Identification of target transcripts regulated by small RNA RyhB homologs in Salmonella: RyhB-2 regulates motility phenotype

Salmonella encodes numerous small RNAs that play important roles in the cellular regulatory network. Recently we have shown that two RyhB homologs of Salmonella Typhimurium, RyhB-1 and RyhB-2, have distinct sets of overlapping targets. In this study, we used microarrays for comprehensive identification of the target genes regulated by these sRNAs. Microarray analysis was conducted with cDNAs generated from total RNAs of three mutants (ΔryhB-1, ΔryhB-2, and ΔryhB-1ΔryhB-2) and wild type S. Typhimurium strain. Candidate targets were first identified by selecting genes with increased transcript signals in a double deletion strain as compared to the wild type. Then those candidates were clustered into the following 4 groups according to the patterns of increased transcript signals as compared to the wild type: (1) greater with both single deletions, (2) reduced or unchanged with both single deletions, and (3) greater only with either the ryhB-1 or (4) ryhB-2 deletion. We found that the transcripts of the three genes (flgJ, cheY, and fliF), involved in motility and chemotaxis, are down-regulated only by RyhB-2. Motility assay confirmed that the ryhB-2 deletion increased motility, which was then reduced back to wild type level by overexpression of ryhB-2 gene. Collectively, our results suggest the regulatory roles of RyhB-1 and RyhB-2 in Salmonella on multiple cellular pathways, including motility, at a post-transcriptional level.