The bacterium Vibrio cholerae, like other human pathogens that reside in environmental reservoirs, survives predation by unicellular eukaryotes. Strains of the O1 and O139 serogroups cause cholera, whereas non-O1͞non-O139 strains cause human infections through poorly defined mechanisms. Using Dictyostelium discoideum as a model host, we have identified a virulence mechanism in a non-O1͞non-O139 V. cholerae strain that involves extracellular translocation of proteins that lack N-terminal hydrophobic leader sequences. Accordingly, we have named these genes ''VAS'' genes for virulence-associated secretion, and we propose that these genes encode a prototypic ''type VI'' secretion system. We show that vas genes are required for cytotoxicity of V. cholerae cells toward Dictyostelium amoebae and mammalian J774 macrophages by a contact-dependent mechanism. A large number of Gram-negative bacterial pathogens carry genes homologous to vas genes and potential effector proteins secreted by this pathway (i.e., hemolysin-coregulated protein and VgrG). Mutations in vas homologs in other bacterial species have been reported to attenuate virulence in animals and cultured macrophages. Thus, the genes encoding the VAS-related, type VI secretion system likely play an important conserved function in microbial pathogenesis and represent an additional class of targets for vaccine and antimicrobial drug-based therapies.Dictyostelium discoideum ͉ type VI secretion ͉ virulence-associated secretion C holera is a severe, life-threatening diarrheal disease caused by Vibrio cholerae strains of the O1 and O139 serogroups. In contrast, non-O1, non-O139 strains of V. cholerae are primarily associated with isolated cases of extra-intestinal infection or gastroenteritis. An exception to this pattern was a large outbreak of a cholera-like illness that occurred in 1968 in Sudan, where an O37 strain of V. cholerae caused 460 cases and 125 deaths (1). The virulence mechanisms of O1 and O139 strains involve the elaboration of extracellular factors such as cholera enterotoxin and toxin coregulated pili. In contrast, the virulence mechanisms used by non-O1, non-O139 strains remain poorly defined (2). Using the social amoeba Dictyostelium discoideum as a model host, we have developed an experimental system designed to identify novel virulence mechanisms from pathogenic non-O1, non-O139 strains.D. discoideum is a eukaryotic organism that seeks out and preys on bacteria through its phagocytic feeding behavior. As such, it has been used as a model eukaryotic cell that mimics a mammalian macrophage in aspects of its cell biology and interaction with microbes. Several environmental pathogenic bacteria, including Legionella pneumophila, Mycobacterium marinum, and Pseudomonas aeruginosa (3), resist Dictyostelium predation by producing factors that either kill amoebae or allow successful intracellular survival and multiplication. In these cases, the same virulence mechanisms operative against mammalian cells have also been implicated in resistance to Dictyosteli...

T4-like myoviruses are ubiquitous, and their genes are among the most abundant documented in ocean systems. Here we compare 26 T4-like genomes, including 10 from non-cyanobacterial myoviruses, and 16 from marine cyanobacterial myoviruses (cyanophages) isolated on diverse Prochlorococcus or Synechococcus hosts. A core genome of 38 virion construction and DNA replication genes was observed in all 26 genomes, with 32 and 25 additional genes shared among the non-cyanophage and cyanophage subsets, respectively. These hierarchical cores are highly syntenic across the genomes, and sampled to saturation. The 25 cyanophage core genes include six previously described genes with putative functions (psbA, mazG, phoH, hsp20, hli03, cobS), a hypothetical protein with a potential phytanoyl-CoA dioxygenase domain, two virion structural genes, and 16 hypothetical genes. Beyond previously described cyanophage-encoded photosynthesis and phosphate stress genes, we observed core genes that may play a role in nitrogen metabolism during infection through modulation of 2-oxoglutarate. Patterns among non-core genes that may drive niche diversification revealed that phosphorus-related gene content reflects source waters rather than host strain used for isolation, and that carbon metabolism genes appear associated with putative mobile elements. As well, phages isolated on Synechococcus had higher genome-wide %G+C and often contained different gene subsets (e.g. petE, zwf, gnd, prnA, cpeT) than those isolated on Prochlorococcus. However, no clear diagnostic genes emerged to distinguish these phage groups, suggesting blurred boundaries possibly due to cross-infection. Finally, genome-wide comparisons of both diverse and closely related, co-isolated genomes provide a locus-to-locus variability metric that will prove valuable for interpreting metagenomic data sets.

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