A myovirus-like temperate phage, ⌽HAP-1, was induced with mitomycin C from a Halomonas aquamarina strain isolated from surface waters in the Gulf of Mexico. The induced cultures produced significantly more virus-like particles (VLPs) (3.73 ؋ 10 10 VLP ml ؊1 ) than control cultures (3.83 ؋ 10 7 VLP ml ؊1 ) when observed with epifluorescence microscopy. The induced phage was sequenced by using linker-amplified shotgun libraries and contained a genome 39,245 nucleotides in length with a G؉C content of 59%. The ⌽HAP-1 genome contained 46 putative open reading frames (ORFs), with 76% sharing significant similarity (E value of <10 ؊3 ) at the protein level with other sequences in GenBank. Putative functional gene assignments included small and large terminase subunits, capsid and tail genes, an N6-DNA adenine methyltransferase, and lysogeny-related genes. Although no integrase was found, the ⌽HAP-1 genome contained ORFs similar to protelomerase and parA genes found in linear plasmid-like phages with telomeric ends. Southern probing and PCR analysis of host genomic, plasmid, and ⌽HAP-1 DNA indicated a lack of integration of the prophage with the host chromosome and a difference in genome arrangement between the prophage and virion forms. The linear plasmid prophage form of ⌽HAP-1 begins with the protelomerase gene, presumably due to the activity of the protelomerase, while the induced phage particle has a circularly permuted genome that begins with the terminase genes. The ⌽HAP-1 genome shares synteny and gene similarity with coliphage N15 and vibriophages VP882 and VHML, suggesting an evolutionary heritage from an N15-like linear plasmid prophage ancestor.

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.Bacteriophage lambda uses a phage-encoded integrase (Int) to catalyze the site-specific recombination reaction that integrates its chromosome into and excises it out of the Escherichia coli chromosome (e.g., see references 2 and 34). We have previously identified and characterized hexapeptides that inhibit site-specific recombination by the phage lambda Int in vitro by binding to the Holliday junction (HJ) intermediates of the reaction and preventing their resolution (4,7,13,22). The most potent of these peptides (WRWYCR, KWWCRW, and related peptides) were subsequently found to be bactericidal, very likely due to their causing the accumulation of DNA breaks and inhibiting chromosome segregation (18; C. Gunderson and A. Segall, unpublished data). In vivo, however, the D-amino acid forms of the peptides (wrwycr and kwwcrw) were more potent than their L-form counterparts, presumably because they resist peptidases (18).The question remains whether these peptides block sitespecific recombination and accumulate HJ inside bacterial cells. Int is the archetype of a large family of site-specific recombinases that use a tyrosine nucleophile for sequential transesterification reactions. The LT2 strain of Salmonella enterica serovar Typhimurium has four naturally occurring prophages (bacteriophages integrated in its chromosome): Gifsy-1, Gifsy-2, Fels-1, and Fels-2 (11, 15, 36). Each of these prophages encodes an Int-like tyrosine recombinase and can be induced to excise and replicate in a manner very similar to that of phage lambda. DNA damage is the predominant signal that leads to ...

The genome for the marine pseudotemperate member of the Siphoviridae HSIC has been sequenced using a combination of linker amplification library construction, restriction digest library construction, and primer walking. HSIC enters into a pseudolysogenic relationship with its host, Listonella pelagia, characterized by sigmoidal growth curves producing >10 9 cells/ml and >10 11 phage/ml. The genome (37,966 bp; G؉C content, 44%) contained 47 putative open reading frames (ORFs), 17 of which had significant BLASTP hits in GenBank, including a ␤ subunit of DNA polymerase III, a helicase, a helicase-like subunit of a resolvasome complex, a terminase, a tail tape measure protein, several phage-like structural proteins, and 1 ORF that may assist in host pathogenicity (an ADP ribosyltransferase). The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. This evidence is consistent with a headful packaging mechanism similar to that of Salmonella phage P22 and Shigella phage Sf6. Because none of the phage-like ORFs were closely related to any existing phage sequences in GenBank (i.e., none more than 62% identical and most <25% identical at the amino acid level), HSIC is unique among phages that have been sequenced to date. These results further emphasize the need to sequence phages from the marine environment, perhaps the largest reservoir of untapped genetic information.

Antibiotics with novel and/or multiple targets are highly desirable in the face of the steady rise of clinical antibiotic resistance. We have screened and identified small molecules, typified by the compound TPI1609-10 (aka SM10), with antibiotic activity against both gram-positive and gram-negative bacteria. SM10 was screened in vitro to bind branched Holliday junction intermediates of homologous recombination and tyrosine recombinase-mediated recombination; thus, the cellular targets of the small molecules were expected to include the RuvABC Holliday junction resolvasome and the XerCD complex involved in proper segregation of replicated chromosomes to daughter cells. SM10 indeed induces DNA damage and filamentation in E. coli. However, SM10 also induces envelope stress and causes increased production of intracellular reactive oxygen species. In addition, SM10 has similar effects to endogenously-induced envelope stress via overproducing outer membrane proteins (OmpC and OmpF), which also induces the SOS response, chromosome fragmentation, and production of reactive oxygen species. The synergy between SM10, and cerulenin, a fatty acid synthesis inhibitor, together with the SM10 hypersensitivity of cpx and rpoE mutants, further support that SM10's mode of action damages membrane damage. The lethality of SM10 treatment and of OmpC overproduction are observed in both aerobically- and anaerobically-grown cells, and is accompanied by substantial DNA damage even anaerobically. Thus, only some DNA damage is due to reactive oxygen. We propose that membrane depolarization and the potential reduction in intracellular pH, leading to abasic site formation, cause a substantial amount of the DNA damage associated with both SM10 treatment and endogenous envelope stress. While it is difficult to completely exclude effects related to envelope damage as the sources of DNA damage, trapping intermediates associated with DNA repair and chromosome segregation pathways remains very likely. Thus SM10 may have distinct but synergistic modes of action.

Eleven Bacillus isolates from the surface and subsurface waters of the Gulf of Mexico were examined for their capacity to sporulate and harbor prophages. Occurrence of sporulation in each isolate was assessed through decoyinine induction, and putative lysogens were identified by prophage induction by mitomycin C treatment. No obvious correlation between ability to sporulate and prophage induction was found. Four strains that contained inducible virus-like particles (VLPs) were shown to sporulate. Four strains did not produce spores upon induction by decoyinine but contained inducible VLPs. Two of the strains did not produce virus-like particles or sporulate significantly upon induction. Isolate B14905 had a high level of virus-like particle production and a high occurrence of sporulation and was further examined by genomic sequencing in an attempt to shed light on the relationship between sporulation and lysogeny. In silico analysis of the B14905 genome revealed four prophage-like regions, one of which was independently sequenced from a mitomycin C-induced lysate. Based on PCR and transmission electron microscopy (TEM) analysis of an induced phage lysate, one is a noninducible phage remnant, one may be a defective phage-like bacteriocin, and two were inducible prophages. One of the inducible phages contained four putative transcriptional regulators, one of which was a SinR-like regulator that may be involved in the regulation of host sporulation. Isolates that both possess the capacity to sporulate and contain temperate phage may be well adapted for survival in the oligotrophic ocean.Lysogeny and sporulation are strategies for phage and host survival, respectively, under adverse conditions. During both processes, the genome of the phage or bacterium is replicated into a form, prophage or endospore, that increases its survival (14). The initiation of both lysogeny and sporulation involves the repression and activation of promoters that are regulated by feedback from their gene products. In coliphage , cI binds to the lytic promoter P R during lysogeny while cro binds to P R during a virulent infection (39). SinR binds to the vegetative promoter P V in the Sin operon under normal cell conditions, and SpoA activates the sporulation promoter (27). The tertiary structures of the DNA-binding domain of SinR from Bacillus subtilis and CI and Cro from the Escherichia coli phage 434 are nearly identical (25). Recent work has also demonstrated that CIII and sporulation control protein SpoVM are both inhibited by the FtsH protease (21). These structural and functional similarities indicate a possible evolutionary relationship between prophage induction and sporulation. Previous work has indicated a possible link between phages and sporulation. Meijer et al. (28) found that lytic development of the virulent Bacillus phage 29 was repressed in sporulating cells through inhibition of transcription of early phage genes by SpoA. A phage-encoded sigma factor in the B. anthracis virulent phage Fah was negatively controlled by a sp...