Identification and Heterologous Expression of Genes Involved in Anaerobic Dissimilatory Phosphite Oxidation by Desulfotignum phosphitoxidans

Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO₂ reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assays.Phosphorus (P) is an important nutrient for all living organisms. The predominant forms of phosphorus in biological systems are inorganic phosphate and its organic esters and acid anhydrides in which P is at its highest oxidation state (+V). The P requirements of living cells can be fulfilled with phosphate in various forms, including reduced organic and inorganic phosphorus compounds (23). Several aerobic bacteria were shown to be able to oxidize hypophosphite (+I) and phosphite (+III) to phosphate (+V) and to incorporate the last into their biomass (5, 15-17, 31, 34). Phosphite can also be oxidized under anaerobic conditions, as shown for an anaerobic Bacillus strain (7) and for Pseudomonas stutzeri which can use phosphite under denitrifying conditions (17, 21). The only bacterium known to oxidize phosphite as the sole source of electrons in lithoautotrophic energy metabolism is Desulfotignum phosphitoxidans (24, 25).Three different metabolic pathways for the use of phosphite as a single P source have been characterized so far. Two of them were discovered and characterized with Escherichia coli and one with Pseudomonas stutzeri. The first pathway in E. coli is mediated by the enzyme carbon phosphorus lyase (C-P lyase), and the second one by the alkaline phosphatase encoded by phoA (16, 34). This alkaline phosphatase not only hydrolyzes phosphate esters but also hydrolyzes phosphite to phosphate and molecular hydrogen (32). This is a particular property only of the E. coli alkaline phosphatase and is not observed with alkaline phosphatases of other bacteria. The third pathway is encoded by the ptxABCDE gene cluster in P. stutzeri (17). In this system, phosphite is transported into the cell by a binding protein-dependent phosphite transporter at the expense of ATP (PtxABC). Phosphite is oxidized by a phosphite:NAD⁺ oxidoreductase (encoded by ptxD), a new member of the 2-hydroxy acid dehydrogenases (8). The ptx operon of P. stutzeri is regulated in response to phosphate starvation by the two-component regulatory system phoBR (28, 29). Furthermore, in Alcaligenes faecalis WM2072, another gene cluster involved in hypophosphite and phosphite uptake and oxidation was characterized: the htxABCD-ptxDE locus (31). The htxABCD-ptxDE genes and their products in A. faecalis WM 2072 have high nucleotide and amino acid sequence identities with those found in the htx and ptx operons in P. stutzeri WM88, which are required for the oxidation of hypophosphite and phosphite, respectively. This unique genetic arrangement of hypophosphite- and phosphite-oxidizing genes in A. faecalis WM2072 suggests a horizontal gene transfer and an ancient evolution of phosphite oxidation.The diversity of pathways used for assimilatory phosphite oxidation and the fact that D. phosphitoxidans is so far the only bacterium known to use phosphite as an electron source caused us to investigate the phosphite uptake and oxidation gene cluster of this bacterium. The aims of our study were (i) to establish enzymatic assays for measurement of phosphite oxidation activity in cell extracts, (ii) to identify the genes involved in phosphite uptake and oxidation, and (iii) to characterize these genes physiologically.