Pathogenicity of Mutants ofErwinia carotovorasubsp.carotovoraDeficient in Aerobactin and Catecholate Siderophore Production

Abstract

Erwinia carotovora subsp. carotovora strain W3C105 produces the hydroxamate siderophore aerobactin and a catecholate metabolite, which previously was not known to function in iron acquisition by the phytopathogen. A region containing catechol biosynthesis genes (cbsEA + ) of strain W3C105 complements EntA- and EntE- mutants of Escherichia coli, which are deficient in biosynthesis of the catecholate siderophore enterobactin. Four plasmids containing the cbsEA region mutagenized with Tn3-Spice complemented either the EntA- or the EntE- mutant of E. coli but not both mutants. Two of the plasmids, in which insertions of Tn3-Spice generated transcriptional fusions of the promoterless ice nucleation reporter gene (inaZ) to a cbs promoter(s), conferred iron-regulated ice nucleation activity on E. coli. One of the Tn3-Spice insertions was introduced into the genome of W3C105 by marker-exchange mutagenesis to generate a mutant deficient in catechol biosynthesis (Cbs-). Introduction of the insertion into a previously-derived mutant of W3C105 deficient in aerobactin biosynthesis (Iuc-) resulted in a double mutant that produced neither the catechol nor aerobactin (Cbs- Iuc-). A catechol-producing mutant (Cbs + Iuc - ) grew on a minimal medium containing higher concentrations of the iron-chelator 2,2'-dipyridyl than did a mutant producing neither siderophore (Cbs - Iuc - ), demonstrating that the catechol functions as a siderophore in strain W3C105. Wild-type W3C105 and an aerobactin-producing mutant (Cbs - Iuc + ), however, grew on a minimal medium containing higher concentrations of 2,2'-dipyridyl than did a mutant producing only the catechol (Cbs + Iuc - ), indicating that aerobactin was superior to the catecholate siderophore in competing with 2,2'-dipyridyl for iron. Mutants deficient in the production of either or both siderophores did not differ from the wild-type strain in the capacity to macerate potato tuber tissue or cause aerial stem rot of potato.