Juárez-Rodríguez María Dolores, Torres-Escobar Ascención, Demuth Donald R
Research Group in Oral Health and Systemic Disease, University of Louisville School of Dentistry, 501 S. Preston Street, Louisville, KY 40202, USA.
Microbiology (Reading). 2014 Dec;160(Pt 12):2583-2594. doi: 10.1099/mic.0.083501-0. Epub 2014 Sep 15.
The QseBC two-component system plays a pivotal role in regulating virulence and biofilm growth of the oral pathogen Aggregatibacter actinomycetemcomitans. We previously showed that QseBC autoregulates the ygiW-qseBC operon. In this study, we characterized the promoter that drives ygiW-qseBC expression. Using lacZ transcriptional fusion constructs and 5'-rapid amplification of cDNA ends, we showed that ygiW-qseBC expression is driven by a promoter that initiates transcription 53 bases upstream of ygiW and identified putative cis-acting promoter elements, whose function was confirmed using site-specific mutagenesis. Using electrophoretic mobility shift assays, two trans-acting proteins were shown to interact with the ygiW-qseBC promoter. The QseB response regulator bound to probes containing the direct repeat sequence CTTAA-N6-CTTAA, where the CTTAA repeats flank the -35 element of the promoter. The ygiW-qseBC expression could not be detected in A. actinomycetemcomitans ΔqseB or ΔqseBC strains, but was restored to WT levels in the ΔqseBC mutant when complemented by single copy chromosomal insertion of qseBC. Interestingly, qseB partially complemented the ΔqseBC strain, suggesting that QseB could be activated in the absence of QseC. QseB activation required its phosphorylation since complementation did not occur using qseB(pho-), encoding a protein with the active site aspartate substituted with alanine. These results suggest that QseB is a strong positive regulator of ygiW-qseBC expression. In addition, integration host factor (IHF) bound to two sites in the promoter region and an additional site near the 5' end of the ygiW ORF. The expression of ygiW-qseBC was increased by twofold in ΔihfA and ΔihfB strains of A. actinomycetemcomitans, suggesting that IHF is a negative regulator of the ygiW-qseBC operon.
QseBC双组分系统在调节口腔病原体伴放线聚集杆菌的毒力和生物膜生长中起关键作用。我们之前表明QseBC可自动调节ygiW - qseBC操纵子。在本研究中，我们对驱动ygiW - qseBC表达的启动子进行了表征。使用lacZ转录融合构建体和5'-cDNA末端快速扩增，我们表明ygiW - qseBC表达由一个在ygiW上游53个碱基处起始转录的启动子驱动，并鉴定了推定的顺式作用启动子元件，其功能通过位点特异性诱变得到证实。使用电泳迁移率变动分析，显示有两种反式作用蛋白与ygiW - qseBC启动子相互作用。QseB应答调节因子与含有直接重复序列CTTAA - N6 - CTTAA的探针结合，其中CTTAA重复序列位于启动子的 - 35元件两侧。在伴放线聚集杆菌ΔqseB或ΔqseBC菌株中未检测到ygiW - qseBC表达，但当通过qseBC的单拷贝染色体插入对ΔqseBC突变体进行互补时，表达恢复到野生型水平。有趣的是，qseB部分互补了ΔqseBC菌株，表明QseB在没有QseC的情况下可以被激活。QseB的激活需要其磷酸化，因为使用编码活性位点天冬氨酸被丙氨酸取代的蛋白质的qseB(pho - )进行互补时未发生互补。这些结果表明QseB是ygiW - qseBC表达的强正调节因子。此外，整合宿主因子（IHF）与启动子区域的两个位点以及ygiW ORF 5'端附近的另一个位点结合。在伴放线聚集杆菌的ΔihfA和ΔihfB菌株中，ygiW - qseBC的表达增加了两倍，表明IHF是ygiW - qseBC操纵子的负调节因子。
