| The first detailed biochemical and genetic analysis of reduced P metabolism in microorganisms is described in this thesis. Pseudomonas stutzeri WM88 is able to oxidize hypophosphite (P valence +1) and phosphite (P valence +3) for their use as the sole source of P. This process requires two operons encoded by htxABCDEFGHIJKLMN and ptxABCDE, which are separated by ca. 15 kbp. Sequence analysis of the htx operon suggests that htrA encodes a novel member of the 2-oxoglutarate dependent dioxygenase enzyme family, htxBCDE encode a binding protein dependent hypophosphite transporter and that htxF-htxN encode the components of a Carbon-Phosphorus lyase for the degradation of phosphonate compounds. Sequence analysis of the ptx operon suggests that ptxABC encode a binding protein dependent phosphite transporter, ptxD encodes a novel NAD dependent dehydrogenase, and that ptxE encodes a putative transcriptional regulator in the LysR family. Recombinant HtxA and PtxD were purified to apparent homogeneity and were biochemically characterized. The biochemical analysis of hypophosphite oxidation indicates that HtxA is a hypophosphite/2-oxoglutarate dependent dioxygenase which catalyzes the direct oxidation of hypophosphite to phosphite and requires 2-oxoglutarate, oxygen, and ferrous ions for activity. PtxD is a novel member of the D-isomer specific 2-hydroxy-acid enzyme family of dehydrogenases and catalyzes the oxidation of phosphite to phosphate while reducing NAD to NADH. Reporter gene fusions were used to examine the regulation of expression of the htx and ptx operons. Under conditions of phosphate starvation, the ptx operon is induced 22 fold and the htx operon is induced 18 fold relative to the expression levels in cultures grown with excess phosphate. In addition, the phosphate starvation inducible expression of these operons was dependent on PhoBR in E. coli, strongly supporting a role for these genes in phosphate acquisition. The htxB-htxN genes were demonstrated to encode a functional C-P lyase that has no role in the oxidation of either hypophosphite or phosphite. Interestingly, an additional C-P lyase is encoded by the phnCDEFGHIJKLMNP operon located elsewhere on the chromosome. This C-P lyase has a broad substrate range, allowing growth on methylphosphonate, aminoethylphosphonate and phosphite. |
