Novel hyperthermophilic crenarchaeon Infirmifilum lucidum gen. nov. sp. nov., reclassification of Thermofilum uzonense as Infirmifilum uzonense comb. nov. and assignment of the family Thermofilaceae to the order Thermofilales ord. nov.

A novel hyperthermophilic crenarchaeon, strain 3507LTT, was isolated from a terrestrial hot spring near Tinguiririca volcano, Chile. Cells were non-motile thin, slightly curved filamentous rods. It grew at 73-93 °C and pH range of 5 to 7.5 with an optimum at 85 °C and pH 6.0-6.7. The presence of culture broth filtrate of another hyperthemophilic archaeon as well as yeast extract was obligatory for growth of the novel isolate. Strain 3507LTT is an anaerobic chemoorganoheterotroph, fermenting monosaccharides, disaccharides and polysaccharides (lichenan, starch, xanthan gum, xyloglucan, alpha-cellulose and amorphous cellulose). No growth stimulation was detected when nitrate, thiosulfate, selenate or elemental sulfur were added as the electron acceptors. The complete genome of strain 3507LTT consisted of a single circular chromosome with size of 1.63 Mbp. The DNA G+C content was 53.9%. According to the 16S rRNA gene sequence as well as conserved protein sequences phylogenetic analyses, strain 3507LTT together with Thermofilum uzonense formed a separate cluster within a Thermofilaceae family (Thermoproteales/Thermoprotei/Crenarchaeota). Based on phenotypic characteristics, phylogeny as well as AAI comparisons, a novel genus and species Infirmifilum lucidum strain 3507LTT (=VKM B-3376T = KCTC 15797T) gen. nov. sp. nov. was proposed. Its closest relative, Thermofilum uzonense strain 1807-2T should be reclassified as Infirmifilum uzonense strain 1807-2T comb. nov. Finally, based on phylogenomic and comparative genome analyses of representatives of Thermofilaceae family and other representatives of Thermoproteales order, a proposal of transfer of the family Thermofilaceae into a separate order Thermofilales ord. nov. was made.

Properties of an amylase from thermophilic Bacillus SP

Alpha-Amylase production by thermophilic Bacillus sp strain SMIA-2 cultivated in liquid cultures containing soluble starch as a carbon source and supplemented with 0.05 percent whey protein and 0.2 percent peptone reached a maximum activity at 32 h, with levels of 37 U/mL. Studies on the amylase characterization revealed that the optimum temperature of this enzyme was 90ºC. The enzyme was stable for 1 h at temperatures ranging from 40-50ºC while at 90ºC, 66 percent of its maximum activity was lost. However, in the presence of 5 mM CaCl2, the enzyme was stable at 90ºC for 30 min and retained about 58 percent residual activity after 1 h. The optimum pH of the enzyme was found to be 8.5. After incubation of enzyme for 2 h at pH 9.5 and 11.0 was observed a decrease of about 6.3 percent and 16.5 percent of its original activity. At pH 6.0 the enzyme lost about 36 percent of its original activity. The enzyme was strongly inhibited by Co2+, Cu2+ and Ba2+, but less affected by Mg2+, Na+ and K+. In the presence of 2.0 M NaCl, 63 percent of amylase activity was retained after 2 h incubation at 45ºC. The amylase exhibited more than 70 percent activity when incubated for 1 h at 50ºC with sodium dodecyl sulphate. However, very little residual activity was obtained with sodium hypochlorite and with hydrogen peroxide the enzyme was completely inhibited. The compatibility of Bacillus sp SMIA-2 amylase with certain commercial detergents was shown to be good as the enzyme retained 86 percent, 85 percent and 75 percent of its activity after 20 min incubation at 50ºC in the presence of the detergent brands Omo®, Campeiro® and Tide®, respectively.

A produção de alfa-amilase por um termofilico, Bacillus sp SMIA-2, cultivado em meio l¨ªquido contendo amido sol¨²vel como fonte de carbono, alcançou uma atividade m¨¢xima de 37 U/mL em 32 horas. Estudos sobre a caracterização da amilase revelaram que a temperatura ¨®tima desta enzima foi 90ºC. A enzima foi est¨¢vel por 1 hora a temperaturas de 40 e 50ºC enquanto a 90ºC, 66 por cento da atividade m¨¢xima foi perdida. Entretanto, na presença de 5 mM de CaClð2, a enzima foi est¨¢vel a 90ºC por 30 minutos e manteve cerca de 58 por cento de sua atividade residual por 1 hora. O pH ¨®timo da enzima encontrado foi de 8.5. Ap¨®s a incubação da enzima por 2 horas a pH 9.5 e 11.0 foi observado um decr¨¦scimo de aproximadamente 6.3 por cento e 16.5 por cento da atividade original. Em pH 6.0 a enzima perdeu cerca de 36 por cento de sua atividade original. A enzima foi fortemente inibida por Co2+, Cu2+, e Ba2+, por¨¦m pouco afetada por Mg2+, Na+ e K+. Na presença de 2.0 M de NaCl, 63 por cento da atividade da amilase foi mantida ap¨®s 2 horas de incubação a temperatura de 45ºC. A amilase exibiu atividade acima de 70 por cento quando incubada por 1 hora a 50ºC em presença de s¨®dio dodecil sufato (SDS). Entretanto, uma baixa atividade residual foi obtida quando na presença do hipoclorito de s¨®dio e uma completa inibição quando a enzima foi incubada em per¨®xido de hidrog¨ºnio. A compatibilidade da amilase produzida pelo Bacillus sp SMIA-2, em relação a alguns detergentes comerciais mostrou que a enzima manteve 86 por cento, 85 por cento, e 75 por cento da atividade ap¨®s 20 minutos de incubação a 50ºC na presença dos detergentes Omo®, Campeiro® e Tide®, respectivamente.

Genome sequence of Thermofilum pendens reveals an exceptional loss of biosynthetic pathways without genome reduction.

We report the complete genome of Thermofilum pendens, a deeply branching, hyperthermophilic member of the order Thermoproteales in the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact, T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features that are common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known previously to utilize peptides as an energy source, but the genome revealed a substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may obtain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogen lyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time that this enzyme has been found outside the Methanosarcinales, and the presence of a presenilin-related protein. The predicted highly expressed proteins do not include proteins encoded by housekeeping genes and instead include ABC transporters for carbohydrates and peptides and clustered regularly interspaced short palindromic repeat-associated proteins.

Microbial life in Champagne Pool, a geothermal spring in Waiotapu, New Zealand.

Surveys of Champagne Pool, one of New Zealand's largest terrestrial hot springs and rich in arsenic ions and compounds, have been restricted to geological and geochemical descriptions, and a few microbiological studies applying culture-independent methods. In the current investigation, a combination of culture and culture-independent approaches were chosen to determine microbial density and diversity in Champagne Pool. Recovered total DNA and adenosine 5'-triphosphate (ATP) content of spring water revealed relatively low values compared to other geothermal springs within New Zealand and are in good agreement with low cell numbers of 5.6 +/- 0.5 x 10(6) cells/ml obtained for Champagne Pool water samples by 4',6-diamidino-2-phenylindole (DAPI) staining. Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA (small-subunit ribosomal nucleic acid) gene clone library analyses of environmental DNA indicated the abundance of Sulfurihydrogenibium, Sulfolobus, and Thermofilum-like populations in Champagne Pool. From these results, media were selected to target the enrichment of hydrogen-oxidizing and sulfur-dependent microorganisms. Three isolates were successfully obtained having 16S rRNA gene sequences with similarities of approximately 98% to Thermoanaerobacter tengcongensis, 94% to Sulfurihydrogenibium azorense, and 99% to Thermococcus waiotapuensis, respectively.