Alginate is an exopolysaccharide synthesized by mucoid Pseudomonas aeruginosa. Its production is transcriptionally regulated involving several chromosomally encoded loci. The alginate system is one of the best characterized in Pseudomonas and offers solid basis for studying gene regulation in these important bacteria. The purpose of this research is to study how environmental and genetic factors influence the emergence of mucoid P. aeruginosa strains exploiting recent findings of the molecular events governing alginate production. Research will include: 1. Analysis of environmental and metabolic factors influencing transcription of the algD gene coding for GDPmannose dehydrogenase, a critical enzyme for mucoid phenotype. This will be accomplished using the well characterized algD-xylE transcription fusion system. 2. Analysis of critical domains of the algD promoter by site directed mutagenesis and monitoring expression using mutant algD-xy1E transcription fusions and S1 nuclease protection analysis. 3. Subcloning, DNA sequencing and expression analysis of the algR and algQ genes, the positive regulatory elements of algD. This will be done using new methods and vectors for rapid gene subcloning and expression, dideoxy chain termination method and RNA-DNA hybridization studies. 4. Studies of the nature of the algR gene product and algD promoter interaction by in vitro and in vivo footprinting and transcription analyses. 5. Surveying potential role of DNA rearrangements in the alg regulatory region for the emergence of mucoid P. aeruginosa strains. 6. Comparative studies of the structure and function of promoters controlling alginate genes. These experiments should provide greater understanding of the molecular events controlling alginate production by mucoid P. aeruginosa. More general, the proposed studies will broaden our knowledge of gene regulation and coordination in Pseudomonas and increase our understanding of the signal transduction in transcriptional activation processes in bacteria.
