Major sites of metal binding in Bacillus licheniformis walls.

Isolated and purified walls of Bacillus licheniformis NCTC 6346 his contained peptidoglycan, teichoic acid, and teichuronic acid (0.36 mumol of diaminopimelic acid, 0.85 mumol of organic phosphorus, and 0.43 mumol of glucuronic acid per mg [dry weight] of walls, respectively). The walls also contained a total of 0.208 mumol of metal per mg. When these walls were subjected to metal-binding conditions (T. J. Beveridge and R. G. E. Murray, J. Bacteriol. 127:1502-1518, 1976) for nine metals, the amount of bound metal above background ranged from 0.910 mumol of Na to 0.031 mumol of Au per mg of walls. Most were in the 0.500-mumol mg-1 range. Electron-scattering profiles from unstained thin sections indicated that the metal was dispersed throughout the wall fabric. Mild alkali treatment extracted teichoic acid from the walls (97% based on phosphorus) but left the peptidoglycan and teichuronic acid intact. This treatment reduced their capacity for all metals but Au. Thin sections revealed that the wall thickness had been reduced by one-third, but metal was still dispersed throughout the wall fabric. Trichloroacetic acid treatment of the teichoic acid-less walls removed 95% of the teichuronic acid (based on glucuronic acid) but left the peptidoglycan intact (based on sedimentable diaminopimelic acid). The thickness of these walls was not further reduced, but little binding capacity remained (usually less than 10% of the original binding). The staining of these walls with Au produced a 14.4-nm repeat frequency within the peptidoglycan fabric. Sedimentation velocity experiments with the extracted teichuronic acid in the presence of metal confirmed it to be a potent metal-complexing polymer. These results indicated that teichoic and teichuronic acids are the prime sites of metal binding in B. licheniformis walls.     

产朊假丝酵母细胞壁对铜离子吸附机理研究

比较了产朊假丝酵母细胞与分离纯化的细胞壁对铜离子吸附能力。观察铜离子浓度、温度和pH值对产朊假丝酵母吸附铜离子的影响，探讨细胞壁在酵母吸附重金属离子过程中的作用机理。结果表明，细胞壁是酵母吸附重金属离子的主要部位。细胞壁的蛋白酶酶解实验证明，对胰蛋白酶不敏感的细胞壁嵌合蛋白是铜离子吸附的主要位点。     

Binding of an inert, cationic osmium probe to walls of Bacillus subtilis.

X-ray fluorescence spectroscopy and electron microscopy of unstained specimens have been used to study the binding of chloropentaammineosmium(III) chloride to isolated walls of Bacillus subtilis. Native walls bound 0.220 mumol of the osmium probe per mg (dry weight) of walls, whereas walls which were chemically treated to neutralize the available carboxylate groups of the peptidoglycan bound only 0.040 mumol. Teichoic acid-depleted walls bound 0.210 mumol. Thin sections of all wall types showed the osmium probe to be scattered throughout the wall matrix as a small staining deposit. The results support the idea that the metal ion-binding capacity of these walls is mediated by the available carboxylate groups in the wall fabric.     

Uptake and distribution of chromium in Saccharomyces cerevisae exposed to Cr(III)-organic compounds

Abstract

The present study investigates the influence of different Cr(III)-organic compounds [Cr(III)-citrate and Cr(III)-histidine] in growth-nonsupportive exposure medium on the uptake and localisation of chromium in the cell structure of the yeast Saccharomyces cerevisae. The amount of total accumulated chromium in yeast cells and the distribution of chromium between the yeast cell walls and spheroplasts were determined by atomic absorption spectroscopy. Chromium accumulation potential was shown to depend on treatment time, metal concentration as well as the nature of the bound ligand. Chromium uptake was characterised by a time-dependent increase of total chromium which suggests that the amount of cell-accumulated chromium also tended to increase over time. Cellular chromium accumulation (mg g^?1 dry wt) of Cr(III)-histidine is higher than Cr(III)-citrate. The pH dependence pattern of chromium accumulation is similar for both of the Cr(III)-organic compounds: pH 6.5>pH 5>pH 8. Substantial differences were found between the two Cr(III)-organic compounds, in the total chromium accumulation as well as in the distribution in yeast cell walls and spheroplasts.     

Physicochemical interaction of Escherichia coli cell envelopes and Bacillus subtilis cell walls with two clays and ability of the composite to immobilize heavy metals from solution.

Isolated Escherichia coli K-12 cell envelopes or Bacillus subtilis 168 cell walls were reacted with smectite or kaolinite clay in distilled deionized water (pH 6.0); unbound envelopes or walls were separated by sucrose density gradient centrifugation, and the extent of adsorption was calculated. At saturation, both clays adsorbed approximately 1.0 mg (dry weight) of envelopes or walls per mg (dry weight) of clay. Clays showed a preference for edge-on orientation with both walls and envelopes, which was indicative of an aluminum polynuclear bridging mechanism between the wall or envelope surface and the clay edge. The addition of heavy metals increased the incidence of planar surface orientations, which suggested that multivalent metal cation bridging was coming into play and was of increasing importance. The metal-binding capacity of isolated envelopes, walls, clays, and envelope-clay or wall-clay mixtures was determined by atomic absorption spectroscopy after exposure to aqueous 5.0 mM Ag+, Cu2+, Cd2+, Ni2+, Pb2+, Zn2+, and Cr3+ nitrate salt solutions at pHs determined by the buffering capacity of wall, envelope, clay, or composite system. The order of metal uptake was walls greater than envelopes greater than smectite clay greater than kaolinite clay for the individual components, and walls plus smectite greater than walls plus kaolinite greater than envelopes plus smectite greater than envelopes plus kaolinite for the mixtures. On a dry-weight basis, the envelope-clay and wall-clay mixtures bound 20 to 90% less metal than equal amounts of the individual components did.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physicochemical interaction of Escherichia coli cell envelopes and Bacillus subtilis cell walls with two clays and ability of the composite to immobilize heavy metals from solution

Isolated Escherichia coli K-12 cell envelopes or Bacillus subtilis 168 cell walls were reacted with smectite or kaolinite clay in distilled deionized water (pH 6.0); unbound envelopes or walls were separated by sucrose density gradient centrifugation, and the extent of adsorption was calculated. At saturation, both clays adsorbed approximately 1.0 mg (dry weight) of envelopes or walls per mg (dry weight) of clay. Clays showed a preference for edge-on orientation with both walls and envelopes, which was indicative of an aluminum polynuclear bridging mechanism between the wall or envelope surface and the clay edge. The addition of heavy metals increased the incidence of planar surface orientations, which suggested that multivalent metal cation bridging was coming into play and was of increasing importance. The metal-binding capacity of isolated envelopes, walls, clays, and envelope-clay or wall-clay mixtures was determined by atomic absorption spectroscopy after exposure to aqueous 5.0 mM Ag+, Cu2+, Cd2+, Ni2+, Pb2+, Zn2+, and Cr3+ nitrate salt solutions at pHs determined by the buffering capacity of wall, envelope, clay, or composite system. The order of metal uptake was walls greater than envelopes greater than smectite clay greater than kaolinite clay for the individual components, and walls plus smectite greater than walls plus kaolinite greater than envelopes plus smectite greater than envelopes plus kaolinite for the mixtures. On a dry-weight basis, the envelope-clay and wall-clay mixtures bound 20 to 90% less metal than equal amounts of the individual components did.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of mercury and organomercurials on cellular glucose utilization: a study using resting mercury-resistant yeast cells

AIMS: Mercury compounds are highly toxic to all types of living cells. Isolated yeast strains of Rhodotorula rubra showed high and low resistance pattern towards mercury and organomercurial compounds. To investigate the basis of differential sensitivity of these two types of strains, glucose utilization was measured in the presence of mercury compounds. METHODS AND RESULTS: Glucose utilization process remained unaffected in resting cells of highly Hg(2+)-resistant strain in the presence of HgCl(2) but not in the presence of phenylmercuric acetate and thimerosal. However, HgCl(2) significantly affected glucose utilization in the case of low-resistant cells. The Hg-retaining ability of the cell wall of highly Hg(2+)-resistant yeast strain was greater than that of the weakly Hg(2+)-resistant strain. The spheroplast-bound Hg(2+) was also significantly less in the highly Hg(2+)-resistant strain than in the weakly Hg(2+)-resistant strain. CONCLUSIONS: Glucose uptake machinery was not affected in the presence of toxic metal ions in the case of high-resistant strains. But in the case of low Hg(2+)-resistant strain, glucose transport system may be affected either by inactivation of sensor proteins containing -SH group associated with glucose uptake. SIGNIFICANCE AND IMPACT OF THE STUDY: Cell wall of mercury-resistant yeast cells may play an important role in heavy metal bioremediation process.     

Ultrastructural localization of heavy metals in the extraradical mycelium and spores of the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi, obligate symbionts of most plant species, are able to accumulate heavy metals, thereby, protecting plants from metal toxicity. In this study, the ultrastructural localization of Zn, Cu, and Cd in the extraradical mycelium and spores of the arbuscular mycorrhizal fungus Glomus intraradices grown in monoxenic cultures was investigated. Zinc, Cu, or Cd was applied to the extraradical mycelium to final concentrations of 7.5, 5.0, or 0.45 mmol/L, respectively. Samples were collected at time 0, 8 h, and 7 days after metal application and were prepared for rapid freezing and freeze substitution. Metal content in different subcellular locations (wall, cytoplasm, and vacuoles), both in hyphae and spores, was determined by energy-dispersive X-ray spectroscopy. In all treatments and fungal structures analysed, heavy metals accumulated mainly in the fungal cell wall and in the vacuoles, while minor changes in metal concentrations were detected in the cytoplasm. Incorporation of Zn into the fungus occurred during the first 8 h after metal addition with no subsequent accumulation. On the other hand, Cu steadily accumulated in the spore vacuoles over time, whereas Cd steadily accumulated in the hyphal vacuoles. These results suggest that binding of metals to the cell walls and compartmentalization in vacuoles may be essential mechanisms for metal detoxification.     

Composition and properties of the outer cell wall layer in Bacillus brevis--the producer of gramicidin S

Two membrane antigens were found by cross immunoelectrophoresis in the cell walls of Bacillus brevis var. G.-B., R form, which started to synthesize gramicidin S (20 mg per 1 ml of cultural broth). The cell wall contained no membrane components in cells at the beginning of the logarithmic growth phase. The protein with a molecular mass of 100 kDa is a component of the cell wall outer layer. The protein is not digested by trypsin or pronase when it comprises the cell walls of cells synthesizing gramicidin S. In the preparation of isolated cell walls, this protein becomes susceptible to the action of the above proteases only when the peptidoglycan layer is broken down by lysozyme. Electron microscopy of cells treated with proteases and shadowed with a metal revealed that many cells lacked the cytoplasm. Therefore, the outer layer of B. brevis R cell wall contains small regions susceptible to the action of protease along with regions composed of the 100 kDa protein and resistant to these enzymes. It is possible that the small regions contain membrane components.     

The use of hollow fiber cross-flow microfiltration in bioaccumulation and continuous removal of heavy metals from solution by Saccharomyces cerevisiae

Cross-flow microfiltration was shown to retain Saccharomyces cerevisiae biomass utilized for heavy metal bioaccumulation. The passage of metal-laden influent through a series of sequential bioaccumulation systems allowed for further reductions in the levels of copper, cadmium, and cobalt in the final effluent than that afforded by a single bioaccumulation process. Serial bioaccumulation systems also allowed for partial separation of metals from dual metal influents. More than one elemental metal cation could be accumulated simultaneously and in greater quantities than when a single metal was present in the effluent (Cu(2+) 0.43 mmol, Cu(2+) + Cd(2+) 0.67 mmol, and Cu(2+) + Co(2+) 0.83 mmol/g yeast dry mass when the initial concentration of each of the metal species was 0.2 mmol.L(-1)). Co-accumulation of two different metal cations allowed higher total levels of bioaccumulation than found with a single metal. The flux rate was 2.9 x 10(2) L.h(-2)mum(-2) using a polypropylene microfiltration membrane (0.1 mum pore size) at 25 degrees C. (c) 1994 John Wiley & Sons, Inc.     

Comparison between lead accumulation of Pomphorhynchus laevis (Palaeacanthocephala) in the intestine of chub (Leuciscus cephalus) and in the body cavity of goldfish (Carassius auratus auratus)

This experimental study assessed the role of the microhabitat in the uptake of metals by adult acanthocephalans. We examined the accumulation of lead by adult Pomphorhynchus laevis in the intestine of chub (Leuciscus cephalus) and compared it with that in goldfish, Carassius auratus auratus, in which the parasites penetrate the intestinal wall and enter the body cavity. Chub and goldfish experimentally infected with adult Pomphorhynchus laevis were exposed to 0.01 mg l(-1) Pb(2+) over 3 weeks. Lead was rapidly accumulated in the intestinal acanthocephalans reaching a mean concentration of 7.3 microg g(-1). This concentration was significantly greater than in the host muscle, liver and intestine and more than 730 times higher than the exposure concentration. Intraperitoneal P. laevis in goldfish exposed to lead did not accumulate the metal. Thus, it was conclusively shown that metal accumulation in acanthocephalans is associated with the intestinal location and does not occur in the body cavity.     

Scavenging and antioxidant properties of compounds synthesized by carotenogenic yeasts stressed by heavy metals--EPR spin trapping study

Free radical scavenging and antioxidant activities of metabolites produced by carotenogenic yeasts of Rhodotorula sp. and Sporobolomyces sp. grown under heavy metal presence were studied using various EPR experiments. The thermally initiated decomposition of K(2)S(2)O(8) coupled with EPR spin trapping was shown to be the best choice to characterize antioxidant properties of yeast's samples. EPR spectroscopy revealed that yeast walls showed higher ability to scavenge free radicals than those from inside the cells. Since carotenogenic yeast differ to each other in resistance against the heavy metals due to their individual protective system, quenching properties and antioxidant activities of carotenogenic yeasts were modulated by Ni(2+) or Zn(2+) ions variously.     

AtHMA1 is a thapsigargin-sensitive Ca2+/heavy metal pump

The Arabidopsis thaliana AtHMA1 protein is a member of the P(IB)-ATPase family, which is implicated in heavy metal transport. However, sequence analysis reveals that AtHMA1 possesses a predicted stalk segment present in SERCA (sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase)-type pumps that is involved in inhibition by thapsigargin. To analyze the ion specificity of AtHMA1, we performed functional complementation assays using mutant yeast strains defective in Ca(2+) homeostasis or heavy metal transport. The heterologous expression of AtHMA1 complemented the phenotype of both types of mutants and, interestingly, increased heavy metal tolerance of wild-type yeast. Biochemical analyses were performed to describe the activity of AtHMA1 in microsomal fractions isolated from complemented yeast. Zinc, copper, cadmium, and cobalt activate the ATPase activity of AtHMA1, which corroborates the results of metal tolerance assays. The outcome establishes the role of AtHMA1 in Cd(2+) detoxification in yeast and suggests that this pump is able to transport other heavy metals ions. Further analyses were performed to typify the active Ca(2+) transport mediated by AtHMA1. Ca(2+) transport displayed high affinity with an apparent K(m) of 370 nm and a V(max) of 1.53 nmol mg(-1) min(-1). This activity was strongly inhibited by thapsigargin (IC(50) = 16.74 nm), demonstrating the functionality of its SERCA-like stalk segment. In summary, these results demonstrate that AtHMA1 functions as a Ca(2+)/heavy metal pump. This protein is the first described plant P-type pump specifically inhibited by thapsigargin.     

Cell Wall Composition and Associated Properties of Methicillin-Resistant Staphylococcus aureus Strains

Methicillin-resistant (MR) Staphylococcus aureus strains have previously been reported to be deficient in surface negative charge; this has been correlated with methicillin resistance and ascribed to a deficiency of teichoic acid at the cell surface (A. W. Hill and A. M. James, Microbios 6:157-167, 1972). Teichoic acid was present in walls of MR organisms as revealed by appreciable phosphate levels and detection of ribitol residues. Phosphate levels in walls from five MR strains (0.54 to 0.77 mumol/mg of wall) were lower than in three unrelated methicillin-sensitive (MS) strains (0.86 to 1.0 mumol/mg of wall). However, two MS strains derived from two of the MR strains had wall phosphate levels very similar to those of the MR strains. No evidence for unusual wall polymers was found. Simple deficiency of wall teichoic acid does not result in methicillin resistance since an independently isolated teichoic acid-deficient strain (0.1 mumol of phosphate per mg of wall) was not methicillin resistant. In studies of biological properties possibly related to wall teichoic acid, it was discovered that walls isolated from MR organisms grown in the presence of methicillin autolyzed more rapidly than those isolated from organisms grown in the absence of the drug. Since methicillin resistance is enhanced by NaCl and suppressed by ethylenediaminetetraacetate, the effects of these compounds on autolysis of isolated walls were studied. NaCl (1.0 M) and ethylenediaminetetraacetate (1.0 mM) inhibited the autolysis of walls isolated from MR and MS strains. An MR strain bound phage 47, 52A, and 3A only slightly less well than their respective propagating strains.     

Biosorption of cadmium,copper and lead by isolated mother cell walls and whole cells of Chlorella fusca

Using a new method for the isolation of released mother cell walls of Chlorella fusca, the biosorption of cadmium, copper and lead by purified cell wall isolates and whole cell suspensions was comparatively characterized. In all cases whole cells accumulated more metal ions than isolated cell walls. Both the Langmuir and Freundlich isotherm models were suitable for describing the short-term adsorption of cadmium, copper and lead by cell walls and the cadmium and copper adsorption by whole cells. However, neither model could sufficiently explain the lead accumulation by whole cells. The feasibility of a practical use of whole cells or isolated cell walls as biosorbents is discussed.     

The effects of copper and zinc on <Emphasis Type="Italic">Spirulina platensis</Emphasis> growth and heavy metal accumulation in its cells

The effects of copper and zinc on Spirulina platensis (Nordst.) Geitl. growth and the capability of this cyanobacterium for accumulation of these heavy metals (HMs) were studied. S. platensis tolerance to HMs was shown to depend on the culture growth phase. When copper was added during the lag phase, its lethal concentration was 5 mg/l, whereas 4 mg/l were lethal during the linear growth phase. Zinc concentration of 8.8 mg/l was lethal during the linear but not lag phase of growth. HM-treated S. platensis cells were capable for accumulation of tenfold more copper and zinc than control cells. Independently of Cu²⁺ content in the medium and of the growth phase, cell cultures accumulated the highest amount of this metal as soon as after 1 h, which may be partially determined by its primary sorption by cell-wall polysaccharides. A subsequent substantial decrease in the intracellular copper content occurred due to it secretion, which was evident from the increased metal concentration in the culturing medium. When zinc was added during the linear growth phase, similar pattern of its accumulation was observed: the highest content after 1 h and its subsequent decrease to the initial level. When the initial density of the culture was low and the cells had much time to adapt to HM, zinc accumulated during the entire linear growth phase, and thereafter the metal was secreted to the medium. The mechanisms of S. platensis tolerance to HM related to both their sorption by the cell walls and secretion of metal excess into the culturing medium and its conversion into the form inaccessible for the cells are discussed.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 259–265.Original Russian Text Copyright © 2005 by Nalimova, Popova, Tsoglin, Pronina.This revised version was published online in April 2005 with a corrected cover date.     

Ultrastructural studies on the cell walls in Fusarium sulphureum.

The cell walls of Fusarium sulphureum have a microfibrillar component that is randomly arranged. X-ray-diffraction diagrams of the microfibrils are consistent with a high degree of crystallinity and show that they are chitin. The chitin microfibrils of the peripheral walls envelop the hyphal apex and extend across the septae. During the first 8h in culture, the conversion of conidial cells to chlamydospores is evidenced by a swelling of the cells and the original microfibrils remain randomly arranged. Within 24h new wall material is deposited as the cells expand and the wall thickens. The new microfibrils are indistinguishable from those of the original conidial cells. After 3 days in culture, the chlamydospores are fully developed and have the characteristic thick wall which is a continuous layer of randomly arranged microfibrils. Chlamydospores maintained in a conversion medium for 8 days have microfibrils identical with those in 3-day-old cultures; thus a further change in the microfibril orientation did not occur during that period. Alkaline hydrolysis of the walls removes most of the electron-dense staining constituents from the inner wall layer and leaves the outer wall layer intact. This treatment also reveals some of the wall microfibrils. An additional treatment of the walls with HAc/H2O2 completely removes the wall components that react positively to heavy metal stains. The results are discussed in relation to the structure of other fungal cell walls.     

Melanin Production by a Filamentous Soil Fungus in Response to Copper and Localization of Copper Sulfide by Sulfide-Silver Staining

Gaeumannomyces graminis var. graminis, a filamentous soil ascomycete, exhibited enhanced cell wall melanin accumulation when exposed to as little as 0.01 mM CuSO(inf4) in minimal broth culture. Because its synthesis was inhibited by tricyclazole, the melanin produced in response to copper was dihydroxynaphthalene melanin. An additional hyphal cell wall layer was visualized by electron microscopy when hyphae were grown in the presence of copper and fixed by cryotechniques. This electron-dense layer was between the outer cell wall and the inner chitin layer and doubled the total wall thickness. In copper-grown cells that were also treated with tricyclazole, this electron-dense layer was absent. Atomic absorption spectroscopy demonstrated that up to 3.5 mg of Cu per g of fungal mycelium was adsorbed or taken up by hyphae grown in 0.06 mM CuSO(inf4). A method for silver enhancement was developed to determine the cellular location of CuS. CuS was present in cell walls and septa of copper-grown hyphae. Electron microscopy of silver-stained cells suggested that CuS was associated with the melanin layer of cell walls.     

Autolytic activities of the cell wall in rice coleoptiles. Effects of nojirimycin

Oryza sativa L. var. bahia coleoptile cell walls show sufficient autolytic activity for the release into the surrounding medium of amounts up to 60 μg of sugars per mg of dry weight of cell wall. The products released elute in Bio-gel P.2 as mono- and polysaccharides with glucose as the sole component. The polysaccharide component releases tri- and tetrasaccharides on treatment with a glucanase specific for β (1–3) (1–4) linkages in the same proportion as that of the mixed glucan of the cell wall. This supports the hypothesis that the polysaccharide component originates from the cell wall glucan and that autolysis is therefore related to the processes of the loss of rigidity of the cell wall. Nojirimycin (a specific glucanase inhibitor and inhibitor of auxin-induced elongation) decreases autolytic activity of the cell walls, reducing it to 30% of its normal value. Bio-gel P. 2 elution of the products released in autolysis in the presence of nojirimycin shows that only the monosaccharide fraction was affected.     

Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins

A novel strategy using synthetic phytochelatins is described for the purpose of developing microbial agents for enhanced bioaccumulation of toxic metals. Synthetic genes encoding for several metal-chelating phytochelatin analogs (Glu-Cys)(n)Gly (EC8 (n = 8), EC11 (n = 11), and EC20 (n = 20)) were synthesized, linked to a lpp-ompA fusion gene, and displayed on the surface of E. coli. For comparison, EC20 was also expressed periplasmically as a fusion with the maltose-binding protein (MBP-EC20). Purified MBP-EC20 was shown to accumulate more Cd(2+) per peptide than typical mammalian metallothioneins with a stoichiometry of 10 Cd(2+)/peptide. Cells displaying synthetic phytochelatins exhibited chain-length dependent increase in metal accumulation. For example, 18 nmoles of Cd(2+)/mg dry cells were accumulated by cells displaying EC8, whereas cells exhibiting EC20 accumulated a maximum of 60 nmoles of Cd(2+)/mg dry cells. Moreover, cells with surface-expressed EC20 accumulated twice the amount of Cd(2+) as cells expressing EC20 periplasmically. The ability to genetically engineer ECs with precisely defined chain length could provide an attractive strategy for developing high-affinity bioadsorbents suitable for heavy metal removal.