Molecular characterization of the eps gene cluster of Pseudomonas solanacearum and its transcriptional regulation at a single promoter

Abstract

Production of EPS I, an unusual exopolysaccharide virulence factor of the phytopathogen Pseudomonas solanacearum, requires the 18 kb eps gene cluster. DNA sequence analysis of the first seven genes of eps (epsAPBCDEF), subcellular localization of their products in maxicells, and phoA fusion analysis showed that: (i) epsA, epsB, epsE, and epsF encode exported or membrane-associated proteins probably involved in polymerization and/or export of EPS I; (ii) epsC and epsD encode soluble enzymes probably involved in synthesis of sugar components of EPS I (N-acetylgalactosaminuronic acid and possibly N-acetyltrideoxygalactose, respectively); and (iii) epsP probably encodes a phosphatase involved in EPS I production in an unknown way. Non-polar insertional mutagenesis showed that most, if not all, of these eps genes are absolutely required for production of EPS I. Using random eps::lacZ fusions and primer extension we located a transcription start site and promoter upstream of epsA. Analysis of a plasmid with this promoter fused to lacZ showed that a 140 bp regulatory region upstream of the eps transcription start site was sufficient for normal regulation of eps transcription by the multicomponent virulence gene regulatory network of P. solanacearum. Deletion of this eps promoter from a plasmid-borne epsAPBCDE::lacZ fusion reduced its expression 10-fold, indicating that this promoter alone is responsible for regulated transcription of an eps operon composed of at least epsAPBCDE. Analysis of genomic and plasmid-borne eps::lacZ fusions suggested that most remaining eps genes are part of this same operon or, and this is less likely, comprise a second co-ordinately regulated eps operon.