Table 1.
Bacterial strains and plasmids.
Table 2.
Primers.
Table 3.
RGVC isolates.
Figure 1.
Ability of RGVC isolates to kill E. coli. Rough RGVC isolates DL2111 and DL2112, and smooth RGVC isolates DL4211 and DL4215 were tested for their ability to confer T6SS-mediated prokaryotic killing.
V52 and V52ΔvasK were used as virulent and avirulent controls, respectively. V. cholerae and E. coli were mixed in a 10∶1 ratio and incubated for 4 hours at 37°C. Bacterial spots were resuspended, serially diluted, and plated on E. coli-selective media to determine the number of surviving E. coli. The averages and standard deviations of two independent experiments, each performed in duplicates are shown.
Figure 2.
RGVC isolates with a constitutive T6SS kill D. discoideum.
103 D. discoideum cells were plated with indicated bacteria on SM/5 agar plates that support bacterial but not amoeboid growth. Plaques formed by D. discoideum were counted on the third day of incubation. The graph summarizes the results of two independent experiments. Standard deviations are shown. KP: Klebsiella pneumoniae.
Figure 3.
RGVC isolates differ in T6SS regulation.
Indicated RGVC isolates and V52 (positive control) were cultured to midlogarithmic phase of growth followed by centrifugal separation of pellets and culture supernatants. Supernatant portions were concentrated by TCA precipitation and both fractions were subjected to SDS-PAGE followed by western blotting using the antibodies indicated. Experiments were repeated at least three times with equivalent results.
Figure 4.
Complementation of a vasK null-mutation restores T6SS-dependent secretion and virulence.
(A) VasK-mutants of smooth RGVC isolates carrying a plasmid for arabinose-induced vasK expression were cultured to midlogarithmic phase of growth in the presence or absence of 0.1% arabinose. V52 and the isogenic vasK mutant were used as positive and negative controls, respectively. Pellets and culture supernatants were separated by centrifugation. The supernatant portions were concentrated by TCA precipitation and both fractions were subjected to SDS-PAGE followed by western blotting using the antibodies indicated. (B) Survival of E. coli MG1655 after mixing with V. cholerae. V. cholerae and E. coli were mixed in a 10∶1 ratio and incubated for 4 hours at 37°C before the resulting spots were resuspended, serially diluted, and plated on E. coli-selective media. Data represent the averages of three independent experiments. Standard deviations are included. (C) Survival of D. discoideum after mixing with V. cholerae. D. discoideum was plated with V. cholerae and the number of plaques formed by surviving D. discoideum were counted after a 3-day incubation at 22°C. Data are representative of three independent experiments. Standard deviations are shown.
Figure 5.
Alignment of VasH polypeptide sequences of RGVC isolates.
VasH of V52, N16961, and four RGVC isolates were aligned. In the rough isolates, a guanine was inserted at position 157 of vasH to restore the open reading frame. Colored bars indicate substitutions compared to VasH from V52.
Figure 6.
VasH complementation restores Hcp synthesis but not secretion in rough RGVC isolates.
V. cholerae isolates were transformed with pBAD18-vasH::myc. The isolates were cultured to midlogarithmic phase of growth in the presence or absence of 0.1% arabinose. Pellets and culture supernatants were separated by centrifugation. The supernatant portions were concentrated by TCA precipitation and both fractions were subjected to SDS-PAGE followed by western blotting using the antibodies indicated. Data are representative of three independent experiments.
Figure 7.
RGVC isolates kill bacterial neighbors.
V. cholerae and prey bacteria were mixed in a 10∶1 ratio and incubated on ½ YTSS agar for 4 hours at 30°C. Bacterial spots were resuspended, serially diluted, and plated on selective YTSS agar to determine the number of surviving prey. The average and standard deviations of three independent experiments, each performed in duplicates, are shown.
Figure 8.
T6SS-dependent competition among V. cholerae isolates.
(A–C) 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 37°C. 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 T6SS− 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 vasK-deficient 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.
Table 4.
Secretion and virulence phenotypes of RGVC isolates.