Extremophiles最新文献

Glaciozyma antarctica PI12 is a psychrophilic yeast isolated from Antarctica. In this work, we describe the heterologous production, biochemical properties and in silico structure analysis of an arginase from this yeast (GaArg). GaArg is a metalloenzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea. The cDNA of GaArg was reversed transcribed, cloned, expressed and purified as a recombinant protein in Escherichia coli. The purified protein was active against L-arginine as its substrate in a reaction at 20 °C, pH 9. At 10-35 °C and pH 7-9, the catalytic activity of the protein was still present around 50%. Mn²⁺, Ni²⁺, Co²⁺ and K⁺ were able to enhance the enzyme activity more than two-fold, while GaArg is most sensitive to SDS, EDTA and DTT. The predicted structure model of GaArg showed a very similar overall fold with other known arginases. GaArg possesses predominantly smaller and uncharged amino acids, fewer salt bridges, hydrogen bonds and hydrophobic interactions compared to the other counterparts. GaArg is the first reported arginase that is cold-active, facilitated by unique structural characteristics for its adaptation of catalytic functions at low-temperature environments. The structure and function of cold-active GaArg provide insights into the potentiality of new applications in various biotechnology and pharmaceutical industries.

The enzymology of the key steps in the archaeal phospholipid biosynthetic pathway has been elucidated in recent years. In contrast, the complete biosynthetic pathways for proposed membrane regulators consisting of polyterpenes, such as carotenoids, respiratory quinones, and polyprenols remain unknown. Notably, the multiplicity of geranylgeranyl reductases (GGRs) in archaeal genomes has been correlated with the saturation of polyterpenes. Although GGRs, which are responsible for saturation of the isoprene chains of phospholipids, have been identified and studied in detail, there is little information regarding the structure and function of the paralogs. Here, we discuss the diversity of archaeal membrane-associated polyterpenes which is correlated with the genomic loci, structural and sequence-based analyses of GGR paralogs.

Small heat shock proteins (HSPs), such as HSP20, represent cellular thermal resistance mechanisms, to avoid protein aggregation at elevated temperatures. Recombinantly expressed HSP20s serve as a molecular tool for improving the tolerance of living cells to various physical and chemical stressors. Here, we aimed to heterologously express 18 HSP20s from 12 thermotolerant bacteria in Escherichia coli and evaluate their effects on various physical and chemical cellular stresses. Seventeen HSP20s were successfully expressed as soluble proteins. Recombinant E. coli cells were subjected to heat, cold, acidic, alkaline, and hyperosmolar stress to evaluate the effects of HSP20 proteins on stress resistance. Notably, the overexpression of 15 HSP20s enhanced the stress resistance of E. coli compared to that of the control strain. In particular, HSPs from Tepidimonas sediminis and Oceanithermus profundus improved the stress tolerance of E. coli under all tested conditions. In addition, E. coli harboring HSP20 from T. sediminis retained cell viability even after heat treatment at 52 °C for 5 days. To our knowledge, this is the first report of E. coli tolerance to prolonged (> 100 h) high-temperature stress. These findings indicate the potential of thermotolerant HSPs as molecular tools for improving stress tolerance in E. coli.

The isolated halophilic bacterial strain Halovibrio variabilis TG-5 showed a good performance in the pretreatment of coal gasification wastewater. With the optimum culture conditions of pH = 7, a temperature of 46 °C, and a salinity of 15%, the chemical oxygen demand and volatile phenol content of pretreated wastewater were decreased to 1721 mg/L and 94 mg/L, respectively. The removal rates of chemical oxygen demand and volatile phenol were over 90% and 70%, respectively. At the optimum salinity conditions of 15%, the total yield of intracellular compatible solutes and the extracellular transient released yield under hypotonic conditions were increased to 6.88 g/L and 3.45 g/L, respectively. The essential compatible solutes such as L-lysine, L-valine, and betaine were important in flocculation mechanism in wastewater pretreatment. This study provided a new method for pretreating coal gasification wastewater by halophilic microorganisms, and revealed the crucial roles of compatible solutes in the flocculation process.

The genera Haloarcula and Halomicroarcula are the most closely related genera within the family Haloarculaceae (class Halobacteria). The respective 16S rRNA genes of type strains from the genus Haloarcula showed 94.7-96.5% similarities to their homologous genes of type strains from the genus Halomicroarcula. The Haloarcula species showed 89.3-92.8% rpoB' gene similarities to Halomicroarcula species. These similarities were higher than the proposed genus boundary. Phylogenomic analysis revealed that these two genera formed a tight cluster separated from Halomicrobium with high bootstrap confidence. The average amino acid identity (AAI) values among Haloarcula and Halomicroarcula were 70.1-74.5%, higher than the cutoff value (67.0%) to differentiate the genera Haloarcula and Halomicroarcula from Halomicrobium. These results indicated that the genus Halomicroarcula should be merged with Haloarcula. Then, six novel species are described based on strains DFY41^T, GDY20^T, SHR3^T, XH51^T, YJ-61-S^T, and ZS-22-S1^T isolated from coarse sea salt, marine solar saltern, and salt lake (China). These six strains formed separate clades (90.1-99.3% 16S rRNA and 89.0-94.9% rpoB' gene similarities) and then clustered with current Haloarcula and Halomicroarcula species (89.4-99.1% 16S rRNA and 87.6-95.0% rpoB' gene similarities), as revealed by phylogenetic analyses. The average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), and AAI values among these six strains and current Haloarcula and Halomicroarcula species were 76.2-89.8%, 25.3-46.0%, and 70.3-89.7%, respectively, clearly below the species demarcation threshold. These six strains were distinguished from current Haloarcula and Halomicroarcula species according to differential phenotypic characteristics. Six novel species, Haloarcula halophila sp. nov., Haloarcula litorea sp. nov., Haloarcula rara sp. nov., Haloarcula halobia sp. nov., Haloarcula pelagica sp. nov., and Haloarcula ordinaria sp. nov., are proposed to accommodate strains DFY41^T, GDY20^T, SHR3^T, XH51^T, YJ-61-S^T, and ZS-22-S1^T, respectively.

Second-generation ethanol, a promising biofuel for reducing greenhouse gas emissions, faces challenges due to the inefficient metabolism of xylose, a pentose sugar. Overcoming this hurdle requires exploration of genes, pathways, and organisms capable of fermenting xylose. Thermoanaerobacterium saccharolyticum is an organism capable of naturally fermenting compounds of industrial interest, such as xylose, and understanding evolutionary adaptations may help to bring novel genes and information that can be used for industrial yeast, increasing production of current bio-platforms. This study presents a deep evolutionary study of members of the firmicutes clade, focusing on adaptations in Thermoanaerobacterium saccharolyticum that may be related to overall fermentation metabolism, especially for xylose fermentation. One highlight is the finding of positive selection on a xylose-binding protein of the xylFGH operon, close to the annotated sugar binding site, with this protein already being found to be expressed in xylose fermenting conditions in a previous study. Results from this study can serve as basis for searching for candidate genes to use in industrial strains or to improve Thermoanaerobacterium saccharolyticum as a new microbial cell factory, which may help to solve current problems found in the biofuels' industry.

Mining activities generate large quantities of wastes that significantly alter the biogeochemistry and ecological structure of entire river basins. Microbial communities that develop in these areas present a variety of survival and adaptation mechanisms. Knowing this diversity at the molecular level is strategic both for understanding adaptive processes and for identifying genomes with potential use in bioremediation and bioprospecting. In this work, prokaryotic and eukaryotic communities were evaluated by meta-taxonomics (16S and 18S amplicons) in sediments and water bodies impacted by acid mine drainage in an important coal mining area in southern Brazil. Five sampling stations were defined on a gradient of impacts (pH 2.7-4.25). Taxon diversity was directly proportional to pH, being greater in sediments than in water. The dominant prokaryotic phyla in the samples were Proteobacteria, Actinobacteria, Acidobacteria, OD1, Nitrospirae, and Euryarchaeota, and among the eukaryotes, algae (Ochrophyta, Chlorophyta, Cryptophyceae), fungi (Basidiomycota, Ascomycota, and Cryptomycota), and protists (Ciliophora, Heterolobosea, Cercozoa). The prokaryotic genera Leptospirillum, Acidithiobacillus, Acidiphilium, Thiomonas, Thermogymnomonas, and Acidobacterium, and the eukaryotic genera Pterocystis and Poteriospumella were associated with more acidic conditions and higher metal concentrations, while the prokaryotic genera Sediminibacterium, Gallionella Geothrix, and Geobacter were more abundant in transitional environments.

Topoisomerases are crucial enzymes in genome maintenance that modulate the topological changes during DNA metabolism. Deinococcus radiodurans, a Gram-positive bacterium is characterized by its resistance to many abiotic stresses including gamma radiation. Its multipartite genome encodes both type I and type II topoisomerases. Time-lapse studies using fluorescently tagged topoisomerase IB (drTopoIB-RFP) and DNA gyrase (GyrA-RFP) were performed to check the dynamics and localization with respect to DNA repair and cell division under normal and post-irradiation growth conditions. Results suggested that TopoIB and DNA gyrase are mostly found on nucleoid, highly dynamic, and show growth phase-dependent subcellular localization. The drTopoIB-RFP was also present at peripheral and septum regions but does not co-localize with the cell division protein, drFtsZ. On the other hand, DNA gyrase co-localizes with PprA a pleiotropic protein involved in radioresistance, on the nucleoid during the post-irradiation recovery (PIR). The topoIB mutant was found to be sensitive to hydroxyurea treatment, and showed more accumulation of single-stranded DNA during the PIR, compared to the wild type suggesting its role in DNA replication stress. Together, these results suggest differential localization of drTopoIB-RFP and GyrA-RFP in D. radiodurans and their interaction with PprA protein, emphasizing the functional significance and role in radioresistance.

This study investigated the metabolism of Geobacillus sp. LC300, a promising biorefinery host organism with high substrate utilization rates. A new defined medium was designed and tested that allows for exponential growth to elevated cell densities suitable for quantitative physiological studies. Screening of the metabolic requirements of G. sp. LC300 revealed prototrophy for all essential amino acids and most vitamins and only showed auxotrophy for vitamin B12 and biotin. The effect of temperature and pH on growth rate was investigated, adjusting the optimal growth temperature to several degrees lower than previously reported. Lastly, studies on carbon source utilization revealed a capability for fast growth on several common carbon sources, including monosaccharides, oligosaccharides, and polysaccharides, and the highest ever reported growth rate in defined medium on glucose (2.20 h^-1) or glycerol (1.95 h^-1). These findings provide a foundation for further exploration of G. sp. LC300's physiology and metabolic regulation, and its potential use in bioproduction processes.