Bhattacharjee M K, Kachlany S C, Fine D H, Figurski D H
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
J Bacteriol. 2001 Oct;183(20):5927-36. doi: 10.1128/JB.183.20.5927-5936.2001.
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.
伴放线放线杆菌是一种革兰氏阴性病原体，可引发侵袭性青少年牙周炎，其细胞能在固体表面形成紧密附着的生物膜。该细菌会产生成束菌毛的长纤丝，这是附着所必需的。flp - 1（编码菌毛主要亚基）或七个下游tad基因（tadABCDEFG）中的任何一个发生突变，都会导致纤丝产生、自聚集和紧密附着方面的缺陷。我们推测，tad基因指定了一种新型分泌系统的一部分，用于Flp菌毛的组装和运输。TadA的预测氨基酸序列（426个氨基酸，47,140 Da）包含分泌型NTP水解酶（NTPase）蛋白中常见的核苷酸结合和水解基序。此外，tadA基因是潜在IV型分泌NTPase基因一个独特亚家族的首个代表。在此我们报告关于TadA功能的研究。tadA基因经改造后表达一种在其C末端融合了11个残基表位（T7 - TAG）的TadA修饰版本。TadA - T7蛋白在互补伴放线放线杆菌tadA突变体的纤丝和附着缺陷能力方面与野生型无异。尽管预测TadA没有跨膜结构域，但该蛋白定位于伴放线放线杆菌细胞的内膜和细胞质部分，表明可能与内膜存在外周关联。TadA - T7经纯化后发现在体外能水解ATP。ATP酶活性受Triton X - 100刺激，在临界胶束浓度时刺激最大。TadA - T7形成多聚体，在非还原条件下的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳过程中稳定，电子显微镜显示TadA - T7能形成与其他IV型分泌NTPase的六聚体环极为相似的结构。采用定点诱变将丙氨酸和谷氨酰胺残基替代沃克A框中用于核苷酸结合的保守赖氨酸残基。发现这两种突变体在互补tadA突变体的能力方面均存在缺陷。我们认为，TadA的ATP酶活性是为伴放线放线杆菌紧密附着而使Flp菌毛组装或分泌获得能量所必需的。
