We have determined that Borrelia burgdorferi strain B31 MI carries 21 extrachromosomal DNA elements, the largest number known for any bacterium. Among these are 12 linear and nine circular plasmids, whose sequences total 610 694 bp. We report here the nucleotide sequence of three linear and seven circular plasmids (comprising 290 546 bp) in this infectious isolate. This completes the genome sequencing project for this organism; its genome size is 1 521 419 bp (plus about 2000 bp of undetermined telomeric sequences). Analysis of the sequence implies that there has been extensive and sometimes rather recent DNA rearrangement among a number of the linear plasmids. Many of these events appear to have been mediated by recombinational processes that formed duplications. These many regions of similarity are reflected in the fact that most plasmid genes are members of one of the genome's 161 paralogous gene families; 107 of these gene families, which vary in size from two to 41 members, contain at least one plasmid gene. These rearrangements appear to have contributed to a surprisingly large number of apparently non‐functional pseudogenes, a very unusual feature for a prokaryotic genome. The presence of these damaged genes suggests that some of the plasmids may be in a period of rapid evolution. The sequence predicts 535 plasmid genes ≥300 bp in length that may be intact and 167 apparently mutationally damaged and/or unexpressed genes (pseudogenes). The large majority, over 90%, of genes on these plasmids have no convincing similarity to genes outside Borrelia, suggesting that they perform specialized functions.
In little more than 30 years, Lyme disease, which is caused by the spirochaete Borrelia burgdorferi, has risen from relative obscurity to become a global public health problem and a prototype of an emerging infection. During this period, there has been an extraordinary accumulation of knowledge on the phylogenetic diversity, molecular biology, genetics and host interactions of B. burgdorferi. In this Review, we integrate this large body of information into a cohesive picture of the molecular and cellular events that transpire as Lyme disease spirochaetes transit between their arthropod and vertebrate hosts during the enzootic cycle.
We have characterized seven different 32-kb circular plasmids carried by Borrelia burgdorferi isolate B31. Restriction endonuclease recognition site mapping and partial sequencing of these plasmids indicated that all seven are probably closely related to each other throughout their lengths and have substantial relationships to cp8.3, an 8.3-kb circular plasmid of B. burgdorferi sensu lato isolate Ip21. With the addition of the seven 32-kb plasmids, this bacterial strain is known to carry at least 10 linear and 9 circular plasmids. Variant cultures of B. burgdorferi B31 lacking one or more of the 32-kb circular plasmids are viable and, at least in some cases, infectious. We have examined a number of different natural isolates of Lyme disease borreliae and found that all of the B. burgdorferi sensu stricto isolates and most of the B. burgdorferi sensu lato isolates tested appear to carry multiple 32-kb circular plasmids related to those of B. burgdorferi B31. The ubiquity of these plasmids suggests that they may be important in the natural life cycle of these organisms. They may be highly conjugative plasmids or prophage genomes, which could prove to be useful in genetically manipulating B. burgdorferi.Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic ailment of humans that is spread through the bite of certain species of Ixodes ticks (13, 61). As a spirochete, B. burgdorferi is a member of a morphologically and phylogenetically distinct order of eubacteria (11,28,(42)(43)(44)70). Although classically defined as gram-negative organisms (28), recent phylogenetic studies based on rRNA sequences indicated that the spirochetes are as distantly related to the gramnegative Escherichia coli as they are to gram-positive bacteria (42,70).One of the most striking differences between B. burgdorferi and other bacteria is its unusual genome, which includes a small (approximately 1 Mb) linear chromosome (12,16,19,21,23,41) and numerous linear and circular plasmids, sometimes approaching 20 different plasmids in one bacterium (7,8,10,22,27,30,35,57,65,71). A curious feature of these different plasmids is that they often appear to share regions of homologous DNA (58,60,65,71,73,74). Homologs of DNA sequences mapped to circular plasmids have even been found on linear plasmids (74), although in a related bacterium, Borrelia hermsii, at least one plasmid exists in both linear and circular forms (24), blurring the distinction between these two forms of DNA. Several of the plasmids that these bacteria harbor appear to be present in all natural isolates; therefore, the term minichromosome may be a more apt description of their nature. For example, a 49-to 54-kb linear plasmid and a 26-kb circular plasmid, which carry the outer surface protein genes ospAB and ospC, respectively, are ubiquitous (7,34,37,52,54,63,71). It is not known whether members of either of these two plasmid families have similar overall gene orders in different bacterial isolates, but their nearly invariant sizes support the idea that they...
We have identified four loci in Borrelia burgdorferi B31 that contain open reading frames capable of encoding six proteins that are related to the antigenic proteins OspE and OspF. We have designated these proteins Erp, for OspEF-related protein, and named their respective genes erp. The erpA and erpB genes are linked, as are erpC and erpD, and the pairs probably constitute two operons. The erpG and erpH genes appear to be monocistronic. The ErpA and ErpC proteins are expressed by B. burgdorferi B31 in culture and are recognized by a polyclonal antiserum raised against the OspE protein of B. burgdorferi N40. The four erp loci are each located on different 32-kb circular plasmids that contain additional DNA sequences that are homologous to each other and to an 8.3-kb circular plasmid of B. burgdorferi sensu lato Ip21. All four 32-kb plasmids can be maintained within a single bacterium, which may provide a model for the study of plasmid replication and segregation in B. burgdorferi.Borrelia burgdorferi is a member of the order Spirochaetales, the spirochetes, an order that is phylogenetically and morphologically distinct from such well-characterized bacteria as Escherichia coli and Bacillus subtilis (8,24,34,35,53). B. burgdorferi bacteria naturally contain a wide variety of plasmids (4,13,25,40,42,45,47,54), yet nothing is known about the mechanisms by which plasmids are maintained in these bacteria. Several observations suggest that the small DNA species of B. burgdorferi and other spirochetes may be fundamentally different from the plasmids of other bacteria, the most significant being that there are linear plasmids in B. burgdorferi (4,5,7,13,22,23,42). Also, two B. burgdorferi genes involved in purine biosynthesis are located on a circular plasmid (29), which appears to be inconsistent with the classical definition of a plasmid as a nonessential extrachromosomal element. Other plasmids of B. burgdorferi appear to be capable of undergoing recombination to form multimeric plasmids (25, 31), and there have been reports of DNA sequences located on several different plasmids within the same bacterium (46,47,(54)(55)(56).B. burgdorferi is the causative agent of Lyme disease, an increasingly common ailment of humans and several other mammals (15,48). Infection of a mammal by B. burgdorferi is generally accompanied by the production of antibodies directed against a limited number of bacterial antigens (17,19,41,52), several of which have been identified as surface-exposed lipoproteins. At least three of these lipoproteins, OspC, OspE, and OspF, are differentially expressed by B. burgdorferi, being produced in greater quantities by cultured bacteria that are shifted from 23 to 32ЊC than by those maintained at the lower temperature (43, 49). For OspC, at least, this appears to be related to induction of specific protein synthesis by the spirochete in ticks following feeding upon a warm-blooded animal (43). Temperature-dependent differential expression of bacterial proteins that are involved in host infection has been o...
The pathogenic spirochete Leptospira interrogans disseminates throughout its hosts via the bloodstream, then invades and colonizes a variety of host tissues. Infectious leptospires are resistant to killing by their hosts' alternative pathway of complement-mediated killing, and interact with various host extracellular matrix (ECM) components. The LenA outer surface protein (formerly called LfhA and Lsa24) was previously shown to bind the host ECM component laminin and the complement regulators factor H and factor H-related protein-1. We now demonstrate that infectious L. interrogans contain five additional paralogs of lenA, which we designated lenB, lenC, lenD, lenE and lenF. All six genes encode domains predicted to bear structural and functional similarities with mammalian endostatins. Sequence analyses of genes from seven infectious L. interrogans serovars indicated development of sequence diversity through recombination and intragenic duplication. LenB was found to bind human factor H, and all of the newly-described Len proteins bound laminin. In addition, LenB, LenC, LenD, LenE and LenF all exhibited affinities for fibronectin, a distinct host extracellular matrix protein. These characteristics suggest that Len proteins together facilitate invasion and colonization of host tissues, and protect against host immune responses during mammalian infection.
Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is gyrB r , a mutated form of the chromosomal gyrB gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumermycin A 1 . The utility of the coumermycin-resistant gyrB r gene for targeted gene disruption is limited by a high frequency of recombination with the endogenous gyrB gene. A kanamycin resistance gene (kan) was introduced into B. burgdorferi, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen. B. burgdorferi transformants with the kan gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with the kan gene expressed from either the B. burgdorferi flaB or flgB promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in B. burgdorferi and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.Borrelia burgdorferi, the spirochetal agent of Lyme disease (4), is maintained in nature by an infectious cycle involving tick vectors and small rodent hosts (13). The genome sequence of B. burgdorferi has provided a wealth of data about the genetic composition of this bacterium (7). However, relatively little is known about the function of most of the deduced proteins encoded by the genome. For example, 41% of the chromosomal open reading frames and 84% of plasmid open reading frames are either homologs to hypothetical proteins in other bacteria or have no match in databases (7). Although many B. burgdorferi gene products have been identified and characterized on the basis of antigenicity, abundance, membrane location, or pattern of synthesis, the functions of most of these proteins are unknown (5,12,20,31,32,36). As a consequence, relatively little is known about the molecular mechanisms mediating B. burgdorferi variation and adaptation and the roles of these processes in the infectious cycle.Identification of genes that encode virulence factors or participate in the transmission cycle of B. burgdorferi is hindered because this pathogen differs considerably from bacteria with well-developed genetic systems. These differences include an atypical outer membrane composition, unique genome structure with an undefined mechanism of replication, and complex, stringent in vitro growth requirements. One major factor that has impeded genetic studies in B. burgdorferi is the lack of an exogenous selectable marker. The only available selectable marker was derived by mutating the B. burgdorferi gene for the B subunit of DNA gyrase (gyrB), yielding a derivative gene (gyrB r ) whose product confers resistance to the antibiotic coumermycin A 1 (26). When the gyrB r gene is used for gene inactivation by allelic exchange or integration into the genome, the most common outcome (Ͼ99.5%) is recombination with the endogenous chr...
SummaryBorrelia burgdorferi, the aetiological agent of Lyme disease, employs sophisticated means to survive in diverse mammalian hosts. Recent studies demonstrated that acquisition of complement regulators factor H and factor H-like protein-1 (FHL-1) allows spirochetes to resist complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASPs) that bind factor H and/or FHL-1. In this study we have identified and characterized one of those B. burgdorferi proteins, named BbCRASP-2. BbCRASP-2 is distinct from the four previously identified factor H/FHL-1-binding CRASPs of B. burgdorferi strains. The single copy of the gene encoding BbCRASP-2, cspZ, is located on the linear plasmid lp28-3. BbCRASP-2 is highly divergent from the factor H/FHL-1-binding protein BbCRASP-1 and from members of the factor H-binding Erp (OspE/F-related) protein family. Peptide mapping analysis revealed that the factor H/FHL-1 binding site is discontinuous and it was found that C-terminal truncations abrogate factor H and FHL-1 binding. The predominant BbCRASP-2 binding site of both host complement regulators was mapped to the short consensus repeat 7 (SCR 7). Factor H and FHL-1 bound to BbCRASP-2 maintain cofactor activity for factor I-mediated C3b inactivation and accelerate the decay of the C3 convertase. Expression of BbCRASP-2 in serum-sensitive B. burgdorferi mutant B313 increased resistance to complementmediated lysis. The characterization of BbCRASP-2 now provides a complete picture of the three diverse complement regulator-binding protein families of B. burgdorferi yielding new insights into the pathogenesis of Lyme disease.
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