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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The 32 kb circular plasmid (cp32) family of Borrelia burgdorferi has been the subject of intensive investigation because its members encode numerous differentially expressed lipoproteins. As many as nine different cp32s appear to be capable of stable replication within a single spirochaete. Here, we show that a construct (pCE310) containing a 4 kb fragment from the putative maintenance region of a B. burgdorferi CA-11.2A cp32 was capable of autonomous replication in both high-passage B. burgdorferi B31 and virulent B. burgdorferi 297. Deletion analysis revealed that only the member of paralogous family 57 and the adjacent non-coding segment were essential for replication. The PF32 ParA orthologue encoded by the pCE310 insert was almost identical to the PF32 orthologues encoded on the B31 and 297 cp32-3 plasmids. The finding that cp32-3 was selectively deleted in both B31 and 297 transformants carrying pCE310 demonstrated the importance of the PF32 protein for cp32 compatibility and confirmed the prediction that cp32 plasmids expressing identical PF32 paralogues are incompatible. A shuttle vector containing the CA-11.2A cp32 plasmid maintenance region was used to introduce green, yellow and cyan fluorescent protein reporters into B. burgdorferi. Flow cytometry revealed that the green fluorescent protein was well expressed by almost 90% of both avirulent and infectious transformants. In addition to enhancing our understanding of B. burgdorferi plasmid biology, our results further the development of genetic systems for dissecting pathogenic mechanisms in Lyme disease.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Integration host factor (IHF) is a small, heterodimeric DNA-binding protein with pleiotropic function. IHF was purified to apparent homogeneity from Neisseria gonorrhoeae. Gel-retardation assays demonstrated binding of IHF to the pilE promoter region. The IHF-binding site was identified by DNase I protection assays and mapped proximal to three previously defined pilE promoters. Removal of the putative IHF-binding domain from pilE promoter DNA negated retardation of the DNA fragment when assessed by gel-shift analysis. Kleinschmidt electron microscopy showed pronounced kinking of pilE promoter DNA following incubation with IHF. Isogenic N. gonorrhoeae strains were constructed that contained either a wild-type pilE locus or a deleted pilE locus where the IHF-binding domain was removed. Primer-extension analysis and Northern blotting of total gonococcal RNA showed that in the absence of IHF binding at the pilE promoter, transcription was reduced 10-fold. Together, these data indicate that IHF is a transcriptional co-activator of pilE.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The 26 to 28 kb circular plasmid of B. burgdorferi sensu lato (cp26) is ubiquitous among bacteria of this group and contains loci implicated in the mouse–tick transmission cycle. Restriction mapping and Southern hybridization indicated that the structure of cp26 is conserved among isolates from different origins and culture passage histories. The cp26 ospC gene encodes an outer surface protein whose synthesis within infected ticks increases when the ticks feed, and whose synthesis in culture increases after a temperature upshift. Previous studies of ospC coding sequences showed them to have stretches of sequence apparently derived from the ospC genes of distantly related isolates by homologous recombination after DNA transfer. We found conservation of the promoter regions of the ospC and guaA genes, which are divergently transcribed. We also demonstrated that the increase in OspC protein after a temperature upshift parallels increases in mRNA levels, as expected if regulatory regions adjoin the conserved sequences in the promoter regions. Finally, we used directed insertion to inactivate the ospC gene of a non-infectious isolate. This first example of directed gene inactivation in B. burgdorferi shows that the OspC protein is not required for stable maintenance of cp26 or growth in culture.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: OspA, OspB and OspC are the major outer surface proteins of Borrelia burgdorferi that are differentially synthesized in response to environmental conditions, including culture temperature. We found that DNA was more negatively supercoiled in B. burgdorferi cultures grown at 23°C compared with cultures grown at 35–37°C. We examined the regulation of ospAB and ospC transcription by temperature and DNA supercoiling. DNA supercoiling was relaxed by adding coumermycin A1, an antibiotic that inhibits DNA gyrase. Syntheses of the major outer surface proteins, expression of the ospA and ospC genes and the activities of the ospAB operon and ospC gene promoters were assayed. ospA product levels decreased, whereas ospC product levels increased after shifting from 23°C to 35°C or after adding coumermycin A1. In addition, OspC synthesis was higher in a gyrB mutant than in wild-type B. burgdorferi. Promoter activity was quantified using cat reporter fusions. Increasing temperature or relaxing supercoiled DNA resulted in a decrease in ospAB promoter activity in B. burgdorferi, but not in Escherichia coli, as well as an increase in ospC promoter activity in both bacteria. ospC promoter activity was increased in an E. coli gyrB mutant with an attenuated DNA supercoiling phenotype. These results suggest that B. burgdorferi senses environmental changes in temperature by altering the level of DNA supercoiling, which then affects the expression of the ospAB operon and the ospC gene. This implies that DNA supercoiling acts as a signal transducer for environmental regulation of outer surface protein synthesis.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1573-4978
    Keywords: Borrelia burgdorferi ; DNA-binding protein ; Lyme disease ; outer membrane protein ; transcriptional regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The synthesis of the major outer surface proteins OspA and OspB inBorrelia burgdorferi varies among strains and duringin vitro cultivation. We examinedB. burgdorferi CA-11.2A for the presence of proteins that bind to theospAB operon promoter region. Three major DNA-protein complexes were detected using a mobility shift assay; one of these complexes was due to sequence-specific binding. These proteins may be involved in the regulation ofospAB transcription and the pathogenesis of Lyme disease.
    Type of Medium: Electronic Resource
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