Molecular genetic analysis of phosphite and hypophosphite oxidation by Pseudomonas stutzeri WM88

Abstract

The first molecular and genetic characterization of a biochemical pathway for oxidation of the reduced phosphorus (P) compounds phosphite and 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 genes 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 intermediate. First, plasmid subclones that conferred oxidation of phosphite, but not hypophosphite, upon heterologous hosts were readily obtained. All plasmid subclones that failed to confer phosphite oxidation also failed to confer hypophosphite oxidation. No subclones that conferred only hypophosphite expression were obtained. Second, various deletion derivatives of the cloned genes were made in vitro and recombined onto the chromosome of P. stutzeri WM88. Two phenotypes were displayed by individual mutants. Mutants with the region encoding phosphite oxidation 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 oxidize hypophosphite. The phenotypes displayed by these mutants also demonstrate that the cloned genes are responsible for the P oxidation phenotypes displayed by the original P. stutzeri WM88 isolate. The DNA sequences of the minimal regions implicated in oxidation of each compound were determined. The region required for oxidation of phosphite to phosphate putatively encodes a binding-protein-dependent phosphite transporter, an NAD+-dependent phosphite dehydrogenase, and a transcriptional activator of the lysR family. The region required for oxidation of hypophosphite to phosphite putatively encodes a binding-protein-dependent hypophosphite transporter and an alpha-ketoglutarate-dependent hypophosphite 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 growth-limiting, nutrient.

FIG. 1

Structures of the broad-host-range plasmids pWM263 and pWM265. The physical maps of the cloning vectors pWM263 and pWM265 are shown. The large number of unique restriction sites in these plasmids greatly facilitates subcloning of DNA inserted into these vectors (see text for details). Only unique restriction sites are shown. Two additional plasmids, pWM264 and pWM266, are similar but with the polylinker in the orientation opposite to that in pWM263 and pWM265, respectively. Genes cloned into pWM263 and pWM264 can be expressed from the tac promoter (ptac) in an IPTG (isopropyl-β-d-thiogalactopyranoside)-dependent manner. The rrnB terminator (trrnB) in these plasmids terminates transcripts originating at ptac. All four plasmids can be mobilized to a variety of recipients from E. coli hosts that carry the tra genes of RP4 in trans. The bla gene encodes resistance to β-lactam antibiotics in pWM263 and pWM264. The tetA gene encodes resistance to tetracycline in pWM265 and pWM266. The lacZα gene of pWM265 and pWM266 is not functional due to stop codons in the large polylinkers of these plasmids.

FIG. 2

Deletion analysis of the hypophosphite- and phosphite-oxidizing functions encoded by the plasmid pWM239. A series of deletion derivatives of the plasmid pWM239 were constructed and tested for expression of the hypophosphite and phosphite oxidation phenotypes in E. coli S17-1 and P. aeruginosa PAK Δpil rif, as described in the text. The ability to confer growth in 0.4% glucose–MOPS medium containing hypophosphite (Hpt) or phosphite (Pt) as the sole phosphorus source is indicative of the ability to oxidize the indicated compound to phosphate. Examination of the P oxidation phenotypes displayed by P. aeruginosa carrying the various deletion plasmids indicates that the shaded region between the KpnI₂ and AseI sites is required for Pt oxidation. Further, these data suggest that oxidation of hypophosphite proceeds via a phosphite intermediate. Thus, P. aeruginosa strains carrying plasmids lacking the Pt region are also defective in hypophosphite oxidation. The ability to oxidize Pt in E. coli hosts, indicated by (+), is not related to the plasmids, because E. coli is a natural phosphite oxidizer. Therefore, deletions of the Pt region do not affect hypophosphite oxidation in E. coli, and the minimal region required for this phenotype can be determined by examination of the complementation pattern in this host. Accordingly, the shaded region between the SstI and NheI sites is required for hypophosphite oxidation in E. coli. The thick line at the top represents the cloned region of the P. stutzeri chromosome, with the restriction sites used for construction of individual deletions shown. The flanking restriction sites used for construction of each deletion were provided by the polylinker of the vector pWM265 (Fig. 1). The thin lines represent the remaining insert region of each deletion plasmid. Not all restriction sites within the insert were mapped for each enzyme; therefore, the sites shown are not necessarily unique.

FIG. 3

P. stutzeri WM88 chromosomal mutations in the region linked to P oxidation phenotypes. The regions of the P. stutzeri WM88 chromosome putatively required for oxidation of hypophosphite (Hpt) and phosphite (Pt) (arrows) were identified by complementation of heterologous hosts (see the text and Fig. 2). Deletion and insertion mutations in clones of this genomic region were constructed in vitro and recombined onto the P. stutzeri WM88 chromosome as described in the text. The P oxidation phenotypes of these mutants were examined by scoring the ability to grow on media containing either Hpt or Pt as the sole P source as described in the text. These phenotypes confirm that the genes carried in this region are responsible for the oxidation of phosphite and hypophosphite by the original isolate (see text for discussion). The thin line at the top indicates the chromosomal region of P. stutzeri under study, with relevant restriction sites shown. Not all sites for each enzyme have been mapped; therefore, the sites shown are not necessarily unique. The thick lines below this represent the extents of the in vitro-constructed deletion mutations. The triangles show the sites of insertion mutations.

FIG. 4

Physical structures of DNA fragments shown to be required for oxidation of phosphite and hypophosphite by P. stutzeri WM88. The complete DNA sequences of both fragments were determined as described in the text. (GenBank accession no. AF061070 and AF061267). (A) Structure of a 5.6-kbp KpnI fragment encoding functions required for oxidation of phosphite to phosphate in P. stutzeri WM88. Seven ORFs, indicated by arrows, were identified within this sequence. The ptxE gene is truncated in this clone, as indicated by the partially shaded arrow. Five of these genes, designated ptxA through ptxE, are likely to be involved in oxidation of phosphite and probably form a single transcriptional unit. PtxABC probably comprise a binding-protein-dependent transport system for the uptake of phosphite. PtxD is probably an NAD⁺-dependent phosphite dehydrogenase, and PtxE is probably a transcriptional regulator for the ptxABCDE operon. (B) Structure of an 8.9-kbp SstI-to-NheI fragment encoding functions required for oxidation of hypophosphite to phosphite in P. stutzeri WM88. Nine ORFs, indicated by arrows and designated htxA through htxI, are likely to form a single transcriptional unit. Relevant restriction sites used for various plasmid constructions are shown. The BglII and AgeI sites shown in boldface were used as insertion sites for gene disruption experiments (Fig. 3). HtxA is a putative α-ketoglutarate-dependent hypophosphite dioxygenase. HtxBCDE comprise a putative binding-protein-dependent hypophosphite transporter. The remaining genes encode subunits of a putative C-P lyase but are not required for oxidation of hypophosphite. The partially shaded arrow indicates that the htxI gene is truncated in this sequence. See the text and Table 3 for details. Approximately 15 kbp separates the two regions. This 15-kbp region is not required for oxidation of either compound and was not characterized.