Genetic regulation, biochemical properties and physiological importance of arginase from Sinorhizobium meliloti

Abstract

In bacteria,l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine tol-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiontSinorhizobium meliloti1021 has two genes annotated as arginases,argI1(smc03091) andargI2(sma1711). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021argI1null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in theargI1mutant genetically complemented with a genomically integratedargI1gene. In the wild-type, arginase activity andargI1transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highestin vitroenzymatic activity at pH 7.5 with Ni2+as cofactor. The enzyme was also active with Mn2+and Co2+, both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator (smc03092) divergently transcribed fromargI1was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to theargI1promoter in a region preceding the predictedargI1transcriptional start. Our results indicate that ArgI1 is the sole arginase inS. meliloti, that it contributes substantially to arginine catabolismin vivoand thatargI1induction by arginine is dependent on ArgIR.