RNA sequencing result summary.

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.

小RNA（small RNA，sRNA）可与全局分子伴侣调控蛋白Hfq结合，正向或负向调控细菌的基因表达。本研究鉴定并初步表征了可结合Hfq的昏睡嗜血杆菌（Histophilus somni）小RNA。研究人员通过抗Hfq抗体进行免疫共沉淀结合小RNA测序，分离并鉴定了该菌中与Hfq结合的小RNA。对小RNA样本的序列分析共鉴定出100个潜在小RNA，其中16个仅存在于致病菌株2336中，而非致病菌株129Pt中未检测到这些小RNA。生物信息学分析显示，小RNA HS9、HS79和HS97可结合众多疑似与毒力/生物膜形成相关的基因。此外，对基因组中小RNA区域的多序列比对分析显示，HS9和HS97可与σ54（Sigma 54）相互作用——σ54是一种与细菌诸多重要性状（包括运动性、毒力及生物膜形成）相关的转录因子。研究采用Northern印迹法（Northern blotting）测定了这些小RNA的近似分子量、表达丰度以及是否存在加工修饰过程。通过体外转录合成小RNA并结合重组Hfq进行电泳迁移率变动分析，证实了部分候选小RNA可与Hfq结合。采用RNA连接酶介导的cDNA末端快速扩增技术（RNA ligase-mediated rapid amplification of cDNA ends，RLM-RACE）结合克隆与测序，确定了候选小RNA的精确转录起始位点。本研究是首次针对昏睡嗜血杆菌小RNA开展的相关研究，结果表明这类小RNA可能在毒力与生物膜形成过程中发挥重要的调控作用。