Molecular genetic analysis of extracellular enzyme secretion by Erwinia carotovora

Abstract

Erwinia carotovora subsp. carotovora (Ecc) secretes a variety of extracellular enzymes, namely pectinases (Pel), cellulases (Cel) and proteases (Prt). Some of these extracellular enzymes are considered to be the major pathogenicity determinants of this bacterium. Using the chemical mutagen ethyl methyl sulphonate (EMS), a range of Ecc mutants defective in extracellular enzyme production have been generated. One class was found to be pleiotropically defective in the production of Pel and Cel but unaffected for Prt production. Pel and Cel were still synthesised in this class of mutant but both enzymes accumulated within the periplasm. Mutants of the Pel-, Cel-, Prt+ class have been termed Out- mutants. A single Out- mutant, RJP190, was partially resistant to infection by two Ecc bacteriophages. Using a cosmid library of wild-type Ecc, 12 of the 14 Out- mutants were complemented to Out+. Further analysis of the complementing cosmids led to the identification of at least six out loci. A 3.7 kb region of DNA containing out genes was sequenced. This fragment of DNA overlapped with other out genes sequenced In this laboratory. The contiguous DNA (5.7 kb) encoded four proteins, OutD, OutE, OutF and OutG, which were visualised using a T7 directed expression system. The predicted Out proteins were found to share homology with other eubacterial proteins involved in macromolecular trafficking. Accumulated findings strongly suggest that this Out-type system is the major pathway used by Gram-negative bacteria for secreting proteins to the extracellular milieu