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The pathogenic Borrelia burgdorferi sensu lato species contain atypically large linear plasmids of 92 to 105 kilobases (kb). Molecular genetic techniques reveal that these plasmids carry the p27 and ospAB genes which in other related species are found in the 50-kb plasmids. Southern blot analysis with plasmid gene probes show that the large linear plasmids are dimers of these 50-kb plasmids.

The use of resistance to coumermycin A1, a DNA gyrase inhibitor, as a genetic marker helps observe the transformation of Borrelia burgdorferi by electroporation. Site-directed mutagenesis of gyrB gene indicates the success of the transformation, which takes place on the chromosome, and proves that B. burgdorferi derives its resistance to coumermycin A1 from the mutations at Arg-133 of DNA gyrase B. gyrB marker and genetic transformation help elucidate the pathogenesis and physiology of the Lyme disease at molecular levels.

Molecular and sequence analysis of the ospD gene reveals that the ospD gene is not universal but is detectable only in a few Lyme disease spirochete (LDS) isolates. The upstream control region of the ospD gene contains different numbers of a 17-bp repeated sequence while a specific region inside the coding sequence exhibits numerous direct repeats, deletions and insertions. The gene tree for ospD gene does not correlate to that of other LDS gene trees. The ospD-encoding plasmids have different sizes.

Borrelia burgdorferi mutants resist antibiotic coumermycin A1 that targets the DNA gyrase B subunit. The resistivity of mutant strains is 100- or 300-fold higher than that of wild type strains. The mutations in gyrB genes activate the conversion of Arg-133 to Ile or Gly. Arg residue mutations of DNA gyrase are involved in the antibiotic-resistivity of mutants.

Borrelia burgdorferi, Borrelia garinii and VS461 group cause Lyme disease, which was originally identified as a type of arthritis. An analysis of Lyme disease spirochactes included a comparison of the size and structure of the chromosomes and the sizes of the linear plasmid with the encoding genes of the major outer-surface proteins OspA and OspB. Size differences in ospA-containing plasmids were observed.

The location of the ospC gene in Lyme disease spirochetes was mapped and its transcriptional start sites were determined. It was demonstrated that the ospC gene is carried on a 26-kb circular plasmid. Northern blot analysis also demonstrated that the transcriptional expression ofthe ospC gene varies among isolates. Moreover, transcriptional start site analyses revealed that the gene may be under the control of multiple promoters.

6S RNA binds to RNA polymerase and regulates gene expression, contributing to bacterial adaptation to environmental stresses. In this study, we examined the role of 6S RNA in murine infectivity and tick persistence of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi. B. burgdorferi 6S RNA (Bb6S RNA) binds to RNA polymerase, is expressed independent of growth phase or nutrient stress in culture, and is processed by RNase Y. We found that rny (bb0504), the gene encoding RNase Y, is essential for B. burgdorferi growth, while ssrS, the gene encoding 6S RNA, is not essential, indicating a broader role for RNase Y activity in the spirochete. Bb6S RNA regulates expression of the ospC and dbpA genes encoding outer surface protein C and decorin binding protein A, respectively, which are lipoproteins important for host infection. The highest levels of Bb6S RNA are found when the spirochete resides in unfed nymphs. ssrS mutants lacking Bb6S RNA were compromised for infectivity by needle inoculation, but injected mice seroconverted, indicating an ability to activate the adaptive immune response. ssrS mutants were successfully acquired by larval ticks and persisted through fed nymphs. Bb6S RNA is one of the first regulatory RNAs identified in B. burgdorferi that controls the expression of lipoproteins involved in host infectivity. [ABSTRACT FROM AUTHOR]

An rpsL, encoding the S12 protein of the small ribosomal subunit, is used as a counterselectable marker in Borrelia burgdorferi, the causative agent of Lyme disease, where the mutations in rpsL have conferred streptomycin resistance. Streptomycin susceptibility is dominant in an rpsL merodiploid and streptomycin is selected for the loss of wild-type rpsL carried in trans.

The Lyme disease spirochete Borrelia (Borreliella) burgdorferi must tolerate nutrient stress to persist in the tick phase of its enzootic life cycle. We previously found that the stringent response mediated by RelBbu globally regulates gene expression to facilitate persistence in the tick vector. Here, we show that RelBbu regulates the expression of a swath of small RNAs (sRNA), affecting 36% of previously identified sRNAs in B. burgdorferi. This is the first sRNA regulatory mechanism identified in any spirochete. Threefold more sRNAs were RelBbu-upregulated than downregulated during nutrient stress and included antisense, intergenic and 5′ untranslated region sRNAs. RelBbu-regulated sRNAs associated with genes known to be important for host infection (bosR and dhhp) as well as persistence in the tick (glpF and hk1) were identified, suggesting potential mechanisms for post-transcriptional regulation of gene expression. [ABSTRACT FROM AUTHOR]