ELECTRON MICROPROBE AND X-RAY MICROFLUORESCENCE ANALYSES OF COPPER BINDING TO ACTIVE AND INACTIVATED CELLS OF MUCOR ROUXII

I. Cano-Aguilera¹, J.L. Gardea-Torresdey¹, N.E. Pingitore Jr.², and R. Webb³

¹Chemistry Department, ²Department of Geological Sciences, and ³Department of Biological Sciences, The University of Texas at El Paso, El Paso, TX, 79968.

ABSTRACT

Electron microprobe and x-ray microfluorescence spectroscopies have been used to study copper binding to active and inactivated Mucor rouxii copper-sensitive and copper-tolerant cells. A better understanding of metal resistance may help in the application of fungal biomass for the treatment of metal-contaminated water, and also in enrichment or recycling of valuable metals. After repeated culturing in progressively higher concentrations of copper sulfate, a copper-tolerant Mucor rouxii strain was obtained. The copper-tolerant strain differed from the sensitive parental strain in both shape and size. Copper binding studies using a laboratory batch technique revealed that the copper-tolerant strain cultured at higher copper levels bound large amounts of this metal. Electron microprobe and x-ray microfluorescence analyses showed that the copper characteristic x-ray signal on the cell surface of the copper-tolerant strain after copper binding was higher than the copper signal in sensitive cells. The copper signal in cross sections of the copper-tolerant cells also showed a statistically significant correlation with the sulfur signal but no correlation with the phosphorus signal. These results suggest that there are several mechanisms for metal detoxification inside and outside of the Mucor rouxii cells and that copper may be binding to sulfur-containing groups.

Keywords: electron microprobe, x-ray microfluorescence, metal binding, fungi.

INTRODUCTION

Microorganisms present several mechanisms of metal tolerance. Metal-microbe interactions can be conveniently divided into three main mechanisms: 1) extracellular interactions, involving extracellular polymers, proteins, acid metabolites and changes in the localized environment due to biochemical processes; 2) cell surface interactions, in which metals bind to microbial cell surfaces as a result of specific functional groups, and 3) intracellular interactions, where metals accumulate in microbial cells due to specific active transport processes. Detoxification occurs through transformation to insoluble or more volatile forms, or incorporation of specific metals into proteins (Ford and Mitchell, 1992).

Microorganisms require metals in trace quantities for metabolism and growth, but higher concentrations can be toxic (Ford, et al., 1995). The toxicity of metals is caused primarily by their ability to denature proteins. This effect can be caused by the blocking of functional groups, displacing an essential metal, or modifying the active conformation of the molecule (Gadd and Griffiths, 1978). Microbial cells require mechanisms for the acquisition of the essential metals to metabolize, grow, and reproduce. However, for survival in environments containing high concentrations of available metals, mechanisms to counter the inherent toxicity of the metal ions are required (Gadd, 1993). The important role played by fungi in biogeochemical cycles make them candidates for studies of metal-microbe interactions. Fungal strains belonging to the class of Zygomycetes are of particular importance due to the presence in their cell walls of such polymers as chitin, chitosan, and glucan (Bartnicki-Garcia, 1968). These polymers are known to be efficient metal ion biosorbents (Muzzarelli, 1985).

A better understanding of the factors responsible for metal resistance may help in the application of fungal biomass for the treatment of metal-contaminated water, and in enrichment or recycling of valuable metals (Gadd, 1993). The potential use of Mucor rouxii biomass for the removal and recovery of heavy metal ions from aqueous solution has been proposed in previous studies (Gardea-Torresdey, et al., 1996). Preliminary studies have been performed on the cellular localization of copper and the mechanisms for copper binding in Mucor rouxii (Ramirez-Salgado, et al., 1996; Gardea-Torresdey, et al., 1996). However, further studies are required to gain a better understanding of metal binding to the fungus, in particular the binding of copper ions. Electron microprobe and x-ray microfluorescence spectroscopic analyses are capable of detecting, in situ, elements at the cellular level (Jansen, et al., 1982; Nott, 1995). Unlike such bulk analytic techniques as atomic absorption (AA) and inductively coupled plasma atomic emission spectroscopy and mass spectrometry (ICP-AES and ICP-MS), electron probe spectroscopy and x-ray microfluorescence permit spatially resolved in situ elemental analysis of biological materials (Goldstein, et al., 1981). Advantages of the microprobe are low detection limits, down to perhaps 100 or few hundred ppm, and high spatial resolution, with an analytic volume or perhaps a few cubic micrometers. The chief disadvantage is the damage to the sample often caused by the electron beam. X-ray microfluorescence uses a stopped-down x-ray beam to excite the sample to fluoresce. This technique is employed at synchrotron sources and is now available in a less powerful form in several laboratory instruments. The advantage of x-ray microfluorescence over the microprobe is the stability of biological materials in the x-ray excitation beam. This permits long excitation and counting times, and therefore better detection limits, especially for the transition metals (perhaps 10 ppm). Of course, the electron microprobe permits much better spatial resolution. We believe that the x-ray microfluorescence technique will have significant applications in environmental analysis of biological materials, as demonstrated in the present paper. Consequently, it has been evident for some time that these methods could in principle provide unique and important information about the metal tolerance mechanisms expressed by living cells.

The purpose of this study was to elucidate the mechanisms used by Mucor rouxii for copper-tolerance. By using electron microprobe and x-ray microfluorescence analyses, we investigated: a) the copper bound by inactivated cells after metal binding, in both the copper-sensitive and the copper-tolerant cells; b) copper uptake by active cells in cross sections of both young and mature copper-tolerant cells and the possible elements associated with the copper binding, and c) the intracellular accumulation and distribution of copper.

METHODOLOGY

Microorganisms

Mucor rouxii strains copper-sensitive (IM-80) and Mucor rouxii copper-tolerant (P1) were utilized in this study. The copper-tolerant strain was derived from the sensitive parental strain by exposure of the cells growing in the presence of increasing and subinhibitory copper concentrations of up to 4.8 mM copper ions (as CuSO₄). The propagation and maintenance of cells have been described previously (Gardea-Torresdey, et al, 1996). The resulting biomasses (after culturing) were washed, freeze-dried, ground, and sieved to pass a 100-mesh screen.

Copper binding capacity studies

For copper binding capacity studies, the biomass of each fungal strain was washed by centrifugation twice with 0.01M HCl to remove any debris or soluble biomolecules that might interact with metal ions. Washings were collected, dried and weighed to account for any biomass weight loss. Each biomass was resuspended in 0.01 M HCl (5 mg per ml) and adjusted to pH 5. After centrifugation, the supernatants were removed and a copper solution of 0.3 mM (CuSO₄) at pH 5 was added to each of the corresponding pellets. The suspensions were shaken for 15 min by continuous agitation and then centrifuged. The same biomasses were resuspended several more times in fresh metal solution until saturation (i.e., no more copper was bound by the biomass). Final pH was measured and the metal content was determined by flame atomic absorption spectroscopy (FAAS) of all the supernatants. The amount of metal ion bound to the biomass was calculated from the total metal accumulated from these separate metal-containing solutions as described in previous copper adsorption studies (Gardea-Torresdey, et al., 1996).

Electron microprobe analysis of copper bound to inactivated Mucor rouxii cells

Electron microprobe spectroscopy was used to analyze Mucor rouxii cells laden with copper. After copper saturation, each one of the biomasses was washed, dried and fixed with varnish on a glass slide. These preparations were then carbon coated (~200 Å) to make the surface electrically conductive. Cells were analyzed for elemental composition using Wavelength Dispersion Spectroscopy (WDS) on a CAMECA SX-50 electron microprobe. Operating conditions of the microprobe were: beam current 25 nA, electron energy 5.0 keV, and analysis time, 60 seconds.

X-ray microfluorescence analysis of copper bound to inactivated Mucor rouxii copper-tolerant cells

For x-ray microfluorescence analysis, the cells from copper saturation studies of the copper-tolerant Mucor rouxii strain cultured at high and trace copper concentrations were washed with distilled water and freeze-dried. The resulting biomasses were placed directly on a glass slide and the cells were analyzed on a Kevex Omicron x-ray microfluorescence spectrometer for elemental composition. We used a 100 micrometer beam and an energy dispersive solid-state detector (EDS) for analysis The operating conditions were: energy Mo tube at 40 keV, and up to 2 min counting time.

Correlation among elements in cross sections of active Mucor rouxii cells by electron microprobe analysis

To attempt to correlate the tungsten signal with the phosphorus signal as well as the copper signal with either the phosphorus or sulfur signal, Mucor rouxii copper-tolerant cells cultured at high copper concentration for 48 h were washed with distilled water and embedded in paraffin (Kierman, 1981). Semi-ultrathin sections of the paraffin embedded cells were then exposed to 10% sodium tungstate in distilled water at pH 5.5 (adjusted with 1 M HCl), and incubated for 24 h at 37^oC (Lillie, 1976). All preparations were washed with distilled water and dried. Elemental signals for W, P, S, and Cu were recorded by electron microprobe analysis and the correlation coefficients were computed.

Copper localization in active Mucor rouxii cells

Mucor rouxii copper-tolerant cells cultured at high copper concentrations for 12 h were prepared by fixing, dehydrating, and embedding them in epoxy resin (Gardea-Torresdey, et al., 1997). Ultrathin sections of resin-embedded cells were then exposed to 0.05% alcoholic rubeanic acid (dithiooxamide) in 10% aqueous sodium acetate (to prevent staining of both cobalt and nickel) and subsequently were incubated for 2 h at room temperature. The former procedure was performed for the localization of copper-colored complexes (Bancroft and Stevens, 1982) inside the cells as copper rubeanate by using electron microprobe analysis.

Copper localization on inactivated Mucor rouxii cells

Inactivated cells of the copper-tolerant Mucor rouxii cultured at high copper concentration, after copper saturation studies, were washed with distilled water, then freeze-dried and fixed with methanol. These preparations were also exposed to rubeanic acid (as described before) for the localization of copper-colored complexes on the surface of the cells.

RESULTS AND DISCUSSION

Metal binding by whole organisms, tissues, or cells is typically determined indirectly by analysis of the metal remaining in the supernatants by Atomic Absorption Spectroscopy or other similar techniques. However, such bulk analysis cannot localize the metal in any particular cell compartment (Kirk and Lee, 1995). In contrast, electron microprobe and x-ray microfluorescence provide spatially resolved in situ analyses which may elucidate the cell components (and elements) that interact with the reactive forms of different metals to drive the processes of uptake and accumulation.

Electron microprobe analysis provides a useful tool to perform in situ elemental analysis while scanning electron images of the cells, since one can control the size of the beam as well as the size of the mass excited. In this way, one can also use cross sections to compare specific subcellular components with each other for their relative metal concentrations. On the other hand, the applicability of x-ray microfluorescence analysis is of interest since it does not require particular fixation techniques that could alter metal distribution or concentration (Goldstein, et al., 1981).

It has been previously reported that it is possible to localize areas of specific metal reactivity by the use of highly metal specific dyes and histochemical agents (Winsome, et al., 1989). For this purpose, sodium tungstate staining was used for rapid detection of nucleic acids in Mucor rouxii cross sections because of tungsten's high affinity for phosphate groups. Therefore, we expect a high correlation between the phosphorus (mainly present in nucleic acids as phosphate groups) and tungsten signals (from sodium tungstate) on the microprobe. This can provide an indirect correlation for copper binding to either phosphorus- or sulfur-containing ligands (Hashemi, et al., 1994). For example, if there is a correlation between copper and sulfur, this may mean that copper is bound to sulfur-containing ligands such as proteins that are localized throughout the cell. In contrast, if there is a correlation between copper and phosphorus, copper binding could be occurring through phosphate-containing ligands localized mainly in the nuclear region. In the same way, rubeanic acid was also used in order to distinguish the copper distribution inside and on the surface of the Mucor rouxii cells. The copper is localized in the greenish-black colored regions developed by the copper rubeanate complex.

Copper binding capacity studies of Mucor rouxii inactivated cells

Experiments were performed to determine the copper-binding capacities of fungal biomasses grown at trace and high concentrations of copper ions. Clearly, as we reported before, the copper-tolerant cells of Mucor rouxii (P1) cultured under high copper levels show almost twice (23 mg/g dry weight) as much binding capacity as the same cells (11 mg/g dry weight) cultured in trace copper concentrations (Gardea-Torresdey et al., 1997). If the results of the copper-binding saturation studies are coupled with elemental analysis, this may confirm the presence of this metal on the cell surface as well as the different abilities of each strain to bind copper.

Electron microprobe analysis of copper bound to inactivated Mucor rouxii cells

The copper signal was determined in different sites of various varnish-fixed and carbon-coated inactivated cells after copper-binding saturation studies, using electron microprobe analysis. Table 1 shows the average of the copper signal detected most likely on the surface of several fungal cells. As we observed in this table, a much higher copper signal is detected in the copper-tolerant cells cultured at higher copper levels. Although the means of the copper signal show a high standard deviation, the compared relationships among strain, exposure, and copper signal are always maintained. These data illustrate that EDS and FAAS provide complementary information in a multiple method approach, because the analysis of the concentrations of copper bound were in accord with the quantitative data obtained by using the batch laboratory technique with FAAS analysis of the supernatants.

These results may also support the previously proposed hypothesis that, upon exposure to high levels of copper in the culture medium, Mucor rouxii develops (as first defense mechanism) molecules with chemical functional groups that may bind copper ions on the surface of the cell (Gardea-Torresdey, et al., 1996, 1997).

X-ray microfluorescence analysis of copper bound to inactivated Mucor rouxii copper-tolerant cells

X-ray microfluorescence spectra were obtained of copper bound to inactivated Mucor rouxii copper-tolerant cells cultured at high-copper and trace-copper concentrations after copper-saturation studies. The spectra show that the copper peak is higher and more pronounced in cells exposed to high-copper concentration (Fig.1) than those exposed to trace-copper concentration (Fig. 2). These results are also in accord with the data obtained with electron microprobe and laboratory batch techniques.

Correlation among elements in cross sections of active Mucor rouxii cells

The presence of a statistically significant correlation between sulfur-containing groups and copper was ascertained by electron microprobe analysis of sodium tungstate and phosphorus-containing groups. Table 2 shows the correlation among copper, sulfur, phosphorus, and tungsten signals in cross sections of paraffin-embedded mature copper-tolerant Mucor rouxii cells cultured at high-copper concentration. This table clearly shows a high correlation coefficient between tungsten and phosphorus signals (as was expected) and a statistically significant correlation coefficient between sulfur and copper signals. No statistically significant correlation coefficient was found either between copper and phosphorus signals or between tungsten and sulfur signals.

These results suggest that copper is mainly bound to sulfur-containing ligands and could represent another possible mechanism responsible for copper binding. We have previously found that carboxyl groups present on the surface of Mucor rouxii cells play a role in copper binding to the fungal cell walls (Gardea-Torresdey, et al., 1996). Further biochemical and chemical studies are necessary in order to clearly identify the sulfur-containing ligands (probably of protein origin) and also for the elucidation of the biochemical mechanisms of copper binding.

Copper localization in Mucor rouxii cells

In the analysis of cross sections and whole cells of Mucor rouxii exposed to rubeanic acid, the cell envelope and nucleoplasm presented evidence of localized copper deposits. The dark-green color developed for the copper-rubeanate complexes was detected in those cell sectors where the copper was high (data not shown). The more intense the staining reaction, the higher the signal. These observations indicated that copper accumulation occurred on the surface and inside the cell, since cell cross sections were used. The results are also in agreement with the results obtained by electron microprobe analysis and the batch laboratory technique. The data, however, did not reveal details of sub-cellular distribution of this element.

CONCLUSIONS

Elevated levels of copper, bound on the cell surface of copper-tolerant Mucor rouxii biomass cultured at high copper-concentration, were documented by electron microprobe and x-ray microfluorescence spectroscopies. In tungsten-treated cross sections of the active cells, the phosphorus x-ray signal detected by electron microprobe analysis showed a statistically significant correlation with the tungsten signal, but not with the copper signal. This indicates that phosphorus is not directly involved in copper binding. On the other hand, the copper signal was higher in the tolerant strain and showed a statistically significant correlation with the sulfur signal. This suggests that copper binds to sulfur-containing groups. Probe analysis of cross sections of active cells showed that the copper is taken up and sequestered by cell walls and nuclei.

These results suggest that there are metal detoxification mechanisms both inside and outside the Mucor rouxii cells and that copper may be binding to sulfur-containing groups. Our findings support the hypothesis that, as a first defense mechanism to ambient high levels of copper, Mucor rouxii develops molecules with chemical functional groups that bind the copper ions to the surface of the cell.

Acknowledgments

The authors acknowledge the collaboration and financial support from the University of Texas at El Paso's Center for Environmental Resource Management. J.L. Gardea-Torresdey acknowledges support from the National Institutes of Health (grant GM08012-25) and R. Webb acknowledges support of the National Institutes of Health (grant RR08124-03). This material is based upon work supported by the National Science Foundation under Grant No. EAR-96011715. The electron microprobe used in this study was obtained with funds made available through the National Science Foundation Grant No. RII-8504371 and the Permanent University Fund of the University of Texas System. We also acknowledge the collaboration of Dr. J. Jesus Gamez and Gloria M. Goytia of the Pathology Department, Plaza Medica, Cd. Juarez, Chih., Mex., for the paraffin thin sections.

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Table 1. Electron Microprobe Analysis of Copper Bound to Inactivated Mucor rouxii Cells.

aCopper Signal

Mucor rouxii	Cultured at 1.6x10-3mM Cu2+	Cultured at 3.2 mM Cu2+
Copper-sensitive (IM-80)	76 ± 9b	294 ± 50b
Copper-tolerant (P1)	387 ± 91b	752 ± 324b

^a Copper characteristic x-ray counts.

^bEach entry is the mean of fifteen analyses ± standard deviation.

Table 2. Correlation Among Copper, Sulfur, Phosphorus, and Tungsten Signals in Cross Sections of Active Mucor rouxii Cells.

ACorrelation Coefficient (r)

Mucor rouxii (Copper-tolerant cells cultured at 3.2 mM Cu 2+ )	Sulfur signal	Phosphorus signal
Copper signal	0.70358b	0.02031
Tungsten signal	0.36806	0.9303b

^aEvery correlation coefficient (r) was derived from fifteen analyses in different cells of x-ray element-characteristic counts.

^b Based on the Student's t-test, r was statistically significant at (P<0.05).

Figure 1. X-ray microfluorescence spectrum obtained from the inactivated copper-tolerant Mucor rouxii cells cultured at high copper concentration after copper binding. Note the intense peak of the copper K characteristic x-ray emission. As and Ca peaks result from penetration of x-rays into the glass mounting slide and fluorescence of these elements in the glass. Large peak at the far right is Compton scattering of Mo source x-rays. The horizontal scale correspond to the x-ray energy and the vertical scale to x-ray counts.

Figure 2. X-ray microfluorescence spectrum obtained from the inactivated copper-tolerant Mucor rouxii cells cultured at trace copper concentration after copper binding. Note that the peak of the copper signal is one-tenth that of the copper signal obtained from the tolerant strain cultured at high copper concentration. As and Ca peaks result from penetration of x-rays into the glass mounting slide and fluorescence of elements in the glass. Large peak at the far right is Compton scattering of Mo source x-rays. The horizontal scale corresponds to the x-ray energy and the vertical scale to x-ray counts.