Sequence and analysis of Actinobacillus actinomycetemcomitans.

Abstract

A. actinomycetemcomitans is a Gram negative coccobacillus, facultative anaerobe that is implicated in the persistence of peridontitis, the destruction of the gum tissues and loss of teeth (Fives-Taylor et al, 1999). In an effort to understand the biochemical events that result in A. actinomycetemcomitans induced oral cavity disease, its genome was sequenced. To date over 99.8% of this ∼ 2 MB genome have been sequenced. Analysis of this data reveals much new information about the biochemical metabolic pathways and the pathogenesis of A. actinomycetemcomitans .

During these studies, several pathogenicity-associated metabolic pathways also were identified. These include the LPS and dTDP-L-rhamanose pathways. In addition, at least 12 new putative proteins related to invasion, iron acquisition, and immunosuppression were discovered.

With the sequence of A. actinomycetemcomitans essentially complete, it now is possible to develop experimental tests of these virulence mechanisms, develop other testable hypothesis of this bacteria's life style, and determine the mechanisms it uses to colonize the human oral cavity.

Multiple sequencing approaches were utilized to obtain the highest accuracy of sequence data. Initially, the preliminary sequence data were collected by the shotgun sequencing method, and then, since no physical map was available for this bacterium, a multiplex PCR sequencing technique was developed to aid in gap closure and finishing. Once six genome equivalents of the sequence were obtained, the A. actinomycetemcomitans genome sequence was analyzed to obtain the predicted gene positions, their functions, and their structural motifs. With this information, PCR-based methods were used for sequencing gap closure and a detailed metabolic profile of A. actinomycetemcomitans was determined. Through homology and motif analysis, putative A. actinomycetemcomitans virulence factors were determined revealing a potential mode of pathogenesis.

Analysis of the predicted intermediary metabolism genes in A. actinomycetemcomitans reveals that this bacterium can synthesize all amino acids except leucine, histidine, and methionine and that the complete glycolytic and pentose phosphate pathways are present to allow for production of both the reducing potentials needed for energy and for the different metabolites needed as substrates for other biosynthetic pathways. These pathways in addition to the several encoded carbohydrate transporters, allow the organism to utilize different carbohydrates from the environment as energy sources. In contrast, fatty acids cannot be utilized from the environment by A. actinomycetemcomitans since several of the enzymes required for beta-oxidation are not present in the genome, and the synthesis of both purines and pyrimidines only can occur via their salvage pathways. In contrast, the genes coding for the biosynthesis of several vitamins and cofactors, such as biotin, riboflavin, and folate, were present in A. actinomycetemcomitans genome.