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The utilization of 4-aminobutylphosphonate as sole nitrogen source by a strain of Kluyveromyces fragilis

Nigel G. Ternan, Geoffrey McMullan
DOI: http://dx.doi.org/10.1111/j.1574-6968.2000.tb09020.x 237-240 First published online: 1 March 2000


A strain of the yeast Kluyveromyces fragilis was screened for its ability to utilize a range of synthetic and natural organophosphonate compounds as the sole source of phosphorus, nitrogen or carbon. Only 4-aminobutylphosphonate was utilized as sole nitrogen source with protein yields increasing proportionally with substrate concentrations up to 10 mM. No 4-aminobutylphosphonate metabolizing enzyme activity was detectable in cell-free extracts prepared from K. fragilis pregrown on 2.5 mM 4-aminobutylphosphonate. None of the organophosphonates tested served as a source of carbon or phosphorus for K. fragilis.

  • Yeast
  • Organophosphonate
  • Biodegradation
  • pho regulon

1 Introduction

While inorganic phosphate (Pi) is the preferred phosphorus source for microbial growth, many microbes are able, under conditions of Pi limitation, to metabolize a range of organophosphorus compounds as the sole phosphorus source. These compounds include both biogenic and xenobiotic members of the organophosphonates, which are characterized by the presence of a stable covalent carbon to phosphorus (C–P) bond. The C–P bond imparts upon these molecules a relative resistance to chemical, thermal, photolytic and biodegradation [1].

Cleavage of the C–P bond is the central requirement for complete organophosphonate mineralization [2] and previous studies of organophosphonate metabolism by environmental microorganisms have shown that in the main, phosphate starvation is required for organophosphonate uptake and induction of C–P bond cleavage enzymes. In the bacterial isolates studied to date these enzymes are part of the pho regulon, a collection of genes whose transcription, under conditions of environmental phosphate limitation, leads to gene products allowing the acquisition of alternative sources of phosphorus, including a diverse range of organophosphonates [3,4]. However, strict control of the pho regulon by inorganic phosphate ensures that bacterial cells can only utilize organophosphonates as sole sources of phosphorus, as the Pi released during the catabolism of their carbon skeletons serves to repress and/or inhibit further mineralization.

More recently bacterial strains with the ability to completely mineralize both natural and xenobiotic organophosphonates as sources of carbon, nitrogen and phosphorus have been isolated [1,5]. These isolates have in the main contained novel, inducible enzymes with a high degree of specificity for a single organophosphonate compound and represent a departure at both a physiological and genetic level from the accepted consensus that organophosphonates are utilized only as sole phosphorus sources [1].

While the biodegradation of organophosphonates by both Gram-positive and Gram-negative bacteria is well documented, little is known of the ability of yeast strains to metabolize these compounds. Indeed, the only documented report is of the ability of a strain of Candida maltosa to utilize the natural organophosphonate, 2-amino-3-phosphonopropionic acid (phosphonoalanine) as a source of nitrogen [6]. In this paper we report for the first time the ability of a strain of Kluyveromyces fragilis to utilize the synthetic organophosphonate 4-aminobutylphosphonate as sole source of nitrogen.

2 Materials and methods

2.1 Chemicals

All chemicals, of highest purity available, and all reagents for phosphate determination were obtained from Sigma-Aldrich Chemical Company (Poole, UK), except for the following compounds: N-[phosphonomethyl]-glycine (Zeneca Agrochemicals, UK); 2-phosphonobutyrate and 3-phosphonopropionate (Lancaster Synthesis Ltd., Morecambe, UK).

2.2 Microorganisms, media and culture conditions

K. fragilis UU1 was provided by Prof. R. Marchant (University of Ulster, Coleraine, UK) and maintained on malt extract agar at 4°C. The organism was routinely grown on complete M1 medium which contained (per l): MgCl2·6H2O, 2.03 g; KCl, 0.2 g; MgSO4·7H2O, 0.2 g; CaCl2·2H2O, 0.01 g; urea, 0.15 g; D-glucose, 30 g; ferric ammonium citrate, 1.0 mg; KH2PO4, 0.068 g; K2HPO4·3H2O, 0.114 g (final PO42− concentration, 1.0 mM), and 1 ml each of trace element solution [7], phosphate-free yeast extract [8] and vitamin solution [9]. Nitrogen and phosphorus-free M1 mineral salts medium was prepared by omitting the urea and inorganic phosphate, respectively, from M1 medium. Filter sterilized (0.2 μm pore size) solutions of urea and organophosphonates were added aseptically after autoclaving. Phosphorus sources were supplied at a final concentration of 1.0 mM, and nitrogen sources to a final concentration of 5.0 mM organic nitrogen except where stated.

Starter cultures (20 ml in 100 ml Erlenmeyer flasks) of K. fragilis were starved in either phosphorus-free or nitrogen-free medium for 48 h prior to inoculation into flasks containing fresh medium supplemented with organophosphonates as sole phosphorus source (1 mM) or as sole nitrogen source (5 mM), respectively, at an initial OD 650 nm of 0.01. Cultures were incubated at 30°C (150 rpm) and growth followed using a Pharmacia Biotech Novaspec II spectrophotometer.

2.3 High performance liquid chromatography (HPLC) analysis

Removal of aminophosphonates from culture supernatants was followed by the method described by Kawai and Uno [10]. HPLC separation was performed using a Phenomenex (Macclesfield, UK) 4.6 mm i.d.×150 mm reverse phase Luna C18 column, a ThermoSeparation Products (San Jose, USA) spectraSYSTEM P4000 pump, a spectraSYSTEM AS3000 autosampler, a spectraSYSTEM UV2000 detector and a Rheodyne model A4169-030 loop injector with a 100 μl loop. The column was equilibrated with 200 mM phosphate phosphoric acid buffer (pH 2.3):acetonitrile (85:15) at a flow rate of 1.0 ml min−1. Derivatization of samples was carried out as follows: to a 0.5 ml sample containing 4-aminobutylphosphonate was added 0.25 ml 400 mM phosphate derivatization buffer (pH 11.0) and 0.1 ml p-toluenesulfonyl chloride solution (10 mg ml−1 in acetonitrile). The mixture was heated at 50°C for 5 min in a water bath, and following filtration through a 0.2 μm filter, a 20 μl aliquot was injected into the HPLC column.

2.4 Cell-free enzyme assays

K. fragilis was grown on 4-aminobutylphosphonate (2.5 mM) as sole nitrogen source (1 l total culture volume). Cells were harvested once they reached mid-log phase (45 h) and disrupted by sonication using the 10 mm diameter probe of an MSE Soniprep 150. Cells were suspended 1:1 (w/v) in 50 mM HEPES–NaOH buffer (pH 7.5) and aliquots were sonicated on full power (30 s sonication with 2 min cooling on ice) in the presence of one volume of glass beads (0.6–0.8 mm diameter). The resultant homogenate was centrifuged at 25 000×g for 30 min and the supernatant then used in assays for 4-aminobutylphosphonate metabolizing activity as previously described by Ternan and Quinn [5]. All assays were carried out in triplicate with suitable controls.

Samples were taken from the assays over the course of 30 min and run on TLC plates using the solvent system 94% ethanol:34% ammonia solution (3:1). Standards of alanine, aspartate and glutamate were included, these being the expected transamination products of pyruvate, oxaloacetate and α-ketoglutarate, respectively. Plates were visualized by staining with 0.2% ninhydrin in ethanol and heating at 90°C on a hot plate until color development occurred.

2.5 Analytical methods

Inorganic phosphate was determined by the colorimetric method of Fiske and SubbaRow [11]. The protein yield from K. fragilis cultures was assayed by the method of Binks et al. [12]; protein concentration in cell extracts was measured by the method of Bradford [13].

3 Results and discussion

The bacterial metabolism of organophosphonates has been extensively studied [1] however little is known of the ability of yeasts to utilize such compounds. It is known that as in bacteria certain yeasts contain a pho regulon [14], however, the involvement of organophosphonate metabolizing enzymes in yeast phosphorus scavenging has yet to be shown. In the only previous reported study, Bode and Birnbaum [6] described the utilization of the natural phosphonate phosphonoalanine as a nitrogen source by C. maltosa. Our current study is the first to report the ability of a yeast, K. fragilis, to metabolize a synthetic organophosphonate, 4-aminobutylphosphonate.

Of eight aminophosphonates tested only 4-aminobutylphosphonate was utilized by K. fragilis as sole nitrogen source, with cell protein yields being comparable to that of the positive control (Table 1). Surprisingly, given its widespread bacterial metabolism [1], there is tentative evidence that 2-aminoethylphosphonate, the most abundant natural organophosphonate, was an inhibitor of K. fragilis (Table 1). Our study also found that phosphonoacetate, phosphonomycin, phosphonoalanine, phosphonomethylglycine, phosphonomethyliminodiacetate, 2-amino-4-phosphonobutyrate, 3-phosphonopropionate, 4-phosphonobutyrate, methyl-, ethyl-, phenyl-, aminomethyl-, 2-aminoethyl-, 3-aminopropyl- and 4-aminobutylphosphonates failed to serve as either a carbon or phosphorus source for the yeast. The failure of these substrates to serve as a carbon source is unsurprising given the experience of previous organophosphonate metabolism studies [1]; however the inability of the yeast to utilize any of the compounds, especially 4-aminobutylphosphonate, as a phosphorus source suggests the absence of C–P bond cleavage enzymes. Our observations for K. fragilis would therefore appear to be similar to those of McMullan and Quinn [15] who described a Gram-negative bacterial isolate capable of utilizing 3-aminopropylphosphonate as a nitrogen but not phosphorus source.

View this table:
Table 1

Utilization of organophosphonates as sole nitrogen source by K. fragilis

Organophosphonate substrate (5.0 mM final concentration)Growth of K. fragilisa (μg protein ml−1)
Positive controlb716
Negative control39
Results are the means of duplicates which on no occasion varied by more than 5%.
  • aMaximum culture growth was measured after 5 days incubation.

  • bPositive control contained 2.5 mM urea.

When provided as sole source of nitrogen HPLC analysis clearly demonstrated that 4-aminobutylphosphonate was removed from the culture medium concomitant with growth of K. fragilis (Fig. 1). K. fragilis cell protein yields were found to be directly proportional to the 4-aminobutylphosphonate concentration within the range of 0 to 10 mM (Fig. 2) with no inhibition of protein yields detectable up to a concentration of 20 mM. Our findings indicate that uptake and utilization of 4-aminobutylphosphonate is independent of the phosphate status of the cell. According to the theories proposed by McMullan and Quinn [15]K. fragilis must therefore possess either a Pi-deregulated phosphonate uptake system or a novel uptake pathway which can transport 4-aminobutylphosphonate in the presence of high levels of Pi. In the only other study of organophosphonate metabolism by a yeast it was also proposed that C. maltosa possessed a general amino acid permease which allowed transport of aminoorganophosphonates such as phosphonoalanine [6].

Figure 1

Growth of K. fragilis on 4-aminobutylphosphonate as sole nitrogen source. 4-Aminobutylphosphonate was supplied at a final concentration of 2.5 mM, with 30 g l−1 glucose carbon source and 1 mM Pi. The experiment was carried out on several occasions, with representative results shown. Symbols: (●) protein yield (μg ml−1); (▪) 4-aminobutylphosphonate remaining in culture supernatant.

Figure 2

Stationary phase protein yield of K. fragilis as a function of 4-aminobutylphosphonate concentration in the medium. The experiment was carried out on several occasions, with representative results shown. All cultures were supplemented with 30 g l−1 glucose carbon source and 1 mM Pi.

The ability to utilize 4-aminobutylphosphonate as sole nitrogen source is a novel finding for any yeast and points to a previously undescribed biodegradation pathway for this compound. Despite a number of attempts, however, no cell-free activity of the enzyme(s) involved could be obtained. Again our findings are similar to those of McMullan and Quinn [15], who were unable to demonstrate transamination of aminoalkylphosphonates by cell-free extracts of environmental bacterial isolates. The identity of the enzyme(s) involved in both reports thus remains unclear but is presumed to be distinct from the narrow specificity 2-aminoethylphosphonate:pyruvate aminotransferase (EC previously described by Dumora et al. [16] and the phosphinothricin transaminase described by Bartsch and Tebbe [17]. What does seem certain is that a number of previously undescribed enzymes exist throughout the microbial world capable of aminoalkylphosphonate metabolism.

The well characterized pho system of Saccharomyces cerevisiae is analogous to that described in various bacteria [1]. The yeast pho system is involved in the acquisition and metabolic integration of inorganic phosphate, and includes both acid and alkaline phosphatases, phosphate permease, polyphosphate kinase and a polyphosphatase [14]. Whilst our current study clearly demonstrates the capabilities of a K. fragilis strain to utilize an organophosphonate as a nitrogen source the presence of C–P bond cleavage enzymes in yeasts, similar to those found in bacteria, remains to be demonstrated.


Sponsors: Zeneca Agrochemicals, Jealott's Hill Research Station, Bracknell, Berkshire, RG42 6ET, UK.


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