The major objective of the proposal is to study the biochemical and genetic mode of biosynthesis of alginic acid, an exopolysaccharide produced by mucoid cells of P. aeruginosa isolated from the lung tissues of cystic fibrosis patients. The production of alginic acid is believed to contribute to the severity of such infections, and our study may lead to the formulation of metabolic inhibitors or sugar analogues that may prevent Pseudomonas infection through inhibition of alginate formation. In particular, we would like to find out what role the keto acids such as Alpha-ketoglutaric or pyruvic acid and sugars such as glucose or gluconate play in the induction of the pathway enzymes or as direct precursors of alginate. The involvement of phosphorylated sugars such as mannose-1-PO4, GDP-mannose, glucose-6-PO4, 6-phosphogluconic acid, etc. by cell free extracts in the formation of alginic acid will be examined. Mutants blocked in the oxidative glucose and gluconate pathways (glucose and gluconate dehydrogenases and the Entner-Doudoroff pathway) as well as those blocked in phosphorylated glucose pathway (but allowing gluconate degradation) will be examined for the formation of alginate from glucose, gluconate and succinate. Mutants blocked specifically in the alginate pathway will be isolated by transposon mutagenesis of a stable alginate-producing FRD mutant and accumulation of biosynthetic intermediates and their subsequent conversion to alginate by cell-free extracts, will be followed. Once the pathway enzymes and intermediates are full characterized, various sugar analogues and metabolic inhibitors will be tried to see if specific inhibitors for alginate formation can be identified. The genes for the various alginate biosynthetic enzymes will then be mapped, and if the structural genes are found clustered, they will be cloned in E. coli, P. putida, etc. to see expression of such genes in heterologous hosts. The genes will also be cloned in other laboratory strains of alginate-negative P. aeruginosa, and the ability of Alg+ and Alg- cells for specific adhesions to CF lung tissues or in a suitable animal model system if or when such a system is developed, will be examined.
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