Selective isolation and rapid identification of members of the genus Micromonospora

Abstract

Improved methods for selective isolation of diverse actinomycetes of the genus Micromonospora and a genus-specific nested PCR for rapid identification of putative Micromonospora isolates were developed. The robustness of both the isolation and the identification approach was underpinned by phylogenetic analysis based on 16S rRNA gene sequences.

FIG. 1.

Average numbers of colonies (10⁴ CFU/g dry weight composite soil) of nonactinomycete bacteria (grey bars), Micromonospora-like actinomycetes (hatched bars), and other actinomycetes (striped bars) recorded on different selective plates seeded with all seven composite samples processed by different pretreatments. Error bars show standard deviations.

FIG. 2.

Representative nucleotide sequence alignments with Micromonospora genus-specific primers M558F and C1028R targeting the 16S rRNA gene and examples of mismatches outside the genus, demonstrating the specificity of the primer pair.

FIG. 3.

Neighbor-joining tree based on 16S rRNA gene sequences of 42 representative Micromonospora isolates and 33 type strains of the genus showing phylogenetic relationships between the isolates and Micromonospora species. The isolates are shown in boldface, and GenBank accession numbers are given in parentheses. Undescribed phylotypes are marked with vertical lines on the right of the sequence items. Percentage bootstrap values based on 1,000 resampled data sets are shown at the nodes; only values above 50% are given. The scale bar indicates 0.01 nucleotide substitution per nucleotide position.