| Popis: |
Background 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 first identified and then partially characterized to initiate understanding their role in regulation of virulence factors, which is unknown in H. somni. Results The Hfq-associated sRNAs in H. somni were first identified by co-immunoprecipitation using anti-Hfq antibody, followed by sRNA sequencing. Sequence analysis of the sRNA samples identified 180 putative sRNAs, out of which 17 were present in pathogenic strain 2336, but not in non-pathogenic strain 129Pt. Bioinformatic analyses depicted that the sRNAs HS9, HS79, and HS97 bound to the 5′-untranslated region (5′-UTR) of many genes involved in virulence/biofilm formation. Furthermore, multi-sequence alignment of these sRNA candidates revealed that these sRNAs 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 alternate splice products of sRNAs. Two bands of ~ 115 bp and ~ 60 bp were identified for sRNA HS9, sRNA HS79 had three bands of ~ 80 bp, ~ 130 bp and ~ 300 bp, and two bands of ~ 140 bp and ~ 200 bp were identified for HS97. All three 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 were determined by RNA ligase-mediated rapid amplification of cDNA ends, followed by cloning and sequencing. Conclusions In this study, a number of Hfq-associated sRNAs were identified and characterized in the bovine pathogen, H. somni 2336. Analysis of these sRNAs showed that they may be driven by environmental factors and mayave important regulatory roles in H. somni virulence and biofilm formation. |
