THE REGULATORY PROTEIN MUCR BINDS TO A SHORT DNA REGION LOCATED UPSTREAM OF THE MUCR CODING REGION IN RHIZOBIUM-MELILOTI

The Rhizobium meliloti MucR protein is known to regulate the biosynthe sis of the two exopolysaccharides, succinoglycan and galactoglucan. Th e mucR gene was successfully overexpressed in Escherichia coli BL21 ce lls by heat shock induction using a two-plasmid system. Cell extracts of the production strain contained about 20% of a polypeptide of 17 kD a apparent molecular mass, corresponding to the size expected for MucR . As shown by an electrophoretic mobility shift assay, these extracts were active in the specific retardation of a 219-bp DNA fragment inclu ding 134-bp of the non-coding region upstream of the mucR gene. Primer extension analysis showed that this DNA fragment was located within t he transcribed region upstream of the mucR gene. Competition experimen ts revealed that a 44-bp sequence present within the 134-bp upstream o f the mucR gene contained the MucR binding site. Although binding of M ucR to this site exhibited a moderate dissociation constant of K-d app roximate to 1.4 x 10(-7) M, the reaction was highly specific since fra gments containing binding sites for the homologous Ros protein from Ag robacterium tumefaciens were not able to compete for MucR binding.