Characterization of a Gene Cluster for Polysaccharide Biosynthesis in Erwinia Stewartii

Abstract

In Erwinia stewartii, three groups of avirulent mutants have been defined by complementation studies using clones from a genomic library constructed in cosmid pVK100. Clone pES2144 (27 kb insert) restored virulence and extracellular polysaccharide synthesis (EPS) to mutants in complementation group III which consists of 14 EPS mutants, two of which are sensitive to galactose (GAL8 and GAL17). A restriction map of pES2144 was constructed. Hybridization of pES2144 probe DNA to Southern blots of genomic DNA from the EPS- mutants revealed deletions of this region in five of the mutants (including GAL8 and GAL17). A subclone, pPD0527, containing a 4 kb HindIII- BamH1 fragment complemented five EPS- mutants and another subclone, pPD011, containing a 6.2 kb HindIII fragment, complemented the galactose-sensitive phenotype of GAL8 and GAL17 but did not restore capsule synthesis. Tn5 lac and Tn3 HoHoI mutagenesis indicated that five separate regions of pES2144 contain EPS genes. Properties of EPS-lac gene fusions will be discussed. Erwinia stewartii causes a leaf blight and vascular wilt of sweet corn and maize. Extracellular polysaccharide (EPS) is produced in the form of both slime and capsule by the bacterium and is probably responsible for wilting. The results of this and the previous study (Coplin et al., these proceedings) suggest that a cell surface polysaccharide, possibly EPS, may also be involved in producing watersoaked lesions. A library of E. stewartii DNA has been constructed in pVK100 and clone pES2144 was found to complement 14 EPS- mutants (Coplin et al., these proceedings). The objective of this study was to identify genes on pES2144 involved in EPS synthesis and/or virulence.