The Role Of Alternate Sigma Factors In The Transmission Cycle Of B. Burgdorferi

伯氏杆菌在自然界中存在于两种截然不同的环境中，硬蜱和哺乳动物宿主。它在这些非常不同的环境中适应和生存的能力归因于它能够感知温度、pH、细胞密度、氧气和/或接触宿主因素的变化，并相应地改变基因表达。细菌调节转录启动的能力允许基因在适当的时间协调表达。以往的报道表明，调节这些反应的中心是Sigma(由rpos编码)和Sigma54(由rpoN编码，也称为NTRA)。此外，依赖于Sigma54的Sigma表达还负责关键毒力因子的表达，如外表面蛋白C(OspC)、OspA和核心蛋白结合蛋白C(DbpC)在感染周期中的感染性和传播性所需。 Sigma54-RNA聚合酶全酶(Sigma54-holozyme)识别的启动子相对于转录起始点分别在Sigma4和Sigma12位具有很好的GG和GC双链保守，而不是Sigma70类启动子中典型的-35/-10盒。普遍地，依赖于Sigma54的转录被证明需要激活蛋白，其中许多是双组分系统的反应调节蛋白。在大多数情况下，Sigma54激活蛋白结合位于启动子转录起始点上游100-200bp的增强子样元件。对伯氏杆菌基因组的分析表明，RRP2(BBO763)是一个预测的依赖于Sigma54的激活子，由N-末端的受体结构域、中心的ATPase结构域和C-末端的DNA结合域组成。 Sigma54-全酶激活剂的活性受环境信号的调节。#M54-全酶的许多激活剂是双组分调节系统中的反应调节器，这些蛋白质的磷酸化导致它们的激活。这些反应调节因子被它们的同源蛋白组氨酸激酶磷酸化，以响应环境信号。一旦被磷酸化，反应调节器就会激活其他基因的转录。伯氏杆菌中Sigma54-全酶的激活子RRP2(由RRP2编码)也是一个双组分系统的反应调节因子，RRP2是一个操纵子，带有编码其同源蛋白组氨酸激酶HK2(由HK2编码)的基因。依赖Sigma54的Sigma激活需要反应调节因子RRP2(BB0763)。在凝胶迁移率改变分析中，纯化的重组RRP2不与伯氏杆菌rpos上游的DNA结合，RRP2-delta123也不结合，RRP2-delta123是RRP2的截短成分活性形式。RRP2-delta123在大肠杆菌中对rpos-lacZ报告基因的转录激活依赖于sigma54，但不需要rpos启动子上游的DNA序列。同样，用Burgdorferi中的CAT报告基因进行的定量RT-PCR实验表明，RRP2不需要依赖于sigma54的rpos启动子上游的DNA序列来激活rpos P/O-CAT报告结构的转录。综上所述，这些发现表明，与大多数依赖sigma54的启动子的表达不同，RRP2不利用增强子样序列来激活rpos的转录。定量RT-PCR结果表明，在伯氏杆菌培养进入稳定期时，rpoN是rpoS表达所必需的，HK2是rpoS在此期间最佳表达所必需的，但不是绝对必需的。这表明，除Hk2外，另一个组氨酸激酶或小分子可以作为RRP2的磷酸供体，提供多种信号通路来调节RPOS和毒力相关蛋白的表达，如OspC。我们这方面的研究的重点是进一步表征这一涉及伯氏杆菌Sigma和Sigma54的调节级联反应，并确定促进该细菌在扁虱中肠和人类宿主中生存的细胞外信号。

B. burgdorferi is maintained in nature in two very distinct environments, the Ixodes tick and mammalian hosts. Its ability to adapt and survive in these very different environments is attributed to its ability to sense changes in temperature, pH, cell density, oxygen and/or exposure to host factors and alter gene expression accordingly. The capability of bacteria to regulate transcription initiation allows for the coordinated expression of genes at appropriate times. Previous reports have demonstrated that central to the regulation of these responses are SigmaS (encoded by rpoS) and Sigma54 (encoded by rpoN also known as ntrA). In addition, Sigma54-dependent expression of SigmaS is responsible for the expression of key virulence factors e.g., outer surface protein C (OspC), OspA and decorin-binding protein C (DbpC) required for infectivity and transmission during the infective cycle. Promoters recognized by Sigma54-RNA polymerase holoenzyme (Sigma54-holoenzyme) have well conserved GG and GC doublets at Sigma4 and Sigma12 positions, respectively, relative to the transcriptional start site, instead of the typical -35/-10 boxes observed in Sigma70 class promoters. Universally, Sigma54-dependent transcription has been shown to require activator proteins, many of which are response regulators of two-component systems. In most cases, Sigma54-activator proteins bind enhancer-like elements located 100-200 bp upstream of the transcriptional start site of the promoter. Analysis of the genome of B. burgdorferi identified Rrp2 (BBO763) as a predicted Sigma54-dependent activator consisting of an N-terminal receiver domain, a central ATPase domain and a C-terminal DNA-binding domain. The activities of activators of Sigma54-holoenzyme are regulated in response to environmental signals. Many of the activators of #m54-holoenzyme are response regulators in two-component regulatory systems, and phosphorylation of these proteins results in their activation. These response regulators are phosphorylated by their cognate protein histidine kinases in response to an environmental signal. Once phosphorylated, the response regulator activates transcription of other genes. An activator of Sigma54-holoenzyme in B. burgdorferi, Rrp2 (encoded by rrp2), is also a response regulator of a two-component system and rrp2 is in an operon with a gene encoding its cognate protein histidine kinase, hk2 (encoded by hk2). The Sigma54-dependent activation of SigmaS requires response regulator, Rrp2 (BB0763). Purified recombinant Rrp2 did not bind to the DNA upstream of B. burgdorferi rpoS in gel-mobility shift assays, nor did Rrp2-delta123, a truncated constitutively active form of Rrp2. Transcriptional activation of a rpoS-lacZ reporter gene in E. coli by Rrp2-delta123 was dependent on sigma54 but did not require DNA sequences upstream of the rpoS promoter. Similarly, quantitative RT-PCR experiments using a cat reporter gene in B. burgdorferi indicated that DNA sequences upstream of the sigma54-dependent rpoS promoter were not needed by Rrp2 to activate transcription of the rpoS P/O-cat reporter construct. Taken together, these findings suggest that unlike expression from most sigma54-dependent promoters, Rrp2 does not utilize an enhancer-like sequence to activate transcription of rpoS. Finally, quantitative RT-PCR showed that rpoN is required for expression of rpoS in B. burgdorferi as cultures enter stationary phase, and that hk2 is needed for optimal expression of rpoS during this time but is not absolutely essential. This suggests that, in addition to Hk2, another histidine kinase or small molecular weight molecule can serve as a phosphate donor to Rrp2, providing multiple signaling pathways for modulating the expression of RpoS and virulence related proteins, such as OspC. The focus of this aspect of our research is to further characterize this regulatory cascade involving SigmaS and Sigma54 in B. burgdorferi and to determine the extracellular signals that promote the survival of the bacterium in the tick midgut and in a human host.