RUI: The Role of AlgZ in the Twitching Motility of Pseudomonas Aeruginosa

铜绿假单胞菌和其他一些常见的土壤细菌利用一种不依赖于鞭毛的运动模式，称为抽动运动（也称为滑动运动）。铜绿假单胞菌具有先进的遗传学和新完成的基因组序列数据库，是研究这种运动形式的良好模式生物。在抽搐运动中，细菌使用纤维极性附属物（4型菌毛）在表面上移动。据认为，这种运动的基础是这些毛的伸展和收缩。通过刺接琼脂平板，观察细菌在平板-琼脂界面上的扩散生长，可以测定其抽搐运动能力。该项目的合作者辛西娅·惠特彻奇博士对铜绿假单胞菌的抽搐运动进行了视频显微镜观察。这可以在http://www.cmcb.uq.edu.au/cmcb/PUBS/twitch.html上查看。抽搐运动是高度复杂的，涉及不同染色体位点上的数十个基因。虽然已经确定了许多抽搐所需的基因，但对抽搐运动的机制和调节的理解是难以捉摸的。一个基因首先被确定为海藻酸盐生产所需的调节因子，algZ，是抽搐运动所必需的。algZ编码一种带状-螺旋-螺旋DNA结合蛋白，该蛋白首先由主要研究者鉴定和克隆。在环境铜绿假单胞菌分离物中，algZ的缺失导致抽搐运动性的丧失。这个项目的目标是确定algZ在抽搐运动中的作用。这些研究将确定algZ缺失菌株的表型。将对菌株进行分析，以确定缺陷是否出现在菌毛表达、加工、出口或功能上。将使用电子显微镜、全细胞ELISA、噬菌体敏感性和视频显微镜。生物化学、遗传和基因组方法将确定algz依赖性和参与抽搐运动的候选基因。生化方法包括利用纯化的AlgZ和基因组DNA进行修饰的SELEX（配体的指数富集系统进化）技术。一个转座启动子探针在一个algz诱导菌株被用于遗传方法。基因组方法包括确定AlgZ与DNA结合的共识（通过DNA足迹和诱变），然后对铜绿假单胞菌基因组数据库进行查询，以寻找可能的AlgZ靶点。候选基因(s)将通过在algZ诱导菌株中构建候选基因融合(s)来验证对algZ的依赖性。候选基因的表达，然后可以监测响应algZ诱导。最后，将进行研究，其中候选基因的表达或中断（或这些基因的某些组合）将能够恢复algZ缺失菌株的抽搐表型。这将证实所有涉及抽搐运动的algz依赖基因都已被确定。这些研究将阐明抽搐运动的遗传控制。本科生将参与这些研究的各个方面。这不仅将增加目前关于海藻酸盐生产和抽搐运动协调调节的知识，而且还将在生物修复方面更好地利用和控制环境细菌。

Pseudomonas aeruginosa and some other common soil bacteria utilize a flagella-independent mode of movement called twitching motility (also called gliding motility). P. aeruginosa is an excellent model organism in which to study this form of motility due to its advanced genetics and newly completed genome sequence database. In twitching motility, bacteria use fibrous polar appendages (type 4 pili) to move across a surface. It is thought that the basis of this movement is the extension and retraction of these pili. Twitching motility may be assayed by stab-inoculation of an agar plate and then observation of spreading growth of the bacteria at the plate-agar interface. Dr. Cynthia Whitchurch, a collaborator on this project, has performed video microscopy of P. aeruginosa twitching motility. This may be viewed at http://www.cmcb.uq.edu.au/cmcb/PUBS/twitch.html. Twitching motility is highly complex and involves dozens of genes at different chromosomal loci. Although many genes required for twitching have been identified, an understanding of the mechanism and regulation of twitching motility is elusive. A gene first identified as a regulator required for alginate production, algZ, is required for twitching motility. algZ encodes a ribbon-helix-helix DNA binding protein that was first identified and cloned by the principle investigator. The deletion of algZ in an environmental P. aeruginosa isolate results in a loss of twitching motility. The goal of this project is to define the role of algZ in twitching motility. These studies will define the phenotype of the algZ deletion strain. The strain will be analyzed to determine whether the defect appears to be in pili expression, processing, export, or function. Electron microscopy, whole cell ELISA, phage sensitivity, and video microscopy will be used. Biochemical, genetic, and genomic approaches will identify genes that are candidates for algZ-dependence and involvement in twitching motility. The biochemical approach involves a modified SELEX (systematic evolution of ligands by exponential enrichment) technique utilizing purified AlgZ and genomic DNA. A transposable promoter probe in an algZ-inducible strain is used in the genetic approach. The genomic approach involves the determination of the consensus for AlgZ binding to DNA (via DNA footprinting and mutagenesis) and then interrogation of the P. aeruginosa genome database for possible AlgZ targets. The candidate gene(s) will then be tested to confirm algZ-dependence by construction of a candidate gene fusion(s) in an algZ inducible strain. The expression of the candidate gene(s) may then be monitored in response to algZ induction. Lastly, studies will be undertaken in which the expression or interruption of a candidate gene(s) (or some combination of these) will be able to restore the algZ deletion strain to a twitching phenotype. This will confirm that all algZ-dependent genes involved in twitching motility have been identified. These studies will elucidate the genetic control of twitching motility. Undergraduate students will be involved in all aspects of these studies. This will not only increase current knowledge regarding the coordinate regulation of alginate production and twitching motility, but also the ways to better use and control environmental bacteria with regard to bioremediation.