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

B。burgdorferi在自然界中维持在两种非常不同的环境中，硬蜱和哺乳动物宿主。其在这些非常不同的环境中适应和生存的能力归因于其感知温度、pH、细胞密度、氧气和/或暴露于宿主因子的变化并相应地改变基因表达的能力。 细菌调节转录起始的能力允许基因在适当的时间协调表达。先前的报道已经证明，这些反应的调节中心是SigmaS（由rpoS编码）和Sigma 54（由rpoN编码，也称为ntrA）。此外，SigmaS的Sigma 54依赖性表达负责关键毒力因子的表达，外表面蛋白C（OspC）、OspA和核心蛋白聚糖结合蛋白C（DbpC）是感染周期中感染和传播所需的。 被Sigma 54-RNA聚合酶全酶（Sigma 54-holoenzyme）识别的启动子分别在相对于转录起始位点的Sigma 4和Sigma 12位置处具有良好保守的GG和GC双联体，而不是在Sigma 70类启动子中观察到的典型的-35/-10盒。 一般来说，Sigma 54依赖性转录已被证明需要激活蛋白，其中许多是双组分系统的反应调节剂。在大多数情况下，Sigma 54激活蛋白结合位于启动子的转录起始位点上游100-200 bp的增强子样元件。B的基因组分析。burgdorferi将Rrp 2（BBO 763）鉴定为预测的Sigma 54依赖性激活剂，由N末端接收器结构域、中央ATP酶结构域和C末端DNA结合结构域组成。 Sigma 54全酶的激活剂的活性响应于环境信号而被调节。 #m54-全酶的许多激活剂是双组分调节系统中的反应调节剂，并且这些蛋白质的磷酸化导致它们的激活。 这些反应调节剂被它们的同源蛋白组氨酸激酶磷酸化以响应环境信号。 一旦磷酸化，反应调节因子激活其他基因的转录。 B中的Sigma 54全酶的激活剂。burgdorferi，Rrp 2（由rrp 2编码），也是双组分系统的应答调节剂，并且rrp 2与编码其同源蛋白组氨酸激酶hk 2（由hk 2编码）的基因一起位于操纵子中。SigmaS的Sigma 54依赖性激活需要反应调节因子Rrp 2（BB 0763）。纯化的重组Rrp 2不与B上游的DNA结合。在凝胶迁移率变动分析中，也没有Rrp 2-delta 123，Rrp 2的截短组成型活性形式。rpoS-lacZ报告基因在大肠杆菌中的转录激活Rrp 2-delta 123对大肠杆菌的诱导依赖于sigma 54，但不需要rpoS启动子上游的DNA序列。类似地，在B. burgdorferi指出，Rrp 2不需要σ 54依赖性rpoS启动子上游的DNA序列来激活rpoS P/O-cat报告基因构建体的转录。总之，这些发现表明，与大多数sigma 54依赖性启动子的表达不同，Rrp 2不利用增强子样序列来激活rpoS的转录。 最后，定量RT-PCR显示rpoN是B中rpoS表达所必需的。因此，在培养物进入稳定期时，hk 2对于rpoS的最佳表达是必需的，但不是绝对必需的。这表明，除了Hk 2之外，另一种组氨酸激酶或小分子量分子可以作为Rrp 2的磷酸供体，为调节RpoS和毒力相关蛋白（如OspC）的表达提供多种信号传导途径。我们这方面研究的重点是进一步表征B中涉及SigmaS和Sigma 54的调节级联。并确定促进细菌在蜱中肠和人类宿主中存活的细胞外信号。

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.