FIG 6.

Essential roles of VqsA in interbacterial competition activity, biofilm formation, exotoxin production, and virulence. (A) CFU of E. coli before (t = 0) and after (t = 4) a 4-h coculture with the indicated V. alginolyticus predator strains or E. coli alone. The Δhcp2 mutant strain deficient in interbacterial competition capacity was used as a control. The data represent the results from three independent experiments. ***, P < 0.001 by an unpaired two-tailed t test. (B) Biofilm formation in glass tubes after 48 h was quantitatively assayed with crystal violet staining of the biofilm cells. For the colorimetric assay of the released crystal violet from the biofilm cells, the OD₅₇₀ was measured and normalized with the OD₆₀₀ of the cultures. **, P < 0.01 based on Student's t test relative to the WT strain. (C) Western blot analysis was performed to assay Asp yields in the ΔvqsA and vqsA⁺ mutant strains. RNAP was used as the loading control. (D) qRT-PCR analysis of the transcriptional levels of the pep and mviN genes. The WT, ΔvqsA mutant, and vqsA⁺ mutant strains were cultured in LBS for 9 h, and the mRNA transcripts were detected by qRT-PCR; 16S rRNA was selected as a control. ***, P < 0.001, Student's t test relative to the vqsA⁺ mutant strain. (E) Groups of 30 fish were infected with the indicated strains. The number of fish that survived was recorded for 4 days. The P values were calculated using a Kaplan-Meier survival analysis with a log rank test with Prism software (version 6.01).