Emerging Infectious Diseases
[Volume 6 No.2 / March - April 2000]

Synopses

The bdr Gene Families of the Lyme Disease and Relapsing Fever
Spirochetes: Potential Influence on Biology, Pathogenesis, and
Evolution

David M. Roberts,* Jason A Carlyon, Michael Theisen, and
Richard T. Marconi*
*Medical College of Virginia at Virginia Commonwealth
University, School of Medicine, Richmond, Virginia, USA; Yale
School of Medicine, Yale University, New Haven, Connecticut ,
USA; and Statens Serum Institute, Copenhagen, Denmark

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
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
bornePediculus humanus) and endemic (tick
borneOrnithodoros 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 a 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 -3 to 1 x 10 -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.
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).

Table 1. Borrelia species carrying bdr-related genes or expressing
proteins immunoreactive with anti-Bdr antisera

Identification of Borrelia Direct Repeat (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.

Table 2. Borrelia Bdr homology groups and gene nomenclature

Figure 1. Restriction fragment length polymorphism pattern
analysis of the rep or bdr genes of the Lyme disease spirochetes.
Total DNA, isolated from Borrelia cultures, was digested with
Xba1, fractionated by electrophoresis, and transferred onto
membranes for hybridization. Hybridization was performed by the
bdrAB-R1 oligonucleotide (46). The species and isolates analyzed
are indicated above each lane. MW markers in kb are indicated.
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 bdr-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).

To assess and compare the composition and complexity of the bdr
gene family among species and isolates of the B. burgdorferi sensu
lato complex, restriction fragment length polymorphism (RFLP)
patterns were determined (Appendix). Genomic DNA digested
with Xba1 was Southern blotted and probed with an
oligonucleotide targeting the bdr genes (Figure 1). A variable
number of hybridizing bands of different size were detected. These
analyses demonstrate that extensive bdr gene families are carried
by B. burgdorferi sensu lato complex isolates and that the RFLP
patterns vary at the inter- and intraspecies level. Hybridization
analyses of other Borrelia species showed that they also carry bdr-
related gene families (24,25,46). bdr-related genes have been
detected by hybridization in B. turicatae, B. hermsii, B. parkeri, B.
coriaceae, and B. anserina (25,46). Isolates of these species also
exhibit substantial variation in their bdr RFLP patterns at the
intraspecies level. Table 1 lists the Borrelia species that carry bdr-
related genes and indicates the methods by which these genes or
proteins were detected.

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 bdrA 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 bdrF 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. General organization of two bdr loci in Borrelia turicatae
and B. burgdorferi. The gene arrangement depicted for B. turicatae
was determined through cloning and sequence analysis of a 2,217
base-pair XbaI restriction fragment. The arrangement for the bdr-
carrying locus of B. burgdorferi was previously determined through
the sequencing of the B. burgdorferi B31 genome (30). The arrows
indicate the direction of transcription. Genes exhibiting homology
are indicated by similar shading or hatch marks. Genes indicated
by unfilled arrows are not homologous. The numbering is indicated
for scale and is not indicative of the positioning of these genes on
the plasmids that carry them.

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 >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 >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 bdrA 1 , bdrA 2 , bdrA 3, and bdrA 4, while members
of the bdrB family are designated bdrB 1 through bdrB 5 . This
revised Bdr nomenclature scheme was modeled after that proposed
for bacterial polysaccharide synthesis genes (47) and is in
accordance with the nomenclature guidelines established by
Demerec (48).

The subfamily affiliation of bdr genes can be readily determined
through comparative sequence analyses of the amino acid segment
preceding the polymorphic repeat motif region of these proteins
(described in detail below) (25). Relationship assessments based on
the genetically stable N terminal domain (vs. complete sequences)
are preferable because the calculated evolutionary distances and
clustering relationships are not artificially skewed by the variable
number of repeat motifs present in the repeat motif domain. Since
the genetically unstable repeat motif domain comprises as much as
50% of the total coding sequence in some alleles, it can have a
substatial impact on inferred relationships. In addition, extensive
sequence variation in the carboxyl termini of the Bdr proteins at
the interspecies level makes it difficult to align this domain with
confidence, which further influences the inferred relationships.

Figure 3. Key features and putative functional domains of the Bdr
proteins. The schematic depicts a prototype Bdr protein with the
characteristics of each domain indicated. The abbreviation, ID%, is
for percentage amino acid identity at either the inter- or intra-
family level as indicated in the figure. Standard amino acid
abbreviations are used in the figure to denote the conserved C-
terminal lysine (K) or asparagine (N) residues, which are thought
to be exposed in the periplasm and the cytoplasmically located core
tripeptide of the repeat motif (lysine-isoleucine-aspartic acid; KID).

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

Molecular Features and Physical Properties of the Bdr Proteins

While early analyses of Borrelia bdr genes demonstrated their
multicopy nature (41,42,46), the full extent of the complexity of
the bdr gene family in the Lyme disease spirochetes was not fully
recognized until the B. burgdorferi genome sequence was
determined (30). B. burgdorferi B31 was found to carry 17 distinct
bdr-related genes (and one truncated variant) distributed among
different linear and circular plasmids. B. turicatae, which carries at
least nine different bdr alleles, carries these genes exclusively on
linear plasmids (24,25,46). Other relapsing fever spirochete species
(B. parkeri and B. hermsii) are similar to the Lyme disease bacteria
in that they carry bdr genes on both linear and circular plasmids
(25). In the Lyme disease spirochetes each of the 32-kb circular
plasmids, with the exception of plasmids M and P, carry two
different bdr genes separated by seven or eight ORFs. Each of
these circular plasmids carries one bdrD subfamily member and
one bdrE subfamily member. The maintenance of genes belonging
to different subfamilies on a single plasmid is consistent with the
possibility that each carries out a different function. In contrast, in
the Lyme disease spirochetes, the bdrF subfamily members are
localized to linear plasmids with only a single bdr gene per
plasmid. These observations suggest that there has been selective
pressure to maintain the association of specific subfamilies with
specific types of plasmids. Less is known about the bdr-carrying
plasmids and the organization of the bdr genes and subfamilies in
the relapsing fever borreliae. However, as in the Lyme disease
spirochetes, in B. turicatae most bdr-carrying plasmids carry two
bdr genes, one from subfamily bdrA and one from subfamily bdrB
(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 numbers of the repeat motif.
The core tripeptide 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
kinase is a descriptive term broadly applied to at least two classes
of ubiquitous protein kinases for which the substrates may include
various enzymes and noncatalytic proteins involved in important
cellular regulatory functions (49). Most proteins phosphorylated by
CK2-like kinases are highly acidic, as are the Borrelia Bdr proteins
(isoelectric points between 5 and 6). The phosphorylation site in
CK2P motifs is either the Ser or Thr residue of the motif. Although
histidine kinases have been known to exist in some bacteria, it has
been widely held that bacteria lack Ser - Thr kinases. However, Ser
-Thr kinases have recently been identified in several bacterial
species, including Myxococcus, Anabeana, Freymella, Yersinia,
and Streptomyces (50). Most importantly, analysis of the B.
burgdorferi genome sequence identified a putative Ser - Thr kinase
designated BB0648 (30,50). This ORF carries a domain that
exhibits homology with the active site of Ser - Thr kinases. B.
burgdorferi also carries a homolog of the PPM family of eucaryotic
protein Ser - Thr phosphatases (30,50). The presence of these
genes in B. burgdorferi suggests that the Borrelia possess the
machinery necessary for Ser - Thr phosphorylation and
dephosphorylation. Another important conserved feature identified
through sequence analyses is the hydrophobic carboxyl terminal
domain of approximately 20 amino acids. Computer analyses
conducted with the TMpred program indicate that this domain has
a high propensity to form a transmembrane helix (24,25). The
Tmpred values for the 20 aa C-terminal domains are 2,000 to
2,600. A value of 500 or greater is considered significant (24,25).
Comparison of the Bdr putative transmembrane domain sequences
from the Lyme disease spirochetes with those from the relapsing
fever spirochetes indicates that, while there is conservation in
physical properties, there is essentially no conservation of primary
sequence. However, sequence conservation does exist at the
subfamily level (24,25). Since the Bdr proteins lack an obvious
export signal, membrane association would most likely be with the
spirochetal inner membrane, with the rest of the protein, which is
hydrophilic, extending into the cytoplasm. The terminal residue of
the protein is in almost all cases a positively charged amino acid
(lysine or asparagine). This residue could extend into the periplasm
and serve to anchor 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 populations has prompted speculation that there may be
differential expression at either the subfamily or individual allele
level, possibly in response to environmental stimuli (46). Limited
studies of bdr expression and production, based on either mRNA
detection or immunoblot analyses, have been performed. Porcella
et al. (42) used Northern hybridization to determine if expression
of B. burgdorferi bdr-related genes occurs during cultivation in the
laboratory under standard culture conditions (33C in BSK media).
Bdr transcripts were not detected by this approach. Similarly, in an
earlier analysis, we also conducted Northern hybridization
experiments to assess bdr expression (46). We detected expression
of B. turicatae OZ1 bdrA subfamily members in bacteria cultivated
under standard laboratory growth conditions (46). However, when
reverse transcriptase (RT)-PCR methods were applied,
transcription of a single bdrA allele was detected (46). B. turicatae
OZ-1 was later demonstrated to carry at least nine bdr alleles, four
of which belong to the bdrA subfamily. Analysis of the sequence of
these alleles showed that all four should have been readily
amplified by the RT-PCR primer set because of the conservation of
the primer binding sites (24). The lack of detection of transcript
derived from these alleles suggested that only a subset of the bdr A
subfamily alleles is expressed. This raised the possibility that other
bdr alleles are either nonfunctional genes or their expression
requires different environmental stimuli. The transcriptional
expression of the bdrB subfamily has not been specifically
assessed. Thorough transcriptional analyses using allele-specific
probes and primers are an important step, since they allow specific
assessment of the expression of individual bdr alleles under
differing environmental conditions. In addition, analyses of the
upstream DNA sequences of individual bdr alleles and their
genomic location may elucidate the molecular basis for bdr
transcriptional regulation.

Figure 4. Immunoblot analyses demonstrating the variation in Bdr
protein expression in Borrelia species and isolates. Bacteria were
cultivated and prepared for analysis as described in the methods.
Proteins were fractionated by SDS-PAGE, immunoblotted and
screened with anti-BdrF 1-B.afzelii DK1 antisera. The species and
isolates analyzed are indicated above each lane in panels A, B and
C. The migration positions of the protein standards are indicated in
each panel.  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 BdrF 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 any event, the fact that immunoreactive bands were not
detected in this species attests to the specificity of the anti-Bdr
antisera. As a further demonstration of the specificity of the
antisera and to highlight the fact that the Bdr proteins are unique to
Borrelia, a cell lysate of Leptospira interrogans was included in the
immunoblot analyses. Immunoreactivity with proteins in the Bdr
size range was not observed with the anti-Bdr antisera in this
spirochete species. Borrelia species that expressed immunoreactive
proteins included B. garinii, B. burgdorferi, B. turdae, B. tanukii,
B. japonica, B. valaisiana, B. afzelii, B. coriaceae, B. bissettii, B.
miyamotoi, B. parkeri, B. hermsii, and B. turicatae (Table 1).
Particularly striking was the extensive variation in the number and
molecular weight of the immunoreactive proteins expressed, with
up to 12 distinct Bdr proteins detected. Variation in expression
patterns was observed at both the inter- and intraspecies level.
Analysis of three B. burgdorferi isolates (B31G, cN40, and CA12)
demonstrated variability in both the size and number of expressed
Bdr proteins. Isolate B31G has been demonstrated by genomic
sequencing to carry 18 distinct bdr alleles. Immunoblot analyses
show that not all alleles are expressed during in vitro cultivation;
therefore, some alleles may be differentially regulated.

The broad immunoreactivity of the antisera with diverse Borrelia
species indicates that some epitopes are conserved genuswide. In
view of the sequence divergence in the N and C terminal domains
of the Bdr proteins derived from different subfamilies, it is likely
that the cross-reactive epitopes reside in the conserved repeat motif
region. Consistent with this, computer analyses of the repeat
domain of all determined Bdr protein sequences predict them to be
alpha helical and to have a surface exposed on the protein and a
positive Jameson-Wolf antigenic index (24,25,44). The
conservation and synthesis of these polymorphic proteins in such a
diverse group of Borrelia 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 characterized in several diverse Borrelia species
(24,25,42-44,46). Comparative sequence analyses, which have
identified conserved putative functional domains, have provided
the basis for the development of hypotheses regarding Bdr function
and cellular location. The Bdr proteins, which lack known
consensus export signals, are likely anchored to the cytoplasmic
membrane through their conserved, hydrophobic, putative
transmembrane spanning domain. The C-terminal positively
charged amino acid may be exposed to the periplasm, where it may
interact with other cellular components that may include the
peptidoglycan. The repeat motif domain, which is predicted by
computer analyses to be hydrophilic and surface exposed on the
protein, likely extends into the cytoplasm. The conserved repeat
motif domain that carries the putative Ser - Thr phosphorylation
motifs may then be accessible for phosphorylation or to interact
with other cytoplasmic proteins or DNA to form a membrane
anchored complex. As with numerous other proteins,
phosphorylation and dephosphorylation could play a regulatory
role, perhaps in signaling or sensing.

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

Acknowledgments

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

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

Mr. Roberts is a 3rd-year Ph.D. student, Department of
Microbiology and Immunology, Medical College of Virginia at
Virginia Commonwealth University. His scientific interests are
centered on the study of the molecular mechanisms of pathogenesis
and adaptive responses of arthropod-carried spirochetes,
specifically on the role of plasmid-carried gene families in Borrelia
biology and pathogenesis.

Address for correspondence: Richard T. Marconi, Department of
Microbiology and Immunology, Medical College of Virginia at
VCU, Richmond, VA 23298-0678, USA; fax: 804-828-9946; e-
mail: Rmarconi@hsc.vcu.edu.

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Appendix

Bacterial Cultivation, DNA Isolation, and Southern Hybridization
Analyses

Isolates belonging to the Borrelia burgdorferi sensu lato complex
were cultivated in complete BSK-H media (Sigma) at 33C. To
cultivate the relapsing fever borreliae and other Borrelia species,
the complete BSK-H media were supplemented with additional
rabbit sera (Sigma) to a final concentration of 12% (vol/vol).
Bacteria were harvested by centrifugation and washed with
phosphate buffered saline (pH 7.0), and DNA was extracted (25).
For Southern hybridization analyses, 5 g of DNA from each
isolate was digested under standard conditions with Xba1 and
fractionated by electrophoresis in 0.8% GTG agarose gels. (The
DNA was transferred onto membranes for hybridization by vacuum
blotting using the VacuGene system as described by the
manufacturer (Pharmacia). All other Southern hybridization
methods were as previously described (39)

Immunoblot Analyses

Bacterial cultures were grown and harvested as described above.
One OD600 equivalent of cells was pelleted and resuspended in
100 l of standard SDS-sample buffer with reducing agents. The
cell lysates (7 l) were fractionated by electrophoresis in 15% SDS-
PAGE gels and electroblotted onto Immobilon P membranes (38).
The immunoblots were blocked overnight in blocking buffer (1X
PBS, 0.2% Tween, 0.002% NaCl, and 5% nonfat dry milk) and
then incubated with a 1:1,000 antisera dilutions. ImmunoPure Goat
antimouse IgG (H+L) peroxidase conjugate served as the
secondary antibody. The secondary antibody was incubated with
the blots for 1 hour at room temperature at a 1:40,000- fold dilution
and then the blots were washed three times with wash buffer. For
chemiluminescent detection, the Supersignal West Pico Stable
Peroxide solution and the Supersignal West Pico
Luminol/Enhancer solution were used. Both reagents were from
Pierce Chemical Company, Rockford, IL and were used as
described by the manufacturer. The immunoblots were exposed to
film for time frames of 5 to 30 seconds.

Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

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