The formation of dental plaque, a highly complex biofilm that causes gingivitis and periodontitis, requires specific adherence among many oral microbes, including the coaggregation of Actinomyces oris with Streptococcus oralis that helps to seed biofilm development. Here, we report the discovery of a key coaggregation factor for this process. This protein, which we named coaggregation factor A (CafA), is one of 14 cell surface proteins with the LPXTG motif predicted in A. oris MG1, whose function was hitherto unknown. By systematic mutagenesis of each of these genes and phenotypic characterization, we found that the Actinomyces/Streptococcus coaggregation is only abolished by deletion of cafA. Subsequent biochemical and cytological experiments revealed that CafA constitutes the tip of a unique form of the type 2 fimbria long known for its role in coaggregation. The direct and predominant role of CafA in adherence is evident from the fact that CafA or an antibody against CafA inhibits coaggregation, whereas the shaft protein FimA or a polyclonal antibody against FimA has no effect. Remarkably, FimA polymerization was blocked by deletion of genes for both CafA and FimB, the previously described tip protein of the type 2 fimbria. Together, these results indicate that some surface proteins not linked to a pilus gene cluster in Gram-positive bacteria may hijack the pilus. These unique tip proteins displayed on a common pilus shaft may serve distinct physiological functions. Furthermore, the pilus shaft assembly in Gram-positive bacteria may require a tip, as is true for certain Gram-negative bacterial pili.

AtxA, the master virulence gene regulator of Bacillus anthracis, is a PRD-Containing Virulence Regulator(PCVR) as indicated by the crystal structure, posttranslational modifications and activity of the protein. PCVRs are transcriptional regulators, named for PTS Regulatory Domains (PRDs) subject to phosphorylation by the phosphoenolpyruvate phosphotransferase system (PEP-PTS) and for their impact on virulence gene expression. Here we present data from experiments employing physiological, genetic and biochemical approaches that support a model in which the PTS proteins HPr and Enzyme I (EI) are required for transcription of the atxA gene, rather than phosphorylation of AtxA. We show that atxA transcription is reduced 2.5-fold in a mutant lacking HPr and EI, and that this change is sufficient to affect anthrax toxin production. Mutants harboring HPr proteins altered for phosphotransfer activity were unable to restore atxA transcription to parent levels, suggesting that phosphotransfer activity of HPr and EI is important for regulation of atxA. In a mouse model for anthrax, a HPr -EImutant was attenuated for virulence. Virulence was restored by expressing atxA from an alternative, PTS-independent, promoter. Our data support a model in which HPr transfers a phosphate to an unidentified downstream transcriptional regulator to influence atxA gene transcription.

Infection with B. anthracis can result in systemic disease with large numbers of the bacterium in multiple tissues. We found that branched-chain amino acid (BCAA) synthesis is insufficient for the robust growth of B. anthracis ; access to BCAAs is necessary for the proliferation of the pathogen during culture and during infection in a murine model for anthrax. B. anthracis produces an unusually large repertoire of BCAA-related transporters.