Cheng-gui Zhang

Isolation and identification of a strain ofLeptospirillum ferriphilum from an extreme acid mine drainage site

Based on a new selective isolation strategy that mimicked physiological characteristics of leptospirilla, such as pH, temperature and its less sensitivity to the high ferric-ferrous iron ratio, a bacterial strain, called strain YSK, was isolated from an extreme acid mine drainage (AMD) site. Cells were Gram-negative, small curved rods measuring 0.27–0.52 by 0.81–3.17 μm. The cell shape suggested that strain YSK was likely a strain ofLeptospirillum ferrooxidans. However, based on the 16S rDNA sequence analysis, strain YSK possessed 100% sequence similarity with that of the typicalLeptospirillum ferriphilum strain Fairview; furthermore, the G+C content, the size of the 16S–23S rRNA gene spacer regions and the ability to grow at 45°C further indicated that strain YSK belonged to the speciesLeptospirillum ferriphilum.

Comparative study on effects of Tween-80 and sodium isobutyl-xanthate on growth and sulfur-oxidizing activities of Acidithiobacillus albertensis BY-05

Abstract Effects of two typical surfactants, Tween-80 and sodium isobutyl-xanthate (NaIBX), with different concentrations on the growth and sulfur-oxidizing activities of a new strain Acidithiobacillus albertensis BY-05, an acidophilic sulfur-oxidizing bacterium, were investigated. The results indicate that both surfactants can enhance the growth and sulfur-oxidizing activities of A. albertensis BY-05 only at some special concentrations, e.g., 10−4–10−8 g/L for NaIBX and lower than 10−8 g/L for Tween-80, but were inhibited and even harmful at higher concentrations. Both surfactants can not be metabolized by A. albertensis BY-05. The contact between the bacteria and the sulfur particles may be dependent upon both the extracellular substance and the surfactants, both of which provide the amphiphilic environment improving the attachment for bacteria to the sulfur particles surface. These data could be significant for enlarging the applications of both A. albertensis BY-05 and some typical surfactants for industrial bioleaching of sulfides minerals.

Jarosite-type precipitates mediated by YN22, Sulfobacillus thermosulfidooxidans, and their influences on strain

Abstract To have a better understanding on the properties of the jarosite-type precipitate synthesized by Sulfobacillus thermosulfidooxidans, the evolution of the S. thermosulfidooxidans-mediated precipitation and the influence of the precipitate on this species, a newly isolated strain (YN22) of S. thermosulfidooxidans was cultured in a medium containing Fe2+ as energy source under optimal conditions (pH 1.5, 53 °C, 0.2 g/L yeast extract, 30 g/L Fe2SO4 · 7H2O and 170 r/min), added with or without glass beads. Remarkable differences were found in the oxidation rate of Fe2+, the precipitate yield of jarosite-type compounds and the population density between the two groups of cultures. The group with glass beads has a 6 h faster Fe2+ oxidation, 6 h earlier precipitation, 78% higher precipitate yield and much lower population density than those without glass beads. XRD, EDS, FTIR and SEM analysis reveals that the precipitates originated from both groups are a mixture of potassium jarosite and ammoniojarosite, with morphological features similar to the latter. The results of the test referring to influence of the precipitates on YN22 show that the precipitate from the group without glass beads has no apparent influence on Fe2+ oxidation rate of YN22 and only a limited influence on growth of the strain, whereas that from the group with glass beads remarkably inhibits the growth and Fe2+ oxidation ability of YN22 when a precipitate content over 4 g/L is used.

Investigation of Elemental Sulfur Speciation Transformation Mediated by Acidithiobacillus ferrooxidans

The speciation transformation of elemental sulfur mediated by the leaching bacterium Acidithiobacillus ferrooxidans was investigated using an integrated approach including scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, energy dispersive X-ray spectroscopy, and X-ray absorption near edge spectroscopy (XANES). Our results showed that when grown on elemental sulfur powder, At. ferrooxidans ATCC23270 cells were first attached to sulfur particles and modified the surface sulfur with some amphiphilic compounds. In addition, part of the elemental sulfur powder might be converted to polysulfides. Furthermore, sulfur globules were accumulated inside the cells. XANES spectra of these cells suggested that these globules consisted of elemental sulfur bound to thiol groups of protein.

Sulfur activation-related extracellular proteins of Acidithiobacillus ferrooxidans

Abstract The fractions of the extracellular proteins of Acidithiobacillus ferrooxidans grown on two different energy substrates, elemental sulfur and ferrous sulfate, were selectively prepared with hot water treatment and distinctly shown by two-dimensional gel electrophoresis. Some protein spots with apparently higher abundance in sulfur energy substrate than in ferrous sulfate energy substrate were identified by using MALDI-TOF/TOF. Based on peptide mass fingerprints and bioinformatical analysis, the extracellular proteins were classified according to their functions as conjugal transfer protein, pilin, vacJ lipoprotein, polysaccharide deacetylase family protein, Ser/Thr protein phosphatase family protein and hypothetical proteins. Several extracellular proteins were found abundant in thiol groups and with CXXC functional motif, these proteins may be directly involved in the sulfur activation by use of their thiol group (Pr-SH) to bond the elemental sulfur.

Insights into Two High Homogenous Genes Involved in Copper Homeostasis in Acidithiobacillus ferrooxidans

Acidithiobacillus ferrooxidans, an important microorganism in bioleaching industry, has been sequenced recently, and from the annotated information, there are four genes involved in copper homeostasis. Sequence analysis showed that two of them, Afe0329 and Afe0663, were high homologous (94.43% identity). With the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) cloning approach, the differential gene expression of these two high homologous genes in a genome was successfully identified for the first time. In comparison with Afe0663, Afe0329 was highly expressed grown in the medium with copper, and the restriction fragment length polymorphism (RFLP) profile showed that 96% of lanes were products of Afe0329. Analysis of the protein sequence encoded by Afe0329 suggested a conserved domain of P1b3-type ATPase, which is a heavy-metal pump, and, to be unexpected, the molecular modeling revealed that the amino acids determining the type of heavy-metal pumps were responsible for the gate of the copper ion channel in the transmembrane area of the protein. The activity of P1b-type ATPase disrupted in Escherichia coli could be partially rescued by complementation by the plasmid-carrying Afe0329 gene. All of these results suggest that a copper homeostasis mechanism including P-type ATPase is of importance for the survival of this extremophilic microorganism.

A new strain Leptospirillum ferriphilum YTW315 for bioleaching of metal sulfides ores

Abstract A new strain named YTW315 was isolated from Dexing area using the double-layer culture technique. The morphological, biochemical and physiological characteristics of YTW315 were studied. Physiological investigation indicates that the strain YTW315 is a strict (obligate) chemolithoautotroph, metabolizing ferrous iron and pyrite. The optimal growth conditions for the strain are 40 °C and pH 1.6. A phylogenetic analysis based on 16S rRNA sequences shows that the isolate is clustered to Leptospirillum ferriphilum with 99.8% similarity to Leptospirillum ferriphilum strain Fairview and ATCC 49881. The molar fraction of DNA (G+C) of the isolate is 58.1%. The strain can tolerate high concentration of Fe(III) and As(V) (500 mmol/L and 50 mmol/L, respectively). Bioleaching experiment indicates that the strain can oxidize Fe(II) efficiently, and after 30 d, 44.56% of copper and 95.31% of iron are extracted from chalcopyrite and pyrite, respectively.

Differential expression of genes encoding sulfur metabolism-related periplasmic proteins of Acidithiobacillus ferrooxidans ATCC 23270

Abstract Reverse-transcription qualitative PCR (RT-qPCR) was used to analyze the changes in transcription levels of the sulfur metabolism-related periplasmic protein genes of Acidithiobacillus ferrooxidans ATCC 23270 grown on sulfur or ferrous. Seven periplasmic proteins with apparently higher abundance grown on elemental sulfur than on ferrous sulfate were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Expression analysis of the corresponding genes by RT-qPCR shows that the constitutive expression of all those genes are more up-regulated grown on sulfur than those grown on ferrous (>10 folder). Study on the corresponding genes of the identified periplasmic proteins by RT-qPCR confirmed the results of two-dimensioned gel electrophoresis, indicating they may be related with sulfur metabolism in A. ferrooxidans .

Full structure building and docking of NifS from extremophile Acidithiobacillus ferrooxidans

The gene iscS-2 from extremophile Acidithiobacillus ferrooxidans may play a crucial role in nitrogenase maturation. To investigate the protein encoded by this gene, a reliable integral three-dimensional molecular structure was built. The obtained structure was further used to search binding sites, carry out the flexible docking with cofactor pyridoxal 5′-phosphate(PLP) and substrate cysteine, and identify its key residues. The docking results of PLP reveal that the residues of Lys203, His100, Thr73, Ser200, His202, Asp177 and Gln180 have large interaction energies and/or hydrogen bonds fixation with PLP. The docking results of cysteine show that the amino group in cysteine is very near His100, Lys203 and PLP, and the interaction energies for cysteine with them are very big. These identified residues are in line with the experimental facts of NifS from other sources. Moreover, the four residues of Asn152, Val179, Ala102 and Met148 in the PLP docking and the two residues of Lys208 and Ala102 in the cysteine docking also have large interaction energies, which are fitly conserved in NifS from all kinds of sources but have not been identified before. According to these results, this gene encodes NifS protein, and the substrate cysteine can be effectively recruited into the active site. Furthermore, all of the above detected key residues are directly responsible for the binding and/or catalysis of PLP and cysteine.

Expression, purification and molecular modeling of iron-containing superoxide dismutase from Acidithiobacillus ferrooxidans

The superoxide dismutase(SOD) from Acidithiobacillus ferrooxidans may play an important role in its tolerance to the extremely toxic and oxidative environment of bioleaching. This gene was cloned and then successfully expressed in Escherichia coli. The expressed protein was finally purified by one-step affinity chromatography to homogeneity and observed to be dimer according to SDS-PAGE and MALDI-TOF-MS. The metal content determination and optical spectra results of the recombinant protein confirmed that the protein was an iron-containing superoxide dismutase. Molecular modeling for the protein revealed that the iron atom was ligated by His26, His75, Asp158 and His162.