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Outer surface lipoprotein C (OspC) is a key virulence factor of Borrelia burgdorferi, ospC is differentially regulated during borrelial transmission from ticks to rodents, and such regulation is essential for maintaining the spirochete in its natural enzootic cycle. Recently, we showed that the expression of ospC in B. burgdorferi is governed by a novel alternative sigma factor regulatory network, the RpoN-RpoS pathway. However, the precise mechanism by which the RpoN-RpoS pathway controls ospC expression has been unclear. In particular, there has been uncertainty regarding whether ospC is controlled directly by RpoS ([[sigma].sup.s]) or indirectly through a transactivator (induced by RpoS). Using deletion analyses and genetic complementation in an OspC-deficient mutant of B. burgdorferi, we analyzed the cis element(s) required for the expression of ospC in its native borrelial background. Two highly conserved upstream inverted repeat elements, previously implicated in ospC regulation, were not required for ospC expression in B. burgdorferi. Using similar approaches, a minimal promoter that contained a canonical -35/-10 sequence necessary and sufficient for [[sigma].sup.s]-dependent regulation of ospC was identified. Further, targeted mutagenesis of a C at position -15 within the extended -10 region of ospC, which is postulated to function like the strategic C residue important for E[[sigma].sup.s] binding in Escherichia coli, abolished ospC expression. The minimal ospC promoter also was responsive to coumermycin [A.sub.1], further supporting its [[sigma].sup.s] character. The combined data constitute a body of evidence that the RpoN-RpoS regulatory network controls ospC expression by direct binding of [[sigma].sup.s] to a [[sigma].sup.s]-dependent promoter of ospC. The implication of our findings to understanding how B. burgdorferi differentially regulates ospC and other ospC-like genes via the RpoN-RpoS regulatory pathway is discussed.

To enhance genetic manipulation of the Lyme disease spirochete Borrelia burgdorferi, we assayed the aadA gene for the ability to confer resistance to the antibiotics spectinomycin and streptomycin. Using the previously described pBSV2 as a backbone, a shuttle vector, termed pKFSS1, which carries the aadA open reading frame fused to the B. burgdorferi flgB promoter was constructed. The hybrid flgB promoter-aadA cassette confers resistance to spectinomycin and streptomycin in both B. burgdorferi and Escherichia coli. pKFSS1 has a replication origin derived from the 9-kb circular plasmid and can be comaintained in B. burgdorferi with extant shuttle vector pCE320, which has a replication origin derived from a 32-kb circular plasmid, or pBSV2, despite the fact that pKFSS1 and pBSV2 have the same replication origin. Our results demonstrate the availability of a new selectable marker and shuttle vector for genetically dissecting B. burgdorferi at the molecular level.

Research has been conducted on Borrelia burgdorferi, Lyme disease causative agent. The up-regulation of GroEL protein and groEL mRNA transcript carried out in bacterium's gyrB mutants is described.

Research demonstrates that the Lyme disease agent Borrelia burgdorferi phage PhiBB-1 can package and transduce DNA into other cells. Results indicate that the phage can be used to transduce a kanamycin resistance marker into B. burgdorferi cells by lateral gene transfer.

Results demonstrate that the A subunit of the DNA gyrase, a small DNA-binding protein is not required for growth of Borrelia and its absence cause no alterations in the DNA related functions.

Short oligonucleotides were utilized to genetically transform Borrelia burgdorferi, the etiologic agent of Lyme disease by means of polymerase chain reaction and electrocorporation. Spontaneous mutants of Borrelia burgdorferi that are resistant to the antibiotic coumermycin A1 have been isolated and found to have single-point mutations in their gyrB genes that converted Arg-133 to either Gly or Ile. Single-stranded oligonucleotides were used to introduce site-directed mutations conferring coumarin resistance into the gyrB gene of Borrelia burgdorferi.

A single gene in Borrelia burgdorferi, called hbb and whose product Hbb complements the defects of lambda DNA packaging found in Escherichia coli strains mutant, was found in the genes for integration host factor (IHF) and HU proteins. The sequence of the predicted protein is similar to those of HU and IHF-like proteins in other bacteria. The gene seems to be in an operon with the order rpsT-hbb-orfH, where the rpsT gene is a homologue of the E. coli gene encoding ribosomal protein S20 and the orfH gene encodes a protein of unknown function. This operon is found upstream of the B. burgdorferi rho homologue.

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.

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.