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)。铜绿假单胞菌由于其先进的遗传学和最新完成的基因组序列数据库，是研究这种运动性的一个很好的模式生物。在抽搐运动中，细菌使用纤维状的极地附属物(4型菌毛)在表面上移动。人们认为，这场运动的基础是这些菌毛的伸展和回缩。用刺法接种琼脂平板，然后观察细菌在平板-琼脂界面的扩散生长，即可测定其抽动能力。Cynthia Whitchurch博士是该项目的合作者，她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.上查看抽动运动是高度复杂的，涉及不同染色体上的数十个基因。虽然已经确定了许多与抽动有关的基因，但对抽动运动的机制和调节的了解还很难理解。一种最先被确定为藻酸盐生产所需的调节基因，algZ，是抽动运动所必需的。AlgZ编码一种带状-螺旋-螺旋DNA结合蛋白，该蛋白是由首席研究员首次发现和克隆的。环境中的铜绿假单胞菌中的algZ基因缺失会导致丧失抽动能力。这个项目的目标是确定algZ在抽动运动中的作用。这些研究将确定algZ缺失菌株的表型。将对该菌株进行分析，以确定缺陷是否出现在菌毛表达、加工、输出或功能上。将使用电子显微镜、全细胞酶联免疫吸附试验、噬菌体敏感性和视频显微镜。生化、遗传学和基因组学方法将确定哪些基因可能依赖algZ并参与抽动运动。生化方法包括一种改进的SELEX(通过指数富集法进行配体的系统进化)技术，利用纯化的ALZ和基因组DNA。在遗传方法中使用了藻Z可诱导菌株中的转座启动子探针。基因组学方法包括确定ALZ与DNA结合的共识(通过DNA足迹和突变)，然后询问铜绿假单胞菌基因组数据库中可能的ALZ靶标。候选基因(S)随后将通过构建候选基因融合(S)在algZ诱导菌株中进行测试，以确认对algZ的依赖性。然后，可以监测候选基因(S)的表达情况，以响应Gal Z的诱导。最后，将进行研究，候选基因(S)的表达或中断(或这些基因的某种组合)将能够将algZ缺失菌株恢复到抽动表型。这将证实参与抽动运动的所有依赖于algZ的基因都已经被鉴定出来。这些研究将阐明抽动运动的基因控制。本科生将参与这些学习的方方面面。这不仅将增加目前关于藻酸盐生产和抽动运动的协调调节的知识，而且还将增加在生物修复方面更好地利用和控制环境细菌的方法。

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.