Levan and levansucrase synthesized by the fireblight pathogen Erwinia amylovora

Abstract

The fireblight pathogen Erwinia amylovora synthesizes two different types of extracellular polysaccharide (EPS) when cultured in vitro, an acidic and a neutral compound. Neutral EPS was prepared from culture supernatants by cross-flow ultrafiltration and molecular washing, and purified by column chromatography. This material represented most of the crude EPS, which was produced in high-sucrose medium. It was entirely composed of fructose. The signals obtained from the EPS-preparation by 13C-NMR spectroscopy were identical with those of levan from Aerobacter levanicum indicating a β-2,6 linkage of the sugar residues. The neutral component of the EPS was thus identified as levan (β-2,6- d-fructofuranan). In the protein fraction associated with crude EPS, the levan polymerizing enzyme levansucrase (β-2,6- d-fructan: d-glucose 6-fructosyltransferase, EC 2.4.1.10) was detected. The enzyme was also produced at similar levels with several carbon sources by the bacterium. In polyacrylamide gel eletrophoresis its molecular weight under completely denaturating conditions was 46 kDa, when purified to apparent homogeneity by membrane-exchange chromatography. However, in isoelectric focussing, the enzyme preparation produced two bands at pI 4·0 and pI 3·6, respectively. Both bands displayed enzyme activity in an in situ levansucrase assay. Secretion of levansucrase may play an important role for multiplication of E. amylovora in sucrose containing plant tissue.