(Using colony direct PCR we established, kan gene (GenBank accession No. AB028210) coding for an aminoglycoside acetyltransferase, AAC(3), was surveyed among strains of Streptomyces griseus and its related taxa (S. anulatus group). The regulatory and ORF regions of kan gene were targeted by the use of original primers. With the regulatory region primers, the target fragment (518 bp) amplification was observed in all the streptomycin (SM)-producing strains of S. griseus and in several strains of S. anulatus group. The other target fragment (930 bp) amplification using the ORF primers was observed only in the SM-producing strains of S. griseus including ‘S. ornatus’ that has substantially identical properties to those of S. griseus. It was thus conclusive that kan gene is highly conserved in SM-producing strains of S. griseus and distributed in taxa related to S. griseus. There were numbers of strains that provided no amplification upon PCR but hybridization bands upon Southern hybridization, suggesting the lack or alteration of kan gene. Characterization by colony direct PCR using appropriate primers will be a big help for taxonomic as well as ecological characterization of actinomycetes.

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Forty-one Actinokineospora isolates from soils and decaying leaves, together with 14 marker strains of Actinokineospora and related genera in the family Actinosynnemataceae, were subjected to a numerical phenetic analysis. Strains were examined using 125 unit characters, and phenetic relationships were determined by the simple matching (SSM) coefficient, with clustering by the unweighted pair group average algorithm. Four major, seven minor and 16 single-member clusters, defined by the SSM coefficient at the 80% similarity (S-) level, were assigned to three cluster-groups, distinguished at the 64% S-level. The first group included all the environmental isolates and marker strains of Actinokineospora and the second group was composed of Saccharothrix and Streptoalloteichus strains. Actinosynnema, Lechevalieria and Lentzea strains constituted the third group. Type strains of all the recognized Actinokineospora species were well-separated from one another, as they fell into different clusters. Eighteen, or 44 percent, of the 41 environmental isolates were placed in the four well-circumscribed major clusters, of which two corresponded to the validated species Actinokineospora globicatena and A. terrae, respectively. These numerical, phenetic data support the integrity of the Actinokineospora genus and its validated species, and suggest that the remaining two major clusters, encompassing only environmental isolates, merit recognition as new taxospecies. Chemotaxonomic and 16S rDNA sequence analyses, as well as DNA-DNA hybridization studies, indicate that these taxa belong to two new species of Actinokineospora. It is proposed that the two novel taxa be assigned to the genus Actinokineospora as A. enzanensis (type strain, YU 924-101T=IFO 16517T) and A. auranticolor sp. nov. (type strain, YU 961-1T=IFO 16518T). The identifying morphological, physiological and chemotaxonomic characters of these new species are described.

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