Perez B A, Planet P J, Kachlany S C, Tomich M, Fine D H, Figurski D H
Department of Microbiology, College of Physicians and Surgeons, Columbia University, 701 West 168th St., New York, NY 10032, USA.
J Bacteriol. 2006 Sep;188(17):6361-75. doi: 10.1128/JB.00496-06.
The tad locus of Actinobacillus actinomycetemcomitans encodes a molecular transport system required for tenacious, nonspecific adherence to surfaces and formation of extremely strong biofilms. This locus is dedicated to the biogenesis of Flp pili, which are required for colonization and virulence. We have previously shown that 11 of the 14 tad locus genes are required for adherence and Flp pilus production. Here, we present genetic and phylogenetic analyses of flp-2, tadV, and rcpB genes in biofilm formation. We show that tadV, predicted to encode prepilin peptidase, is required for adherence. In contrast, targeted insertional inactivation of flp-2, a gene closely related to the prepillin gene flp-1, did not abrogate biofilm formation. Expression studies did not detect Flp2-T7 protein under standard laboratory conditions. We present phylogenetic data showing that there is no significant evidence for natural selection in the available flp-2 sequences from A. actinomycetemcomitans, suggesting that flp-2 does not play a significant role in the biology of this organism. Mutants with insertions at the 3' end of rcpB formed biofilms equivalent to wild-type A. actinomycetemcomitans. Surprisingly, 5' end chromosomal insertion mutants in rcpB were obtained only when a wild-type copy of the rcpB gene was provided in trans or when the Tad secretion system was inactivated. Together, our results strongly suggest that A. actinomycetemcomitans rcpB is essential in the context of a functional tad locus. These data show three different phenotypes for the three genes.
伴放线放线杆菌的tad位点编码一种分子转运系统，该系统对于牢固、非特异性地黏附于表面以及形成极强的生物膜是必需的。此位点专门负责Flp菌毛的生物合成，而Flp菌毛对于定殖和毒力是必需的。我们之前已经表明，14个tad位点基因中的11个对于黏附和Flp菌毛的产生是必需的。在此，我们展示了flp - 2、tadV和rcpB基因在生物膜形成中的遗传学和系统发育分析。我们发现，预测编码前菌毛肽酶的tadV对于黏附是必需的。相比之下，与前菌毛基因flp - 1密切相关的基因flp - 2的靶向插入失活并未消除生物膜的形成。表达研究在标准实验室条件下未检测到Flp2 - T7蛋白。我们展示的系统发育数据表明，在来自伴放线放线杆菌的现有flp - 2序列中，没有显著的自然选择证据，这表明flp - 2在该生物体的生物学过程中不发挥重要作用。在rcpB的3'端插入的突变体形成的生物膜与野生型伴放线放线杆菌相当。令人惊讶的是，只有当rcpB基因的野生型拷贝通过反式提供或当Tad分泌系统失活时，才能获得rcpB的5'端染色体插入突变体。总之，我们的结果强烈表明，在功能性tad位点的背景下，伴放线放线杆菌的rcpB是必需的。这些数据展示了这三个基因的三种不同表型。
