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Journal of Bacteriology, September 2006, p. 6124-6134, Vol. 188, No. 17
0021-9193/06/$08.00+0     doi:10.1128/JB.00459-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Comparative Genome Hybridization Reveals Substantial Variation among Clinical Isolates of Borrelia burgdorferi Sensu Stricto with Different Pathogenic Properties

Darya Terekhova,1 Radha Iyer,1 Gary P. Wormser,2 and Ira Schwartz1,2*

Departments of Microbiology and Immunology,1 Division of Infectious Diseases, Department of Medicine, New York Medical College, Valhalla, New York 105952

Received 3 April 2006/ Accepted 16 June 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Clinical and murine studies suggest that there is a differential pathogenicity of different genotypes of Borrelia burgdorferi, the spirochetal agent of Lyme disease. Comparative genome hybridization was used to explore the relationship between different genotypes. The chromosomes of all studied isolates were highly conserved (>93%) with respect to both sequence and gene order. Plasmid sequences were substantially more diverse. Plasmids lp54, cp26, and cp32 were present in all tested isolates, and their sequences and gene order were conserved. The majority of linear plasmids showed variation both in terms of presence among different isolates and in terms of sequence and gene order. The data strongly imply that all B. burgdorferi clinical isolates contain linear plasmids related to each other, but the structure of these replicons may vary substantially from isolate to isolate. These alterations include deletions and presumed rearrangements that are likely to result in unique plasmid elements in many isolates. There is a strong correlation between complete genome hybridization profiles and other typing methods, which, in turn, also correlate to differences in pathogenicity. Because there is substantially less variation in the chromosomal and circular plasmid portions of the genome, the major differences in open reading frame content and genomic diversity among isolates are linear plasmid driven.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Borrelia burgdorferi is the spirochetal pathogen of Lyme disease (53), the most frequently reported arthropod-borne infection in the United States (41). Lyme disease has a worldwide distribution, with cases reported throughout the northern hemisphere (39, 52, 57). There are 11 related Borrelia species within the B. burgdorferi sensu lato complex, but only three, i.e., B. burgdorferi sensu stricto (referred to simply as B. burgdorferi), B. afzelii, and B. garinii are known to cause a substantial number of human disease cases (57). All three genospecies occur and cause disease in Europe (39, 57), whereas all human infection in the United States is caused by B. burgdorferi sensu stricto (57).

The only complete Borrelia genome sequence reported to date is that of B. burgdorferi sensu stricto strain B31MI (17). Its linear chromosome and 21 linear and circular plasmids carry a total of 853 chromosomal and 898 plasmid genes and pseudogenes (9, 17). Biological roles were assigned to 59% of the chromosomal and only 4% of the plasmid open reading frames (ORFs) (9, 17). Thus, there is no information regarding function for a majority of the genes carried in the B. burgdorferi genome.

B. burgdorferi isolates can be differentiated by several genotypic parameters (6, 27, 32, 33, 38, 61). We have classified isolates obtained from patients with early Lyme disease on the basis of rRNA gene spacer sequences and have shown that the distribution of genotypes varies between isolates obtained from erythema migrans (EM) skin lesions and blood of these patients (35). There is a highly significant association between B. burgdorferi rRNA gene spacer type (RST) and hematogenous dissemination in this patient population (59). This association was confirmed in mice and demonstrated that isolates designated as having the RST1 genotype produced a more severe, disseminated infection than did isolates of the RST3 genotype (56).

Differences in genomic content between clinical isolates of different RST genotypes may correlate to differences in pathogenicity, and genome comparisons may identify potential virulence-encoding elements. Comparative genome hybridization (CGH) employing microarrays has been used to assess genome plasticity, population structure, and evolutionary trends and to reveal elements encoding potential virulence factors in a number of bacterial pathogens (3, 5, 12, 13, 15, 24, 25, 31, 49, 51, 58, 60). Some of the putative virulence-encoding regions identified by CGH studies, such as the RD1 region in Mycobacterium tuberculosis (3), have subsequently been proven to contain genes responsible for pathogenicity (22). Furthermore, comparison between genomes of B. burgdorferi isolates should reveal genes that are conserved and thus may encode essential functions, perhaps providing clues to the roles of ORFs with unknown function. To begin such an analysis, genome arrays based on the sequence of strain B31MI were produced and total genomic DNAs of 15 B. burgdorferi isolates representing different genotypes were compared to that of strain B31MI by CGH.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
B. burgdorferi isolate sources and cultivation. The B. burgdorferi isolates used in the study are listed in Table 1. Clinical isolates were obtained from either skin biopsy of EM lesions or blood of patients presenting with EM at the Lyme Disease Practice of the Westchester Medical Center. All clinical isolates were used at passage 4 or less. Cultures were grown in Barbour-Stoenner-Kelly H medium (Sigma, St. Louis, MO) supplemented with 6% rabbit serum (Sigma) for 5 days to 2 weeks at 34°C.


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  		TABLE 1. Isolates employed in this study

 
DNA isolation. DNA was isolated from 10 ml of B. burgdorferi culture (1 x 107 to 1 x 108 cells/ml) by using the IsoQuick nucleic acid extraction kit (Orca Research, Bothell, WA). Isolated DNA was resuspended in 50 µl of nuclease-free water and kept at 4°C. DNA concentrations were determined by measuring optical density at 260 nm.

B. burgdorferi microarray construction. Oligonucleotides representing all annotated ORFs and pseudogenes of B. burgdorferi B31MI in the TIGR or NCBI genome databases were synthesized by QIAGEN-Operon (Alameda, CA) according to a proprietary algorithm that optimizes the melting temperature (Tm) for all oligonucleotides within 5°C of each other. The target Tm was 70°C, and only five oligonucleotides (BB0150, BB0609, BBH39, BBK001, and BBQ83) failed to have a minimum Tm of ≥65°C. All oligonucleotides are 70 nucleotides in length and are at least 40 nucleotides from the 3' end of the ORF sequence (except for a number of short ORFs, where these criteria could not be met). In addition, 19 random-sequence 70-mers were synthesized as negative controls.

The oligonucleotides were resuspended in Pronto printing ink (Corning Inc., Corning, NY) to a concentration of 40 µM and printed on UltraGap gamma amino propyl silane slide surfaces (Corning Inc., Corning, NY), using a Bio-Rad VersaArray arrayer. Each oligonucleotide was printed in triplicate on each array, with spot-to-spot separation of 350 µm. Following printing, the arrays were cross-linked with 600 mJ of UV energy by using a Stratalinker cross-linker and stored in a desiccator prior to hybridization.

Several quality control and quality assurance tests were performed for each array print run. These included nonspecific staining of the printed oligonucleotides, hybridization with random cyanine-3-dUTP (Cy3)-labeled 9-mers, and experimental B. burgdorferi DNA hybridizations. These tests allowed for assessment of the overall quality of the arrays.

DNA labeling and hybridization. Total genomic DNA was digested with HindIII (New England Biolabs, Beverly, MA) for 2 h at 37°C, followed by heat inactivation at 65°C for 15 min. Digested DNA samples were stored at –80°C until use. DNA of reference strain B31MI was labeled with cyanine-5-dUTP (Cy5) (Perkin-Elmer, Boston, MA), and genomic DNAs from all other isolates were labeled with Cy3 (Perkin Elmer). Two micrograms of digested genomic DNA was labeled by incubation with 10 µg of random hexamers (Invitrogen, Carlsbad, CA), 4 µg of random hexamers (Promega, San Luis Obispo, CA), 30 U of the Klenow fragment of DNA polymerase I (New England Biolabs), 3 nmol of Cy3 or Cy5 (Perkin-Elmer), and 12 nmol of deoxynucleoside triphosphates (Roche, Indianapolis, IN) in a total volume of 24 µl at 37°C for 3 h. Klenow fragment was subsequently inactivated by addition of 1.5 µmol of EDTA (EMD, Darmstadt, Germany) to the labeling mixture. Nine micromoles of sodium acetate and 2 volumes of ice-cold ethanol were added to the labeling reaction mixture, which was incubated for 1 h at –20°C. The resulting precipitate was recovered by centrifugation, washed with 75% ethanol, and dissolved in 50 µl of nuclease-free water. Cy3- and Cy5-labeled DNAs were combined and further purified using the QIAquick PCR purification kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. Purified DNAs were dried and resuspended in 45 µl of nuclease-free water. The following reagents were added to the purified DNA solution: 0.5 µg of salmon sperm DNA, 24 µl of formamide (Acros Organics, Belgium), 24 µl of 4x RNA hybridization buffer (Amersham Biosciences, Piscataway, NJ), and water to a final volume of 100 µl. The mixture was then denatured by heating at 95°C for 5 min, cooled on ice for 1 min, and applied to arrays. The slides were prehybridized in a solution of 30% formamide, 5x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate (SDS), and 0.1% bovine serum albumin (Sigma) for 45 to 60 min at 42°C. They were then washed in distilled water twice, dipped in isopropanol, and dried immediately by centrifugation at 2,700 rpm for 3 min. After application of the labeled DNA sample, slides were incubated overnight at 42°C and washed twice with 2x SSC-0.1% SDS for 10 min at 42°C, twice with 0.1x SSC-0.1% SDS for 10 min at room temperature, and four times with 0.1x SSC for 1 min at room temperature. Arrays were immediately dried by centrifugation at 2,700 rpm for 3 min.

Microarray scanning and data analysis. The hybridized microarray slides were scanned on a GenePix 4000B microarray scanner (Axon Instruments, Union City, CA) at 10-µm resolution. The resulting dual-color images were recorded in 16-bit multi-image TIFF files which were analyzed by GenePix Pro software (Axon Instruments). GenePix data were input to Microsoft Excel for background correction, normalization, and filtering. Means and standard deviations of the fluorescence intensities were calculated for all blank control spots in Cy3 and Cy5 channels separately; the mean plus two standard deviations was considered to be background, and this value was subtracted from each intensity value in the appropriate channel. Background correction was performed separately for each slide. Spots yielding signals below background for B31MI DNA hybridization and/or with irregular morphology were excluded from analysis. The background-adjusted intensity values were normalized across all arrays. The resultant values were used to calculate log2(Cy3/Cy5) for each spot, and six log2(Cy3/Cy5) values for each ORF from two separate microarrays were averaged to yield a final value. These mean log2(Cy3/Cy5) values for each ORF in each isolate were visualized using GeneTraffic (Iobion, La Jolla, CA). Conservation and divergence of ORFs were determined using GACK (Charlie Kim, Stanford University) (29). Binary output with estimated probability of presence of ≥95% for conserved ORFs was used to distinguish between conserved and divergent ORFs. ORFs predicted as divergent by GACK analysis were verified individually for spot morphology in GeneTraffic.

PCR amplification and DNA sequence analysis. The nature of selected divergent chromosomal ORFs was determined by PCR amplification and subsequent sequencing. PCR primers were designed for regions flanking the ORFs to be analyzed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and the sequence of B. burgdorferi B31MI. All primers are listed in Table 2. Each PCR mixture contained 10 ng of DNA, 250 µM deoxynucleoside triphosphates (Roche), 10 ng of each primer, and 1 U of Taq DNA polymerase (Roche) in PCR buffer (Roche). PCR conditions were optimized for each pair of primers, and PCR products were analyzed by electrophoresis in 1.5% agarose gels. PCR products were purified using the QIAquick PCR purification kit (QIAGEN) according to the manufacturer's instructions and were sequenced by Davis Sequencing (Davis, CA). Sequence comparisons were performed by means of ClustalW (http://www.ebi.ac.uk/clustalw/).


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  		TABLE 2. Primers used in this study

 
Southern blot analysis. Bacterial cells were harvested, washed, mixed with 1% pulsed-field grade agarose, and lysed with proteinase K in agarose blocks (Bio-Rad, Hercules, CA) as previously described (54). Total genomic content was resolved by pulsed-field gel electrophoresis (PFGE) on a CHEF DR II apparatus (Bio-Rad) at constant voltage (6 V/cm) for 19 h with a switch time of 0.7 to 2.2 s in 0.5x Tris-borate-EDTA at 14°C. DNA was visualized by staining with ethidium bromide, after which DNA was transferred to positively charged nylon membranes (Boehringer Manheim) and hybridized with digoxigenin-labeled probes as previously described (26).

Phylogenetic analysis. For whole-genome hybridization analysis, individual ORFs were coded as binary characters of 0 (divergent) or 1 (conserved) as determined by GACK. These values were used as input for maximum-parsimony analysis using PAUP version 4.0b10 (Sinauer Associates, Sunderland, Mass.). Parsimony analysis was based on the subset of characters that were phylogenetically informative. Characters were unordered and given equal weight. A heuristic search with 100 replicates of random-sequence addition and tree-bisection-reconnection branch swapping resulted in four most-parsimonious trees. A bootstrap analysis with 1,000 replicates was conducted to determine the statistical stability of each node of the tree. A high-resolution image of the phylogram was generated by using TreeView version 1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).

Nucleotide sequence and microarray data accession numbers. Fully annotated microarray data have been deposited in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) with accession numbers E-MEXP-735 Schwartz-B.burgdorferis.s.-CGH (for the experiment) and A-MEXP-505 B.burgdorferi 70mer oligoarray v.3 and A-MEXP-506 B.burgdorferi B31MI 70mer oligo v.2 (for the oligoarray designs used). Nucleotide sequences for different alleles of BB0227, BB0419, BB0535, BB0570, and BB0766 have been deposited in GenBank (http://www.ncbi.nlm.nih.gov) with accession numbers DQ662896 to -9, DQ662903 to -5, DQ662900 to -2, DQ662906 to -17, and DQ662918 to -31, respectively.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Oligonucleotide microarrays as a tool for B. burgdorferi CGH. Microarray quality was determined by preliminary hybridizations with reference B31MI DNA labeled with both Cy3 and Cy5 (Fig. 1). Seventy-nine ORFs (4.5%) represented on the arrays were noninformative due to irregular spot morphology and/or B31MI hybridization signals that were below background. These ORFs are listed in Table S1 at http://www.nymc.edu/iraschwartz/gene_arrays.asp and were excluded from all further analyses. In addition, since the clone of reference strain B31MI used in the study lacks cp9, the 12 ORFs carried on this plasmid were not included in the analysis.


Figure 1
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  		FIG. 1. Complete genome hybridization analysis of 17 B. burgdorferi isolates. Cy3-labeled tester DNA (from each isolate) and Cy5-labeled B31 MI DNA were cohybridized to B. burgdorferi 70-mer oligonucleotide arrays and analyzed on a GenePix array scanner. GenePix data were imported to GeneTraffic v. 3.2. Shown is a genomic comparison generated in GeneTraffic. Each column represents an isolate. Each row represents a different ORF. Rows are arranged by replicon as indicated in the right margin. Black areas represent ORFs conserved in the isolate, red indicates divergent ORFs compared to B31MI, and white indicates that no interpretable data are available.

 
To assess the specificity of the arrays, Cy3-labeled DNA from strain B314, a B31 derivative lacking linear plasmids (48), was cohybridized with Cy5-labeled B31MI DNA (Fig. 1). As expected, Cy3 and Cy5 signals were essentially equal for virtually all the ORFs except those carried on linear plasmids, for which the Cy3 signal was below background. The only linear-plasmid-carried ORFs which produced hybridization signals above background were those that have paralogous genes carried on circular plasmids or the chromosome (9, 17).

Distribution of conserved and divergent ORFs in the B. burgdorferi genome. B. burgdorferi isolates can be differentiated into three RSTs (35) and nine intergenic spacer (IGS) types (6) based on rRNA gene spacer sequence and into 20 OspC types (57) based on ospC sequence. The isolates differ in pathogenicity, and a correlation between RST and pathogenicity was observed: the most invasive of the isolates are associated with RST1 and the least invasive with RST3 (55, 56, 59). To assess whether variability on a genomic scale correlates with typing of single loci and may be responsible for differences in pathogenicity, isolates representing all three RSTs, seven IGS types, and seven OspC types (Table 1) were compared by genome hybridization to the custom B. burgdorferi oligonucleotide arrays. Total genomic DNA for each isolate was labeled with Cy3 and hybridized simultaneously with Cy5-labeled B31MI DNA as a reference. Each hybridization was performed in duplicate. Complete hybridization profiles for 17 B. burgdorferi isolates are presented in Fig. 1. All ORFs were categorized as either conserved or divergent relative to B31MI by GACK analysis (29). Conservation of ORFs among the isolates is summarized in Table 3 and shown schematically in Fig. 2 for the plasmids.


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  		TABLE 3. Conserved ORFs identified by microarray analysis

 

Figure 2
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  		FIG. 2. Conservation of ORFs on B. burgdorferi plasmids. All plasmids are presented in linear form beginning with the gene numbered 01 according to Fraser et al. and Casjens et al. (9, 17). Plasmid lengths are scaled according to the number of carried ORFs (not total size). Conserved ORFs are presented as black and divergent ORFs as red. White indicates that no interpretable data were available. The bracketed region on lp56 indicates insertion of cp32 sequence. Note that ORFs carried on plasmids lp28-4 and lp38 are conserved among all isolates containing the plasmid, but plasmid is completely absent from some isolates.

 
The chromosome is highly conserved, with 93 to 99% ORF identity relative to B31MI (Table 3). Among the plasmids, three patterns were observed. Some plasmids are present in all isolates and are highly conserved (lp54, cp26, and most cp32). Certain plasmids are absent from some isolates but when present show low ORF divergence (lp28-4, lp38, and some cp32). The remaining plasmids show substantial variation among isolates. The presence or absence of a specific plasmid can be assessed by scoring for positive hybridization to plasmid-specific partition loci. Plasmids absent from certain isolates are listed in Table 4. B. burgdorferi genomic variation is described in more detail below.


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  		TABLE 4. Plasmids absent from certain isolates

 
(i) The chromosome. The chromosome is the most conserved portion of the B. burgdorferi genome. Of those ORFs that could be analyzed by array, 33 and 62 were found to be divergent in RST2 and RST3 isolates, respectively (see Table S2 at http://www.nymc.edu/iraschwartz/gene_arrays.asp). Of these, 16 were located at the right telomeres of several isolates (Fig. 1; see Table S2 at http://www.nymc.edu/iraschwartz/gene_arrays.asp), which is likely due to the size variation relative to B31MI as previously reported (8). The remaining ORFs were distributed along the length of the chromosome (see Table S2 at http://www.nymc.edu/iraschwartz/gene_arrays.asp), with no clustering of divergent ORFs. Thus, absence of hybridization was not due to large-scale deletion events.

Lack of hybridization could be due to the total absence of a gene or to sequence variation. In order to clarify the nature of the divergent chromosomal ORFs, PCR amplification using primers from the regions flanking four of these ORFs was performed. Figure 3 shows representative results for BB0570. For the 10 isolates studied, PCR products of the same size as that for B31MI were obtained (Fig. 3A), suggesting that weak hybridization was due to sequence variation rather than deletion. Sequencing of the PCR products showed five nucleotide changes in the region covered by the 70-mer representing BB0570 on the array (Fig. 3B); similar results were obtained with BB0419 and BB0766. Thus, 7% sequence variation was sufficient to abrogate detectable hybridization to the array. Sequencing of the entire BB0570 ORF revealed a total of 13 single-nucleotide polymorphisms (SNPs), i.e., 3.5% sequence divergence. Twelve of these resulted in synonymous codon changes, and one would result in a conservative lysine-to-arginine amino acid change. Similar results were obtained for BB0419 and BB0766, which showed 17 and 15 SNPs distributed across the entire ORF, resulting in synonymous or conservative amino acid changes representing 1.9% and 3.1% sequence divergence, respectively. By contrast, similar analysis of two conserved ORFs (BB0227 and BB0535) revealed one and two SNPs in the 70-mer oligonucleotide region representing the ORF on the arrays and totals of four and four SNPs distributed across the entire ORF, representing 0.6% and 0.5% sequence divergence, respectively. Thus, oligonucleotides affixed to the microarrays used in the study appear to be representative of the complete ORFs. Taken together, these results demonstrate few, if any, gene deletions in the chromosome (other than at the right telomere) and show that when SNPs do occur among different isolates, they would result in little variation in protein sequence.


Figure 3
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  		FIG. 3. Analysis of BB0570, a divergent chromosomal ORF. (A) PCR amplification of BB0570 with primers from regions flanking the ORF. PCR products of a size equal to that of B31MI were obtained for all isolates analyzed. (B) Comparison of BB0570 sequences for 12 B. burgdorferi strains to that of a 70-mer oligonucleotide representing BB0570 on the microarray. Asterisks indicate positions of sequence identity.

 
(ii) cp26 and lp54. Circular plasmid cp26 and linear plasmid lp54 are the most conserved B. burgdorferi plasmids. They are present in all tested isolates and contain little ORF divergence. ORF comparisons for these plasmids are presented in Fig. 4 (also see Fig. 2 and Table 3). All lp54-carried ORFs except BBA70 were conserved in all isolates analyzed (Fig. 2 and 4 and Table 3). In addition, BBA10 and BBA24 were found to be divergent in isolate JD1 (Fig. 2 and 4 and Table 3). cp26 was also present in all isolates (Tables 3 and 4). Only five cp26-carried ORFs were divergent in more than two isolates. These were the cluster of BBB19 to -22, which includes the polymorphic ospC (BBB19), and BBB29 (Fig. 2 and 4).


Figure 4
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  		FIG. 4. Genomic comparison for linear plasmid lp54 (left) and circular plasmid cp26 (right). Microarray analysis was performed as described in the legend to Fig. 1. Positions of ORFs discussed in the text are indicated. Black areas represent ORFs conserved in the isolate, and red indicates divergent ORFs compared to B31MI.

 
(iii) Circular cp32 plasmids. B. burgdorferi B31MI contains seven copies of highly paralogous circular cp32 plasmids and a complete cp32 sequence integrated into linear plasmid lp56 (9, 10, 17). These replicons were among the most conserved in the isolates analyzed (Fig. 1 and 2 and Table 3). Although virtually all of the cp32-carried ORFs appear to be conserved, the high density of paralogous sequences found on all these plasmids complicates definitive conclusions regarding the presence of all ORFs on all cp32 plasmids. For example, most cp32-carried ORFs share high sequence homology among all cp32 plasmids within isolates (9), and the presence of only a single copy of such an ORF would result in a positive hybridization signal for all paralogues. The partition loci and ospE/ospF/elp (erp) genes are among the most divergent sequences on cp32 plasmids (9). Thus, specific hybridization to oligonucleotide probes for each of these loci could be taken to indicate the presence of a specific cp32 plasmid. On this basis, the majority of isolates tested harbored all B31MI-like cp32s; cp32-1 and cp32-8 were absent individually from some of RST1 isolates (Table 4), consistent with previous studies (26). cp32 content was more variable in RST2 and RST3 than in RST1 isolates (Tables 3 and 4).

Linear plasmid lp56 contains a complete cp32 sequence (9). Several of the isolates analyzed have been shown previously to lack a B31MI-like lp56 (26), and this is confirmed in the present study (Table 4). The non-cp32 portion of lp56 is highly conserved among RST1 isolates but showed substantial variation in the remaining isolates (Fig. 1 and 2 and Table 3).

(iv) Linear plasmids. With the exception of lp54, linear plasmids constitute the most divergent portion of the B. burgdorferi genome (Fig. 1 and 2 and Tables 3 and 4). This variation can be the result of sequence divergence, deletion of individual ORFs, or absence of entire plasmids. Partition loci for some plasmids were not detected for several isolates, suggesting the absence of these plasmids (Table 4), but when present there was virtually complete sequence conservation. This was the case for lp28-4 and lp38 (Fig. 2). For the remaining linear plasmids, even when plasmid partition loci were present, extensive variability was observed in other plasmid-carried ORFs. These divergent ORFs were often found in clusters (Fig. 1 and 2).

The divergent portion of lp36 (BBK35 to -50) was analyzed further in order to determine the nature of this variability. PCR amplification of selected ORFs in this region with B31MI-based primers was unsuccessful, suggesting that these regions may be deleted. To confirm this possibility, total genomic DNA was separated by PFGE, the DNA was transferred to nylon membranes, and Southern blotting was performed with probes corresponding to three different regions of lp36: probes BBK11-12, BBK21-23, and BBK45. The BBK11-12 region was previously used to determine the presence of lp36 in clinical isolates by PCR (26) and corresponds to a region which appears to be conserved based on the current microarray analysis (Fig. 5A). Probe BBK21-23 corresponds to the partition locus of lp36 (17), and probe BBK45 corresponds to a putative divergent region (Fig. 5A). The Southern blot results were in complete agreement with the data obtained by microarray analysis (Fig. 5B). All three probes hybridized to a 36-kb band in all RST1 isolates. Hybridization was absent with strain B314, which is known to lack all linear plasmids (48). Variable results were obtained for the remaining isolates. Probe BBK11-12 hybridized to all isolates except B356 and B376, and probe BBK21-23 hybridized to bands in all isolates except of B356 (Fig. 5B). However, the sizes of the bands varied among the isolates. In those cases where hybridization was observed with both probes, the two hybridizing bands were always the same size, but this size was invariably not 36 kbp as in B31MI. By contrast, probe BBK45 did not hybridize to most of the non-RST1 isolates examined (Fig. 5B). BBK45 probe hybridization was observed only for isolates B348, B418, and N40, but the bands were smaller than those to which the two other probes hybridized (Fig. 5B). This suggests that BBK45 is not located on an lp36-like replicon in these isolates. These findings suggest that RST1 isolates contain an lp36 plasmid essentially identical to that of B31MI. Non-RST1 isolates contain an lp36-like plasmid (based on the presence of the B31MI-like partition locus), but this replicon is substantially different from that found in B31MI. In particular, sequences corresponding to BBK35 to -50 are absent in most of these isolates. Finally, B356 appears to lack an lp36-related plasmid entirely. Similar Southern blot studies were performed for plasmids lp28-1 and lp56 and corroborated the microarray findings. Taken together, these data strongly imply that B. burgdorferi isolates contain linear plasmids related to those characterized in strain B31MI (based on the presence of plasmid-specific partition loci), but the structure of these replicons is substantially different from that of those found in strain B31MI. These alterations include deletions and presumed rearrangements that are likely to result in unique plasmid elements in many of these isolates.


Figure 5
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  		FIG. 5. Analysis of linear plasmid lp36. (A) Genomic hybridization to oligonucleotides representing lp36 ORFs. Black areas represent conserved ORFs, and red represents divergent ORFs compared to B31MI. The positions of ORFs discussed in the text are indicated in the right margin. (B) Southern blotting of PFGE-separated total genomic DNAs of B. burgdorferi isolates with lp36-specific probes BBK11-12 (upper panel), BBK21-23 (middle panel), and BBK45 (lower panel). Arrows indicate the position of B31MI lp36.

 
Correlation of CGH profiles with other typing methods. All RST1 isolates studied to date are IGS type 1 and either OspC type A or B (data not shown). The RST1 clinical isolates employed in the current study are all IGS type 1 and OspC type A. The genomes of RST1 isolates were the most similar to that of B31MI and to each other, and they formed a single homogenous group distinct from the other isolates (Fig. 1). This was expected, since the microarray is based on the B31MI sequence and B31MI is an RST1 isolate (35). Genomes of RST1 isolates constitute the most conserved group, with 98% of ORFs being conserved (Table 3). The only exception to this conservation was the absence of certain plasmids in individual isolates. All three RST1 isolates apparently lacked lp5 and lp21, and single isolates lacked a particular cp32 (Table 4). By contrast, the hybridization profiles of the remaining isolates were substantially more heterogeneous (Fig. 1 and Table 3). Thus, 4% and 7% of chromosomal ORFs and 19% and 26% of plasmid ORFs were found to be divergent in RST2 and RST3 isolates, respectively (Table 3).

The microarray data were employed for assessment of phylogenetic relationships among the isolates. RTS1 isolates formed a distinct monophyletic clade with 98% confidence at the node between RST1 and the other isolates (Fig. 6). The remaining isolates formed a stepwise topology rather than a distinct branching pattern. The major differences in ORF content and diversity among isolates are linear plasmid driven, since there is substantially less variation in the chromosomal and circular plasmid portions of the genome. Despite this, the phylogenetic distinctiveness of RST1 isolates initially characterized on the basis of rRNA gene spacer sequence is largely upheld by complete genome comparisons.


Figure 6
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  		FIG. 6. Phylogenetic relationships of B. burgdorferi isolates based on CGH data. GACK-determined binary characters (for conserved or divergent ORFs) were input for parsimony analysis using PAUP version 4.0b10. The analysis yielded four most-parsimonious trees, a representative of which is shown. Statistical stability of each node of the tree was determined by bootstrap analysis with 1,000 replicates. Bootstrap values higher than 50 are shown. The resulting phylogram was visualized with TreeView version 1.6.6. The branch lengths reflect the number of character changes. The bracket indicates RST1 isolates.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To date, only one complete genome sequence is available for the Lyme disease spirochete, B. burgdorferi (9, 17). A second, partial sequence has been reported for a related B. garinii isolate (18). The annotated genome of B. burgdorferi strain B31MI has yielded few insights into potential virulence factors and mechanisms of pathogenesis (9, 17). Comparative genomic sequencing has been a valuable tool for uncovering virulence determinants in other pathogens (14, 16, 20, 43, 46, 47). In the absence of complete genome sequence information, we have undertaken analysis of the genomic contents of 15 B. burgdorferi isolates by microarray analysis.

Most of the CGH studies to date have been performed using PCR product microarrays (3, 5, 15, 24, 31, 49). In the present investigation, 70-mer oligonucleotide arrays were employed. Such microarrays have a number of advantages over PCR product arrays, including uniform Tm and minimal secondary structure of the probes (19), reduced cost and time for repeated fabrication (28), and equivalent sensitivity and potentially higher specificity (19, 23, 28). Specificity is a particularly important issue in B. burgdorferi CGH because the genome contains 175 paralogous gene families with 83% of plasmid ORFs belonging to such families (9). When genomic DNA from strain B314, a B31 derivative lacking linear plasmids (48), was hybridized to PCR product microarrays in the current study (data not shown), substantial hybridization was observed for linear plasmid sequences. These false-positive signals were the result of hybridization to paralogous sequences located elsewhere in the genome, i.e., either the chromosome or circular plasmids (data not shown). Such false-positive hybridization was rarely observed with the 70-mer oligonucleotide microarrays. Thus, oligonucleotide microarrays are more specific and better suited for CGH studies of B. burgdorferi.

There are, however, potential limitations with oligonucleotide microarrays. First, use of a single probe to represent the entire ORF limits detection of sequence variability to that particular 70-mer nucleotide region. Thus, nucleotide sequence differences outside this region will not be detected. In order to assess the extent of this problem, five ORFs that were scored as either conserved or divergent were fully sequenced for several isolates. The results indicated that the extent of sequence variation in the 70-mer region was similar to that found for the entire ORF. This suggests that 70-mer oligonucleotides are representative of the sequence of the entire ORF. The high sensitivity of oligonucleotide probes to single-nucleotide mismatches makes it impossible to distinguish between complete ORF deletions and the presence of isolated SNPs. Therefore, in this study ORFs were designated as either conserved or divergent, where the latter term includes both SNPs and indels.

The genomic variability of North American B. burgdorferi isolates has been previously assessed by restriction fragment length polymorphism (RFLP) analysis and/or PFGE (8, 27, 32, 33, 35, 61, 62), nucleotide sequence analysis of coding and noncoding regions (6, 32, 38), and plasmid content (26, 42). These studies demonstrated the existence of substantial genomic heterogeneity among these isolates and provided markers for genotyping. This is supported by partial genome sequence comparison of B31MI and two other tick isolates (45). However, no complete genome sequences of B. burgdorferi clinical isolates are available, and thus comparative genomic analysis of clinical isolates has not yet been performed. In this study of 15 B. burgdorferi strains, the extent of genomic variability was remarkably different between two subgroups that corresponded to genotypes previously characterized by sequence analysis of the rRNA gene spacer and ospC. RST1 isolates (IGS type 1 and OspC type A) were virtually homogenous, with 98% of all ORFs conserved. On the other hand, the remaining isolates were more heterogeneous and divergent compared to B31MI (88 and 83% of ORFs conserved, respectively). Phylogenetic trees constructed from the CGH data support the monophyletic nature of RST1 isolates.

Twelve of the isolates analyzed were obtained from Lyme disease patients in a narrow geographic area (Westchester County, NY). The striking extent of genome variation in this set of isolates suggests that analysis of B. burgdorferi isolates with a broader geographic representation may reveal even greater variation than that found in the current study. Previous investigations have revealed an association between RST and dissemination in mice and Lyme disease patients; RST1 isolates were uniformly more invasive than RST3 isolates (55, 56, 59). The fact that there is substantial genomic variability among isolates with various pathogenic properties suggests that B. burgdorferi pathogenesis is a complex phenomenon resulting from a combination of gene products rather than being determined by a single locus.

The observed variation is the result of SNPs distributed throughout the genome, the variable presence of certain plasmids, and gene deletions on some plasmids. The chromosome and plasmids lp54, cp26, and cp32 represent the essential part of the B. burgdorferi genome. The results demonstrate that there is very low ORF divergence on the chromosome and that such changes are the result of SNPs rather than deletions. This is consistent with previous physical and genetic mapping of B. burgdorferi sensu lato isolates that indicated conservation of gene order on the chromosome (8, 9, 17, 27, 40, 50). These findings were confirmed by sequencing of the B. garinii PBi chromosome (18). lp54 and cp26 were previously reported to be present and conserved in size in all isolates analyzed (2, 26, 37, 42) and were proposed to be minichromosomes (9). cp26 was shown to be required for B. burgdorferi viability (7), and although lp54 apparently is not required for in vitro growth (48), it may be required in the natural B. burgdorferi life cycle. Of the cp32 plasmids, at least several copies are present in all isolates as well (10, 26). In the current study, all of these replicons were found to be remarkably conserved, which supports the idea that genes carried on these genomic elements are indispensable in the natural life cycle of B. burgdorferi.

It was shown in previous studies that certain plasmids may be absent from some isolates (26, 42). Indeed, in the current study several plasmids were characterized as absent from some isolates based on the lack of hybridization to probes representing the putative plasmid partition loci (Table 4). Thus, lp5 and lp21 were absent from almost all isolates studied (Fig. 1 and Tables 3 and 4), consistent with previous findings (42). Therefore, these plasmids may be considered of minor importance in B. burgdorferi biology compared to the other genomic elements.

Other linear plasmids are usually present in the natural isolates, based on the presence of their unique partition loci. They can be divided into three classes with regard to the presence of ORFs linked to the unique partition locus as found in the sequenced strain B31MI. Thus, lp54 is stable, and all B31-like lp54 genes are present in all natural isolates. On the other hand, plasmids lp38 and lp28-4 were absent from certain isolates (Fig. 1 and 2 and Tables 3 and 4). However, when they were present, all uniquely linked ORFs (based on comparison to B31MI) were also present in the genome. This is likely to be the result of physical linkage of these ORFs on the same plasmid, although location of these ORFs on other replicons cannot be ruled out by microarray analysis. The fact that the ORFs carried on these plasmids are either absent or present as a unit suggests that they are not absolutely required for the B. burgdorferi life cycle but may be functionally linked.

The remaining linear plasmids (lp17, lp25, lp28-1, lp28-2, lp28-3, lp36, and lp56) represent a third class. Partition loci for these plasmids were present in essentially all isolates, but the presence of linked ORFs (based on comparison to B31MI) was highly variable. These divergent ORFs were found in clusters which were often located at either the right or left ends of the replicons, suggesting a mosaic structure for these plasmids. The location of clustered divergent ORFs may suggest that ResT telomere resolvase may play role in linear plasmid rearrangement, as recently proposed (11, 30). Interestingly, the right end of lp54 is the only telomeric region that does not have substantial similarity with another B31MI telomere (9), which may contribute to the stability of lp54. Linear plasmid alterations may include deletions and/or rearrangement of fragments between plasmids, as initially suggested by Casjens et al. (9), and were observed for lp17, lp25, lp28-1, lp28-2, lp28-3, and lp36 by Palmer et al. (42). Plasmid dimerization is also possible, as was described for lp54-like plasmids in B. burgdorferi sensu lato (36). Thus, in this latter plasmid class, ORFs located on a specific plasmid in B31MI may either be linked to the same partition locus, be present on a different replicon, or be completely absent from other isolates.

Since linear plasmids constitute the most variable portion of the B. burgdorferi genome, they are the most likely genomic location for potential selection-driven virulence-encoding elements. Only 16% (38/235) of all divergent plasmid ORFs identified in this analysis have assigned functions (see Table S3 at http://www.nymc.edu/iraschwartz/gene_arrays.asp); all but four of these are predicted to encode surface-exposed proteins. These include some genes that have been previously described as polymorphic and implicated or suggested to have a role in B. burgdorferi pathogenesis, e.g., ospC and members of the ospE/ospF/elp (erp) families (1, 4, 21). ORFs carried on lp17, lp25, lp28-1, lp28-2, lp36, and lp56 that are either conserved or divergent in clusters are of special interest as additional potential candidate virulence genes or sites of surface antigen variation. Some of these clustered ORFs had hybridization signals equal to those of the reference strain B31MI in the most invasive isolates, but no hybridization was observed in the remaining isolates (Fig. 1 and 5A). For example, BBK35 to BBK50, which are carried on lp36, are conserved in RST1 isolates but divergent in RST2 and RST3 isolates (Fig. 5A). Indeed, lp36 and its associated ORFs are apparently completely absent from isolate B356 (Fig. 5), which does not disseminate in mice (55, 56). Based on these results, it is reasonable to suggest that lp36 may encode factors that contribute to B. burgdorferi dissemination from the initial site of infection.

In general, the phylogenetic tree generated from the CGH data supports previous genotyping studies that were based on analysis of single loci. For example, both rRNA gene spacer and ospC sequence typing demonstrate that RST1 and OspC type A isolates are essentially monophyletic and form a deeply branching clade (56). This is also the case for the CGH-based tree (Fig. 6). However, the tree topologies are distinctly different. Those based on the rRNA gene spacer and ospC have deeply branched tree topologies. By contrast, the CGH-based tree had a ladder-like topology. Plasmid variation, both presence and sequence divergence, is the major contributor to differences among isolates in the CGH analysis and is the likely reason for the distinctions in tree topologies. Thus, phylogenetic trees based on complete genome analysis, which are a composite of many different loci, appear to provide a unique perspective on evolutionary relationships among isolates compared with those constructed from single genetic loci.

In summary, there is substantial genomic variation among B. burgdorferi clinical isolates, particularly in the linear plasmid portion of the genome. This and the fact that a substantial portion (90%) of divergent linear plasmid ORFs with assigned functions encode surface-exposed proteins suggest that B. burgdorferi isolates may evolve under selective immune pressure by sequence variation in linear-plasmid-carried genetic elements. By contrast, the chromosome and plasmids lp54, cp26, and cp32 are highly conserved, which may imply that these genomic elements encode functions required for B. burgdorferi viability. There is a strong correlation between complete genome hybridization profiles and other typing methods, which also correlate to differences in pathogenicity. Comparison of genomic differences between the least and the most invasive isolates predicts potential virulence-encoding elements. Further studies of these regions should facilitate identification of factors responsible, in part, for B. burgdorferi pathogenicity.


   ACKNOWLEDGMENTS
 
We thank P. Rosa, N. Parveen, L. Bockenstedt, R. Gilmore, and J. Radolf for providing strains; A. Brooks for printing of microarrays and many helpful discussions; D. A. Robinson for many enlightening discussions on phylogenetics; D. Liveris for primer design and discussions; and D. Liveris, S. Sandigursky, and K. Hanincova for sharing IGS and OspC typing results.

This work was supported by NIH grant AI45801.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, New York Medical College, BSB Room 308, Valhalla, NY 10595. Phone: (914) 594-4658. Fax: (914) 594-4176. E-mail: schwartz{at}nymc.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, September 2006, p. 6124-6134, Vol. 188, No. 17
0021-9193/06/$08.00+0     doi:10.1128/JB.00459-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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