d -Alanine-d -alanine ligase gene ( ddl ) of Erwinia chrysanthemi strain EC16 II: analysis of pectate lyase regulation using ddl ? mutant

 Erwinia chrysanthemi (Ech) triggers soft rot disease mainly by secreting pectate lyase (Pel), which is regulated in a complex manner by many regulatory genes. In a previous study, we used a gene dosage method to show that the ddl gene, which encodes d-alanine-D-alanine ligase, reduced Pel production and tissue maceration by Ech strain EC16n. In this study, the ddl ⁻ marker-exchanged mutant was shown to overcome the long growth lag caused by various salts in the growth medium and to increase Pel production over that by EC16n, especially in a medium containing magnesium salts. Thus, ddl seems to regulate Pel production in a negative manner. Because the profiles of a gel shift assay using the pelE promoter region as the target DNA with crude extracts of EC16n and ddl ⁻ mutant were distinguishable, Ddl is thought to affect the binding of other regulatory proteins. Expression of the ddl gene was induced in the medium containing a low-molecular-weight fraction of potato extract, but it was reduced in that containing both polygalacturonic acid (PGA) and the fraction. The repression of ddl expression by PGA should contribute in part to the in planta hyperinduction of Pel. Received: May 15, 2002 / Accepted: June 20, 2002     

D-Alanine-D-Alanine Ligase Gene (ddl) of Erwinia chrysanthemi Strain EC16 I. Isolation and Gene Dosage Effect on Pectate Lyase Synthesis

New regulatory gene for pectate lyase (Pel) production of Erwinia chrysanthemi strain EC16 was searched by observing the gene dosage effect of each cosmid library in Pel production. From this survey, a cosmid clone, p5A, had reduced in Pel production under both inducing and non-inducing conditions and caused less tissue maceration of potato tubers. The 2.64-kb HindIII fragment from p5A was found to be responsible for this phenotype and to contain one major ORF consisting of 1107 nucleotides, which had homology with ddlA (49.6%) and ddlB (49.6%) of Escherichia coli and with ddlA (44.6%) of Salmonella typhimurium. These genes had been shown to encode D-alanine-D-alanine ligase (Ddl), an enzyme which is involved in the synthesis of peptidoglycan. This ORF encodes 368 amino acid residues and its molecular weight is estimated to be 43 kDa. This ORF of EC16 could complement ddl deficient mutant of Escherichia coli. Received 15 May 2002/ Accepted in revised form 20 June 2002