The nucleotide sequence of a linear plasmid of Borrelia burgdorferi reveals similarities to those of circular plasmids of other prokaryotes.
Ontology highlight
ABSTRACT:
SUBMITTER: Barbour AG
PROVIDER: S-EPMC178553 | biostudies-other | 1996 Nov
REPOSITORIES: biostudies-other
Publications
The nucleotide sequence of a linear plasmid of Borrelia burgdorferi reveals similarities to those of circular plasmids of other prokaryotes.
Barbour A G AG Carter C J CJ Bundoc V V Hinnebusch J J
Journal of bacteriology 19961101 22
A linear plasmid of Borrelia burgdorferi had 16,927 bp, a G+C content of 23.1%, a relative deficiency of CpG dinucleotides, and open reading frames A to O. The OrfC and OrfE proteins were similar to hypothetical proteins encoded by circular plasmids of B. burgdorferi. The OrfM and OrfN proteins were similar to replication proteins of circular plasmids of other bacteria. ...[more]
Similar Datasets
Project description:The genome of Borrelia burgdorferi, the etiologic agent of Lyme disease, is composed of a linear chromosome and more than 20 linear and circular plasmids. Typically, plasmid content analysis has been carried out by pulsed-field gel electrophoresis and confirmed by Southern hybridization. However, multiple plasmids of virtually identical sizes (e.g., lp28 and cp32) complicate the interpretation of such data. The present study was undertaken to investigate the complete plasmid complements of B. burgdorferi clinical isolates cultivated from patients from a single region where early Lyme disease is endemic. A total of 21 isolates obtained from the skin biopsy or blood samples of Lyme disease patients were examined for their complete plasmid complements by Southern hybridization and plasmid-specific PCR analysis. All clinical isolates harbored at least six of the nine previously characterized cp32s. Fourteen isolates harbored all B31-like linear plasmids, and seven isolates simultaneously lacked lp56, lp38, and some segments of lp28-1. The distinctive plasmid profile observed in these seven isolates was specific to organisms that had ribosomal spacer type 2 and pulsed-field gel type A, which implies a clonal origin for this genotype. The presence of nearly identical complements of multiple linear and circular plasmids in all of the human isolates suggests that these plasmids may be particularly necessary for infection, adaptation, and/or maintenance in the infected host.
| S-EPMC161973 | biostudies-literature
Project description:The Lyme disease agent Borrelia burgdorferi has a genome composed of a linear chromosome and a series of linear and circular plasmids. We previously mapped the oriC of the linear chromosome to the center of the molecule, where a pronounced switch in CG skew occurs. In this study, we analyzed B. burgdorferi plasmid sequences for AT and CG skew in an effort to similarly identify plasmid replication origins. Cumulative skew diagrams of the plasmids suggested that they, like the linear chromosome, replicate bidirectionally from an internal origin. The B. burgdorferi linear chromosome contains homologs to partitioning protein genes soj and spoOJ, which are closely linked to oriC at the minimum cumulative skew point of the 1-Mb molecule. A soj/parA homolog also maps to cumulative skew minima of the B. burgdorferi linear and circular plasmids, further suggesting that these regions contain the replication origin. The heterogeneity in these genes and in the nucleotide sequences of the putative origin regions could account for the mutual compatibility of the multiple DNA elements in B. burgdorferi.
| S-EPMC310945 | biostudies-literature
Project description:We have determined the complete nucleotide sequence of a small circular plasmid from the spirochete Borrelia burgdorferi Ip21, the agent of Lyme disease. The plasmid (cp8.3/Ip21) is 8,303 bp long, has a 76.6% A+T content, and is unstable upon passage of cells in vitro. An analysis of the sequence revealed the presence of two nearly perfect copies of a 184-bp inverted repeat sequence separated by 2,675 bp containing three closely spaced, but nonoverlapping, open reading frames (ORFs). Each inverted repeat ends in sequences that may function as signals for the initiation of transcription and translation of flanking plasmid sequences. A unique oligonucleotide probe based on the repeated sequence showed that the DNA between the repeats is present predominantly in a single orientation. Additional copies of the repeat were not detected elsewhere in the Ip21 genome. An analysis for potential ORFs indicates that the plasmid has nine highly probable protein-coding ORFs and one that is less probable; together, they occupy almost 71% of the nucleotide sequence. Analysis of the deduced amino acid sequences of the ORFs revealed one (ORF-9) with features in common with Borrelia lipoproteins and another (ORF-2) having limited homology with a replication protein, RepC, from a gram-positive plasmid that replicates by a rolling circle (RC) mechanism. Known collectively as RC plasmids, such plasmids require a double-stranded origin at which the Rep protein nicks the DNA to generate a single-stranded replication intermediate. cp8.3/Ip21 has three copies of the heptameric motif characteristically found at a nick site of most RC plasmids. These observations suggest that cp8.3/Ip21 may replicate by an RC mechanism.
| S-EPMC205412 | biostudies-other
Project description:Plasmid cp8.3 of Borrelia afzelii IP21 carries several open reading frames (ORFs) and a 184-bp inverted repeat (IR) element. It has been speculated that this plasmid may encode factors involved in virulence or infectivity. In this report, we have characterized the distribution, molecular variability, and organization of ORFs 1, 2, and 4 and the IR elements among isolates of the Borrelia burgdorferi sensu lato complex. ORFs 1 and 2 are contained within a segment of cp8.3 that is bordered by the IR elements, while ORF 4 resides just outside of the IR-bordered region. By PCR, ORF 4 was amplified from most isolates while ORFs 1 and 2 were amplified from only some B. afzelii isolates. However, Southern hybridization analyses with ORF 1, 2, and 4 probes detected related sequences even in some isolates that were PCR negative. The ORF restriction fragment length polymorphism patterns varied widely even among isolates of the same species. Two-dimensional contour-clamped homogeneous electric field-pulsed-field gel electrophoresis and Southern hybridization detected ORF 1-, 2-, and 4-related sequences on linear and circular plasmids. In addition, an ORF 4-related sequence was detected on a previously uncharacterized, circular plasmid that is greater than 70 kb in size. The IR elements originally identified on plasmid cp8.3 of B. afzelii IP21 were also analyzed by Southern hybridization. Related sequences were detected in some but not all B. burgdorferi sensu lato isolates. These sequences are carried on plasmids in addition to cp8.3 in some isolates. Single-primer PCR analyses demonstrated that in some isolates these sequences exist with IR orientation. The data presented here demonstrate that the IR elements and the ORF 1-, 2-, and 4-related sequences are multicopy and are variable in organization and in genomic location among isolates of the B. burgdorferi sensu lato complex. These analyses provide additional evidence for the highly variable organization of the plasmid component of the B. burgdorferi sensu lato genome.
| S-EPMC108028 | biostudies-literature
Project description:Studies of the biology of Borrelia burgdorferi and the pathogenesis of Lyme disease are severely limited by the current lack of genetic tools. As an initial step toward facile genetic manipulation of this pathogenic spirochete, we have investigated gene inactivation by allelic exchange using a mutated borrelial gyrB gene that confers resistance to the antibiotic coumermycin A1 as a selectable marker. We have transformed B. burgdorferi by electroporation with a linear fragment of DNA in which this selectable marker was flanked by sequences from a native borrelial 26-kb circular plasmid. We have identified coumermycin A1-resistant transformants in which gyrB had interrupted the targeted site on the 26-kb plasmid via homologous recombination with the flanking sequences. Antibiotic resistance conferred by the mutated gyrB gene on the plasmid is dominant, and transformed spirochetes carrying this plasmid do not contain any unaltered copies of the plasmid. Coumermycin A1 resistance can be transferred to naive B. burgdorferi by transformation with borrelial plasmid DNA from the initial transformants. This work represents the first example of a directed mutation in B. burgdorferi whereby a large segment of heterologous DNA (gyrB) has been inserted via homologous recombination with flanking sequences, thus demonstrating the feasibility of specific gene inactivation by allelic exchange.
| S-EPMC178451 | biostudies-other
Project description:Borrelia burgdorferi, the aetiological agent of Lyme disease, follows a life cycle that involves passage between the tick vector and the mammalian host. To investigate the role of the 36 kb linear plasmid, lp36 (also designated the B. burgdorferi K plasmid), in the infectious cycle of B. burgdorferi, we examined a clone lacking this plasmid, but containing all other plasmids known to be required for infectivity. Our results indicated that lp36 was not required for spirochete survival in the tick, but the clone lacking lp36 demonstrated low infectivity in the mammal. Restoration of lp36 to the mutant strain confirmed that the infectivity defect was due to loss of lp36. Moreover, spirochetes lacking lp36 exhibited a nearly 4-log increase in ID(50) relative to the isogenic lp36(+) clone. The infectivity defect of lp36-minus spirochetes was localized, in part, to loss of the bbk17 (adeC) gene, which encodes an adenine deaminase. This work establishes a vital role for lp36 in the infectious cycle of B. burgdorferi and identifies the bbk17 gene as a component of this plasmid that contributes to mammalian infectivity.
| S-EPMC1974800 | biostudies-literature
Project description:The Borrelia genome is composed of a linear chromosome and a number of variable circular and linear plasmids. Atypically large linear plasmids of 92 to 105 kb have been identified in several Borrelia burgdorferi sensu lato isolates and characterized. These plasmids carry the p27 and ospAB genes, which in other isolates reside on a 50-kb plasmid. Here we demonstrate that these plasmids are dimers of the 50-kb ospAB plasmid (pAB50). The 94-kb plasmid from isolate VS116, pVS94, was an exception and did not hybridize with any plasmid gene probes. When this plasmid was used as a probe, homologous sequences in other isolates were not detected, suggesting that it is unique to isolate VS116. These analyses provide insight into the mechanism of linear plasmid replication and the mechanisms by which plasmid variability can arise.
| S-EPMC178094 | biostudies-other
Project description: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 Ip2l. 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.
| S-EPMC178120 | biostudies-other
Project description:The loss of linear plasmid lp28-1, which contains the vls antigenic variation locus, is associated with reduced infectivity of Borrelia burgdorferi in immunocompetent mice. The recombinant shuttle vector pBBE22, which includes the virulence determinant BBE22 from lp25 and restores infectivity to readily transformable B. burgdorferi lacking lp25 and lp56, was used to determine the effect of trans expression of vlsE on virulence. Spirochetes lacking lp28-1 were complemented with the plasmid pBBE22:vlsE, containing both BBE22 and vlsE. VlsE protein produced by this construct was expressed and surface accessible in in vitro-cultured B. burgdorferi, as determined by surface proteolysis and immunoblot analysis. Clones lacking lp25 but containing lp28-1 and either pBBE22 or pBBE22:vlsE were reisolated consistently from immunocompetent mice 8 weeks after infection. In contrast, a clone lacking both lp25 and lp28-1 and complemented with pBBE22:vlsE was isolated from only a single tissue of one of six C3H/HeN mice 8 weeks postinfection. These results indicate that either an intact vls antigenic variation locus or another determinant on lp28-1 is required to restore complete infectivity. In addition, an isogenic clone that retained lp28-1 was complemented with the vlsE shuttle plasmid and was examined for vlsE sequence variation and infectivity. Sequence variation was not observed for the shuttle plasmid, indicating that the cis arrangement of vlsE and the vls silent cassettes in lp28-1 facilitate vlsE gene conversion. Lack of vlsE sequence variation on the shuttle plasmid thus did not result in clearance of the trans-complemented strain in immunocompetent mice under the conditions tested.
| S-EPMC523020 | biostudies-literature
Project description:Borrelia burgdorferi, the bacterium that causes Lyme disease, has a unique segmented genome consisting of numerous linear and circular plasmids and a linear chromosome. Many of these genetic elements have been found to encode factors critical for B. burgdorferi to complete the infectious cycle. However, several plasmids remain poorly characterized, and their roles during infection with B. burgdorferi have not been elucidated. To more fully characterize the role of one of the four 28-kb linear plasmids, lp28-3, we generated strains specifically lacking lp28-3 and assayed the contribution of genes carried by lp28-3 to B. burgdorferi infection. We found that lp28-3 does not carry any genes that are strictly required for infection of a mouse or tick and that lp28-3-deficient spirochetes are competent at causing a disseminated infection. Interestingly, spirochetes containing lp28-3 were at a selective advantage compared to lp28-3-deficient spirochetes when coinjected into a mouse, and this advantage was reflected in the population of spirochetes acquired by feeding ticks. Our data demonstrate that genes carried by lp28-3, although not essential, contribute to the fitness of B. burgdorferi during infection.
| S-EPMC3719586 | biostudies-literature