ResT, a telomere resolvase encoded by the Lyme disease spirochete.

The genus Borrelia includes the causative agents of Lyme disease and relapsing fever. An unusual feature of these bacteria is a segmented genome consisting mostly of a number of linear DNA molecules with covalently closed hairpin ends or telomeres. In this study we show that the BBB03 locus encodes the B. burgdorferi telomere resolvase, ResT. The purified protein catalyzes telomere resolution in vitro through a unique reaction: breakage of two phosphodiester bonds in a single DNA duplex (one on each strand) and joining of each end with the opposite DNA strand to form covalently closed hairpin telomeres. Telomere resolution by ResT occurs through a two-step transesterification reaction involving the formation of a covalent protein-DNA intermediate at a position three nucleotides from the axis of symmetry in each strand of the substrate.     

rRNA gene organization in the Lyme disease spirochete, Borrelia burgdorferi.

Lyme disease is the most common vector-borne disease in the United States. The causative agent is the spirochete Borrelia burgdorferi. The copy number and organization of the genes encoding the rRNAs of this organism were determined. There is a single gene for 16S rRNA and two copies each of the 23S rRNA and 5S rRNA genes. All of the genes are located within a chromosomal fragment of approximately 9.5 to 10.0 kb. The 23S and 5S rRNA genes are tandemly duplicated in the order 23S-5S-23S-5S and are apparently not linked to the 16S rRNA gene, which is situated over 2 kb upstream from the 23S-5S duplication. The individual copies of the 23S-5S duplication are separated by a 182-bp spacer. Within each 23S-5S unit, an identical 22-bp spacer separates the 23S and 5S rRNA sequences from each other. The genome organization of the 23S-5S gene cluster in a number of different B. burgdorferi isolates obtained at a number of different geographical locations, as well as in several other species of Borrelia, was investigated. All isolates of B. burgdorferi tested displayed the tandem duplication, whereas the closely related species B. hermsii, B. anserina, and B. turicatae all contained a single copy of each of the genes. In addition, different geographical isolates of B. burgdorferi can be differentiated on the basis of a restriction fragment length polymorphism associated with the 23S-5S gene cluster. This polymorphism can be a useful tool for the determination of genetic relatedness between different isolates of B. burgdorferi.     

The histopathology of experimentally infected hamsters with the Lyme disease spirochete, Borrelia burgdorferi

Seven hamsters, experimentally infected with Borrelia burgdorferi, were examined by both cultural and histological techniques at 1 to 9 months postinfection. Spirochetes were detected in the spleen, kidney, or eye of all animals by culture and in the spleen, kidney, eye, liver, or heart blood of five of seven animals by histological examination. Two animals showed nonspecific hepatic portal lymphocytic infiltration, while five of the hamsters displayed no significant histologic signs of inflammation or granuloma formation in the major organ systems. Synovitis and arthropathy did not occur. All animals showed some degree of follicular lymphoid hyperplasia of the spleen. Spirochetes were predominantly extracellular with a rare organism appearing to be partially within a macrophage.     

The structure of a pyrophosphate-dependent phosphofructokinase from the Lyme disease spirochete Borrelia burgdorferi

The structure of the 60 kDa pyrophosphate (PP(i))-dependent phosphofructokinase (PFK) from Borrelia burgdorferi has been solved and refined (R(free) = 0.243) at 2.55 A resolution. The domain structure of eubacterial ATP-dependent PFKs is conserved in B. burgdorferi PFK, and there are three large insertions relative to E. coli PFK, including a helical domain containing a hairpin structure that interacts with the active site. Asp177, conserved in all PP(i) PFKs, negates the binding of the alpha-phosphate group of ATP and likely contacts the essential Mg(2+) cation via a water molecule. Asn181 blocks the binding of the adenine moiety of ATP. Lys203 hydrogen bonds to a sulfate anion that likely mimics PP(i) substrate binding.     

Defining the plasmid-borne restriction-modification systems of the Lyme disease spirochete Borrelia burgdorferi

The restriction-modification (R-M) systems of many bacteria present a barrier to the stable introduction of foreign DNA. The Lyme disease spirochete Borrelia burgdorferi has two plasmid-borne putative R-M genes, bbe02 and bbq67, whose presence limits transformation by shuttle vector DNA from Escherichia coli. We show that both the bbe02 and bbq67 loci in recipient B. burgdorferi limit transformation with shuttle vector DNA from E. coli, irrespective of its dam, dcm, or hsd methylation status. However, plasmid DNA purified from B. burgdorferi transformed naïve B. burgdorferi much more efficiently than plasmid DNA from E. coli, particularly when the bbe02 and bbq67 genotypes of the B. burgdorferi DNA source matched those of the recipient. We detected adenine methylation of plasmid DNA prepared from B. burgdorferi that carried bbe02 and bbq67. These results indicate that the bbe02 and bbq67 loci of B. burgdorferi encode distinct R-M enzymes that methylate endogenous DNA and cleave foreign DNA lacking the same sequence-specific modification. Our findings have basic implications for horizontal gene transfer among B. burgdorferi strains with distinct plasmid contents. Further characterization and identification of the nucleotide sequences recognized by BBE02 and BBQ67 will facilitate efficient genetic manipulation of this pathogenic spirochete.Borrelia burgdorferi sensu lato is a zoonotic pathogen whose natural infectious cycle alternates between a tick vector and rodent or bird reservoir hosts (1, 7, 8, 14, 32, 33, 36). Transmission of B. burgdorferi to humans occurs through the bite of an infected tick and can lead to Lyme disease, which is a major public health concern in areas of North America and Europe where B. burgdorferi is endemic (8, 53).The genomic structure of the spirochete B. burgdorferi is unique, consisting of a linear chromosome of approximately 900 kb and more than 20 linear (lp) and circular (cp) plasmids, ranging in size from ∼5 kb to 56 kb, in the type strain B31 (9, 10, 11, 19, 42). The plasmids of B. burgdorferi are present at unit copy number relative to the chromosome (22), and some are relatively unstable during in vitro propagation (52, 57). The loss of linear plasmids lp25, lp28-1, and lp36 by strain B31 was found to correlate with the loss of infectivity in mice (20, 31, 45, 56), leading to the identification of genes carried on these plasmids that are dispensable in vitro but required in vivo during an experimental infectious cycle (21, 26, 35, 44, 47). The loss of two linear plasmids, lp25 and lp56, was shown to correlate with enhanced shuttle vector transformation, suggesting that specific lp25 and lp56 gene products present a barrier to stable introduction of foreign DNA (34). Further studies linked the transformation phenotype of B. burgdorferi strain B31 with the bbe02 and bbq67 genes on lp25 and lp56, respectively, and the putative restriction-modification (R-M) enzymes that they encode (11, 27, 29, 34). The recent demonstration by Chen and colleagues of enhanced transformation of B. burgdorferi following in vitro methylation of DNA (13) further supports the hypothesis that these B. burgdorferi plasmids encode R-M enzymes that degrade foreign DNA lacking the appropriate modification.The barrier to foreign DNA presented by the bbe02 and bbq67 loci of B. burgdorferi implies that genomic DNA should be modified in spirochetes carrying these plasmid genes. To test this hypothesis, we compared the transformation of B. burgdorferi with shuttle vector DNA isolated from either Escherichia coli or B. burgdorferi, as outlined in Fig. ​Fig.1.1. We also examined whether and how the presence of putative R-M genes in either the donor or recipient B. burgdorferi strain influenced transformation. Finally, we analyzed the type of modification present on DNA isolated from B. burgdorferi with different plasmid or gene contents. Our data indicate that the bbe02 and bbq67 loci of B. burgdorferi encode enzymes that both methylate endogenous DNA and restrict foreign DNA lacking these modifications. These findings have basic implications regarding horizontal gene transfer among B. burgdorferi strains with distinct plasmid contents. These results also help elucidate the molecular mechanisms underlying the relative inefficiency of genetic transformation of B. burgdorferi and suggest ways in which genetic manipulation of this pathogenic spirochete could be enhanced.Open in a separate windowFIG. 1.Shuttle vector transformations. Schematic representation of the various DNA sources, strains and methods used to assess the contributions of bbe02 and bbq67 to the restriction-modification (R-M) systems of B. burgdorferi.     

Carbohydrate utilization by the Lyme borreliosis spirochete, Borrelia burgdorferi

Growth kinetic analyses of Borrelia burgdorferi indicated that this bacterium can utilize a limited number of carbon sources for energy: the monosaccharides glucose, mannose, and N-acetylglucosamine, the disaccharides maltose and chitobiose, and glycerol. All of these carbohydrates are likely to be available to B. burgdorferi during infection of either vertebrate and arthropod hosts, enabling development of a model describing energy sources potentially used by the Lyme borreliosis spirochete during its natural infectious cycle.     

Identification of a candidate glycosaminoglycan-binding adhesin of the Lyme disease spirochete Borrelia burgdorferi

Binding of glycosaminoglycans (GAGs) by Borrelia burgdorferi, the Lyme disease spirochete, has the potential to promote the colonization of diverse tissues. GAG binding by B. burgdorferi is associated with haemagglutination and we have identified a 26 kDa protein, which we have termed Bgp (Borrelia GAG-binding protein), on the basis of its ability to bind to heparin and erythrocytes. Bgp was found in outer membrane fractions of B. burgdorferi and on the surface of intact bacteria, as assayed by labelling with a membrane-impermeable biotinylating agent or anti-Bgp antibodies. Purified recombinant Bgp agglutinated erythrocytes, binds to the same spectrum of GAGs as the B. burgdorferi strain from which the cloned bgp sequence was obtained, and inhibited B. burgdorferi binding to purified GAGs and to cultured mammalian cells. Thus, Bgp is a strong candidate for a GAG-binding adhesin of B. burgdorferi.     

Identification and function of the RNA chaperone Hfq in the Lyme disease spirochete Borrelia burgdorferi

Hfq is a global regulatory RNA‐binding protein. We have identified and characterized an atypical Hfq required for gene regulation and infectivity in the Lyme disease spirochete Borrelia burgdorferi. Sequence analyses of the putative B. burgdorferi Hfq protein revealed only a modest level of similarity with the Hfq from Escherichia coli, although a few key residues are retained and the predicted tertiary structure is similar. Several lines of evidence suggest that the B. burgdorferi bb0268 gene encodes a functional Hfq homologue. First, the hfq_Bb gene (bb0268) restores the efficient translation of an rpoS::lacZ fusion in an E. coli hfq null mutant. Second, the Hfq from B. burgdorferi binds to the small RNA DsrA_Bb and the rpoS mRNA. Third, a B. burgdorferi hfq null mutant was generated and has a pleiotropic phenotype that includes increased cell length and decreased growth rate, as found in hfq mutants in other bacteria. The hfq_Bb mutant phenotype is complemented in trans with the hfq gene from either B. burgdorferi or, surprisingly, E. coli. This is the first example of a heterologous bacterial gene complementing a B. burgdorferi mutant. The alternative sigma factor RpoS and the outer membrane lipoprotein OspC, which are induced by increased temperature and required for mammalian infection, are not upregulated in the hfq mutant. Consequently, the hfq mutant is not infectious by needle inoculation in the murine model. These data suggest that Hfq plays a key role in the regulation of pathogenicity factors in B. burgdorferi and we hypothesize that the spirochete has a complex Hfq‐dependent sRNA network.     

Natural exposure of Wisconsin dogs to the Lyme disease spirochete (Borrelia burgdorferi)

Lyme disease, a tick-borne disease caused by Borrelia burgdorferi, has been described recently in dogs. In a serologic survey of specimens obtained from March to September 1984, 53% of 380 dogs from two USDA licensed vendors in Wisconsin were positive for indirect immunofluorescent antibodies to B. burgdorferi at a serum dilution of 1:64 or greater. B. burgdorferi was cultured from the blood of 8/111 dogs. These findings suggest that exposure to this spirochete is common in endemic areas and that this zoonotic disease is of importance to veterinarians and researchers.     

Incompetence of catbirds as reservoirs for the Lyme disease spirochete (Borrelia burgdorferi)

We compared the relative infectivity to vector ticks of gray catbirds (Dumetella carolinensis) and white-footed mice (Peromyscus leucopus) for the Lyme disease spirochete (Borrelia burgdorferi). Of 28 catbirds captured in a site enzootic for this agent, 18 were infested by immature Ixodes dammini, the tick vector. By comparison, each of 32 mice sampled concurrently from the same site was infested, and by about 10 times as many ticks as were found infesting the 3 most commonly netted bird species. Although 76% of noninfected larval ticks placed on these mice in a xenodiagnosis became infected, none of the ticks similarly placed on 12 catbirds did so. Spirochetes were detected in ticks derived from 2 Carolina wrens (Thryothorus ludovicianus) and a common yellowthroat (Geothlypis trichas), but these species' potential contribution to infecting ticks does not compare with that of mice. Thus, although birds may help establish new foci of ticks, catbirds, at least, do not appear to contribute as reservoirs of infection.     

Vector competence of the Australian paralysis tick, Ixodes holocyclus, for the Lyme disease spirochete Borrelia burgdorferi

Clinical and serologic evidence of Lyme disease in Australia, including the typical rash, erythema migrans, has been reported. The vector tick transmitting Borrelia burgdorferi in Australia, however, has not been determined. The Australian paralysis tick, Ixodes holocyclus, is a logical candidate vector of the Lyme disease spirochete in Australia; therefore, we tested the ability of I. holocyclus to acquire and maintain a North American isolate of B. burgdorferi. Larval I. holocyclus ingested spirochetes, but none of 84 derived nymphs were infected. These experiments should be repeated with Australian strains of spirochetes.     

Changes in antigenic reactivity of Borrelia burgdorferi, the Lyme disease spirochete, during persistent infection in mice

Adult laboratory mice, Mus musculus, were shown to be suitable experimental animals for studying infectivity, persistent infection, and in vivo antigenic changes of Borrelia burgdorferi. Sixteen mice were inoculated intraperitoneally with a low-passage culture of an uncloned strain of B. burgdorferi and 16 months later spirochetes were reisolated from the urinary bladder of 15 (94%) of the mice. Spirochetes recovered from the urinary bladder of one persistently infected mouse were tested for infectivity and found to be infectious when passaged into four laboratory mice. Western blot analysis of immune serum from each of the persistently infected mice demonstrated that spirochetes used to infect the mice reacted differently when compared with the spirochetes subsequently reisolated from the mice, demonstrating for the first time that changes in antigenic reactivity had occurred in the spirochete populations during persistent infection.     

Crystal structure of outer surface protein C (OspC) from the Lyme disease spirochete, Borrelia burgdorferi

Outer surface protein C (OspC) is a major antigen on the surface of the Lyme disease spirochete, Borrelia burgdorferi, when it is being transmitted to humans. Crystal structures of OspC have been determined for strains HB19 and B31 to 1.8 and 2.5 A resolution, respectively. The three-dimensional structure is predominantly helical. This is in contrast to the structure of OspA, a major surface protein mainly present when spirochetes are residing in the midgut of unfed ticks, which is mostly beta-sheet. The surface of OspC that would project away from the spirochete's membrane has a region of strong negative electrostatic potential which may be involved in binding to positively charged host ligands. This feature is present only on OspCs from strains known to cause invasive human disease.     

Investigation of venereal, transplacental, and contact transmission of the Lyme disease spirochete, Borrelia burgdorferi, in Syrian hamsters.

A hamster was inoculated with the SI-1 strain of Borrelia burgdorferi and subsequently served as a host to larval Ixodes scapularis Say. Approximately 68% of the nymphs resulting from the fed larvae were infected. Nymphs from this group were fed on uninfected hamsters, and 3 of 4 males and 6 of 6 females became infected. The infected hamsters were allowed to mate with uninfected partners to test for venereal transmission. Six infected females were mated with 6 uninfected males, whereas 3 infected males were mated with 6 uninfected females. None of the uninfected hamsters became infected after mating. Two protocols were used to determine if transplacental transmission of B. burgdorferi occurred. One group included 6 nonpregnant infected females that were subsequently mated and became pregnant. Three of the females were allowed to carry to full term, whereas the other 3 were killed prior to parturition. All fetuses and offspring were negative for B. burgdorferi based on cultures and monoclonal antibody assays. Another group of 6 females was infected via tick bite after becoming pregnant; those females were allowed to carry fetuses to birth and all were negative. Attempts at contact transmission of B. burgdorferi from 2 infected females to 2 uninfected male and 2 uninfected female hamsters and from 2 infected males to 2 uninfected male and uninfected female hamsters via urine or feces failed.     

Synthesis of autoinducer 2 by the lyme disease spirochete, Borrelia burgdorferi

Defining the metabolic capabilities and regulatory mechanisms controlling gene expression is a valuable step in understanding the pathogenic properties of infectious agents such as Borrelia burgdorferi. The present studies demonstrated that B. burgdorferi encodes functional Pfs and LuxS enzymes for the breakdown of toxic products of methylation reactions. Consistent with those observations, B. burgdorferi was shown to synthesize the end product 4,5-dihydroxy-2,3-pentanedione (DPD) during laboratory cultivation. DPD undergoes spontaneous rearrangements to produce a class of pheromones collectively named autoinducer 2 (AI-2). Addition of in vitro-synthesized DPD to cultured B. burgdorferi resulted in differential expression of a distinct subset of proteins, including the outer surface lipoprotein VlsE. Although many bacteria can utilize the other LuxS product, homocysteine, for regeneration of methionine, B. burgdorferi was found to lack such ability. It is hypothesized that B. burgdorferi produces LuxS for the express purpose of synthesizing DPD and utilizes a form of that molecule as an AI-2 pheromone to control gene expression.     

Perpetuation of the Lyme disease spirochete Borrelia lusitaniae by lizards

To determine whether the Lyme disease spirochete Borrelia lusitaniae is associated with lizards, we compared the prevalence and genospecies of spirochetes present in rodent- and lizard-associated ticks at a site where this spirochete frequently infects questing ticks. Whereas questing nymphal Ixodes ricinus ticks were infected mainly by Borrelia afzelii, one-half of the infected adult ticks harbored B. lusitaniae at our study site. Lyme disease spirochetes were more prevalent in sand lizards (Lacerta agilis) and common wall lizards (Podarcis muralis) than in small rodents. Although subadult ticks feeding on rodents acquired mainly B. afzelii, subadult ticks feeding on lizards became infected by B. lusitaniae. Genetic analysis confirmed that the spirochetes isolated from ticks feeding on lizards are members of the B. lusitaniae genospecies and resemble type strain PotiB2. At our central European study site, lizards, which were previously considered zooprophylactic for the agent of Lyme disease, appear to perpetuate B. lusitaniae.     

Functional analysis of a lipid galactosyltransferase synthesizing the major envelope lipid in the Lyme disease spirochete Borrelia burgdorferi

One of the major lipids in the membranes of Borrelia burgdorferi is monogalactosyl diacylglycerol (MGalDAG), a glycolipid recently shown to carry antigenic potency. Herein, it is shown that the gene mgs (TIGR designation bb0454) of B. burgdorferi encodes for the protein bbMGS that, when expressed in Escherichia coli, catalyzes the glycosylation of 1,2-diacylglycerol with specificity for the donor substrate UDP-Gal yielding MGalDAG. Related lipid enzymes were found in many Gram-positive bacteria. The presence of this galactosyltransferase activity and synthesis of a cholesteryl galactoside by another enzyme were verified in B. burgdorferi cell extract. Besides MGalDAG, phosphatidylcholine, phosphatidylglycerol, and cholesterol were also found as major lipids in the cell envelope. The high isoelectric point of bbMGS and clustered basic residues in its amino acid sequence suggest that the enzyme interacts with acidic lipids in the plasma membrane, in agreement with strong enzymatic activation of bbMGS by phosphatidylglycerol. The membrane packing and immunological properties of MGalDAG are likely to be of great importance in vivo.