The use of molecular beacons to directly measure bacterial mRNA abundances and transcript degradation.
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.
Kuechenmeister
Lisa J
LJ
University of Nebraska Medical Center, Omaha, NE 68198-6495, USA.
Anderson
Kelsi L
KL
Morrison
John M
JM
Dunman
Paul M
PM
D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015342:DNA Probes; D005456:Fluorescent Dyes; D015964:Gene Expression Regulation, Bacterial; D015336:Molecular Probe Techniques; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D020871:RNA Stability / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism*; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics*; D037521:Virulence Factors / Q000032:analysis / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D015342:DNA Probes; D005456:Fluorescent Dyes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012333:RNA, Messenger; D013205:Staphylococcal Protein A; D037521:Virulence Factors; C075040:NorA protein, Staphylococcus