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

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 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 Ea1/79-YA. 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.

Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State

“…However, cells maintained in the presence of copper had an altered morphology with a partial increase in the thickness of the external layer. It has been reported that copper ions increase the level of the exopolysaccharide (EPS) amylovoran (4) and that these ions are accumulated on the surface of E. amylovora cells (41). Moreover, it is known that bacterial EPSs have a cation-binding capacity (18).…”

“…Their use is one of the most common methods for controlling bacterial plant diseases, but it has led many bacteria to develop different strategies against copper ions (31). Until now, very little information on the interaction between E. amylovora and Cu 2ϩ ions has been available (4,15,41). Copper treatments have traditionally been considered as bactericides in agriculture (29,34,35), their effectiveness often being measured by the absence of bacterial growth on a solid medium (12,13).…”

Identification and genetics of 6-thioguanine secreted by Erwinia species and its interference with the growth of other bacteria

“…This strain and an artificial insertion mutant of tgsA, Ea1/79-YA, form white colonies on MM2Cu and show no 6-TG in culture supernatants. A complementation of both strains was possible by plasmid expression of tgsA (Zhang et al 2000). In plant assays on detached apple flowers, pear slice assays and shoot infection on apple and pear seadlings (MM106 and Kirchensaller Mostbirne) both 6-TG mutants still induced symptom formation.…”

“…This phenotype can be used as selection criterion for isolation from infected plant material. The yellow color formation has been linked to a substance with an absorption maximum of 340 nm (CP340) at neutral pH that can be detected in culture supernatants (Zhang et al 2000). An open reading frame (ORF) tentatively annotated as ycfA (yellow color formation) could be linked to biosynthesis of this compound.…”

Supervivencia de Erwinia amylovora en condiciones de estrés: influencia de la presencia de cobre y la limitación de nutrientes

“…Their use is one of the most common methods for controlling bacterial plant diseases, but it has led many bacteria to develop different strategies against copper ions [Saxena et al, 2002]. Until now, very little information on the interaction between E. amylovora and Cu 2+ ions is available [Bereswill et al, 1998;Geider, 1999;Zhang et al, 2000].…”