Price Family Foundation Institute of Structural Biology, University of Oklahoma, Norman, Oklahoma, USA.
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA.
J Bacteriol. 2018 Nov 6;200(23). doi: 10.1128/JB.00351-18. Print 2018 Dec 1.
The R20291 genome encodes 57 response regulator proteins that, as part of two-component signaling pathways, regulate adaptation to environmental conditions. Genomic and transcriptomic studies in have been limited, due to technical challenges, to the analysis of either high-throughput screens or high-priority targets, such as primary regulators of toxins or spore biology. We present the use of several technically accessible and generally applicable techniques to elucidate the putative regulatory targets of a response regulator, RR_1586, involved in sporulation of the hypervirulent strain R20291. A DNA-binding specificity motif for RR_1586 was determined using a bacterial one-hybrid assay originally developed for transcription factors. Comparative bioinformatics approaches identified and experiments confirmed RR_1586 binding sites upstream of putative target genes, including those that encode phosphate ion transporters, spermidine/putrescine biosynthesis and transport pathways, ABC type transport systems, known regulators of sporulation, and genes encoding spore structural proteins. Representative examples of these regulatory interactions have been tested and confirmed in -based reporter assays. Finally, evidence of possible regulatory mechanisms is also presented. A working model includes self-regulation by RR_1586 and phosphorylation-dependent and -independent DNA binding at low- and high-fidelity binding sites, respectively. Broad application of this and similar approaches is anticipated to be an important catalyst for the study of gene regulation by two-component systems from pathogenic or technically challenging bacteria. spores survive under harsh conditions and can germinate into actively dividing cells capable of causing disease. An understanding of the regulatory networks controlling sporulation and germination in could be exploited for therapeutic advantage. However, such studies are hindered by the challenges of working with an anaerobic pathogen recalcitrant to genetic manipulation. Although two-component response regulators can be identified from genetic sequences, identification of their downstream regulatory networks requires further development. This work integrates experimental and bioinformatic approaches, which provide practical advantages over traditional transcriptomic analyses, to identify the putative regulon of the response regulator RR_1586 by first screening for protein-DNA interactions in and then predicting regulatory outputs in .
R20291 基因组编码 57 种应答调节蛋白，这些蛋白作为双组分信号通路的一部分，调节对环境条件的适应。由于技术挑战，对 的基因组学和转录组学研究仅限于高通量筛选或高优先级目标的分析，例如毒素或孢子生物学的主要调节剂。我们展示了几种技术上可及且普遍适用的技术的使用，以阐明参与高毒力 R20291 菌株孢子形成的应答调节子 RR_1586 的假定调节靶标。使用最初为 转录因子开发的细菌单杂交测定法确定 RR_1586 的 DNA 结合特异性基序。比较生物信息学方法鉴定并 实验证实了 RR_1586 在假定靶基因上游的结合位点，包括那些编码磷酸盐离子转运体、腐胺/亚精胺生物合成和转运途径、ABC 型转运系统、孢子形成的已知调节剂以及编码孢子结构蛋白的基因。这些调控相互作用的代表性例子已在基于 的报告基因测定中进行了测试和验证。最后，还提出了可能的调控机制的证据。一个工作模型包括 RR_1586 的自我调节以及在低保真度和高保真度结合位点处分别依赖和不依赖磷酸化的 DNA 结合。预计广泛应用这种和类似的方法将成为研究致病性或技术上具有挑战性的细菌的双组分系统基因调控的重要催化剂。孢子在恶劣条件下存活，并能萌发生长为能够引起疾病的活跃分裂细胞。了解控制 孢子形成和萌发的调控网络可能会带来治疗优势。然而，由于难以对厌氧病原体进行遗传操作，因此此类研究受到阻碍。尽管可以从遗传序列中鉴定出双组分应答调节子，但需要进一步开发才能鉴定其下游调控网络。这项工作整合了实验和生物信息学方法，通过首先在 中筛选蛋白-DNA 相互作用，然后在 中预测调控输出，为鉴定 应答调节子 RR_1586 的假定调控子提供了实用优势。
