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 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.