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PMID: 11566992 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

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

Journal of bacteriology ·Vol. 183 ·No. 20 ·2001-10-00 ·Pages 5927-36

Bhattacharjee MK, Kachlany SC, Fine DH, Figurski DH

Abstract

Cells of Actinobacillus actinomycetemcomitans, a gram-negative pathogen responsible for an aggressive form of juvenile periodontitis, form tenaciously adherent biofilms on solid surfaces. The bacteria produce long fibrils of bundled pili, which are required for adherence. Mutations in flp-1, which encodes the major subunit of the pili, or any of seven downstream tad genes (tadABCDEFG) cause defects in fibril production, autoaggregation, and tenacious adherence. We proposed that the tad genes specify part of a novel secretion system for the assembly and transport of Flp pili. The predicted amino acid sequence of TadA (426 amino acids, 47,140 Da) contains motifs for nucleotide binding and hydrolysis common among secretion NTP hydrolase (NTPase) proteins. In addition, the tadA gene is the first representative of a distinct subfamily of potential type IV secretion NTPase genes. Here we report studies on the function of TadA. The tadA gene was altered to express a modified version of TadA that has the 11-residue epitope (T7-TAG) fused to its C terminus. The TadA-T7 protein was indistinguishable from the wild type in its ability to complement the fibril and adherence defects of A. actinomycetemcomitans tadA mutants. Although TadA is not predicted to have a transmembrane domain, the protein was localized to the inner membrane and cytoplasmic fractions of A. actinomycetemcomitans cells, indicating a possible peripheral association with the inner membrane. TadA-T7 was purified and found 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-polyacrylamide gel electrophoresis in nonreducing conditions, and electron microscopy revealed that TadA-T7 can form structures closely resembling the hexameric rings of other type IV secretion NTPases. Site-directed mutagenesis was used 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 defective 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 pili for tight adherence of A. actinomycetemcomitans.

MeSH Terms
Acid Anhydride Hydrolases/genetics Adenosine Triphosphatases/physiology Aggregatibacter actinomycetemcomitans/physiology,ultrastructure Amino Acid Sequence Bacterial Adhesion/physiology Bacterial Proteins/metabolism Conserved Sequence Fimbriae, Bacterial/metabolism Genetic Complementation Test Mutation Nucleoside-Triphosphatase
Chemicals
Bacterial Proteins Acid Anhydride Hydrolases Adenosine Triphosphatases TadA protein, Actinobacillus actinomycetemcomitans Nucleoside-Triphosphatase
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Bhattacharjee M K
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
Kachlany S C
Fine D H
Figurski D H
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/JB.183.20.5927-5936.2001
Published
2001-10-00
Pages
5927-36
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC99671
Subset
IM
External Links
PubMed source DOI: 10.1128/JB.183.20.5927-5936.2001 PMC Full Text View journal details
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