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Organophosphonate metabolism by a moderately halophilic bacterial isolate

Velma E.A. Hayes, Nigel G. Ternan, Geoffrey McMullan
DOI: http://dx.doi.org/10.1111/j.1574-6968.2000.tb09099.x 171-175 First published online: 1 May 2000

Abstract

A Gram-negative halophile isolated from soil beneath a road gritting salt pile grew optimally at 10% (w/v) NaCl and was shown most likely to be Chromohalobacter marismortui or Pseudomonas beijerinckii on the basis of 16S rRNA analysis. The strain utilised phosphonoacetate, 2-aminoethyl-, 3-aminopropyl-, 4-aminobutyl-, methyl- and ethyl-phosphonates as phosphorus sources for growth. Differences were observed in the growth rate on different phosphonates and the range of phosphonates utilised at elevated NaCl concentrations, possibly as a result of differentially-induced transport mechanisms. An assay of cell-free extracts of 2-aminoethylphosphonate (2AEP) grown cells showed no detectable 2AEP:pyruvate aminotransferase or phosphonoacetaldehyde hydrolase activity.

Keywords
  • Organophosphonate
  • Biodegradation
  • Halophilic
  • Pho regulon
  • C–P lyase

1 Introduction

The organophosphonates are a class of organophosphorus compounds characterised by the presence of a stable, covalent carbon to phosphorus (C–P) bond which imparts upon these molecules a relative resistance to chemical, thermal, photolytic and enzymatic (bio)degradation. While the existence of organophosphonates was first described in 1944 with the synthesis of aminomethylphosphonic acid, the existence of natural organophosphonates was not demonstrated until 1959, when 2-aminoethylphosphonic acid (2AEP) was identified in an extract of rumen protozoa [1].

Both natural and synthetic organophosphonates are of importance, with the latter being utilised extensively in the chemical industry as detergents, plasticisers, coolant additives and pesticides, with the result that thousands of tonnes of these compounds enter the environment annually [2]. By far the most important use of synthetic organophosphonates, however, is as herbicides, with glyphosate (N-phosphonomethyl-glycine) and phosphinothricin (Basta™) being leading examples. Indeed, glyphosate, the world's leading agrochemical, is worth in excess of US$1 billion per year to its manufacturer, Monsanto Agricultural Products Company, St. Louis, MO, USA. Natural organophosphonates of significance include antimicrobial compounds such as phosphonomycin and natural herbicides such as Bialaphos (L-alanyl-L-alanyl-phosphinothricin) and phosphonothrixin [1].

Once organophosphonates enter the soil environment, microbial activity has been found to be almost exclusively responsible for their removal [3]. However, the vast majority of microorganisms studied to date are only capable of utilising organophosphonates as sole phosphorus sources for growth via cleavage of the C–P bond. In these isolates, organophosphonate biodegradation has been shown to be under control of the pho (or analogous) regulon which is expressed only under conditions of environmental phosphate limitation [4]. Recently a number of novel bacteria have been described which are capable of mineralising organophosphonates as carbon, nitrogen and phosphorus sources in a phosphate starvation independent manner [1]. Subsequently, additional reports have described the mineralisation of phosphonoalanine by a strain of Burkholderia cepacia[5] and the natural antibiotic, phosphonomycin, was found by McGrath et al. [6] to be mineralised by a strain of Rhizobium huakuii. In all such cases, the presence of inorganic phosphate did not affect the uptake or metabolism of the organophosphonates, a significant departure from the accepted consensus.

The metabolism of organophosphonates has traditionally been studied in greatest detail within soil and soil microorganisms, largely due to the interest within the scientific community as to the environmental fate of the herbicide glyphosate. Few attempts have been made to investigate the biodiversity of microorganisms capable of degrading organophosphonates [1], with the exception of investigations into the ability of filamentous fungi and streptomycete strains to degrade these compounds [7,8]. The microbial diversity held within more extreme environments to those previously investigated could offer novel biodegradative pathways for organophosphonates, including previously uncharacterised enzymes and modes of regulation. We now report for the first time the isolation of a halophilic microorganism with the ability to cleave the C–P bond of a number of synthetic and natural organophosphonate compounds.

2 Materials and methods

2.1 Chemicals

Chemicals, of the highest purity available, reagents for phosphate determination and organophosphonates were obtained from Sigma (Poole, UK), except for the following compounds: methylphosphonate and ethylphosphonate (Lancaster Synthesis, Morecambe, UK), N-(phosphonomethyl)-iminodiacetic acid (PIA; Aldrich, Poole, UK) and 2-phosphonoacetaldehyde (QUCHEM, Belfast, UK).

2.2 Media

Selective halophile medium [9] (pH 7.4) contained (per l): bacto-casitone (Difco), 7.5 g; yeast extract, 10.0 g; trisodium citrate, 3.0 g; KCl, 2.0 g; MgSO4·7H2O, 20.0 g; FeCl3, 0.023 g. NaCl was added at a concentration between 0 and 25% (w/v) to this medium, which was then made up to 80% of the final volume, autoclaved at 121°C for 5 min, filtered through a Whatman No. filter paper and made up to full volume with deionised water prior to autoclaving at 121°C for 15 min.

Defined halophile medium (pH 7.4) contained (per l): Tris–HCl, 6.61 g; Tris–base, 0.97 g; trisodium citrate, 3.0 g; KCl, 2.0 g; MgSO4·7H2O, 20.0 g; FeCl3, 0.023 g, sodium pyruvate, 2.5 g; sodium succinate, 2.5 g; MEM-amino acids solution (Sigma), 20.0 ml; phosphate-free yeast extract [10], 0.1 g; vitamin solution [10], 1.0 ml, and trace element solution [10], 1.0 ml. As per the selective halophile medium, NaCl was added prior to autoclaving at concentrations between 0 and 25% (w/v). Heat labile substrates (amino acids, yeast extract, vitamins, sodium succinate, sodium pyruvate) were filter sterilised (0.2-μm pore size) and added aseptically after autoclaving. Filter-sterilised (0.2 μm pore size) solutions of organophosphonates were added aseptically after autoclaving as either a sole phosphorus source (1.0 mM) or as a sole nitrogen source (5.0 mM).

2.3 Isolation of microorganisms

The isolation of halophilic microorganisms was undertaken using an inoculum of soil from underneath a road gritting salt pile located at Rasharkin, Northern Ireland. To a 15% (w/v) NaCl solution was added 20% (w/v) soil; the resulting suspension was mixed continuously on a roller for 30 min. The resultant homogeneous suspension was used as an inoculum (0.5%) in Erlenmeyer flasks (250 ml) containing 50 ml selective halophile medium with 15% (w/v) NaCl and incubated at 37°C (175 rpm).

2.4 Effect of NaCl concentration on growth

Selective halophile medium containing a range of NaCl concentrations was employed to determine the optimum NaCl concentration for growth. Cells taken from the mid-exponential phase of growth in halophile medium containing 5% (w/v) NaCl were used as an inoculum and growth was followed as the OD650 using a NovaSpec II spectrophotometer (Pharmacia).

2.5 Screening for organophosphonate utilisation

The ability of isolates to utilise a range of organophosphonates as sole phosphorus source (final concentration, 1.0 mM) or as sole nitrogen source (final concentration, 5.0 mM) in defined halophile medium at NaCl concentrations of 5.0, 10.0 and 15.0% (w/v) was investigated. Mid-exponential phase cells grown in phosphate-free basal salts medium with 5, 10 or 15% (w/v) NaCl were used as inocula. Phosphate release during growth was assayed for by the method of Fiske and SubbaRow [11]. Maximal growth was expressed as the yield of protein ml−1 from cultures as determined by the method of Binks et al. [12].

2.6 Preparation of cell extracts

Cells were grown in defined halophile medium containing 5% (w/v) NaCl with 1.0 mM 2AEP as sole phosphorus source and harvested in the mid-exponential phase of growth by centrifugation at 9000×g at 4°C for 10 min. Cells were washed once in 50 mM Tris–NaOH buffer (pH 7.5) containing 5 mM MgCl2 and resuspended at 25% w/v in the same buffer containing 0.5 mM dithiothreitol and 0.5 mM mercaptoethanol. Cell extracts were prepared by sonicating the cell suspension using the microtip of an MSE Soniprep 150 at full power for 5 min (30-s sonication followed by 2-min cooling). The resultant homogenate was centrifuged at 25 000×g at 4°C for 30 min and the supernatant was used as crude cell extract. The protein concentration in the cell extract was determined by the method of Bradford [13].

2.7 Cell-free enzyme assays

Cell extracts were assayed for the presence of 2AEP:pyruvate aminotransferase and phosphonatase by the method of Ternan and Quinn [14] at 30°C. Transaminase assay supernatants were analysed qualitatively by thin layer chromatography [15]. Phosphonatase activity was expressed as nmol inorganic phosphate released min−1 mg−1 cell extract protein. Phosphate release was determined by the method of Fiske and SubbaRow [11]. Bacterial alkaline phosphatase activity in cell extracts was assayed by the method of Ternan and Quinn [14].

3 Results and discussion

Hypersaline environments are a typical type of ‘extreme’ habitat in which microbial diversity can be found and include marine biotopes, salt mines and terrestrial salt dumps. The spectrum of organisms that inhabit these saline conditions is regulated by such factors as ionic composition, temperature, pH, O2 solubility and, most obviously, salinity [16]. Here, we describe a bacterium isolated from a hypersaline environment capable of metabolising both biogenic and synthetic organophosphonates.

Following the inoculation of selective halophile medium, growth was observed in the flasks after 7 days incubation at 37°C. Individual isolates were obtained by plating onto solid selective halophile medium containing 15% (w/v) NaCl. Morphologically distinct colonies were selected and streaked to purity on the same solid medium. One such isolate, designated VH1, was selected at random for further investigation. VH1 was a Gram-negative, oxidase- and catalase-positive isolate capable of esculin hydrolysis which after 16S rRNA gene analysis (performed by NCIMB, UK) and a FASTA search [17] was found to be most similar to Pseudomonas beijerinckii (98.715%) and Chromohalobacter marismortui (98.034%). It is interesting that the FASTA search results indicate that our isolate is most similar to two unrelated organisms. We have no firm idea as to why this should be but it is noteworthy that both C. marismortui and P. beijerinckii are, like VH1, salt-adapted organisms having been isolated from the dead sea and canned salted beans, respectively.

The isolate was capable of growth between 2.5 and 25% (w/v) NaCl with the optimum growth rate occurring at 10% (w/v) NaCl which, using the definition of Kushner [18], classifies VH1 as a moderate halophile. Our findings compare favourably with the growth characteristics previously reported for a moderately halophilic strain of P. beijerinckii by Unemoto et al. [19].

In the presence of 10 and 15% (w/v) NaCl, isolate VH1 was capable of utilising methyl-, ethyl-, 3-aminopropyl- and 4-aminobutyl-phosphonate as the sole source of phosphorus for growth (Table 1). When the NaCl concentration was reduced to 5% (w/v), VH1 was also capable of growth on phosphonoacetate and 2AEP. In the presence of 5% (w/v) NaCl, methylphosphonate was utilised almost as readily as inorganic phosphate whereas only after approximately 50 h was significant growth observed upon 2AEP or phosphonoacetate (Fig. 1). No growth of isolate VH1 was observed above that of a negative control lacking amino acids when it was screened for its ability to utilise a range of aminophosphonates (2AEP, 3-aminopropylphosphonate, 4-aminobutylphosphonate, phosphonoalanine, PIA and glyphosate) as sole nitrogen source for growth at 5, 10 and 15% (w/v) NaCl concentrations.

View this table:
Table 1

Range of organophosphonate substrates utilised as sole phosphorus source at a final concentration of 1.0 mM by isolate VH1 at varying NaCl concentrations

Substrate (1 mM)Growth (μg of protein ml−1)
5% NaCl10% NaCl15% NaCl
Phosphate240240160
P-free control351515
Methylphosphonate22027088
Ethylphosphonate18510087
Phenylphosphonate303030
Glyphosate1055
Phosphonomethyliminodiacetate10510
Phosphonoformate303530
Phosphonoacetate2201510
Aminomethylphosphonate201010
2-Aminoethylphosphonate2332010
3-Aminopropylphosphonate300230110
4-Aminobutylphosphonate200170130
Phosphonoalanine201010
Phosphonomycin102010
Results were scored negative if the protein yield, as measured by the method of Binks et al. [12], was less than 20% of that of the positive control containing 1 mM inorganic phosphate. Results are means of duplicates which on no occasion varied by more than 5%.
Figure 1

Growth of isolate VH1 on a range of organophosphonates as sole phosphorus source. The isolate was grown in defined halophile medium containing 5% (w/v) NaCl with organophosphonates supplied at a final concentration of 1.0 mM. Symbols: (●) inorganic phosphate; (○) phosphate-free control; (▪) methylphosphonate; (▾) 2-aminoethylphosphonate and (▿) phosphonoacetate.

Microbial metabolism of 2AEP has been widely reported with bacteria being divided into two distinct groups [1], i.e. those regulated by inorganic phosphate and capable of utilising 2AEP only as sole phosphorus source and those capable of utilising the compound as a carbon, nitrogen and phosphorus source in the presence of inorganic phosphate. It has been demonstrated at a molecular level that the genes for 2AEP biodegradation in both Salmonella typhimurium and Enterobacter aerogenes IFO 12010 are under the control of the phosphate starvation-inducible pho regulon [20,21]. In the second group of bacteria, represented by strains of Pseudomonas putida and Pseudomonas fluorescens 23F, 2AEP is mineralised with the excretion of large quantities of inorganic phosphate into the culture supernatant [14,22], although as yet little is understood of how the 2AEP degradation pathway is regulated in such bacteria.

Our moderate halophile would appear to belong to the first group of bacteria as it was unable to utilise 2AEP as a source of carbon or nitrogen. In addition, growth on any of the phosphonate substrates, or the incubation of cells in phosphorus-free controls, was not accompanied by the release of phosphate. Surprisingly, no 2AEP:aminotransferase [EC 2.6.1.37] nor phosphonatase [EC 2.6.1.37] activity was detectable in cell-free extracts prepared from cells of VH1 grown on 2AEP. Previously, Wackett et al. [23] had proposed that in addition to the pathway described above, 2AEP would be a suitable substrate for the ‘C–P lyase’ cluster of proteins, a viewpoint supported by Ternan [24] who failed to detect any 2AEP:aminotransferase or phosphonatase activity in a number of bacteria capable of 2AEP metabolism. In cells grown under phosphate-free conditions or on 2AEP as sole (w/v) source, alkaline phosphatase activity levels were elevated some 50% over that in cells grown in phosphate-replete medium (results not shown). This induction under phosphate limiting conditions further points towards the presence of a C–P lyase pathway for 2AEP biodegradation in this strain. It is highly likely that this is the method by which the other organophosphonates utilised by isolate VH1 are metabolised.

The effect of NaCl concentration upon the ability of VH1 to utilise organophosphonates was surprising. It is clear that VH1 must contain a C–P lyase-like enzyme for catabolism of the phosphonates given the absence of phosphonatase activity in cell extracts. In addition, no phosphate release was observed during growth on phosphonoacetate in liquid culture, suggesting that VH1 did not possess phosphonoacetate hydrolase. Given that C–P lyase is capable of dephosphonylating methyl- and ethyl-phosphonates, phosphonoacetate and 2AEP [23], it is likely that differences in organophosphonate transporter functionality are the cause of the observed growth responses on phosphonates of the isolate at varying NaCl concentrations. The transporter is likely to be similar to the phoE porin of Escherichia coli described by Wanner [4], which is the only organophosphonate transport protein characterised to date. This protein, induced under conditions of phosphate starvation, allows bacterial uptake of alternative sources of environmental phosphorus, including phosphonates. In the case of 2AEP and phosphonoacetate, both of which have been shown to be metabolised under phosphate-replete conditions, transport protein(s) other than phoE must exist which may be more sensitive to elevated levels of NaCl.

In this study we have shown for the first time that halophilic bacteria are capable of metabolising organophosphonates as sole phosphorus source. This observation has implications for the biogeochemical cycling of phosphorus in saline biotopes, particularly marine environments which have been reported to contain a variety of organisms which are capable of synthesising organophosphonates [25]. The isolation of VH1 demonstrates that the diversity of organophosphonate-degrading bacteria is greater than that currently reported in the literature.

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