Analysis of a growth-phase-regulated two-component regulatory system in the periodontal pathogen Treponema denticola
Analysis of a growth-phase-regulated two-component regulatory system in the periodontal pathogen Treponema denticola
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DOI：
10.1128/jb.00046-08
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发表时间：
2008-09-15
影响因子：
3.2
通讯作者：
Marconi, Richard T.
中科院分区：
文献类型：
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作者：
Frederick, Jesse R.;Rogers, Elizabeth A.;Marconi, Richard T.
目前对齿密螺旋体和其他口腔螺旋体的全球调控网络一无所知。在这份报告中，我们评估了齿纹夜蛾可能的双组分调控系统(TCS)的性质和潜在的磷转移能力，该系统由开放阅读框tde0032(一种传感器激酶)和tde0033(一种反应调节因子)的产物组成，此后分别命名为ATCs和AtcR。通过PCR和DNA序列分析，证明atcs和atcR在齿纹夜蛾分离株中广泛分布和保守。逆转录-聚合酶链式反应(RT-PCR)分析表明，这些基因是共转录的，也可能作为一个更大的操纵子的一部分表达，该操纵子包括几个侧翼基因。利用5‘末端快速扩增的分析确定了这些操纵子的转录起始点，并提供了证据表明其中一些基因可能是从内部启动子独立转录的。实时定量RT-PCR和Western印迹分析显示，atcRs在生长后期显著上调，表明其表达依赖于生长阶段。最后，利用重组蛋白对AtcRS系统的磷光传递能力进行了评估和验证。ATCs被发现经历了自动磷酸化，并将磷酸盐转移到ATcR。这些分析首次描述了口腔螺旋体中的功能TCS，并为这些重要细菌的转录调控机制提供了洞察。
Nothing is currently known regarding the global regulatory networks of Treponema denticola and other oral spirochetes. In this report, we assess the properties and potential phosphotransfer capability of a putative two-component regulatory system (TCS) of T. denticola that is formed by the products of open reading frames tde0032 (a sensor kinase) and tde0033 (a response regulator), henceforth designated AtcS and AtcR, respectively. Using PCR and DNA sequence analyses, atcS and atcR were demonstrated to be widely distributed and conserved among T. denticola isolates. Reverse transcription-PCR (RT-PCR) analyses revealed that these genes are cotranscribed and may also be expressed as part of a larger operon that includes several flanking genes. Analyses using 5' rapid amplification of cDNA ends identified the transcriptional start sites for these operons and provided evidence that some of these genes may be independently transcribed from internal promoters. Real-time RT-PCR and Western blot analysis revealed significant upregulation of atcRS during late-stage growth, indicating growth-phase-dependent expression. Lastly, the phosphorelay capability of the AtcRS system was assessed and demonstrated using recombinant proteins. AtcS was found to undergo autophosphorylation and to transfer phosphate to AtcR. These analyses represent the first description of a functional TCS in an oral spirochetes and provide insight into the transcriptional regulatory mechanisms of these important bacteria.