Yuandong Liu

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.

Genomic and transcriptomic analyses reveal adaptation mechanisms of an Acidithiobacillus ferrivorans strain YL15 to alpine acid mine drainage

Acidithiobacillus ferrivorans is an acidophile that often occurs in low temperature acid mine drainage, e.g., that located at high altitude. Being able to inhabit the extreme environment, the bacterium must possess strategies to copy with the survival stress. Nonetheless, information on the strategies is in demand. Here, genomic and transcriptomic assays were performed to illuminate the adaptation mechanisms of an A. ferrivorans strain YL15, to the alpine acid mine drainage environment in Yulong copper mine in southwest China. Genomic analysis revealed that strain has a gene repertoire for metal-resistance, e.g., genes coding for the mer operon and a variety of transporters/efflux proteins, and for low pH adaptation, such as genes for hopanoid-synthesis and the sodium:proton antiporter. Genes for various DNA repair enzymes and synthesis of UV-absorbing mycosporine-like amino acids precursor indicated hypothetical UV radiation—resistance mechanisms in strain YL15. In addition, it has two types of the acquired immune system–type III-B and type I-F CRISPR/Cas modules against invasion of foreign genetic elements. RNA-seq based analysis uncovered that strain YL15 uses a set of mechanisms to adapt to low temperature. Genes involved in protein synthesis, transmembrane transport, energy metabolism and chemotaxis showed increased levels of RNA transcripts. Furthermore, a bacterioferritin Dps gene had higher RNA transcript counts at 6°C, possibly implicated in protecting DNA against oxidative stress at low temperature. The study represents the first to comprehensively unveil the adaptation mechanisms of an acidophilic bacterium to the acid mine drainage in alpine regions.

Purification and enzymatic properties of arsenic resistance protein ArsH from heterogeneous expression in E.coli BL21

Abstract Four arsenic-resistance genes (arsB, arsC, arsH, arsR) have been discovered in Acidithiobacillus ferrooxidans. Their gene sequences have been identified and three different arsenic-resistance mechanisms have been elucidated. However, the function of the arsH gene in At. ferrooxidans remains unclear. In order to evaluate the function of the arsH gene, we cloned it and expressed it in Escherichia coli. The protein was purified and its relative molecular mass was determined by SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The results indicated that the relative molecular mass of the purified ArsH was approximately 29 kDa. The purified protein ArsH from E.coli BL21 was a flavoprotein that oxidized in vitro NADPH with an optimal pH of 6.4.

Expression, purification, and characterization of a [Fe2S2] cluster containing ferredoxin from Acidithiobacillus ferrooxidans.

The [2Fe-2S] cluster containing ferredoxin has attracted much attention in recent years. Genetic analyses show that it has an essential role in the maturation of various iron–sulfur (Fe-S) proteins and functions as a component of the complex machinery responsible for the biogenesis of Fe-S clusters. The gene of ferredoxin from A. ferrooxidans ATCC 23270 was cloned, successfully expressed in Escherichia coli, and purified by one-step affinity chromatography to homogeneity. The MALDI-TOF MS and spectra results of the recombinant protein confirmed that the iron–sulfur cluster was correctly inserted into the active site of the protein. Site-directed mutagenesis results revealed that Cys42, Cys48, Cys51, and Cys87 were ligating with the [Fe2S2] cluster of the protein.

Oct-2-en-4-ynoyl-CoA as a specific inhibitor of acyl-CoA oxidase.

Oct-2-en-4-ynoyl-CoA was found to be a specific inhibitor of acyl-CoA oxidase in fatty acid oxidation in peroxisomes that has no inhibitory effect on acyl-CoA dehydrogenase in mitochondria. The inhibition reaction involves a nucleophilic attack of Glu421 to the delta carbon of the inhibitor. The result indicates that acyl-CoA oxidase and acyl-CoA dehydrogenase have certain differences in active-site structure, which makes it possible to control fatty acid oxidation selectively in either mitochondria or peroxisomes with different enzyme inhibitors.

Expression, purification and characterization of an iron-sulfur cluster assembly protein, IscU, from Acidithiobacillus ferrooxidans.

An iron-sulfur cluster assembly protein, IscU, is encoded by the operon iscSUA in Acidithiobacillus ferrooxidans. The gene of IscU was cloned and expressed in Escherichia coli. The protein was purified by one-step affinity chromatography to homogeneity. The protein was in apo-form, the [Fe2S2] cluster could be assembled in apoIscU with Fe2+ and sulfide in vitro, and in the presence of IscA and IscS, the IscU could utilize l-cysteine and Fe2+ to synthesize [Fe2S2] cluster in the protein. Site-directed mutagenesis for the protein revealed that Cys37, Asp39, Cys63 and Cys106 were involved in ligating with the [Fe2S2] cluster.

Evolution of Sulfobacillus thermosulfidooxidans secreting alginate during bioleaching of chalcopyrite concentrate

To explore the distribution disciplinarian of alginate on the chalcopyrite concentrate surface during bioleaching.

Geography Plays a More Important Role than Soil Composition on Structuring Genetic Variation of Pseudometallophyte Commelina communis

Pseudometallophytes are excellent models to study microevolution and local adaptation to soil pollution, as they can grow both on metalliferous and contrasting non-metalliferous soils. Although, there has been accumulating evidence for the effects of edaphic conditions and geographical isolation on the genetic structure of pesudometallophytes, it is still a difficult problem in evolutionary biology to assess their relative importance. In this study, we investigated the spatial patterns of genetic variability, population differentiation and genetic groups in pseudometallophyte Commelina communis with 12 microsatellite loci. Eight metallicolous and six non-metallicolous populations of C. communis were sampled from cupriferous sites and surrounding non-contaminated areas in China. Neither significant reduction in genetic diversity nor apparent founder and bottleneck effects were observed in metallicolous populations of C. communis. Based on Bayesian and Neighbor-Joining clustering analyses and a principal coordinates analysis, all sampled populations were found to be mainly separated into three genetic groups, corresponding well to their geographical locations rather than edaphic origins. Moreover, a significant and strong correlation between population genetic divergence and geographical distance were detected by Mantel test (r = 0.33; P < 0.05) and multiple matrix regression with randomization (MMRR; βD = 0.57, P < 0.01). However, the effect of copper concentration on genetic patterns of C. communis was not significant (MMRR; βE = -0.17, P = 0.12). Our study clearly demonstrated that the extreme edaphic conditions in metalliferous areas had limited effects on the genetic variability in C. communis. Geographic distance played a more important role in affecting the genetic structure of C. communis than soil composition did. In C. communis, the geographically disjunctive populations on metalliferous soils had multiple origins and evolved independently from nearby non-metallicolous populations.

Differential Gene Expression and Bioinformatics Analysis of Copper Resistance Gene afe_1073 in Acidithiobacillus ferrooxidans

Copper resistance of acidophilic bacteria is very significant in bioleaching of copper ore since high concentration of copper are harmful to the growth of organisms. Copper resistance gene afe_1073 was putatively considered to be involved in copper homeostasis in Acidithiobacillus ferrooxidans ATCC23270. In the present study, differential expression of afe_1073 in A. ferrooxidans strain DY26 and DC was assessed with quantitative reverse transcription polymerase chain reaction. The results showed the expression of afe_1073 in two strains increased with the increment of copper concentrations. The expression of DY26 was lower than that of DC at the same copper concentration although A. ferrooxidans strain DY26 possessed higher copper resistance than strain DC. In addition, bioinformatics analysis showed AFE_1073 was a typical transmembrane protein P1b1-ATPase, which could reduce the harm of Cu+ by pumping it out from the cell. There were two mutation sites in AFE_1073 between DY26 and DC and one may change the hydrophobicity of AFE_1073, which could enhance the ability of DY26 to pump out Cu+. Therefore, DY26 needed less gene expression of afe_1073 for resisting copper toxicity than that of DC at the same copper stress. Our study will be beneficial to understanding the copper resistance mechanism of A. ferrooxidans.

Expression, Purification, and Characterization of an Iron Chaperon Protein CyaY from Acidithiobacillus ferrooxidans

CyaY is the bacterial homolog of frataxin, proposed to be involved in the assembly of iron–sulfur clusters. While, the physiological iron donor for the iron–sulfur clusters assembly remains controversial. In this study, the gene of CyaY from Acidithiobacillus ferrooxidans was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The CyaY protein can bind ferric iron and serve as an iron donor for the biogenesis of iron–sulfur clusters on the scaffold protein IscU in the presence of IscS and l-cysteine in vitro.