Genetic transfer of amylovoran and stewartan synthesis between Erwinia amylovora and Erwinia stewartii

Abstract

DNA fragments with ams genes of Erwinia amylovora and cps genes of Erwinia stewartii were transferred to exopolysaccharide (EPS)-deficient mutants of the other species. The resulting EPSs were characterized by sensitivity to EPS-dependent bacteriophages, staining with amylovoran-specific fluorescein-isothiocyanate-labelled lectin and chemical techniques, such as determination of the sugar composition and methylation analysis in order to distinguish between amylovoran and stewartan. Degradation by the stewartan-dependent phage ø-K9 was used to detect stewartan production, and staining with a lectin from Abrus precatorius detected amylovoran capsules. The patterns of sugar linkages were determined by methylation analysis. Stewartan contained a significantly higher glucose to galactose ratio than amylovoran and produced a characteristic signal from 6-linked glucose residues. By these criteria, most E. stewartii cps mutants displayed exclusively amylovoran synthesis when complemented with the ams cluster, and E. amylovora ams mutants complemented with E. stewartii cps genes produced stewartan. The complementation to an EPS-positive phenotype may require most genes of the ams or the cps operon. An exception was an E. stewartii cpsK mutant that made predominantly stewartan when complemented with the ams cosmid. IR spectra showed that both amylovoran and stewartan were acylated when synthesized in E. amylovora, but not in E. stewartii. The amylovoran-producing E. stewartii merodiploids regained virulence to corn seedlings when mucoidy was restored by the ams cluster, but the stewartan-producing E. amylovora ams-lcps + strains were weakly virulent on pear slices and avirulent on apple seedlings.

The genetic and structural basis of two distinct terminal side branch residues in stewartan and amylovoran exopolysaccharides and their potential role in host adaptation

“…However, we showed that virulence of either pathogen is not influenced by the nature of the EPS produced when the host plants are infected by manual inoculation (Table 1). This is consistent with the finding by Bernhard et al . (1996) showing that the P. stewartii strain producing a hybrid stewartan was virulent when assayed by manual inoculation.…”

“…Bernhard et al . (1996) showed that expression of the ams‐I gene cluster in P. stewartii led to the production of partially pyruvylated stewartan.…”

Interaction of the regulator proteins RcsA and RcsB with the promoter of the operon for amylovoran biosynthesis in Erwinia amylovora

“…Ew. amylovora mainly produces the acidic heteropolymer amylovoran, composed of repeating pentamer units, each with four galactose residues and one glucuronic acid residue Mol Gen Genet (1997) 256: 72±83 (Smith et al 1990;Bernhard et al 1996). The complete ams gene cluster responsible for the biosynthesis of amylovoran has been cloned and sequenced (Bernhard et al 1993;Bugert and Geider 1995).…”

“…Transfer of the ams cluster into Ew. stewartii mutants de®cient in capsule synthesis results in the production of amylovoran, as shown by sugar analysis and the use of EPS-speci®c phage depolymerases (Bernhard et al 1996). The ams gene cluster of Ew.…”

Genetics of biosynthesis and structure of the capsular exopolysaccharide from the Asian pear pathogen Erwinia pyrifoliae The GenBank accession number for the sequence reported in this paper is AJ300463.

“…In addition to some differences in the acetylation pattern and substitution by succinyl groups, this glucose residue is a major difference for EPS of E. pyrifoliae and amylovoran of E. amylovora. Amylovoran produced in E. stewartii cps mutants carrying pEA109 is not acetylated, in contrast to stewartan synthesized in E. amylovora ams mutants with pES2144, a cosmid with the cps genes of Pantoea (Erwinia) stewartii (Bernhard et al, 1996). Genes for acetylation should reside apart from the ams region inserted in plasmid pEA109.…”