Molecular analysis of the rlsA gene regulating levan production by the fireblight pathogen Erwinia amylovora

Abstract

Natural Erwinia amylovora can display a reduced level of levan synthesis. A gene (rlsA) responsible for the suppression of low levansucrase synthesis by E. amylovora was cloned by complementation of natural levan-deficient strains lacking levansucrase expression. The rlsA region was sequenced, and an open reading frame of 266 amino acids was found. Sequence analysis revealed that RlsA resembles a transcriptional activator and may be a member of the LysR family. Overexpression of rlsA from a high copy number plasmid led to a two-fold increase of levan production, but had no influence on amylovoran synthesis. A mutant created by site-directed mutagenesis of wild type strain Eal/79 had the same phenotype as natural levan-deficient strains. The rlsA gene of natural levan deficient strains (with the exception of strain PD494) could be amplified by PCR, yielding the same DNA fragments as wild type strains. The gene was absent in a strain with a large deletion affecting the hrp region of E. amylovora . A match of the nucleotide sequence of rlsA with a sequence database revealed its identity with an incomplete open reading frame located downstream of two recently characterised dsp genes.