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The marine pathogenic bacterium  Photobacterium damselae  subsp.  damselae  causes septicemia in marine animals and in humans. The pPHDD1 plasmid-encoded hemolysins damselysin (Dly) and phobalysin P (PhlyP), and the chromosome-encoded hemolysin phobalysin C (PhlyC) constitute its main virulence factors. However, the mechanisms by which expression of these three hemolysins is regulated remain unknown. Here we report the isolation of a mini-Tn 10  transposon mutant which showed a strong impairment in its hemolytic activity. The transposon disrupted a putative sensor histidine kinase gene  vda_000600  ( rstB ), which together with  vda_000601  ( rstA ) is predicted to encode a putative two-component regulatory system. This system showed to be homologous to the  Vibrio cholerae  CarSR/VprAB and  Escherichia coli  RstAB systems. Reconstruction of the mutant by allelic exchange of  rstB  showed equal impairment in hemolysis, and complementation with a plasmid expressing  rstAB  restored hemolysis to wild-type levels. Remarkably, we demonstrated by promoter expression analyses that the reduced hemolysis in the  rstB  mutant was accompanied by a strong decrease in transcription activities of the three hemolysin genes  dly  (damselysin),  hlyA pl   (phobalysin P) and  hlyA ch   (phobalysin C). Thus, RstB, encoded in the small chromosome, regulates plasmid and chromosomal virulence genes. We also found that reduced expression of the three virulence genes correlated with a strong decrease in virulence in a sea bass model, demonstrating that RstB constitutes a master regulator of the three  P. damselae  subsp.  damselae  hemolysins and plays critical roles in the pathogenicity of this bacterium. This study represents the first evidence of a direct role of a RstAB-like system in the regulation of bacterial toxins.

rstB Regulates Expression of the Photobacterium damselae subsp. damselae Major Virulence Factors Damselysin, Phobalysin P and Phobalysin C