Springer Science and Business Media LLC

Three strains of sulfate-reducing bacteria (M1T, D, and E) were isolated from acidic sediments (White river and Tinto river) and characterized phylogenetically and physiologically. All three strains were obligately anaerobic, mesophilic, spore-forming straight rods, stained Gram-negative and displayed variable motility during active growth. The pH range for growth was 3.8–7.0, with an optimum at pH 5.5. The temperature range for growth was 15–40 °C, with an optimum at 30 °C. Strains M1T, D, and E used a wide range of electron donors and acceptors, with certain variability within the different strains. The nominated type strain (M1T) used ferric iron, nitrate, sulfate, elemental sulfur, and thiosulfate (but not arsenate, sulfite, or fumarate) as electron acceptors, and organic acids (formate, lactate, butyrate, fumarate, malate, and pyruvate), alcohols (glycerol, methanol, and ethanol), yeast extract, and sugars (xylose, glucose, and fructose) as electron donors. It also fermented some substrates such as pyruvate and formate. Strain M1T tolerated up to 50 mM ferrous iron and 10 mM aluminum, but was inhibited by 1 mM copper. On the basis of phenotypic, phylogenetic, and genetic characteristics, strains M1T, D, and E represent a novel species within the genus Desulfosporosinus, for which the name Desulfosporosinus acididurans sp. nov. is proposed. The type strain is M1T (=DSM 27692T = JCM 19471T). Strain M1T was the first acidophilic SRB isolated, and it is the third described species of acidophilic SRB besides Desulfosporosinus acidiphilus and Thermodesulfobium narugense.

We successfully expressed the l-aspartate oxidase homolog gene (accession no: OCC_06611) of Thermococcus litoralis DSM 5473 in the soluble fraction of Escherichia coli BL21 (DE3) using a pET21b vector with 6X His tag at its C-terminus. The gene product (Tl-LASPO) showed l-aspartate oxidase activity in the presence of FAD in vitro, and this report is the first that details an l-aspartate oxidase derived from a Thermococcus species. The homologs of Tl-LASPO existed mainly in archaea, especially in the genus of Thermococcus, Pyrococcus, Sulfolobus, and Halobacteria. The quaternary structure of Tl-LASPO was homotrimeric with a subunit molecular mass of 52 kDa. The enzyme activity of Tl-LASPO increased with temperature up to 70 °C. Tl-LASPO was active from pH 6.0 to 9.0, and its highest activity was at pH 8.0. Tl-LASPO was stable at 80 °C for 1 h. The highest k cat/K m value was observed in assays at 70 °C. Tl-LASPO was highly specific for l-aspartic acid. Tl-LASPO utilized fumaric acid, 2,6-dichlorophenolindophenol, and ferricyanide in addition to FAD as a cofactor under anaerobic conditions. The absorption spectrum of holo-Tl-LASPO exhibited maxima at 380 and 450 nm. The FAD dissociation constant, K d, of the FAD-Tl-LASPO complex was determined to be 5.9 × 10−9 M.

Một phiên bản đột biến của gen phosphotransferase hygromycin B (hph mut ) từ Escherichia coli, được tách lọc qua quá trình tiến hóa hướng đích ở 75°C trong các biến thể của một chủng Sulfolobus solfataricus nhiệt cực, đã được đặc trưng về độ ổn định di truyền cả trong chủng mesophilic nguyên bản và chủng nhiệt cực mới. Gen này được chứng minh có khả năng biểu hiện kiểu hình kháng hygromycin B và duy trì ổn định cũng như được nhân lên trong các chủng S. solfataricus nhiệt cực khác, tức là ở nhiệt độ lên tới 82°C. Hơn nữa, nó có thể được chuyển vào tế bào S. solfataricus qua đồng biến đổi với pKMSD48, một yếu tố ngoài nhiễm sắc thể khác được phân lập từ virus SSV1 của Sulfolobus shibatae, mà không có mất mát độ ổn định và không ảnh hưởng đến khả năng sao chép và lây nhiễm của DNA virus này. Các sản phẩm của gen hph mut và gen тип đã được biểu hiện ở mức cao hơn trong E. coli và được tinh chế bằng sắc ký ái lực đặc hiệu trên hygromycin B đã được cố định. Việc so sánh đặc trưng cho thấy enzyme đột biến đã có khả năng kháng nhiệt đáng kể và thể hiện hoạt động nhiệt cao hơn với hiệu suất xúc tác tăng.

The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation. Members of Halococcus spp. are known for their rigid cell walls, and are thus difficult to lyse and could potentially be overlooked in an environment. Furthermore, the lack of a suitable lysis method hinders subsequent molecular analysis. The effects of six different DNA extraction methods were tested on Halococcus hamelinensis, Halococcus saccharolyticus and Halobacterium salinarum NRC-1 as well as on an organic rich, highly carbonated sediment from stromatolites spiked with Halococcus hamelinensis. The methods tested were based on physical disruption (boiling and freeze/thawing), chemical lysis (Triton X-100, potassium ethyl xanthogenate (XS) buffer and CTAB) and on enzymatic lysis (lysozyme). Results showed that boiling and freeze/thawing had little effect on the lysis of both Halococcus strains. Methods based on chemical lysis (Triton X-100, XS-buffer, and CTAB) showed the best results, however, Triton X-100 treatment failed to produce visible DNA fragments. Using a combination of bead beating, chemical lysis with lysozyme, and thermal shock, lysis of cells was achieved however DNA was badly sheared. Lysis of cells and DNA extraction of samples from spiked sediment proved to be difficult, with the XS-buffer method indicating the best results. This study provides an evaluation of six commonly used methods of cell lysis and DNA extraction of Halococcus spp., and the suitability of the resulting DNA for molecular analysis.

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.

Site-directed mutagenesis were done in Haloarcula marismortui using the strategy that Khorana and coworkers devised for deleting the bacteriorhodopsin gene from Halobacterium halobium [Krebs et al. Proc Natl Acad Sci USA 90:1987–1991 (1993)]. Strains have been prepared from H. marsimortui, which normally has three rRNA operons, that are missing either its rrnB operon or both its rrnB and rrnC operons. In rich media, both strains grow at about the same rate as wild type. The G2099 in the 23S rRNA gene of the single operon strain was changed to A, and a three amino acid deletion was introduced into the gene for ribosomal protein L22 of the wild-type organism. The structural consequences of these and other such mutations can be determined with unusual accuracy because crystals of the large ribosomal subunit of H. marismortui diffract to atomic resolution.

Genetic relationships and diversity of 101 Thermus isolates from different geothermal regions in Iceland were investigated by using multilocus enzyme electrophoresis (MLEE) and small subunit ribosomal rRNA (SSU rRNA) sequence analysis. Ten polymorphic enzymes were used and seven distinct and genetically highly divergent lineages of Thermus were observed. Six of seven lineages could be assigned to species whose names have been validated. The most diverse lineage was Thermus scotoductus. In contrast to the other lineages, this lineage was divided into very distinct genetic sublineages that may represent subspecies with different habitat preferences. The least diverse lineage was Thermus brockianus. Phenotypic and physiological analysis was carried out on a subset of the isolates. No relationship was found between growth on specific single carbon source to the grouping obtained by the isoenzyme analysis. The response to various salts was distinguishing in a few cases. No relationship was found between temperature at the isolation site and the different lineages, but pH indicated a relation to specific lineages.