L neighbors and potential eukaryotic predators.Rough RGVC Isolates Carry Unique

L neighbors and potential eukaryotic predators.Rough RGVC Isolates Carry Unique vasH SequencesWe previously showed that the global transcriptional activator VasH is essential for expression of hcp and other T6SS genes. As the rough isolates failed to synthesize Hcp (Figure 3), we tested whether these isolates carried a nonfunctional vasH allele. The 1594 nucleotide-long vasH sequences of V52 and RGVC isolates were PCR-amplified and their polypeptide sequences aligned. The rough RGVC 1655472 isolates were missing a 25033180 guanine in codon 157 (DG157) which resulted in a frameshift. To include vasH of the rough isolates in our comparative analysis, we restored the vasH Title Loaded From File reading frame by in-silico insertion of G157. We found that all RGVC VasH sequences aligned with V52 and N16961 as well as with each other (Figure 5). Therefore, vasH is conserved in environmental (RGVC), pandemic (N16961), and endemic (V52) V. cholerae strains. The repaired vasH open reading frame closely resembled vasH from N16961 with only two unique substitutions (Q278L and T456I). Smooth RGVC isolate DL4211 carried an intact VasH gene identical to N16961; DL4215 differed from N16961 and V52 by three and four residues, respectively (Table 3). Substitutions of histidine to aspartic acid at position 116 (H116D) and threonine to alanine at position 449 (T449A) appear to be common substitutions that are also present in N16961 (Figure 5). In conclusion, RGVC isolates carry a VasH gene related to the El Tor version with the characteristic D116 and A449 residues (Figure 5). However, rough V. cholerae isolates carried a nonsense mutation and are Title Loaded From File likely to produce a truncated 63 amino acid-long VasH mutant protein.Smooth RGVC Isolates Use Their T6SS for Intraspecific CompetitionV. cholerae O37 strain V52 kills E. coli and S. Typhimurium, but is unable to kill other V. cholerae, including the O1 serogroup N16961 (El Tor) and O395 (classical biotype) strains [6]. Accordingly, the T6SS+ isolates V52, DL4211 and DL4215 also exhibited immunity, because we did not observe a decline in viable CFUs when we recovered these isolates from single-isolate spots on LB agar plates after a 4-hour incubation (data not shown). We hypothesized that V. cholerae employs an immunity system that provides protection against T6SS-mediated toxicity. A functional link between T6SS and toxin/antitoxin systems has been established in Pseudomonas aeruginosa and Burkholderia species [28,29], which employ antitoxin proteins to counteract T6SS effectors [28]. VCA0124, an open reading frame downstream of the T6SS effector gene vgrG3 (VCA0123), has been implicated as an antitoxin gene in V. cholerae [30]. As RGVCs killed close relatives such as V. harveyi (Figure 7), we wondered if the RGVC isolates have the ability to kill each other. We hypothesized that if RGVC isolates use different toxins (and antitoxins), the T6SS might be used for intraspecific competition. We predicted that immunity of an RGVC isolate would be lost when approached by a V. cholerae bacterium with a different set of T6SS toxins to which the former lacks the corresponding antitoxin gene. To test this hypothesis, we mixed V52, DL4211, and DL4215 (predators) with smooth and rough RGVC isolates as prey bacteria. To eliminate the killing activity of smooth T6SS+ prey, we used vasK-deficient mutants with a disabled T6SS as prey. Rough wild-type RGVC isolates were used as prey since they do not express Hcp (Figure 3)VasH Complementation in Rough RGVC IsolatesWe.L neighbors and potential eukaryotic predators.Rough RGVC Isolates Carry Unique vasH SequencesWe previously showed that the global transcriptional activator VasH is essential for expression of hcp and other T6SS genes. As the rough isolates failed to synthesize Hcp (Figure 3), we tested whether these isolates carried a nonfunctional vasH allele. The 1594 nucleotide-long vasH sequences of V52 and RGVC isolates were PCR-amplified and their polypeptide sequences aligned. The rough RGVC 1655472 isolates were missing a 25033180 guanine in codon 157 (DG157) which resulted in a frameshift. To include vasH of the rough isolates in our comparative analysis, we restored the vasH reading frame by in-silico insertion of G157. We found that all RGVC VasH sequences aligned with V52 and N16961 as well as with each other (Figure 5). Therefore, vasH is conserved in environmental (RGVC), pandemic (N16961), and endemic (V52) V. cholerae strains. The repaired vasH open reading frame closely resembled vasH from N16961 with only two unique substitutions (Q278L and T456I). Smooth RGVC isolate DL4211 carried an intact VasH gene identical to N16961; DL4215 differed from N16961 and V52 by three and four residues, respectively (Table 3). Substitutions of histidine to aspartic acid at position 116 (H116D) and threonine to alanine at position 449 (T449A) appear to be common substitutions that are also present in N16961 (Figure 5). In conclusion, RGVC isolates carry a VasH gene related to the El Tor version with the characteristic D116 and A449 residues (Figure 5). However, rough V. cholerae isolates carried a nonsense mutation and are likely to produce a truncated 63 amino acid-long VasH mutant protein.Smooth RGVC Isolates Use Their T6SS for Intraspecific CompetitionV. cholerae O37 strain V52 kills E. coli and S. Typhimurium, but is unable to kill other V. cholerae, including the O1 serogroup N16961 (El Tor) and O395 (classical biotype) strains [6]. Accordingly, the T6SS+ isolates V52, DL4211 and DL4215 also exhibited immunity, because we did not observe a decline in viable CFUs when we recovered these isolates from single-isolate spots on LB agar plates after a 4-hour incubation (data not shown). We hypothesized that V. cholerae employs an immunity system that provides protection against T6SS-mediated toxicity. A functional link between T6SS and toxin/antitoxin systems has been established in Pseudomonas aeruginosa and Burkholderia species [28,29], which employ antitoxin proteins to counteract T6SS effectors [28]. VCA0124, an open reading frame downstream of the T6SS effector gene vgrG3 (VCA0123), has been implicated as an antitoxin gene in V. cholerae [30]. As RGVCs killed close relatives such as V. harveyi (Figure 7), we wondered if the RGVC isolates have the ability to kill each other. We hypothesized that if RGVC isolates use different toxins (and antitoxins), the T6SS might be used for intraspecific competition. We predicted that immunity of an RGVC isolate would be lost when approached by a V. cholerae bacterium with a different set of T6SS toxins to which the former lacks the corresponding antitoxin gene. To test this hypothesis, we mixed V52, DL4211, and DL4215 (predators) with smooth and rough RGVC isolates as prey bacteria. To eliminate the killing activity of smooth T6SS+ prey, we used vasK-deficient mutants with a disabled T6SS as prey. Rough wild-type RGVC isolates were used as prey since they do not express Hcp (Figure 3)VasH Complementation in Rough RGVC IsolatesWe.