Genus-specific primers targeting the 16S rRNA gene for PCR detection of members of the genus Verrucosispora

Abstract

Little is known about the genus Verrucosispora though it does contain organisms which produce novel antibiotics. A set of genus-specific oligonucleotide primers was generated to gain an insight into the presence, distribution and taxonomic diversity of members of this genus in diverse samples taken from marine habitats. In silico and pure culture studies showed that the primers matched perfectly with target sequences of the 16S rRNA genes of representatives of the genus Verrucosispora. The primers, designated S-G-Verr-0195-a-S-20 and S-G-Verr-1152-a-A-18, amplified an ≈960 bp stretch of the 16S rRNA genes of Verrucosispora strains but not those of representatives of other genera classified in the family Micromonosporaceae. Genus-specific amplicons were detected from 17 out of 20 community DNA samples prepared from diverse marine sediments and coastal soils. Phylogenetic analysis of over 40% of clones derived from five of the samples indicated they belonged to novel Verrucosispora species. The primers were also used to confirm the identity of Verrucosispora-like strains isolated from two of the environmental samples. The primers can be used to facilitate the isolation of novel Verrucosispora strains by allowing prescreening of environmental samples and the subsequent identification of verrucosisporae on selective isolation plates. For this purpose, a novel medium facilitating the recovery of Verrucosispora strains was formulated and used to recover novel isolates validated using the novel PCR primers. This medium may be useful as the basis for development of a selective medium.