Nonspecific adherence and fibril biogenesis by Actinobacillus actinomycetemcomitans: TadA protein is an ATPase

Cells of Actinobacillus actinomycetemcomitans, a gram-negative pathogen res ponsible for an aggressive form of juvenile periodontitis, form tenaciously adherent biofilms on solid surfaces. The bacteria produce long fibrils of bundled pill, which are required for adherence. Mutations in flp-1, which e ncodes the major subunit of the pili, or any of seven downstream tad genes (tadABCDEFG) cause defects in fibril production, autoaggregation, and tenac ious adherence. We proposed that the tad genes specify part of a novel secr etion system for the assembly and transport of Flp pili. The predicted amin o acid sequence of TadA (426 amino acids, 47,140 Da) contains motifs for nu cleotide binding and hydrolysis common among secretion NTP hydrolase (NTPas e) proteins, In addition, the tadA gene is the first representative of a di stinct subfamily of potential type IV secretion NTPase genes. Here we repor t studies on the function of TadA. The tadA gene was altered to express a m odified version of TadA that has the 11-residue epitope (T7-TAG) fused to i ts C terminus. The TadA-T7 protein was indistinguishable from the wild type in its ability to complement the fibril and adherence defects of A. actino mycetemcomitans tadA mutants. Although TadA is not predicted to have a tran smembrane domain, the protein was localized to the inner membrane and cytop lasmic fractions of A. actinomycetemcomitans cells, indicating a possible p eripheral association with the inner membrane. TadA-T7 was purified and fou nd to hydrolyze ATP in vitro. The ATPase activity is stimulated by Triton X -100, with maximal stimulation at the critical micellar concentration. TadA -T7 forms multimers that are stable during sodium dodecyl sulfate-polyacryl amide gel electrophoresis in nonreducing conditions, and electron microscop y revealed that TadA-T7 can form structures closely resembling the hexameri c rings of other type IV secretion NTPases. Site-directed mutagenesis was u sed to substitute Ala and Gln residues for the conserved Lys residue of the Walker A box for nucleotide binding. Both mutants were found to be defecti ve in their ability to complement tadA mutants. We suggest that the ATPase activity of TadA is required to energize the assembly or secretion of Flp p ili for tight adherence of A. actinomycetemcomitans.