Bonch-Osmolovskaya, Elizaveta A.

Abstract This protocol describes a rapid protein extraction method for the archaeon Candidatus Vulcanisaeta moutnovskia, which can be also implemented for other archaea. The utilization of two different methods for protein extraction constitute the main step of the protocol. Method I involves the extraction with a multi-chaotropic lysis buffer containing a non-denaturing zwitterionic detergent, most efficient for extracting cytosolic proteins. Method II involves a denaturing anionic detergent allowing total disruption of the membranes and capable of extracting both membrane (hydrophobic) and non-membrane (water-soluble, hydrophilic) proteins. The big advantage of the methods is to use general laboratory chemicals to make powerful extraction buffers, resulting in high quality and quantity of proteins. The methods probably are usable for any other archaea or microbial cells, and takes about 14-22 h. Following extraction and further protein digestion, 1D-nano Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MSMS) analysis with Triple TOF 5600 and Orbitrap technologies were used for protein identification and further quantification.

ABSTRACT Strain 768-28 was isolated from a hot spring in Kamchatka, Russia, and represents a novel member of the Vulcanisaeta genus. The complete genome sequence of this thermoacidophilic anaerobic crenarchaeon reveals genes for protein and carbohydrate-active enzymes, the Embden-Meyerhof and Entner-Doudoroff pathways for glucose metabolism, the tricarboxylic acid cycle, beta-oxidation of fatty acids, and sulfate reduction.

Two novel thermophilic and slightly acidophilic strains, Kam940Tand Kam1507b, which shared 99 % 16S rRNA gene sequence identity, were isolated from terrestrial hot springs of the Uzon caldera on the Kamchatka peninsula. Cells of both strains were non-motile, regular cocci. Growth was observed between 55 and 85 °C, with an optimum at 65–70 °C (doubling time, 6.1 h), and at pH 4.5–7.5, with optimum growth at pH 5.5–6.0. The isolates were strictly anaerobic organotrophs and grew on a narrow spectrum of energy-rich substrates, such as beef extract, gelatin, peptone, pyruvate, sucrose and yeast extract, with yields above 107cells ml−1. Sulfate, sulfite, thiosulfate and nitrate added as potential electron acceptors did not stimulate growth when tested with peptone. H2at 100 % in the gas phase inhibited growth on peptone. Glycerol dibiphytanyl glycerol tetraethers (GDGTs) with zero to four cyclopentyl rings were present in the lipid fraction of isolate Kam940T. The G+C content of the genomic DNA of strain Kam940Twas 37 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolates were archaea of the phylumCrenarchaeota, only distantly related to the cultured members of the classThermoprotei(no more than 89 % identity), and formed an independent lineage adjacent to the ordersDesulfurococcalesandAcidilobalesand clustering only with uncultured clones from hot springs of Yellowstone National Park and Iceland as the closest relatives. On the basis of their phylogenetic position and novel phenotypic features, isolates Kam940Tand Kam1507b are proposed to be assigned to a new genus and species,Fervidicoccus fontisgen. nov., sp. nov. The type strain ofFervidicoccus fontisis strain Kam940T(=DSM 19380T=VKM B-2539T). The phylogenetic data as well as phenotypic properties suggest that the novel crenarchaeotes form the basis of a new family,Fervidicoccaceaefam. nov., and order,Fervidicoccalesord. nov., within the classThermoprotei.

A novel anaerobic, thermophilic, alkalitolerant bacterium, strain 2204T, was isolated from a hot spring of the Baikal Lake region. The cells of strain 2204Twere straight rods of variable length, Gram-positive with an S-layer, motile with one to two lateral flagella, and often formed aggregates of 3–15 cells. The isolate was shown to be an obligate anaerobe oxidizing CO and producing equimolar quantities of H2and CO2according to the equation CO+H2O→CO2+H2. No organic substrates were used as energy sources. For lithotrophic growth on CO, 0·2 g acetate or yeast extract l−1was required but did not support growth in the absence of CO. Growth was observed in the temperature range 37–68 °C, the optimum being 55 °C. The pH range for growth was 6·7–9·5, the optimum pH being 8·0. The generation time under optimal conditions was 1·3 h. The DNA G+C content was 45 mol%. Penicillin, erythromycin, streptomycin, rifampicin, vancomycin and tetracycline completely inhibited both growth and CO utilization by strain 2204T. Thus, isolate 2204Twas found to be the first known moderately thermophilic and alkalitolerant H2-producing anaerobic carboxydotroph. The novel bacterium fell within the cluster of the familyPeptococcaceaewithin the low-G+C-content Gram-positive bacteria, where it formed a separate branch. On the basis of morphological, physiological and phylogenetic features, strain 2204Tshould be assigned to a novel genus and species, for which the nameThermincola carboxydiphilagen. nov., sp. nov. is proposed. The type strain is strain 2204T(=DSM 17129T=VKM B-2283T=JCM 13258T).