Ww. Metcalf et Rs. Wolfe, MOLECULAR-GENETIC ANALYSIS OF PHOSPHITE AND HYPOPHOSPHITE OXIDATION BY PSEUDOMONAS-STUTZERI WM88, Journal of bacteriology (Print), 180(21), 1998, pp. 5547-5558
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.