Characterization of an activator gene upstream of lsc, involved in levan synthesis of Erwinia amylovora

Abstract

In addition to activation by rlsA, a second gene for positive regulation of levansucrase expression of the fire blight pathogen Erwinia amylovora has been detected. The gene rlsB was cloned from a genomic library by suppression of a levan-deficient strain and is located adjacent to the start of lsc in the opposite orientation. The gene rlsB presumably encodes a 6.9kDa basic protein and is preceded by a potential promoter and a ribosome binding site. RlsB belongs to the phage P2 Ogr protein superfamily, which includes other transcriptional activators containing the zinc-finger CCCC motif. An rlsB mutant was created from the wild-type strain Ea1/79 by gene disruption, which was reduced in levan synthesis and was complemented by the intact rlsB gene. Expression of rlsB also restored extracellular levan synthesis of several natural levan-deficient E. amylovora strains, but did not affect amylovoran synthesis. The lsc promoter inserted in an intermediate-copy number plasmid diminished levan synthesis, which could be restored by high expression of rlsB. PCR primers, deduced from rlsB, produced a specific DNA fragment only with E. amylovora strains. The genes rlsB and lsc of E. amylovora were not detected in the chromosome of Escherichia coli, but the genes at both borders localized in the complete genomic map of E. coli. It can be assumed that the lsc -gene cluster was introduced into the fire blight pathogen at a late stage of evolution.