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 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.     

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.     

Involvement of the phosphate regulon and the psiD locus in carbon-phosphorus lyase activity of Escherichia coli K-12.

Escherichia coli K-12 can readily mutate to use methylphosphonic acid as the sole phosphorus source by a direct carbon-to-phosphorus (C-P) bond cleavage activity that releases methane and Pi. The in vivo C-P lyase activity is both physiologically and genetically regulated as a member of the phosphate regulon. Since psiD::lacZ(Mu d1) mutants cannot metabolize methylphosphonic acid, psiD may be the structural gene(s) for C-P lyase.     

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     

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.     

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.     

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.     

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     

Rhizobium (Sinorhizobium) meliloti phn Genes: Characterization and Identification of Their Protein Products

In Escherichia coli, the phn operon encodes proteins responsible for the uptake and breakdown of phosphonates. The C-P (carbon-phosphorus) lyase enzyme encoded by this operon which catalyzes the cleavage of C-P bonds in phosphonates has been recalcitrant to biochemical characterization. To advance the understanding of this enzyme, we have cloned DNA from Rhizobium (Sinorhizobium) meliloti that contains homologues of the E. coli phnG, -H, -I, -J, and -K genes. We demonstrated by insertional mutagenesis that the operon from which this DNA is derived encodes the R. meliloti C-P lyase. Furthermore, the phenotype of this phn mutant shows that the C-P lyase has a broad substrate specificity and that the organism has another enzyme that degrades aminoethylphosphonate. A comparison of the R. meliloti and E. coli phn genes and their predicted products gave new information about C-P lyase. The putative R. meliloti PhnG, PhnH, and PhnK proteins were overexpressed and used to make polyclonal antibodies. Proteins of the correct molecular weight that react with these antibodies are expressed by R. meliloti grown with phosphonates as sole phosphorus sources. This is the first in vivo demonstration of the existence of these hitherto hypothetical Phn proteins.     

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.     

膦酸天然产物中碳磷键生物合成机制的研究进展

膦酸天然产物是指生物体产生的结构中含有碳磷键的小分子化合物,它因为与细胞中的羧酸和磷酸化的化合物结构相似,常作为某些生命活动必需的酶抑制剂,表现出良好的生物抑制活性。已知的膦酸天然产物碳磷键合成机制可以分为四类:磷酸烯醇式丙酮酸变位酶(PepM)催化类、磷甲基转移酶(PhpK)催化类、由S-2-羟基丙基膦酸环氧化酶(HppE)催化类和一种未知的碳磷键合成机制。本文就膦酸天然产物中碳磷键生物合成机制进行综述。     

Degradation of organophosphonates by Penicillium citrinum

Utilization of organophosphonates as the sole source of phosphorus, carbon or nitrogen by a soil isolate of Penicillium citrinum was studied. Penicillium citrinum was found to utilize 2-aminoethylphosphonic and 2-oxoalkylphosphonic acids as the sole phosphorus source whereas 1-hydroxyalkylphosphonates as well as 1-aminoalkylphosphonates and their dipeptides did not support the growth of the fungus. The mould did not metabolize any of the phosphonates tested, when they served as the sole carbon or nitrogen source.
Penicillium citrinum is perhaps the first mould strain isolated from soil, shown to be capable of organophosphonate degradation.     

Phosphonate utilization by bacteria.

Bacteria able to use at least one of 13 ionic alkylphosphonates of O-alkyl or O,O-dialkyl alkylphosphonates as phosphorus sources were isolated from sewage and soil. Four of these isolates used 2-aminoethylphosphonic acid (AEP) as a sole carbon, nitrogen, and phosphorus source. None of the other phosphonates served as a carbon source for the organisms. One isolate, identified as Pseudomonas putida, grew with AEP as its sole carbon, nitrogen, and phosphorus source and released nearly all of the organic phosphorus as orthophosphate and 72% of the AEP nitrogen as ammonium. This is the first demonstration of utilization of a phosphonoalkyl moiety as a sole carbon source. Cell-free extracts of P. putida contained an inducible enzyme system that required pyruvate and pyridoxal phosphate to release orthophosphate from AEP; acetaldehyde was tentatively identified as a second product. Phosphite inhibited the enzyme system.     

Cloning of genes encoding for C-P lyase fromPseudomonas isolates PG2982 and GLC11: Identification of a cryptic allele on the chromosome ofP. aeruginosa

Two isolates ofPseudomonas sp., GLC11 and PG2982, can use glyphosate as a sole source of phosphorus. This ability is indicative of enzymatic cleavage of a carbon-phosphorus bond, and the enzyme has been named C-P lyase. We have cloned, inEscherichia coli, gene/s coding for C-P lyase on a broad host range cosmid pLA2917. Restriction fragment arrangement of cloned fragments of PG2982 and GLC11 has been established. Analysis by Southern hybridization between two clones revealed a strong homology between threePstI fragments of pPG-CP-14 (derived from PG2982) and pGC-CP-4 (derived from GLC11). With the construct pGC-CP-4 as a probe, the presence of a cryptic allele for C-P lyase has been demonstrated on the chromosome of the parent isolate,pseudomonas aeruginosa PAO1. It is suggested that genetic rearrangement such as frame shift or a point mutation activated the cryptic C-P lyase gene. Metabolism of glyphosate byE. coli carrying pPG-CP-14 or pGC-CP-4 has been demonstrated by radiometric experiments.     

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.     

Evidence for two phosphonate degradative pathways in Enterobacter aerogenes.

We screened mini-Mu plasmid libraries from Enterobacter aerogenes IFO 12010 for plasmids that complement Escherichia coli phn mutants that cannot use phosphonates (Pn) as the sole source of phosphorus (P). We isolated two kinds of plasmids that, unexpectedly, encode genes for different metabolic pathways. One kind complements E. coli mutants with both Pn transport and Pn catalysis genes deleted; these plasmids allow degradation of the 2-carbon-substituted Pn alpha-aminoethylphosphonate but not of unsubstituted alkyl Pn. This substrate specificity is characteristic of a phosphonatase pathway, which is absent in E. coli. The other kind complements E. coli mutants with Pn catalysis genes deleted but not those with both transport and catalysis genes deleted; these plasmids allow degradation of both substituted and unsubstituted Pn. Such a broad substrate specificity is characteristic of a carbon-phosphorus (C-P) lyase pathway, which is common in gram-negative bacteria, including E. coli. Further proof that the two kinds of plasmids encode genes for different pathways was demonstrated by the lack of DNA homology between the plasmids. In particular, the phosphonatase clone from E. aerogenes failed to hybridize to the E. coli phnCDEFGHIJKLMNOP gene cluster for Pn uptake and degradation, while the E. aerogenes C-P lyase clone hybridized strongly to the E. coli phnGHIJKLM genes encoding C-P lyase but not to the E. coli phnCDE genes encoding Pn transport. Specific hybridization by the E. aerogenes C-P lyase plasmid to the E. coli phnF, phnN, phnO, and phnP genes was not determined. Furthermore, we showed that one or more genes encoding the apparent E. aerogenes phosphonatase pathway, like the E. coli phnC-to-phnP gene cluster, is under phosphate regulon control in E. coli. This highlights the importance of Pn in bacterial P assimilation in nature.     

The ability of soil-borne fungi to degrade organophosphonate carbon-to-phosphorus bonds

The ability of a wide variety of soil-borne fungal strains to degrade four structurally different com pounds containing PC bonds, namely the naturally occurring amino acid ciliatine, the popular herbicide glyphosate, phosphonoacetic acid and 2-amino-3-phosphonopropionic acid, was studied in order to show that soil fungi may play an important role in the biodegradation of organophosphonates. Most of the strains appeared to utilize ciliatine as the sole source of phosphorus for growth. Only a limited number of strains were able to grow on the other phosphonates used in this work. The strains of Trichoderma harzianum, Scopulariopsis sp. and Aspergillus niger chosen for more detailed study show the ability to degrade ciliatine, glyphosate and also amino(3-methoxyphenyl)mehtylphosphonic acid effectively. Received: 14 May 1997 / Received revision: 10 June 1997 / Accepted: 14 June 1997