Molecular typing of papillomatous digital dermatitis-associated Treponema isolates based on analysis of 16S-23S ribosomal DNA intergenic spacer regions.

Papillomatous digital dermatitis (PDD), an emerging infectious disease of cattle, is characterized by painful, ulcerative foot lesions. The detection of high numbers of invasive spirochetes in PDD lesions suggests an important role for these organisms in the pathogenesis of PDD. PDD-associated spirochetes have phenotypic characteristics consistent with members of the genus TREPONEMA: Partial 16S ribosomal DNA (rDNA) sequence analysis of clonal isolates from California cattle showed that they comprise three phylotypes which cluster closely with human-associated Treponema spp. of the oral cavity (T. denticola and T. medium/T. vincentii) or genital area (T. phagedenis). The goal of our study was to apply 16S-23S rDNA intergenic spacer region (ISR) sequence analysis to the molecular typing of U.S. PDD-associated Treponema isolates. This methodology has potentially greater discriminatory power for differentiation of closely related bacteria than 16S rDNA analysis. We PCR amplified, cloned, and sequenced the ISRs from six California PDD-associated Treponema isolates and, for comparative purposes, one strain each of T. denticola, T. medium, T. vincentii, and T. phagedenis. Two ISRs that varied in length and composition were present in all the PDD-associated Treponema isolates and in T. denticola, T. medium, and T. phagedenis. ISR1 contained a tRNA(Ala) gene, while ISR2 contained a tRNA(Ile) gene. Only a single ISR (ISR1) was identified in T. vincentii. Comparative analyses of the ISR1 and ISR2 sequences indicated that the California PDD-associated Treponema isolates comprised three phylotypes, in agreement with the results of 16S rDNA analysis. PCR amplification of the 16S-tRNA(Ile) region of ISR2 permitted rapid phylotyping of California and Iowa PDD-associated Treponema isolates based on product length polymorphisms.

Molecular characterization of the Treponema denticola fliQ region.

A Treponema denticola 4.2 kb DNA region containing four complete genes (orfl, fliQ, fliR, and flhB) and a truncated gene (flhA') was sequenced and analyzed. The deduced amino acid sequences of FliQ, FliR, FlhB and FlhA' have significant homology with bacterial proteins associated with the flagellar export apparatus, whereas the deduced amino acid sequence of Orf1 has homology with an E. coli alcohol dehydrogenase. A putative sigma70-like promoter was identified upstream of fliQ. RT-PCR analysis indicated that fliQ, fliR, flhB and flhA' are co-transcribed independently of orfl, suggesting that the motility-associated genes are components of an operon. The location of the T. denticola fliQ-flhA' genes differs from that of the corresponding T. pallidum and Borrelia burgdorferi genes which are present in the large fla or flgB flagellar operons, respectively.

The sequence-variable, single-copy tprK gene of Treponema pallidum Nichols strain UNC and Street strain 14 encodes heterogeneous TprK proteins.

Syphilis is a chronic infection with early relapses that are hypothesized to result from the emergence of phenotypic variants of Treponema pallidum. Recent studies demonstrated that TprK, a target of protective immunity, is heterogeneous in several T. pallidum strains, but not in Nichols strain Seattle (A. Centurion-Lara, C. Godornes, C. Castro, W. C. Van Voorhis, and S. A. Lukehart, Infect. Immun. 68:824-831, 2000). Analysis of PCR-amplified tprK from Nichols strain UNC and Street strain 14 treponemes showed that TprK has seven regions of intrastrain heterogeneity resulting from amino acid substitutions, insertions, and deletions. In contrast, analysis of PCR-amplified tprJ showed little intrastrain or interstrain heterogeneity. Reverse transcriptase PCR analysis demonstrated that mRNA transcripts representing unique polymorphic TprK proteins are present during syphilitic infection. Southern hybridization confirmed that Nichols strain UNC and Street strain 14 each contain a single copy of tprK, indicating that intrastrain heterogeneity is due to the presence of multiple treponemal subpopulations which contain a variant form of tprK.

Molecular characterization of a flagellar (fla) operon in the oral spirochete Treponema denticola ATCC 35405.

A Treponema denticola 9.6-kb motility locus containing 11 genes was identified, sequenced and analyzed. The genes were designated tap1, flgD, flgE, orf4, motA, motB, fliL, fliM, fliY, orf10 and fliP. The order of these genes is identical to that of the corresponding region of the Treponema pallidum fla operon. Seven of the deduced Fla proteins share significant homology with both Escherichia coli and Bacillus subtilis proteins associated with flagellar structure and function. Reverse transcription-PCR analysis indicated that the T. denticola fla genes are transcribed as a single unit. A putative sigma(28)-like promoter, virtually identical to the T. pallidum fla promoter, was identified upstream of tap1. These results showed that the T. denticola and T. pallidum fla operons are highly conserved, supporting the proposed phylogenetic relatedness of these spirochetes.

Identification and sequence analysis of Treponema pallidum tprJ, a member of a polymorphic multigene family.

TnphoA mutagenesis was used to identify genes encoding exported proteins in a genomic DNA library of Treponema pallidum, the syphilis agent. The nucleotide sequence of an open reading frame (tprJ) that encodes a 755-amino acid protein with a predicted molecular mass of 81.1 kDa was determined. The deduced amino acid sequence of TprJ has homology to the major surface protein of Treponema denticola, a periodontal pathogen. Southern hybridization and genomic DNA sequence analysis indicate that tprJ is a member of a polymorphic multigene family. RT-PCR data showed that tprJ is expressed in treponemes during syphilitic infection. A putative tprJ gene was sequenced from T. pertenue, the closely related yaws agent. The deduced amino acid sequence of T. pertenue TprJ is 87.3% identical to that of T. pallidum TprJ. This is the first report of significant sequence differences within homologous genes of T. pallidum and T. pertenue.

Identification and characterization of a Treponema pallidum subsp. pallidum gene encoding a DNA adenine methyltransferase.

The nucleotide sequence of a DNA adenine methyltransferase gene (dam) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The dam gene encodes a 303 amino acid protein whose deduced sequence has significant homology with DNA (N6-adenine) methyltransferases. T. pallidum Dam can be assigned to group alpha DNA amino methyltransferases based on the order of nine conserved motifs that are present in the protein. Digests of T. pallidum chromosomal DNA performed with isoschizomer restriction endonucleases (Sau3AI, DpnI, and MboI) confirmed the presence of methylated adenine residues in GATC sequences (Dam+ phenotype).