Accumulation of Intermediates of the Carbon-Phosphorus Lyase Pathway for Phosphonate Degradation in phn Mutants of Escherichia coli

The catabolism of phosphonic acids occurs in Escherichia coli by the carbon-phosphorus lyase pathway, which is governed by the 14-cistron phn operon. Here, several compounds are shown to accumulate in strains of E. coli with genetic blocks in various phn cistrons when the strains are fed with phosphonate.Phosphonates (Pn), which contain the carbon-phosphorus bond, are quite abundant in nature, primarily as components of phosphonolipids, where 2-aminoethyl phosphonate (AEPn) is analogous to ethanolamine phosphate as the constituent of phospholipids. In addition, Pn are constituents of polysaccharides, glycoproteins, glycolipids, and several antibiotics (25). Furthermore, large amounts of manmade Pn enter the environment (24). Pn utilization polypeptides in Escherichia coli are specified by the phnCDEFGHIJKLMNOP operon (15). Of the 14 cistrons, one (phnF) encodes a repressor protein, as inferred from sequence alignment (6); three (phnCDE) encode an ABC transport system for Pn (22); seven (phnGHIJKLM) have been postulated to encode the carbon-phosphorus (CP) lyase activity (26); and three (phnNOP) have been postulated to encode “auxiliary enzymes.” Among the latter genes, the phnO gene has been shown to specify an enzyme with aminoalkylphosphonate N-acetyltransferase activity (5). We previously identified the product of the phnN gene as an enzyme capable of catalyzing the phosphorylation of ribose 1,5-bisphosphate to 5-phosphoribosyl α-d-1-diphosphate (ribose 1,5-bisphosphate phosphokinase; EC2.7.4.23) (11). The relation of these two compounds to Pn catabolism remains to be established. Since both the substrate and the product of ribose 1,5-bisphosphate phosphokinase are phosphorylated compounds, we inferred that at least some of the other intermediates of the pathway might be also phosphate esters. It might therefore be possible to identify these intermediates by labeling cells with radioactive phosphate ion in the presence of Pn followed by visualization by appropriate thin-layer chromatography (TLC) procedures. In addition, we employed mutants defective in individual genes of the pathway, and we looked for the accumulation of radiolabeled compounds as candidates for members of the phn pathway. Finally, the mutant strains used harbored the ΔpstS605 allele to render the expression of the phn operon constitutive and, thus, independent of the phosphate supply.