Biofilm Formation by Variovorax paradoxus (pilot)

该项目将研究一种广泛存在的革兰氏阴性需氧细菌的生物被膜形成， Variovorax paroxus。该项目的具体目标将是了解胞外聚合物(EPS)产生的遗传和生理基础，以及这种生物合成途径如何影响表面稳定聚集结构(生物膜)的形成。该项目将通过插入突变识别EPS生物合成所必需的基因和相应的产物，并同时识别不再能够形成生物膜的突变体。进一步的工作将表征不同的基因在EPS生物合成和生物膜形成中的作用，通过互补敲除的基因，以及 利用无启动子绿色荧光蛋白构建分析显影胶片中的基因表达。 Variovorax paroxus广泛存在于环境中，此前已被鉴定为异源污染物的降解者。因此，利用这种微生物的发育良好的遗传和生理系统可以在生物修复环境中用于从受污染的地点去除污染物。生物被膜的生活方式与许多革兰氏阴性细菌感染的过程以及抗生素耐药性密切相关。因此，使用多种生物被膜形成的模型系统，以便发现可能抑制各种生物体生物被膜形成的潜在治疗靶点，是极其重要的。在许多系统中，EPS的生物合成途径与生物膜的形成和发病密切相关。通过鉴定异常弧菌生物膜形成和EPS合成所必需的基因，并与已有的相关植物和人类病原体的基因组序列进行比较，我们可以在细胞中识别这些靶点用于化疗。

This project will investigate biofilm formation by a widespread gram-negative aerobic bacterium, Variovorax paradoxus. The specific aims of this project will be to understand the genetic and physiologic basis for extracellular polymeric substance (EPS) production and how this biosynthetic pathway influences the formation of stable aggregated structures (biofilms) on surfacs. The project will identify the genes and corresponding products essential for EPS biosynthesis by insertional mutagenesis, and will simultaneously identify mutants that are no longer capable of forming biofilms. Further work will characterize the roles of various genes in EPS biosynthesis and biofilm formation by complementation of the knocked out genes, and analysis of gene expression in the developing film using a promoterless GFP construct. Variovorax paradoxus is widespread in the environment, and has previously been identified as a degrader of xenobiotic contaminants. Well developed genetic and physiological systems utilizing this microbe may thus be utilized in bioremediative settings to remove contaminants from polluted sites. The biofilm lifestyle has been strongly implicated in the course of many gram negative bacterial infections, as well as in antibiotic resistance. It is therefore extremely important to use multiple model systems for biofilm formation that allow for the discovery of potential therapeutic targets that might inhibit biofilm formation in a variety of organisms. The EPS biosynthesis pathways are strongly linked to biofilm formation and pathogenesis in a number of systems. By identifying genes essential to biofilm formation and EPS synthesis in V. paradoxus, and comparison to the available sequenced genomes of related plant and human pathogens, we may identify such targets in the cell for chemotherapy.