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。     

Swimming motility, a virulence trait of Ralstonia solanacearum, is regulated by FlhDC and the plant host environment

Swimming motility allows the bacterial wilt pathogen Ralstonia solanacearum to efficiently invade and colonize host plants. However, the bacteria are essentially nonmotile once inside plant xylem vessels. To determine how and when motility genes are expressed, we cloned and mutated flhDC, which encodes a major regulator of flagellar biosynthesis and bacterial motility. An flhDC mutant was nonmotile and less virulent than its wild-type parent on both tomato and Arabidopsis; on Arabidopsis, the flhDC mutant also was less virulent than a nonmotile fliC flagellin mutant. Genes in the R. solanacearum motility regulon had strikingly different expression patterns in culture and in the plant. In culture, as expected, flhDC expression depended on PehSR, a regulator of early virulence factors; and, in turn, FlhDC was required for fliC (flagellin) expression. However, when bacteria grew in tomato plants, flhDC was expressed in both wild-type and pehR mutant backgrounds, although PehSR is necessary for motility both in culture and in planta. Both flhDC and pehSR were significantly induced in planta relative to expression levels in culture. Unexpectedly, the fliC gene was expressed in planta at cell densities where motile bacteria were not observed, as well as in a nonmotile flhDC mutant. Thus, expression of flhDC and flagellin itself are uncoupled from bacterial motility in the host environment, indicating that additional signals and regulatory circuits repress motility during plant pathogenesis.     

Two novel flagellar components and H-NS are involved in the motor function of Escherichia coli

A mutation in H-NS results in non-flagellation of Escherichia coli due to a reduced expression of the flhDC master operon. We found that the hns-negative strain restored its flagellation in the presence of flhDC, although the resulting strain was still non-motile. Since the intracelluar levels of motor components MotA, MotB, and FliG in the Deltahns strain were unaltered, the non-motility indicates that H-NS affects flagellar function as well as biogenesis. We obtained an insertion in ycgR, a putative gene encoding a protein of 244 amino acid residues, which suppresses the motility defect of hns-deficient cells. The abnormally low swimming speed of hns mutant cells was fully restored by an insertion in ycgR, as assessed with computer-assisted motion analysis. A similar suppressor phenotype was observed with a multicopy expression of yhjH, a putative gene encoding a polypeptide of 256 amino acid residues. Since the flagella of most hns-deficient cells were not rotating, except a few with reduced speed, the suppression appears to increase the number of rotating flagella as observed with tethered bacteria. The ycgR and yhjH genes contain the consensus sequence found among the class III promoters of the flagellar regulon, and their expression monitored with a lacZ fusion requires FlhDC. These findings suggest that ycgR and yhjH, together with H-NS, are involved in the motor function and constitute new members of the flagellar regulon.     

The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility

The ability to move over and colonize surface substrata has been linked to the formation of biofilms and to the virulence of some bacterial pathogens. Results from this study show that the gastrointestinal pathogen Yersinia enterocolitica can migrate over and colonize surfaces by swarming motility, a form of cooperative multicellular behavior. Immunoblot analysis and electron microscopy indicated that swarming motility is dependent on the same flagellum organelle that is required for swimming motility, which occurs in fluid environments. Furthermore, motility genes such as flgEF, flgMN, flhBA, and fliA, known to be required for the production of flagella, are essential for swarming motility. To begin to investigate how environmental signals are processed and integrated by Y. enterocolitica to stimulate the production of flagella and regulate these two forms of cell migration, the motility master regulatory operon, flhDC, was cloned. Mutations within flhDC completely abolished swimming motility, swarming motility, and flagellin production. DNA sequence analysis revealed that this locus is similar to motility master regulatory operons of other gram-negative bacteria. Genetic complementation and functional analysis of flhDC indicated that it is required for the production of flagella. When flhDC was expressed from an inducible ptac promoter, flagellin production was shown to be dependent on levels of flhDC expression. Phenotypically, induction of the ptac-flhDC fusion also corresponded to increased levels of both swimming and swarming motility.