Jianqiang Lin

Acidithiobacillus caldus Sulfur Oxidation Model Based on Transcriptome Analysis between the Wild Type and Sulfur Oxygenase Reductase Defective Mutant

Background Acidithiobacillus caldus (A. caldus) is widely used in bio-leaching. It gains energy and electrons from oxidation of elemental sulfur and reduced inorganic sulfur compounds (RISCs) for carbon dioxide fixation and growth. Genomic analyses suggest that its sulfur oxidation system involves a truncated sulfur oxidation (Sox) system (omitting SoxCD), non-Sox sulfur oxidation system similar to the sulfur oxidation in A. ferrooxidans, and sulfur oxygenase reductase (SOR). The complexity of the sulfur oxidation system of A. caldus generates a big obstacle on the research of its sulfur oxidation mechanism. However, the development of genetic manipulation method for A. caldus in recent years provides powerful tools for constructing genetic mutants to study the sulfur oxidation system. Results An A. caldus mutant lacking the sulfur oxygenase reductase gene (sor) was created and its growth abilities were measured in media using elemental sulfur (S0) and tetrathionate (K2S4O6) as the substrates, respectively. Then, comparative transcriptome analysis (microarrays and real-time quantitative PCR) of the wild type and the Δsor mutant in S0 and K2S4O6 media were employed to detect the differentially expressed genes involved in sulfur oxidation. SOR was concluded to oxidize the cytoplasmic elemental sulfur, but could not couple the sulfur oxidation with the electron transfer chain or substrate-level phosphorylation. Other elemental sulfur oxidation pathways including sulfur diooxygenase (SDO) and heterodisulfide reductase (HDR), the truncated Sox pathway, and the S4I pathway for hydrolysis of tetrathionate and oxidation of thiosulfate in A. caldus are proposed according to expression patterns of sulfur oxidation genes and growth abilities of the wild type and the mutant in different substrates media. Conclusion An integrated sulfur oxidation model with various sulfur oxidation pathways of A. caldus is proposed and the features of this model are summarized.

Arsenic resistance operon structure in Leptospirillum ferriphilum and proteomic response to arsenic stress.

The response of Leptospirillum ferriphilum ML-04 to arsenic stress was analyzed using two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Thirty-eight of 65 significantly differentially expressed arsenic response proteins were identified, and 25 of them have known functions. Three proteins are arsenic resistance system (ARS) member proteins. Two ars operons appear to be present in this strain. In addition to the ARS system, phosphate regulation and glutathione (GSH) synthesis appear involved in As[V] and As[III] tolerance, respectively. These findings provide information potentially useful for the genetic engineering of arsenic resistant organisms.

Complete genome of Leptospirillum ferriphilum ML-04 provides insight into its physiology and environmental adaptation

Leptospirillum ferriphilum has been identified as the dominant, moderately thermophilic, bioleaching microorganism in bioleaching processes. It is an acidic and chemolithoautrophic bacterium that gains electrons from ferrous iron oxidation for energy production and cell growth. Genetic information about this microorganism has been limited until now, which has hindered its further exploration. In this study, the complete genome of L. ferripilum ML-04 is sequenced and annotated. The bacterium has a single circular chromosome of 2,406,157 bp containing 2,471 coding sequences (CDS), 2 rRNA operons, 48 tRNA genes, a large number of mobile genetic elements and 2 genomic islands. In silico analysis shows L. ferriphilum ML-04 fixes carbon through a reductive citric acid (rTCA) cycle, and obtains nitrogen through ammonium assimilation. The genes related to “cell envelope biogenesis, outer membrane” (6.9%) and “DNA replication, recombination and repair” (5.6%) are abundant, and a large number of genes related to heavy metal detoxification, oxidative and acidic stress defense, and signal transduction pathways were detected. The genomic plasticity, plentiful cell envelope components, inorganic element metabolic abilities and stress response mechanisms found the base for this organism’s survival in the bioleaching niche.

Development of a Markerless Gene Replacement System for Acidithiobacillus ferrooxidans and Construction of a pfkB Mutant

ABSTRACT The extremely acidophilic, chemolithoautotrophic Acidithiobacillus ferrooxidans is an important bioleaching bacterium of great value in the metallurgical industry and environmental protection. In this report, a mutagenesis system based on the homing endonuclease I-SceI was developed to produce targeted, unmarked gene deletions in the strain A. ferrooxidans ATCC 23270. A targeted phosphofructokinase (PFK) gene (pfkB) mutant of A. ferrooxidans ATCC 23270 was constructed by homologous recombination and identified by PCR with specific primers as well as Southern blot analysis. This potential pfkB gene (AFE_1807) was also characterized by expression in PFK-deficient Escherichia coli cells, and heteroexpression of the PFKB protein demonstrated that it had functional PFK activity, though it was significantly lower (about 800-fold) than that of phosphofructokinase-2 (PFK-B) expressed by the pfkB gene from E. coli K-12. The function of the potential PFKB protein in A. ferrooxidans was demonstrated by comparing the properties of the pfkB mutant with those of the wild type. The pfkB mutant strain displayed a relatively reduced growth capacity in S0 medium (0.5% [wt/vol] elemental sulfur in 9K basal salts solution adjusted to pH 3.0 with H2SO4), but the mutation did not completely prevent A. ferrooxidans from assimilating exogenous glucose. The transcriptional analysis of some related genes in central carbohydrate metabolism in the wild-type and mutant strains with or without supplementation of glucose was carried out by quantitative reverse transcription-PCR. This report suggests that the markerless mutagenesis strategy could serve as a model for functional studies of other genes of interest from A. ferrooxidans and multiple mutations could be made in a single A. ferrooxidans strain.

Microbial transformation of phytosterol in corn flour and soybean flour to 4-androstene-3,17-dione by Fusarium moniliforme Sheld.

A strain that was capable of transforming the phytosterol in corn flour and soybean flour was isolated from soil and identified as Fusarium moniliforme Sheld. The main transformation product was purified by high performance liquid chromatography (HPLC), and was characterized by nuclear magnetic resonance (NMR), mass spectrum (MS), and infrared spectrum (IR). The results indicated that the product was 4-androstene-3,17-dione (AD). The production of AD was increased with the increase of initial concentration of corn flour while the yield of AD was decreased. The yield of AD was lower in the media with only soybean flour. Sulfate-phosphate-ferric method (SPF) was first used for determination of the total phytosterol content in corn flour or soybean flour. The measured value by SPF method matched reasonably well with that by HPLC, which indicated the validity of SPF method.

Cd(II) and As(III) bioaccumulation by recombinant Escherichia coli expressing oligomeric human metallothioneins.

Metallothioneins (MTs) are a family of metal binding proteins. Recombinant Escherichia coli expressing the human MT (hMT-1A) gene was constructed for bioaccumulation of heavy metals. In order to increase protein stability, the glutathione S-transferase (GST) gene was fused with the hMT-1A gene and coexpressed. In order to increase MT expression efficiency and metal binding capacity, two, three or four hMT-1A genes were integrated in series and overexpressed in E. coli. The recombinant E. coli expressing the GST fused trimeric hMT-1A protein exhibited the highest Cd(II) and As(III) bioaccumulation ability, 6.36 mg Cd/g dry cells and 7.59 mg As/g dry cells, respectively.

Overexpression of Rusticyanin in Acidithiobacillus ferrooxidans ATCC19859 Increased Fe(II) Oxidation Activity

A wide-host-range plasmid pTRUS containing a homologous rus gene under the control of Ptac promoter was constructed and transferred into Acidithiobacillus ferrooxidans ATCC19859 using conjugation gene transfer method to generate the engineered strain of A. ferrooxidans(pTRUS). The plasmid-based recombinant rus gene was successfully expressed. According to the results of real-time quantitative PCR assay, not only the transcription level of recombinant rus gene but also the transcription levels of the other genes of rus operon (cyc1, orf, coxB, coxA) were increased in A. ferrooxidans(pTRUS). The ferrous ion (Fe2+) oxidation activity was increased in A. ferrooxidans(pTRUS).

Kinetics and Modeling of Chemical Leaching of Sphalerite Concentrate Using Ferric Iron in a Redox-controlled Reactor

Abstract This work presents a study for chemical leaching of sphalerite concentrate under various constant Fe3+ concentrations and redox potential conditions. The effects of Fe3+ concentration and redox potential on chemical leaching of sphalerite were investigated. The shrinking core model was applied to analyze the experimental results. It was found that both the Fe3+ concentration and the redox potential controlled the chemical leaching rate of sphalerite. A new kinetic model was developed, in which the chemical leaching rate of sphalerite was proportional to Fe3+ concentration and Fe3+/Fe2+ ratio. All the model parameters were evaluated from the experimental data. The model predictions fit well with the experimental observed values.

Construction of recombinant mercury resistant Acidithiobacillus caldus

A mercury-resistant plasmid of pTMJ212 which was able to shuttle between Acidithiobacillus caldus and Escherichia coli was constructed by inserting the mercury resistant determinants, the mer operon of Acidithiobacillus ferrooxidans, into the IncQ plasmid of pJRD215. pTMJ212 was transferred from Escherichia coli into Acidithiobacillus caldus through conjugation. Furthermore, pTMJ212 was transferred back from Acidithiobacillus caldus into Escherichia coli, thereby confirming the initial transfer of pTMJ212 from Escherichia coli to Acidithiobacillus caldus. Compared to the control, the cell growth of the recombinant Acidithiobacillus caldus increased markedly under mercury (Hg(2+)) stress especially at Hg(2+) concentrations ranging from 2.0 to 4.5 μg/ml.

Construction of small plasmid vectors for use in genetic improvement of the extremely acidophilic Acidithiobacillus caldus

The genetic improvement of biomining bacteria including Acidithiobacillus caldus could facilitate the bioleaching process of sulfur-containing minerals. However, the available vectors for use in A. caldus are very scanty and limited to relatively large broad-host-range IncQ plasmids. In this study, a set of small, mobilizable plasmid vectors (pBBR1MCS-6, pMSD1 and pMSD2) were constructed based on plasmid pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups. The function of the tac promoter on 5.8-kb pMSD2 was determined by inserting a kanamycin-resistant reporter gene. The resulting recombinant pMSD2-Km was successfully transferred by conjugation into A. caldus MTH-04 with transfer frequency of 1.38±0.64×10(-5). The stability and plasmid copy number of pMSD2-Km in A. caldus MTH-04 were 75±2.7% and 5-6 copies per cell, respectively. By inserting an arsABC operon into pMSD2, an arsenic-resistant recombinant pMSD2-As was constructed and transferred into A. caldus MTH-04 by conjugation. The arsenic tolerance of A. caldus MTH-04 containing pMSD2-As was obviously increased up to 45mM of NaAsO2. These vectors could be applied in genetic improvement of A. caldus as well as other bioleaching bacteria.