Nonspecific Adherence byActinobacillus actinomycetemcomitansRequires Genes Widespread inBacteriaandArchaea

Abstract

The gram-negative coccobacillus, Actinobacillus actinomycetemcomitans, is the putative agent for localized juvenile periodontitis, a particularly destructive form of periodontal disease in adolescents. This bacterium has also been isolated from a variety of other infections, notably endocarditis. Fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms, a property likely to be critical for colonization of teeth and other surfaces. Here we report the identification of a locus of seven genes required for nonspecific adherence of A. actinomycetemcomitans to surfaces. The recently developed transposon IS903kan was used to isolate mutants of the rough clinical isolate CU1000 that are defective in tight adherence to surfaces (Tad ؊ ). Unlike wild-type cells, Tad ؊ mutant cells adhere poorly to surfaces, fail to form large autoaggregates, and lack long, bundled fibrils. Nucleotide sequencing and genetic complementation analysis revealed a 6.7-kb region of the genome with seven adjacent genes (tadABCDEFG) required for tight adherence. The predicted TadA polypeptide is similar to VirB11, an ATPase involved in macromolecular transport. The predicted amino acid sequences of the other Tad polypeptides indicate membrane localization but no obvious functions. We suggest that the tad genes are involved in secretion of factors required for tight adherence of A. actinomycetemcomitans. Remarkably, complete and highly conserved tad gene clusters are present in the genomes of the bubonic plague bacillus Yersinia pestis and the human and animal pathogen Pasteurella multocida. Partial tad loci also occur in strikingly diverse Bacteria and Archaea. Our results show that the tad genes are required for tight adherence of A. actinomycetemcomitans to surfaces and are therefore likely to be essential for colonization and pathogenesis. The occurrence of similar genes in a wide array of microorganisms indicates that they have important functions. We propose that tad-like genes have a significant role in microbial colonization.

Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification

“…We made a Ϫ1 frame shift at nucleotide 846 in the sequence where similarity of the predicted product with other TadA proteins broke down to restore a separate 450-aa protein product whose C terminus was similar to other TadA proteins. Haemophilus aphrophilus tadA was sequenced after PCR amplification by using primers designed for A. actinomycetemcomitans (27). We attempted to include the functionally analogous cytoplasmic NTPases of type III secretion, but we were unable to convincingly align these with the putative NTPases of type II͞IV secretion.…”

“…Preliminary (27). A compilation of intein sequences is found in the New England BioLabs Intein Database (http:͞͞www.neb.com͞inteins).…”

“…We recently described a cluster of genes (tadABCDEFG) in Actinobacillus actinomycetemcomitans required for nonspecific, tenacious adherence of the bacterium to surfaces (27). The predicted product of the tadA gene showed significant sequence similarity to the putative NTPases of type II and type IV secretion systems, and recent work has confirmed that TadA hydrolyzes ATP (M. K. Battacharjee, S.C.K., and D.H.F., unpublished data).…”

“…The predicted product of the tadA gene showed significant sequence similarity to the putative NTPases of type II and type IV secretion systems, and recent work has confirmed that TadA hydrolyzes ATP (M. K. Battacharjee, S.C.K., and D.H.F., unpublished data). ORFs bearing significant sequence similarities to tadA are present in many other Bacteria and Archaea (27)(28)(29).…”

tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

“…Clinical isolates autoaggregate and secrete long, bundled pili (Flp fibrils) (Holt et al, 1980;Scannapieco et al, 1987;Rosan et al, 1988;Inoue et al, 1998;Kachlany et al, 2001b;Henderson et al, 2003;Fine et al, 2006). In vitro A. actinomycetemcomitans can produce rare, stable, nonadherent, and nonaggregative genetic variants that form smooth, large colonies and fail to produce Flp fibrils (Wyss, 1989;Inouye et al, 1990;Fine et al, 1999Fine et al, , 2006Kachlany et al, 2000Kachlany et al, , 2001aHenderson et al, 2003).…”