DNA-DNA relatedness of all validly described Thermoactinomyces species was determined to infer the genetic relationships between them. The levels of DNA-DNA relatedness among the type strains of Thermoactinomyces species ranged from 2-5 to 92.8%. Based on DNA relatedness data, the type strains of Thermoactinomyces intermedius, Thermoactinomyces putidus, Thermoactinomyces dichotomicus and Thermoactinomyces peptonophilus were considered to be distinct species of the genus Thermoactinomyces. However, the relationship between the type strains of Thermoactinomyces vulgaris and Thermoactinomyces candidus and the relationship between the type strains of Thermoactinomyces sacchari and Thermoactinomyces thalpophilus were reevaluated from levels of DNA-DNA relatedness. The independent DNA relatedness values between Thermoactinomyces vulgaris KCTC 9076T and Thermoactinomyces candidus KCTC 9557T were 90.8 and 92.8%. Thermoactinomyces thalpophilus KCTC 9789T and Thermoactinomyces sacchari KCTC 9790T exhibited independent values of 85.6 and 87.3%. Accordingly, on the basis of DNA-DNA relatedness data together with 16S rDNA sequence data determined recently, it is proposed that Thermoactinomyces candidus should be considered as a synonym of Thermoactinomyces vulgaris and Thermoactinomyces thalpophilus be considered as a synonym of Thermoactinomyces sacchari.

Phylogenetic analysis based on 16S rRNA gene sequences revealed that Thermoactinomyces species with validly published names can be assigned to four clusters or lineages. The type strains of Thermoactinomyces sacchari and Thermoactinomyces putidus were differentiated from the type strains of Thermoactinomyces vulgaris and Thermoactinomyces intermedius by the predominant menaquinone and fatty acid profiles. The type strains of Thermoactinomyces dichotomicus and Thermoactinomyces peptonophilus formed lines of descent distinct from other Thermoactinomyces species. Thermoactinomyces dichotomicus KCTC 3667T was distinguishable from the type strains of Thermoactinomyces vulgaris and Thermoactinomyces intermedius by the contents of two fatty acids, iso-C(16 : 0) and iso-C(17 : 0). Thermoactinomyces dichotomicus could be distinguished from other Thermoactinomyces species by DNA G+C content and some phenotypic properties, particularly its property of forming a yellow colour. The type strain of Thermoactinomyces peptonophilus was distinguishable from other Thermoactinomyces species by differences in menaquinone profile, major fatty acids, DNA G+C content and some physiological properties including optimal growth temperature. On the basis of these data, the creation of three new genera, Laceyella, Thermoflavimicrobium and Seinonella, is proposed in addition to the genus Thermoactinomyces sensu stricto. The genus Laceyella gen. nov. is proposed to accommodate Thermoactinomyces sacchari and Thermoactinomyces putidus as Laceyella sacchari comb. nov. and Laceyella putida comb. nov., the genus Thermoflavimicrobium gen. nov. is proposed for Thermoactinomyces dichotomicus as Thermoflavimicrobium dichotomicum comb. nov. and the genus Seinonella gen. nov. is proposed for Thermoactinomyces peptonophilus as Seinonella peptonophila comb. nov.