Thermoflexus hugenholtzii gen. nov., sp. nov., a thermophilic, microaerophilic, filamentous bacterium representing a novel class in the Chloroflexi, Thermoflexia classis nov., and description of Thermoflexaceae fam. nov. and Thermoflexales ord. nov. , a member of an as-yet uncultivated lineage that is present and sometimes abundant in some hot springs worldwide, was isolated from sediment of Great Boiling Spring in Nevada, USA. Cells had a mean diameter of 0.3 mm and length of 4.0 mm, and formed filaments that typically ranged in length from 20 to 200 mm. Filaments were negative for the Gram stain reaction, spores were not formed and motility was not observed. The optimum temperature for growth was 72.5-75 6C, with a range of 67.5-75 6C, and the optimum pH for growth was 6.75, with a range of pH 6.5-7.75. Peptone, tryptone or yeast extract were able to support growth when supplemented with vitamins, but no growth was observed using a variety of defined organic substrates. Strain JAD2 T was microaerophilic and facultatively anaerobic, with optimal growth at 1 % (v/v) O 2 and an upper limit of 8 % O 2 . The major cellular fatty acids (.5 %) were C 16 : 0 , C 19 : 0 , C 18 : 0 , C 20 : 0 and C 19 : 1 . The genomic DNA G+C content was 69.3 mol%. Phylogenetic and phylogenomic analyses using sequences of the 16S rRNA gene and other conserved genes placed JAD2 T within the phylumChloroflexi, but not within any existing class in this phylum. These results indicate that strain JAD2 T is the first cultivated representative of a novel lineage within the phylum Chloroflexi, for which we propose the name Thermoflexus hugenholtzii gen. nov., sp. nov., within Thermoflexia classis nov., Thermoflexales ord. nov. and Thermoflexaceae fam. nov. The type strain of Thermoflexus hugenholtzii is JAD2 T (5JCM 19131 T 5CCTCC AB-2014030 T ).The GenBank/EMBL/DDBJ accession numbers for the sequences of the 16S rRNA gene and the dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplK, rplL, rplS, rplT, rpmA, rpoB, rpsB, rpsI, smpB and galE genes of strain JAD2
