Detection of mRNA in Streptomyces Cells by Whole-Cell Hybridization with Digoxigenin-Labeled Probes

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.

[1]  K. Schleifer,et al.  Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides. , 1991, Journal of General Microbiology.

[2]  A. Riggs,et al.  Genomic sequencing. , 1993, Methods in molecular biology.

[3]  Deshpande Bs,et al.  Biologically active secondary metabolites from Streptomyces , 1988 .

[4]  B. Horváth,et al.  In situ localization of Rhizobium mRNAs in pea root nodules: nifA and nifH localization. , 1991 .

[5]  K. Schleifer,et al.  Identification in situ and phylogeny of uncultured bacterial endosymbionts , 1991, Nature.

[6]  T. Bisseling,et al.  Sequential induction of nodulin gene expression in the developing pea nodule. , 1990, The Plant cell.

[7]  E. Viégas-Péquignot,et al.  Genes occupy a fixed and symmetrical position on sister chromatids , 1991, Cell.

[8]  C. Cremer,et al.  PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization. , 1992, BioTechniques.

[9]  E. Delong,et al.  Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. , 1989, Science.

[10]  William Wisden,et al.  In situ hybridization with oligonucleotides: a simplified method to detect Drosophila transcripts , 1991, Nucleic Acids Res..

[11]  D. Hopwood.  Genetic manipulation in Streptomyces , 1983 .

[12]  C. Kessler,et al.  Non-radioactive labeling of RNA transcripts in vitro with the hapten digoxigenin (DIG); hybridization and ELISA-based detection. , 1990, Nucleic acids research.

[13]  G J Olsen,et al.  Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells , 1988, Journal of bacteriology.

[14]  H. Scherthan,et al.  Chromosomal in situ hybridization with double-labeled DNA: signal amplification at the probe level. , 1992, Cytogenetics and cell genetics.

[15]  B. Horváth,et al.  The ENOD12 gene product is involved in the infection process during the pea-rhizobium interaction , 1990, Cell.

[16]  D A Stahl,et al.  Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology , 1990, Journal of bacteriology.

[17]  T. Bisseling,et al.  Nodulin Gene Expression and ENOD2 Localization in Effective, Nitrogen-Fixing and Ineffective, Bacteria-Free Nodules of Alfalfa. , 1990, The Plant cell.

[18]  C. Thompson,et al.  Biochemical characterization of resistance determinants cloned from antibiotic-producing streptomycetes , 1982, Journal of bacteriology.

[19]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[20]  E. Cundliffe.  How antibiotic-producing organisms avoid suicide. , 1989, Annual review of microbiology.

[21]  K. Schleifer,et al.  Detection of micro-organisms in soil after in situ hybridization with rRNA-targeted, fluorescently labelled oligonucleotides. , 1992, Journal of general microbiology.

[22]  E. Delong,et al.  Phylogenetic characterization and in situ localization of the bacterial symbiont of shipworms (Teredinidae: Bivalvia) by using 16S rRNA sequence analysis and oligodeoxynucleotide probe hybridization , 1991, Applied and environmental microbiology.

[23]  R. Amann,et al.  Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms , 1992, Applied and environmental microbiology.