Summary auto-generated
This study evaluated methods for discriminating between Streptomyces albidoflavus strains, which are important producers of antifungal antibiotics. Researchers analyzed 30 Streptomyces strains using 16S ribosomal DNA (rDNA) sequencing and examination of 16S-23S rDNA intergenic spacer regions. While 16S rDNA sequence analysis showed 100% similarity among 27 strains, including the S. albidoflavus type strain, this method proved insufficient for strain-level discrimination. However, analysis of the 16S-23S rDNA intergenic spacer regions revealed significant variation in size and sequence composition both between and within strains. Using high-resolution polyacrylamide gel electrophoresis with fluorescently labeled PCR products and GeneScan software, researchers identified 19 distinct band patterns among the 27 strains with identical 16S sequences. The spacer region contains six highly conserved regions and five variable regions with multiple sequence types. This approach successfully discriminated between strains that were indistinguishable by 16S rDNA analysis, establishing a rapid and reproducible method for strain-level identification applicable to Streptomyces species and potentially other bacteria.
Key findings
- 16S rDNA sequencing alone cannot discriminate among S. albidoflavus strains, as 27 of 30 examined strains showed identical sequences
- The 16S-23S rDNA intergenic spacer region exhibits extensive length and sequence polymorphism both within and between strains, making it suitable for strain discrimination
- High-resolution polyacrylamide gel electrophoresis combined with GeneScan software successfully identified 19 distinct band patterns, providing a rapid and highly reproducible method for strain typing
- Individual spacer regions contain six conserved and five variable regions, with multiple sequence types present within single strains
- This approach is applicable beyond Streptomyces for strain-level identification across bacterial taxa
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Abstract
In an attempt to develop a rapid and accurate method for discrimination of streptomycetes at the strain level, 21 strains identified by fatty acid analysis as Streptomyces albidoflavus and the type strains of nine subjective synonyms of S. albidoflavus were selected for a full or partial 16S ribosomal DNA (rDNA) sequence analysis and an investigation of the 16S-23S rDNA intergenic spacer region. 16S rDNA sequence analysis showed that 27 of the strains exhibited 100% sequence similarity; these strains included the type strain of S. albidoflavus and the type strains of the subjective synonyms Streptomyces canescens, Streptomyces coelicolor, Streptomyces felleus, Streptomyces limosus, Streptomyces odorifer, and Streptomyces sampsonii. The type strains of the other subjective synonyms of S. albidoflavus (i.e., Streptomyces gougerotii, Streptomyces intermedius, and Streptomyces rutgersensis) were found to have levels of 16S rDNA sequence difference of 1.0 to 1.1% when they were compared to the type strain of S. albidoflavus. In order to discriminate between the strains which had identical 16S rDNA sequences, the 16S-23S rDNA intergenic spacer regions were amplified and cloned, and the sequences of the spacer regions were determined for four S. albidoflavus strains, including the type strain. The 16S-23S rDNA intergenic spacer region was found to vary in length and sequence composition among the strains and within each strain. The sizes and numbers of 16S-23S rDNA intergenic spacer regions for the 27 strains with identical 16S rDNA sequences were determined by high-resolution electrophoresis of FAM-lbeled PCR products and a subsequent size analysis with GeneScan 672 software. This was shown to be a useful method for discrimination of S. albidoflavus strains, Strains with the same 16S-23S rDNA intergenic spacer band pattern, as determined by high-resolution electrophoresis of FAM-labeled PCR products, could be further discriminated on the basis of the sequence composition of the spacer region.