Xanthan gum (xanthan) is an exopolysaccharide that is produced by the gram-negative pseudomonad, Xanthomonas campestris. Xanthan is composed of repeating penta-saccharide subunits that are polymerized through B-1-4 linkages between adjacent glucose residues to create a polymer with a glucose backbone, having the same structure as cellulose; alternate glucose molecules contain trisaccharide side-chains composed of one molecule of glucuronate plus two molecules of mannose substituted with an acetyl or pyruvate group. This project will study xanthan production in X.campestris as a model system to obtain an understanding of the genetics and biochemistry that determine the ability of bacteria to synthesize and export exopolysaccharides. The study of xanthan production is important for two reasons: the ability of bacteria to colonize hosts is often associated with polysaccharide production; thus investigations into the production of polysaccharides should contribute significant new knowledge about bacterial-host relationships. Also, these studies of xanthan biosynthesis will contribute to our understanding of macromolecules cross the molecularly complex cellular envelope. A cluster of 12 gum genes codes for proteins that function in the assembly and polymerization of the xanthan repeat units. The expression of this gum gene cluster will be delineated, and the presence of additional, as yet, undiscovered gum genes that function in the cellular export of xanthan will be investigated. A collection lacZ and phoA fusions to each of the clustered gum genes will be isolated using a combination of in vivo and in vitro procedures. These fusions will be used to: (i) characterized the cellular location of the gum proteins, (ii) determine the level of in vivo expression of each gum gene, and (iii) define the transcriptional director gum transcripton unit. The region of DNA that contains the gum cluster promoter will be fused to lacZ; the effect of deletion and spontaneous "leaky" mutations on the expression of lacZ will be used to clearly define and characterized the gum promoter. Gene products that are needed to export xanthan from the cell are virtually certain to be envelope proteins. Since alkaline phosphatase (product of phoA) activity is restricted to the periplasm, putative export mutants will be isolated as TnphoA insertions that are Gum-PhoA+. These mutants will be characterized to begin to define the genes involved in the export process.
