A Singular Case of Prophage Complementation in Mutational Activation of recET Orthologs in Salmonella enterica Serovar Typhimurium

Abstract

A class of mutations that suppress the recombination defects of recB mutants in Salmonella enterica serovar Typhimurium strain LT2 activates the normally silent recET module of the Gifsy-1 prophage. Allele sbcE21 is a 794-bp deletion within the immunity region of the prophage. Concomitant with activating recET, sbcE21 stimulates Gifsy-1 excision, resulting in unstable suppression. Early studies found both recB suppression and its instability to depend on the presence of the related Gifsy-2 prophage elsewhere in the chromosome. In cells lacking Gifsy-2, the sbcE21 allele became stable but no longer corrected recB defects. Here, we show that a single Gifsy-2 gene is required for Gifsy-1 recET activation in the sbcE21 background. This gene encodes GtgR, the Gifsy-2 repressor. Significantly, the sbcE21 deletion has one end point within the corresponding gene in the Gifsy-1 genome, gogR, which in strain LT2 is a perfect duplicate of gtgR. The deletion truncates gogR and places the Gifsy-1 left operon, including the recET and xis genes, under the control of the gogR promoter. The ability of GtgR to trans-activate this promoter therefore implies that GtgR and GogR normally activate the transcription of their own genes. Consistent with the symmetry of the system, a similar deletion in Gifsy-2 results in a Gifsy-1-dependent sbc phenotype (sbcF24). Two additional Gifsy-1 deletions (sbcE23 and sbcE25) were characterized, as well. The latter causes all but the last codon of the gogR gene to fuse, in frame, to the second half of recE. The resulting hybrid protein appears to function as both a transcriptional regulator and a recombination enzyme.Some classic contributions to the study of homologous recombination in Escherichia coli have come from the analysis of mutations that bypass the requirement for the RecBCD enzyme in the recombinational repair of damaged DNA. This approach led to the discovery of recombination proteins and was critical to the development of the concept that recombination can proceed through different pathways, depending on the nature of the DNA substrates (9, 29, 36). Mutations were selected as suppressors of the hypersensitivity of recB or recC mutants to mitomycin C and hence were designated sbc (for suppressor of recB and recC mutation) (2). The first class of alleles, sbcA, was mapped in the Rac prophage and a defective prophage found in the chromosomes of many E. coli strains (28). sbcA mutations cause the constitutive activation of the prophage's recombination module, which comprises the recE and recT genes. The RecE protein (deoxyexonuclease VIII) is a double-stranded-DNA-specific exonuclease, which degrades linear DNA in a 5Ј-to-3Ј direction (23, 24). Thus, the enzyme leaves a protruding 3Ј single-stranded tail that presumably acts in the strand invasion step of homologous recombination. The RecT protein binds single-stranded DNA and, acting in conjunction with RecE, can promote homologous pairing and strand exchange in vitro (21,26). Therefore, the recET module is functionally equivalent to th...

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