INTERACTION OF THE REGULATOR PROTEINS RCSA AND RCSB WITH THE PROMOTEROF THE OPERON FOR AMYLOVORAN BIOSYNTHESIS IN ERWINIA-AMYLOVORA

The RcsA and RcsB proteins of Erwinia amylovora and Escherichia coli w ere expressed in E. coli and purified. Their DNA-binding activity was examined using a 1-kb DNA region containing the putative promoter of t he ams operon of Ew. amylovora, which is responsible for the biosynthe sis of the exopolysaccharide amylovoran. Mobility shift assays indicat ed specific binding of RcsA and RcsB to a region of 78 bp spanning nuc leotide positions -578 to -501 relative to the translational start of the first open reading frame of the operon. This region includes stret ches of homology to E. coli sigma(70) promoter consensus sequences and to the E. coli cps promoter region. Binding of the Rcs proteins was n ot found at a JUMPstart consensus, typical for various promoters of po lysaccharide gene clusters. DNA-binding activity was not detected for RcsA alone and only high concentrations of RcsB were able to interact with the ams promoter in our assay. The two proteins bind cooperativel y at the indicated region of the ams promoter and further evidence is provided showing that the DNA-protein complex formed involves a hetero dimer of RcsA and RcsB. The specific activity of RcsA, but not of RcsB , was enhanced when the protein was expressed in E. coli at 28 degrees C, relative to expression at 37 degrees C. In addition, DNA-protein c omplex formation is affected by temperature. The E. coli RcsA/RcsB pro teins bind to the same region of the ams promoter and are able to inte ract with the Res proteins from Ew. amylovora.