Creation and genetic restoration of Erwinia amylovora strains with low levan synthesis

Erwinia amylovora synthesizes the enzyme levansucrase, which was found unprocessed after secretion. Its synthesis depends on several factors including rlsA, located in the hrp region and rlsB, located upstream of the levansucrase gene and a region downstream of lsc. A novel protein of 63 amino acids, RlsC, was identified as another regulator. Levan production of several natural levan-deficient strains and of an rlsC-mutant was strongly increased by expression of the cloned rlsC gene, but also of rlsA and rlsB. In addition, mutants in rlsA and rlsB were restored by overexpression of either one of the three Rls-proteins. A levan-deficient mutant downstream of lsc was only restored for levan synthesis by RlsA. In PCR assays with primers from an internal region of rlsA, a signal was obtained for Erwinia pyrifoliae and Erwinia strains from Japan, which do not produce levan, but not with rlsB or rlsC primers. Since only levansucrase expression but not the activity of the lsc-promoter was enhanced by RlsC and by RlsB, we assume interference of these factors on a post-transcriptional level.