Cloning and characterization of a Rhizobium meliloti nonspecific acid phosphatase

Abstract

Nodulated legumes require high levels of phosphorus for optimal symbiotic performance. However, the basis for this elevated phosphorus requirement is poorly understood, and very little information regarding bacteroid phosphorus metabolism is available. To develop an understanding of the relative importance of organic and inorganic phosphorus sources for bacteroids, we investigated phosphatase activity in Rhizobium meliloti. An R. meliloti plasmid library clone that complemented an Escherichia coli phosphatase mutant was isolated, and the clone was sequenced. The complementing fragment contained a 337-amino-acid open reading frame that has a potential leader sequence and processing sites characteristic of periplasmic proteins. The phosphatase activity was located in the periplasm of R. meliloti and of E. coli containing the cloned gene. The subunit molecular mass of the cloned phosphatase was 33 kDa, and gel filtration indicated the active enzyme was a 66-kDa homodimer. Lack of substrate specificity suggests the cloned gene, napD, encodes a nonspecific acid phosphatase with a pH optimum of approximately 6.5. An R. meliloti napD transposon-insertion mutant was constructed, and its symbiotic phenotype was determined to be Fix⁺ regardless of the level of phosphorus provided to the host plant.