Rapid characterization by colony direct PCR of distribution specificity in Streptomyces of kan gene encoding a specific aminoglycoside-3-N-acetyltransferase.

Abstract

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.

A Comparative Study of Malaysian and Japanese Actinomycetes Using a Simple Identification Method Based on Partial 16S rDNA Sequence

“…However, for the treatment of many isolates, DNA sequence analysis is costly in terms of money, time and effort. Recently, Tsuchizaki et al have reported that colony direct PCR may also be applied to actinomycetes 8) . Independently, we have devised direct PCR method using a culture broth.…”

Development of a simple-identification method for actinomycetes based on partial 16S rDNA sequences as exemplified by a comparative study of Malaysian and Japanese actinomycetes

“…It may be said that this method is in concept similar to DNA barcoding. In previous reports of direct PCR amplification of Streptomyces DNA, intact mycelia grown on the agar media were used as the DNA template source (Ishikawa et al, 2000;Tsuchizaki et al, 2001). We employed liquid cultures in 96-well format plates as templates.…”