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。我们的主要目标是使用分子遗传学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。我们灭活了染色体编码的sigma因子RpoS。与野生型相比，伯氏疏螺旋体RpoS突变体表现出固定相反应的改变和蛋白质组成的多重差异。用二维凝胶分离的总细菌蛋白的质谱已被用于鉴定由RpoS调节的假定基因。RpoS突变体的进一步表征将提供关于伯氏疏螺旋体如何适应可变环境条件的信息。伯氏疏螺旋体的基因组序列还发现了一种双糖壳聚糖转运体的同源物，这种转运体可能有助于蜱在生长过程中获取营养。我们已经开始对壳聚糖转运体基因（chbA， chbB和chbC）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。我们灭活了chbB基因，编码了假定的膜跨越蛋白，并将突变体在各种培养基中的生长与其等基因亲本的生长进行了比较。突变体不能利用壳聚糖，而突变体和野生型细菌都不能利用纤维素糖。我们还发现chbB基因受生长温度和培养基组成的调控。对野生型伯氏疏螺旋体的研究表明，壳聚糖可以以一小部分摩尔浓度取代n -乙酰氨基葡萄糖，而n -乙酰氨基葡萄糖以前被认为是必需的。由于壳聚糖是蜱虫表皮的一种成分，我们预测chbB基因对宿主细菌的生长应该是重要的。

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 molecular 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 the osp genes. We inactivated the chromosomally encoded sigma factor RpoS. The B. burgdorferi RpoS mutant exhibited an altered stationary phase response and multiple differences in protein composition relative to wild type. Mass spectroscopy of total bacerial proteins separated by 2-dimensional gels has been used to identify putative genes regulated by RpoS. Further characterization of the RpoS mutant will provide information about how B. burgdorferi adapts to variable environmental conditions. The genomic sequence of B. burgdorferi also identified a homolog of a transporter for the disaccharide chitobiose, which could be useful for nutrient acquisition during growth in the tick. We have begun a genetic analysis of the chitobiose transporter genes (chbA, chbB and chbC) to determine their role and function in B. burgdorferi. We inactivated the chbB gene, encoding the putative membrane-spanning protein, and have compared growth of the mutant in various media to that of its isogenic parent. The mutant is unable to utiize chitobiose, whereas neither the mutant nor wild type bacteria can utilize cellobiose. We have also found that the chbB gene is regulated by growth temperature and medium composition. Studies with wild type B. burgdorferi demonstrated that chitobiose can replace N-acetyl glucosamine, which was previously considered essential, at a fraction of the molar concentration. Since chitobiose is a component of tick cuticle, we predict that the chbB genes should be important for bacterial growth in the host.