Articles
BIOSYNTHESIS AND CHARACTERIZATION OF CAPSULAR EXOPOLYSACCHARIDES FROM ERWINIA AMYLOVORA AND ERWINIA STEWARTH
Article number
489_53
Pages
307 – 314
Language
Abstract
The fire blight pathogen Erwinia amylovora synthesizes an acidic extracellular polysaccharide (EPS), named amylovoran, and the related corn pathogen Erwinia stewartii produces a structurally similar EPS named stewartan.
Biosynthesis of amylovoran was examined in an in vitro system using EDTA-treated cells and following incorporation of radio-labeled UDP-galactose into lipid-linked oligosaccharides.
The oligosaccharide ‘repeating units’ were analyzed by HPLC-gel permeation chromatography.
A model for function of each ams gene in sequential addition of the sugar residues to the repeating unit is presented.
The molecular weight of amylovoran and stewartan was determined by SEC-DRI/LALLS. The chain length depended on temperature, carbon source, and the genetic background of the strains. E. stewartii cpsH and cpsJ mutants produced increased amounts of a higher molecular weight EPS. An amsD mutant E. amylovora was complemented with a DNA fragment carrying the genes cpsF and cpsG of E. stewartii and synthesized an EPS composed of a stewartan-like backbone and an amylovoran-like side chain.
The function of amsM, which has high homology to galU/F genes of E. coli, was characterized by genetic and biochemical assays.
Chromosomal amsM mutants could not grow on galactose and were EPS negative.
Unexpectedly, the UDP-glucose pyrophosphorylase activity of these mutants was higher than in the parental strain; this activity was reduced after overexpression of amsM. Since AmsM presumably has no enzymatic activity by its own, it may modulate the intracellular concentration of UDP-sugars by interaction with other proteins.
Biosynthesis of amylovoran was examined in an in vitro system using EDTA-treated cells and following incorporation of radio-labeled UDP-galactose into lipid-linked oligosaccharides.
The oligosaccharide ‘repeating units’ were analyzed by HPLC-gel permeation chromatography.
A model for function of each ams gene in sequential addition of the sugar residues to the repeating unit is presented.
The molecular weight of amylovoran and stewartan was determined by SEC-DRI/LALLS. The chain length depended on temperature, carbon source, and the genetic background of the strains. E. stewartii cpsH and cpsJ mutants produced increased amounts of a higher molecular weight EPS. An amsD mutant E. amylovora was complemented with a DNA fragment carrying the genes cpsF and cpsG of E. stewartii and synthesized an EPS composed of a stewartan-like backbone and an amylovoran-like side chain.
The function of amsM, which has high homology to galU/F genes of E. coli, was characterized by genetic and biochemical assays.
Chromosomal amsM mutants could not grow on galactose and were EPS negative.
Unexpectedly, the UDP-glucose pyrophosphorylase activity of these mutants was higher than in the parental strain; this activity was reduced after overexpression of amsM. Since AmsM presumably has no enzymatic activity by its own, it may modulate the intracellular concentration of UDP-sugars by interaction with other proteins.
Publication
Authors
C. Langlotz, H. Ullrich, K. Geider, A. Huber, M. Nimtz, D.L. Coplin
Keywords
amylovoran, stewartan, Ams-proteins, Cps proteins, biosynthesis of repeating unit, UDP-glucose pyrophosphorylase
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