Sulfide oxidation by spheroplasts of Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans is an acidophilic organism important to metal leaching of low-grade ores. The aforementioned importance is related to the ability of the bacterium to oxidize reduced iron and sulfur, principally found in nature as pyrite (FeS2). The present study dealt with sulfide oxidation at low pH values and the involvement of the cell envelope in the process of the inorganic oxidations. Sulfide oxidation was noted in spheroplasts of T. ferrooxidans prepared by enzymatic and chemical treatments and partially purified by differential centrifugation. No enzyme activities were noted in membrane fractions containing enrichments of lipopolysaccharide symbolic of outer membrane material or in membrane vesicles containing (or associated with) higher levels of proteins. Results to date indicate that in an acid milieu the envelope structure containing both the outer membrane and the intact inner cytoplasmic membrane is required for sulfide oxidation.     

A novel gene encoding a sulfur-regulated outer membrane protein in Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans is a Gram-negative chemolithotrophic bacterium able to oxidize ferrous iron, elemental sulfur and inorganic sulfur compounds. The oxidation of sulfur by T. ferrooxidans resulted in an expression of some outer membrane proteins (OMPs) at a level higher than that observed during ferrous iron oxidation. Among these OMPs, a protein with a molecular mass of 54 kDa was purified and 18 amino acids of the N-terminal sequence determined. Using a 54 bp PCR generated DNA product as a probe for the protein, we isolated a 4.5 kb Pst I DNA chromosomal fragment containing the corresponding gene. Sequencing 2169 bp of this fragment revealed the open reading frame codifying for the protein, consisting of 467 amino acids and a molecular mass of 49,674 Da. The mature protein was produced by the removal of a 32 amino acid signal peptide-like sequence from the N-terminus of a 499 amino acid peptide. Although no significant homology with any known protein has been found and its physiological role remains unclear, its high expression on sulfur substrates suggests a role in sulfide mineral oxidation.     

Mineral and iron oxidation at low temperatures by pure and mixed cultures of acidophilic microorganisms

An enrichment culture from a boreal sulfide mine environment containing a low-grade polymetallic ore was tested in column bioreactors for simulation of low temperature heap leaching. PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing revealed the enrichment culture contained an Acidithiobacillus ferrooxidans strain with high 16S rRNA gene similarity to the psychrotolerant strain SS3 and a mesophilic Leptospirillum ferrooxidans strain. As the mixed culture contained a strain that was within a clade with SS3, we used the SS3 pure culture to compare leaching rates with the At. ferrooxidans type strain in stirred tank reactors for mineral sulfide dissolution at various temperatures. The psychrotolerant strain SS3 catalyzed pyrite, pyrite/arsenopyrite, and chalcopyrite concentrate leaching. The rates were lower at 5 degrees C than at 30 degrees C, despite that all the available iron was in the oxidized form in the presence of At. ferrooxidans SS3. This suggests that although efficient At. ferrooxidans SS3 mediated biological oxidation of ferrous iron occurred, chemical oxidation of the sulfide minerals by ferric iron was rate limiting. In the column reactors, the leaching rates were much less affected by low temperatures than in the stirred tank reactors. A factor for the relatively high rates of mineral oxidation at 7 degrees C is that ferric iron remained in the soluble phase whereas, at 21 degrees C the ferric iron precipitated. Temperature gradient analysis of ferrous iron oxidation by this enrichment culture demonstrated two temperature optima for ferrous iron oxidation and that the mixed culture was capable of ferrous iron oxidation at 5 degrees C.     

Purification and characterization of sulfide:quinone oxidoreductase from an acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans

Sulfide:quinone oxidoreductase (SQR) was purified from membrane of acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans NASF-1 cells grown on sulfur medium. It was composed of a single polypeptide with an apparent molecular mass of 47 kDa. The apparent K(m) values for sulfide and ubiquinone were 42 and 14 muM respectively. The apparent optimum pH for the SQR activity was about 7.0. A gene encoding a putative SQR of A. ferrooxidans NASF-1 was cloned and sequenced. The gene was expressed in Escherichia coli as a thioredoxin-fusion protein in inclusion bodies in an inactive form. A polyclonal antibody prepared against the recombinant protein reacted immunologically with the purified SQR. Western blotting analysis using the antibody revealed an increased level of SQR synthesis in sulfur-grown A. ferrooxidans NASF-1 cells, implying the involvement of SQR in elemental sulfur oxidation in sulfur-grown A. ferrooxidans NASF-1 cells.     

Inhibition of growth, iron, and sulfur oxidation in Thiobacillus ferrooxidans by simple organic compounds.

Iron and sulfur oxidation by Thiobacillus ferrooxidans as well as growth on ferrous iron were inhibited by a variety of low molecular weight organic compounds. The influences of chemical structure of the organic inhibitors, pH, temperature, physical treatment of cells, and added inhibitory or stimulatory inorganic ions and iron oxidation suggest that a major factor contributing to the inhibitory effects on iron oxidation is the relative electronegativity of the organic molecule. The data also suggest that inhibitory organic compounds may (i) directly affect the iron-oxidizing enzyme system, (ii) react abiologically with ferrous iron outside the cell, (iii) interfere with the roles of phosphate and sulfate in iron oxidation, and (iv) nonselectively disrupt the cell envelope or membrane.     

Oxidation of gallium sulfides by Thiobacillus ferrooxidans.

The bacterial oxidation of naturally occurring gallium-bearing chalcopyrite concentrate and a pure synthetic gallium (III) sulfide has been investigated at pH 1.8 and 35 degree C, using an active culture of Thiobacillus ferrooxidans. This oxidation process may proceed by direct or by indirect bacterial action. The highest dissolved gallium and copper concentrations were about 2.2 and 40.2 g/l, respectively. The order of the specific rate of oxygen uptake by T. ferrooxidans in approximately CuFES2 greater than or equal to gallium-bearing CuFeS2 greater than FeS2 greater than Cu2S greater than Cu2S greater than Ga2S3.     

Purification of protein A, an outer membrane component missing in Escherichia coli K-12 ompA mutants.

Outer membrane materials prepared from an Escherichia coli ompA (tolG) strain do not contain one of the major outer membrane proteins found in ompA+ strains. This protein has been purified in high yield from detergent-solubilized cell envelope material prepared from an ompA+ strain by preparative electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. The purified protein is homogeneous in three electrophoretic systems, contains 2 mol of reducing sugar/mol of peptide and has alanine as the N-terminal amino acid. The amino acid composition is nearly identical to outer membrane protein II or B purified by others from incompletely solubilized cell envelope material. Thus, the fraction of outer membrane protein II or B that is difficult to solubilize is identical with the more readily solubilized fraction.     

Strain polymorphism of the plasmid profiles in Acidithiobacillus ferrooxidans

Plasmid profiles were studied in 27 Acidithiobacillus ferrooxidans strains isolated from different geographic zones and substrates differing in the composition of the main sulfide minerals, and also in experimentally obtained strains with acquired enhanced resistance to the ions of heavy metals (Fe, Ni, Cu, Zn, As). In 16 out of 20 strains isolated from different substrates, one to four 2- to 20-kb and larger plasmids were revealed. Plasmids were found in all five strains isolated from gold-containing pyrite-arsenopyrite ores and concentrates, in nine of 11 strains isolated from the ores and concentrates containing nonferrous metals, and in two of four strains isolated from the oxidation substrates of simple composition (mine waters, pyritized coals, active sludge). Changes in the plasmid profiles in some A. ferrooxidans strains (TFZ, TFI-Fe, TFV-1-Cu) with experimentally enhanced resistance to Zn2+, Fe3+, and Cu2+, respectively, were noted as compared with the initial strains. After 30 passages on S0-containing medium, strain TFBk showed changes in the copy number of plasmids. The role of plasmids in the processes of oxidation of energy substrates and in the acquired enhanced resistance to the heavy metal ions is discussed.     

Effect of anions on selective solubilization of zinc and copper in bacterial leaching of sulfide ores

Bacterial leaching of sulfide ores using Thiobacillus ferrooxidans, Thiobacillus thiooxidans, or a combination of the two was studied at various concentrations of specific anions. Selective zinc and copper solubilization was obtained by inhibiting iron oxidation without affecting sulfur/sulfide oxidation. Phosphate reduced iron solubilization from a pyrite (FeS(2))-sphalerite (ZnS) mixture without significantly affecting zinc solubilization. Copper leaching from a chalcopyrite (CuFeS(2))-sphalerite mixture was stimulated by phosphate, whereas chloride accelerated zinc extraction. In a complex sulfide ore containing pyrite, chalcopyrite, and sphalerite, both phosphate and chloride reduced iron solubilization and increased copper extraction, whereas only chloride stimulated zinc extraction. Maximum leaching obtained was 100% zinc and 50% copper. Time-course studies of copper and zinc solubilization suggest the possibility of selective metal recovery following treatment with specific anions.     

Plasmid profiles of Acidithiobacillus ferrooxidans strains adapted to different oxidation substrates

Plasmid profiles were studied in five Acidithiobacillus ferrooxidans strains of various origin cultivated on medium with Fe2+, as well as adapted to such oxidation substrates as S0, FeS2, and sulfide concentrate. The method used revealed plasmids in all A. ferrooxidans strains grown on medium with Fe2+. One plasmid was found in strain TFL-2, two plasmids, in strains TFO, TFBk, and TFV-1, and three plasmids were detected in strain TFN-d. The adaptation of strain TFN-d to sulfide concentrate and the adaptation of strain TFV-1 to S0, FeS2, or sulfide concentrate resulted in a change in the number of plasmids occurring in cells. In cells of strain TFN-d adapted to sulfide concentrate, the number of plasmids decreased from three to two. The number of plasmids in cells of strain TFV-1 adapted to different substrates varied from three to six depending on the energy source present in the medium: three plasmids were found after growth on FeS2, four after growth on S0, and six after growth on sulfide concentrate. The possible role of plasmids in the adaptation of A. ferrooxidans to new energy substrates and in the regulation of the intensity of their oxidation is discussed.     

Lipid composition of outer leaflet of chloroplast outer envelope determines topology of OEP7.

OEP7, a 6.7-kDa outer envelope protein of spinach chloroplasts inserts into the outer envelope of the organelle independent of a classical cleavable targeting signal. The insertion of OEP7 was studied to describe the determinants for association with, integration into, and orientation of the protein in the outer envelope of chloroplasts. The insertion of OEP7 into the membrane is independent of outer membrane channel proteins and can be reconstituted with the use of protein-free liposomes. In situ, the binding of OEP7 to the membrane surface is not driven by electrostatic interaction because reduction of phosphatidylglycerol or phosphatidylinositol did not reduce the association with the liposomes. The positively charged amino acids flanking the transmembrane domain at the C terminus are essential to retain the native N(in)-C(out) orientation during insertion into chloroplasts. OEP7 inserts with reversed orientation into liposomes containing the average lipid composition of the outer envelopes. The native like N(in)-C(out) orientation is achieved by reduction of the phoshpatidylglycerol concentration mimicking the composition of the outer leaflet of the outer envelope of chloroplasts. We conclude that the unique lipid composition of the outer leaflet due to lipid asymmetry of the outer envelope is essential for the correct topology of OEP7.     

Herpes simplex virus nucleocapsids mature to progeny virions by an envelopment --> deenvelopment --> reenvelopment pathway

Herpes simplex virus (HSV) nucleocapsids acquire an envelope by budding through the inner nuclear membrane, but it is uncertain whether this envelope is retained during virus maturation and egress or whether mature progeny virions are derived by deenvelopment at the outer nuclear membrane followed by reenvelopment in a cytoplasmic compartment. To resolve this issue, we used immunogold electron microscopy to examine the distribution of glycoprotein D (gD) in cells infected with HSV-1 encoding a wild-type gD or a gD which is retrieved to the endoplasmic reticulum (ER). In cells infected with wild-type HSV-1, extracellular virions and virions in the perinuclear space bound approximately equal amounts of gD antibody. In cells infected with HSV-1 encoding an ER-retrieved gD, the inner and outer nuclear membranes were heavily gold labeled, as were perinuclear enveloped virions. Extracellular virions exhibited very little gold decoration (10- to 30-fold less than perinuclear virions). We conclude that the envelope of perinuclear virions must be lost during maturation and egress and that mature progeny virions must acquire an envelope from a post-ER cytoplasmic compartment. We noted also that gD appears to be excluded from the plasma membrane in cells infected with wild-type virus.     

Chemolithotrophic Bacteria in Copper Ores Leached at High Sulfuric Acid Concentration

Extensive bacterial growth was observed when copper sulfide ores were leached with 0.6 N sulfuric acid. The bacterial population developed in this condition was examined by characterization of the spacer regions between the 16S and 23S rRNA genetic loci obtained after PCR amplification of the DNA extracted from the leached ore. The spacers observed had the sizes found in strains of "Leptospirillum ferrooxidans" and Thiobacillus thiooxidans, except for a larger one, approximately 560 bp long, that was not observed in any of the strains examined, including those of Thiobacillus ferrooxidans. The bacteria with this last spacer were selected after culturing in mineral and elemental sulfur media containing 0.7 N sulfuric acid. The spacer and the 16S ribosomal DNA of this isolate were sequenced and compared with those in species commonly found in bioleaching processes. Though the nucleotide sequence of the spacer showed an extensive heterologous region with T. thiooxidans, the sequence of its 16S rDNA gene indicated a close relationship (99.85%) with this species. These results indicate that a population comprised of bacterial strains closely related to T. thiooxidans and of another strain, possibly related to "L. ferrooxidans," can develop during leaching at high sulfuric acid concentration. Iron oxidation in this condition is attributable to "L. ferrooxidans" and not T. ferrooxidans, based on the presence of spacers with the "L. ferrooxidans" size range and the absence of spacers characteristic of T. ferrooxidans.     

Electron microscopy of the cell envelope of Ferrobacillus ferrooxidans prepared by freeze-etching and chemical fixation techniques

Remsen, C. C. (Swiss Federation Institute of Technology, Zurich, Switzerland), and D. G. Lundgren. Electron microscopy of the cell envelope of Ferrobacillus ferrooxidans prepared by freeze-etching and chemical fixation techniques. J. Bacteriol. 92:1765-1771. 1966.-A comparison was made of the fine structure of the cell envelope of the gram-negative bacterium Ferrobacillus ferrooxidans when cells were prepared for microscopy by freeze-etching and chemical fixation techniques. Cell envelopes of chemically fixed cells appeared as five separate layers distinguishable by their location and electron density. Frozen-etched cells showed a three-layered complex with each layer measuring approximately 100 A in thickness. The latter technique is considered to be "artifact-free" and, as a technique, yields purely morphological information on the natural state. The three layers revealed by freeze-etching are: the outer layer, a lipoprotein-lipopolysaccharide layer; the middle layer, a layer composed of globular protein attached to fibrillar mucopeptide; and the innermost layer, the cytoplasmic membrane. The latter was covered with 100 to 120 A particles. The relationship of the aforementioned layers to those seen in chemically fixed cells is discussed.     

Bacterial leaching of a sulfide ore by Thiobacillus ferrooxidans and Thiobacillus thiooxidans: I. Shake flask studies

Bacterial leaching of a sulfide ore containing pyrite, chalcopyrite, and sphalerite was studied in shake flask experiments using Thiobacillus ferrooxidans and Thiobacillus thiooxidans strains isolated from mine sites. The Fe(2+)grown T. ferrooxidans isolates solubilized sphalerite preferentially over chalcopyrite leaching 7-10% Cu, 68-76% Zn, and 10-22% Fe from the ore in 18 days. The sulfur grown T. thiooxidans isolates leached Zn much more slowly and very little Fe, with a Cu-Zn extraction ratio twice the value obtained with T. ferrooxidans. The ore adapted T. ferrooxidans started solubilizing Cu and Zn without a lag period. The ore-adapted T. thiooxidans extracted Cu as well as T. ferrooxidans, but the extraction of Zn or Fe was still much slower in the low-phosphate medium, while in the high-phosphate medium it approached the value obtained with T. ferrooxidans. A high Cu-Zn extraction ratio of 0.34 was obtained with T. thiooxidans in the low phosphate medium. In the mixed-culture experiments with T. ferrooxidans and T. thiooxidans, the culture behaved as T. thiooxidans in the low-phosphate medium with a higher Cu-Zn extraction ratio and as T. ferrooxidans in the high-phosphate medium with a lower Cu-Zn extraction ratio. It is concluded that T. ferrooxidans and T. thiooxidans solubilize sulfide minerals by different mechanisms.     

Herpes simplex virus 1 envelopment follows two diverse pathways

Herpesvirus envelopment is assumed to follow an uneconomical pathway including primary envelopment at the inner nuclear membrane, de-envelopment at the outer nuclear membrane, and reenvelopment at the trans-Golgi network. In contrast to the hypothesis of de-envelopment by fusion of the primary envelope with the outer nuclear membrane, virions were demonstrated to be transported from the perinuclear space to rough endoplasmic reticulum (RER) cisternae. Here we show by high-resolution microscopy that herpes simplex virus 1 envelopment follows two diverse pathways. First, nuclear envelopment includes budding of capsids at the inner nuclear membrane into the perinuclear space whereby tegument and a thick electron dense envelope are acquired. The substance responsible for the dense envelope is speculated to enable intraluminal transportation of virions via RER into Golgi cisternae. Within Golgi cisternae, virions are packaged into transport vacuoles containing one or several virions. Second, for cytoplasmic envelopment, capsids gain direct access from the nucleus to the cytoplasm via impaired nuclear pores. Cytoplasmic capsids could bud at the outer nuclear membrane, at membranes of RER, Golgi cisternae, and large vacuoles, and at banana-shaped membranous entities that were found to continue into Golgi membranes. Envelopes originating by budding at the outer nuclear membrane and RER membrane also acquire a dense substance. Budding at Golgi stacks, designated wrapping, results in single virions within small vacuoles that contain electron-dense substances between envelope and vacuolar membranes.     

Toc64--a preprotein-receptor at the outer membrane with bipartide function

Protein translocation across membranes is assisted by translocation machineries present in the membrane targeted by the precursor proteins. Translocon subunits can be functionally divided into receptor proteins warranting the specificity of this machine and a translocation channel. At the outer envelope of chloroplasts two sets of receptor proteins regulate protein translocation facing the cytosol or acting in the intermembrane space. One, Toc64 is a receptor of the translocon at the outer envelope of chloroplasts (Toc complex) with dual function. Toc64 recognizes Hsp90 delivered precursor proteins via a cytosolic exposed domain containing three tetratrico-peptide repeat motifs and as demonstrated in here, Toc64 functions also as a major component of a complex facing the intermembrane space. The latter complex is composed of an Hsp70 localized in the intermembrane space, its interaction partner Toc12, a J-domain containing protein and the intermembrane space protein Tic22. We analyzed the intermembrane space domain of Toc64. This domain is involved in preprotein recognition and association with the Toc-complex independent of the cytosolic domain of the Toc64 receptor. Therefore, Toc64 is involved in preprotein translocation across the outer envelope at both sites of the membrane.     

Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. II. Biochemical characterization

In the previous paper (Block, M. A., Dorne, A.-J., Joyard, J., and Douce, R. (1983) J. Biol. Chem. 258, 13273-13280), we have described a method for the separation of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. The two envelope membranes have a different weight ratio of acyl lipid to protein (2.5-3 for the outer envelope membrane and 0.8-1 for the inner envelope membrane). The two membranes also differ in their polar lipid composition. However, in order to prevent the functioning of the galactolipid:galactolipid galactosyltransferase during the course of envelope membrane separation, we have analyzed the polar lipid composition of each envelope membrane after thermolysin treatment of the intact chloroplasts. The outer envelope membrane is characterized by the presence of high amounts of phosphatidylcholine and digalactosyldiacylglycerol whereas the inner envelope membrane has a polar lipid composition almost identical with that of the thykaloids. No phosphatidylethanolamine or cardiolipin could be detected in either envelope membranes, thus demonstrating that the envelope membranes, and especially the outer membrane, do not resemble extrachloroplastic membranes. No striking differences were found in the fatty acid composition of the polar lipids from either the outer or the inner envelope membrane. The two envelope membranes also differ in their carotenoid composition. Among the different enzymatic activities associated with the chloroplast envelope, we have shown that the Mg2+-dependent ATPase, the UDP-Gal:diacylglycerol galactosyltransferase, the phosphatidic acid phosphatase, and the acyl-CoA thioesterase are associated with the inner envelope from spinach chloroplasts whereas the acyl-CoA synthetase is located on the outer envelope membrane.     

Export of hybrid proteins FhuA''-''LacZ and FhuA''-''PhoA to the cell envelope of Escherichia coli K-12.

The fhuA gene of Escherichia coli K-12 encodes an outer membrane protein that acts as the ferrichrome-iron(III) receptor. To determine the export signals and sorting information within FhuA, gene fusions of fhuA'-'lacZ and fhuA'-'phoA were constructed. Although a FhuA'-'LacZ hybrid protein was detected in the Triton X-100-insoluble fraction of the cell envelope, direct immunoelectron microscopic observation showed that this protein remained in the cytoplasm. FhuA'-'PhoA hybrid proteins were all exported across the cytoplasmic membrane. Those hybrids containing up to 88 amino acids of FhuA (FhuA88) fused to PhoA were released along with other periplasmic proteins. Hybrids containing 180 or more amino acids of FhuA (FhuA180) fused to PhoA were associated with the outer membrane. It is proposed that some information inherent in the sequences between FhuA88 and FhuA180 confers stable association with the outer membrane.