The complete genome sequence of Treponema pallidum was determined and shown to be 1,138,006 base pairs containing 1041 predicted coding sequences (open reading frames). Systems for DNA replication, transcription, translation, and repair are intact, but catabolic and biosynthetic activities are minimized. The number of identifiable transporters is small, and no phosphoenolpyruvate:phosphotransferase carbohydrate transporters were found. Potential virulence factors include a family of 12 potential membrane proteins and several putative hemolysins. Comparison of the T. pallidum genome sequence with that of another pathogenic spirochete, Borrelia burgdorferi, the agent of Lyme disease, identified unique and common genes and substantiates the considerable diversity observed among pathogenic spirochetes.

We have identified and characterized an elaborate genetic system in the Lyme disease spirochete Borrelia burgdorferi that promotes extensive antigenic variation of a surface-exposed lipoprotein, VlsE. A 28 kb linear plasmid of B. burgdorferi B31 (lp28-1) was found to contain a vmp-like sequence (vls) locus that closely resembles the variable major protein (vmp) system for antigenic variation of relapsing fever organisms. Portions of several of the 15 nonexpressed (silent) vls cassette sequences located upstream of vlsE recombined into the central vlsE cassette region during infection of C3H/HeN mice, resulting in antigenic variation of the expressed lipoprotein. This combinatorial variation could potentially produce millions of antigenic variants in the mammalian host.

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 .

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We present the complete 2,843,201-bp genome sequence of Treponema denticola (ATCC 35405) an oral spirochete associated with periodontal disease. Analysis of the T. denticola genome reveals factors mediating coaggregation, cell signaling, stress protection, and other competitive and cooperative measures, consistent with its pathogenic nature and lifestyle within the mixed-species environment of subgingival dental plaque. Comparisons with previously sequenced spirochete genomes revealed specific factors contributing to differences and similarities in spirochete physiology as well as pathogenic potential. The T. denticola genome is considerably larger in size than the genome of the related syphiliscausing spirochete Treponema pallidum. The differences in gene content appear to be attributable to a combination of three phenomena: genome reduction, lineage-specific expansions, and horizontal gene transfer. Genes lost due to reductive evolution appear to be largely involved in metabolism and transport, whereas some of the genes that have arisen due to lineage-specific expansions are implicated in various pathogenic interactions, and genes acquired via horizontal gene transfer are largely phagerelated or of unknown function.

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The bacterial flagellar motor is a remarkable nanomachine that provides motility through flagellar rotation. Prior structural studies have revealed the stunning complexity of the purified rotor and C-ring assemblies from flagellar motors. In this study, we used high-throughput cryo-electron tomography and image analysis of intact Borrelia burgdorferi to produce a three-dimensional ( Flagellum-based motility plays a critical role in the biology and pathogenesis of many bacteria (3,6,17,31). The wellconserved flagellum is commonly divided into three physical parts: the flagellar motor, the helically shaped flagellar filament, and the hook which provides a universal joint between the motor and the filament. In most bacteria, counterclockwise rotation of the flagella results in bundling of the helical flagella and propulsion of the cell through liquid or viscous environments. Clockwise rotation of the flagellar motor results in random turning of the cell with little translational motion ("tumbling"). Bacterial motility is thus a zigzag pattern of runs and tumbles, in which chemotactic signals favor running toward attractants and away from repellents (3).Borrelia burgdorferi and other closely related spirochetes are the causative agents of Lyme disease, which is transmitted to humans via infected Ixodes ticks (40). Spirochetes have a distinctive morphology in that the flagella are enclosed within the outer membrane sheath and are thus called periplasmic flagella (6). The flagellar motors are located at both ends of the cell and are coordinated to rotate in opposite directions during translational motion and in the same direction (i.e., both clockwise or both counterclockwise) during the spirochete equivalent of tumbling, called "flexing" (6,15). Spirochetes are also capable of reversing translational motion by coordinated reversal of the direction of motor rotation at both ends of the cell. Rotation of the flagella causes a serpentine movement of the entire cell body, allowing B. burgdorferi to efficiently bore its way through tissue and disseminate throughout the mammalian host, resulting in manifestations in the joints, nervous system, and heart (40).The flagellar motor is an extraordinary nanomachine powered by the electrochemical potential of specific ions across the cytoplasmic membrane (3). Current knowledge of the flagellar motor structure and rotational mechanisms is based primarily on studies of Escherichia coli and Salmonella enterica and is summarized in several recent comprehensive reviews (3,22,31,39,42). The flagellar motor is constructed from at least 20 different kinds of proteins. The approximate location of these flagellar proteins has been determined by a variety of approaches and appears to be relatively consistent in a wide variety of bacteria. It can be divided into several morphological domains: the MS ring (FliF, the base for the flagellar motor); the C ring (FliG, FliM, and FliN, the switch complex regulating motor rotation); the export apparatus (multiple-protein complex located at the cytop...