Microarray Studies of Vibro cholerae EPS Regulation

描述(申请人提供)：O1霍乱弧菌在自然界的主要宿主是河流、河口和海洋栖息地。霍乱弧菌O1的El Tor生物型在光滑和皱纹两种形态之间表现出不同的相变。其中，四聚糖的变体被认为特别适合在天然水源中长期生存：它产生一种胞外多糖(EPS)，使其能够形成三维生物膜，并对氯和过氧化氢具有抵抗力。EPS的表达受VpsR的正调控，VpsR是双组分信号转导调节因子类的反应调节因子，HapR是哈维氏弧菌LuxR调节因子的同系物，HapR是负调节因子。这项建议的主要目的是利用最近公布的霍乱弧菌O1 El Tor基因组序列，利用微阵列表达谱和生物信息学工具捕捉和分析光滑和皱纹菌落形态的转录本，并定义VpsR和HapR调节子。这将极大地促进母基金(AI43422，“EPS：结构、调节和功能”)的目标，特别是该基金侧重于EPS表达调控的具体目标#2。利用P.I.实验室制作的霍乱弧菌全基因组DNA微阵列，将获得光滑和皱纹菌落类型以及hapR和vpsR突变体的表达谱。聚类算法将被用来识别在每种菌落类型中差异调控的基因，以及其调控依赖于HapR或VpsR的基因。一种新的生物信息学工具BioProspector将被用于识别HapR或VpsR共同序列，这些序列位于被认为构成HapR和VpsR调节子的基因的上游启动子区域。将进行凝胶延迟分析以证实BioProspector得出的预测。这一项目的可能意义在于，它可能会提供更深入的了解，以了解“粗糙”的性质，从而了解O1El Tor霍乱弧菌在环境水库中持续存在的能力。这一建议与R21机制的探索/发展性质是一致的，因为它探索了微阵列表达谱和生物信息学的组合使用来定义调节子--这可能是这一方法的一种非常强大但未被完全检验的应用。

DESCRIPTION (provided by applicant): The principal reservoirs of Vibrio cholerae O1 in nature are rivers, estuaries and marine habitats. The El Tor biotype of V. cholerae O1 exhibits phase variation between the smooth and rugose morphotypes. Of these, the rugose variant is thought be particularly well adapted for long-term survival in natural water sources: it produces an extracellular polysaccharide (EPS) that enables it to form three-dimensional biofilms and it confers resistance to chlorine and hydrogen peroxide. EPS expression is positively regulated by VpsR, a response regulator of the two-component signal transduction regulator class and negatively regulated by HapR, a homologue of the LuxR regulator of V. harveyi. The main purpose of this proposal is capitalize on the recent publication of the V. cholerae O1 El Tor genome sequence by using microarray expression profiling and bioinformatic tools to capture and analyze the transcriptomes of the smooth and rugose colonial morphotypes and to define the VpsR and HapR regulons. This will greatly facilitate the goals of the parent grant (AI43422, "EPS: structure, regulation and function") and in particular Specific Aim #2 of that grant which focuses on the regulation of EPS expression. Using a whole-genome DNA V. cholerae microarray fabricated in the laboratory of the P.I., expression profiles will be obtained from the smooth and rugose colony types and from hapR and vpsR mutants. Clustering algorithms will be used to identify genes that are differentially regulated in each colony type and genes whose regulation is HapR or VpsR-dependent. A novel bioinformatic tool, BioProspector, will be employed to identify HapR or VpsR consensus sequences in the upstream promoter regions of the genes believed to compose the HapR and VpsR regulons. Gel retardation assays will be performed to corroborate the BioProspector-derived predictions. The possible significance of this project comes from the likelihood that it will provide a deeper understanding of the nature of "rugosity" and thus of the capacity of V. cholerae O1 El Tor to persist in environmental reservoirs. This proposal is consistent with the exploratory/development nature of the R21 mechanism because it explores the combined use of microarray expression profiling and bioinformatics to define regulons--potentially a very powerful, but incompletely examined application of this methodology.