Characterization of an ATP-dependent DNA ligase from the acidophilic archaeon “<Emphasis Type="Italic">Ferroplasma acidarmanus</Emphasis>” Fer1

Analysis of the genome of "Ferroplasma acidarmanus" Fer1, an archaeon that is an extreme acidophile, identified an open reading frame encoding a putative ATP-dependent DNA ligase, which we termed FaLig. The deduced amino acid sequence of FaLig contains 595 amino acids, with a predicted molecular mass of 67.8 kDa. "F. acidarmanus" Fer1 is classified as a Euryarchaeote, but phylogenetic analysis using amino acid sequences showed that FaLig is more similar to DNA ligases from Crenarchaeota, suggesting that lateral transfer of these genes has occurred among archaea. The gene sequence encoding FaLig was cloned into a bacterial expression vector harbouring an upstream His-tag to aid purification. Conditions for expression and purification from Escherichia coli were identified and recombinant FaLig was confirmed to be an ATP-dependent DNA ligase. Optimal conditions for nick-joining by the protein were pH 6-7, 0.5 mM ATP, in the presence of either Mg(2+) or Mn(2+). Using a range of nicked, double-stranded nucleic acids, ligation was detected with the same substrates as previously determined for other DNA ligases. Although FaLig is the DNA ligase from one of the most extreme acidophilic organism yet studied, this characterization suggests that its biochemical mechanism is analogous to that of enzymes from other cellular systems.     

Isolation and characterization of Ferroplasma thermophilum sp. nov., a novel extremely acidophilic, moderately thermophilic archaeon and its role in bioleaching of chalcopyrite

Aims:  To isolate Ferroplasma thermophilum L1^T from a low pH environment and to understand its role in bioleaching of chalcopyrite.
Methods and Results:  Using serial dilution method, a moderately thermophilic and acidophilic ferrous iron-oxidizing archaeon, named L1^T, was isolated from a chalcopyrite-leaching bioreactor. The morphological, biochemical and physiological characteristics of strain L1^T and its role in bioleaching of chalcopyrite were studied. Strain L1^T was a nonmotile coccus that lacked cell wall. Strain L1^T had a temperature optimum of 45°C and the optimum pH for growth was 1·0. Strain L1^T was capable of chemomixotrophic growth on ferrous iron and yeast extract. Results of fatty acid analysis, DNA–DNA hybridization, G+C content, and analysis based on 16S rRNA gene sequence indicated that strain L1^T should be grouped in the genus Ferroplasma , and represented a new species, Ferroplasma thermophilum . Ferroplasma thermophilum in combination with Acidithiobacillus caldus and Leptospirillum ferriphilum could improve the copper dissolution in bioleaching of chalcopyrite.
Conclusions:  A novel extremely acidophilic, moderately thermophilic archaeon isolated from a bioleaching reactor has been identified as F. thermophilum that played an important role in bioleaching of chalcopyrite at low pH.
Significance and Impact of the Study:  This study contributes to understand the characteristics of F. thermophilum L1^T and its role in bioleaching of sulfide ores.     

嗜中高温嗜酸古菌Ferroplasma thermophilum的培养条件优化

在中高温和较低pH条件下, Ferrroplasma spp. 是进行硫化矿生物浸出的重要微生物。Ferroplasma spp.为古菌,无细胞壁, 对矿浆浓度、搅拌剪切力以及溶液中的重金属离子等敏感, 很难得到高密度的纯培养, 给大规模的工业应用带来了一定难度。研究了F. thermophilum摇瓶培养时的最佳生长条件, 单因素考察结果表明最适培条件为: 温度50oC, 初始pH 0.5, 250 mL的摇瓶装液量为50 mL, 无机氮源(NH4)2SO4。通过正交试验确定了FeSO4·7H2O、酵母粉和蛋白胨最适组合为FeSO4·7H2O 40 g/L, 酵母粉0.3 g/L, 蛋白胨0.2 g/L。优化培养后, F. thermophilum 浓度达到了6.3×107个/mL, 40 g/L的FeSO4·7H2O在72 h内全部氧化完全。该结果可为该类古菌的扩大培养以及工业应用提供参考。     

Molybdate treatment and sulfate starvation decrease ATP and DNA levels in Ferroplasma acidarmanus

Sulfate is a primary source of sulfur for most microbes and in some prokaryotes it is used an electron acceptor. The acidophile Ferroplasma acidarmanus (strain fer1) requires a minimum of 150 mM of a sulfate-containing salt for growth. Sulfate is assimilated by F. acidarmanus into proteins and reduced to form the volatile organic sulfur compounds methanethiol and dimethyldisulfide. In the absence of sulfate, cell death occurs by an unknown mechanism. In this study, cell viability and genomic DNA and ATP contents of F. acidarmanus were monitored in response to the absence of sulfate or the presence of sulfate and the sulfate analog molybdate ( MoO42- ). Cellular DNA and ATP contents were monitored as markers of cell viability. The absence of sulfate led to a decrease in viable cell numbers of greater than 7 log₁₀ within 5 days, a > 99% reduction in genomic DNA within 3 days, and a > 60% decrease in ATP within 6 h. Likewise, cells incubated with lost viability (decreased by > 2 log₁₀ in 5 days), extractable genomic DNA (reduction of > 60% in 2 days), and ATP (reduction of > 70 % in 2 hours). These results demonstrate that sulfate deprivation or the presence of molybdate have similar impacts on cell viability and essential biomolecules. Sulfate was coupled to cellular ATP content and maintenance of DNA integrity in F. acidarmanus, a finding that may be applicable to other acidophiles that are typically found in sulfate-rich biotopes.