Regulation of the cytotoxic enterotoxin gene in Aeromonas hydrophila: Characterization of an iron uptake regulator

The cytotoxic enterotoxin Act from a diarrheal isolate, SSU, of Aeromonas h ydrophila is aerolysin related and crucial to the pathogenesis of Aeromonas infections. To elucidate the role of environmental signals which influence the expression of the cytotoxic enterotoxin gene (act), a portion of the a ct gene, including the putative promoter region, was fused in frame to a tr uncated alkaline phosphatase gene (phoA) of Escherichia coli. The act::phoA reporter gene was then introduced into the chromosome of A. hydrophila by using the suicide vector pJQ200SK, allowing the fusion protein to be secret ed out into the culture medium. Western blot analysis demonstrated the pres ence of a correctly size 110-kDa fusion protein in the culture supernatant, which reacted with both anti-Act and anti-alkaline phosphatase antibodies. Based on alkaline phosphatase (PhoA) activity in the culture supernatant, we demonstrated that calcium significantly increased the activity of the ac t promoter but that glucose and iron repressed its activity in a dose-depen dent fashion. The act promoter exhibited optimal activity at pH 7.0 and at 37 degreesC, and maximal PhoA activity was noted when the culture was aerat ed. Using a Vibrio cholerae iron uptake regulator gene (fur) as a probe, a 2.6-kb SalI/HindIII DNA fragment from an A. hydrophila chromosome was clone d and sequenced. The DNA sequence revealed a 429-bp open reading frame that exhibited 69% homology at the DNA level with the fur gene and 79% homology at the amino acid level with the iron uptake regulator (Fur) protein of V. cholerae. Complementation experiments demonstrated that the A. hydrophila fur gene could restore iron regulation in an E. coli fur-minus mutant. Usin g the suicide vector pDMS197, we generated a fur isogenic mutant of wild-ty pe A. hydrophila SSU. Northern blot analysis data indicated that the repres sion in the transcription of the act gene by iron was relieved in the fur i sogenic mutant. Further, iron regulation in the fur isogenic mutant of A. h ydrophila could be restored by complementation. These results are important in understanding the regulation of the act gene under in vivo conditions.