Genes of Erwinia amylovora involved in yellow color formation and release of a low-molecular-weight compound during grow
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O R I GI N A L P A P E R
Y. Zhang á S. Jock á K. Geider
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 Received: 1 April 1999 / Accepted: 10 May 2000 / Published online: 5 July 2000 Ó Springer-Verlag 2000
Abstract 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 ORF132 (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 aect 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. Key words Fire blight á Erwinia amylovora á Yellow colonies á Cu2+ transport á Secretion mechanisms
Introduction Fire blight, which is caused by the gram-negative bacterium Erwinia amylovora, is a destructive disease of pome trees and other rosaceous plants. The pathogen can be speci®cally detected and identi®ed using molecular tools such as DNA hybridization and PCR assays (Falkenstein et al. 1988; Bereswill et al. 1992, 1995), and by screening on a minimal medium containing a copper salt (Bereswill et al. 1998). E. amylovora forms yellow mucoid colonies on MM2 agar containing asparagine and copper sulfate (MM2Cu). In a previous study, out of more than 100 strains of E. amylovora isolated from various geographic regions and dierent host plants, only strain Ea25/82 formed white colonies on MM2Cu; all other strains produced yellow colonies. Yellow pigments are found in epiphytic bacteria such as E. herbicola (now Pantoea agglomerans) and E. stewartii (now P. stewartii subsp. stewartii). Induction of silent genes in E. amylovora by copper ions could also explain the yellow color. Soft-rot Erwinias (now Pectobacterium spp.) secrete an array of
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