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Applied and Environmental Microbiology, September 2006, p. 6414-6416, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.01084-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Seleno-L-Methionine Is the Predominant Organic Form of Selenium in Cupriavidus metallidurans CH34 Exposed to Selenite or Selenate{dagger}

Laure Avoscan,1 Richard Collins,1,{ddagger} Marie Carriere,1 Barbara Gouget,1* and Jacques Covès2*

Laboratoire Pierre Süe, CEA/CNRS UMR 9956, 91191 Gif sur Yvette,1 Laboratoire des Protéines Membranaires, Institut de Biologie Structurale—Jean-Pierre Ebel, UMR 5075 CNRS-CEA-UJF, 41, rue Jules Horowitz, 38027 Grenoble Cedex, France2

Received 5 May 2006/ Accepted 21 June 2006


   ABSTRACT
Top
 Abstract
Introduction
 References
 
The accumulated organic form of selenium previously detected by X-ray absorption near-edge structure (XANES) analyses in Cupriavidus metallidurans CH34 exposed to selenite or selenate was identified as seleno-L-methionine by coupling high-performance liquid chromatography to inductively coupled plasma-mass spectrometry.


   INTRODUCTION
Top
 Abstract
 Introduction
References
 
Accumulation of selenium from natural or anthropogenic sources generates toxic environmental conditions (1, 9, 17). Microorganisms can be involved in the geochemical cycle of selenium and thus can potentially be used for bioremediation processes (4-7, 13, 16). This is the case for Cupriavidus metallidurans CH34 (formerly Ralstonia metallidurans CH34), which has been demonstrated to resist high concentrations of selenite and to reduce it to elemental selenium immobilized by the biomass as electron-dense granules (14). C. metallidurans CH34 is a facultatively autotrophic gram-negative bacterium characteristic of metal-contaminated biotopes (10, 11) that has already been used to remove heavy metals from soil or liquid waste (2, 3, 10). Its use to target selenium-polluted environments still requires a better understanding of the molecular events leading to the incorporation of the selenium oxyanions, selenite [Se(IV)] and selenate [Se(VI)], and their subsequent reduction. In recent studies, we have identified a gene encoding a putative selenite transporter (8) and compared the kinetics of selenite and selenate accumulation to identify by X-ray absorption near-edge structure (XANES) spectroscopy the possible chemical intermediates during the transformation of these oxyanions (15). We observed that one, or numerous, organic species of Se (alkyl selenide) were produced by this bacterium regardless of the initial oxidation state of Se [Se(IV) or Se(VI)]. In cases of selenite exposure, this organic species is both transient and minor compared to the dominating chemical species, Se0, while in cases of selenate exposure, Se0 occurred as a minor species and the major accumulated form was alkyl selenide. However, it was impossible to unequivocally identify this organic selenium species by XANES spectroscopy, and recourse to other analytical techniques is required. Here we have developed the direct coupling of high-performance liquid chromatography (HPLC) to inductively coupled plasma-mass spectrometry (ICP-MS) to identify the compound of interest after extraction of the endogenous Se species.

Cells were grown aerobically at 29°C in Tris salt mineral medium with 2% gluconate as a carbon source as previously described (15). The experiments were initiated by adding Se(IV) or Se(VI) to the culture medium when the absorbance at 600 nm reached 0.3. The final Se oxyanion concentration was 2 mM. The culture medium was sampled every 24 h over a 144-h period, and total accumulated selenium was quantified for each sample as previously reported (15). As expected, Se(IV) induced an extended lag phase (14), while Se(VI) did not cause any change in growth (Fig. 1) compared to the growth of a culture without any added selenium oxyanion (not shown). The concentrations of Se accumulated by the two cultures were similar after 24 h, but the accumulation patterns diverged rapidly thereafter. At the end of the experiment (144 h), the bacteria exposed to Se(IV) had accumulated Se to a concentration of 290 mg g of protein–1, compared to 13 mg Se g of protein–1 for Se(VI) exposure (Fig. 1).


Figure 1
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  		FIG. 1. Mean bacterial Se accumulation and culture medium absorbance at 600 nm during the exposure of C. metallidurans CH34 to Se(IV) or Se(VI) (initial concentration, 2 mM). •, absorbance at 600 nm [Se(IV)-exposed cultures]; {blacktriangleup}, absorbance at 600 nm [Se(VI)-exposed cultures]; {circ}, Se accumulation [Se(IV)-exposed cultures]; {triangleup}, Se accumulation [Se(VI)-exposed cultures]. Note that the scale of the right y axis is logarithmic. Bacterial growth was also assayed by quantification of the protein contents and confirmed the A600 data. As previously observed (14), the presence of particulate Se0 did not disturb the measurement of the absorbance.

 
Bacterial pellets from 5-ml culture medium samples were obtained by centrifugation, washed three times with high-purity water, and finally resuspended in 1 ml of a solution containing 10 mM Tris-HCl (pH 7.2) and 2 mM CaCl2. Then cells were lysed and the proteins digested by addition of 50 µl of a solution containing 72 g liter–1 of both lysozyme and protease XIV (12). The samples were incubated at 37°C for 24 h until complete digestion of proteins to their mono-amino acids. The supernatant of a new centrifugation was removed and identified to species level by HPLC-ICP-MS analyses after dilution by a fivefold factor with high-purity water. This dilution step is required to prevent HPLC-ICP-MS peak retention time shifts induced by high ionic strength. As a control, the entire procedure was applied to seleno-L-methionine, seleno-DL-cystine, seleno-methylseleno-L-cysteine, Se(IV), Se(VI), and Se0. It was observed, by use of HPLC-ICP-MS, that the extraction procedure neither transformed these Se species nor solubilized Se0 into Se(IV) or Se(VI). Besides, these controls did not reveal any relevant contamination of selenite by selenate, or vice versa.

The percentage of Se recovered was variable, depending on whether the bacteria had been exposed to Se(IV) or Se(VI) (Table 1). Between 24 and 144 h of exposure to Se(IV), very little of the Se associated with the bacteria was extracted by using the enzymatic digestion protocol (<5% in terms of the total concentration of accumulated Se). This was expected, because insoluble Se0 becomes the predominant biotransformation product (>95%) at later sampling times (14, 15). The extraction procedure was very effective for the Se(VI)-exposed bacteria, with an average Se recovery, at all sampling times, of 82% (Table 1). Therefore, the majority of Se accumulated by C. metallidurans CH34, during exposure to 2 mM concentrations of Se(VI), is found in soluble and/or proteinogenic forms, a finding that is also consistent with our previous XANES spectroscopy results (15).


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  		TABLE 1. Percentage of recovery of total accumulated Se by C. metallidurans CH34 after enzymatic digestion of bacterial pellets

 
Separation of the extracted Se species was achieved with a 4-mm AS11 anion-exchange column (Dionex, Sunnyvale, CA) using a flow rate of 2 ml min–1 and the following NaOH gradient elution: 0.8 mM NaOH (0 to 1 min), 0.8 to 40 mM NaOH (1 to 4 min), and 0.8 mM NaOH (4 to 7 min). An injection volume of 25 µl was used for all samples and standards. The column was coupled directly to the nebulizer of the ICP-MS, via PEEK tubing, and chromatograms of counts per second against time were recorded for each isotope ion of Se. Detailed ICP-MS conditions for total Se quantification and seleno-L-methionine identification are described in the supplemental material. A typical HPLC-ICP-MS chromatogram showing the separation of five reference compounds is shown in Fig. 2a. Upon HPLC-ICP-MS analysis of the enzymatic extraction solutions of the Se(IV)-exposed bacteria, only one peak was observed, with a retention time corresponding to that of seleno-L-methionine (Fig. 2b). As a control, the seleno-L-methionine standard was added to the sample solution and found to elute at the same time as the bacterially derived seleno-L-methionine. Similarly, seleno-L-methionine was the sole organic species of Se in the enzymatic extraction solutions of the Se(VI)-exposed bacteria (Fig. 2c), regardless of the time of sampling. It must be noted that any seleno-L-cystine that could be produced by oxidation of seleno-L-cysteine putatively present in the samples was not detected in any of the extraction solutions.


Figure 2
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  		FIG. 2. Stacked HPLC-ICP-MS chromatograms of various solutions. Line a, standard solution containing 50 µg of each of the following Se species liter–1: seleno-methylseleno-L-cysteine (peak 1), seleno-L-methionine (peak 2), Se(IV) (peak 3), seleno-DL-cystine (peak 4), and Se(VI) (peak 5). When the entire procedure, i.e., extraction plus identification to species level and quantification by HPLC-ICP-MS, was applied to these Se-containing chemical forms, no other species besides the initial chemical standards was detected, and their concentrations remained identical before and after extraction. Lines b and c, enzymatic extraction solution of C. metallidurans CH34 exposed for 24 h to 2 mM Se(IV) and 2 mM Se(VI), respectively. Peaks are relative to counts per second for the 80Se+ isotope ion.

 
These results indicate that seleno-L-methionine is the accumulated proteinogenic (and organic) form of Se in this bacterium, whether it is exposed to 2 mM concentrations of Se(IV) or Se(VI). We have previously shown that transport and reduction of selenite are slowly activated upon selenite exposure (8, 15), while this is not the case for selenate exposure (15). From our previously published data (15), one can calculate that the now identified seleno-L-methionine represented about 25 mg g of protein–1 after 50 h in the presence of Se(IV) (about 10% of the added selenium), while it represented only 11 mg g of protein–1 in the presence of Se(VI) (95% of the added selenium). One can speculate that a threshold in the seleno-L-methionine concentration could be the signal to set up a resistance pathway. This threshold should be reached after about 50 h in the presence of 2 mM selenite, triggering the phase of fast Se(IV) uptake.


   ACKNOWLEDGMENTS
 
This work was supported in part by the French National Program of Environmental Nuclear Toxicology (ToxNucE).


   FOOTNOTES
 
* Corresponding author. Mailing address for J. Covès: Laboratoire des Protéines Membranaires, Institut de Biologie Structurale—Jean-Pierre Ebel, UMR 5075 CNRS-CEA-UJF, 41, rue Jules Horowitz, 38027 Grenoble Cedex, France. Phone: 33 (0) 4-38-78-24-03. Fax: 33 (0) 4-38-78-54-94. E-mail: jacques.coves{at}ibs.fr. Mailing address for B. Gouget: Laboratoire Pierre Süe, CEA/CNRS UMR 9956, 91191 Gif sur Yvette, France. Phone: 33 (0) 1-69-08-33-13. Fax: 33 (0) 1-69-08-69-23. E-mail: barbara.gouget{at}cea.fr. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back

{ddagger} Present address: Centre for Water and Waste Technology, School of Civil and Environmental Engineering, The University of New South Wales, Sydney 2052, Australia. Back


   REFERENCES
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Applied and Environmental Microbiology, September 2006, p. 6414-6416, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.01084-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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