Identification of a fliG homologue in Treponema denticola.

Using a bacteriophage lambda library of Treponema denticola (Td) ATCC 35405 DNA, and, as a reagent, sera derived from individuals with advanced adult periodontal disease, a variety of recombinant clones producing antigens of this oral spirochete have been isolated. Nucleotide sequence analysis of a clone expressing three immunoreactive antigens has revealed the presence of an open reading frame highly homologous to the flagellar switch/motor protein, FliG, which is known to be essential for flagellar assembly and rotation, and chemotaxis in enteric bacteria. The deduced amino-acid sequence of the treponemal FliG protein had 73% similarity (55% identity) to the Bacillus subtilis FliG protein, and showed significant, but lesser homologies to Gram- FliG proteins. Sequence analysis of regions flanking fliG indicated that this gene is immediately preceded by a fliF homologue, further supporting that the cloned DNA encodes FliG of Td. The findings imply that although the signals for control of chemotaxis may be distinctly different in spirochetes, at least some of the molecules involved in torque generation, control of flagellar rotation and signal transduction are highly conserved with other bacteria. The stronger homology of the spirochete FliG with those of Gram+ bacteria is also consistent with recent analyses of other spirochetal genes.

Genetic and transcriptional analysis of flgB flagellar operon constituents in the oral spirochete Treponema denticola and their heterologous expression in enteric bacteria.

Oral spirochetes possess many potential virulence factors, including the capacity for tissue invasion and persistence despite a vigorous host immune response. In an attempt to identify treponemal immunoreactive components, sera derived from individuals with advanced periodontal disease were used as a reagent to isolate recombinant bacteriophage lambda clones expressing antigens of the oral spirochete Treponema denticola ATCC 35405. Nucleotide sequence analysis of a clone expressing three immunoreactive products has revealed seven T. denticola genes which appear to encode homologs of flagellar basal body constituents, FlgB, FlgC, FliE, and FliF, a flagellar switch component, FliG, and the putative flagellar export proteins, FliH and FliI, initially characterized in Salmonella typhimurium. Also identified was a gene resembling fliJ. Primer extension analysis identified a transcriptional start site 5' to the treponemal flgB gene. Appropriately spaced with respect to this start site was a sigma28 binding motif. The absence of additional identifiable sigma factor binding motifs within the treponemal sequence and the proximity of adjacent genes suggested operonic arrangement, and reverse transcriptase PCR provided evidence of cotranscription. Supporting the identification of these genes as flagellar components, heterologous expression in enteric bacteria of the putative switch basal body genes from T. denticola interfered with motility. Specifically, the presence of a plasmid expressing treponemal fliG reduced swarming motility in S. typhimurium, while in Escherichia coli, this plasmid conferred a nonmotile phenotype and a reduction in flagellar number. Thus, while spirochetal flagella are subject to unique synthetic and functional constraints, the organization of flagellar genes and the presence of sigma28-like elements are reminiscent of the flagellar systems of other bacteria, and there appears to be sufficient conservation of constituent proteins to allow interaction between T. denticola switch-basal body proteins and the flagellar machinery of gram-negative bacteria.