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.     

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.     

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     

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.     

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     

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.     

The role of three-tandem Pho Boxes in the control of the C-P lyase operon in a thermophilic cyanobacterium

Cyanobacteria have an inherited advantage in phosphonate phytoremediation. However, studies on phosphonate metabolism in cyanobacteria are rare and mostly focus on physiology and ecology. Here, C-P lyase gene cluster regulation in an undomesticated thermophilic Synechococcus OS-B′ was examined in Synechocystis sp. PCC6803, a unicellular cyanobacterial model. Phylogenetic and cluster synteny analysis of C-P lyase genes revealed a closer relationship between Syn OS-B′ and Thermus thermophilus, than with other cyanobacteria. Pho boxes were identified in the 5′-end-flanking region of the C-P lyase gene cluster, through which the downstream gene expression was regulated in a phosphate concentration-dependent manner. Unexpectedly, the phosphate concentration that thoroughly inhibited Pho boxes was almost two orders of magnitude higher than that of any natural or anthropogenic wastewater reported so far. The Pho boxes mediated regulation was achieved through the Pho regulon two-component system, and the absence of either SphS or SphR ablated the cell's ability to sense ambient phosphate changes. The three tandems of Pho boxes maintained inequivalent roles, of which the third tandem was not essential; however, it played a role in adjusting Pho boxes response in both positive and negative manner under phosphorus limitation.     

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.     

Identification of a copper-responsive two-component system on the chromosome of Escherichia coli K-12

Using a genetic screen we have identified two chromosomal genes, cusRS (ylcA ybcZ), from Escherichia coli K-12 that encode a two-component, signal transduction system that is responsive to copper ions. This regulatory system is required for copper-induced expression of pcoE, a plasmid-borne gene from the E. coli copper resistance operon pco. The closest homologs of CusR and CusS are plasmid-borne two-component systems that are also involved in metal responsive gene regulation: PcoR and PcoS from the pco operon of E. coli; CopR and CopS from the cop operon, which provides copper resistance to Pseudomonas syringae; and SilR and SilS from the sil locus, which provides silver ion resistance to Salmonella enterica serovar Typhimurium. The genes cusRS are also required for the copper-dependent expression of at least one chromosomal gene, designated cusC (ylcB), which is allelic to the recently identified virulence gene ibeB in E. coli K1. The cus locus may comprise a copper ion efflux system, because the expression of cusC is induced by high concentrations of copper ions. Furthermore, the translation products of cusC and additional downstream genes are homologous to known metal ion antiporters.     

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     

Identification of an operon required for ferrichrome iron utilization in Vibrio cholerae

Mutagenesis of Vibrio cholerae with TnphoA, followed by screening for fusions that were activated under low-iron conditions, led to the identification of seven independent fusion strains, each of which was deficient in the ability to utilize ferrichrome as a sole iron source for growth in a plate bioassay and had an insertion in genes encoding products homologous to Escherichia coli FhuA or FhuD. Expression of the gene fusions was independent of IrgB but regulated by Fur. We report here a map of the operon and the predicted amino acid sequence of FhuA, based on the nucleotide sequence. Unlike those of the E. coli fhu operon, the V. cholerae ferrichrome utilization genes are located adjacent and opposite in orientation to a gene encoding an ATP-binding cassette transporter homolog, but this gene, if disrupted, does not affect the utilization of ferrichrome in vitro.     

Molecular genetics of carbon-phosphorus bond cleavage in bacteria

Phosphonates (Pn) are a large class of organophosphorus molecules that have direct carbon-phosphorus (C - P) bonds in place of the carbon-oxygen-phosphorus ester bond. In bacteria two pathways exist for Pn breakdown for use as a P source: the phosphonatase and C - P lyase pathways. These pathways differ both in regard to their substrate specificity and their cleavage mechanism. The phosphonatase pathway acts on the natural Pn -aminoethylphosphonate(AEPn). In a two-step process it leads to cleavage of the C - P bond by a hydrolysis reaction requiring an adjacent carbonyl group. In contrast the C - P lyase pathway has a broad substrate specificity. It leads to cleavage of substituted Pn (such as AEPn) as well as unsubstituted Pn by a mechanism involving redox or radical chemistry. Due to its broad substrate specificity, the C - P lyase pathway is generally thought to be responsible for the breakdown of Pn herbicides (such as glyphosate) by bacteria. As a way to gain a more in-depth understanding of these Pn degradative pathways, their respective genes have been isolated and characterized. In the absence of a biochemical assay for the C - P lyase pathway such molecular approaches have been especially valuable. The roles of individual genes have been inferred from DNA sequence analysis and mutational effects. Genes for the C - P lyase pathway exist in a fourteen-gene operon that appears to encode both a binding protein-dependent Pn transporter and a C - P lyase. Genes for the phosphonatase pathway also exist in a gene cluster containing Pn uptake and degradative genes. A combination of biochemistry, molecular biology, and molecular genetics approaches has provided more detailed understanding of the mechanisms of C - P bond cleavage. Such basic information may provide a new handle for improvement of Pn degradation capabilities in bacteria, or in other cells in which the respective genes may be introduced and expressed.Abbreviations AEPn -aminoethylphosphonate - C carbon - kbp kilobase pair - kDa kilodalton - MPn methylphosphonate - P phosphorus - P _i inorganic phosphate - Pn phosphonate - psi phosphate starvation inducible     

Evidence that the Bacteroides thetaiotaomicron chondroitin lyase II gene is adjacent to the chondro-4-sulfatase gene and may be part of the same operon.

The chondroitin lyase II gene from Bacteroides thetaiotaomicron has previously been cloned in Escherichia coli on a 7.8-kilobase (kb) fragment (pA818). In E. coli, the chondroitin lyase II gene appeared to be expressed from a promoter that was about 0.5 kb from the beginning of the gene. However, when a subcloned 5-kb fragment from pA818 which contained the chondroitin lyase II gene and the promoter from which the gene is expressed in E. coli was introduced into B. thetaiotaomicron on a multicopy plasmid (pEG800), the chondroitin lyase specific activity of B. thetaiotaomicron was not altered. Further evidence that the promoter that is recognized in E. coli may not be the promoter from which the chondroitin lyase II gene is transcribed in B. thetaiotaomicron was obtained by making an insertion in the B. thetaiotaomicron chromosome at a point which is 1 kb upstream from the chondroitin lyase II gene. This insertion stopped synthesis of the chondroitin lyase II gene product, as would be predicted if the gene was part of an operon and was transcribed in B. thetaiotaomicron from a promoter that was at least 1 kb upstream from the chondroitin lyase II gene. A region of pA818 which was adjacent to the chondroitin lyase II gene and which included the region used to make the insertional mutation was found to code for chondro-4-sulfatase, an enzyme that breaks down one of the products of the chondroitin lyase reaction. The upstream insertion mutant of B. thetaiotaomicron which stopped synthesis of chondroitin lyase II had no detectable chondro-4-sulfatase activity. This mutant was still able to grow on chondroitin sulfate, although the rate of growth was slower than that of the wild type.     

Cloning and sequencing of the genes involved in glyphosate utilization by Pseudomonas pseudomallei.

Thirty-four strains of Pseudomonas pseudomallei isolated from soil were selected for their ability to degrade the phosphonate herbicide glyphosate. All strains tested were able to grow on glyphosate as the only phosphorus source without the addition of aromatic amino acids. One of these strains, P. pseudomallei 22, showed 50% glyphosate degradation in 40 h in glyphosate medium. From a genomic library of this strain constructed in pUC19, we have isolated a plasmid carrying a 3.0-kb DNA fragment which confers to E. coli the ability to use glyphosate as a phosphorus source. This 3.0-kb DNA fragment from P. pseudomallei contained two open reading frames (glpA and glpB) which are involved in glyphosate tolerance and in the modification of glyphosate to a substrate of the Escherichia coli carbon-phosphorus lyase. glpA exhibited significant homology with the E. coli hygromycin phosphotransferase gene. It was also found that the hygromycin phosphotransferase genes from both P. pseudomallei and E. coli confer tolerance to glyphosate.     

Phosphonate metabolism in Helicobacter pylori

Helicobacter pylori has been shown to degrade two phosphonates, N-phosphonoacetyl-l-aspartate and phosphonoacetate; however, the bacterium does not have any genes homologous to those of the known phosphonate metabolism pathways suggesting that H. pylori may have a novel phosphonate metabolism pathway. Growth of H. pylori on phosphonates was studied and the catabolism of these compounds was measured employing ¹H-nuclear magnetic resonance spectroscopy. The specificity of the catabolic enzymes was elucidated by assaying the degradation of several phosphonates and through substrate competition studies. H. pylori was able to utilise phenylphosphonate as a sole source of phosphate for growth. Three strains of H. pylori showed sigmoidal enzyme kinetics of phenylphosphonate catabolism. Allosteric kinetics were removed when lysates were fractionated into cytosolic and membrane fractions. Catabolic rates increased with the addition of DTT, Mg²⁺ and phosphate and decreased with the addition of EDTA. The physiological properties of H. pylori phosphonate metabolism were characterised and the presence of at least two novel phosphonate catabolism pathways that do not require phosphate starvation growth conditions for activity has been established.