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Long-lived T-dependent B cell responses fail to develop during persistent infection of mice with Borrelia burgdorferi, the causative agent of Lyme disease, raising questions about the induction and/or functionality of anti-B. burgdorferi adaptive immune responses. Yet, a lack of reagents has limited investigations into B. burgdorferi-specific T and B cells. We attempted two approaches to track B. burgdorferi-induced CD4 T cells. First, a B. burgdorferi mutant was generated with an influenza hemagglutinin (HA) peptide, HA111-119, inserted into the B. burgdorferi arthritis-related protein (Arp) locus. Although this B. burgdorferi arp::HA strain remained infectious, peptide-specific TCR transgenic CD4 T cells in vitro, or adoptively transferred into B. burgdorferi arp::HA-infected BALB/c mice, did not clonally expand above those of recipients infected with the parental B. burgdorferi strain or a B. burgdorferi mutant containing an irrelevant peptide. Some expansion, however, occurred in B. burgdorferi arp::HA-infected BALB/c SCID mice. Second, a (to our knowledge) newly identified I-Ab-restricted CD4 T cell epitope, Arp152-166, was used to generate Arp MHC class II tetramers. Flow cytometry showed small numbers of Arp-specific CD4 T cells emerging in mice infected with B. burgdorferi but not with Arp-deficient Borrelia afzelii. Although up to 30% of Arp-specific CD4 T cells were ICOS+PD-1+CXCR5+BCL6+ T follicular helper cells, their numbers declined after day 12, before germinal centers (GCs) are prominent. Although some Arp-specific B cells, identified using fluorochrome-labeled rArp proteins, had the phenotype of GC B cells, their frequencies did not correlate with anti-Arp serum IgG. The data suggest a failure not in the induction, but in the maintenance of GC T follicular helper and/or B cells to B. burgdorferi.

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.

Research has been conducted on the plasmid family of Borrelia burgdorferi packaged into a bacteriophage particle. Identification and characterization of a nonstructural product involved in the bacteriophage function from spirochete bacteriophage have been examined.

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.

Research has been conducted on the chloroform-resistant DNA molecule from the cell-free supernatant of a Borrelia burgdorferi culture. Results indicate that the prophage can be induced with MNNG from Borrelia isolates.

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.

Targeted insertion of the antibiotic resistance marker gyrB(super r) into a 26-kb circular plasmid (cp26) is described. It is the first example of a directed mutation in Borrelia burgdorferi, whereby a large segment of heterologous DNA has been inserted via homologous recombination with flanking sequences. The process confirms the feasibility of specific gene inactivation by allelic exchange.

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.

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.

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.

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]

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]