Infectivity-associated plasmids were identified in Borrelia burgdorferi B31 by using PCR to detect each of the plasmids in a panel of 19 clonal isolates. The clones exhibited high-, low-, and intermediate-infectivity phenotypes based on their frequency of isolation from needle-inoculated C3H͞HeN mice. Presence or absence of 21 of the 22 plasmids was determined in each of the clones by using PCR primers specific for regions unique to each plasmid, as identified in the recently available genome sequence. Southern blot hybridization results were used to confirm the PCR results in some cases. Plasmid lp25 exhibited a direct correlation with infectivity in that it was consistently present in all clones of high or intermediate infectivity and was absent in all low-infectivity clones. lp28 -1, containing the vmp-like sequence locus, also correlated with infectivity; all clones that lacked lp28 -1 but contained lp25 had an intermediate infectivity phenotype, in which infection was primarily restricted to the joints. Plasmids cp9, cp32-3, lp21, lp28 -2, lp28 -4, and lp56 apparently are not required for infection in this model, because clones lacking these plasmids exhibited a high-infectivity phenotype. Plasmids cp26, cp32-1, cp32-2 and͞or cp32-7, cp32-4, cp32-6, cp32-8, cp32-9, lp17, lp28 -3, lp36, lp38, and lp54 were consistently present in all clones examined. On the basis of these results, lp25 and lp28 -1 appear to encode virulence factors important in the pathogenesis of B. burgdorferi B31.

SummaryBorrelia burgdorferi , a spirochaete that causes Lyme borreliosis, contains 21 linear and circular plasmids thought to be important for survival in mammals or ticks. Our results demonstrate that the gene BBE22 encoding a nicotinamidase is capable of replacing the requirement for the 25 kb linear plasmid lp25 during mammalian infection. Transformation of B. burgdorferi lacking lp25 with a shuttle vector containing the lp25 gene BBE22 (pBBE22) restored infectivity in mice to a level comparable to that of wild-type Borrelia . This complementation also restored the growth and host adaptation of lp25 -B. burgdorferi in dialysis membrane chambers (DMCs) implanted in rats. A single Cys to Ala conversion at the putative active site of BBE22 abrogated the ability of pBBE22 to re-establish infectivity or growth in DMCs. Additional Salmonella typhimurium complementation studies and enzymatic analysis demonstrated that the BBE22 gene product has nicotinamidase activity and is most probably required for the biosynthesis of NAD. These results indicate that some plasmid-encoded products fulfil physiological functions required in the enzootic cycle of pathogenic Borrelia .

The presence of the linear plasmids lp25 and lp56 of Borrelia burgdorferi B31 was found to dramatically decrease the rate of transformation by electroporation with the shuttle vector pBSV2, an autonomously replicating plasmid that confers kanamycin resistance (P. E. Stewart, R. Thalken, J. L. Bono, and P. Rosa, Mol. Microbiol. 39:714-721, 2001). B. burgdorferi B31 clones had transformation efficiencies that were either low, intermediate, or high, and this phenotype correlated with the presence or absence of lp25 and lp56. Under the conditions utilized in this study, no transformants were detected in clones that contained both lp25 and lp56; the few kanamycin-resistant colonies isolated did not contain pBSV2, indicating that the resistance was due to mutation. Intermediate electroporation rates (10 to 200 colonies per g of DNA) were obtained with B31 clones that were either lp25 ؊ and lp56 ؉ or lp25 ؉ and lp56 ؊ . Clones in this group that initially contained lp25 lacked this plasmid in pBSV2 transformants, a finding consistent with selective transformation of lp25 ؊ variants. High transformation rates (>1,000 colonies per g of DNA) occurred in clones that lacked both lp25 and lp56. Sequence analysis indicated that lp25 and lp56 contain genes that may encode restriction and/or modification systems that could result in the low transformation rates obtained with strains containing these plasmids. The previously reported correlation between lp25 and infectivity in mice, coupled with the barrier lp25 presents to transformation, may explain the difficulty in obtaining virulent transformants of B. burgdorferi.Lyme disease is a multistage, systemic disease caused by members of the spirochete genus Borrelia and is transmitted to humans by Ixodes ticks. Borrelia burgdorferi is the principal causative agent of Lyme disease in the United States, whereas B. burgdorferi, B. afzelii, and B. garinii have each been shown to cause human disease in regions of Europe and Asia. The genome of B. burgdorferi B31 has been sequenced (7) and is composed of a linear chromosome, nine circular plasmids (cp) and 12 linear plasmids (lp). These plasmids range in size from 9 kb (cp9) to 56 kb (lp56). In vitro passage of organisms leads to spontaneous loss of plasmids. The loss of lp25 and lp28-1 correlates with reduced infectivity of B. burgdorferi B31 in needle-inoculated mice (11,17,27), indicating that factors important in the virulence of Lyme disease borrelia are encoded by these plasmids.Efforts to identify virulence-associated genes in B. burgdorferi have been hampered by the lack of efficient methods for genetic manipulation of this bacterium. In recent years, great strides have been made in the development of genetic tools. Initial success in transformation and genetic exchange in B. burgdorferi was achieved by the electroporation of DNA segments containing gyrB with point mutations that conferred resistance to coumermycin (20). Unfortunately, the high rate of recombination into the native gyrB site on the chromosome and the occurrence...

The genome of Borrelia burgdorferi, the etiologic agent of Lyme disease, is composed of a linear chromosome and more than 20 linear and circular plasmids. Typically, plasmid content analysis has been carried out by pulsed-field gel electrophoresis and confirmed by Southern hybridization. However, multiple plasmids of virtually identical sizes (e.g., lp28 and cp32) complicate the interpretation of such data. The present study was undertaken to investigate the complete plasmid complements of B. burgdorferi clinical isolates cultivated from patients from a single region where early Lyme disease is endemic. A total of 21 isolates obtained from the skin biopsy or blood samples of Lyme disease patients were examined for their complete plasmid complements by Southern hybridization and plasmid-specific PCR analysis. All clinical isolates harbored at least six of the nine previously characterized cp32s. Fourteen isolates harbored all B31-like linear plasmids, and seven isolates simultaneously lacked lp56, lp38, and some segments of lp28-1. The distinctive plasmid profile observed in these seven isolates was specific to organisms that had ribosomal spacer type 2 and pulsed-field gel type A, which implies a clonal origin for this genotype. The presence of nearly identical complements of multiple linear and circular plasmids in all of the human isolates suggests that these plasmids may be particularly necessary for infection, adaptation, and/or maintenance in the infected host.