College of Veterinary Medicine, Long Island University, Brookville, New York, United States of America.
College of Science, Virginia Tech, Blacksburg, VA, United States of America.
PLoS One. 2023 May 23;18(5):e0286158. doi: 10.1371/journal.pone.0286158. eCollection 2023.
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（sRNA）与全局伴侣调节因子 Hfq 结合，可正向或负向调节细菌中的基因表达。在本研究中，鉴定了与 Hfq 结合的亨氏尼亚菌 sRNA，并对其进行了部分特征分析。通过使用抗 Hfq 抗体进行共免疫沉淀，分离并鉴定了亨氏尼亚菌中的 Hfq 相关 sRNA，随后进行 sRNA 测序。sRNA 样本的序列分析鉴定出 100 个推定的 sRNA，其中 16 个存在于致病性菌株 2336 中，但不存在于非致病性菌株 129Pt 中。生物信息学分析表明，sRNA HS9、HS79 和 HS97 可能与许多假定与毒力/生物膜形成有关的基因结合。此外，对基因组中 sRNA 区域的多序列比对表明，HS9 和 HS97 可以与 sigma 54 相互作用，sigma 54 是与重要细菌特性（包括运动性、毒力和生物膜形成）相关的转录因子。Northern 印迹用于确定 sRNA 的大致大小、丰度和任何归因于 sRNA 的加工事件。通过使用体外转录合成的 sRNA 和重组 Hfq 进行电泳迁移率变动分析，确定了选定的 sRNA 候选物与 Hfq 的结合。通过 RNA 连接酶介导的 cDNA 末端快速扩增，确定了 sRNA 候选物的确切转录起始位点，随后进行克隆和测序。这是首次对亨氏尼亚菌 sRNA 的研究，表明它们可能在毒力和生物膜形成中具有重要的调节作用。
