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Identification of genes coding for exported proteins of Actinobacillus actinomycetemcomitans.

ABSTRACT: Random fusions of genomic DNA fragments to a partial gene encoding a signal sequence-deficient bacterial alkaline phosphatase were utilized to screen for exported proteins of Actinobacillus actinomycetemcomitans in Escherichia coli. Twenty-four PhoA(+) clones were isolated and sequenced. Membrane localization signals in the form of signal sequences were deduced from most of these sequences. Several of the deduced amino acid sequences were found to be homologous to known exported or membrane-associated proteins. The complete genes corresponding to two of these sequences were isolated from an A. actinomycetemcomitans lambda phage library. One gene was found to be homologous to the outer membrane lipoprotein LolB. The second gene product had homology with a Haemophilus influenzae protein and was localized to the inner membrane of A. actinomycetemcomitans.

SUBMITTER: Mintz KP

PROVIDER: S-EPMC97021 | biostudies-literature | 1999 Nov

REPOSITORIES: biostudies-literature

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Identification of genes coding for exported proteins of Actinobacillus actinomycetemcomitans.

Mintz K P KP Fives-Taylor P M PM

Infection and immunity 19991101 11

Random fusions of genomic DNA fragments to a partial gene encoding a signal sequence-deficient bacterial alkaline phosphatase were utilized to screen for exported proteins of Actinobacillus actinomycetemcomitans in Escherichia coli. Twenty-four PhoA(+) clones were isolated and sequenced. Membrane localization signals in the form of signal sequences were deduced from most of these sequences. Several of the deduced amino acid sequences were found to be homologous to known exported or membrane-asso ...[more]

PMID: 10531295

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Project description: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 IS903phikan 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.

Project description:The cell density-dependent control of gene expression is employed by many bacteria for regulating a variety of physiological functions, including the generation of bioluminescence, sporulation, formation of biofilms, and the expression of virulence factors. Although periodontal organisms do not appear to secrete acyl-homoserine lactone signals, several species, e.g., Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, have recently been shown to secrete a signal related to the autoinducer II (AI-2) of the signal system 2 pathway in Vibrio harveyi. Here, we report that the periodontal pathogen Actinobacillus actinomycetemcomitans expresses a homolog of V. harveyi luxS and secretes an AI-2-like signal. Cell-free conditioned medium from A. actinomycetemcomitans or from a recombinant Escherichia coli strain (E. coli AIS) expressing A. actinomycetemcomitans luxS induced luminescence in V. harveyi BB170 >200-fold over controls. AI-2 levels peaked in mid-exponential-phase cultures of A. actinomycetemcomitans and were significantly reduced in late-log- and stationary-phase cultures. Incubation of early-log-phase A. actinomycetemcomitans cells with conditioned medium from A. actinomycetemcomitans or from E. coli AIS resulted in a threefold induction of leukotoxic activity and a concomitant increase in leukotoxin polypeptide. In contrast, no increase in leukotoxin expression occurred when cells were exposed to sterile medium or to conditioned broth from E. coli AIS(-), a recombinant strain in which luxS was insertionally inactivated. A. actinomycetemcomitans AI-2 also induced expression of afuA, encoding a periplasmic iron transport protein, approximately eightfold, suggesting that LuxS-dependent signaling may play a role in the regulation of iron acquisition by A. actinomycetemcomitans. Finally, A. actinomycetemcomitans AI-2 added in trans complemented a luxS knockout mutation in P. gingivalis by modulating the expression of the luxS-regulated genes uvrB and hasF in this organism. Together, these results suggest that LuxS-dependent signaling may modulate aspects of virulence and the uptake of iron by A. actinomycetemcomitans and induce responses in other periodontal organisms in mixed-species oral biofilm.

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Identification of genes coding for exported proteins of Actinobacillus actinomycetemcomitans.

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Identification of genes coding for exported proteins of Actinobacillus actinomycetemcomitans.

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