Department of Microbiology, Faculty of Science, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
Microbiology (Reading). 2013 Mar;159(Pt 3):475-492. doi: 10.1099/mic.0.062117-0. Epub 2013 Jan 3.
Legionella pneumophila, an intracellular parasite of protozoa, possesses a distinct dimorphic life cycle that alternates between the vegetative replicative form and the resilient but highly infectious cyst form. Previously, temporally expressed heterodimeric integration host factor (IHF) was shown to be required for differentiation into the cyst form. However, the precise regulatory mechanisms controlling the expression of IHF have not been identified. Microplate kinetic assays with GFP reporter promoter fusion constructs in wild-type, Δihf, ΔrpoS and ΔletA mutant strain backgrounds were employed to assess differences in expression levels of ihfA, ihfB, rsmY and rsmZ. Loss of IHF, RsmY and RsmZ expression in various mutant strain backgrounds was confirmed by quantitative PCR. Here we report that the stationary phase sigma factor RpoS is a positive regulator of IHF, whereas IHF appears to act as a positive autoregulator assisting RpoS. Bioinformatic analyses identified a set of IHF binding sites upstream of one RpoS binding site in the promoter region for both ihfA and ihfB. Recombinant IHF protein bound ihfA and ihfB promoter regions in vitro, confirming the functionality of these IHF binding sites that may assist in the bending of the promoter DNA to facilitate transcription activation of ihfA and ihfB by RpoS. Interestingly, the consensus binding site for IHF is very similar to that of the two-component response regulator LetA. LetA negatively regulates transcription of ihfA and ihfB, implying titrational regulatory control by LetA and IHF. Along with LetA, IHF was found to positively regulate expression of the non-coding regulatory RNAs RsmY and RsmZ responsible for the de-repression of CsrA-repressed transcripts associated with cyst formation, and coordinated post-exponential virulent phenotypes. Taken together, these observations indicate that IHF may have more of an integral role in the global regulatory system governing the transition from replicative to cyst forms than previously thought.
嗜肺军团菌是一种原生动物的细胞内寄生虫，具有独特的二态生命周期，在营养复制形式和有弹性但高度感染的囊形式之间交替。以前，已经显示出时间表达的异二聚体整合宿主因子（IHF）对于分化为囊形式是必需的。然而，控制 IHF 表达的精确调节机制尚未确定。在野生型、Δihf、ΔrpoS 和 ΔletA 突变株背景下，使用 GFP 报告启动子融合构建的微量板动力学测定来评估 ihfA、ihfB、rsmY 和 rsmZ 的表达水平差异。通过定量 PCR 确认了 IHF、RsmY 和 RsmZ 在各种突变株背景下的表达缺失。在这里，我们报告静止期 σ 因子 RpoS 是 IHF 的正调控因子，而 IHF 似乎作为一个正自调控因子协助 RpoS。生物信息学分析鉴定了一个 IHF 结合位点集，位于 ihfA 和 ihfB 启动子区域的一个 RpoS 结合位点上游。重组 IHF 蛋白在体外结合 ihfA 和 ihfB 启动子区域，证实了这些 IHF 结合位点的功能，这些结合位点可能有助于弯曲启动子 DNA，促进 RpoS 对 ihfA 和 ihfB 的转录激活。有趣的是，IHF 的保守结合位点与双组分响应调节剂 LetA 非常相似。LetA 负调控 ihfA 和 ihfB 的转录，暗示 LetA 和 IHF 的滴定调节控制。与 LetA 一起，IHF 被发现正向调节非编码调节 RNA RsmY 和 RsmZ 的表达，这负责解除与囊形成相关的 CsrA 抑制转录物的抑制，并协调指数后毒力表型。总之，这些观察结果表明，与之前的想法相比，IHF 在调节从复制到囊形式的过渡的全局调控系统中可能具有更重要的作用。
