Ure 2) depends on a functional T6SS, we performed killing assays and plaque assays with DL4211DvasK and DL4215DvasK as a predator. VasK is an inner membrane protein believed to provide the energy for T6SS-mediated secretion[26,27]. VasK is, therefore, crucial for a functional T6SS. As shown in figure 4A, parental V52, DL4211, and DL4215 constitutively produced and secreted Hcp, while deletion of vasK blocked secretion but not synthesis of Hcp. To complement the vasK chromosomal deletion, vasK from V52 was cloned downstream of an arabinose-inducible promoter in the plasmid pBAD24 and introduced into DL4211DvasK (DL4211DvasK/ pvasK) and DL4215DvasK (DL4215DvasK/pvasK). Trans complementation of vasK restored Hcp secretion in V52 and the two smooth order HIF-2��-IN-1 isolates (Figure 4A). To assess the role of T6SS in killing E. coli, we incubated E. coli with various V. cholerae isolates and determined the number of surviving E. coli after a 4-hourCompetition Mechanisms of V. choleraeFigure 8. T6SS-dependent competition among V. cholerae isolates. (A ) Smooth V. cholerae isolates successfully competed with each other and outcompeted the rough isolates in a T6SS-dependent manner. All combinations among the isolates and their isogenic vasK mutants were tested in a killing assay: Predator- and prey-V. cholerae were mixed in a 10:1 ratio and incubated for 4 hours at 37uC. Bacterial spots were resuspended, serially diluted, and plated on selective media to determine the number of surviving prey. The number of surviving prey in the presence of T6SS+ or T6SS2 predator are shown. (D) Arrows indicate the competitive relationship between isolates such that the arrow points from the predator towards the prey. Arrow thickness indicates relative killing efficiency. T6SS-dependence of the killing phenotype was confirmed by employing the vasKdeficient predator of each V. cholerae isolate examined. To avoid killing of the predator, vasK-deficient prey of smooth T6SS+ isolates were used. The average and 80-49-9 standard deviations of two independent experiments, each performed in duplicates, are shown. doi:10.1371/journal.pone.0048320.gincubation at 37uC (Figure 4B). VasK mutants of V52, DL4211, and DL4215 lost their ability to kill E. coli, but providing vasK in trans restored virulence. Furthermore, amoebae were unable toform plaques in lawns of V52, DL4211, and DL4215, but did so in lawns of V52DvasK, DL4211DvasK and DL4215DvasK (Figure 4C). Complemented isolates, V52DvasK/pvasK, DL4211DvasK/pvasKCompetition Mechanisms of V. choleraeTable 4. Secretion and virulence phenotypes of RGVC isolates.Isolate V52 DL2111 DL2112 DL4211 DLHcp Pellet +++ 2 2 +++ +++Hcp Supernatant +++ 2 2 +++ +++Eukaryotic Killing +++ 2 2 ++ +++Prokaryotic Killing +++ 2 2 +++ +++doi:10.1371/journal.pone.0048320.tand DL4215DvasK/pvasK, regained virulence towards D. discoideum (Figure 4C). Although the wild-type phenotype of DL4211 could not be fully complemented by episomal expression of vasK, the complemented phenotype is statistically significant (unpaired ttest, p = 0.0116). We conclude that smooth RGVC 
isolates conferred T6SS-mediated virulence towards E. coli and D. discoideum, demonstrating that the virulence phenotype described in Figures 1 and 2 is T6SS-dependent.Smooth RGVC Isolates Use Their T6SS to Compete with Natural NeighborsBecause RGVC isolates with active T6SSs kill E. coli, we hypothesized that RGVC isolates use their T6SS to compete with other bacteria in their environmental niche. To test t.Ure 2) depends on a functional T6SS, we performed killing assays and plaque assays with DL4211DvasK and DL4215DvasK as a predator. VasK is an inner membrane protein believed to provide the energy for T6SS-mediated secretion[26,27]. VasK is, therefore, crucial for a functional T6SS. As shown in figure 4A, parental V52, DL4211, and DL4215 constitutively produced and secreted Hcp, while deletion of vasK blocked secretion but not synthesis of Hcp. To complement the vasK chromosomal deletion, vasK from V52 was cloned downstream of an arabinose-inducible promoter in the plasmid pBAD24 and introduced into DL4211DvasK (DL4211DvasK/ pvasK) and DL4215DvasK (DL4215DvasK/pvasK). Trans complementation of vasK restored Hcp secretion in V52 and the two smooth isolates (Figure 4A). To assess the role of T6SS in killing E. coli, we incubated E. coli with various V. cholerae isolates and determined the number of surviving E. coli after a 4-hourCompetition Mechanisms of V. choleraeFigure 8. T6SS-dependent competition among V. cholerae isolates. (A ) Smooth V. cholerae isolates successfully competed with each other and outcompeted the rough isolates in a T6SS-dependent manner. All combinations among the isolates and their isogenic vasK mutants were tested in a killing assay: Predator- and prey-V. cholerae were mixed in a 10:1 ratio and incubated for 4 hours at 37uC. Bacterial spots were resuspended, serially diluted, and plated on selective media to determine the number of surviving prey. The number of surviving prey in the presence of T6SS+ or T6SS2 predator are shown. (D) Arrows indicate the competitive relationship between isolates such that the arrow points from the predator towards the prey. Arrow thickness indicates relative killing efficiency. T6SS-dependence of the killing phenotype was confirmed by employing the vasKdeficient predator of each V. cholerae isolate examined. To avoid killing of the predator, vasK-deficient prey of smooth T6SS+ isolates were used. The average and standard deviations of two independent experiments, each performed in 
duplicates, are shown. doi:10.1371/journal.pone.0048320.gincubation at 37uC (Figure 4B). VasK mutants of V52, DL4211, and DL4215 lost their ability to kill E. coli, but providing vasK in trans restored virulence. Furthermore, amoebae were unable toform plaques in lawns of V52, DL4211, and DL4215, but did so in lawns of V52DvasK, DL4211DvasK and DL4215DvasK (Figure 4C). Complemented isolates, V52DvasK/pvasK, DL4211DvasK/pvasKCompetition Mechanisms of V. choleraeTable 4. Secretion and virulence phenotypes of RGVC isolates.Isolate V52 DL2111 DL2112 DL4211 DLHcp Pellet +++ 2 2 +++ +++Hcp Supernatant +++ 2 2 +++ +++Eukaryotic Killing +++ 2 2 ++ +++Prokaryotic Killing +++ 2 2 +++ +++doi:10.1371/journal.pone.0048320.tand DL4215DvasK/pvasK, regained virulence towards D. discoideum (Figure 4C). Although the wild-type phenotype of DL4211 could not be fully complemented by episomal expression of vasK, the complemented phenotype is statistically significant (unpaired ttest, p = 0.0116). We conclude that smooth RGVC isolates conferred T6SS-mediated virulence towards E. coli and D. discoideum, demonstrating that the virulence phenotype described in Figures 1 and 2 is T6SS-dependent.Smooth RGVC Isolates Use Their T6SS to Compete with Natural NeighborsBecause RGVC isolates with active T6SSs kill E. coli, we hypothesized that RGVC isolates use their T6SS to compete with other bacteria in their environmental niche. To test t.
