The toxR gene of Vibrio (Listonella) anguillarum controls expression of the major outer membrane proteins but not virulence in a natural host model

To examine the hypothesis that the ancestral role of the toxR gene in the f amily Vibrionaceae is control of the expression of outer membrane protein ( OMP)-encoding genes for adaptation to environmental change, we investigated the role of the toxR gene in Vibrio anguillarum, an important fish pathoge n. The toxR gene of V. angullarum (Va-toxR) was cloned from strain PT-87050 isolated from diseased ayu (Plecoglossus altivelis), and the sequence was analyzed. The toxR sequence was 63 to 51% identical to those reported for o ther species of the family Vibrionaceae. Distribution of the Va-toxR gene s equence in V. anguillarum strains of various serotypes was confirmed by usi ng DNA probe and PCR methods. An isogenic toxR mutant of V anguillarum PT-2 4, isolated from diseased ayu, was constructed by using an allelic exchange method. The wild-type strain and the toxR mutant did not differ in the abi lity to produce a protease(s) and a hemolysin(s) or in pathogenicity for ay u when examined by the intramuscular injection and immersion methods. A 35- kDa major OMP was not produced by the toxR mutant. However, a 46-kDa OMP wa s hardly detected in the wild-type strain but was produced as the major ONO by the toxR mutant. For the toxR mutant, the MICs of two P-lactam antibiot ics were higher and the minimum bactericidal concentration of sodium dodecy l sulfate was lower than for the wild-type strain. Analysis of the N-termin al amino acid sequences of the 35- and 46-kDa OMPs indicated that these pro teins are the porin-like OMPs and are related to the toxR-regulated major O MPs of the family Vibrionaceae. The results indicate that the toxR gene is not involved in virulence expression in V. anguillarum PT-24 and that toxR regulation of major OMPs is universal in the family Vibrionaceae. These res ults support the hypothesis that the ancestral role of the toxR gene is reg ulation of OMP gene expression and that only in some Vibrio species has Tox R been appropriated for the regulation of a virulence gene(s).