The field of bacterial phosphorus (P) metabolism has undergone a significant transformation in the past decade owing to the elucidation of widespread and diverse pathways for the metabolism of reduced P compounds. The characterization of these pathways dramatically changes the current and narrow view of P metabolism and our understanding of the forms in which P is produced and available in the environment. In this review, recent investigations into the biochemical pathways and molecular genetics of reduced P metabolism in bacteria are discussed. Particular attention is paid to recently elucidated metabolic reactions and the genetic characterization of biosynthesis of organic reduced P compounds and to the pathways for oxidation of the inorganic reduced P compounds hypophosphite and phosphite.

The ptxD gene from Pseudomonas stutzeri WM88 encoding the novel phosphorus oxidizing enzyme NAD:phosphite oxidoreductase (trivial name phosphite dehydrogenase, PtxD) was cloned into an expression vector and overproduced in Escherichia coli. The heterologously produced enzyme is indistinguishable from the native enzyme based on mass spectrometry, amino-terminal sequencing, and specific activity analyses. Recombinant PtxD was purified to homogeneity via a two-step affinity protocol and characterized. The enzyme stoichiometrically produces NADH and phosphate from NAD and phosphite. The reverse reaction was not observed. Gel filtration analysis of the purified protein is consistent with PtxD acting as a homodimer. PtxD has a high affinity for its substrates with K m values of 53.1 ؎ 6.7 M and 54.6 ؎ 6.7 M, for phosphite and NAD, respectively. V max and k cat were determined to be 12.2 ؎ 0.3 mol min ؊1 mg ؊1 and 440 min ؊1. NADP can substitute poorly for NAD; however, none of the numerous compounds examined were able to substitute for phosphite. Initial rate studies in the absence or presence of products and in the presence of the dead end inhibitor sulfite are most consistent with a sequential ordered mechanism for the PtxD reaction, with NAD binding first and NADH being released last. Amino acid sequence comparisons place PtxD as a new member of the D-2-hydroxyacid NAD-dependent dehydrogenases, the only one to have an inorganic substrate. To our knowledge, this is the first detailed biochemical study on an enzyme capable of direct oxidation of a reduced phosphorus compound.Phosphorus is widely reported to be a redox conservative element in biological systems, with the sum total of phosphorus biochemistry consisting of the formation and hydrolysis of phosphate-ester bonds. These reports imply that reduced phosphorus compounds are not important in living systems and that enzymatically catalyzed redox reactions of phosphorus compounds do not occur; however, an increasing body of evidence indicates that this is not the case. Although it is true that inorganic phosphate (P valence ϩ5) is the principal form of phosphorus in living systems and that phosphate-esters play a critical role in phosphate biochemistry, it is now clear that reduced phosphorus compounds of both natural and xenobiotic origin play important roles in numerous biological systems. Accordingly, many organisms have been shown to possess metabolic pathways for reduction of phosphate to a variety of reduced phosphorus compounds (1-3); others have been shown to possess metabolic pathways for oxidation of reduced phosphorus compounds (4 -9). Among the most striking of these is a recently isolated sulfate-reducing bacterium that obtains all of the energy it requires for growth from the oxidation of phosphite (ϩ3 valence) to phosphate (10).Unfortunately, detailed studies examining the mechanisms of biological phosphorus oxidation and reduction are scarce. This is particularly true with regard to the biochemical characterization of enzymes involved in reduced p...

DNA sequencing and analysis of two distinct COP lyase operons in Pseudomonas stutzeri WM88 were completed. The htxABCDEFGHIJKLMN operon encodes a hypophosphite-2-oxoglutarate dioxygenase (HtxA), whereas the predicted amino acid sequences of HtxB to HtxN are each homologous to the components of the Escherichia coli phn operon, which encodes COP lyase, although homologs of E. coli phnF and phnO are absent. The genes in the htx operon are cotranscribed based on gene organization, and the presence of the intergenic sequences is verified by reverse transcription-PCR with total RNA. Deletion of the htx locus does not affect the ability of P. stutzeri to grow on phosphonates, indicating the presence of an additional COP lyase pathway in this organism. To identify the genes comprising this pathway, a ⌬htx strain was mutagenized and one mutant lacking the ability to grow on methylphosphonate as the sole P source was isolated. A ca.-10.6-kbp region surrounding the transposon insertion site of this mutant was sequenced, revealing 13 open reading frames, designated phnCDEFGHIJKLMNP, which were homologous to the E. coli phn genes. Deletion of both the htx and phn operons of P. stutzeri abolishes all growth on methylphosphonate and aminoethylphosphonate. Both operons individually support growth on methylphosphonate; however, the phn operon supports growth on aminoethylphosphonate and phosphite, as well. The substrate ranges of both COP lyases are limited, as growth on other phosphonate compounds, including glyphosate and phenylphosphonate, was not observed.

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We report here the first use of directed mutagenesis in Methanosarcina acetivorans C2A. The method employs homologous recombination-mediated gene replacement and was used to construct a variety of proline auxotrophs with mutations in the proABC locus. Each mutation was also complemented in trans with autonomously replicating Methanosarcina-Escherichia plasmid shuttle vectors.Studies of methane-producing archaea have been hampered by a lack of genetic tools; however, in recent years this has been changing rapidly. Useful genetic tools have now been developed for organisms in two genera, Methanococcus and Methanosarcina. These tools include selectable markers, transformation methods, plasmids, reporter genes, and a variety of mutagenesis protocols (reviewed in references 13 and 23). Despite these advances, serious deficiencies in the genetic tools available for many methanoarchaea are still evident. In particular, robust protocols for directed mutagenesis of Methanosarcina species, which are the most metabolically diverse of the methanoarchaea, are still lacking, while plasmid-based complementation of mutations has yet to be reported for any methanogen.Directed mutagenesis of Methanosarcina species is clearly possible. Transformation and integration of foreign DNA into the chromosome via homologous recombination have been demonstrated for Methanosarcina mazei S-6 (6). In that study, the first to demonstrate the transformation of any Methanosarcina species, a selectable marker inserted into the grpE-dnaK intergenic region was recombined onto the M. mazei chromosome after chemical transformation. The process was, however, inefficient, requiring 50 g of DNA to produce transformed cultures. More recently, a very efficient liposome-mediated transformation method for Methanosarcina species was developed. This method allows isolation of up to 10 8 transformants per g of DNA in Methanosarcina acetivorans C2A. The availability of this high-frequency transformation method led us to attempt directed mutagenesis in this species. To examine the factors involved in this type of experiment and to verify that the process of homologous recombination occurs in a reliable and predictable manner, we decided to mutagenize genes with known functions and predictable phenotypes. For this purpose, we chose to examine genes involved in the biosynthesis of proline.Cloning the proline biosynthetic genes of M. acetivorans. Previous studies demonstrated the feasibility of cloning proline biosynthetic genes from methanoarchaea by complementation of Escherichia coli mutants (10). Therefore, we used the same method to clone the proC gene from each of the three Methanosarcina species frequently used in our laboratory. Chromosomal DNAs, isolated as previously described (3) from M. acetivorans C2A, Methanosarcina thermophila TM1, and Methanosarcina barkeri Fusaro, were used to construct genomic libraries in the single-copy, dual-cos vector pWM357 (Table 1). The libraries contain ca. 330,000, 14,000, and 50,000 independent clones, respectively, and w...