Identification of two outer membrane proteins involved in the oxidation of sulphur compounds in Thiobacillus ferrooxidans

Abstract: Shift of three Thiobacillus ferrooxidans strains from Fe(II) to S⁰ or thiosulphate liquid medium caused distinctive changes in the outer membrane protein profile. In addition to a new 55-kDa protein which was synthesized only in the presence of sulphur compounds, a higher expression of a 47-kDa protein was observed. This latter protein appeared to be constitutively synthesized, since it was detectable in small amounts even in tile presence of ferrous iron as sole energy source, but its expression was greatly enhanced when elemental sulphur or thiosulphate were present in the growth medium.     

Thiobacillus ferrooxidans pili

The surface structures of Thiobacillus ferrooxidans were studied. When growing on a medium containing elemental sulphur, the cells possess peritrichously located filaments (piles) whose diameter varies from 4.5 to 7.0 nm and length, from 0.7 to 3.0 mcm. The cells of T. ferrooxidans do not have piles on a medium with ferrous iron. The physiological role of these structures for thiobacilli is discussed.     

Extension of logarithmic growth of Thiobacillus ferrooxidans by potential controlled electrochemical reduction of Fe(III).

In this study, we demonstrated that the period of logarithmic growth for Thiobacillus ferrooxidans could be extended when optimal conditions for cell growth were maintained using potential controlled electrochemical cultivation with sufficient aeration. The optimal pH and Fe(II) concentration for the electrolytic cultivation were determined to be 2.0 and 150 mM, respectively. When the potential was set to 0.0V vs Ag/AgCl, the Pt electrode reduced Fe(III) to Fe(II) with an efficiency of 95%. A porous glass microbubble generator was used to maintain adequate levels of dissolved oxygen, which was the electron acceptor for T. ferrooxidans when the cell density in the medium was high. Under these conditions, cells at an initial density of 10(7) cells/mL grew logarithmically for 4days until the cell density was 4 x 10(9) cells/mL. This corresponded to a period of logarithmic growth that was 3 times longer than was observed in batch cultures without electrolysis. In addition, the final cell density reached 10(10) cells/mL after 6 days of electrochemical cultivation, which was a 50-fold increase over conventional batch culture. Under conditions of increasing cell density, potentiostatic electrolysis made it possible to remove Fe(III), which causes product inhibition, at an increasing rate and to correspondingly increase the production rate of Fe(II), which is the electron donor for T. ferrooxidans. Thus, our cultivation system provides a sufficient supply of electron donor and acceptor for T. ferrooxidans, thereby elongating the period of logarithmic growth and producing very high cell densities.     

Detection of regional changes in protein levels in the in vivo canine model of acute heart failure following ischemia-reperfusion injury: functional proteomics studies

In this study, we demonstrate the use of proteomics to detect regional differences in protein levels between the reperfused ischemic zone (IZ) and the nonischemic zone (NIZ) of dog hearts which were subjected to in vivo ischemia-reperfusion injury. Using the two-dimensional gel electrophoresis (2-DE) technique, we identified five proteins that were differentially expressed in the IZ versus NIZ: (1) the alpha subunit of ATP synthase isoform precursor was decreased 1.71-fold; (2) creatine kinase M chain was decreased 1.72-fold; (3) NAD+-isocitrate dehydrogenase, alpha subunit was increased 8.34-fold; (4) ATP synthase D chain, mitochondrial was increased 3.02-fold; (5) ventricular myosin light chain-1 was decreased 2.02-fold. Additionally, we found that the level of actin was decreased 2.6-times in the IZ compared to the NIZ on Western blot analysis but was unchanged on 2-DE.     

Reduction of Mo6+ with elemental sulfur by Thiobacillus ferrooxidans.

In the presence of phosphate ions, molybdic ions (Mo6+) were reduced enzymatically with elemental sulfur by washed intact cells of Thiobacillus ferrooxidans to give molybdenum blue. The whole-cell activity that reduced Mo6+ was totally due to cellular sulfur:ferric ion oxidoreductase (SFORase) (T. Sugio, W. Mizunashi, K. Inagaki, and T. Tano, J. Bacteriol. 169:4916-4922, 1987). The activity of M06+ reduction with elemental sulfur was competitively inhibited by Fe3+, Cu2+, and Co2+. The Michaelis constant of SFORase for Mo6+ was 7.6 mM, and the inhibition constants for Fe3+, Cu2+, and Co2+ were 0.084, 0.015, and 0.17 mM, respectively, suggesting that SFORase can reduce not only Fe3+ and Mo6+ but also Cu2+ and Co2+ with elemental sulfur.     

氧化亚铁硫杆菌分离复壮及固定化的研究

用稀释涂布平板法从已退化的氧化亚铁硫杆菌（Thiobacillus ferrooxidans）菌液中分离出氧化活性较高、生命力强的氧化亚铁硫杆菌T1。以H2软性填料作为氧化亚铁硫杆菌的固定化载体,构建了固定床生物反应器。考察了固定床生物反应器氧化Fe2+的情况：Fe2+最大氧化速率达7.67g/(L·h)。并对固定床生物反应器运行过程中在载体表面形成的沉淀物进行了研究,通过X衍射证明此沉淀物为黄钾铁矾［Kfe3(SO4)2(OH)6］。     

Heat-shock-induced proteins from Myxococcus xanthus

Optimal conditions for two-dimensional gel electrophoresis of total cellular proteins from Myxococcus xanthus were established. Using these conditions, we analyzed protein patterns of heat-shocked M. xanthus cells. Eighteen major spots and 15 minor spots were found to be induced by heat shock. From N-terminal sequences of 15 major spots, DnaK, GroEL, GroES, alkyl hydroperoxide reductase, aldehyde dehydrogenase, succinyl coenzyme A (CoA) synthetase, 30S ribosomal protein S6, and ATP synthase alpha subunit were identified. Three of the 18 major spots had an identical N-terminal sequence, indicating that they may be different forms of the same protein. Although a DnaK homologue, SglK, has been identified in M. xanthus (R. M. Weimer, C. Creghton, A. Stassinopoulos, P. Youderian, and P. L. Hartzell, J. Bacteriol. 180:5357-5368, 1998; Z. Yang, Y. Geng, and W. Shi, J. Bacteriol. 180:218-224, 1998), SglK was not induced by heat shock. In addition, there were seven substitutions within the N-terminal 30-residue sequence of the newly identified DnaK. This is the first report to demonstrate that succinyl CoA synthetase, 30S ribosomal protein S6, and ATP synthase alpha subunit are heat shock inducible.     

Bulk and surface characterization of crystalline and plastic sulphur oxidized by two Thiobacillus species

This work studies the surface interaction between Thiobacillus ferrooxidans and Thiobacillus thiooxidans with crystalline and plastic elemental sulphur. The interaction mechanisms were analysed by fractal geometry which describes textural modifications of the substrate caused by bacterial action. The results demonstrated that the bacteria are able to produce two different effects depending on the substrates. Only surface smoothing (decrease on fractal dimension values) was detected on crystalline sulphur (this effect being stronger with T. ferrooxidans than with T. thiooxidans), but, perforation of the bulk was also observed in plastic sulphur     

Use of the respiration activity ofThiobacillus ferrooxidans for the specific determination of iron(II,III)

A method is described for the determination of Fe2+ and Fe3+ after its reduction to Fe2+ on the basis of oxygen uptake rate as a function of Fe2+ concentration. By using substrate-specific Thiobacillus ferrooxidans in combination with the standard addition method a specific determination of iron(II, III) is possible with the determination limit of 3 mumol/L.     

Proteomic profiling of proteins associated with methamphetamine-induced neurotoxicity in different regions of rat brain

It is well documented that methamphetamine (MA) can cause obvious damage to the brain, but the exact mechanism is still unknown. In the present study, proteomic methods of two-dimensional gel electrophoresis in combination with mass spectrometry analysis were used to identify global protein profiles associated with MA-induced neurotoxicity. For the first time, 30 protein spots have been found differentially expressed in different regions of rat brain, including 14 in striatum, 12 in hippocampus and 4 in frontal cortex. The proteins identified by tandem mass spectrometry were Cu, Zn superoxide dismutase, dimethylarginine dimethylaminohydrolase 1, alpha synuclein, ubiquitin-conjugating enzyme E2N, stathmin 1, calcineurin B, cystatin B, subunit of mitochondrial H-ATP synthase, ATP synthase D chain, mitochondrial, NADH dehydrogenase(ubiquinone) Fe-S protein 8, glia maturation factor, beta, Ash-m, neurocalcin delta, myotrophin, profiling IIa, D-dopachrome tautomerase, and brain lipid binding protein. The known functions of these proteins were related to the pathogenesis of MA-induced neurotoxicity, including oxidative stress, degeneration/apoptosis, mitochontrial/energy metabolism and others. Of these proteins, alpha-synuclein was up-regulated, and ATP synthase D chain, mitochondrial was down-regulated in all brain regions. Two proteins, Cu, Zn superoxide dismutase, subunit of mitochondrial H-ATPsynthase were down-regulated and Ubiquitin-conjugating enzyme E2N, NADH dehydrogenase (ubiquinone) Fe-S protein 8 were up-regulated simultaneously in striatum and hippocaltum. The expression of dimethylarginine dimethylaminohydrolase 1 (DDAH 1) increased both in striatum and frontal cortex. The parallel expression patterns of these proteins suggest that the pathogenesis of MA neurotoxicity in different brain regions may share some same pathways.     

Identification and characterization of GroEL and DnaK homologues in Thiobacillus ferrooxidans

The major heat shock proteins from Thiobacillus ferrooxidans were identified as DnaK and GroEL equivalents by Western blotting and analysis of the N-terminal amino acid sequence of spots isolated from dried 2-D polyacrylamide electrophoresis gels. The T. ferrooxidans chaperonins showed 70% and 80% identity with the Escherichia coli GroEL and DnaK, respectively. By using electrophoresis with a transverse pore gradient of cross-linked polyacrylamide and nondenaturing conditions followed by Western blotting, we found that the GroEL proteins from both bacteria formed a 14-mer, whereas E. coli DnaK protein existed partially as a dimer and the T. ferrooxidans DnaK-equivalent showed only a monomeric nature under our experimental conditions.     

Crystallization and preliminary X-ray crystallographic studies of rusticyanin from Thiobacillus ferrooxidans.

Rusticyanin is a 16.5 kDa type I blue copper protein isolated from Thiobacillus ferrooxidans. This organism can grow on Fe2+ as its sole energy source. Rusticyanin is thought to be a principal component in the iron respiratory electron transport chain of T. ferrooxidans. As a component of the periplasmic space of an acidophilic bacterium, rusticyanin is remarkably stable at acidic pH. It is redox-active down to pH 0.2. Crystals of rusticyanin have been grown from solutions of PEG 8000 by the hanging-drop vapor diffusion method. The crystals are orthorhombic, space group P2(1)2(1)2(1), with unit cell dimensions a = 32.36 A, b = 60.37 A, c = 74.60 A. The crystals diffract to 2.0 A resolution and they are stable in the X-ray beam for at least two days.     

Optimal conditions for bio-oxidation of ferrous ions to ferric ions using Thiobacillus ferrooxidans

A chemo-biochemical process using Thiobacillus ferrooxidans for desulphurization of gaseous fuels and emissions containing hydrogen sulphide (H2S) has been developed. In the first stage, H2S present in fuel gas and emissions is selectively oxidized to elemental sulphur using ferric sulphate. The ferrous sulphate produced in the first stage of the process is oxidized to ferric sulphate using Thiobacillus ferrooxidans for recycle and reuse in the process. The effects of process variables, temperature, pH, total dissolved solids (TDS), elemental sulphur, ferric and magnesium ions on bio-oxidation of ferrous ions to ferric ions were investigated using flask culture experiments. The bio-oxidation of ferrous ions to ferric ions could be achieved efficiently in the temperature range of 20(+/-1)-44(+/-1) degrees C. A pH range of 1.8(+/-0.02)-2.2(+/-0.02) was optimum for the growth of culture and effective bio-oxidation of ferrous ions to ferric ions. The effect of TDS on bio-oxidation of ferrous ions indicated that a preacclimatized culture in a growth medium containing high dissolved solid was required to achieve effective bio-oxidation of ferrous ions. Elemental sulphur ranging from 1000 to 100,000 mg/l did not have any effect on efficiency of ferrous ion oxidation. The efficiency of bio-oxidation of ferrous ions to ferric ions was not affected in the presence of ferric ions up to a concentration of 500 mg/l while 3 mg/l of magnesium ion was optimal for achieving effective bio-oxidation.     

Sulfite oxidation by iron-grown cells of Thiobacillus ferrooxidans at pH 3 possibly involves free radicals, iron, and cytochrome oxidase

Thiobacillus ferrooxidans cells grown on ferrous iron oxidized sulfite to sulfate at pH 3, possibly by a free radical mechanism involving iron and cytochrome oxidase. A purely chemical system with low concentrations of Fe3+ simulated the T. ferrooxidans system. Metal chelators, ethylenediamine tetraacetic acid (EDTA), 4,5-dihydroxy-1-3-benzene disulfonic acid (Tiron), o-phenanthroline, and 2,2'-dipyridyl, inhibited both sulfite oxidation systems, but the T. ferrooxidans system was inhibited only after the initial brief oxygen consumption. EDTA and Tiron, strong chelators of Fe3+, inhibited the oxidation at lower concentrations than o-phenanthroline and 2,2'-dipyridyl, strong chelators of Fe2+. Inhibition of Fe3+-catalyzed sulfite oxidation by EDTA and Tiron was instant, but the inhibition by o-phenanthroline and dipyridyl was briefly delayed, presumably for the reduction of Fe3+ to Fe2+. Mannitol, a free radical scavenger, inhibited both systems to the same extent. Cyanide and azide inhibited only the T. ferrooxidans system, suggesting a role of cytochrome oxidase. It is proposed that sulfite is oxidized by a free radical mechanism initiated by Fe3+ on the cell surface of T. ferrooxidans. Cytochrome oxidase is possibly involved in the regeneration of Fe3+ from Fe2+ by the normal Fe2+-oxidizing system of T. ferrooxidans.     

基于质谱和生物信息学分析的小菜蛾蛋白质鉴定

本研究以非模式昆虫小菜蛾Plutella xylostella为材料, 对比2, 3, 4龄幼虫的蛋白质组双向电泳图谱, 得到24个蛋白质差异点, 从中选取了编号为1111的差异表达蛋白质点进行质谱鉴定和生物信息学分析. 采用胶内酶解的多肽进行MALDI-TOF/TOF分析, 获得该点的肽质量指纹图谱（PMF）及串联质谱（MS/MS）图谱。将获得的PMF分别用MASCOT和ProFound等常用软件在NCBInr的Metazoa蛋白质数据库进行搜索, 匹配结果不理想. 进一步用PMF+MS/MS谱图搜索NCBInr的Metazoa蛋白质数据库, 以及小菜蛾EST数据库。 在NCBInr库中匹配结果为拟暗果蝇Drosophila pseudoobscura中的一种假定蛋白GA18218-PA, 而用EST库搜索的结果为家蚕Bombyx mori的ATP合酶的亚基。为验证搜索结果, 将该蛋白质点进行磺基异硫氰酸苯酯（SPITC）化学衍生后de novo测序, 最后确认该点可能为ATP合酶的一个亚基。最后着重讨论了蛋白质的质谱鉴定与生物信息学分析的联合使用, 希望据此选择出最适合于非模式昆虫蛋白质组学鉴定的方法。     

Thiobacillus ferrooxidans, a facultative hydrogen oxidizer

The type strain (ATCC 23270) and two other strains of Thiobacillus ferrooxidans were able to grow by hydrogen oxidation, a feature not recognized before. When cultivated on H2, a hydrogenase was induced and the strains were less extremely acidophilic than during growth on sulfidic ores. Cells of T. ferrooxidans grown on H2 and on ferrous iron showed 100% DNA homology. Hydrogen oxidation was not observed in eight other species of the genus Thiobacillus and in Leptospirillum ferrooxidans.     

Thiobacillus ferrooxidans, a facultative hydrogen oxidizer.

The type strain (ATCC 23270) and two other strains of Thiobacillus ferrooxidans were able to grow by hydrogen oxidation, a feature not recognized before. When cultivated on H2, a hydrogenase was induced and the strains were less extremely acidophilic than during growth on sulfidic ores. Cells of T. ferrooxidans grown on H2 and on ferrous iron showed 100% DNA homology. Hydrogen oxidation was not observed in eight other species of the genus Thiobacillus and in Leptospirillum ferrooxidans.     

Nucleotide sequence of the gene encoding the nitrogenase iron protein of Thiobacillus ferrooxidans.

The DNA sequence was determined for the cloned Thiobacillus ferrooxidans nifH and part of the nifD genes. A putative T. ferrooxidans nifH promoter was identified whose sequences showed perfect consensus with those of the Klebsiella pneumoniae nif promoter. Two putative consensus upstream activator sequences were also identified. The amino acid sequence was deduced from the DNA sequence. In a comparison of nifH DNA sequences from T. ferrooxidans and eight other nitrogen-fixing microbes, a Rhizobium sp. isolated from Parasponia andersonii showed the greatest homology (74%) and Clostridium pasteurianum (nifH 1) showed the least homology (54%). In a comparison of the amino acid sequences of the Fe proteins, the Rhizobium sp. and Rhizobium japonicum showed the greatest homology (both 86%) and C. pasteurianum (nifH 1 gene product) demonstrated the least homology (56%) to the T. ferrooxidans Fe protein.     

Numerical simulation for electrochemical cultivation of iron oxidizing bacteria

A numerical simulation model was constructed for electrochemical cultivation of iron oxidizing bacterium, Thiobacillus ferrooxidans, based on Monod's dual limitation equation. In this model, two limiting factors were examined, low supply of Fe(II) ion and dissolved oxygen, from empirical viewpoints. The simulation model was constructed taking into consideration the energy balance based on the amount of the electronic flow from the electrode to bacteria via an iron ion, and then to oxygen. The model consisted of a logarithmic bacterial growth phase during the first three days, followed by a plateau and growth limitation thereafter. The predicted results were in agreement with the actual growth under electrochemical cultivation. It was predicted the growth limiting factor would be changed from insufficient supply of Fe(II) ions to that of oxygen by decreasing the value of oxygen transfer constant K, which correlated with the aeration rate. The optimum aeration rate was determined for the ideal electrochemical cultivation. The algorithm described here can be used in any electrochemical cultivation by modifying the parameters for each system.