Two C-P lyase operons in Pseudomonas stutzeri and their roles in the oxidation of phosphonates, phosphite, and hypophosphite

Abstract

DNA sequencing and analysis of two distinct C-P 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 C-P 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 C-P lyase pathway in this organism. To identify the genes comprising this pathway, a Deltahtx 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 C-P lyases are limited, as growth on other phosphonate compounds, including glyphosate and phenylphosphonate, was not observed.

FIG. 1.

Structure of the htx operon of P. stutzeri WM88. Black arrows indicate genes with no homology to phn genes. Blue arrows indicate htx genes that are homologous to phn genes, which comprise all of the components of a complete C—P lyase. The sequences of the genes lying within the shaded gray box had previously been determined (22).

FIG. 2.

RT-PCR of total RNA prepared from P. stutzeri WM567 grown on hypophosphite as the sole P source to determine the operon structure of htx. Lanes a show complete RT reactions; lanes b contain a negative control, with which no reverse transcriptase was added to the reaction mixture; and lanes c contain a PCR-positive control, for which chromosomal DNA was used as the template. Lanes L, 100-bp ladder. The junction sequences amplified are indicated above each set of reactions. For a list of the primers used and the predicted PCR product sizes, refer to Table 2.

FIG. 3.

Organization of the genes of the htx and phn operons involved in the metabolism of phosphonates in P. stutzeri compared to that of the phn operon of E. coli. The black arrow represents genes within the htx operon that are not homologous to any phn gene, green arrows represent genes likely involved in phosphonate transport, red arrows represent genes with putative regulatory function, blue arrows represent genes thought to encode the catalytic components of the C—P lyase, and gold arrows represent genes believed to encode accessory proteins to C—P lyase. The percentages of predicted amino acid sequence identity between each of the homologous proteins are indicated.

FIG. 4.

Growth of P. stutzeri htx, ptx, and phn deletion mutants on minimal media with various P sources after 4 days of growth. (A) Fresh cultures were streaked from MOPS minimal solid agar containing 0.1 mM P_i onto MOPS minimal agar containing the indicated P sources (phosphite [Pt], hypophosphite [Hpt], inorganic phosphate [Pi], aminoethylphosphonate [Aepn], and methylphosphonate [Mpn]) at 0.5 mM. The schematic on the plate lacking phosphorus (No P) represents the order in which the deletion mutants were streaked: 1, Δhtx Δphn mutant (WM3616); 2, Δphn mutant (WM3614); 3, Δhtx mutant (WM1926); 4, wild type (WM567); 5, Δphn Δptx mutant (WM3748); 6, Δhtx Δptx mutant (WM3747); 7, Δptx mutant (WM3746); and 8, Δptx-htx Δphn mutant (WM3617) (B and C). Representative Δhtx Δptx and Δptx mutant colonies showing enhanced growth that were grown on phosphite (B) and hypophosphite (C) are indicated by white arrows.