![]() For DNA extraction, the strain was enriched at 80☌ in M236 medium, using a serum bottle. ![]() 236 (M236) (containing per liter salt base solution: 2.94 g trisodium citrate dihydrate, 0.5 g yeast extract, 10.0 ml trace vitamins, 1.0 mg resazurin, 0.5 g Na 2S♹H 2O, and 20 mM thiosulfate). thermophila CBA1501 T from the solfataric soil of the Mayon volcano in the Republic of the Philippines ( Yim et al., 2015) and cultivated it on modified JCM medium no. Materials and Methods Culture Conditions and DNA Extraction thermophila CBA1501 T has been reported and information of hyperthermophilic enzymes of high biotechnological value has been provided. thermophila CBA1501 T (= ATCC BAA-2415 T = JCM 17228 T) was isolated from solfataric soil in the Republic of the Philippines ( Yim et al., 2015). These enzymes can be studied using model systems to elucidate enzyme mechanisms and evolution of proteins stable at high temperatures and to determine the higher temperature limit for enzyme stability ( Vieille and Zeikus, 2001). Hyperthermophilic enzymes are stable and active at high temperatures of >70☌ ( Vieille et al., 1996). distributa and “ Vulcanisaeta moutnovskia” ( Mavromatis et al., 2010 Gumerov et al., 2011), have been reported for the genus Vulcanisaeta, as per the NCBI genome database ( ). To date, 15 genomes, including two complete genomes, V. Members of the genus Vulcanisaeta are rod-shaped, anaerobic, hyperthermophilic, and acidophilic ( Itoh et al., 2002). thermophila ( Yim et al., 2015), as per the List of Prokaryotic Names with Standing in Nomenclature database ( Parte, 2014). It currently includes 3 validly named species, that is, Vulcanisaeta distributa ( Itoh et al., 2002), V. The genus Vulcanisaeta belongs to the family Thermoproteaceae, order Thermoproteales, phylum Crenarchaeota, and was first proposed by Itoh et al. Binding to mutated probes could also be efficiently competed by adding a non-labeled WT-probe.Hyperthermophilic archaea have been isolated from high-temperature environments such as geothermally heated soils, sulfur-rich hot springs, and submarine volcanic habitats optimal growth of these organisms occurs above 80☌ ( Stetter, 1999, 2006, 2013). Probes mut-m1 and mut-m2 harboring mutations in motifs 1 and 2 are also bound by Ros1WOPR-His, but the interaction results in a less pronounced shift than observed for the WT-probe and an even smaller shift when both motifs are mutated (mut-m1+2). Ros1WOPR-His did not bind a probe of the same length corresponding to a part of the ros1 coding sequence (ORF-probe). When incubated with Ros1WOPR-His, the WT-probe was shifted and this could be competed by addition of a non-labeled WT-probe (competitor). coli was used in EMSA assays with the probe shown in B (WT-probe). Ros1WOPR-His expressed and purified from E. (C) In vitro binding of Ros1WOPR-His to the ros1 promoter. Putative binding sites (m1, m2 and m3) for Ros1 are boxed and mutations introduced in the respective sites are indicated (mut-m1, mut-m2). (B) Sequence of the probe fragment used for EMSA assays. The location of the fragment (WT-probe) used as a probe for EMSA is indicated by a black arrow. Peaks with significant peak shape scores are indicated in the ChIP peaks lane by dark red arrows. ![]() (A) The graph generated with the CLC Genomics Workbench 7.5 software (CLC bio) shows the ChIP-seq read distribution in the genomic region containing ros1 in output DNA from the sample where maize was infected with FB1Δros1-Ros1HA x FB2Δros1-Ros1HA (Output Ros1HA) and in the control sample where the infection was done with FB1Δros1-Ros1 x FB2Δros1-Ros1 (output Ros1) Open reading frames are represented by yellow arrows. The WOPR Protein Ros1 Is a Master Regulator of Sporogenesis and Late Effector Gene Expression in the Maize Pathogen Ustilago maydis Fig 9
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