Microarray Studies of Vibro cholerae EPS Regulation

描述（申请人提供）：霍乱弧菌O 1在自然界的主要储存库是河流、河口和海洋生境。霍乱弧菌O 1的El Tor生物型表现出光滑和皱纹形态型之间的相位变化。其中，皱纹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，并赋予对氯和过氧化氢的抗性。EPS表达受VpsR（双组分信号转导调节剂类的反应调节剂）正调节，并受HapR（哈维氏弧菌LuxR调节剂的同源物）负调节。该提案的主要目的是利用最近发表的霍乱弧菌O 1 El Tor基因组序列，通过使用微阵列表达谱和生物信息学工具来捕获和分析光滑和皱纹菌落形态型的转录组，并定义VpsR和HapR调节子。这将极大地促进母基金的目标（AI 43422，“EPS：结构、调节和功能”），特别是该基金的具体目标#2，其集中于EPS表达的调节。使用P.I.实验室制作的全基因组DNA霍乱弧菌微阵列，从光滑和皱纹菌落类型以及hapR和vpsR突变体获得表达谱。将使用聚类算法来鉴定在每种集落类型中差异调节的基因和其调节依赖于HapR或VpsR的基因。一种新的生物信息学工具，BioProspector，将被用来确定HapR或VpsR的共识序列的上游启动子区的基因被认为是组成的HapR和VpsR调节子。将进行凝胶阻滞试验，以证实BioProspector得出的预测。该项目的可能意义来自于它将提供对“rugosity”性质的更深入理解的可能性，从而了解V. cholesterol O 1 El Tor在环境水库中持续存在的能力。这个提议与R21机制的探索/开发性质是一致的，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-可能是这种方法的一个非常强大但不完全检查的应用。

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.