A chromosomally encoded regulator is required for expression of the Yersinia enterocolitica inv gene and for virulence

The primary invasion factor of Yersinia enterocolitica, invasin, is encoded by inv. inv expression is regulated in response to pH, growth phase and te mperature. In vitro, inv is maximally expressed at 26 degrees C, pH 8.0, or 37 degrees C, pH 5.5, in early stationary phase. At 37 degrees C, pH 8.0, inv is weakly expressed. To identify which gene(s) are required for inv reg ulation, we screened for transposon insertions that decreased expression of an inv-'phoA chromosomal reporter at 26 degrees C, Of 30 000 mutants scree ned, two were identified that had negligible inv expression in all conditio ns tested. Both of these independent mutants had an insertion into the same gene, designated rovA (regulator of virulence). RovA has 77% amino acid id entity to-the Salmonella typhimurium transcriptional regulator SlyA. Comple mentation with the wild-type rovA allele restores wild-type inv expression as monitored by Western blot analysis, tissue culture invasion assay and al kaline phosphatase assay. There is also a significant decrease in invasin l evels in bacteria recovered from mice infected with the rovA mutant; theref ore, RovA regulates inv expression in vivo as well as in vitro. In the mous e infection model, an inv mutant has a wild-type LD50, even though the kine tics of infection is changed. In contrast, the rovA mutant has altered kine tics, as well as a 70-fold increase in the LD50 compared with wild type. Fu rthermore, because the rovA mutant is attenuated in the mouse model, this s uggests that RovA regulates other virulence factors in addition to inv. Ana lysis of other proposed virulence factors such as Ail, YadA and the Yop pro teins shows no regulatory role for RovA. The more severe animal phenotype c ombined with the lack of impact on known virulence genes aside from inv sug gests RovA regulates potentially novel virulence genes of Y. enterocolitica during infection.