Characterization of Circular Plasmid Dimers in Borrelia burgdorferi

We have inactivated the ospC, oppAIV, and guaB genes on the 26-kb circular plasmid of Borrelia burgdorferi (cp26) by allelic exchange. On several occasions following such transformations, the cp26 of transformants had an aberrant mobility through agarose gels. Characterization of these cp26 molecules showed that the plasmid had dimerized. These dimers were quite stable during either selective or nonselective passage. Subsequent transformations with dimer DNA supported the hypothesis that in B. burgdorferi, transforming cp26 DNA most likely does not displace the resident homologous plasmid but rather must recombine in order to donate sequences that it carries. These serendipitous findings provide a mechanism for obtaining heterozygous complemented control strains when mutant phenotypes are characterized.     

lacZ Reporter System for Use in Borrelia burgdorferi

Regulation of gene expression is critical for the ability of Borrelia burgdorferi to adapt to different environments during its natural infectious cycle. Reporter genes have been used successfully to study gene regulation in multiple organisms. We have introduced a lacZ gene into B. burgdorferi, and we show that B. burgdorferi produces a protein with detectable β-galactosidase activity in both liquid and solid media when lacZ is expressed from a constitutive promoter. Furthermore, when lacZ is expressed from the ospC promoter, β-galactosidase activity is detected only in B. burgdorferi clones that express ospC, and it accurately monitors endogenous gene expression. The addition of lacZ to the repertoire of genetic tools available for use in B. burgdorferi should contribute to a better understanding of how B. burgdorferi gene expression is regulated during the infectious cycle.Borrelia burgdorferi sensu lato, the pathogen that causes Lyme disease (7), alternates between two distinct environments, an arthropod vector and a vertebrate host. As B. burgdorferi moves from one milieu to the other, its ability to adapt and survive requires dramatic changes in gene expression. Many studies have shown that different B. burgdorferi gene products are upregulated or downregulated at specific times during the infectious cycle (19, 31) and in response to host and environmental signals (6, 8a, 15, 24, 25). Although it is clear that B. burgdorferi alters gene expression to adapt to different environments, the genetic tools for studying gene regulation in B. burgdorferi are limited.Within the last 2 decades, the complete genomic sequence of B. burgdorferi strain B31 was published (10, 14) and techniques for basic genetic manipulation of B. burgdorferi became available (5, 11, 13, 27-29, 36). A chloramphenicol acetyltransferase (CAT) gene was the first reporter gene that was fused to B. burgdorferi promoters for analysis of promoter strength (33). The development of luciferase (4) and multiple fluorescent proteins (9, 11, 30) as reporter systems in B. burgdorferi followed. Although these systems have value, there are limitations with each. β-Galactosidase, encoded by lacZ, has been used extensively as a convenient reporter gene in Escherichia coli and is still applicable to a broad range of organisms, both prokaryotic and eukaryotic, but has not yet been used with B. burgdorferi. β-Galactosidase activity can be monitored easily and quickly by simple colorimetric assays in both liquid and solid media, neither of which require expensive or specialized equipment. Additionally, a wide variety of substrates for β-galactosidase allow for different levels of sensitivity in either in vitro or in vivo detection formats (17). Having lacZ available as a genetic tool for B. burgdorferi would enhance investigation of the complex regulatory events that are integral to the spirochete''s infectious cycle. To this end, we developed lacZ as a reporter gene in B. burgdorferi and demonstrated its utility.     

Complementation of a Nonmotile flaB Mutant of Borrelia burgdorferi by Chromosomal Integration of a Plasmid Containing a Wild-Type flaB Allele

With the recent identification of antibiotic resistance phenotypes, the use of reporter genes, the isolation of null mutants by insertional inactivation, and the development of extrachromosomal cloning vectors, genetic analysis of Borrelia burgdorferi is becoming a reality. A previously described nonmotile, rod-shaped, kanamycin-resistant B. burgdorferi flaB::Km null mutant was complemented by electroporation with the erythromycin resistance plasmid pED3 (a pGK12 derivative) containing the wild-type flaB sequence and 366 bp upstream from its initiation codon. The resulting MS17 clone possessed erythromycin and kanamycin resistance, flat-wave morphology, and microscopic and macroscopic motility. Several other electroporations with plasmids containing wild-type flaB and various lengths (198, 366, or 762 bp) of sequence upstream from the flaB gene starting codon did not lead to functional restoration of the nonmotile flaB null mutant. DNA hybridization, PCR analysis, and sequencing indicated that the wild-type flaB gene in nonmotile clones was present in the introduced extrachromosomal plasmids, while the motile MS17 clone was a merodiploid containing single tandem chromosomal copies of mutated flaB::Km and wild-type flaB with a 366-bp sequence upstream from its starting codon. Complementation was thus achieved only when wild-type flaB was inserted into the borrelial chromosome. Several possible mechanisms for the failure of complementation for extrachromosomally located flaB are discussed.     

TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi

The Lyme disease agent Borrelia burgdorferi naturally persists in a cycle that primarily involves ticks and mammals. We have now identified a tick receptor (TROSPA) that is required for spirochetal colonization of Ixodes scapularis. B. burgdorferi outer surface protein A, which is abundantly expressed on spirochetes within the arthropod and essential for pathogen adherence to the vector, specifically bound to TROSPA. TROSPA mRNA levels in ticks increased following spirochete infestation and decreased in response to engorgement, events that are temporally linked to B. burgdorferi entry into and egress from the vector. The blockade of TROSPA by TROSPA antisera or by the repression of TROSPA expression via RNA interference reduced B. burgdorferi adherence to the I. scapularis gut in vivo, thereby preventing efficient colonization of the vector and subsequently reducing pathogen transmission to the mammalian host. Identification of an I. scapularis receptor for B. burgdorferi is the first step toward elucidating arthropod ligands that are required for survival of spirochetes in nature.     

Chemotaxis in Borrelia burgdorferi

Borrelia burgdorferi is a motile spirochete which has been identified as the causative microorganism in Lyme disease. The physiological functions which govern the motility of this organism have not been elucidated. In this study, we found that motility of B. burgdorferi required an environment similar to interstitial fluid (e.g., pH 7.6 and 0.15 M NaCl). Several methods were used to detect and measure chemotaxis of B. burgdorferi. A number of chemical compounds and mixtures were surveyed for the ability to induce positive and negative chemotaxis of B. burgdorferi. Rabbit serum was found to be an attractant for B. burgdorferi, while ethanol and butanol were found to be repellents. Unlike some free-living spirochetes (e.g., Spirochaeta aurantia), B. burgdorferi did not exhibit any observable chemotaxis to common sugars or amino acids. A method was developed to produce spirochete cells with a self-entangled end. These cells enabled us to study the rotation of a single flagellar bundle in response to chemoattractants or repellents. The study shows that the frequency and duration for pausing of flagella are important for chemotaxis of B. burgdorferi.     

Requirements for Borrelia burgdorferi plasmid maintenance

Borrelia burgdorferi has multiple linear and circular plasmids that are faithfully replicated and partitioned as the bacterium grows and divides. The low copy number of these replicons implies that active partitioning contributes to plasmid stability. Analyzing the requirements for plasmid replication and partition in B. burgdorferi is complicated by the complexity of the genome and the possibility that products may act in trans. Consequently, we have studied the replication-partition region (bbb10-13) of the B. burgdorferi 26kb circular plasmid (cp26) in Escherichia coli, by fusion with a partition-defective miniF plasmid. Our analysis demonstrated that bbb10, bbb11, and bbb13 are required for stable miniF maintenance, whereas bbb12 is dispensable. To validate these results, we attempted to inactivate two of these genes in B. burgdorferi. bbb12 mutants were obtained at a typical frequency, suggesting that the bbb12 product is dispensable for cp26 maintenance as well. We could not directly measure cp26 stability in the bbb12 mutant, because cp26 carries essential genes, and bacteria that have lost cp26 are inviable. Conversely, we were unable to inactivate bbb10 on cp26 of B. burgdorferi. Our results suggest that bbb12 is dispensable for cp26 maintenance, whereas bbb10, bbb11, and bbb13 play crucial roles in that process.     

Detection of glycoproteins in Borrelia burgdorferi

The presence of carbohydrates on proteins of Borrelia burgdorferi, the causative agent of Lyme disease, was investigated by using a digoxigenin labeling method together with Schiff staining and N-glycosidase F assay. The two major outer surface exposed proteins of 31 kDa and 34 kDa showed to be glycosylated and gel filtration high pressure liquid chromatography (HPLC) of proteins of B. burgdorferi metabolically labeled with ¹⁴C-N-acetylglucosamine revealed the incorporation of the carbohydrate into the glycosyl residue of these proteins.Abbreviations N-glycosidase F peptide-N-glycosidase F (EC 3.5.1.52) - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - WB Western blotting - HPLC high pressure liquid chromatography - SDS sodium dodecyl sulphate - mAb monoclonal antibody - MIAF mouse immune ascitic fluid - SS Schiff staining - Osp Outer surface protein     

Penicillin-binding proteins in Borrelia burgdorferi.

Penicillin-binding proteins were identified in Borrelia burgdorferi membranes. A 94-kilodalton penicillin-binding protein was the first to be labeled with tritiated penicillin and was the first band to disappear in a competition experiment. Its binding ability was destroyed when membranes were preboiled. In addition, several of these penicillin-binding proteins comigrated with bands previously identified as surface proteins.     

Whole genome sequence of an unusual Borrelia burgdorferi sensu lato isolate

Human Lyme disease is caused by a number of related Borrelia burgdorferi sensu lato species. We report here the complete genome sequence of Borrelia sp. isolate SV1 from Finland. This isolate is to date the closest known relative of B. burgdorferi sensu stricto, but it is sufficiently genetically distinct from that species that it and its close relatives warrant its candidacy for new-species status. We suggest that this isolate should be named "Borrelia finlandensis."     

The many faces of Borrelia burgdorferi

In this review we describe several genetic regulatory mechanisms adopted by the agent of Lyme disease, Borrelia burgdorferi, to sense and adapt to different host and environmental conditions either in vitro or in vivo. This regulation results in the increased or decreased synthesis of several proteins whose levels are believed to play key roles in the ability of B. burgdorferi to cycle between both arthropod and mammalian hosts. Moreover, the differential synthesis of these proteins serves to modulate the response of B. burgdorferito signals in the requisite host and may also, in some cases, function as virulence determinants of this spirochete. Elucidation of these mechanisms will help in the understanding of the pathogenicity of B. burgdorferi as well as aid in identifying proteins that are important during different stages of infection.     

Functional properties of Borrelia burgdorferi recA

Functions of the Borrelia burgdorferi RecA protein were investigated in Escherichia coli recA null mutants. Complementation with B. burgdorferi recA increased survival of E. coli recA mutants by 3 orders of magnitude at a UV dose of 2,000 microJ/cm(2). The viability at this UV dose was about 10% that provided by the homologous recA gene. Expression of B. burgdorferi recA resulted in survival of E. coli at levels of mitomycin C that were lethal to noncomplemented hosts. B. burgdorferi RecA was as effective as E. coli RecA in mediating homologous recombination in E. coli. Furthermore, E. coli lambda phage lysogens complemented with B. burgdorferi recA produced phage even in the absence of UV irradiation. The level of phage induction was 55-fold higher than the level in cells complemented with the homologous recA gene, suggesting that B. burgdorferi RecA may possess an enhanced coprotease activity. This study indicates that B. burgdorferi RecA mediates the same functions in E. coli as the homologous E. coli protein mediates. However, the rapid loss of viability and the absence of induction in recA expression after UV irradiation in B. burgdorferi suggest that recA is not involved in the repair of UV-induced damage in B. burgdorferi. The primary role of RecA in B. burgdorferi is likely to be a role in some aspect of recombination.     

Incorporation of cysteine by Borrelia burgdorferi and Borrelia hermsii.

The growth rate of Borrelia burgdorferi and Borrelia hermsii in BSK II medium prepared with cysteine-free or cysteine-containing (0.185-5.92 mM) CMRL 1066 medium was studied. In media with cysteine-free CMRL 1066, growth of borreliae was detectable, although it was reduced by approximately 80%. Bacterial growth was maximal when the concentration of cysteine in CMRL 1066 reached 1.48 mM, which represents the standard cysteine concentrations of the medium; higher concentrations inhibited the growth of borreliae. Cysteine incorporation, measured by the uptake of radiolabeled cysteine, showed that cysteine enters B. burgdorferi and B. hermsii cells by passive diffusion. Labeling studies of borreliae with [35S]cysteine indicated that B. burgdorferi has several cysteine-containing proteins, including ones at 22, 30 (OspA), and 34 kDa (OspB), whereas B. hermsii showed only two [35S]cysteine-incorporating proteins, at 22 and 24 kDa, which were exposed onto the outer cell surface. In addition, most of the cysteine-incorporating proteins could be biosynthetically radiolabeled when bacterial cells were grown in vitro with [3H]palmitate, and the differences in cysteine incorporation observed between B. burgdorferi and B. hermsii were found to be correlated with differences in lipoproteins.     

aadA confers streptomycin resistance in Borrelia burgdorferi

To enhance genetic manipulation of the Lyme disease spirochete Borrelia burgdorferi, we assayed the aadA gene for the ability to confer resistance to the antibiotics spectinomycin and streptomycin. Using the previously described pBSV2 as a backbone, a shuttle vector, termed pKFSS1, which carries the aadA open reading frame fused to the B. burgdorferi flgB promoter was constructed. The hybrid flgB promoter-aadA cassette confers resistance to spectinomycin and streptomycin in both B. burgdorferi and Escherichia coli. pKFSS1 has a replication origin derived from the 9-kb circular plasmid and can be comaintained in B. burgdorferi with extant shuttle vector pCE320, which has a replication origin derived from a 32-kb circular plasmid, or pBSV2, despite the fact that pKFSS1 and pBSV2 have the same replication origin. Our results demonstrate the availability of a new selectable marker and shuttle vector for genetically dissecting B. burgdorferi at the molecular level.     

Molecular evidence for a new bacteriophage of Borrelia burgdorferi

We have recovered a DNase-protected, chloroform-resistant molecule of DNA from the cell-free supernatant of a Borrelia burgdorferi culture. The DNA is a 32-kb double-stranded linear molecule that is derived from the 32-kb circular plasmids (cp32s) of the B. burgdorferi genome. Electron microscopy of samples from which the 32-kb DNA molecule was purified revealed bacteriophage particles. The bacteriophage has a polyhedral head with a diameter of 55 nm and appears to have a simple 100-nm-long tail. The phage is produced constitutively at low levels from growing cultures of some B. burgdorferi strains and is inducible to higher levels with 10 microg of 1-methyl-3-nitroso-nitroguanidine (MNNG) ml(-1). In addition, the prophage can be induced with MNNG from some Borrelia isolates that do not naturally produce phage. We have isolated and partially characterized the phage associated with B. burgdorferi CA-11.2A. To our knowledge, this is the first molecular characterization of a bacteriophage of B. burgdorferi.     

Seroepidemiological survey for antibody to Borrelia burgdorferi in cows.

Antibody to Borrelia burgdorferi was examined in 380 healthy and 38 clinical cases of cows from Hokkaido and Shizuoka in Japan. In healthy animals, IgG and IgM antibody to B. burgdorferi HO14 strain were found in 44 cows (14.6%) and 24 cows (8.0%) from Hokkaido. In contrast, antibody-positive case was not observed except for only 1 case which was IgM positive (1/79: 1.3%) in cows from Shizuoka. Mean antibody levels of healthy animals in Hokkaido and Shizuoka were 0.651 and 0.263 (IgG antibody to HO14 strain), 0.642 and 0.169 (IgG to HP3 strain), 0.613 and 0.367 (IgM to HO14 strain) and 0.582 and 0.286 (IgM to HP3 strain). The differences of the antibody levels between cows from Hokkaido and Shizuoka were significant. Seasonal difference was found in seropositive cows from Hokkaido. The rate of seropositive cows was high in summer (23.4% in June and 11.8% in July) but low in winter (0% in January and February). The pattern was discussed to be associated with activation of ticks. One of 4 cows with arthritis showed significantly higher IgG antibody level than that of healthy cows and cows with some disease, although the serum was collected from Shizuoka where antibody-positive animals for B. burgdorferi were rare among healthy cows. This high IgG antibody may suggest that the arthritis of such cows was caused by infection with B. burgdorferi. Two of 7 cows with unclassified abortion showed positive antibody reaction in Hokkaido. These cases, however, may not be related to the B. burgdorferi infection because the positive rate was similar to those of healthy cows in the same season.     

Positive IgG Western blot for Borrelia burgdorferi in Colombia.

In order to evaluate the presence of specific IgG antibodies to Borrelia burgdorferi in patients with clinical manifestations associated with Lyme borreliosis in Cali, Colombia, 20 serum samples from patients with dermatologic signs, one cerebrospinal fluid (CSF) sample from a patient with chronic neurologic and arthritic manifestations, and twelve serum samples from individuals without clinical signs associated with Lyme borreliosis were analyzed by IgG Western blot. The results were interpreted following the recommendations of the Centers for Diseases Control and Prevention (CDC) for IgG Western blots. Four samples fulfilled the CDC criteria: two serum specimens from patients with morphea (localized scleroderma), the CSF from the patient with neurologic and arthritic manifestations, and one of the controls. Interpretation of positive serology for Lyme disease in non-endemic countries must be cautious. However these results suggest that the putative "Lyme-like" disease may correlate with positivity on Western blots, thus raising the possibility that a spirochete genospecies distinct from B. burgdorferi sensu stricto, or a Borrelia species other than B. burgdorferi sensu lato is the causative agent. Future work will focus on a survey of the local tick and rodent population for evidence of spirochete species that could be incriminated as the etiologic agent.     

Identification of Specific Chemoattractants and Genetic Complementation of a Borrelia burgdorferi Chemotaxis Mutant: Flow Cytometry-Based Capillary Tube Chemotaxis Assay

Measuring the chemotactic response of Borrelia burgdorferi, the bacterial species that causes Lyme disease, is relatively more difficult than measuring that of other bacteria. Because these spirochetes have long generation times, enumerating cells that swim up a capillary tube containing an attractant by using colony counts is impractical. Furthermore, direct counts with a Petroff-Hausser chamber is problematic, as this method has a low throughput and necessitates a high cell density; the latter can lead to misinterpretation of results when assaying for specific attractants. Only rabbit serum and tick saliva have been reported to be chemoattractants for B. burgdorferi. These complex biological mixtures are limited in their utility for studying chemotaxis on a molecular level. Here we present a modified capillary tube chemotaxis assay for B. burgdorferi that enumerates cells by flow cytometry. Initial studies identified N-acetylglucosamine as a chemoattractant. The assay was then optimized with respect to cell concentration, incubation time, motility buffer composition, and growth phase. Besides N-acetylglucosamine, glucosamine, glucosamine dimers (chitosan), glutamate, and glucose also elicited significant chemoattractant responses, although the response obtained with glucose was weak and variable. Serine and glycine were nonchemotactic. To further validate and to exploit the use of this assay, a previously described nonchemotactic cheA2 mutant was shown to be nonchemotactic by this assay; it also regained the wild-type phenotype when complemented in trans. This is the first report that identifies specific chemical attractants for B. burgdorferi and the use of flow cytometry for spirochete enumeration. The method should also be useful for assaying chemotaxis for other slow-growing prokaryotic species and in specific environments in nature.