CHARACTERIZATION OF THE CYTOPLASMIC FILAMENT PROTEIN GENE (CFPA) OF TREPONEMA-PALLIDUM SUBSP PALLIDUM

Treponema pallidum and other members of the genera Treponema, Spirocha eta, and Leptonema contain multiple cytoplasmic filaments that run the length of the organism just underneath the cytoplasmic membrane. Thes e cytoplasmic filaments have a ribbon-like profile and consist of a ma jor cytoplasmic filament protein subunit (CfpA, formerly called TpN83) with a relative molecular weight of similar to 80,000. Degenerate DNA primers based on N-terminal and CNBr cleavage fragment amino acid seq uences of T. pallidum subsp. pallidum (Nichols) CfpA were utilized to amplify a fragment of the encoding gene (cfpA). A 6.8-kb EcoRI fragmen t containing all but the 5' end of cfpA was identified by hybridizatio n with the resulting PCR product and cloned into Lambda ZAP II. The 5' region was obtained by inverse PCR, and the complete gene sequence wa s determined, The cfpA sequence contained a 2,034-nucleotide coding re gion, a putative promoter with consensus sequences (5'-TTTACA-3' for - 35 and 5'-TACAAT-3' for -10) similar to the sigma(70) recognition sequ ence of Escherichia coli and other organisms, and a putative ribosome- binding site (5'-AGGAG-3'), The deduced amino acid sequence of CfpA in dicated a protein of 678 residues with a calculated molecular mass of 78.5 kDa and an estimated pI of 6.15. No significant homology to known proteins or structural motifs was found among known prokaryotic or eu karyotic sequences. Expression of a LacZ-CfpA fusion protein in E. col i was detrimental to survival and growth of the host strain and result ed in the formation of short, irregular filaments suggestive of partia l self-assembly of CfpA. The cytoplasmic filaments of T. pallidum and other spirochetes appear to represent a unique form of prokaryotic int racytoplasmic inclusions.