The bdr gene families of the Lyme disease and relapsing fever spirochetes: potential influence on biology, pathogenesis, and evolution.

Species of the genus Borrelia cause human and animal infections, including Lyme disease, relapsing fever, and epizootic bovine abortion. The borrelial genome is unique among bacterial genomes in that it is composed of a linear chromosome and a series of linear and circular plasmids. The plasmids exhibit significant genetic redundancy and carry 175 paralogous gene families, most of of unknown function. Homologous alleles on different plasmids could influence the organization and evolution of the Borrelia genome by serving as foci for interplasmid homologous recombination. The plasmid-carried Borrelia direct repeat (bdr) gene family encodes polymorphic, acidic proteins with putative phosphorylation sites and transmembrane domains. These proteins may play regulatory roles in Borrelia. We describe recent progress in the characterization of the Borrelia bdr genes and discuss the possible influence of this gene family on the biology, pathogenesis, and evolution of the Borrelia genome.

Species of the genus Borrelia cause human and animal infections (1). In North America, Lyme disease and endemic relapsing fever pose the greatest threat to human health and have received the most attention of the borrelial diseases. Approximately 14,000 cases of Lyme disease are reported in the United States each year; however, the actual number of cases may be 10-fold higher (2). Lyme disease was not recognized as a distinct clinical entity in North America until the 1970s (3). The causative agent, a previously uncharacterized spirochete transmitted through the bite of infected ticks of the Ixodes ricinus complex (I. scapularis in the Northeast and Midwest and I. pacificus on the West Coast) (4,5); was classified in the genus Borrelia and named B. burgdorferi. With the emergence of Lyme disease and the identification of its etiologic agent, Borrelia research focused on the development of reliable Lyme disease diagnostic assays and vaccines, and the phenotypic and genotypic diversity of Borrelia was thoroughly analyzed. Through modern molecular taxonomic techniques, several newly described species of Borrelia have emerged as possible causative agents of Lyme disease or at least as agents genetically related to B. burgdorferi (6-15). The B. burgdorferi sensu lato complex is composed of the following species: B. turdae, B. tanukii, B. bissettii, B. valaisiana, B. lusitaniae, B. bissettii, B. andersonii, B. japonica, B. garinii, and B. afzelii. Of these, B. burgdorferi, B. garinii, and B. afzelii are the dominant species associated with infection in humans.

Relapsing fever has been studied not only for its impact on human health but also as a model system for antigenic variation. There are two general forms of relapsing fever, epidemic (louse borne--Pediculus humanus) and endemic (tick borne--Ornithodoros spp.) (1). Epidemic relapsing fever tends to be associated with poor living conditions and social disruption (famine and war) and is rare in the United States. Endemic relapsing fever is more prevalent, predominantly in the western regions. Three closely related Borrelia species, B. hermsii, B. turicatae, and B. parkeri, are associated with this disease. Hallmark features of relapsing fever include cyclic fever and spirochetemia. The molecular basis for these features can be attributed to the differential production of dominant variable surface antigens of the Vmp protein families (16). The 40 or so plasmid-carried vmp related genes in the B. hermsii genome are expressed only one at a time. A single expression locus exists, and genes not at this site lack a promoter element and are therefore not transcribed (17). The expressed Vmp becomes primary target of a vigorous humoral immune response that kills most of the spirochetal population. However, at a frequency of approximately of 1 x [10.sup.-3] to 1 x [10.sup.-4] per generation, the identity of the expressed Vmp changes (18) through gene conversion (19). The net effect of this nonreciprocal event is to replace the gene located in the expression locus with one that was previously silent. The production of a new antigenically distinct Vmp allows evasion of the humoral immune response. This ongoing change in Vmp synthesis allows the relapsing fever spirochete population to reestablish itself in the host, thus leading to spirochetemia and the relapse of fever. Antigenic variation systems have also been identified in the Lyme disease spirochetes; however, they appear to exert a more subtle effect (20).

While clinical relapsing fever and Lyme disease differ from each other in many ways, their causative agents share many similarities at both the biologic and genetic levels. At the biologic level, they are host associated and undergo similar environmental transitions in the course of cycling between mammals and arthropods. In view of the distinctly different characteristics of these environments, the spirochetes must be able to adapt rapidly. Evidence suggests that the relapsing fever and Lyme disease spirochetes use related proteins to adapt to or carry out similar functions in changing environments. For example, homologs of the plasmid-carried ospC gene of the Lyme disease spirochetes are carried by several other Borrelia species, including the relapsing fever spirochetes (21). Both ospC and its relapsing fever spirochete homolog (vmp33) are selectively expressed during the early stages of infection, which suggests that they play a common functional role (22,23). The B. burgdorferi Rep or Bdr protein family is also distributed genuswide. Members of this polymorphic protein family possess highly conserved putative functional motifs and structural properties, which suggests that they may also carry out an important genuswide role (24,25).

The Borrelia Genome

At the molecular level, a unique feature of Borrelia is the unusual organization and structure of their genome. Unlike most bacteria, which carry their genetic material in the form of a single, circular DNA molecule, Borrelia have a segmented genome (26-28). Most genetic elements carried by these bacteria are linear with covalently closed termini or telomeres (27). The telomeres are characterized by short hairpin loops of DNA (29). If heat denatured, these linear molecules relax to form a single-stranded circular molecule. If reannealed, they base-pair upon themselves to form a double-stranded linear molecule that by physical necessity possesses a short single-stranded hairpin loop at each telomere. Genetic elements of this structure are rare in bacteria and are reminiscent of certain viral genomes. In B. burgdorferi (isolate B31), the largest of the linear genomic elements is the 911-kb chromosome (30). The chromosome carries 853 putative ORFs, most of which are thought to encode housekeeping functions. The remaining 12 linear and 8 circular genetic elements are plasmids. The plasmids might best be thought of as mini-chromosomes, since as a group they are indispensable in situ and may carry genes encoding proteins involved in housekeeping functions (31). In addition, they may further deviate from the true definition of a plasmid in that their replication may not be independent and may instead be tightly coordinated with the replication of the chromosome (32,33).

Nearly 50% of the plasmid-carried ORFs lack homology with known sequences, which suggests that their encoded proteins may define the unique biologic and pathogenetic aspects of Borrelia (30). Several of the proteins derived from these plasmid-carried genes of unknown function are antigenic or selectively expressed during infection, which indicates that they function in the mammalian environment (20,34-37). A striking feature of the plasmid-carried ORFs is that they are organized into 175 paralogous gene families of two or more members (30). Hence, the DNA content of the plasmids is highly redundant. Since the maintenance of DNA is energetically expensive, it is likely that this redundant DNA is of biologic importance to Borrelia. The paralogous gene families of Borrelia have been the focus of intensive research as they are thought to play important roles in pathogenesis and to influence genome organization and evolution (20,30,35,38-40).

Identification of Borrelia Direct Repear (bdr) Related Genes

The bdr gene family is a large, polymorphic, plasmid-carried, paralogous gene family of unknown function that was originally identified in B. burgdorferi (41,42). Members of this gene family have been characterized in several Borrelia species and isolates (Table 1) and have been assigned various gene names (25,41-44) (Table 2).We have adopted the bdr designation in the context of a nomenclature system (25), summarized below. Genes belonging to the bdr gene family were first identified through the analysis of repeated DNA sequences in B. burgdorferi sensu lato complex isolates (41,42). Seven nonidentical but closely related copies of a plasmid-carried repeated element were identified in B. burgdorferi 297 (42). Three additional copies of this repeated sequence were further identified in B. burgdorferi 297 (45). These loci carry several ORFs that were designated as rep+, rep-, LPA, LPB (the LP genes have recently been redesignated as mlp for multicopy lipoprotein [45]), rev, and the orfABCD operon (note: ORFs A and B have been redesignated as blyA and blyB). Some of these genes, particularly rep and mlp, exhibit allelic variation and encode polymorphic proteins, the functions of which are under investigation. Focusing specifically on the rep or bdr genes, the rep designation was originally chosen to reflect a central repeat motif carrying domains in the deduced amino acid sequences. The + and - designations were assigned to indicate that the overlapping rep+ and rep- genes are located on opposing DNA strands. Plasmid-carried repeated DNA sequences were also identified in B. burgdorferi B31 and found to carry either all or a subset of seven ORFs, designated A through G (.41). Of relevance to this discussion are the ORF-E sequences that are rep or bdr homologs. A bdt-related gene was also identified in B. afzelii DK1 and designated as p21 (43). B. afzelii causes Lyme disease in Europe and Asia. The rep+, ORF-E, and p21 designations have recently been replaced with bdr gene designations (24,25,44).

[TABULAR DATA 1-2 NOT REPRODUCIBLE IN ASCII]

To assess and compare the composition and complexity of the bdr gene family a isolates of the B. burgdorferi sensu lato complex, restriction fragment length pol (RFLP) patterns were determined (Appendix). Genomic DNA digested with Xba blotted and probed with an oligonucleotide targeting the bdr genes (Figure 1). A hybridizing bands of different size were detected. These analyses demonstrate th gene families are carried by B. burgdorferi sensu lato complex isolates and that t vary at the inter- and intraspecies level. Hybridization analyses of other Borrelia that they also carry bdr-related gene families (24,25,46). bdr-related genes have hybridization in B. turicatae, B. hermsii, B. parkeri, B. coriaceae, and B. anserin of these species also exhibit substantial variation in their bdr RFLP patterns at th level. Table 1 lists the Borrelia species that carry bdr-related genes and indicates which these genes or proteins were detected.

[Figure 1 ILLUSTRATION OMITTED]

Sequences flanking some bdr alleles also appear to be distributed genus wide. Some bdr alleles of B. turicatae, B. parkeri, and B. hermsii are flanked by genes that are homologs of genes carried by the Lyme disease spirochetes (24,25). As a specific example, the B. turicatae bdr[A.sub.1] gene is flanked by ORFs that are homologs of the BBG34 and BBG30 genes of B. burgdorferi (24,25). In the Lyme disease spirochetes, BBG34 is part of a three-member paralogous gene family, while BBG30 is a single-copy gene (30). Located between BBG30 and BBG34 is BBG33, a member of the bdr gene family (recently redesignated as bdr[F.sub.2]) (25). Although these divergent Borrelia species carry related genes, their organization differs (24), which indicates that rearrangement has taken place in the ancestral plasmid that carried these homologs. Figure 2 compares the organization of two bdr loci from B. turicatae and B. burgdorferi.

[Figure 2 ILLUSTRATION OMITTED]

Evolutionary Analyses of bdr-Related Sequences: Revised Nomenclature for the Bdr-Related Proteins

To simplify the complicated nomenclature of bdr-related genes, a bdr nomenclature system has been developed that assigns gene names on the basis of phylogenetic relationships inferred from comparative analysis of genetically stable regions of the bdr genes (25). This system, which is applicable genuswide, allows for a ready assessment of relationships among bdr paralogs and orthologs. The rationale for this system stemmed from the results of a comprehensive evolutionary analysis of [is greater than] 50 bdr-related sequences from five Borrelia species that demonstrated that bdr sequences are organized into six distinct subfamilies, designated A through F (25). Subfamilies are not necessarily species specific; some contain bdr alleles from different Borrelia species (25). Since members of a given subfamily are closely related to one another with identity values for the N terminal domain being [is greater than] 95%, each member is assigned the same gene name being [is greater than] 95%, each member is assigned the same gene name designation, and paralogs are distinguished by a numerical subscript. In B. turicatae OZ-1, two bdr subfamilies, bdrA and bdrB, contain at least four and five members, respectively (24). Members of the bdrA subfamily are designated bdr[A.sub.1], bdr[A.sub.2], bdr[A.sub.3], and bdr[A.sub.4], while members of the bdrB family are designated bdr[B.sub.1] through bdr[B.sub.5]. This revised Bdr nomenclature scheme was modeled after that proposed for bacterial polysaccharide synthesis genes (47) and is in accordance with the [ILLEGIBLE TEXT] established by Demerec (48).

The subfamily affiliation of bdr genes can be readily determined through [ILLEGIBLE TEXT] analyses of the amino acid segment preceding the polymorphic repeat motif [ILLEGIBLE TEXT] (described in detail below) (25). Relationship assessments based on the [ILLEGIBLE TEXT] terminal domain (vs. complete sequences) are preferable because the calculated [ILLEGIBLE TEXT] distances and clustering relationships are not artificially skewed by the variable [ILLEGIBLE TEXT] motifs present in the repeat motif domain. Since the genetically unstable repeat comprises as much as 50% of the total coding sequence in some alleles, it can [ILLEGIBLE TEXT] impact on inferred relationships. In addition, extensive sequence variation in the of the Bdr proteins at the inter-species level makes it difficult to align this [ILLEGIBLE TEXT] which further influences the inferred relationships.

bdr evolutionary analyses show that Borrelia species carry members of at least [ILLEGIBLE TEXT] (25,44). In fact, B. burgdorferi carries three distinct subfamilies. Multiple Bdr [ILLEGIBLE TEXT] diverse Borrelia species suggest that there has been selective pressure to [ILLEGIBLE TEXT] alleles and bdr genetic diversity. This genetic diversity may increase the function Bdr proteins.

Molecular Features and Physical Properties of the Bdr Prote

While early analyses of Borrelia bdr genes demonstrated their multicopy nature extent of the complexity of the bdr gene family in the Lyme disease spirochetes recognized until the B. burgdorferi genome sequence was determined (30). B. [ILLEGIBLE TEXT] found to carry 17 distinct bdr-related genes (and one truncated variant) distribute linear and circular plasmids. B. turicatae, which carries at least nine different bdr these genes exclusively on linear plasmids (24,25,46). Other relapsing fever [ILLEGIBLE TEXT] parkeri and B. hermsii) are similar to the Lyme disease bacteria in that they carry linear and circular plasmids (25). In the Lyme disease spirochetes each of the 32-plasmids, with the exception of plasmids M and P, carry two different bdr genes or eight ORFs. Each of these circular plasmids carries one bdrD subfamily [ILLEGIBLE TEXT] subfamily member. The maintenance of genes belonging to different subfamilies plasmid is consistent with the possibility that each carries out a different function Lyme disease spirochetes, the bdrF subfamily members are localized to linear [ILLEGIBLE TEXT] single bdr gene per plasmid. These observations suggest that there has been [ILLEGIBLE TEXT] maintain the association of specific subfamilies with specific types of plasmids about the bdr-carrying plasmids and the organization of the bdr genes and [ILLEGIBLE TEXT] relapsing fever borreliae. However, as in the Lyme disease spirochetes, in B. [ILLEGIBLE TEXT] carrying plasmids carry two bdr genes, one from subfamily bdrA and one from [ILLEGIBLE TEXT] (24).

The sequence of more than 50 bdr alleles from five different Borrelia species has been determined (Table 2) (24,25,41-43,46). These extensive comparative sequence analyses led to the identification of conserved features that provide insight into the possible biologic roles of the Bdr proteins. For example, all bdr alleles carry centrally located repeat motif domains (Figure 3). Although conserved in sequence, these domains vary in length among alleles as a result of varying these domains vary in length among alleles as a result of varying numbers of the repeat motif. The core tripeptide of the repeat is the sequence KID. The repeat motifs encode consensus casein kinase 2 phosphorylation (CK2P)motifs of the sequence T/SKID/E (43). While it may appear somewhat paradoxical for bacteria to carry casein kinases, casein [ILLEGIBLE TEXT] descriptive term broadly applied to at least two classes of ubiquitous protein [ILLEGIBLE TEXT] substrates may include various enzymes and noncatalytic proteins involved in [ILLEGIBLE TEXT] regulatory functions (49). Most proteins phosphorylated by CK2-1ike kinases are are the Borrelia Bdr proteins (isoelectric points between 5 and 6). The phosphor CK2P motifs is either the Ser or Thr residue of the motif. Although histidine [ILLEGIBLE TEXT] known to exist in some bacteria, it has been widely held that bacteria lack Ser - [ILLEGIBLE TEXT] However, Ser-Thr kinases have recently been identified in several bacterial [ILLEGIBLE TEXT] Myxococcus, Anabeana, Freymella, Yersinia, and Streptomyces (50). Most [ILLEGIBLE TEXT] the B. burgdorferi genome sequence identified a putative Ser - Thr kinase design (30,50). This ORF carries a domain that exhibits homology with the active site [ILLEGIBLE TEXT] B. burgdorferi also carries a homolog of the PPM family of eucaryotic protein S phosphatases (30,50). The presence of these genes in B. burgdorferi suggests [ILLEGIBLE TEXT] possess the machinery necessary for Ser - Thr phosphorylation and dephosphoryl [ILLEGIBLE TEXT]

[Figure 3 ILLUSTRATION OMITTED]

Another important conserved feature identified through sequence analyses is the carboxyl terminal domain of approximately 20 amino acids. Computer analyses TMpred program indicate that this domain has a high propensity to form a [ILLEGIBLE TEXT] (24,25). The Tmpred values for the 20 aa C-terminal domains are 2,000 to 2,600. greater is considered significant (24,25). Comparison of the Bdr putative [ILLEGIBLE TEXT] sequences from the Lyme disease spirochetes with those from the relapsing fever indicates that, while there is conservation in physical properties, there is essential of primary sequence. However, sequence conservation does exist at the [ILLEGIBLE TEXT] Since the Bdr proteins lack an obvious export signal, membrane association [ILLEGIBLE TEXT] with the spirochetal inner membrane, with the rest of the protein, which is [ILLEGIBLE TEXT] into the cytoplasm. The terminal residue of the protein is in almost all cases a [ILLEGIBLE TEXT] amino acid (lysine or asparagine). This residue could extend into the periplasm a the Bdr proteins to other cellular components, such as the peptidoglycan.

Immunologic Analyses of the Bdr Proteins

The presence of multiple bdr alleles and bdr subfamilies within isogeneic [ILLEGIBLE TEXT] speculation that there may be differential expression at either the subfamily or in level, possibly in response to environmental stimuli (46). Limited studies of bdr production, based on either mRNA detection or immunoblot analyses, have been Porcella et al. (42) used Northern hybridization to determine if expression of B. [ILLEGIBLE TEXT] related genes occurs during cultivation in the laboratory under standard culture [ILLEGIBLE TEXT] BSK media). Bdr transcripts were not detected by this approach. Similarly, in an we also conducted Northern hybridization experiments to assess bdr expression ([ILLEGIBLE TEXT] expression of B. turicatae OZ1 bdrA subfamily members in bacteria cultivated [ILLEGIBLE TEXT] laboratory growth conditions (46). However, when reverse transcriptase (RT)-PC applied, transcription of a single bdrA allele was detected (46). B. turicatae OZ-1 demonstrated to carry at least nine bdr alleles, four of which belong to the bdrA [ILLEGIBLE TEXT] Analysis of the sequence of these alleles showed that all four should have been [ILLEGIBLE TEXT] the RT-PCR primer set because of the conservation of the primer binding sites (2 detection of transcript derived from these alleles suggested that only a subset of [ILLEGIBLE TEXT] subfamily alleles is expressed. This raised the possibility that other bdr alleles [ILLEGIBLE TEXT] nonfunctional genes or their expression requires different environmental [ILLEGIBLE TEXT] transcriptional expression of the bdrB subfamily has not been specifically assess transcriptional analyses using allele-specific probes and primers are an important allow specific assessment of the expression of individual bdr alleles under [ILLEGIBLE TEXT] conditions. In addition, analyses of the upstream DNA sequences of individual [ILLEGIBLE TEXT] genomic location may elucidate the molecular basis for bdr transcriptional [ILLEGIBLE TEXT]

Immunologic analyses have provided a somewhat different overall picture regarding Bdr production. Immunologic analyses described in this report and elsewhere (44) demonstrate that several members of the bdr gene family are expressed during in vitro cultivation. We conducted a comprehensive analysis of the expression of Bdr proteins among Borrelia species. When antisera raised against recombinant B. afzelii Bdr[F.sub.1] (24) were used in immunoblot analyses, several immunoreactive proteins were detected in cell lysates of all Borrelia species tested (Figure 4). The only exception was B. anserina, a causative agent of avian spirochetosis. Although bdr-related sequences have been detected in B. anserina by hybridization techniques (46), immunoreactive proteins were not detected in immunoblot analyses. Additional analyses are required to determine if this indicates absence of translational expression or the lack of epitope conservation in this species. In a that immunoreactive bands were not detected in this species attests to the [ILLEGIBLE TEXT] antisera. As a further demonstration of the specificity of the antisera and to [ILLEGIBLE TEXT] the Bdr proteins are unique to Borrelia, a cell lysate of Leptospira interrogans [ILLEGIBLE TEXT] immunoblot analyses. Immunoreactivity with proteins in the Bdr size range was the anti-Bdr antisera in this spirochete species. Borrelia species that expressed [ILLEGIBLE TEXT] proteins included B. garinii, B. burgdorferi, B. turdae, B. tanukii, B. japonica, B. afzelii, B. coriaceae, B. bissettii, B. miyamotoi, B. parkeri, B. hermsii, and B. [ILLEGIBLE TEXT] Particularly striking was the extensive variation in the number and molecular [ILLEGIBLE TEXT] immunoreactive proteins expressed, with up to 12 distinct Bdr proteins detected expression patterns was observed at both the inter- and intraspecies level. Analysis burgdorferi isolates (B31 G, cN40, and CA12) demonstrated variability in both [ILLEGIBLE TEXT] of expressed Bdr proteins. Isolate B31G has been demonstrated by genomic [ILLEGIBLE TEXT] distinct bdr alleles. Immunoblot analyses show that not all alleles are expressed cultivation; therefore, some alleles may be differentially regulated.

[Figure 4 ILLUSTRATION OMITTED]

The broad immunoreactivity of the antisera with diverse Borrelia species [ILLEGIBLE TEXT] epitopes are conserved genuswide. In view of the sequence divergence in the N a domains of the Bdr proteins derived from different subfamilies, it is likely that [ILLEGIBLE TEXT] epitopes reside in the conserved repeat motif region. Consistent with this, [ILLEGIBLE TEXT] repeat domain of all determined Bdr protein sequences predict them to be [ILLEGIBLE TEXT] a surface exposed on the protein and a positive Jameson-Wolf antigenic index (2 conservation and synthesis of these polymorphic proteins in such a diverse group species suggest that they play an important role in Borrelia biology genuswide.

The Bdr Proteins and Borrelia Biology: An Overview

Bdr genes and extensive bdr gene families have now been identified and [ILLEGIBLE TEXT] diverse Borrelia species (24,25,42-44,46). Comparative sequence analyses, [ILLEGIBLE TEXT] conserved putative functional domains, have provided the basis for the [ILLEGIBLE TEXT] regarding Bdr function and cellular location. The Bdr proteins, which lack know signals, are likely anchored to the cytoplasmic membrane through their conserve putative transmembrane spanning domain. The C-terminal positively charged am exposed to the periplasm, where it may interact with other cellular components [ILLEGIBLE TEXT] peptidoglycan. The repeat motif domain, which is predicted by computer analyse and surface exposed on the protein, likely extends into the cytoplasm. The [ILLEGIBLE TEXT] domain that carries the putative Ser - Thr phosphorylation motifs may then be [ILLEGIBLE TEXT] phosphorylation or to interact with other cytoplasmic proteins or DNA to form a anchored complex. As with numerous other proteins, phosphorylation and [ILLEGIBLE TEXT] play a regulatory role, perhaps in signaling or sensing.

Multiple polymorphic bdr alleles may increase the functional range and diversity proteins. Functional partitioning among Bdr proteins could offer a possible [ILLEGIBLE TEXT] Borrelia expend such biologic energy to maintain these genes in large gene [ILLEGIBLE TEXT] variants of these proteins. The homology among bdr alleles may also allow or [ILLEGIBLE TEXT] modification of these genes through homologous recombination. In fact, the [ILLEGIBLE TEXT] repeat motif region, which is clearly not evolutionarily stable, has likely arisen [ILLEGIBLE TEXT] mispairing, recombination, or rearrangement. In view of the extensive genetic [ILLEGIBLE TEXT] plasmid component of the Borrelia genome, recombination in and among related different plasmids could affect the organization and evolution of the genome and pathogen interaction. Inter- or intra-plasmid exchange of DNA sequences could mechanistic basis for the extensive genetic variability that has been widely [ILLEGIBLE TEXT] plasmids (28,29,51-59). In spite of the apparent necessity for at least most of the survival, as inferred from their ubiquitous distribution among Borrelia isolates, [ILLEGIBLE TEXT] able to tolerate remarkable genomic variability. Diversity in the plasmids and the may actually be exploited as a tool for phenotypic diversity and rapid [ILLEGIBLE TEXT]

Acknowledgments

We thank Michael Norgard, Steve Porcella, Justin Radolf, and the Molecular Pat group at Virginia Commonwealth University for helpful discussions.

This work was supported in part by a grant from the Jeffress Memorial Trust.

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Appendix

Bacterial Cultivation, DNA Isolation, and Southern Hybridization An

Isolates belonging to the Borrelia burgdorferi sensu lato complex were cultivate BSK-H media (Sigma) at 33 [degrees] C. To cultivate the relapsing fever borreliae and [ILLEGIBLE TEXT] species, the complete BSK-H media were supplemented with additional rabbit [ILLEGIBLE TEXT] final concentration of 12% (vol/vol). Bacteria were harvested by centrifugation a phosphate buffered saline (pH 7.0), and DNA was extracted (25). For Southern [ILLEGIBLE TEXT] analyses, 5[micro]g of DNA from each isolate was digested under standard conditions fractionated by electrophoresis in 0.8% GTG agarose gels. The DNA was [ILLEGIBLE TEXT] membranes for hybridization by vacuum blotting using the VacuGene system as manufacturer (Pharmacia). All other Southern hybridization methods were as [ILLEGIBLE TEXT] (39).

Immunoblot Analyses

Bacterial cultures were grown and harvested as described above. One OD600 [ILLEGIBLE TEXT] was pelleted and resuspended in 100[micro]l of standard SDS-sample buffer with [ILLEGIBLE TEXT] cell lysates (7[micro]l) were fractionated by electrophoresis in 15% SDS-PAGE gels a onto Immobilon P membranes (38). The immunoblots were blocked overnight in (1X PBS, 0.2% Tween, 0.002% NaCl, and 5% nonfat dry milk) and then [ILLEGIBLE TEXT] antisera dilutions. ImmunoPure Goat anti-mouse IgG (H+L) peroxidase [ILLEGIBLE TEXT] secondary antibody. The secondary antibody was incubated with the blots for 1 [ILLEGIBLE TEXT] temperature at a 1:40,000-fold dilution and then the blots were washed three [ILLEGIBLE TEXT] buffer. For chemiluminescent detection, the Supersignal West Pico Stable [ILLEGIBLE TEXT] Supersignal West Pico Luminol/Enhancer solution were used. Both reagents [ILLEGIBLE TEXT] were used as described by the manufacturer. The immunoblots were exposed to [ILLEGIBLE TEXT] frames of 5 to 30 seconds.

David M. Roberts,(*) Jason A Carlyon,([dagger]) Michael Theisen,([double dagger]) and Richard T. Marconi(*)

(*) Medical College of Virginia at Virginia Commonwealth University, School of Medicine, Richmond, Virginia, USA; ([dagger]) Yale School of Medicine, Yale University, New Haven, Connecticut, USA; and ([double dagger]) Statens Serum Institute, Copenhagen, Denmark

Mr. Roberts is a 3rd-year Ph.D. student, Department of Microbiology and Immu College of Virginia at Virginia Commonwealth University. His scientific interest the study of the molecular mechanisms of pathogenesis and adaptive responses [ILLEGIBLE TEXT] spirochetes, specifically on the role of plasmid-carried gene families in Borrelia pathogenesis.

Address for correspondence: Richard T. Marconi, Department of Microbiology a Medical College of Virginia at VCU, Richmond, VA 23298-0678, USA; fax: 80 mail: [e-mail]