The major objective of this proposal is to study the organization and regulation of chromosomal genes specifying biosynthesis of alginic acid in Pseudomonas aeruginosa. Mucoid alginate-producing strains of P. aeruginosa are known for their ability to cause infection and proliferate in the lung enviornment of Cystic Fibrosis (CF) patients. It has been demonstrated that an enzymatic activity similar to the Escherichia coli phosphomannose isomerase (but catalyzing only fructose 6-phosphate to mannose 6-phosphate conversion and not the reverse reaction) is essential for alginate synthesis. Because the evidence for unidirectional activity has basically been genetic, attempts would be made to purify the enzymatic activity and study its biochemical properties to find out if indeed the enzyme catalyzes a unidirectional phosphomannose isomerase reaction or might turn out to be a new enzyme. Various properties of this enzyme, such as subunit structure, substrate specificity, nature of inhibitors, etc., will be examined and compared with E. coli phosphomannose isomerase. The gene for this enzyme has been mapped at around 45 min. of the P. aeruginosa chromosome. Within 5 kilobase pairs of this gene is a cluster of at least five other genes which are essential for alginate synthesis. Attempts would be made to subclone and finally sequence all these genes and identify and overproduce their gene products. The nature of their upstream sequences would be examined to look for the presence of Pseudomonas promoter elements. Using individual genes as probes and hybridizing with cellular RNA from mucoid and non-mucoid cells, dot and Northern hybridization experiments will be conducted to see if all or some of these genes remain unexpressed in spontaneous non-mucoid mutants of mucoid CF isolates or in normally non-mucoid strains such as P. aeruginosa PAO. Since preliminary experiments demonstrate a lack of transcript formation for this gene cluster in spontaneous non-mucoid cells, a combination of lack of transcript formation for individual genes and lack of the presence of promoter elements in the upstream region of each individual gene should clearly show whether the genes are regulated singly or as one or more operons.
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