Phosphonate utilization by bacterial cultures and enrichments from environmental samples

A selection of axenic microbial strains and a variety of environmental samples were investigated with respect to the utilization of a series of natural and xenobiotic phosphonates as the sole phosphorus source for growth. Phosphonate degradation was observed only with bacteria and not with eucaryotic microorganisms. All representatives of the phosphonates examined supported bacterial growth, with the exception of methylphosphonate diethylester. Yet, distinctly different phosphonate utilization patterns were noted between phosphonate-positive strains. C-P bond cleavage by a photosynthetic bacterium is reported for the first time; growing photoheterotrophically, Rhodobacter capsulatus ATCC 23782 was able to utilize 2-aminoethylphosphonate and alkylphosphonates. Bacteria with the potential to utilize at least one of the phosphonate moieties from the xenobiotic phosphonates Dequest 2010, Dequest 2041, and Dequest 2060 were detected in all environments, with only two exceptions for Dequest 2010. Phosphonate P utilization to an extent of 94 and 97%, for Dequest 2010 and Dequest 2041, respectively, provided evidence that a complete breakdown of these compounds with respect to the C-P bond cleavage can be achieved by some bacteria. The results suggest that phosphonate-utilizing bacteria are ubiquitous, and that selected strains can degrade phosphonates that are more complex than those described previously.     

Phosphonate utilization by bacteria in the presence of alternative phosphorus sources

Batch and continuous culture experiments were carried out to investigate the effect of orthophosphate and p-nitrophenylphosphate on the utilization of various phosphonates as a P source by bacteria. Detailed tests with methylphosphonate as a model phosphonate and the phosphonate-degrading Pseudomonas paucimobilis strain MMM101a revealed that, in contrast with the majority of literature data, the phosphates did not suppress phosphonate utilization. Under conditions of P stress, strain MMM101a simultaneously took up both P-sources, with a preference for the phosphate-P. Study of the kinetic parameters for strain MMM101a, growing on the different P sources revealed similar, rather low, maximum growth rates (ca. 0.15 h^-1). However, the affinity for orthophosphate (K_s: 0.17 M), was more than two orders of magnitude higher than for methylphosphonate (K_s: 66 M), which might account for the preferential uptake of orthophosphate. Cellular phosphorus yields in continuous cultures varied considerably with the conditions applied. The results suggest that phosphonate degradation can occur also in environments with substantial backgrounds of phosphate.Abbreviations D2010 Dequest 2010, Hydroxyethane diphosphonic acid - D2041 Dequest 2041, Ethylenediamine tetramethylphosphonic acid - Gly Glyphosate - N Phosphonomethyl glycine - HEPES N-2-Hydroxy-Ethylpiperazine-N-2-EthaneSulfonic acid - MeP Methylphosphonate - OD₆₅₀, OD₄₁₃ Optical densities respectively at 650 and 413 nm - Pi Orthophosphate - Pn Phosphonate-phosphorus - pNPP para-Nitrophenylphosphate - pNP paranitrophenol - D dilution rate of chemostat, day^-1     

Bacterial carbon-phosphorus lyase: products, rates, and regulation of phosphonic and phosphinic acid metabolism.

Carbon-phosphorus bond cleavage activity, found in bacteria that utilize alkyl- and phenylphosphonic acids, has not yet been obtained in a cell-free system. Given this constraint, a systematic examination of in vivo C-P lyase activity has been conducted to develop insight into the C-P cleavage reaction. Six bacterial strains were obtained by enrichment culture, identified, and characterized with respect to their phosphonic acid substrate specificity. One isolate, Agrobacterium radiobacter, was shown to cleave the carbon-phosphorus bond of a wide range of substrates, including fosfomycin, glyphosate, and dialkyl phosphinic acids. Furthermore, this organism processed vinyl-, propenyl-, and propynylphosphonic acids, a previously uninvestigated group, to ethylene, propene, and propyne, respectively. A determination of product stoichiometries revealed that both C-P bonds of dimethylphosphinic acid are cleaved quantitatively to methane and, furthermore, that the extent of C-P bond cleavage correlated linearly with the specific growth rate for a range of substrates. The broad substrate specificity of Agrobacterium C-P lyase and the comprehensive characterization of the in vivo activity make this an attractive system for further biochemical and mechanistic experiments. In addition, the failure to observe the activity in a group of gram-positive bacteria holds open the possibility that a periplasmic component may be required for in vivo expression of C-P lyase activity.     

Bacterial growth on aminoalkylphosphonic acids

Harkness, Donald R. (University of Miami School of Medicine, Miami, Fla.). Bacterial growth on aminoalkylphosphonic acids. J. Bacteriol. 92:623-627. 1966.-Of 10 bacterial strains tested, 9 were found to be able to utilize the phosphorus of at least one of eight different aminoalkylphosphonic acids for growth, indicating that the ability to catabolize the carbon-phosphorus (C-P) bond is widespread among bacteria. Several organisms gave comparable growth rates as well as cell yields when an equimolar amount of either P(i) or 2-aminoethylphosphonic acid (2-AEP) was added to the medium. No compounds containing C-P bonds were detected in Escherichia coli B grown on 2-AEP(32)-orthophosphate. No degradation of phosphonates by cell-free extracts or suspensions of dried cells was demonstrated. The direct involvement of alkaline phosphatases in cleaving the C-P bond was excluded.     

Global and seasonal variation of marine phosphonate metabolism

Marine microbial communities rely on dissolved organic phosphorus (DOP) remineralisation to meet phosphorus (P) requirements. We extensively surveyed the genomic and metagenomic distribution of genes directing phosphonate biosynthesis, substrate-specific catabolism of 2-aminoethylphosphonate (2-AEP, the most abundant phosphonate in the marine environment), and broad-specificity catabolism of phosphonates by the C-P lyase (including methylphosphonate, a major source of methane). We developed comprehensive enzyme databases by curating publicly available sequences and then screened metagenomes from TARA Oceans and Munida Microbial Observatory Time Series (MOTS) to assess spatial and seasonal variation in phosphonate metabolism pathways. Phosphonate cycling genes were encoded in diverse gene clusters by 35 marine bacterial and archaeal classes. More than 65% of marine phosphonate cycling genes mapped to Proteobacteria with production demonstrating wider taxonomic diversity than catabolism. Hydrolysis of 2-AEP was the dominant phosphonate catabolism strategy, enabling microbes to assimilate carbon and nitrogen alongside P. Genes for broad-specificity catabolism by the C-P lyase were far less widespread, though enriched in the extremely P-deplete environment of the Mediterranean Sea. Phosphonate cycling genes were abundant in marine metagenomes, particularly from the mesopelagic zone and winter sampling dates. Disparity between prevalence of substrate-specific and broad-specificity catabolism may be due to higher resource expenditure from the cell to build and retain the C-P lyase. This study is the most comprehensive metagenomic survey of marine microbial phosphonate cycling to date and provides curated databases for 14 genes involved in phosphonate cycling.Subject terms: Water microbiology, Microbial ecology, Microbial biooceanography, Metagenomics     

Bioconversion of sunflower oil,rapeseed oil and ricinoleic acid by Candida tropicalis M25

On the basis of mutational analysis, the genes for phosphonate uptake and degradation in Escherichia coli were shown to be organized in a 10.9-kb operon of 14 genes (named phnC to phnP) and induced by phosphate (P_i) starvation [Metcalf and Wanner (1993) J Bacteriol 175: 3430–3442]. The repression of phosphonate utilization by P_i has hindered both the biochemical characterization of the carbon-phosphorus (C-P) lyase activity and the development of improved methods for phosphonate biodegradation in biotechnology. We have cloned the genes phnG to phnP (associated with C-P lyase activity) with the lac promoter to provide expression of C-P lyase in the presence of P_i. A number of strains lacking portions of the phn operon have been constructed. In vivo complementation of the strains, in which phnC to phnP (including both Pn transport and catalysis genes) or phnH to phnP (including only catalysis genes) was deleted, with plasmids carrying various fragments of the phn operon revealed that the expression of phnC-phnP gene products is essential to restore growth on minimal medium with phosphonate as the sole phosphorus source, while phnG-phnM gene products are required for C-P lyase activity as assessed by in vivo methane production from methylphosphonic acid. The minimum size of the DNA required for the whole-cell C-P lyase activity has been determined to be a 5.8-kb fragment, encompassing the phnG to phnM genes. Therefore, there is no requirement for the phnCDE-encoded phosphonate transport system, suggesting that cleavage of the C-P bond may occur on the outer surface of the inner membrane of E. coli cells, releasing the carbon moiety into the periplasm. These data are in agreement with the observation that phosphonates cannot serve as the carbon source for E.␣coli growth. Received: 23 September 1997 / Received revision: 5 January 1998 / Accepted: 24 January 1998     

Isolation of a glyphosate-metabolising Pseudomonas: detection,partial purification and localisation of carbon-phosphorus lyase

A Pseudomonas isolate (GLC11) capable of growth in the presence of up to 125 mM glyphosate [N-phosphonomethyl glycine (PMG)] has been isolated. Unlike the previously isolated Pseudomonas PG2982 and other bacterial strains, isolate GLC11 grows equally well in commercial formulation and analytical grade PMG. Utilisation of PMG as a phosphorus source is repressed by inorganic phosphate (Pi) in both isolates. Enzymatic activity responsible for carbon-phosphorus bond cleavage (C-P lyase) was detected in cell-free extracts of both isolates and was partially purified. Resolution on DE-52 anion exchange chromatography yielded a single peak of C-P lyase activity. The molecular mass of C-P lyase as analysed by gel permeation chromatography is approximately 200 kDa. The enzyme activity was localised in the periplasmic space of bacteria. The specific activity of C-P lyase was different for different phosphonates when used as substrates. Correspondence to: R. K. Bhatnagar     

Molecular genetic studies of a 10.9-kb operon in Escherichia coli for phosphonate uptake and biodegradation

Bacteria that use phosphonates as a phosphorus source must be able to break the stable carbon-phosphorus bond. In Escherichia coli phosphonates are broken down by a C-P lyase that has a broad substrate specificity. Evidence for a lyase is based on in vivo studies of product formation because it has been proven difficult to detect the activity in vitro. By using molecular genetic techniques, we have studied the genes for phosphonate uptake and degradation in E. coli, which are organized in an operon of 14 genes, named phnC to phnP. As expected for genes involved in P acquisition, the phnC-phnP operon is a member of the PHO regulon and is induced many hundred-fold during phosphate limitation. Three gene products (PhnC, PhnD and PhnE) comprise a binding protein-dependent phosphonate transporter, which also transports phosphate, phosphite, and certain phosphate esters such as phosphoserine; two gene products (PhnF and PhnO) may have a role in gene regulation; and nine gene products (PhnG, PhnH, PhnI, PhnJ, PhnK, PhnL, PhnM, PhnN, and PhnP) probably comprise a membrane-associated C-P lyase enzyme complex. Although E. coli can degrade many different phosphonates, the ability to use certain phosphonates appears to be limited by the specificity of the PhnCDE transporter and not by the specificity of the C-P lyase.     

Bioremediation potential of glyphosate-degrading microorganisms in eutrophicated Ecuadorian water bodies

Phosphonate compounds are the basis of many xenobiotic pollutants, such as Glyphosate (N-(phosphonomethyl-glycine). Only procaryotic microorganisms and the lower eukaryotes are capable of phosphonate biodegradation through C–P lyase pathways. Thus, the aim of this study was to determine the presence of C–P lyase genes in Ecuadorian freshwater systems as a first step towards assessing the presence of putative glyphosate degraders. To that end, two Nested PCR assays were designed to target the gene that codifies for the subunit J (phnJ), which breaks the C-P bond that is critical for glyphosate mineralization. The assays designed in this study led to the detection of phnJ genes in 7 out of 8 tested water bodies. The amplified fragments presented 85–100% sequence similarity with phnJ genes that belong to glyphosate-degrading microorganisms. Nine sequences were not reported previously in the GenBank. The presence of phosphonate degraders was confirmed by isolating three strains able to grow using glyphosate as a unique carbon source. According to the 16S sequence, these strains belong to the Pantoea, Pseudomonas, and Klebsiella genera. Performing a Nested PCR amplification of phnJ genes isolated from eutrophicated water bodies, prior to isolation, may be a cost-effective strategy for the bioprospection of new species and/or genes that might have new properties for biotech industries, laying the groundwork for additional research.     

Microbial metabolism of sulfurand phosphorus-containing xenobiotics

Abstract: The enzymes involved in the microbial metabolism of many important phosphorus- or sulfur-containing xenobiotics, including organophosphate insecticides and precursors to organosulfate and organosulfonate detergents and dyestuffs have been characterized. In several instances their genes have been cloned and analysed. For phosphonate xenobiotics, the enzyme system responsible for the cleavage of the carbon-phosphorus bond has not yet been observed in vitro, though much is understood on a genetic level about phosphonate degradation. Phosphonate metabolism is regulated as part of the Pho regulon, under phosphate starvation control. For organophosphorothionate pesticides the situation is not so clear, and the mode of regulation appears to depend on whether the compounds are utilized to provide phosphorus, carbon or sulfur for cell growth. The same is true for organosulfonate metabolism, where different (and differently regulated) enzymatic pathways are involved in the utilization of sulfonates as carbon and as sulfur sources, respectively. Observations at the protein level in a number of bacteria suggest that a regulatory system is present which responds to sulfate limitation and controls the synthesis of proteins involved in providing sulfur to the cell and which may reveal analogies between the regulation of phosphorus and sulfur metabolism.     

Utilisation of structurally diverse organophosphonates by Streptomycetes

A group of streptomycete strains was found able to utilise a wide range of structurally diverse phosphonates as a sole phosphorus source. No relation could be observed between ability to synthesise compounds containing a direct carbon-to-phosphorus (C-P) bond and biodegradative potential towards phosphonates in the strains studied. Streptomyces morookaensis DSM 40565 could degrade 2-amino-4-phosphonobutyrate as a sole nitrogen and phosphorus source in a stereoselective-like manner. This result suggests the existence of a new metabolic pathway for C-P bond breakage.     

Crystal structure of PhnH: an essential component of carbon-phosphorus lyase in Escherichia coli

Organophosphonates are reduced forms of phosphorous that are characterized by the presence of a stable carbon-phosphorus (C-P) bond, which resists chemical hydrolysis, thermal decomposition, and photolysis. The chemically inert nature of the C-P bond has raised environmental concerns as toxic phosphonates accumulate in a number of ecosystems. Carbon-phosphorous lyase (CP lyase) is a multienzyme pathway encoded by the phn operon in gram-negative bacteria. In Escherichia coli 14 cistrons comprise the operon (phnCDEFGHIJKLMNOP) and collectively allow the internalization and degradation of phosphonates. Here we report the X-ray crystal structure of the PhnH component at 1.77 Å resolution. The protein exhibits a novel fold, although local similarities with the pyridoxal 5′-phosphate-dependent transferase family of proteins are apparent. PhnH forms a dimer in solution and in the crystal structure, the interface of which is implicated in creating a potential ligand binding pocket. Our studies further suggest that PhnH may be capable of binding negatively charged cyclic compounds through interaction with strictly conserved residues. Finally, we show that PhnH is essential for C-P bond cleavage in the CP lyase pathway.     

Molecular cloning, mapping, and regulation of Pho regulon genes for phosphonate breakdown by the phosphonatase pathway of Salmonella typhimurium LT2.

Two pathways exist for cleavage of the carbon-phosphorus (C-P) bond of phosphonates, the C-P lyase and the phosphonatase pathways. It was previously demonstrated that Escherichia coli carries genes (named phn) only for the C-P lyase pathway and that Enterobacter aerogenes carries genes for both pathways (K.-S. Lee, W. W. Metcalf, and B. L. Wanner, J. Bacteriol. 174:2501-2510, 1992). In contrast, here it is shown that Salmonella typhimurium LT2 carries genes only for the phosphonatase pathway. Genes for the S. typhimurium phosphonatase pathway were cloned by complementation of E. coli delta phn mutants. Genes for these pathways were proven not to be homologous and to lie in different chromosomal regions. The S. typhimurium phn locus lies near 10 min; the E. coli phn locus lies near 93 min. The S. typhimurium phn gene cluster is about 7.2 kb in length and, on the basis of gene fusion analysis, appears to consist of two (or more) genes or operons that are divergently transcribed. Like that of the E. coli phn locus, the expression of the S. typhimurium phn locus is activated under conditions of Pi limitation and is subject to Pho regulon control. This was shown both by complementation of the appropriate E. coli mutants and by the construction of S. typhimurium mutants with lesions in the phoB and pst loci, which are required for activation and inhibition of Pho regulon gene expression, respectively. Complementation studies indicate that the S. typhimurium phn locus probably includes genes both for phosphonate transport and for catalysis of C-P bond cleavage.     

Carbon-phosphorus bond cleavage by Gram-positive and Gram-negative soil bacteria

Summary Five soil bacterial isolates, originally selected for their ability to utilize the herbicide glyphosate as sole phosphorus source, were characterized with respect to their ability to use a range of other structurally-diverse phosphonates. Most showed broad substrate specificity and strains of Pseudomonas and of Bacillus megaterium were capable of degrading 14 of the other 15 phosphonates investigated. However no isolate was able to utilize isopropyl phosphanate, nor the phosphinate herbicide phosphinothricin. Growth rates on most phosphonates were significantly lower than those sustained by inorganic phosphate, and evidence was obtained for preferential utilization of the latter. In addition, the length of lag phase preceing growth on phosphonates varied widely. These characteristics are believed to reflect the diversity of routes by which such molecules enter bacterial cells and are metabolized.     

Phenotypic characterization of some strains of Bacillus sphaericus

Strains of Pseudomonas and of Bacillus megaterium , originally isolated from soil by their ability to cleave the carbon-phosphorus bond of the phosphonate herbicide glyphosate, were not only resistant to the broad-spectrum phosphonate antibiotics alafosfalin and fosfomycin at concentrations in excess of 2 mmol/1 but could also utilize each as sole phosphorus source. The extent to which their resistance is dependent upon antibiotic detoxification through C-P lyase activity is unclear.     

Carbon-phosphorus bond cleavage activity in cell-free extracts of Enterobacter aerogenes ATCC 15038 and Pseudomonas sp. 4ASW.

Carbon-phosphorus bond cleavage activity was investigated in cell-free extracts of Enterobacter aerogenes ATCC 15038 (IFO 12010) and Pseudomonas sp. 4ASW, strains known to utilize a range of phosphonates as sole phosphorus source. In vitro phosphonatase activity was detected in extracts of both organisms; however extensive analysis failed to detect any organic product from phosphonates other than phosphonoacetal dehyde. Non-specific liberation of phosphate was observed in Pseudomonas sp. 4ASW, associated with a single fraction of FPLC-purified extract, and is believed to result from the activity of cellular phosphatases.     

The herbicide glyphosate

Glyphosate has broad spectrum herbicidal activity against a wide range of annual and perennial weeds. The environmental properties of this herbicide such as its soil immobility, rapid soil inactivation and soil biodegradation are outstanding. This herbicide is practically non-toxic to non-plant life forms such as aquatic and avian species, animals and man. Metabolism studies with pure bacterial cultures indicate that glyphosate is metabolized to either aminomethylphosphonate and glyoxylate or sarcosine and phosphate in most bacteria. The enzyme C-P lyase, which catalyzes the cleavage of the carbon-phosphorus bond of phosphonates including glyphosate, appears to be complex, containing multiple subunits. Mode of action studies have demonstrated that glyphosate kills plants by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase, involved in the biosynthesis of aromatic compounds. The status of our understanding of these aspects of glyphosate is reviewed.     

Specific HIV gp120-cleaving antibodies induced by covalently reactive analog of gp120

We report the results of efforts to strengthen and direct the natural nucleophilic activity of antibodies (Abs) for the purpose of specific cleavage of the human immunodeficiency virus-1 coat protein gp120. Phosphonate diester groups previously reported to form a covalent bond with the active site nucleophile of serine proteases (Paul, S., Tramontano, A., Gololobov, G., Zhou, Y. X., Taguchi, H., Karle, S., Nishiyama, Y., Planque, S., and George, S. (2001) J. Biol. Chem. 276, 28314-28320) were placed on Lys side chains of gp120. Seven monoclonal Abs raised by immunization with the covalently reactive analog of gp120 displayed irreversible binding to this compound (binding resistant to dissociation with the denaturant SDS). Catalytic cleavage of biotinylated gp120 by three monoclonal antibodies was observed. No cleavage of albumin and the extracellular domain of the epidermal growth factor receptor was detected. Cleavage of model peptide substrates occurred on the C-terminal side of basic amino acids, and Km for this reaction was approximately 200-fold greater than that for gp120 cleavage, indicating Ab specialization for the gp120 substrate. A hapten phosphonate diester devoid of gp120 inhibited the catalytic activity with exceptional potency, confirming that the reaction proceeds via a serine protease mechanism. Irreversible binding of the hapten phosphonate diester by polyclonal IgG from mice immunized with gp120 covalently reactive analog was increased compared with similar preparations from animals immunized with control gp120, indicating induction of Ab nucleophilicity. These findings suggest the feasibility of raising antigen-specific proteolytic antibodies on demand by covalent immunization.