The Rhizobium leguminosarum bv. trifolii RosR: Transcriptional Regulator Involved in Exopolysaccharide Production

Abstract

The acidic exopolysaccharide is required for the establishment of symbiosis between the nitrogen-fixing bacterium Rhizobium leguminosarum bv. trifolii and clover. Here, we describe RosR protein from R. leguminosarum bv. trifolii 24.2, a homolog of transcriptional regulators belonging to the family of Ros/MucR proteins. R. leguminosarum bv. trifolii RosR possesses a characteristic Cys2His2 type zincfinger motif in its C-terminal domain. Recombinant (His)6RosR binds to an RosR-box sequence located upstream of rosR. Deletion analysis of the rosR upstream region resulted in identification of two -35 to -10 promoter sequences, two conserved inverted palindromic pentamers that resemble the cAMP-CRP binding site of Escherichia coli, inverted repeats identified as a RosR binding site, and other regulatory sequence motifs. When assayed in E. coli, a transcriptional fusion of the cAMP-CRP binding site containing the rosR upstream region and lacZ gene was moderately responsive to glucose. The sensitivity of the rosR promoter to glucose was not observed in E. coli ΔcyaA. A rosR frame-shift mutant of R. leguminosarum bv. trifolii formed dry, wrinkled colonies and induced nodules on clover, but did not fix nitrogen. In the rosR mutant, transcription of pssA-lacZ fusion was decreased, indicating positive regulation of the pssA gene by RosR. Multiple copies of rosR in R. leguminosarum bv. trifolii 24.2 increased exopolysaccharide production.