Genetic divergence and evolutionary instability in ospE-related members of the upstream homology box gene family in Borrelia burgdorferi sensu lato complex isolates - PubMed (original) (raw)

Comparative Study

S Y Sung et al. Infect Immun. 1998 Oct.

Abstract

A series of related genes that are flanked at their 5' ends by a conserved upstream sequence element called the upstream homology box (UHB) have been identified in Borrelia burgdorferi. These genes have been referred to as the UHB or erp gene family. We previously demonstrated that among a limited number of B. burgdorferi isolates, the UHB gene family is variable in composition and organization. Prior to this report the UHB gene family in other species of the B. burgdorferi sensu lato complex had not been studied, and if this family is important in the pathogenesis or biology of the Lyme disease spirochetes, then a wide distribution among species and isolates of the B. burgdorferi sensu lato complex would be expected. To assess this, we screened for the UHB element by Southern hybridization and determined its restriction fragment length polymorphism (RFLP) patterns. The UHB element was found to be carried by all B. burgdorferi sensu lato complex species tested (B. burgdorferi, B. garinii, B. afzelii, B. japonica, B. valaisiana sp. nov., and B. andersonii), but the RFLP patterns varied widely at both the inter- and intraspecies levels. Variation in both the number and size of the hybridizing restriction fragments was evident. PCR analyses also revealed the presence of polymorphic, ospE-related alleles in many isolates. Sequence analyses identified the molecular basis of the polymorphisms as being primarily insertions and deletions. Sequence variation and the insertions and deletions were found to be clustered in two distinct domains (variable domains 1 and 2). In many isolates variable domain 1 is flanked by direct repeat elements, some as long as 38 bp. Computer analyses of the deduced amino acid sequences encoded within variable domain 1 predict them to be hydrophilic, surface exposed, and antigenic. The analyses conducted here suggest that the UHB gene family, as evidenced by the variable UHB RFLP patterns, is not evolutionarily stable and that the polymorphic ospE alleles are derived from a common ancestral gene which has been modified through mutation or recombination events. The characterization of ospE-related genes of the UHB gene family among B. burgdorferi sensu lato species will prove important in attempts to construct a model for UHB gene family organization and in deciphering the role of the UHB gene family in the biology and pathogenesis of the Lyme disease spirochetes.

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Figures

FIG. 1

FIG. 1

RFLP patterns of the UHB element among B. burgdorferi sensu lato complex isolates. _Hae_III-digested DNA was transferred onto a Hybond N membrane and hybridized with the uhb(+) oligonucleotide under conditions described in the text. The isolates analyzed are indicated above the lanes, and molecular size standards (in kilobases) are indicated on the right. Isolates N34, G25, VSBP, Pbi, FRG, B4-91, and B487 are B. garinii; VS116 is B. valaisiana; ECM1 and UMO1 are B. afzelii; IKA2 and HO14 are B. japonica; 21038 is B. andersonii; and N40, CA13, CA3, CA8, IP89, CA9, LP3, VS307, VS134, and T2 are B. burgdorferi.

FIG. 2

FIG. 2

PCR analyses of ospE and hybridization analyses of the amplicons with oligonucleotide probes targeting various ospE paralogs. PCR with the uhb(+)-E470(−) primer set was performed on various isolates. Ten microliters of each reaction mixture was analyzed in a 1.2% agarose gel and then stained with ethidium bromide. (Top panel) Representative PCR data; (lower panels) hybridization results with the amplicons from the top panel. The isolates analyzed are indicated at the top, and the oligonucleotide probes used are indicated at the right. Isolates LP3, LP4, LP7, CA2, CA3, CA4, CA12, R100, JD1, 297, CA9, B31, T2, 272, B31T, NY186, and 25015 are B. burgdorferi; 21038 is B. andersonii; IP90, B491, and B691 are B. garinii; VS116 is B. valaisiana; Pko, UMO1, ECM1, Pbo, and Pgau are B. afzelii; and IKA2 is B. japonica.

FIG. 3

FIG. 3

Alignment of the uhb(+)-E470(−) amplicon nucleotide sequences. Sequences were aligned by using the PILEUP program with some manual adjustment. The isolates analyzed are indicated at the left. For comparative purposes some previously determined gene sequences (ospE, erpC, erpA, and p21) were included, and these are indicated by their designated gene names (for accession numbers, see Table 1). Gaps are indicated by dashes. The binding sites of some oligonucleotide probes and primers (indicated above the alignment) are in boldface. Repeat elements are indicated by underlining, and mismatches are indicated by capital letters. Putative ribosomal binding sites (RBS) and translational start codons are also indicated. Since some of these sequences are partial and lack their 3′ termini, stop codons are not shown for all sequences. The two different sequences from B. japonica IKA2 are the sequences determined for each of the two amplicons (ika2-2 and ika2-3) obtained from this isolate.

FIG. 3

FIG. 3

Alignment of the uhb(+)-E470(−) amplicon nucleotide sequences. Sequences were aligned by using the PILEUP program with some manual adjustment. The isolates analyzed are indicated at the left. For comparative purposes some previously determined gene sequences (ospE, erpC, erpA, and p21) were included, and these are indicated by their designated gene names (for accession numbers, see Table 1). Gaps are indicated by dashes. The binding sites of some oligonucleotide probes and primers (indicated above the alignment) are in boldface. Repeat elements are indicated by underlining, and mismatches are indicated by capital letters. Putative ribosomal binding sites (RBS) and translational start codons are also indicated. Since some of these sequences are partial and lack their 3′ termini, stop codons are not shown for all sequences. The two different sequences from B. japonica IKA2 are the sequences determined for each of the two amplicons (ika2-2 and ika2-3) obtained from this isolate.

FIG. 3

FIG. 3

Alignment of the uhb(+)-E470(−) amplicon nucleotide sequences. Sequences were aligned by using the PILEUP program with some manual adjustment. The isolates analyzed are indicated at the left. For comparative purposes some previously determined gene sequences (ospE, erpC, erpA, and p21) were included, and these are indicated by their designated gene names (for accession numbers, see Table 1). Gaps are indicated by dashes. The binding sites of some oligonucleotide probes and primers (indicated above the alignment) are in boldface. Repeat elements are indicated by underlining, and mismatches are indicated by capital letters. Putative ribosomal binding sites (RBS) and translational start codons are also indicated. Since some of these sequences are partial and lack their 3′ termini, stop codons are not shown for all sequences. The two different sequences from B. japonica IKA2 are the sequences determined for each of the two amplicons (ika2-2 and ika2-3) obtained from this isolate.

FIG. 3

FIG. 3

Alignment of the uhb(+)-E470(−) amplicon nucleotide sequences. Sequences were aligned by using the PILEUP program with some manual adjustment. The isolates analyzed are indicated at the left. For comparative purposes some previously determined gene sequences (ospE, erpC, erpA, and p21) were included, and these are indicated by their designated gene names (for accession numbers, see Table 1). Gaps are indicated by dashes. The binding sites of some oligonucleotide probes and primers (indicated above the alignment) are in boldface. Repeat elements are indicated by underlining, and mismatches are indicated by capital letters. Putative ribosomal binding sites (RBS) and translational start codons are also indicated. Since some of these sequences are partial and lack their 3′ termini, stop codons are not shown for all sequences. The two different sequences from B. japonica IKA2 are the sequences determined for each of the two amplicons (ika2-2 and ika2-3) obtained from this isolate.

FIG. 3

FIG. 3

Alignment of the uhb(+)-E470(−) amplicon nucleotide sequences. Sequences were aligned by using the PILEUP program with some manual adjustment. The isolates analyzed are indicated at the left. For comparative purposes some previously determined gene sequences (ospE, erpC, erpA, and p21) were included, and these are indicated by their designated gene names (for accession numbers, see Table 1). Gaps are indicated by dashes. The binding sites of some oligonucleotide probes and primers (indicated above the alignment) are in boldface. Repeat elements are indicated by underlining, and mismatches are indicated by capital letters. Putative ribosomal binding sites (RBS) and translational start codons are also indicated. Since some of these sequences are partial and lack their 3′ termini, stop codons are not shown for all sequences. The two different sequences from B. japonica IKA2 are the sequences determined for each of the two amplicons (ika2-2 and ika2-3) obtained from this isolate.

FIG. 3

FIG. 3

Alignment of the uhb(+)-E470(−) amplicon nucleotide sequences. Sequences were aligned by using the PILEUP program with some manual adjustment. The isolates analyzed are indicated at the left. For comparative purposes some previously determined gene sequences (ospE, erpC, erpA, and p21) were included, and these are indicated by their designated gene names (for accession numbers, see Table 1). Gaps are indicated by dashes. The binding sites of some oligonucleotide probes and primers (indicated above the alignment) are in boldface. Repeat elements are indicated by underlining, and mismatches are indicated by capital letters. Putative ribosomal binding sites (RBS) and translational start codons are also indicated. Since some of these sequences are partial and lack their 3′ termini, stop codons are not shown for all sequences. The two different sequences from B. japonica IKA2 are the sequences determined for each of the two amplicons (ika2-2 and ika2-3) obtained from this isolate.

FIG. 4

FIG. 4

Alignment of the deduced amino acid sequences. The amino acid alignment was generated as described in the text with some manual adjustment. The isolate from which a sequence was obtained is indicated at the left. Some previously determined sequences (6, 8, 20, 25) were included in the alignment, and the corresponding protein names are indicated on the left. Repeat motifs are indicated by boldface. In cases where the repeats are tandem, alternating copies are indicated by italics and underlining. Mismatches among the repeat elements are indicated by lowercase letters.

FIG. 5

FIG. 5

Computer analysis of the possible physical properties of the deduced amino acid sequence of the B. burgdorferi JD1 _ospE_-related amplicon. The output from analyses conducted by using the Genetics Computer Group, Wisconsin, Sequence Analysis Package, version 9.0, as described in the text, is shown. In all cases default values were used. Variable domain 1 spans residues 20 through 75. KD, Kyte-Doolittle; Prob., probability.

FIG. 6

FIG. 6

Phylograms of _ospE_-related genes. Phylograms were constructed by using the translated amplicon sequences determined in this study and other, previously determined sequences as described in Materials and Methods. The isolate from which a particular amplicon was obtained is indicated at the terminus of the branch. The designations p21 (26), ospE (14), and erpA and erpC (25) indicate gene names, and these sequences were previously determined by others. The phylogram on the left was constructed by using the alignment presented in Fig. 4, while the phylogram on the right was constructed by using the same alignment after deletion of variable domains 1 and 2. Note that some clustering relationships changed as a result of deletion of the variable domains.

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