Negative osmoregulation of the Salmonella ompS1 porin gene independently of OmpR in an hns background

The ompS1 gene encodes a quiescent porin in Salmonella enterica serovars Typhi and Typhimurium. By using random mariner transposon mutagenesis, mutations that caused derepression of ompS1 expression were isolated, one in S. enterica serovar Typhi and two in S. enterica serovar Typhimurium. All of them mapped in the hns gene in the region coding for the carboxy terminus of the H-NS nucleoid protein. The derepressed ompS1 expression was subject to negative regulation at high osmolarity, both in the presence and in the absence of OmpR. This observation was possible due to the fact that there are two promoters: P1, which is OmpR dependent, and P2, which does not require OmpR for activation (rather, OmpR represses P2). The sequences upstream from position -88, a region previously shown to be involved in the negative regulation of ompS1, can form a static bend, and the integrity of this region was required for function and binding of H-NS and for osmoregulation, as determined with gene reporter fusions of different lengths and with a 31-bp deletion mutant. This is consistent with the notion that this region determines a structure required for repression. Hence, ompS1 shares negative regulation by H-NS with other loci, such as the bgl operon and the ade gene.     

副溶血弧菌拟核相关蛋白H-NS间接抑制VP1687-1686的启动子区转录

为了探讨副溶血性弧菌拟核相关蛋白H-NS对Ⅲ型分泌系统(T3SS) VP1687-1686基因位点的转录调控,本研究提取副溶血弧菌hns突变株(Δhns)和野生株(WT)的总RNA,采用引物延伸实验研究靶基因的转录起始位点,并根据产物的丰度判断H-NS对靶基因的调控关系;采用实时定量RT-PCR研究靶基因mRNA在WT和Δhns中转录丰度,以判定H-NS对靶基因的转录调控关系;将靶基因启动子区域DNA序列克隆至lacZ基因上游,将重组质粒转入WT和Δhns中,得到相应的LacZ菌株,通过LacZ报告基因融合实验研究H-NS对靶基因的调控关系;用PCR扩增靶基因的启动子区DNA序列,并纯化His-H-NS蛋白,通过凝胶阻滞实验(EMSA)研究His-H-NS是否对靶基因启动子区具有直接的结合作用。研究结果显示,T3SS的VP1687-1686只含有一个转录起始位点,位于翻译起始位点上游82 bp处,且H-NS能够抑制其转录活性,但不能直接结合到VP1687-1686区的启动子区。另外,H-NS对calR的转录无调控作用,His-H-NS也不能结合到其启动子区。本研究的结果初步说明,H-NS能够间接抑制VP1687-1686的转录,该抑制机制与CalR无关联。