Tetrathionate-Forming Thiosulfate Dehydrogenase from the Acidophilic,Chemolithoautotrophic Bacterium Acidithiobacillus ferrooxidans

Thiosulfate dehydrogenase is known to play a significant role in thiosulfate oxidation in the acidophilic, obligately chemolithoautotroph, Acidithiobacillus ferrooxidans. Enzyme activity measured using ferricyanide as the electron acceptor was detected in cell extracts of A. ferrooxidans ATCC 23270 grown on tetrathionate or sulfur, but no activity was detected in ferrous iron-grown cells. The enzyme was enriched 63-fold from cell extracts of tetrathionate-grown cells. Maximum enzyme activity (13.8 U mg⁻¹) was observed at pH 2.5 and 70°C. The end product of the enzyme reaction was tetrathionate. The enzyme reduced neither ubiquinone nor horse heart cytochrome c, which serves as an electron acceptor. A major protein with a molecular mass of ∼25 kDa was detected in the partially purified preparation. Heme was not detected in the preparation, according to the results of spectroscopic analysis and heme staining. The open reading frame of AFE_0042 was identified by BLAST by using the N-terminal amino acid sequence of the protein. The gene was found within a region that was previously noted for sulfur metabolism-related gene clustering. The recombinant protein produced in Escherichia coli had a molecular mass of ∼25 kDa and showed thiosulfate dehydrogenase activity, with maximum enzyme activity (6.5 U mg⁻¹) observed at pH 2.5 and 50°C.     

氧化亚铁硫杆菌亚铁氧化系统的研究进展

氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)为无机化能自养菌,革兰氏阴性,能在极端酸性环境中生长.由于在生物冶金中的应用及特殊的生理学效应,该菌受到研究者的广泛关注.A.ferrooxidans能氧化亚铁、元素硫及还原态硫化物获得电子,并通过一系列电子载体将电子传递给氧生成水,同时释放能量供生命活动需要.目前对A.ferrooxidans电子传递系统的研究主要集中于亚铁氧化电子传递系统,已发现多种与亚铁氧化电子传递相关电子载体和操纵子,如电子载体铜蓝蛋白(Rustocyanin,Rus)、细胞色素C(Cytochrome C,Cyc)、细胞色素C氧化酶(Cytochrome Coxidase,Cox)、亚铁氧化酶(Iro)、细胞色素bc1复合物(cytochrome bc1 complex,bc1)等,以及rus操纵子和pet操纵子.综述了近年来有关A.ferrooxidans 亚铁氧化电子传递链相关蛋白载体,rus和pet操纵子结构与功能及表达调控等方面的研究进展.     

Functional dissection of a mercuric ion transporter, MerC, from Acidithiobacillus ferrooxidans

Topological analysis with a phoA gene fusion suggested that Acidithiobacillus ferrooxidans MerC, a mercury transporter, has two periplasmic loops and four transmembrane domains. Cys-23 and Cys-26 of the protein were involved in Hg(2+)-recognition/uptake, but Cys-132 and Cys-137 were not. Escherichia coli cells producing the MerC were hypersensitive to CdCl(2). In this case, mutation of His72 rendered the host cells less CdCl(2) sensitive, whereas none of the Cys residues affected it. E. coli cells expressing the gene encoding a mercuric ion transporter (merC)-deletion mutant, in which the coding-sequence of the carboxy-terminal cytoplasmic region was removed, retained Hg(2+) hypersensitivity and showed about 55% HgCl(2) uptake ability compared to that of the one expressing the intact merC, indicating that the region is not essential for Hg(2+) uptake. Coexpression of A. ferrooxidans the gene encoding mercuric reductase (merA) and the merC deletion mutation conferred HgCl(2) tolerance to E. coli host cells. Under this condition, the merC deletion gene product was exclusively present as a monomer.     

Evidence for a Functional O-Linked N-Acetylglucosamine (O-GlcNAc) System in the Thermophilic Bacterium Thermobaculum terrenum

Post-translational modification of proteins is a ubiquitous mechanism of signal transduction in all kingdoms of life. One such modification is addition of O-linked N-acetylglucosamine to serine or threonine residues, known as O-GlcNAcylation. This unusual type of glycosylation is thought to be restricted to nucleocytoplasmic proteins of eukaryotes and is mediated by a pair of O-GlcNAc-transferase and O-GlcNAc hydrolase enzymes operating on a large number of substrate proteins. Protein O-GlcNAcylation is responsive to glucose and flux through the hexosamine biosynthetic pathway. Thus, a close relationship is thought to exist between the level of O-GlcNAc proteins within and the general metabolic state of the cell. Although isolated apparent orthologues of these enzymes are present in bacterial genomes, their biological functions remain largely unexplored. It is possible that understanding the function of these proteins will allow development of reductionist models to uncover the principles of O-GlcNAc signaling. Here, we identify orthologues of both O-GlcNAc cycling enzymes in the genome of the thermophilic eubacterium Thermobaculum terrenum. The O-GlcNAcase and O-GlcNAc-transferase are co-expressed and, like their mammalian orthologues, localize to the cytoplasm. The O-GlcNAcase orthologue possesses activity against O-GlcNAc proteins and model substrates. We describe crystal structures of both enzymes, including an O-GlcNAcase·peptide complex, showing conservation of active sites with the human orthologues. Although in vitro activity of the O-GlcNAc-transferase could not be detected, treatment of T. terrenum with an O-GlcNAc-transferase inhibitor led to inhibition of growth. T. terrenum may be the first example of a bacterium possessing a functional O-GlcNAc system.     

Identification of a gene encoding a tetrathionate hydrolase in Acidithiobacillus ferrooxidans

Tetrathionate is one of the most important intermediates in dissimilatory sulfur oxidation and can itself be utilized as a sole energy source by some sulfur-oxidizing microorganisms. Tetrathionate hydrolase (4THase) plays a significant role in tetrathionate oxidation and should catalyze the initial step in the oxidative dissimilation when sulfur-oxidizing bacteria are grown on tetrathionate. 4THase activity was detected in tetrathionate-grown Acidithiobacillus ferrooxidans ATCC 23270 cells but not in iron-grown cells. A 4THase having a dimeric structure of identical 50kDa polypeptides was purified from tetrathionate-grown cells. The 4THase showed the maximum activity at pH 3.0 and high stability under acidic conditions. An open reading frame (ORF) encoding the N-terminal amino acid sequence of the purified 4THase was identified by a BLAST search using the database for the A. ferrooxidans ATCC 23270 genome. Heterologous expression of the gene in Escherichia coli resulted in the formation of inclusion bodies of the protein in an inactive form. Antisera against the recombinant protein clearly recognized the purified native 4THase, indicating that the ORF encoded the 4THase.     

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.     

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 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 a 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 heavy metal ions is discussed.     

A Lux-like quorum sensing system in the extreme acidophile Acidithiobacillus ferrooxidans

The genome of the acidophilic, proteobacterium Acidithiobacillusferrooxidans, contains linked but divergently oriented genes, termed afel and afeR, whose predicted protein products are significantly similar to the LuxI and LuxR families of proteins. A possible promoter and Lux box are predicted upstream of afel. A cloned copy of afel, expressed in E. coli, encodes an enzyme that catalyzes the production of a diffusible compound identified by gas chromatography and mass spectrometry as an unsubstituted N-acyl homoserine lactone (AHL) of chain length C14. This AHL can be detected by a reporter strain of Sinorhizobium meliloti Rm41 suggesting that it is biologically active. The reporter strain also responds to extracts of the supernatant of A. ferrooxidans grown to early stationary phase in sulfur medium indicating that a diffusible AHL is produced by this microorganism. Semi-quantitative RT-PCR experiments indicate that afeI and afeR are expressed maximally in early stationary phase and are more expressed when A. ferrooxidans is grown in sulfur--rather than iron-containing medium. Given the predicted amino acid sequence and functional properties of AfeI and AfeR it is proposed that A. ferrooxidans has a quorum sensing system similar to the LuxI-LuxR paradigm.     

Bioleaching of chalcopyrite concentrate using Leptospirillum ferriphilum, Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans in a continuous bubble column reactor

To estimate the bioleaching performance of chalcopyrite for various hydraulic residence times (HRTs), laboratory-scale bioleaching of chalcopyrite concentrate was carried out in a continuous bubble column reactor with three different HRTs of 120, 80 and 40 h, respectively. An extraction rate and ratio of 0.578 g Cu l⁻¹ h⁻¹ and 39.7%, respectively, were achieved for an HRT of 80 h at a solids concentration of 10% (w/v). Lower bioleaching performances than this were obtained for a longer HRT of 120 h and a shorter HRT of 40 h. In addition, there was obvious competition between Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans to oxidize ferrous iron, causing large compositional differences between the microbial communitys obtained for the different HRTs. Leptospirillum ferriphilum and Acidithiobacillus thiooxidans were found to be the dominant microbes for the longer HRT (120 h). Acidithiobacillus ferrooxidans became the dominant species when the HRT was decreased. The proportion of Acidithiobacillus thiooxidans was comparatively constant in the microbial community throughout the three process stages.     

氧化亚铁硫杆菌中铜代谢相关两个基因的差异

目的:氧化亚铁硫杆菌(Acidithiobocllius ferrooxidans)在微生物冶金中发挥着重要的作用,研究其铜代谢机理有着十分典型的意义.在A.ferrooxidans全基因组序列数据库中,4个基因被注释与铜代谢相关.其中两个基因,Afe0454和Afe1073目前为止未发现有实验报道.本文旨在研究Afe0454和Afe1073与铜代谢的相关性.方法:通过一系列的方法如实时定量PER、反转录PER、序列分析,将基因导入抗铜基因缺陷的大肠杆菌(Escherichia coli)菌株中等,研究了Afe0454和Afe1073.结果:与Afe0454相比,Afe1073的表达对铜压力更敏感;Afe1073作为一个转录子单独转录,而Afe0454与Afe0453一起转录;序列分析显示Afe1073表达一种典型的重金属离子泵P1b1型ATP酶,而Afe0454表达一种未知功能的跨膜蛋白;不像Afe0454,Afe1073能使P1b型ATP酶敲除的大肠杆菌菌株铜抗性提高.结论:单独转录的Afe1073比Afe0454在铜代谢中发挥的作用更加明显.     

Volatilization of mercury by an iron oxidation enzyme system in a highly mercury-resistant Acidithiobacillus ferrooxidans strain MON-1

A highly mercury-resistant strain Acidithiobacillus ferrooxidans MON-1, was isolated from a culture of a moderately mercury-resistant strain, A. ferrooxidans SUG 2-2 (previously described as Thiobacillus ferrooxidans SUG 2-2), by successive cultivation and isolation of the latter strain in a Fe2+ medium with increased amounts of Hg2+ from 6 microM to 20 microM. The original stain SUG 2-2 grew in a Fe2+ medium containing 6 microM Hg2+ with a lag time of 22 days, but could not grow in a Fe2+ medium containing 10 microM Hg2+. In contrast, strain MON-1 could grow in a Fe2+ medium containing 20 microM Hg2+ with a lag time of 2 days and the ability of strain MON-1 to grow rapidly in a Fe2+ medium containing 20 microM Hg2+ was maintained stably after the strain was cultured many times in a Fe2+ medium without Hg2+. A similar level of NADPH-dependent mercury reductase activity was observed in cell extracts from strains SUG 2-2 and MON-1. By contrast, the amounts of mercury volatilized for 3 h from the reaction mixture containing 7 microM Hg2+ using a Fe(2+)-dependent mercury volatilization enzyme system were 5.6 nmol for SUG 2-2 and 67.5 nmol for MON-1, respectively, indicating that a marked increase of Fe(2+)-dependent mercury volatilization activity conferred on strain MON-1 the ability to grow rapidly in a Fe2+ medium containing 20 microM Hg2+. Iron oxidizing activities, 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD) oxidizing activities and cytochrome c oxidase activities of strains SUG 2-2 and MON-1 were 26.3 and 41.9 microl O2 uptake/mg/min, 15.6 and 25.0 microl O2 uptake/mg/min, and 2.1 and 6.1 mU/mg, respectively. These results indicate that among components of the iron oxidation enzyme system, especially cytochrome c oxidase activity, increased by the acquisition of further mercury resistance in strain MON-1. Mercury volatilized by the Fe(2+)-dependent mercury volatilization enzyme system of strain MON-1 was strongly inhibited by 1.0 mM sodium cyanide, but was not by 50 nM rotenone, 5 microM 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), 0.5 microM antimycin A, or 0.5 microM myxothiazol, indicating that cytochrome c oxidase plays a crucial role in mercury volatilization of strain MON-1 in the presence of Fe2+.