MOLECULAR MECHANISMS OF VARIATION AND ADAPTATION IN BORRELIA BURGDORFERI

伯氏疏螺旋体是莱姆病的病原体，在自然界中是通过野生哺乳动物和扁虱之间的感染循环维持的。像许多细菌病原体一样，伯氏杆菌必须应对一系列不断变化的环境条件，才能成功地持续、增殖并在宿主之间传播。细菌外表面是与宿主相互作用的主要部位。伯氏杆菌外表面蛋白(OSPs)的排列已被证明随着感染周期的不同而变化。这些不同的OSP很可能赋予了螺旋体与其必须生存的不同环境相关的独特性质。我们的广泛目标是使用分子遗传学方法来阐明伯氏杆菌适应的分子机制及其在感染循环中的作用。我们以前已经证明，温度代表一个重要的环境变量，在培养温度上升后，许多OSP的合成都会增加。蛋白质水平与转录水平平行，与基因表达水平的调控一致。伯氏杆菌的全基因组序列确定了3个转录因子的同源物：sigma 70、sigma 54和rpos。我们感兴趣的是rpos和sigma 54在温度诱导和OSP基因转录调控中的作用。我们灭活了染色体编码的西格玛因子rpos。伯氏杆菌rpos突变体与野生型相比，表现出不同的静止期反应和多种蛋白质组成差异。用二维凝胶分离的细菌总蛋白的质谱学已经被用来鉴定受rpos调控的推测基因。对rpos突变体的进一步鉴定将提供有关伯氏杆菌如何适应不同环境条件的信息。伯氏杆菌的基因组序列还确定了二糖壳二糖的转运体的同源基因，这可能有助于硬蜱生长过程中的营养获取。我们已经开始了对壳二糖转运蛋白基因(chba、chbB和chbC)的遗传分析，以确定它们在伯氏杆菌中的作用和功能。我们灭活了编码假定的跨膜蛋白的chbB基因，并比较了突变体在不同介质中的生长情况与其同基因亲本的生长情况。突变体不能利用壳二糖，而突变体和野生型细菌都不能利用纤维二糖。我们还发现chbB基因受生长温度和培养基组分的调控。对野生型Burgdorferi的研究表明，壳二糖可以在摩尔浓度的一小部分取代以前被认为是必需的N-乙酰氨基葡萄糖。由于壳二糖是硬壳层的一种成分，我们预测chbB基因对细菌在宿主中的生长可能是重要的。

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.