Rhizobium (Sinorhizobium) meliloti phn genes: Characterization and identification of their protein products

In Escherichia coli, the phn operon encodes proteins responsible for the up take and breakdown of phosphonates. The C-P (carbon-phosphorus) lyase enzym e encoded by this operon which catalyzes the cleavage of C-P bonds in phosp honates has been recalcitrant to biochemical characterization. To advance t he understanding of this enzyme, we have cloned DNA from Rhizobium (Sinorhi zobium) meliloti that contains homologues of the E. coli phnG, -H, -I, -J, and -K genes. We demonstrated by insertional mutagenesis that the operon fr om which this DNA is derived encodes the R. meliloti C-P lyase. Furthermore , the phenotype of this phn mutant shows that the C-P lyase has a broad sub strate specificity and that the organism has another enzyme that degrades a minoethylphosphonate. A comparison of the R, meliloti and E, coli phn genes and their predicted products gave new information about C-P lyase. The put ative R, meliloti PhnG, PhnH, and PhnK proteins were overexpressed and used to make polyclonal antibodies. Proteins of the correct molecular weight th at react with these antibodies are expressed by R. meliloti grown with phos phonates as sole phosphorus sources. This is the first in vivo demonstratio n of the existence of these hitherto hypothetical Phn proteins.