Genome Differences between Treponema pallidum subsp. pallidum Strain Nichols and T. paraluiscuniculi Strain Cuniculi A

Isolation of T. pallidum and T. paraluiscuniculi chromosomal DNA. T. pallidum subsp. pallidum Nichols and T. paraluiscuniculi Cuniculi A were maintained by rabbit inoculation and purified by Hypaque gradient centrifugation as described previously (2, 8). Chromosomal DNA was prepared as described by Fraser et al. (8).

DNA labeling, microarray hybridization, and data analysis.Preparations of T. pallidum subsp. pallidum and T. paraluiscuniculi chromosomal DNA (0.25 to 0.75 μg) were labeled fluorescently using the Klenow enzyme (New England Biolabs, Ipswich, MA) and random nonamers with a CyScribe First-Strand cDNA labeling kit (Amersham Pharmacia Biotech, Piscataway, NJ) according to the protocol described previously (24). Microarrays containing PCR products representing the 1,039 T. pallidum subsp. pallidum Nichols open reading frames (ORFs) were prepared as described by Šmajs et al. (24). The pretreated slides (24) were hybridized simultaneously with labeled DNA using the CyScribe First-Strand cDNA labeling kit (Amersham Pharmacia Biotech). Quantitation of hybridization, exclusion of outliers, and data normalization were performed using the TIGR Spotfinder and TIGR MIDAS software (21). Combining the results of four independent experiments, including dye swapping in two separate hybridizations, yielded 12 possible values for each gene. From these data points, average signal ratios (ASR) and standard deviations were calculated. From these data, a set of genes with mean ASR of labeled T. paraluiscuniculi Cuniculi A DNA to labeled T. pallidum subsp. pallidum Nichols DNA (ASR_{Cuniculi A/Nichols}) less than 0.7 (average log₂ ratio less than −0.51) was derived. This set comprised 22 genes that are likely to contain deletions and/or major sequence changes. No genes with a mean ASR greater than 1.43 (average log₂ ratio greater than 0.51) were identified.

WGF.WGF was performed as described previously (27). The chromosomal DNA was amplified in 97 overlapping regions with a median length of 12,307 bp (range, 1,778 to 24,758 bp) using a GeneAmp XL PCR kit (Applied Biosystems, Foster City, CA). The primer pairs used for these amplifications are shown in Table S1 in the supplemental material. Each PCR product was digested with BamHI, EcoRI, or HindIII or combinations of these enzymes. To thoroughly assess the possible presence of deletions and insertions in restriction fragments, additional digestions were performed as needed to reduce the length of each restriction fragment to ≤4 kb. This was achieved by additional digestion with AccI, ClaI, EcoRV, KpnI, MluI, NcoI, NheI, RsrII, SacI, SpeI, XbaI, or XhoI (NEB) or combinations of these enzymes. The resulting fingerprints for T. pallidum subsp. pallidum Nichols were compared to those for the T. paraluiscuniculi Cuniculi A genome.

PCR amplification and DNA sequencing.Standard methods were used for PCR amplification from a chromosomal DNA template and agarose gel electrophoresis (22). For sequencing of PCR products, XL PCR was used to minimize the number of PCR errors. Oligonucleotide primers were designed with Primer3 software (20). The resulting PCR products were purified using a QIAquick PCR purification kit (QIAGEN) and were sequenced using a Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems). Complete sequences of amplified regions were finished using specifically designed synthetic oligonucleotides as primers. Computer-assisted sequence analysis was performed using the LASERGENE program package (DNASTAR, Madison, WI). Three XL PCR products comprising regions TPI12, TPI25A, and TPI25B (see Table S1 in the supplemental material) were purified and subjected to mechanical shearing to obtain smaller fragments (0.5 to 1 kb) that were cloned into the pUC18 vector. The resulting recombinant plasmids (96 plasmids for each XL PCR product) of the small insert library were isolated and sequenced using forward and reverse pUC18 primers to obtain multiple coverage (i.e., 2 × 96 sequencing reactions per XL PCR product).

Nucleotide sequence accession numbers.The nucleotide sequences reported in this study have been deposited in the GenBank database under accession numbers EF057750, EF137736 to EF137743, and EF419245 to EF419253.

DNA microarray genome comparison.In this analysis, we used a T. pallidum subsp. pallidum Nichols DNA microarray containing PCR products corresponding to all 1,039 predicted ORFs (24). T. pallidum subsp. pallidum Nichols and T. paraluiscuniculi Cuniculi A chromosomal DNA were labeled with the Cy3 and Cy5 dyes and hybridized simultaneously on individual arrays. The hybridizations were performed four times, including dye swapping in two hybridizations, resulting in a total of 12 hybridizations for each ORF. Hybridization of labeled T. pallidum subsp. pallidum Nichols DNA probes yielded a detectable signal in ≤6 of the 12 reactions for 11 ORFs (TP0161, TP0224, TP0490, TP0573, TP0645, TP0753, TP0777, TP0795, TP0818, TP0932, and TP1032), and therefore analysis of these ORFs was not performed. All of these ORFs are relatively short (93, 105, 189, 93, 177, 285, 225, 159, 153, 93, and 432 bp, respectively) and code for a conserved hypothetical protein (TP0490) or hypothetical proteins (TP0161, TP0224, TP0573, TP0645, TP0753, TP0777, TP0795, TP0818, TP0932, and TP1032). Thus, data were calculated for 1,028 of 1,039 genes (99%) by determining the ASR_{Cuniculi A/Nichols} representing the average, normalized ratio of T. paraluiscuniculi Cuniculi A DNA fluorescent signals to T. pallidum subsp. pallidum Nichols DNA fluorescent signals for replicate spots on each microarray and in replicate experiments. A value of 1.0 corresponded to the mean signal for all genes of the array.

The results of the DNA microarray hybridizations are shown in Table 1. Use of the Cuniculi A probe yielded significantly lower signals for 22 genes, with ASR_{Cuniculi A/Nichols} ranging from 0.14 to 0.7 (Table 1). These genes were not randomly distributed throughout the genome (Fig. 1) and tended to be clustered in regions containing tpr genes and genes in the vicinity of tpr genes. All but four putative genes (TP0128, TP0129, TP0896, and TP0970) belonged to paralogous gene family 2 (PGF2), PGF14, and PGF15. PGF2 represents tpr genes encoding Tpr proteins, which are T. pallidum-specific proteins of unknown function with similarity to the Treponema denticola membrane protein Msp (7). Eight tpr genes (tprC, tprD, tprF, tprG, tprI, tprJ, tprK and tprL) had significantly lower ASR_{Cuniculi A/Nichols} values, indicating that there were deletions or sequence diversity in the T. paraluiscuniculi Cuniculi A genes. In contrast, signals for the tprA, tprB, tprE, and tprH genes were similar in the two genomes, indicating that these genes are present in the Cuniculi A genome and that the Cuniculi A genes are highly homologous to their Nichols counterparts. Genes belonging to PGF14 and PGF15 encode hypothetical proteins with unknown functions encoded by genes in the vicinity of tpr genes. The ASR_{Cuniculi A/Nichols} values for PGF14 and PGF15 are considerably lower (range, 0.14 to 0.56) than the ASR_{Cuniculi A/Nichols} values for tpr genes (0.49 to 0.68). The average lengths of the tpr genes and the PGF14 and PGF15 genes listed in Table 1 were 1,779 bp and 658 bp, respectively, so the PCR products used in the microarray were longer for the tpr genes. Therefore, this approach may be less sensitive to detection of sequence changes when larger genes are examined.

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FIG. 1.

Schematic representation of genes detected by lower-microarray-signal (DNA microarray) analysis, indels detected by WGF, and genes selected for sequencing in the T. paraluiscuniculi Cuniculi A genome. Deletions and sequentially diverse genes are indicated by vertical bars, and insertions are indicated by vertical lines ending with triangles. Open arrows indicate chromosomal regions that were sequenced in the Cuniculi A genome.

WGF of Nichols and Cuniculi A genomes.The genomes of T. pallidum subsp. pallidum strain Nichols and T. paraluiscuniculi strain Cuniculi A were analyzed using 97 overlapping XL PCR amplicons covering the entire treponemal genomes. Restriction mapping of 97 XL PCR products revealed 20 chromosomal regions (in 18 TPI intervals [Table 2]) with detectable insertions or deletions (indels) in the Cuniculi A genome (Table 2). Subsequent sequencing of heterologous parts of these TPI intervals revealed 10 deletions ranging from 15 to 2,609 bp and eight insertions ranging from 41 to 885 bp, as well as two regions (TPI12-13 and TPI66A) with both deletions and insertions. In addition, eight genes were found to contain small indels and multiple single nucleotide polymorphisms (SNPs) (TP0133, TP0136, TP0137, TP0315, TP0548, TP0617, TP0618, and TP1031 [Table 3]), and three tpr genes contained multiple SNPs and single nucleotide indels that resulted in frameshift gene mutations (TP0131, TP0313, and TP0621 [Table 3]). The largest deletions comprised TP0127 to TP0129, TP0133 to TP0135, tprF (TP0316) and tprG (TP0317), and TP0619 and tprI (TP0620). Deletions in the tprF and tprG genes (Table 2) and a frameshift mutation in tprG (Table 3) resulted in a new ORF encoding a 429-amino-acid TprI-like protein. A similar finding in the T. paraluiscuniculi Cuniculi A genome was described by Giacani et al. (9). The largest insertion was localized in the intergenic region (IGR) between the TP0126 and TP0129 loci (the TP0127 and TP0128 genes are missing in the Cuniculi A genome) and included 1,451 bp containing three ORFs showing similarity to the tprK sequence. Another large insertion (885 bp) comprising 14 complete repetitions (length, 60 bp) and one incomplete repetition (45 bp) was found in overlapping parts of the TP0433 and TP0434 genes. The expansion of repetitive sequences was shown by gel electrophoresis and draft sequencing of tandem repeats (data not shown). Because of the tandem repeats, this region was not sequenced completely, and the number of repeats was estimated based on gel electrophoresis. The described insertion resulted in fusion of the two genes (TP0433 and TP0434) to form a single acidic repeat protein gene (arp) (19). Except for deletions of TP0104 (encoding a 5′ nucleotidase), the tprF, tprG, and tprI genes, and TP0545 (encoding MglB-1, a methylgalactoside ABC transporter, periplasmic galactose-binding protein), all other indels were localized in genes encoding hypothetical proteins or in intergenic regions.

Taken together, the indels identified resulted in complete deletion of five genes (TP0127, TP0128, TP0134, TP0619, and TP0620), 13 partial gene deletions (TP0104, TP0129, TP0135, TP0136, TP0315, TP0316, TP0317, TP0470, TP0545, TP0733, TP0865, TP0967, and TP1029), seven gene elongations (TP0133, TP0487, TP0548, TP0616, TP0860, TP0923, and TP1031), two gene fusions (TP0433-TP0434 and TP0617-TP0618), two deletions in IGR (TP0135-TP0136 and TP0545-TP0546), and four insertions in IGR (TP0009-TP0010, TP0126-TP0129, TP0135-TP0136, and TP0548-TP0549). In addition, 14 frameshifts were identified in TP0126, TP0131, TP0132, TP0309, TP0310, TP0311, TP0313, TP0317, TP0318, TP0487, TP0621, TP0922, TP0968, and TP1030 leading to premature termination of 10 genes, elongation of three genes, and a reading frame change in one gene (Table 3).

Hypothetical proteins were characterized by searching the InterPro and Pfam databases and constructing hydrophobicity plots. Of the 25 hypothetical genes described in this study (Tables 1 to 3), 17 were completely sequenced in both the Cuniculi A and Nichols strains. The corresponding 17 hypothetical proteins were analyzed to predict cellular localization. Signal sequences were predicted for six Nichols proteins (encoded by TP0133, TP0134, TP0135, TP0136, TP0548, and TP0733) and five Cuniculi A proteins (encoded by TP0315, TP0470, TP0548, fused genes TP0617 and TP0618, and TP0733). In both strains, transmembrane regions were predicted for TP0733. Three putative protein domains (UPF0164, TPR, and DbpA) were found in five hypothetical proteins (encoded by TP0470, TP0548, TP0860, TP0865, and TP1029). No differences between the Nichols and Cuniculi A strains were found in domain distribution.

DNA sequencing of Cuniculi A chromosomal regions.To determine the level of sequence identity between the Nichols and Cuniculi A genomes, three chromosomal regions that also included IGR were sequenced. Analysis of these regions (5,289 bp; 0.46% of the genome), comprising genes TP0798 to TP0800 (accession number EF419251), TP0933 and TP0934 (accession number EF419252), and TP0961 and TP0962 (accession number EF419253), revealed 37 SNPs that resulted in 11 amino acid substitutions in the corresponding proteins. The average density of SNPs represented one nucleotide change per 143 bp (99.3% identity).