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Micro-X-ray absorption near edge structure as a suitable probe to monitor live organisms
Spectrochimica Acta Part B 63 (2008) 512 – 517 www.elsevier.com/locate/sab
Research note
Micro-X-ray absorption near edge structure as a suitable probe to monitor live organisms Phil M. Oger ⁎, I. Daniel, A. Simionovici 1 , A. Picard Laboratoire de Sciences de la Terre, UMR CNRS 5570, Ecole Normale Supérieure de Lyon-Université Claude Bernard Lyon 1, Lyon, F-69364, France Received 20 June 2007; accepted 19 December 2007 Available online 9 January 2008
Abstract X-ray spectroscopies are very powerful tools to determine the chemistry of complex dilute solutes in abiotic and biotic systems. We have assayed their suitability to monitor the chemistry of complex solutions in a live biotic system. The impact of the probe on cells was quantified for 4 different cellular organisms differing in their resistance level to environmental stresses. We show that none of the organisms tested can survive the radiation doses needed for the acquisition of meaningful spectroscopic data. Therefore, on one hand, X-ray spectroscopy cannot be applied to the monitoring of single cells, and cellular damages have to be taken into account in the interpretation of the evolution of such systems. On the other hand, due to the limited extension of X-ray induced cellular damages in the culture volume, it is possible to probe a population of live cells provided that the culture to beam probe ratio is large enough to minimize the impact of mortality on the evolution of the biological system. Our results suggest that it could be possible to probe the volume in the close vicinity of a cell without affecting its activity. Using this setup we could monitor the reduction of selenite by the X-ray sensitive bacterium, Agrobacterium tumefaciens strain C58, for 24 h. This method has a great potential to monitor the respiration of various metals, such as iron, manganese and arsenic, in situ under relevant environmental conditions by live microorganisms. © 2008 Elsevier B.V. All rights reserved. Keywords: XANES; XRF; Dissimilatory metal reduction; in situ
1. Introduction X-ray spectroscopy, and in particular X-ray Absorption Near Edge Spectroscopy (XANES), have been used to monitor elemental speciation in biological tissues of plants [1–4] and animals [5,6], in plant, animal or bacterial cells [2,7–9], as well as in biominerals [10,11]. It has become a central analytical tool to determine the speciation of contaminating metals in polluted sites, and to monitor their fate in the environment. X-ray Fluorescence (XRF) has recently been used to determine the cellular concentration and spatial distribution of calcium, iron in ⁎ Corresponding author. Current address: Laboratoire de Sciences de la Terre, Ecole Normale Supérieure, 46, Allée d'Italie, 69364 Lyon cedex 07, France. Tel.: +33 472 728 792; fax: +33 472 728 677. E-mail address: [email protected] (P.M. Oger). 1 Present address: Laboratoire de Géophysique Interne et Tectonophysique, UMR CNRS 5559, Univ. J. Fourier, BP 53, 38041 Grenoble. 0584-8547/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.sab.2007.12.013
eukaryotic cells, for which the technique holds interesting perspectives. Indeed, several authors have proposed that it could be used to monitor the cellular trafficking of these important ions on live tissues [7,8]. However, unlike other spectroscopic techniques involving visible, infrared or ultraviolet light, the X-ray probe may induce severe damages to the cells under scrutiny, which in turn might interfere with 1) their survival, 2) their metabolism and 3) the trafficking one wants to monitor. Unfortunately, there is virtually no information on the sensitivity of living organisms to X-ray irradiation. Damages induced by other types of radiations, such as ultraviolet and gamma radiation, have been studied in far more details, while most X-ray data derive from medical studies linked to imaging or cancer treatments. In superior Eukaryotes the tolerance to irradiation is extremely low. An irradiation of 2 Gray kills foetal, ovary or bone marrow cells. One Gray (Gy) corresponds to 1.0 J kg− 1 of absorbed radiation; a focussed synchrotron X-ray beam delivers several kGy to the sample per
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second. Bacteria, to which the most radiation tolerant organisms belong, can withstand a few hundred Gy [12]. The most radiation tolerant organism, e.g. the bacterium Deinococcus radiodurans, tolerates irradiations up to 10 kGy without loss of viability [13]. The tolerance level of a specific organism to irradiation has been linked to its ability to repair the damages induced to cellular macromolecules, in particular nucleic acids [14,15]. Although it remains difficult to extrapolate the above data to the irradiation conditions of a synchrotron experiment with beam intensity several orders of magnitude higher than that used in standard procedures, the induced damages are likely to be important. Therefore, before any convincing data can be obtained by X-ray spectroscopy on live organisms, it was crucial to determine 1) the extent of the X-ray induced damages during analysis, 2) the kinetics of cell death in the conditions typical of a XANES spectrum acquisition to assess the feasibility of in situ monitoring of live cells activity by X-ray spectroscopy. In this paper, we report the survival rates to X-ray exposure of 4 strains of bacteria, differing in their resistance level to environmental stresses. Although, none of the strains studied can withstand the conditions required for the acquisition of a single XANES spectrum we demonstrate that the limited extension of radiation damages in the culture allows the use of X-ray spectroscopy to monitor microbial processes in situ provided a sufficiently low beam to culture volume ratio is employed. This is illustrated by monitoring of the reduction of selenium by an X-ray sensitive bacterium.
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(Fig. 1). Air was kept between the stoppers and the bacterial culture to prevent contamination. The irradiations were performed at the CRG beamline BM30B of the European Synchrotron Radiation Facility (ESRF). The incident beam was focussed into a 150 µm × 300 µm spot (vert. × hor.) by mirrors in the Kirkpatrick–Baez geometry. The incident flux ca. 3 1010 ph/s, used for normalization, was measured using a Si photodiode located before the sample. To achieve a homogenous irradiation of the culture, the entire volume of the capillary was scanned in
2. Materials and methods 2.1. Bacterial strains and growth conditions Four different strains with variable tolerance to UVand gamma ray exposure were assayed: E. coli strain DH5α [16] and Pseudomonas syringae strain PPI203 [17] are sensitive strains. Rhodococcus erythropolis strain W2 is desiccation and heavy metal tolerant [18]. Last, D. radiodurans strain R1 is the most radiotolerant bacterium known to date [13]. Escherichia coli strain DH5α, P. syringae pv. pisi strain PPI203 and R. erythropolis strain W2 are from our bacterial culture collection. D. radiodurans strain R1 was obtained from the American Type Culture Collection. A fifth strain, Agrobacterium tumefaciens strain C58 [19] which can reduce selenite into elemental selenium [17] was chosen as a test organism to monitor the microbiologically mediated reduction of metals by XANES spectroscopy [8]. Bacteria were grown at 30 °C in low salt Luria-Bertani broth (LB low salt, GIBCO®), for 24 h under vigorous shaking. Cells were harvested by centrifugation, washed twice in LB low salt and adjusted to a final concentration of 109 cells/ml. 2.2. Experimental set up Ultra clean glass capillaries (Mark Röhrlichen™, Glass, 10 µm thick, 1.5 mm diameter) were cut open at their smaller end. The larger extremity of the capillaries was connected to a micropipette tip, and used to pipette 10 µl of the cell suspension. The capillary was sealed at both ends to prevent evaporation
Fig. 1. A: Experimental setup. B: Monte-Carlo simulation of the absorption of the X-ray beam by the glass capillary. The portion of the capillary that receives a significantly lower X-ray dose is shaded gray. This corresponds to a volume lower than 7% of the total experimental culture volume, and to doses ranging between 97 and 95% of that deposited at the center of the capillary.
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five runs at a constant speed (Fig. 1A). The intensity of the incident flux was adjusted by using a set of aluminium filters, and by varying the scanning speed between 2 and 17 µm s− 1. Each experiment consisted of two identical capillaries, one irradiated as described above, and one control kept in the experimental hutch away from the beam. 2.3. Irradiation dose calculation The X-ray doses deposited in the experimental volume are presented throughout the manuscript in Gy to allow an easy comparison between treatments and strains and data from the literature. The Irradiation Dose (ID) was calculated as follows: ID ¼ PhtE1:6 1019 MA
ð1Þ
Where ID is in Gy, Ph is the average number of photons per second during irradiation, t is the duration of irradiation (s), E is the beam energy (eV), M is the mass normalization factor, A is the correction factor for the absorption of the glass capillary. In this experiment M is constant and equal to 105 since the sample volume is 10 µl. The correction factor for the absorption of the glass capillary is constant for a given type of capillary. The value of A is estimated to 0.95 in the present experiment, as detailed below. The absorption of the incident beam by the empty glass capillary was measured to be 10%. In order to check for spatial heterogeneity, we performed a Monte-Carlo simulation (Fig. 1B). This showed that the absorption by the capillary varied between 5 and 10% from the center to the edge of the capillary. However, only ca. 7% of the sampled volume received a significantly lower irradiation dose (Fig. 1B), ranging between 97 and 95% of that received at the center of the capillary. Thus, the irradiation dose received by the microorganisms was calculated as the total beam energy corrected for a homogeneous absorption of 5% by the front wall of the glass capillary. The irradiation doses for the different experiments are reported in Table 1.
2.4. Cell survival determination Cell survival was estimated by the colony forming unit method [20]. The cells from the irradiated and control capillaries
Fig. 2. Estimation of bacterial survival following irradiation. The survival of the bacteria was estimated by their ability to form colonies. Single isolated colonies are visible as dark spots for the higher dilutions. Left: Irradiation dose (Gy); Top: Dilution factor. For example, for a calculated dose of 173,360 Gy, we enumerate 2 108 surviving Deinococcus radiodurans cells. Enumerations are performed in quadriplicates.
were transferred to a clean sterile tube using a micropipette. A 10 fold serial dilution series was realized in sterile 0.9% NaCl solution. 10 µl drops of each dilution were spotted in quadruplicate onto a LB agar plate and incubated at 30 °C. Colonies formed on the agar surface were counted after 24 to 72 h depending on the bacterial strain (Fig. 2). Using this method, survival is accurately quantified down to 10− 5 survivor per original cell. 2.5. Membrane integrity determination
Table 1 Irradiation exposure doses for each bacterial strain in Gy E. coli
P. syringae
R. erythropolis
D. radiodurans
1180 2100 8650 31,100 34,300 55,300 142,000
920 1940 5330 8200 31,000 33,600 35,400 57,800 157,000
22200 26400 118,000
2250 5300 24,400 28,400 33,000 54,300 173,000 214,000
Potential damages to the cell envelop were estimated with the LIVE/DEAD coloration kit (Molecular Probe, France). This kit includes two fluorescent dyes. The first dye is soluble in lipids, and can therefore cross the cell membrane and accumulates in the cells, which are stained green. The second dye is insoluble in lipids, therefore does not cross the membrane when the cells are intact, and only accumulate in the cells when the integrity of the cell envelop is compromised. Consequently, doubly stained-cells have compromised membranes, e.g. are dead.
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3. Results and discussion 3.1. Survival of bacteria to high intensity X-ray irradiation The radiation sensitive bacterial cultures E. coli and P. syringae, and the most resistant strain D. radiodurans were submitted to irradiation up to 150 kGy and 214 kGy, respectively (Table 1). The survival to irradiation of the two sensitive species, E. coli and P. syringae, was extremely similar. Viability was severely affected even at the lowest irradiation doses, with survival rates ca. 10% for both strains (Fig. 3). Their survival decreased rapidly to reach values close to the detection limit of our setup at the highest exposure dose (150 kGy). The observed irradiation dose leaving 10% survival, hereafter named DL10, is 1.0 and 1.9 kGy for E. coli and P. syringae, respectively. In contrast, no loss of viability was detected at irradiation doses lower than 50 kGy for the radiation resistant species D. radiodurans, a dose that already induces a 3 log drop in viability for the two sensitive strains. At the highest dose tested in our setup (214 kGy), the decrease in viability of D. radiodurans does not exceed 1.3 logs. The calculated DL10 for D. radiodurans in this setup is 195 kGy. The observed tolerance of R. erythropolis is intermediate between the sensitive (E. coli and P. syringae) and the resistant (D. radiodurans) bacteria, showing a 2 log drop in viability at 120 kGy. The DL10 for this strain is 15 kGy. The DL10 values established in this study are at least one order of magnitude higher than those reported in the literature for comparable bacterial species [12], indicating that the present irradiation strategy induces less damage to the cells. In this experiment, the cells were exposed to a bright focused monochromatic 12.5 keV X-ray beam generating irradiation intensities ranging from 2 to 40 Gy s− 1. Under conventional irradiation conditions, cells are most often exposed to a 60Co source (1.25 MeV, γ-rays), a 137 Cs source (0.662 MeV, γ-rays) or a medical X-ray irradiation devices operating between 20 and 200 keV. These irradiation conditions correspond to irradiation intensities ranging from 1 to 50 Gy mn− 1, ca. 50 times less at
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least than the intensity to which the bacteria were exposed in the present experiment. Thus, in conventional conditions, the same irradiation dose is obtained by exposing the cell to the source for longer periods. The impact of beam energy on cell survival has been studied to some extent to determine the survival of selected human cells to different medical treatments. It was shown that twice more damage occur at 20 keV vs. 200 keV, and four times more at 20 keV vs. 1.25 MeV [21,22]. So the apparent cell damage is negatively correlated to the X-ray energy. The impact of the spectral composition of the incident beam on the survival after irradiation showed a two fold reduction for irradiations using monochromatic X-rays vs. filtered X-rays for a mean energy of 20 keV or 40 keV [23]. This increased morbidity was attributed to the low energy X-rays present in the spectrum of the filtered X-rays. Thus, we would expect that an exposure to a low energy (12.5 keV) monochromatic X-ray beam should induce at least as much damage at the same dose than those reported in the literature for γ-ray tolerance. Therefore, we propose that the reduced mortality observed in the present study stems from the short duration of irradiations imposed to the microorganisms. Nevertheless, the corresponding physical or biological mechanisms remain to be elucidated. Cell death following the exposure to γ-, UV- or X-rays results mainly from the formation of double-strand breaks in the DNA [14,24]. The cells can only repair a percentage of these, which therefore accumulate to a level that inhibits cell division. Although X-rays do not directly affect the DNA molecules; they generate free radicals from water and oxygen, which in turn create the nicks in the DNA molecules. This is visible as a sharp decrease in the ability of the irradiated culture to form colonies, but does not lead to the apparition of spontaneous mutations or cellular damages. We evaluated the apparition of colony morphology mutants on culture plates, as well as cell membrane damages under the microscope after selective LIVE/DEAD staining. In this experiment, we did not observe any colony morphology mutant for any of the four species, regardless of the beam intensity. Furthermore, in the preparations of all strains a constant proportion of dead cells (10%) was enumerated in the irradiated samples, regardless of the exposure time and dose. The same proportion of dead cells was also observed in the unirradiated samples, and is typical of the cellular turnover in a stationary phase bacterial culture. Thus, the increased mortality after irradiation in our setup cannot be attributed to an increase in physical damages to the cell envelop. Last, the possibility that the absorption of the incident X-ray beam and subsequent release of energy by the metal under scrutiny might influence the survival of the microbial culture was assayed. Duplicate cultures containing 5 mM of Na2SeO3 for each bacterial strain were irradiated at the same dose below (12.5 keV) or above (12.8 keV) the Se K edge. The survival was estimated as above. No difference could be detected for any of the 4 strains (Fig. 4 and data not shown). 3.2. Impact of partial irradiations on survival
Fig. 3. Bacterial survival as a function of X-ray irradiation dose. Enumeration error bars are comprised within the dots. DL10, 10% lethality limit. The curves are guides for the eye.
The above observations show that only D. radiodurans may be able to survive the conditions required for a µXANES
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spectrum acquisition, although suffering extensive mortality, since the acquisition of a single spectrum would lead to a viability loss larger than 90%. This would preclude any kinetic measurement of metal reduction over several hours. Nevertheless, hypothesizing that the motion of the free radicals responsible for cellular damages in the cell culture would be very short, we propose that the damage done to the microbial culture could be limited to the beam and its immediate environment. The hypothesis was assayed by partially irradiating a cell culture of the sensitive strain PPI203. A capillary containing a PPI203 cell culture was allowed to sediment. Afterwards ca. 20% of the bacteria remain in solution, while 80% deposit in a pellet at the bottom of the capillary. The sole bacterial pellet was irradiated at a dose of 50 kGy, conditions leading to a cell survival below 0.1% for PPI203. After irradiation, the whole culture, including the pellet and the suspension, was recovered and cultured. The survival was measured to be 18.5%, which matches closely the proportion of bacteria in the suspension. This confirms that the expansion of the damages caused by the X-rays is spatially limited to the irradiated volume. Therefore, we propose that a large enough ratio between the size of the incubator and the size of the beam should lead to a cellular viability decrease which is compatible with the acquisition of meaningful X-ray data. This ratio likely depends on the type of organism under scrutiny, on the energy of the beam, as well as on the exposure time to X-rays. 3.3. Applicability to in situ metabolic studies In the present study, we have used a culture to beam size ratio of 500. We monitored the metabolism of selenite by the X-ray sensitive strain of A. tumefaciens strain C58, which reduces selenite (SeO32−, IV) into elemental selenium (Se, 0) [17] and dimethyl selenide (CH3–Se–CH3, II) by a combination of
Fig. 4. Impact of X-ray absorption on the survival of bacteria following irradiation. The Impact of X-ray absorption on the survival of bacteria following irradiation. The cells were incubated in the presence of 5 mM selenium and irradiated above (12.8 keV), or below (12.5 keV) the absorption K edge of selenium to quantify the impact of X-ray absorption during spectrum acquisition.
Fig. 5. Selenite reduction by strain C58. The conversion from selenite to selenium was monitored over a 24 hour period by XANES. Se(IV): at the start of the experiment, all the selenium is present as selenite. Se(0) + Se(II): At the end of the experiment, selenium is present as a mixture of one third metallic selenium [Se(0)] and two third dimethyl selenide [Se(II)].
µXANES and µXRF analyses [8]. In A. tumefaciens, several pathways have been proposed for the reduction of selenite, which potentially involve an uncharacterized intermediate. A total of 72 XANES spectra of 100 points each, for a total irradiation duration of 21,600 s at an average beam intensity ca. 1011 ph/s were acquired over a 24 hour period, at the same location in the center of the incubator. Under these experimental conditions, we observed a survival rate of ca. 5% in the incubator, while the same cell type only survives a few seconds in the beam. As shown in Fig. 5, the spectrum obtained at 0 h is typical of selenite. Spectra acquired for intermediate incubation times clearly show a gradual replacement of selenite by reduced selenium species in solution. After 24 h, the reaction is complete, and the XANES spectra no longer show any contribution of selenite. Quantitative linear combinations of different redox species of selenium demonstrate the contribution of only 3 selenium species to explain the experimental data, selenite, selenium and dimethyl selenide, and exclude the formation of other species of selenium, such as selenium dioxide or selenate. During the reduction of selenite, selenite is reduced to one third fully reduced amorphous metallic selenium (Se, 0) and 2 thirds dimethyl selenide (CH3–Se–CH3, II). In the control experiment, which consisted of the same medium but devoid of bacteria, no reduction could be observed indicating clearly that the reported activity is of biotic origin. Thus, despite the X-ray-induced physical damages to the microbial cells and the low survival rate, the present results clearly demonstrate that our experimental design allows monitoring the fate of metal during microbiologically-induced reduction, including the identification and quantification of the end products of the reduction pathway. 4. Conclusions The main outcome of the present study is the demonstration that, due to the limited extension of X-ray induced damages in the cultures, the metabolism of bacteria can be monitored in situ
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by X-ray spectroscopy, regardless of their sensitivity to X-rays, provided the culture to beam size ratio is large enough. Furthermore, we show that the resistance level of bacteria to irradiation by X-rays at a synchrotron beamline is linked to their ability to resist other environmental stresses. Neither mutations nor physical damages to the membranes were observed, which support the view that the high-energy focussed beam induces cell death through the damaging of the DNA molecules. This result however, should not conceal the fact that the cells directly exposed to the synchrotron beam dye rapidly during or after exposure. Therefore, kinetic in situ analyses on live material are possible only if a large culture to beam size ratio can be obtained. Thus, in the current state of the art, this still precludes kinetic studies on single cells. This setup allowed us to demonstrate that the X-ray sensitive bacterium, A. tumefaciens strain C58, can metabolize selenite while irradiated with a culture to beam size ratio of 500. We could further demonstrate that selenite is reduced by the bacterium into one third elemental selenium and two thirds methylated selenide species, in absence of other selenium species intermediates. This method can be applied to the monitoring of other metals relevant to dissimilatory metal reduction, such as iron, manganese and arsenic, under conditions typical of the deep-sea hydrothermal vent systems, e.g. under high temperature and high pressure, in high pressure cells. This will allow getting environmentally relevant data on the chemical reactions fuelling these ecosystems. Acknowledgements The authors wish to thank Jean-Louis Hazemann for the opportunity to conduct this series of experiments on beamline BM30B of the CRG at the ESRF. We are grateful to two anonymous reviewers whose comments helped us improve the quality of this manuscript. This work was supported by the interdisciplinary program GEOMEX of the CNRS to ID and PO. References [1] D. Thavarajah, A. Vandenberg, G.N. George, I.J. Pickering, Chemical form of selenium in naturally selenium-rich lentils (Lens culinaris L.) from Saskatchewan, J. Agric. Food Chem. 55 (2007) 7337–7341. [2] G. Sarret, E. Harada, Y.E. Choi, M.P. Isaure, N. Geoffroy, S. Fakra, M.A. Marcus, M. Birschwilks, S. Clemens, A. Manceau, Trichomes of tobacco excrete zinc as zinc-substituted calcium carbonate and other zinccontaining compounds, Plant Physiol. 141 (2006) 1021–1034. [3] E. Harada, G. Sarret, M.P. Isaure, N. Geoffroy, S. Fakra, M.A. Marcus, M. Birschwilks, S. Clemens, A. Manceau, Y.E. Choi, Detoxification of zinc in tobacco (Nicotiana tabacum L.) plants: exudation of Zn as Ca-containing grains through the trichomes, Plant Cell Physiol. 47 (2006) S155. [4] T.G. Sors, D.R. Ellis, G.N. Na, B. Lahner, S. Lee, T. Leustek, I.J. Pickering, D.E. Salt, Analysis of sulfur and selenium assimilation in Astragalus plants with varying capacities to accumulate selenium, Plant J. 42 (2005) 785–797. [5] B.F.G. Popescu, I.J. Pickering, G.N. George, H. Nichol, The chemical form of mitochondrial iron in Friedreich's ataxia, J. Inorg. Biochem. 101 (2007) 957–966.
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Research note
Micro-X-ray absorption near edge structure as a suitable probe to monitor live organisms Phil M. Oger ⁎, I. Daniel, A. Simionovici 1 , A. Picard Laboratoire de Sciences de la Terre, UMR CNRS 5570, Ecole Normale Supérieure de Lyon-Université Claude Bernard Lyon 1, Lyon, F-69364, France Received 20 June 2007; accepted 19 December 2007 Available online 9 January 2008
Abstract X-ray spectroscopies are very powerful tools to determine the chemistry of complex dilute solutes in abiotic and biotic systems. We have assayed their suitability to monitor the chemistry of complex solutions in a live biotic system. The impact of the probe on cells was quantified for 4 different cellular organisms differing in their resistance level to environmental stresses. We show that none of the organisms tested can survive the radiation doses needed for the acquisition of meaningful spectroscopic data. Therefore, on one hand, X-ray spectroscopy cannot be applied to the monitoring of single cells, and cellular damages have to be taken into account in the interpretation of the evolution of such systems. On the other hand, due to the limited extension of X-ray induced cellular damages in the culture volume, it is possible to probe a population of live cells provided that the culture to beam probe ratio is large enough to minimize the impact of mortality on the evolution of the biological system. Our results suggest that it could be possible to probe the volume in the close vicinity of a cell without affecting its activity. Using this setup we could monitor the reduction of selenite by the X-ray sensitive bacterium, Agrobacterium tumefaciens strain C58, for 24 h. This method has a great potential to monitor the respiration of various metals, such as iron, manganese and arsenic, in situ under relevant environmental conditions by live microorganisms. © 2008 Elsevier B.V. All rights reserved. Keywords: XANES; XRF; Dissimilatory metal reduction; in situ
1. Introduction X-ray spectroscopy, and in particular X-ray Absorption Near Edge Spectroscopy (XANES), have been used to monitor elemental speciation in biological tissues of plants [1–4] and animals [5,6], in plant, animal or bacterial cells [2,7–9], as well as in biominerals [10,11]. It has become a central analytical tool to determine the speciation of contaminating metals in polluted sites, and to monitor their fate in the environment. X-ray Fluorescence (XRF) has recently been used to determine the cellular concentration and spatial distribution of calcium, iron in ⁎ Corresponding author. Current address: Laboratoire de Sciences de la Terre, Ecole Normale Supérieure, 46, Allée d'Italie, 69364 Lyon cedex 07, France. Tel.: +33 472 728 792; fax: +33 472 728 677. E-mail address: [email protected] (P.M. Oger). 1 Present address: Laboratoire de Géophysique Interne et Tectonophysique, UMR CNRS 5559, Univ. J. Fourier, BP 53, 38041 Grenoble. 0584-8547/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.sab.2007.12.013
eukaryotic cells, for which the technique holds interesting perspectives. Indeed, several authors have proposed that it could be used to monitor the cellular trafficking of these important ions on live tissues [7,8]. However, unlike other spectroscopic techniques involving visible, infrared or ultraviolet light, the X-ray probe may induce severe damages to the cells under scrutiny, which in turn might interfere with 1) their survival, 2) their metabolism and 3) the trafficking one wants to monitor. Unfortunately, there is virtually no information on the sensitivity of living organisms to X-ray irradiation. Damages induced by other types of radiations, such as ultraviolet and gamma radiation, have been studied in far more details, while most X-ray data derive from medical studies linked to imaging or cancer treatments. In superior Eukaryotes the tolerance to irradiation is extremely low. An irradiation of 2 Gray kills foetal, ovary or bone marrow cells. One Gray (Gy) corresponds to 1.0 J kg− 1 of absorbed radiation; a focussed synchrotron X-ray beam delivers several kGy to the sample per
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second. Bacteria, to which the most radiation tolerant organisms belong, can withstand a few hundred Gy [12]. The most radiation tolerant organism, e.g. the bacterium Deinococcus radiodurans, tolerates irradiations up to 10 kGy without loss of viability [13]. The tolerance level of a specific organism to irradiation has been linked to its ability to repair the damages induced to cellular macromolecules, in particular nucleic acids [14,15]. Although it remains difficult to extrapolate the above data to the irradiation conditions of a synchrotron experiment with beam intensity several orders of magnitude higher than that used in standard procedures, the induced damages are likely to be important. Therefore, before any convincing data can be obtained by X-ray spectroscopy on live organisms, it was crucial to determine 1) the extent of the X-ray induced damages during analysis, 2) the kinetics of cell death in the conditions typical of a XANES spectrum acquisition to assess the feasibility of in situ monitoring of live cells activity by X-ray spectroscopy. In this paper, we report the survival rates to X-ray exposure of 4 strains of bacteria, differing in their resistance level to environmental stresses. Although, none of the strains studied can withstand the conditions required for the acquisition of a single XANES spectrum we demonstrate that the limited extension of radiation damages in the culture allows the use of X-ray spectroscopy to monitor microbial processes in situ provided a sufficiently low beam to culture volume ratio is employed. This is illustrated by monitoring of the reduction of selenium by an X-ray sensitive bacterium.
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(Fig. 1). Air was kept between the stoppers and the bacterial culture to prevent contamination. The irradiations were performed at the CRG beamline BM30B of the European Synchrotron Radiation Facility (ESRF). The incident beam was focussed into a 150 µm × 300 µm spot (vert. × hor.) by mirrors in the Kirkpatrick–Baez geometry. The incident flux ca. 3 1010 ph/s, used for normalization, was measured using a Si photodiode located before the sample. To achieve a homogenous irradiation of the culture, the entire volume of the capillary was scanned in
2. Materials and methods 2.1. Bacterial strains and growth conditions Four different strains with variable tolerance to UVand gamma ray exposure were assayed: E. coli strain DH5α [16] and Pseudomonas syringae strain PPI203 [17] are sensitive strains. Rhodococcus erythropolis strain W2 is desiccation and heavy metal tolerant [18]. Last, D. radiodurans strain R1 is the most radiotolerant bacterium known to date [13]. Escherichia coli strain DH5α, P. syringae pv. pisi strain PPI203 and R. erythropolis strain W2 are from our bacterial culture collection. D. radiodurans strain R1 was obtained from the American Type Culture Collection. A fifth strain, Agrobacterium tumefaciens strain C58 [19] which can reduce selenite into elemental selenium [17] was chosen as a test organism to monitor the microbiologically mediated reduction of metals by XANES spectroscopy [8]. Bacteria were grown at 30 °C in low salt Luria-Bertani broth (LB low salt, GIBCO®), for 24 h under vigorous shaking. Cells were harvested by centrifugation, washed twice in LB low salt and adjusted to a final concentration of 109 cells/ml. 2.2. Experimental set up Ultra clean glass capillaries (Mark Röhrlichen™, Glass, 10 µm thick, 1.5 mm diameter) were cut open at their smaller end. The larger extremity of the capillaries was connected to a micropipette tip, and used to pipette 10 µl of the cell suspension. The capillary was sealed at both ends to prevent evaporation
Fig. 1. A: Experimental setup. B: Monte-Carlo simulation of the absorption of the X-ray beam by the glass capillary. The portion of the capillary that receives a significantly lower X-ray dose is shaded gray. This corresponds to a volume lower than 7% of the total experimental culture volume, and to doses ranging between 97 and 95% of that deposited at the center of the capillary.
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five runs at a constant speed (Fig. 1A). The intensity of the incident flux was adjusted by using a set of aluminium filters, and by varying the scanning speed between 2 and 17 µm s− 1. Each experiment consisted of two identical capillaries, one irradiated as described above, and one control kept in the experimental hutch away from the beam. 2.3. Irradiation dose calculation The X-ray doses deposited in the experimental volume are presented throughout the manuscript in Gy to allow an easy comparison between treatments and strains and data from the literature. The Irradiation Dose (ID) was calculated as follows: ID ¼ PhtE1:6 1019 MA
ð1Þ
Where ID is in Gy, Ph is the average number of photons per second during irradiation, t is the duration of irradiation (s), E is the beam energy (eV), M is the mass normalization factor, A is the correction factor for the absorption of the glass capillary. In this experiment M is constant and equal to 105 since the sample volume is 10 µl. The correction factor for the absorption of the glass capillary is constant for a given type of capillary. The value of A is estimated to 0.95 in the present experiment, as detailed below. The absorption of the incident beam by the empty glass capillary was measured to be 10%. In order to check for spatial heterogeneity, we performed a Monte-Carlo simulation (Fig. 1B). This showed that the absorption by the capillary varied between 5 and 10% from the center to the edge of the capillary. However, only ca. 7% of the sampled volume received a significantly lower irradiation dose (Fig. 1B), ranging between 97 and 95% of that received at the center of the capillary. Thus, the irradiation dose received by the microorganisms was calculated as the total beam energy corrected for a homogeneous absorption of 5% by the front wall of the glass capillary. The irradiation doses for the different experiments are reported in Table 1.
2.4. Cell survival determination Cell survival was estimated by the colony forming unit method [20]. The cells from the irradiated and control capillaries
Fig. 2. Estimation of bacterial survival following irradiation. The survival of the bacteria was estimated by their ability to form colonies. Single isolated colonies are visible as dark spots for the higher dilutions. Left: Irradiation dose (Gy); Top: Dilution factor. For example, for a calculated dose of 173,360 Gy, we enumerate 2 108 surviving Deinococcus radiodurans cells. Enumerations are performed in quadriplicates.
were transferred to a clean sterile tube using a micropipette. A 10 fold serial dilution series was realized in sterile 0.9% NaCl solution. 10 µl drops of each dilution were spotted in quadruplicate onto a LB agar plate and incubated at 30 °C. Colonies formed on the agar surface were counted after 24 to 72 h depending on the bacterial strain (Fig. 2). Using this method, survival is accurately quantified down to 10− 5 survivor per original cell. 2.5. Membrane integrity determination
Table 1 Irradiation exposure doses for each bacterial strain in Gy E. coli
P. syringae
R. erythropolis
D. radiodurans
1180 2100 8650 31,100 34,300 55,300 142,000
920 1940 5330 8200 31,000 33,600 35,400 57,800 157,000
22200 26400 118,000
2250 5300 24,400 28,400 33,000 54,300 173,000 214,000
Potential damages to the cell envelop were estimated with the LIVE/DEAD coloration kit (Molecular Probe, France). This kit includes two fluorescent dyes. The first dye is soluble in lipids, and can therefore cross the cell membrane and accumulates in the cells, which are stained green. The second dye is insoluble in lipids, therefore does not cross the membrane when the cells are intact, and only accumulate in the cells when the integrity of the cell envelop is compromised. Consequently, doubly stained-cells have compromised membranes, e.g. are dead.
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3. Results and discussion 3.1. Survival of bacteria to high intensity X-ray irradiation The radiation sensitive bacterial cultures E. coli and P. syringae, and the most resistant strain D. radiodurans were submitted to irradiation up to 150 kGy and 214 kGy, respectively (Table 1). The survival to irradiation of the two sensitive species, E. coli and P. syringae, was extremely similar. Viability was severely affected even at the lowest irradiation doses, with survival rates ca. 10% for both strains (Fig. 3). Their survival decreased rapidly to reach values close to the detection limit of our setup at the highest exposure dose (150 kGy). The observed irradiation dose leaving 10% survival, hereafter named DL10, is 1.0 and 1.9 kGy for E. coli and P. syringae, respectively. In contrast, no loss of viability was detected at irradiation doses lower than 50 kGy for the radiation resistant species D. radiodurans, a dose that already induces a 3 log drop in viability for the two sensitive strains. At the highest dose tested in our setup (214 kGy), the decrease in viability of D. radiodurans does not exceed 1.3 logs. The calculated DL10 for D. radiodurans in this setup is 195 kGy. The observed tolerance of R. erythropolis is intermediate between the sensitive (E. coli and P. syringae) and the resistant (D. radiodurans) bacteria, showing a 2 log drop in viability at 120 kGy. The DL10 for this strain is 15 kGy. The DL10 values established in this study are at least one order of magnitude higher than those reported in the literature for comparable bacterial species [12], indicating that the present irradiation strategy induces less damage to the cells. In this experiment, the cells were exposed to a bright focused monochromatic 12.5 keV X-ray beam generating irradiation intensities ranging from 2 to 40 Gy s− 1. Under conventional irradiation conditions, cells are most often exposed to a 60Co source (1.25 MeV, γ-rays), a 137 Cs source (0.662 MeV, γ-rays) or a medical X-ray irradiation devices operating between 20 and 200 keV. These irradiation conditions correspond to irradiation intensities ranging from 1 to 50 Gy mn− 1, ca. 50 times less at
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least than the intensity to which the bacteria were exposed in the present experiment. Thus, in conventional conditions, the same irradiation dose is obtained by exposing the cell to the source for longer periods. The impact of beam energy on cell survival has been studied to some extent to determine the survival of selected human cells to different medical treatments. It was shown that twice more damage occur at 20 keV vs. 200 keV, and four times more at 20 keV vs. 1.25 MeV [21,22]. So the apparent cell damage is negatively correlated to the X-ray energy. The impact of the spectral composition of the incident beam on the survival after irradiation showed a two fold reduction for irradiations using monochromatic X-rays vs. filtered X-rays for a mean energy of 20 keV or 40 keV [23]. This increased morbidity was attributed to the low energy X-rays present in the spectrum of the filtered X-rays. Thus, we would expect that an exposure to a low energy (12.5 keV) monochromatic X-ray beam should induce at least as much damage at the same dose than those reported in the literature for γ-ray tolerance. Therefore, we propose that the reduced mortality observed in the present study stems from the short duration of irradiations imposed to the microorganisms. Nevertheless, the corresponding physical or biological mechanisms remain to be elucidated. Cell death following the exposure to γ-, UV- or X-rays results mainly from the formation of double-strand breaks in the DNA [14,24]. The cells can only repair a percentage of these, which therefore accumulate to a level that inhibits cell division. Although X-rays do not directly affect the DNA molecules; they generate free radicals from water and oxygen, which in turn create the nicks in the DNA molecules. This is visible as a sharp decrease in the ability of the irradiated culture to form colonies, but does not lead to the apparition of spontaneous mutations or cellular damages. We evaluated the apparition of colony morphology mutants on culture plates, as well as cell membrane damages under the microscope after selective LIVE/DEAD staining. In this experiment, we did not observe any colony morphology mutant for any of the four species, regardless of the beam intensity. Furthermore, in the preparations of all strains a constant proportion of dead cells (10%) was enumerated in the irradiated samples, regardless of the exposure time and dose. The same proportion of dead cells was also observed in the unirradiated samples, and is typical of the cellular turnover in a stationary phase bacterial culture. Thus, the increased mortality after irradiation in our setup cannot be attributed to an increase in physical damages to the cell envelop. Last, the possibility that the absorption of the incident X-ray beam and subsequent release of energy by the metal under scrutiny might influence the survival of the microbial culture was assayed. Duplicate cultures containing 5 mM of Na2SeO3 for each bacterial strain were irradiated at the same dose below (12.5 keV) or above (12.8 keV) the Se K edge. The survival was estimated as above. No difference could be detected for any of the 4 strains (Fig. 4 and data not shown). 3.2. Impact of partial irradiations on survival
Fig. 3. Bacterial survival as a function of X-ray irradiation dose. Enumeration error bars are comprised within the dots. DL10, 10% lethality limit. The curves are guides for the eye.
The above observations show that only D. radiodurans may be able to survive the conditions required for a µXANES
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spectrum acquisition, although suffering extensive mortality, since the acquisition of a single spectrum would lead to a viability loss larger than 90%. This would preclude any kinetic measurement of metal reduction over several hours. Nevertheless, hypothesizing that the motion of the free radicals responsible for cellular damages in the cell culture would be very short, we propose that the damage done to the microbial culture could be limited to the beam and its immediate environment. The hypothesis was assayed by partially irradiating a cell culture of the sensitive strain PPI203. A capillary containing a PPI203 cell culture was allowed to sediment. Afterwards ca. 20% of the bacteria remain in solution, while 80% deposit in a pellet at the bottom of the capillary. The sole bacterial pellet was irradiated at a dose of 50 kGy, conditions leading to a cell survival below 0.1% for PPI203. After irradiation, the whole culture, including the pellet and the suspension, was recovered and cultured. The survival was measured to be 18.5%, which matches closely the proportion of bacteria in the suspension. This confirms that the expansion of the damages caused by the X-rays is spatially limited to the irradiated volume. Therefore, we propose that a large enough ratio between the size of the incubator and the size of the beam should lead to a cellular viability decrease which is compatible with the acquisition of meaningful X-ray data. This ratio likely depends on the type of organism under scrutiny, on the energy of the beam, as well as on the exposure time to X-rays. 3.3. Applicability to in situ metabolic studies In the present study, we have used a culture to beam size ratio of 500. We monitored the metabolism of selenite by the X-ray sensitive strain of A. tumefaciens strain C58, which reduces selenite (SeO32−, IV) into elemental selenium (Se, 0) [17] and dimethyl selenide (CH3–Se–CH3, II) by a combination of
Fig. 4. Impact of X-ray absorption on the survival of bacteria following irradiation. The Impact of X-ray absorption on the survival of bacteria following irradiation. The cells were incubated in the presence of 5 mM selenium and irradiated above (12.8 keV), or below (12.5 keV) the absorption K edge of selenium to quantify the impact of X-ray absorption during spectrum acquisition.
Fig. 5. Selenite reduction by strain C58. The conversion from selenite to selenium was monitored over a 24 hour period by XANES. Se(IV): at the start of the experiment, all the selenium is present as selenite. Se(0) + Se(II): At the end of the experiment, selenium is present as a mixture of one third metallic selenium [Se(0)] and two third dimethyl selenide [Se(II)].
µXANES and µXRF analyses [8]. In A. tumefaciens, several pathways have been proposed for the reduction of selenite, which potentially involve an uncharacterized intermediate. A total of 72 XANES spectra of 100 points each, for a total irradiation duration of 21,600 s at an average beam intensity ca. 1011 ph/s were acquired over a 24 hour period, at the same location in the center of the incubator. Under these experimental conditions, we observed a survival rate of ca. 5% in the incubator, while the same cell type only survives a few seconds in the beam. As shown in Fig. 5, the spectrum obtained at 0 h is typical of selenite. Spectra acquired for intermediate incubation times clearly show a gradual replacement of selenite by reduced selenium species in solution. After 24 h, the reaction is complete, and the XANES spectra no longer show any contribution of selenite. Quantitative linear combinations of different redox species of selenium demonstrate the contribution of only 3 selenium species to explain the experimental data, selenite, selenium and dimethyl selenide, and exclude the formation of other species of selenium, such as selenium dioxide or selenate. During the reduction of selenite, selenite is reduced to one third fully reduced amorphous metallic selenium (Se, 0) and 2 thirds dimethyl selenide (CH3–Se–CH3, II). In the control experiment, which consisted of the same medium but devoid of bacteria, no reduction could be observed indicating clearly that the reported activity is of biotic origin. Thus, despite the X-ray-induced physical damages to the microbial cells and the low survival rate, the present results clearly demonstrate that our experimental design allows monitoring the fate of metal during microbiologically-induced reduction, including the identification and quantification of the end products of the reduction pathway. 4. Conclusions The main outcome of the present study is the demonstration that, due to the limited extension of X-ray induced damages in the cultures, the metabolism of bacteria can be monitored in situ
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by X-ray spectroscopy, regardless of their sensitivity to X-rays, provided the culture to beam size ratio is large enough. Furthermore, we show that the resistance level of bacteria to irradiation by X-rays at a synchrotron beamline is linked to their ability to resist other environmental stresses. Neither mutations nor physical damages to the membranes were observed, which support the view that the high-energy focussed beam induces cell death through the damaging of the DNA molecules. This result however, should not conceal the fact that the cells directly exposed to the synchrotron beam dye rapidly during or after exposure. Therefore, kinetic in situ analyses on live material are possible only if a large culture to beam size ratio can be obtained. Thus, in the current state of the art, this still precludes kinetic studies on single cells. This setup allowed us to demonstrate that the X-ray sensitive bacterium, A. tumefaciens strain C58, can metabolize selenite while irradiated with a culture to beam size ratio of 500. We could further demonstrate that selenite is reduced by the bacterium into one third elemental selenium and two thirds methylated selenide species, in absence of other selenium species intermediates. This method can be applied to the monitoring of other metals relevant to dissimilatory metal reduction, such as iron, manganese and arsenic, under conditions typical of the deep-sea hydrothermal vent systems, e.g. under high temperature and high pressure, in high pressure cells. This will allow getting environmentally relevant data on the chemical reactions fuelling these ecosystems. Acknowledgements The authors wish to thank Jean-Louis Hazemann for the opportunity to conduct this series of experiments on beamline BM30B of the CRG at the ESRF. We are grateful to two anonymous reviewers whose comments helped us improve the quality of this manuscript. This work was supported by the interdisciplinary program GEOMEX of the CNRS to ID and PO. References [1] D. Thavarajah, A. Vandenberg, G.N. George, I.J. Pickering, Chemical form of selenium in naturally selenium-rich lentils (Lens culinaris L.) from Saskatchewan, J. Agric. Food Chem. 55 (2007) 7337–7341. [2] G. Sarret, E. Harada, Y.E. Choi, M.P. Isaure, N. Geoffroy, S. Fakra, M.A. Marcus, M. Birschwilks, S. Clemens, A. Manceau, Trichomes of tobacco excrete zinc as zinc-substituted calcium carbonate and other zinccontaining compounds, Plant Physiol. 141 (2006) 1021–1034. [3] E. Harada, G. Sarret, M.P. Isaure, N. Geoffroy, S. Fakra, M.A. Marcus, M. Birschwilks, S. Clemens, A. Manceau, Y.E. Choi, Detoxification of zinc in tobacco (Nicotiana tabacum L.) plants: exudation of Zn as Ca-containing grains through the trichomes, Plant Cell Physiol. 47 (2006) S155. [4] T.G. Sors, D.R. Ellis, G.N. Na, B. Lahner, S. Lee, T. Leustek, I.J. Pickering, D.E. Salt, Analysis of sulfur and selenium assimilation in Astragalus plants with varying capacities to accumulate selenium, Plant J. 42 (2005) 785–797. [5] B.F.G. Popescu, I.J. Pickering, G.N. George, H. Nichol, The chemical form of mitochondrial iron in Friedreich's ataxia, J. Inorg. Biochem. 101 (2007) 957–966.
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