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Cloning and molecular characterization of a multicopy, linear plasmid-carried, repeat motif-containing gene from Borrelia turicatae, a causative agent of relapsing fever.

ABSTRACT: Borrelia turicatae is one of several spirochete species that can cause relapsing fever. Here, we describe the identification and characterization of a gene from B. turicatae and other relapsing-fever spirochetes that exhibits homology with the rep+ and ORF-E gene families of the Lyme disease spirochetes. This gene, which we have designated repA, encodes a putative protein of 30.2 kDa with an isoelectric point of 4.69. The central region of RepA harbors a series of amino acid repeat motifs which exhibit homology with casein kinase 2 phosphorylation sites. Through Southern hybridization analyses, we demonstrate that repA (or a closely related sequence) is multicopy in the relapsing-fever spirochetes and is carried on variably sized linear plasmids in both Borrelia parkeri and B. turicatae. Transcriptional analyses demonstrate that repA is expressed, albeit at low levels, during in vitro cultivation of B. turicatae. Transcriptional start site analysis revealed that repA is preceded by a consensus ribosomal binding site and an appropriately spaced promoter element. The sequence conservation, unique features, and multicopy status of repA and its homologs suggest that RepA may play an important genus-wide role in the biology of the Borrelia.

SUBMITTER: Carlyon JA

PROVIDER: S-EPMC107528 | biostudies-literature | 1998 Sep

REPOSITORIES: biostudies-literature

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Cloning and molecular characterization of a multicopy, linear plasmid-carried, repeat motif-containing gene from Borrelia turicatae, a causative agent of relapsing fever.

Carlyon J A JA Marconi R T RT

Journal of bacteriology 19980901 18

Borrelia turicatae is one of several spirochete species that can cause relapsing fever. Here, we describe the identification and characterization of a gene from B. turicatae and other relapsing-fever spirochetes that exhibits homology with the rep+ and ORF-E gene families of the Lyme disease spirochetes. This gene, which we have designated repA, encodes a putative protein of 30.2 kDa with an isoelectric point of 4.69. The central region of RepA harbors a series of amino acid repeat motifs which ...[more]

PMID: 9733706

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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.

Project description:Vector competence refers to the ability of an arthropod to acquire, maintain, and successfully transmit a microbial pathogen. Tick-borne relapsing fever (TBRF) spirochetes are globally distributed pathogens, and most species are transmitted by argasid ticks of the genus Ornithodoros. A defining characteristic in vector competence is an apparent specificity of a species of TBRF spirochete to a given tick species. In arid regions of the southern United States, Borrelia turicatae is the primary cause of TBRF. Interestingly, there are two populations of the tick vector distributed throughout this region. Ornithodoros turicata is a western population that ranges from California to Texas. There is a gap through Louisiana, Mississippi, and Alabama where the tick has not been identified. An isolated eastern population exists in Florida and was designated a subspecies, O. turicata americanus. A knowledge gap that exists is the poor understanding of vector competence between western and eastern populations of ticks for B. turicatae. In this study, we generated uninfected colonies of O. turicata that originated in Texas and Kansas and of O. turicataamericanus. B. turicatae acquisition, maintenance through the molt, and subsequent transmission were evaluated. Our findings revealed significant differences in murine infection after feeding infected O. turicata and O. turicataamericanus ticks on the animals. Interestingly, the salivary glands of both tick populations were colonized with B. turicatae to similar densities. Our results suggest that the salivary glands of the tick colonies assessed in this study impact vector competence of the evaluated B. turicatae isolates.IMPORTANCE Several knowledge gaps exist in the vector competence of various geographical populations of O. turicata that transmit B. turicatae A western population of this tick is distributed from California to Texas, and an eastern population exists in Florida. Utilizing western and eastern populations of the vector, we studied acquisition and transmission of two B. turicatae isolates. Regardless of the isolate used, infection frequencies were poor in mice after the eastern population feeding on them. Since salivary gland colonization is essential for B. turicatae transmission, these tissues were further evaluated. Interestingly, the salivary glands from the two populations were similarly colonized with B. turicatae. These findings suggest the role of tick saliva in the establishment of infection and that the salivary glands may be a bottleneck for successful transmission.

Dataset Information

Cloning and molecular characterization of a multicopy, linear plasmid-carried, repeat motif-containing gene from Borrelia turicatae, a causative agent of relapsing fever.

Publications

Cloning and molecular characterization of a multicopy, linear plasmid-carried, repeat motif-containing gene from Borrelia turicatae, a causative agent of relapsing fever.

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