Complementation of a Treponema denticola flgE mutant with a novel coumermycin A1-resistant T. denticola shuttle vector system.

Treponema denticola has been shown to be associated with the severity of periodontal disease (18). Many potential virulence factors of T. denticola have been identified, and mutants of the corresponding virulence factors have been constructed and studied (5). Some of these putative virulence factors include the methyl-accepting chemotaxis DmcA (13) and DmcB (14) proteins; CheA (17), the central kinase in chemotactic signal transduction; Tap1, which might be involved in monitoring the flagellar hook length (16); the outer membrane-associated chymotrypsinlike protease Prtp (11); and Msp, which is an adhesin with pore-forming activity (6). However, studies using both the mutants and the complemented mutants would provide more accurate information on the role of the genes in the virulence of T. denticola. Previously, only an erythromycin resistance (Emr) marker could be used as a selective marker in T. denticola (14). Therefore, a second antibiotic marker is required for complementation of the Ermr mutants. DNA gyrase is a type II topoisomerase which catalyzes the introduction of negative supercoils into DNA at the expense of ATP hydrolysis to ADP and phosphate (7, 20, 24). This enzyme plays an important role in DNA replication, transcription, and recombination. The enzyme is a tetramer composed of two A and two B subunits which are encoded by the gyrA and gyrB genes, respectively. The coumarin antibiotic coumermycin A1 inhibits DNA gyrase by competing with ATP for binding to the B subunits and blocks introduction of supercoils into relaxed DNA (1, 3, 19, 23). The gyrB gene of the T. denticola ATCC 35405 strain has recently been cloned and sequenced (S. R. Greene and L. V. Stamm, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., p. 257, 1999). Five conserved type II topoisomerase motifs, an ATP binding site (Walker A), and amino acid residues that putatively interact with a nonhydrolyzable ATP analog are highly conserved in T. denticola GyrB. Spontaneous coumermycin A1-resistant T. denticola 35405 mutants were also isolated, and a single point mutation in the gyrB gene, changing Lys-136 to Glu or Thr, confers >20-fold higher resistance in the mutants relative to that in wild-type T. denticola (8). Therefore, we examined the possibility that the mutated gyrB gene which confers coumermycin resistance in T. denticola could serve as a second selectable marker in these spirochetes. The periplasmic flagella, which contribute to the motility of spirochetes, are located between the outer and cytoplasmic membranes (9). In spite of its unique location, the general structure of the flagella is similar to that of other bacteria and contains filaments, a flagellar hook, a rod, and a basal body (21, 22). Previously in our laboratory, a mutant of the flagellar hook gene, flgE, of T. denticola 35405 was constructed (14). The mutant HL51 lost motility, and electron microscopy showed that the mutant lacks both the flagellar hook and filaments. In this study, we report the construction of a coumermycin A1-resistant E. coli-T. denticola shuttle vector based on our previously reported vector pKMR4PE (2) and the functional complementation of the T. denticola ATCC 33520 flgE mutant (14).