Detection of mRNA in Streptomyces cells by whole-cell hybridization with digoxigenin-labeled probes

Abstract

Detection of mRNA of the thiostrepton resistance gene (tsr) harbored by plasmid pIJ673 in Streptomyces violacelatus was achieved by whole-cell hybridization with digoxigenin-labeled in vitro transcripts followed by an antibody-alkaline phosphatase detection of the digoxigenin reporter molecule. Prior to hybridization, the cells had to be permeabilized by lysozyme, the detergent Nonidet P-40, and toluene. The permeability of the S. violacelatus cells for probes and the antibody-alkaline phosphatase conjugate was demonstrated by hybridization with digoxigenin-labeled, 16S rRNA-targeted oligonucleotides.