Organization, transcription, and expression of the 5' region of the fla operon of Treponema phagedenis and Treponema pallidum

A locus encoding polypeptides associated with flagellar structure and function was identified, sequenced, and characterized in Treponema phagedenis and Treponema pallidum. This locus includes homologs of the FlgD, FlgE, MotA, MotB, FliL, and FliM polypeptides found in Salmonella typhimurium and Bacillus subtilis. These polypeptides are extensively conserved between the two treponemes. Several additional polypeptides of unknown function, including Tap1, located upstream of FlgD, and ORF4, located between FlgE and MotA, were also identified, Transcription analysis using RNA PCR indicated that these genes are likely transcribed as part of a single operon and comprise the 5' region of the treponemal fla operon, Primer extension analysis identified a putative promoter, P-fla, preceding T. phagedenis tap1 in a region of divergent transcription, P-fla resembles the class II or class III motility-related promoters of S. typhimurium. FlgE and Tap1 were further characterized, Western blotting (immunoblotting) indicated that T. pallidum FlgE exhibited an unusual polypeptide ladder that was similar but not identical to that of T. phagedenis. Triton X-114 phase partitioning of T. phagedenis cells coupled with Western blotting revealed that Tap1 was located in the aqueous phase, Computer analysis indicated that Tap1 had no significant membrane spanning regions, suggesting that it resides primarily in the cytoplasm. The organization and expression of this operon are similar in both treponemes but different from those of previously described motility-related operons. These results indicate that despite extensive amino acid sequence conservation, the expression of spirochete flagellar polypeptides is different from that in other bacteria.