gingivalis(2,64). multifunctional role of VimA in modulating virulence possibly through its involvement in acetyl-CoA transfer and lipid A synthesis and possibly by protein sorting. == INTRODUCTION == Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, is one of the main etiological brokers of adult periodontitis. While several virulence factors, including fimbriae (28), hemagglutinin (17), capsule (4), and lipopolysaccharide (68), have been implicated in the pathogenicity ofP. gingivalis, the strong proteolytic abilities of this organism are considered to be important for its survival and thus play a significant role in virulence (12,63). The major proteases, called gingipains, consist of arginine-specific (Arg-gingipain [Rgp]) and lysine-specific (Lys-gingipain [Kgp]) proteases that are both extracellular and cell membrane associated (32). The activation of these gingipains is associated with several genes, includingvimA,vimE, andvimF, that modulate the posttranslational glycosylation of those proteins (44,6264). These genes are part of the 6.15-kbbcp-recA-vimA-vimE-vimF-aroGlocus which has previously been shown to be important to the pathogenic potential ofP. gingivalis(1,26,44,6264). We have exhibited that thebcpandrecAgenes play the expected role in oxidative stress resistance and DNA repair, respectively (26). The association of these genes with thevimgenes on the same transcriptional unit could be considered an important strategy forP. gingivalisto coordinate its oxidative stress and proteolytic activities. A response to oxidative stress will involve binding of oxygen and its harmful derivatives to iron RAC1 accumulated on the surface of the cell (1). The bound heme can be involved in the catalytic destruction of the harmful oxygen derivative species (55). Because the specific functional functions of thevimgenes are not yet fully defined, we cannot rule out the possibility that their modulation of other proteins could have comparable mechanistic properties. Protein-protein conversation studies using the purified recombinant VimA (rVimA) showed that this protein can interact with the gingipains, HtrA, RegT, sialidase, and other proteins inP. gingivalis(2,47,61,64). Also, in addition to its role in glycosylation, the VimA protein can affect capsular synthesis, fimbrial phenotypic expression, and anchorage of several cell surface proteins (44). Collectively, these observations may implicate a multifunctional role for the VimA protein. In this study, we further confirmed the functional role for VimA in a different genetic background ofP. gingivalisand used anin silicoapproach to further explore the functional domains of VimA. Here we statement that VimA plays a role in acetyl coenzyme A (acetyl-CoA) transfer and modulates lipid A and its associated proteins. Further, VimA may also be involved in protein sorting and transport. == MATERIALS AND METHODS == == Bioinformatic analysis. == The DNA and amino acid sequences were aligned using Bioedit software (http://www.mbio.ncsu.edu/bioedit/bioedit.html). The phylogenetic associations of these sequences between the oral pathogens were analyzed using the MEGA program, version 4.0 (57). The phylogenetic distance was calculated using the Kimura 2-parameter model (29). For clustering, the neighbor-joining method used bootstrap values based on 1,000 replicates (49). The amino acid sequences were analyzed using OSI-420 the ClustalW program, version 2.0 (http://www.ebi.ac.uk/). The secondary structure prediction and modeling of the protein were performed using the Modeler software package (13). The models were validated using the WHATIF program (65). The transmission peptide and potential cleavage sites were predicted using both the neural network and hidden Markov models (25). Metabolic pathway analysis was carried out using theKyoto Encyclopedia of Genes and Genomes(KEGG) (www.genome.jp/kegg/) (27), based on the information from the online databases Biosilico (24), BRENDA (7), and ExPASy Enzyme (3). == Bacterial strains and growth conditions. == Strains and plasmids used in this study are OSI-420 outlined inTable 1.P. gingivalisstrains were grown in brain heart infusion (BHI) broth (Difco) supplemented with hemin (5 g/ml), vitamin K (0.5 g/ml), and cysteine (0.1%). Experiments with hydrogen peroxide were performed in BHI without cysteine. All cultures, unless otherwise stated, were incubated at 37C.P. gingivalisstrains were maintained in an anaerobic chamber (Coy Manufacturing) in 10% H2, 10% CO2, 80% N2. Growth rates forP. gingivalisstrains were decided spectrophotometrically (optical density at 600 nm [OD600]). Antibiotics were used at the following concentrations: clindamycin, 0.5 g/ml; erythromycin, 10 g/ml. == Table 1. == Strains and plasmids used in this OSI-420 study == DNA isolation and analysis. == P. gingivalischromosomal OSI-420 DNA was prepared as previously explained (37). For plasmid DNA analysis,.