Peptide wrwycr inhibits the excision of several prophages and traps holliday junctions inside bacteria

Abstract

Peptide inhibitors of phage lambda site-specific recombination were previously isolated by screening synthetic combinatorial peptide libraries. These inhibitors cause the accumulation of complexes between the recombinase and the Holliday junction intermediate of several highly divergent tyrosine recombinases. Peptide WRWYCR and its d-amino acid derivative bind to the center of protein-free junctions and prevent their resolution either by site-specific recombinases or by junction resolvases or helicases. With lesser affinity, the peptides also bind to branched DNA molecules that mimic replication forks. The peptides are bactericidal to both gram-positive and gram-negative bacteria, presumably because they can interfere with DNA repair and with chromosome dimer resolution by the XerC and XerD tyrosine recombinases. In order to test the correspondence between their mechanism in vivo and in vitro, we have tested and shown peptide wrwycr's ability to inhibit the excision of several prophages (lambda, P22, Gifsy-1, Gifsy-2, Fels-1, Fels-2) and to trap Holliday junction intermediates of phage lambda site-specific recombination in vivo. In addition, we found that the peptide inhibits replication of the Salmonella prophage Fels-1 while integrated in the chromosome. These findings further support the proposed mechanistic basis for the antimicrobial activity of the peptide and its use as a tool to dissect strand exchange-dependent DNA repair within cells.

FIG. 1.

Effect of peptide and/or MMC on chromosome copy numbers of rpe and sulA (A) and viability (B) of treated Salmonella cultures. The wild-type LT2 and LT2 lacking resident prophages were compared to assess the potential contribution of prophage induction to the observed copy number and viability changes. As described in the text, qPCRs were done with a constant 5 μg of isolated genomic DNA.

FIG. 2.

Peptide wrwycr inhibits excision of Salmonella prophage. (A) Chloroform lysates of LT2 P22 lysogen cultures treated with MMC, peptide, or both (as indicated on the left). (B) Chloroform lysates of wild-type LT2 cultures treated with MMC or peptide (as indicated). Lysates were serially diluted 10-fold and plated onto a lawn of MA8508, an LT2-derived strain cured of three of its four naturally occurring prophages and deleted for a large fraction of the fourth prophage (Gifsy-1, Gifsy-2, Fels-1, and Fels-2). Two microliters of each lysate, either undiluted or subsequent 10-fold dilutions, was spotted onto the bacterial lawns. These plates are representative of four to eight independent induction experiments.

FIG. 3.

Trapping of phage lambda excisive recombination junction intermediates inside E. coli. The left side of the figure shows the structure of plasmid pLR110 and the orientation of the attL and attR sites. The right panel displays the results of a Southern analysis of plasmid DNA isolated from cells in which Int and Xis expression was induced or not, which were treated with the indicated amounts of peptide wrwyrggrywrw. The probe used was a 496-bp fragment encoding attL from pHN872 (32). The position of the markers for recombination substrate and product fragments and HJs is based on a parallel in vitro reaction. prod, products; rec, recombination; MW, molecular weight; +, present; −, absent.