MOLECULAR-GENETIC ANALYSIS OF PHOSPHITE AND HYPOPHOSPHITE OXIDATION BY PSEUDOMONAS-STUTZERI WM88

The first molecular and genetic characterization of a biochemical path way for oxidation of the reduced phosphorus (P) compounds phosphite an d hypophosphite is reported. The pathway was identified in Pseudomonas stutzeri WM88, which was chosen for detailed studies from a group of organisms isolated based on their ability to oxidize hypophosphite (+1 valence) and phosphite (+3 valence) to phosphate (+5 valence). The ge nes required for oxidation of both compounds by P: stutzeri WM88 were cloned on a single ca. 30-kbp DNA fragment by screening for expression in Escherichia coli and Pseudomonas aeruginosa. Two lines of evidence suggest that hypophosphite is oxidized to phosphate via a phosphite i ntermediate. First, plasmid subclones that conferred oxidation of phos phite, but not hypophosphite, upon heterologous hosts were readily obt ained. All plasmid subclones that failed to confer phosphite oxidation also failed to confer hypophosphite oxidation. No subclones that conf erred only hypophosphite expression were obtained. Second, various del etion derivatives of the cloned genes were made in vitro and recombine d onto the chromosome of P. stutzeri WM88. Two phenotypes were display ed by individual mutants. Mutants with the region encoding phosphite o xidation deleted (based upon the subcloning results) lost the ability to oxidize either phosphite or hypophosphite. Mutants with the region encoding hypophosphite oxidation deleted lost only the ability to oxid ize hypophosphite. The phenotypes displayed by these mutants also demo nstrate that the cloned genes are responsible for the P oxidation phen otypes displayed by the original P. stutzeri WM88 isolate. The DNA seq uences of the minimal regions implicated in oxidation of each compound were determined. The region required for oxidation of phosphite to ph osphate putatively encodes: a binding-protein-dependent phosphite tran sporter, an NAD(+)-dependent phosphite dehydrogenase, and a transcript ional activator of the lysR family. The region required for oxidation of hypophosphite to phosphite putatively encodes a binding-protein-dep endent hypophosphite transporter and an ar-ketoglutarate-dependent hyp ophosphite dioxygenase. The finding of genes dedicated to oxidation of reduced P compounds provides further evidence that a redox cycle for P may be important in the metabolism of this essential, and often grow th-limiting, nutrient.