination that they perform is independent of Chi sites, and the exchanges are localized near double-stranded ends (9,55,56).A model wh...

Repair of DNA Damage Induced by Bile Salts in Salmonella enterica

“…The sbcB21 allele, originally in the background of strain LT2, was introduced into ATCC 14028 by P22-mediated cotransduction with the zeb-6314TTn10dTc insertion. The recombinationdeficient phenotypes of newly constructed strains were verified using tests described elsewhere (Mahan and Roth 1989;Benson and Roth 1994;Miesel and Roth 1994;Garzó n et al 1996).…”

“…The alleles recA1 and recF522T Tn5 were provided by J. R. Roth (Section of Microbiology, University of California, Davis, CA). Other recombination alleles used in this study are recB497T MudJ and recC498TMudJ (Mahan and Roth 1989), recD543T Tn10dTc (Miesel and Roth 1994), and recJ504TMudCm (Mahan et al 1992). Transfer of transposon-tagged mutations to ATCC 14028 was performed by transductional crosses with phage P22 HT 105/1 int201 (Schmieger 1972), followed by lysogen disposal on green plates (Chan et al 1972).…”

Role of the RecBCD Recombination Pathway in Salmonella Virulence

“…Growth of Salmonella inside phagocytes is a strict requirement for systemic infection (8 (34), followed by lysogen disposal on green plates (5). The recombinationdeficient phenotypes of the newly constructed strains were checked using tests described elsewhere (11,22,23,26,27). a Bacterial suspensions were prepared as described elsewhere (9).…”

A cryptic proline permease in Salmonella typhimurium

“…Each plasmid was linearized with the restriction enzyme BcZI, then the linear DNA fragments were introduced into a re&: : TnlO strain by electroporation, selecting for the Kan' phenotype of Mud J. The recD mutation inactivates exonuclease V, allowing homologous recombination between the linear DNA fragments and the corresponding gene(s) on the chromosome (Biek & Cohen, 1986;Miesel & Roth, 1994;Russell et aZ., 1989). Phage lysates grown on the resulting strains were then used to transduce the chromosomal Mud J insertions into strains MST3205 (iZvC ZivA 6mQ) and MST3237 (putP proZ) .…”