In this study, we cloned and sequenced a virulence-associated gene (vacB) from a clinical isolate SSU of Aeromonas hydrophila. We identified this gene based on our recently annotated genome sequence of the environmental isolate ATCC 7966 T of A. hydrophila and the vacB gene of Shigella flexneri. The A. hydrophila VacB protein contained 798 amino acid residues, had a molecular mass of 90.5 kDa, and exhibited an exoribonuclease (RNase R) activity. The RNase R of A. hydrophila was a cold-shock protein and was required for bacterial growth at low temperature. The vacB isogenic mutant, which we developed by homologous recombination using marker exchange mutagenesis, was unable to grow at 4°C. In contrast, the wild-type (WT) A. hydrophila exhibited significant growth at this low temperature. Importantly, the vacB mutant was not defective in growth at 37°C. The vacB mutant also exhibited reduced motility, and these growth and motility phenotype defects were restored after complementation of the vacB mutant. The A. hydrophila RNase R-lacking strain was found to be less virulent in a mouse lethality model (70% survival) when given by the intraperitoneal route at as two 50% lethal doses (LD 50 ). On the other hand, the WT and complemented strains of A. hydrophila caused 80 to 90% of the mice to succumb to infection at the same LD 50 dose. Overall, this is the first report demonstrating the role of RNase R in modulating the expression of A. hydrophila virulence.A human diarrheal isolate SSU of Aeromonas hydrophila possesses various pathogenic mechanisms that contribute to the overall virulence of this bacterium in the host. Most importantly, our laboratory has described type II, type III, and type VI secretion system effector proteins that are involved in the disease state (gastroenteritis or septicemia) in animal models (11,15,17,21,32,(44)(45)(46)48). However, to better understand the full virulence potential of any pathogen, it is important to identify new pathogenic factors and/or mechanisms that could be involved in their virulence. This is crucial since the expression of different virulence genes could be contributing factors leading to disease depending upon the anatomical niche where the organisms colonize and the environment in those regions which dictates the differential expression of genes (18).Studies by Tobe et al. (50) identified a novel chromosomal gene, vacB, which was required for the production of S. flexneri virulence factors (e.g., IpaB, IpaC, IpaD, and VirG) from the large plasmid (230 kb) of this microorganism. More recently, it was shown that the vacB gene in S. flexneri and Escherichia coli strains encoded the 3Ј-5Ј exoribonuclease RNase R (10). The deduced size of VacB (virulence-associated) protein is approximately 92 kDa, and it is located at 95 min on the E. coli chromosome, a position consistent with the earlier mapping studies of the gene encoding RNase R (10). These investigators provided evidence that RNase R has an essential cell function, in addition to its role in bacterial virulence, an...