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

A levansucrase gene (lsrA) from Rahnella aquatilis ATCC33071 was isolated f rom a genomic library and the nucleotide sequence of the Isr A structural g ene was determined. Isl A 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 r eported levansucrases and of the conserved regions thought to be implicated in the enzyme activity, comparison of the deduced amino acid sequences cer tified the dissimilarity of the proteins from Gram-negative and Gram-positi ve bacteria. The Is,A gene was expressed from its own promoter in Escherich ia coli in an active form. The lsrA expression in E. coli-pRL1CPR was affec ted by the growth phase of cells: it was repressed in the early phase of gr owth, but was significantly stimulated during the entrance of cells into th e late phase of growth. The growth-phase-dependent fashion of Isi A express ion was altered in a constitutive-like fashion by the deletion of an upstre am region of lsrA (pNd137), suggesting that the growth-phase dependent expr ession of lsrA was mediated by the deleted upstream region. (C) 2000 Elsevi er Science B.V. All rights reserved.