Kram Karin E, Hovel-Miner Galadriel A, Tomich Mladen, Figurski David H
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
J Bacteriol. 2008 Jun;190(11):3859-68. doi: 10.1128/JB.00128-08. Epub 2008 Mar 28.
The tad (tight adherence) locus of Aggregatibacter actinomycetemcomitans includes genes for the biogenesis of Flp pili, which are necessary for bacterial adhesion to surfaces, biofilm formation, and pathogenesis. Although studies have elucidated the functions of some of the Tad proteins, little is known about the regulation of the tad locus in A. actinomycetemcomitans. A promoter upstream of the tad locus was previously identified and shown to function in Escherichia coli. Using a specially constructed reporter plasmid, we show here that this promoter (tadp) functions in A. actinomycetemcomitans. To study expression of the pilin gene (flp-1) relative to that of tad secretion complex genes, we used Northern hybridization analysis and a lacZ reporter assay. We identified three terminators, two of which (T1 and T2) can explain flp-1 mRNA abundance, while the third (T3) is at the end of the locus. T1 and T3 have the appearance and behavior of intrinsic terminators, while T2 has a different structure and is inhibited by bicyclomycin, indicating that T2 is probably Rho dependent. To help achieve the appropriate stoichiometry of the Tad proteins, we show that a transcriptional-termination cascade is important to the proper expression of the tad genes. These data indicate a previously unreported mechanism of regulation in A. actinomycetemcomitans and lead to a more complete understanding of its Flp pilus biogenesis.
伴放线聚集杆菌的紧密黏附（tad）位点包含弗林蛋白菌毛生物合成所需的基因，这些基因对于细菌黏附于表面、生物膜形成及致病作用而言是必需的。尽管已有研究阐明了部分Tad蛋白的功能，但对于伴放线聚集杆菌中tad位点的调控机制却知之甚少。之前已鉴定出tad位点上游的一个启动子，并证明其在大肠杆菌中发挥功能。在此，我们利用一个特别构建的报告质粒表明，该启动子（tadp）在伴放线聚集杆菌中也发挥功能。为了研究菌毛蛋白基因（flp-1）相对于tad分泌复合体基因的表达情况，我们采用了Northern杂交分析及lacZ报告基因检测法。我们鉴定出三个终止子，其中两个（T1和T2）能够解释flp-1 mRNA的丰度，而第三个（T3）位于该位点的末端。T1和T3具有固有终止子的外观及行为特征，而T2具有不同的结构且受双环霉素抑制，这表明T2可能依赖于Rho因子。为了有助于实现Tad蛋白的适当化学计量，我们发现转录终止级联反应对于tad基因的正确表达至关重要。这些数据揭示了伴放线聚集杆菌中一种此前未报道的调控机制，并使我们对其弗林蛋白菌毛生物合成有了更全面的理解。
