Genes of Erwinia amylovora involved in yellow color formation and release of a low-molecular-weight compound during growth in the presence of copper ions

Most Erwinia amylovora strains form yellow mucoid colonies on solid minimal medium containing asparagine and copper sulfate (MM2Cu). One exception is the strain Ea25/82, which produces white colonies on MM2Cu agar. This strain was transformed with a genomic library of E. amylovora and yellow colonies were recovered. A 1.5-kb fragment was found to complement strain Ea25/82 for color formation, and subsequent sequencing revealed two ORFs. The smaller ORF 132 (ycfB) overlapped with the end of the larger ORF253 (ycfA). The putative protein YcfA shows low homology with K+/Na+ channel transporter ATPases. Resistance genes were inserted in both ORFs, and the E. amylovora strains Ea1/79-YA and Ea1/79-YB were created by site-directed mutagenesis. The mutation in ycfB did not affect color formation, whereas the ycfA mutant formed white colonies on MM2Cu. Sequence analysis of the ycf region in strain Ea25/82 revealed a 1-bp alteration in ycfA and no change in ycfB. Stable complementation of Ea25/82 and Ea1/79-YA, however, required both genes. Carotenoids were not detected in E. amylovora grown in the presence of copper ions. On the other hand, copper-independent secretion of a low-molecular-weight compound with an absorption maximum at 340 nm (CP340) was found for strain Ea1/79, but not for Ea25/82 or the mutant Ea1/79YA. CP340 formed a complex with copper ions, and complementation with plasmids carrying both ycfA and ycfB restored its release from mutant strains. The compound may be connected with the yellow pigment or function in sensing bacterial population densities.