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Free keywords:
EPS, extracellular polysaccharide; Km, kanamycin; Nal, nalidixic acid
Abstract:
Levan, a β-2,6-fructofuranan, produced by the fireblight pathogen Erwinia amylovora , is synthesized from sucrose by the extracellular enzyme levansucrase. Levansucrase mutants, created by Tn5 transposon mutagenesis, showed retarded development of necrotic symptoms on inoculated pear seedlings. By complementation experiments the levansucrase gene (lsc) was located in a 1·4 kb PstI-EcoRV chromosomal DNA fragment, isolated from a genomic library of E. amylovora. An open reading frame of 415 amino acids, promotor sequences and a ribosome binding site were identified by DNA sequencing. The cloned lsc gene of E. amylovora was expressed in E. coli from its own promotor. The enzyme accumulated in E. coli in the periplasm and caused an altered colony morphology on sucrose containing solid media. When analysed by SDS-PAGE and isoelectric focusing, the enzyme purified from E. coli was found to be identical with the protein isolated from E. amylovora.
