Molecular cloning of a gene encoding the thermoactive levansucrase from Rahnella aquatilis and its growth phase-dependent expression in Escherichia coli

Abstract

A levansucrase gene (lsrA) from Rahnella aquatilis ATCC33071 was isolated from a genomic library and the nucleotide sequence of the lsrA structural gene was determined. lsrA is composed of 1248 bp and encodes 415 amino acid residues with a calculated molecular mass of 45.9 kDa. Although the amino acid sequence of lsrA gene showed good conservation with the sequences of reported levansucrases and of the conserved regions thought to be implicated in the enzyme activity, comparison of the deduced amino acid sequences certified the dissimilarity of the proteins from Gram-negative and Gram-positive bacteria. The lsrA gene was expressed from its own promoter in Escherichia coli in an active form. The lsrA expression in E. coli-pRL1CPR was affected by the growth phase of cells: it was repressed in the early phase of growth, but was significantly stimulated during the entrance of cells into the late phase of growth. The growth-phase-dependent fashion of lsrA expression was altered in a constitutive-like fashion by the deletion of an upstream region of lsrA (pNd137), suggesting that the growth-phase dependent expression of lsrA was mediated by the deleted upstream region.