Interaction of the Gifsy-1 Xis Protein with the Gifsy-1
            <i>attP</i>
            Sequence

Flanigan, Asa; Gardner, Jeffrey F.

doi:10.1128/jb.00577-07

Cited by 8 publications

(15 citation statements)

References 39 publications

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“…The above data are consistent with the idea that Gifsy-1 instability in cells harboring sbcE21 reflects the upregulation of the prophage's xis gene (19). The Gifsy-1 int gene, whose product is also required for prophage excision, is directed in the opposite orientation and is expected to be expressed independently ( Fig.…”

Section: Resultssupporting

confidence: 78%

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

2008

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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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Section: Resultssupporting

confidence: 78%

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

2008

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“…This 210 bp region could be a novel sort of attachment site ( attP ) for phage integration into the bacterial genome. AttP sites often contain perfect or imperfect direct repeats which interact with the phage integrase and excisionase proteins (Groth and Calos, 2004; Flanigan and Gardner, 2007; Mattis et al. , 2008).…”

Section: Resultsmentioning

confidence: 99%

Isolation and genomic characterization of the first phage infecting Iodobacteria: ϕPLPE, a myovirus having a novel set of features

Leblanc

Caumont-Sarcos

Comeau

et al. 2009

Environ Microbiol Rep

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The aquatic phage ϕPLPE infects a bacterium of the genus Iodobacter that are common inhabitants of rivers, streams and canals that produce violacein-like pigments. Our characterization of ϕPLPE reveals it to be a small, contractile-tailed phage whose 47.5 kb genome sequence is phylogenetically distant from all previously characterized phages. The genome has a generally modular organization (e.g. replication/recombination, structure/morphogenesis, lysis/lysogeny) and approximately half of its 84 open reading frames have no known homologues. It behaves as a virulent phage under the host growth conditions we have employed and, with the exception of an anti-repressor (ant) homologue, the genome lacks all the genes associated with a lysogenic lifestyle. Thus, either ϕPLPE was once a temperate phage that has lost most of its lysogeny cassette or it is a virulent phage that acquired an ant-like gene presumably for some function other than the control of lysogeny. The ϕPLPE genome has few bacterial gene homologues with the interesting exception of a putative acylhydrolase (acylase). This function has been implicated in bacterial quorum sensing since it degrades homoserine-lactone signalling molecules and can disrupt or modulate quorum signalling from either the emitter or its competitors. ϕPLPE may be an example of a phage co-opting components of the bacterial quorum-sensing apparatus to its own advantage.

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“…Although these values should be regarded with caution, they compare reasonably with estimates for other DNA-binding proteins. In gel-shift experiments, a KD of 1 M was estimated for P22 Xis binding to a DNA with a single operator (Mattis, Gumport and Gardner, 2008), and weak but detectable binding to single operators at nM concentrations was seen for λ Xis, HP1 Cox and Gifsy-1 Xis (Yin, Bushman and Landy, 1985) (Flanigan and Gardner, 2007) suggesting single site affinities that are substantially higher than Apl. Thus, it seems that Apl's affinity for its specific sites is relatively low, hence more binding sites may be needed in order to compensate.…”

Section: Apl's Dna Binding Modementioning

confidence: 99%

“…RDFs appear to share a mode of DNA binding in which protomers bind with high cooperativity in a head-to-tail manner to tandem DNA repeats spaced one DNA turn apart, shown for λ Xis (Sam et al, 2002), Gifsy-1 Xis (Flanigan and Gardner, 2007) and Pukovnik Xis (Singh et al, 2014). The crystal structure of P2 Cox has been solved in the absence of DNA, revealing an extensive interaction with neighbouring cox protomers (i+1) and also interactions with i+2 (Berntsson et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

Cutts

Egan

Dodd

et al. 2019

Preprint

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The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA, and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

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Interaction of the Gifsy-1 Xis Protein with the Gifsy-1 attP Sequence

Cited by 8 publications

References 39 publications

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

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

Isolation and genomic characterization of the first phage infecting Iodobacteria: ϕPLPE, a myovirus having a novel set of features

A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

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