| The gram-negative oral pathogen A. actinomycetemcomitans forms a very strong biofilm that is necessary for virulence. 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. I have shown that at least 12, and probably 13 of the 14-tad locus genes are required for adherence and Flp-pilus production. Here we present molecular genetic analysis of the flp-2, tadV, rcpB, and tadZ genes of A. actinomycetemcomitans. I show that tadV, a predicted prepilin peptidase, is required for adherence. In contrast, targeted insertional inactivation of flp-2, a gene closely related to the prepillin gene flp-1 , does not abrogate biofilm formation. Our studies indicate that flp-2 is a vestigal gene in A. actinomycetemcomitans. Mutants with insertions at the 3' end of rcpB form biofilms equivalent to wild-type A. actinomycetemcomitans. Surprisingly, 5'-end chromosomal insertion mutants in rcpB are only obtained when a wild-type copy of the rcpB gene is provided in trans or when the Tad secretion system was inactivated. Together our results strongly suggest that A. actinomycetemcomitans rcpB is essential for viability in the context of a functional tad locus. Functional characterization of the TadZ protein of A. actinomycetemcomitans revealed that TadZ localizes to the cell poles of A. actinomycetemcomitans and E. coli, which does not harbor a tad locus. We find that TadZ localizes to the cell poles independent of septum formation, cell division, and production of the other Tad proteins. Furthermore, we identified two putative TadZ membrane-targeting domains that are predicted to form amphipathic helices and are required for localization and adherence by A. actinomycetemcomitans. Results from the lambda cl dimerization assay indicated that TadZ forms dimers. The Walker-like A motif is required for the function of TadZ in adherence, but not required for TadZ dimerization or localization. Localization of the TadA protein to the cell poles of A. actinomycetemcomitans provides evidence for localization of the entire Tad apparatus and secretion of Flp pili at the cell poles. |
