MOLECULAR MECHANISMS OF VARIATION AND ADAPTATION IN BORRELIA BURGDORFERI

伯氏疏螺旋体是莱姆病的病原体，通过野生哺乳动物和蜱虫之间的传染循环在自然界中得以维持。像许多细菌病原体一样，伯氏疏螺旋体必须应对一系列不断变化的环境条件，才能成功地持续存在、增殖并在宿主之间传播。细菌的外表面是与宿主相互作用的主要部位。伯氏疏螺旋体外表面蛋白（Osps）的排列已被证明随感染周期而变化。很可能是这些不同的Osps赋予了螺旋体与它必须生存的不同环境相关的独特特性。我们的主要目标是利用遗传方法来阐明伯氏疏螺旋体适应的分子机制及其在感染周期中的作用。我们之前已经证明温度是一个重要的环境变量，并且在培养温度升高后，许多Osps的合成增加。蛋白水平与转录水平平行，与基因表达水平的调控一致。完整的伯氏疏螺旋体基因组序列鉴定出三个转录因子sigma 70、sigma 54和RpoS的同源物。我们对RpoS和sigma 54在温度诱导和osp基因转录调控中的作用感兴趣。我们克隆了博氏螺旋体sigma 54和rpoS基因，并证明了福氏志贺氏菌rpoS突变体与伯氏疏螺旋体rpoS基因的部分互补。我们灭活了伯氏疏螺旋体rpoS基因，并证明该突变体在固定期表现出降低的渗透阻力。通过二维凝胶对细菌总蛋白进行比较，发现野生型和rpoS突变螺旋体之间存在一些差异。正在进行的研究旨在鉴定这些蛋白质。伯氏疏螺旋体的基因组序列还确定了双糖壳聚糖（细胞）转运体的同源物。几丁质（蜱表皮成分之一）降解为壳聚糖，可为蜱体内的伯氏疏螺旋体提供重要的碳水化合物来源。我们已经开始对细胞基因进行遗传分析，以确定它们在伯氏疏螺旋体中的作用和功能。我们已经灭活了其中一个基因，细胞b，并表明这个突变体在复杂的生长培养基中与野生型伯氏疏螺旋体一样生长。初步结果表明，在缺乏n -乙酰氨基葡萄糖（NAG）的培养基中，与野生型伯氏疏螺旋体相比，突变体细胞b的生长受到损害，这表明通过细胞转运体摄取壳聚糖可能满足了NAG的需求。-伯氏疏螺旋体，螺旋体，莱姆病，分子遗传学，蜱虫传播，适应性反应

Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature through an infectious cycle between wild mammals and ticks. Like many bacterial pathogens, B. burgdorferi must cope with an array of changing environmental conditions to successfully persist, proliferate and be transmitted between hosts. The bacterial outer surface represents the primary site for interactions with the host. The array of B. burgdorferi outer surface proteins (Osps) has been shown to vary with the infectious cycle. It is likely that these different Osps endow the spirochete with distinct properties relevant to the disparate environments in which it must survive. Our broad objective is to use a genetic approach to elucidate the molecular mechanisms of adaptation in B. burgdorferi and their roles in the infectious cycle. We have previously demonstrated that temperature represents an important environmental variable, and that synthesis of a number of Osps is increased after a rise in culture temperature. Protein levels parallel transcript levels, consistent with regulation at the level of gene expression. The complete genomic sequence of B. burgdorferi identified homologs of three transcription factors, sigma 70, sigma 54, and RpoS. We are interested in the roles of RpoS and sigma 54 in the temperature induction and transcriptional regulation of osp genes. We have cloned the borrelial sigma 54 and rpoS genes and demonstrated partial complementation of a Shigella flexneri rpoS mutant with the B. burgdorferi rpoS gene. We have inactivated the B. burgdorferi rpoS gene and demonstrated that this mutant exhibits decreased osmotic resistance during stationary phase. A comparison of total bacterial proteins by two-dimensional gels revealed several differences between wild type and rpoS mutant spirochetes. Ongoing studies are aimed at the identification of these proteins. The genomic sequence of B. burgdorferi also identified a homolog of a transporter for the disaccharide chitobiose (cel). The degradation of chitin (a component of tick cuticle) to chitobiose could provide an important source of carbohydrate for B. burgdorferi in the tick. We have begun a genetic analysis of the cel genes to determine their role and function in B. burgdorferi. We have inactivated one of these genes, celB, and shown that this mutant grows as well as wild type B. burgdorferi in complex growth medium. Preliminary results indicate that growth of the celB mutant is impaired relative to wild type B. burgdorferi in medium lacking N-acetyl glucosamine (NAG), suggesting that uptake of chitobiose via the Cel transporter may fulfill the requirement for NAG. - Borrelia burgdorferi, spirochete, Lyme disease, molecular genetics, tick transmission, adaptive response