The use of molecular beacons to directly measure bacterial mRNA abundances and transcript degradation

• Published in: Journal of microbiological methods 2009-02, Vol.76 (2), p.146-151
• Main Authors: Kuechenmeister, Lisa J., Anderson, Kelsi L., Morrison, John M., Dunman, Paul M.
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - analysis; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; DNA Probes; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Genetics; Gram positive; Microbiology; Molecular beacon; Molecular Probe Techniques; mRNA abundance; mRNA degradation; Multidrug Resistance-Associated Proteins - analysis; Multidrug Resistance-Associated Proteins - genetics; Multidrug Resistance-Associated Proteins - metabolism; Reverse Transcription; RNA Stability - physiology; RNA, Messenger - analysis; RNA, Messenger - metabolism; Sensitivity and Specificity; Staphylococcal Protein A - analysis; Staphylococcal Protein A - genetics; Staphylococcal Protein A - metabolism; Staphylococcus aureus; Staphylococcus aureus - genetics; Virulence Factors - analysis; Virulence Factors - genetics; Virulence Factors - metabolism
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2008.10.004

The regulation of mRNA turnover is a dynamic means by which bacteria regulate gene expression. Although current methodologies allow characterization of the stability of individual transcripts, procedures designed to measure alterations in transcript abundance/turnover on a high throughput scale are lacking. In the current report, we describe the development of a rapid and simplified molecular beacon-based procedure to directly measure the mRNA abundances and mRNA degradation properties of well-characterized Staphylococcus aureus pathogenicity factors. This method does not require any PCR-based amplification, can monitor the abundances of multiple transcripts within a single RNA sample, and was successfully implemented into a high throughput screen of transposon mutant library members to detect isolates with altered mRNA turnover properties. It is expected that the described methodology will provide great utility in characterizing components of bacterial RNA degradation processes and can be used to directly measure the mRNA levels of virtually any bacterial transcript.