RNA-Seq Analysis Identifies New Genes Regulated by the Histone-Like Nucleoid Structuring Protein (H-NS) Affecting <i>Vibrio cholerae</i> Virulence, Stress Response and Chemotaxis

The histone-like nucleoid structuring protein (H-NS) functions as a transcriptional silencer by binding to AT-rich sequences at bacterial promoters. However, H-NS repression can be counteracted by other transcription factors in response to environmental changes. The identification of potential toxic factors, the expression of which is prevented by H-NS could facilitate the discovery of new regulatory proteins that may contribute to the emergence of new pathogenic variants by anti-silencing. Vibrio cholerae hns mutants of the El Tor biotype exhibit altered virulence, motility and environmental stress response phenotypes compared to wild type. We used an RNA-seq analysis approach to determine the basis of the above hns phenotypes and identify new targets of H-NS transcriptional silencing. H-NS affected the expression of 18% of all predicted genes in a growth phase-dependent manner. Loss of H-NS resulted in diminished expression of numerous genes encoding methyl-accepting chemotaxis proteins as well as chemotaxis toward the attractants glycine and serine. Deletion of hns also induced an endogenous envelope stress response resulting in elevated expression of rpoE encoding the extracytoplamic sigma factor E (σE). The RNA-seq analysis identified new genes directly repressed by H-NS that can affect virulence and biofilm development in the El Tor biotype cholera bacterium. We show that H-NS and the quorum sensing regulator HapR silence the transcription of the vieSAB three-component regulatory system in El Tor biotype V. cholerae. We also demonstrate that H-NS directly represses the transcription of hlyA (hemolysin), rtxCA (the repeat in toxin or RTX), rtxBDE (RTX transport) and the biosynthesis of indole. Of these genes, H-NS occupancy at the hlyA promoter was diminished by overexpression of the transcription activator HlyU. We discuss the role of H-NS transcriptional silencing in phenotypic differences exhibited by V. cholerae biotypes.类组蛋白拟核结构蛋白（histone-like nucleoid structuring protein，H-NS）可通过结合细菌启动子区域的AT富集序列，发挥转录沉默因子的功能。然而，H-NS介导的转录抑制可响应环境变化，被其他转录因子所拮抗。鉴定受H-NS抑制表达的潜在毒性因子，将有助于发现新型调控蛋白——这类蛋白可通过抗沉默作用参与新致病变种的形成。与野生型菌株相比，埃尔托生物型霍乱弧菌（Vibrio cholerae）的hns基因突变株表现出毒力、运动性及环境应激反应表型的改变。本研究采用RNA测序（RNA-seq）分析方法，旨在解析上述hns突变株表型的分子机制，并鉴定H-NS转录沉默的新型靶基因。H-NS可呈生长阶段依赖性，调控全基因组18%的预测基因的表达。H-NS的缺失会导致大量编码甲基接受趋化蛋白的基因表达下调，同时削弱细菌对趋化诱导物甘氨酸与丝氨酸的趋化能力。hns基因的缺失还会诱导内源性包膜应激反应，使编码胞外σ因子E（σE）的rpoE基因表达水平显著升高。本次RNA-seq分析鉴定出了一批受H-NS直接抑制的新型基因，这些基因可影响埃尔托生物型霍乱弧菌的毒力与生物被膜形成能力。本研究证实，H-NS与群体感应调控蛋白HapR可共同沉默埃尔托生物型霍乱弧菌中vieSAB三组分调控系统的转录。本研究同时证实，H-NS可直接抑制hlyA（溶血素，hemolysin）、rtxCA（毒素重复序列或RTX毒素）、rtxBDE（RTX转运系统）的转录，以及吲哚的生物合成。在上述基因中，转录激活因子HlyU的过表达会减弱H-NS在hlyA启动子区域的结合。本研究还讨论了H-NS转录沉默在霍乱弧菌不同生物型表型差异中的作用。