Identification and initial characterization of Hfq-associated sRNAs in Histophilus somni strain 2336

Small RNAs (sRNA), in association with the global chaperone regulator Hfq, positively or negatively regulate gene expression in bacteria. For this study, Histophilus somni sRNAs that bind to Hfq were identified and then partially characterized. The Hfq-associated sRNAs in H. somni were isolated and identified by co-immunoprecipitation using anti-Hfq antibody, followed by sRNA sequencing. Sequence analysis of the sRNA samples identified 100 putative sRNAs, out of which 16 were present in pathogenic strain 2336, but not in non-pathogenic strain 129Pt. Bioinformatic analyses suggested that the sRNAs HS9, HS79, and HS97 could bind to many genes putatively involved in virulence/biofilm formation. Furthermore, multi-sequence alignment of the sRNA regions in the genome revealed that HS9 and HS97 could interact with sigma 54, which is a transcription factor linked to important bacterial traits, including motility, virulence, and biofilm formation. Northern blotting was used to determine the approximate size, abundance and any processing events attributed to the sRNAs. Selected sRNA candidates were confirmed to bind Hfq, as determined by electrophoretic mobility shift assays using sRNAs synthesized by in vitro transcription and recombinant Hfq. The exact transcriptional start site of the sRNA candidates was determined by RNA ligase-mediated rapid amplification of cDNA ends, followed by cloning and sequencing. This is the first investigation of H. somni sRNAs that show they may have important regulatory roles in virulence and biofilm formation.