Identification and molecular analysis of rough-colony-specific outer membrane proteins of Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans, a gram-negative bacterium isolated fr om the human mouth, has been implicated in the pathogenesis of early-onset periodontitis. Primary isolates cultured from subgingival plaque exhibit an adherent, rough colony phenotype which spontaneously converts to a nonadhe rent, smooth phenotype upon in vitro subculture. The rough colony variant p roduces abundant fimbriae and autoaggregates, while the smooth colony varia nt is planktonic and produces scant fimbriae. To begin to understand the si gnificance of colony variation in biofilm formation by A. actinomycetemcomi tans, outer membrane protein profiles of four isogenic rough and smooth col ony variants were compared by sodium dodecyl sulfate-polyacrylamide gel ele ctrophoresis. Two proteins with relative molecular masses of 43 and 20 kDa were expressed by the rough colony variants exclusively. Expression of thes e proteins was not found to be dependent on growth phase, oxygen tension, o r type of complex medium. N-terminal amino acid sequences of these proteins obtained by Edman degradation were compared with sequences from the Univer sity of Oklahoma A. actinomycetemcomitans genome database. Two contiguous o pen reading frames (ORFs) encoding proteins having sequence homology with t hese proteins were identified. The 43-kDa protein (RcpA [rough colony prote in Al]) was similar to precursor protein D of the general secretion pathway of gram-negative bacilli, while the 20-kDa protein (RcpB [rough colony pro tein B]) appeared to be unique. The genes encoding these proteins have been cloned from A. actinomycetemcomitans 283 and sequenced. A BLASTX (gapped B LAST) search of the surrounding ORFs revealed homology with other fimbria-r elated proteins. These data suggest that the genes encoding the 43-kDa (rcp A) and 20-kDa (rcpB) proteins may be functionally related to each other and to genes that may encode fimbria-associated proteins.