A role for carbon catabolite repression in the metabolism of phosphonoacetate by Agromyces fucosus Vs2

A strain of Agromyces fucosus, designated Vs2, metabolized a range of organophosphonate compounds as sole phosphorus sources for growth and metabolized phosphonoacetate as a sole carbon, energy and phosphorus source for growth. With phosphonoacetate as the sole phosphorus source and a pyruvate carbon source, transient phosphate release to the medium was observed, in contrast to cultures grown with glucose and phosphonoacetate, where no phosphate release to the medium was observed. Carbon catabolite repression, specifically by means of inducer exclusion of phosphonoacetate, was proposed as the mechanism responsible, and phosphonoacetate hydrolase enzyme assays carried out on cell extracts confirmed that induced phosphonoacetate hydrolase activities were indeed higher in cells grown on pyruvate with phosphonoacetate as sole phosphorus source. This phenomenon has not previously been demonstrated in vivo, and must represent a significant metabolic control of organophosphonate metabolism. The catabolite repression phenomenon was also evident when A. fucosus grew on 2-aminoethylphosphonate as sole phosphorus source, allowing demonstration of a third mode of control for biodegradation of this compound. Excision of stained zymogram gel pieces, followed by tryptic digestion and mass spectrometric analysis, allowed the identification of phosphonoacetate hydrolase-derived peptides.     

Utilization of organophosphonates by environmental micro-organisms

A survey of the utilization by environmental micro-organisms of a range of compounds containing the carbon–phosphorus (C–P) bond was carried out. Elective culture studies indicated that 15 of 19 alkylphosphonates tested served only as a sole source of phosphorus for microbial growth. Their metabolism did not lead to the extracellular release of inorganic phosphate. However, four organophosphonates—phosphonoacetate, phosphonoalanine, 2-aminoethylphosphonate and phosphonomycin—supported microbial growth when supplied as either a phosphorus source or as a carbon and energy source, with near-quantitative inorganic phosphate release. Four of five aminoalkylphosphonates tested were also utilized as a nitrogen source in the presence of 1 mmol l⁻¹ inorganic phosphate. In a subsequent screening programme, 99% of bacterial isolates tested were able to utilize 2-aminoethylphosphonate as a sole phosphorus source, 61% as a nitrogen source, 10% as a source of nitrogen and phosphorus, and 2% as a source of carbon, nitrogen and phosphorus ; 2% of isolates used phosphonoalanine as a nitrogen source. These results suggest that the uptake and metabolism of organophosphonates by bacteria is less `tightly' regulated by phosphorus starvation than has previously been supposed.     

Detection of phosphonoacetate degradation and phnA genes in soil bacteria from distinct geographical origins suggest its possible biogenic origin

Phosphonoacetate is regarded as an antiviral xenobiotic whose mineralization can be catalysed by an enzyme, phosphonoacetate hydrolase, encoded by the phnA gene. To date the enzyme's activity has been detected in only a limited number of bacteria. Its expression has been shown to occur in a manner independent of the phosphate status of the cell, in direct contrast to the general rule of organophosphonate metabolism being under the control of the pho regulon. In this study the environmental occurrence of the phnA gene was evaluated by polymerase chain reaction amplification of DNA extracts obtained directly from various soil environments. Sensitivity of this method was improved such that a positive result was routinely obtained with soil spiked with as few as 6 colony-forming units (cfu) per gram of soil of Pseudomonas fluorescens 23F (phnA(+)). When total DNA from a variety of Northern Irish, Greek and Bolivian soils was tested, all were positive for phnA. Bacteria capable of utilizing phosphonoacetate as sole carbon, energy and phosphorus source, with the release of essentially equimolar concentrations of phosphate to the culture supernatant, were isolated from all soil samples tested. Analysis of three such isolates revealed all to be species of Pseudomonas sensu stricto, possessing phosphonoacetate hydrolase activity in cell-free extracts. Sequence determination of the phnA gene revealed a similarity of the putative protein sequences at levels of 98.3-99.3% between the Pseudomonas strains. This is the first study to use molecular methods to investigate the distribution of a gene encoding organophosphonate metabolism, and indicates that the phnA gene is ubiquitous within soils from geographically distinct regions. Such an observation supports the proposition that phosphonoacetate is a compound that may also have a biogenic origin.     

Importance of Gram-positive naphthalene-degrading bacteria in oil-contaminated tropical marine sediments

AIMS: The aim of this study was to isolate, characterize and evaluate the importance of naphthalene-degrading bacterial strains from oil-contaminated tropical marine sediments. METHODS AND RESULTS: Three Gram-positive naphthalene-degrading bacteria were isolated from oil-contaminated tropical intertidal marine sediments by direct isolation or enrichment using naphthalene as the sole source of carbon and energy. Bacillus naphthovorans strain MN-003 can also grow on benzene, toluene, xylene and diesel fuel while Micrococcus sp. str. MN-006 can also grow on benzene. Staphylococcus sp. str. MN-005 can only degrade naphthalene and was not able to use the other aromatic hydrocarbons tested. Strain MN-003 possessed the highest maximal specific growth rate with naphthalene as sole carbon source. An enrichment culture fed with naphthalene as sole carbon source exhibited a significant increase in the relative abundances of the three isolates after 21 days of incubation. The three isolates constituted greater than 69% of the culturable naphthalene-degrading microbial community. Strain MN-003 outcompeted and dominated the other two isolates in competition studies involving batch cultures inoculated with equal cell densities of the three isolates and incubated with between 1 and 10 mg l-1 of naphthalene. CONCLUSIONS: Three Gram-positive naphthalene-degrading bacteria were successfully isolated from oil-contaminated tropical marine sediments. Gram-positive bacteria might play an important role in naphthalene degradation in the highly variable environment of oil-contaminated tropical intertidal marine sediments. Among the three isolates, strain MN-003 has the highest maximal specific growth rate when grown on naphthalene, and outgrew the other two isolates in competition experiments. SIGNIFICANCE AND IMPACT OF THE STUDY: This research will aid in the development of bioremediation schemes for oil-contaminated marine environments. Strain MN-003 could potentially be exploited in such schemes.     

Divergence of chemical function in the alkaline phosphatase superfamily: structure and mechanism of the P-C bond cleaving enzyme phosphonoacetate hydrolase

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and (32)P-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group.     

A comparison of three bacterial phosphonoacetate hydrolases from different environmental sources

Cleavage of the carbon–phosphorus bond of the xenobiotic phosphonoacetate by phosphonoacetate hydrolase represents a novel route for the microbial metabolism of organophosphonates, and is unique in that it is substrate-inducible and its expression is independent of the phosphate status of the cell. The enzyme has previously only been demonstrated in cell extracts of Pseudomonas fluorescens 23F. Phosphonoacetate hydrolase activity is now reported in extracts of environmental Curtobacterium sp. and Pseudomonas sp. isolates capable of the phosphate-insensitive mineralization of phosphonoacetate as the sole source of carbon, energy and phosphorus at concentrations up to 40 mmol l⁻¹ and 100 mmol l⁻¹, respectively. The enzymes in both strains were similarly inducible by phosphonoacetate and had a unique specificity for this substrate. However, they differed significantly from each other, and from the previously described Ps. fluorescens 23F enzyme, in respect of their apparent molecular masses, temperature optima, thermostability, sensitivity to inhibition by chelating agents and by structural analogues of phosphonoacetate, and in their affinities for the substrate.     

Utilization of hexamethylenetetramine (urotropine) by bacteria and yeasts

A slow growing bacterial population able to utilize hexamethylelenetetramine (urotropine) as sole source of carbon, nitrogen and energy was isolated from soil. From this crude enrichment culture two bacteria were isolated and identified as Brevundimonas diminuta and a Phyllobacterium sp. by sequencing of 16S ribosomal DNA. These bacteria also grew on urotropine but at a lower rate than the enrichment culture. Addition of glucose to the latter resulted in growth of some yeasts that overgrew the bacteria. Assimilation of urotropine as sole nitrogen source is very common among yeasts, 46 out of 60 species tested showed this characteristic.     

Isolation of dieldrin- and endrin-degrading bacteria using 1,2-epoxycyclohexane as a structural analog of both compounds

This report describes the selective isolation of dieldrin- and endrin-degrading bacteria from soil with high degradation activity toward dieldrin and endrin. Several enrichment cultures from the soil were arranged with several structural analogs of dieldrin and endrin as a growth substrate and examined for their degradation activities toward dieldrin and endrin. An enrichment culture with 1,2-epoxycyclohexane (ECH) was found to aerobically degrade dieldrin and endrin. Denaturing gradient gel electrophoresis (DGGE) indicated that three types of bacteria were predominant in the ECH enrichment culture. Of the three major bacteria, two isolates, Burkholderia sp. strain MED-7 and Cupriavidus sp. strain MED-5, showed high degradation activity toward dieldrin and endrin. The degradation efficiencies of strain MED-7 and MED-5 were 49% and 38% toward dieldrin, respectively, and 51% and 40% toward endrin, respectively, in the presence of ECH for 14 days. These results indicated that ECH was a useful substrate for selective and efficient isolation of dieldrin- and endrin-degrading bacteria from soil containing numerous bacteria. Interestingly, the two isolates could also degrade dieldrin and endrin even in the absence of ECH. These are the first microorganisms demonstrated to grow on dieldrin and endrin as the sole carbon and energy source under aerobic conditions.     

Monitoring of an Alkaline 2,4,6-Trichlorophenol-Degrading Enrichment Culture by DNA Fingerprinting Methods and Isolation of the Responsible Organism, Haloalkaliphilic Nocardioides sp. Strain M6

A site situated near Alkali Lake (Oregon) and highly contaminated by chloroaromatic compounds was chosen for isolation of alkaliphilic chlorophenol-degrading bacteria. Prolonged cultivation of an enrichment culture followed by successive transfers resulted in a strong increase in the 2,4,6-trichlorophenol (2,4,6-TCP) degradation rate. Repetitive extragenic palindromic PCR and amplified ribosomal DNA restriction analysis were applied to distinguish members of the enrichment culture and monitor them during the enrichment procedure. Comparison of the fingerprints of the isolates obtained from the enrichment culture and its total DNA fingerprint indicated the presence of an unidentified bacterium in the enrichment culture, assisting in its isolation. The 2,4,6-TCP-degrading isolate, M6, was tentatively identified as a Nocardioides sp. strain based on its partial 16S RNA sequence and fatty acid profile. Strain M6 was capable of utilizing up to 1.6 g of 2,4,6-TCP per liter as a sole carbon and energy source and could also grow on 2,4-dichlorophenol and 2,4,5-trichlorophenol. A high-cell-density suspension of this strain degraded a wide range of chlorinated phenols from di- to pentachlorophenol while showing a clear preference for phenols containing chlorine substituents in positions 2 plus 4. Based on its optimal pH (9.0 to 9.4) and sodium ion concentration (0.2 to 0.4 M) for growth, Nocardioides sp. strain M6 is a slightly halophilic alkaliphile.     

Isolation of isoproturon-degrading bacteria from treated soil via three different routes

Three different isolation routes (flask enrichment/flask degradation assay, flask enrichment/microplate degradation assay, MPN assay/microplate degradation assay) were used to obtain pure cultures of bacteria which degraded isoproturon (3-(4-isopropylphenyl)-1,1-dimethylurea) as sole carbon and nitrogen source in a mineral salts medium from a field soil treated with isoproturon in the laboratory. All three isolation routes were successful, but the microplate assay of degradation was more successful than the flask assay. Characterization of 36 isolates indicated that they formed 16 distinct phenotypes (10 Gram-positive phenotypes, six Gram-negative phenotypes) which are likely to represent distinct species. Low concentrations of the degradation product 3-(4-isopropylphenyl)-1-methylurea (IPPMU) were occasionally found in the culture solutions. When provided as the sole source of carbon and nitrogen, the monomethyl degradation product was itself rapidly degraded by several of the isolates. Some isolates were also able to use the demethylated degradation product 3-(4-isopropylphenyl)-urea (IPPU) as sole source of carbon and nitrogen, although there was occasionally an extended lag-phase before rapid degradation commenced. One isolate was particularly active and degraded isoproturon, the monomethyl and demethylated degradation products of isoproturon, and demethylated the related phenylureas diuron and linuron.     

Detection and isolation of chloromethane-degrading bacteria from the Arabidopsis thaliana phyllosphere, and characterization of chloromethane utilization genes

Chloromethane gas is produced naturally in the phyllosphere, the compartment defined as the aboveground parts of vegetation, which hosts a rich bacterial flora. Chloromethane may serve as a growth substrate for specialized aerobic methylotrophic bacteria, which have been isolated from soil and water environments, and use cmu genes for chloromethane utilization. Evidence for the presence of chloromethane-degrading bacteria on the leaf surfaces of Arabidopsis thaliana was obtained by specific quantitative PCR of the cmuA gene encoding the two-domain methyltransferase corrinoid protein of chloromethane dehalogenase. Bacterial strains were isolated on a solid mineral medium with chloromethane as the sole carbon source from liquid mineral medium enrichment cultures inoculated with leaves of A. thaliana. Restriction analysis-based genotyping of cmuA PCR products was used to evaluate the diversity of chloromethane-degrading bacteria during enrichment and after strain isolation. The isolates obtained, affiliated to the genus Hyphomicrobium based on their 16S rRNA gene sequence and the presence of characteristic hyphae, dehalogenate chloromethane, and grow in a liquid culture with chloromethane as the sole carbon and energy source. The cmu genes of these isolates were analysed using new PCR primers, and their sequences were compared with those of previously reported aerobic chloromethane-degrading strains. The three isolates featured a colinear cmuBCA gene arrangement similar to that of all previously characterized strains, except Methylobacterium extorquens CM4 of known genome sequence.     

Cytosolic CP bond cleavage activity in bacterial cells isolated from soil

Three kinds of bacteria (CP1, CP9 and CP10), able to accumulate inorganic phosphate (Pi) in a growth medium containing phosphonoacetate as a sole source of phosphorus, were isolated from two hundred soil samples. CP bond cleavage activity in these strains was determined using extracts prepared from cells grown on a medium containing phosphonoacetate. The activity was not found in cell extracts of CP1. Cell extracts prepared from CP9 catalyzed the liberation of Pi only from phosphonoacetate and 2-aminoethylphosphonate. The cell size of CP10 was abnormally large compared with that of CP1 and CP9, and the extracts of CP10 catalyzed the cleavage of CP bonds in methylphosphonate, phosphonoacetate, phenylphosphonate, 2-amino-ethylphosphonate, 2-amino-4-phosphonobutyrate, glyphosate and in phosphonomycine.     

Phosphate removal from the returned liquor of municipal wastewater treatment plant using iron-reducing bacteria

AIM: The application of iron-reducing bacteria (IRB) to phosphate removal from returned liquor (liquid fraction after activated sludge digestion and anaerobic sludge dewatering) of municipal wastewater treatment plant (WWTP) was studied. METHODS AND RESULTS: An enrichment culture and two pure cultures of IRB, Stenotrophomonas maltophilia BK and Brachymonas denitrificans MK identified by 16S rRNA gene sequencing, were produced using returned liquor from a municipal WWTP as carbon and energy source, and iron hydroxide as oxidant. The final concentration of phosphate increased from 70 to 90 mg l(-1) in the control and decreased from 70 to 1 mg l(-1) in the experiment. The mass ratio of removed P to produced Fe(II) was 0.17 g P g(-1) Fe(II). The strain S. maltophilia BK showed the ability to reduce Fe(III) using such xenobiotics as diphenylamine, m-cresol, 2,4-dichlorphenol and p-phenylphenol as sole sources of carbon under anaerobic conditions. CONCLUSIONS: Bacterial reduction of ferric hydroxide enhanced the phosphate removal from the returned liquor. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of the facultative anaerobes S. maltophilia BK and B. denitrificans MK to reduce Fe(III) was shown. These micro-organisms can be used for anaerobic removal of phosphate and xenobiotics by bacterial reduction of ferric ions.     

Effect of Different Carbon Sources on Community Composition of Bacterial Enrichments from Soil

Soil is a highly heterogeneous matrix, which can contain thousands of different bacterial species per gram. Only a small component of this diversity (maybe <1%) is commonly captured using standard isolation techniques, although indications are that a larger proportion of the soil community is in fact culturable. Better isolation techniques yielding greater bacterial diversity would be of benefit for understanding the metabolic activity and capability of many soil microorganisms. We studied the response of soil bacterial communities to carbon source enrichment in small matrices by means of terminal restriction fragment length polymorphism (TRFLP) analysis. The community composition of replicate enrichments from soil displayed high variability, likely attributable to soil heterogeneity. An analysis of TRFLP data indicated that enrichment on structurally similar carbon sources selected for similar bacterial communities. The same analysis indicated that communities first enriched on glucose or benzoate and subsequently transferred into medium containing an alternate carbon source retained a distinct community signature induced by the carbon source used in the primary enrichment. Enrichment on leucine presented a selective challenge that was able to override the imprint left by primary enrichment on acetate. In a time series experiment community change was most rapid 18 hours after inoculation, corresponding to exponential growth. Community composition did not stabilize even 4 days after secondary enrichment. Four different soil types were enriched on four different carbon sources. TRFLP analysis indicated that in three out of four cases communities enriched on the same carbon source were more similar regardless of which soil type was used. Conversely, the garden soil samples yielded similar enrichment communities regardless of the enrichment carbon source. Our results indicate that in order to maximize the diversity of bacteria recovered from the environment, multiple enrichments should be performed using a chemically diverse set of carbon sources.     

Effect of different carbon sources on community composition of bacterial enrichments from soil

Soil is a highly heterogeneous matrix, which can contain thousands of different bacterial species per gram. Only a small component of this diversity (maybe <1%) is commonly captured using standard isolation techniques, although indications are that a larger proportion of the soil community is in fact culturable. Better isolation techniques yielding greater bacterial diversity would be of benefit for understanding the metabolic activity and capability of many soil microorganisms. We studied the response of soil bacterial communities to carbon source enrichment in small matrices by means of terminal restriction fragment length polymorphism (TRFLP) analysis. The community composition of replicate enrichments from soil displayed high variability, likely attributable to soil heterogeneity. An analysis of TRFLP data indicated that enrichment on structurally similar carbon sources selected for similar bacterial communities. The same analysis indicated that communities first enriched on glucose or benzoate and subsequently transferred into medium containing an alternate carbon source retained a distinct community signature induced by the carbon source used in the primary enrichment. Enrichment on leucine presented a selective challenge that was able to override the imprint left by primary enrichment on acetate. In a time series experiment community change was most rapid 18 hours after inoculation, corresponding to exponential growth. Community composition did not stabilize even 4 days after secondary enrichment. Four different soil types were enriched on four different carbon sources. TRFLP analysis indicated that in three out of four cases communities enriched on the same carbon source were more similar regardless of which soil type was used. Conversely, the garden soil samples yielded similar enrichment communities regardless of the enrichment carbon source. Our results indicate that in order to maximize the diversity of bacteria recovered from the environment, multiple enrichments should be performed using a chemically diverse set of carbon sources.     

Cloning of the phosphonoacetate hydrolase gene from Pseudomonas fluorescens 23F encoding a new type of carbon–phosphorus bond cleaving enzyme and its expression in Escherichia coli and Pseudomonas putida

The phnA gene encoding a novel carbon–phosphorus bond cleavage enzyme, phosphonoacetate hydrolase, from Pseudomonas fluorescens 23F was cloned and expressed in Escherichia coli and Pseudomonas putida. It conferred on the latter host the ability to mineralize phosphonoacetate but on the former the ability to utilize it as sole phosphorus source only. The nucleotide and deduced amino acid sequences of the phnA gene showed no significant homology with any data bank accessions.     

In vitro characterization of a phosphate starvation-independent carbon-phosphorus bond cleavage activity in Pseudomonas fluorescens 23F.

A novel, metal-dependent, carbon-phosphorus bond cleavage activity, provisionally named phosphonoacetate hydrolase, was detected in crude extracts of Pseudomonas fluorescens 23F, an environmental isolate able to utilize phosphonoacetate as the sole carbon and phosphorus source. The activity showed unique specificity toward this substrate; its organic product, acetate, was apparently metabolized by the glyoxylate cycle enzymes of the host cell. Unlike phosphonatase, which was also detected in crude extracts of P. fluorescens 23F, phosphonoacetate hydrolase was inducible only in the presence of its sole substrate and did not require phosphate starvation.     

Hydrogen-using bacteria in a methanogenic acetate enrichment culture

A rcher , D.B. 1984. Hydrogen-using bacteria in a methanogenic acetate enrichment culture. Journal of Applied Bacteriology 56 , 125–129.
In a study of the anaerobic utilization of acetate, an enrichment culture of sewage sludge organisms was initiated with calcium acetate as the sole carbon and energy source. A mixed bacterial population became established from which 14 anaerobic species were isolated. Two of the isolates were methanogenic bacteria but only one of these, Methanosarcina barkeri , utilised acetate as an energy source in axenic culture. The other methanogenic isolate, a Methanobacterium sp., utilised H₂/CO₂ but not acetate. A third methanogen, which was morphologically identical to Methanothrix soehngenii , was detected in the enrichment but was not obtained in monoculture. 2-Bromoethanesulphonate, a specific inhibitor of methanogenesis. completely inhibited the enrichment at a concentration of 10 μmol/1. Addition of H₂ formate or methanol to the enrichment did not affect the rate of methanogenesis. An H₂-utilizing Desulfovibrio sp. was also isolated from the enrichment.     

Metabolism of Phosphonoacetate as the Sole Carbon and Phosphorus Source by an Environmental Bacterial Isolate

A gram-negative bacterium isolated from activated sludge was able to utilize up to 25 mM phosphonoacetate as the sole carbon and phosphorus source, with simultaneous excretion of virtually equimolar levels of phosphate. 2-Aminoethylphosphonate was similarly utilized with equivalent growth rates and cellular yields, while 3-aminopropyl-, 4-aminobutyl-, methyl-, ethyl-, and phenylphosphonates served only as phosphorus sources.