mRNA-targeted fluorescent in situ hybridization (FISH) of Gram-negative bacteria without template amplification or tyramide signal amplification.
Technologies are needed to study gene expression at the level of individual cells within a population or microbial community. Fluorescent in situ hybridization (FISH) supplies high-resolution spatial information and has been widely applied to study microbial communities at the rRNA level. While mRNA-targeted FISH has been popular for studying gene expression in eukaryotic cells, very little success has been achieved with prokaryotes. At present, detection of specific mRNAs in individual prokaryotic cells requires the use of in situ RT-PCR or tyramide signal amplification (TSA). In this study we used DNA oligonucleotide probes labeled with a single near-infrared dye in FISH assays to detect multi-copy plasmid-based and endogenous mRNA molecules in Escherichia coli and Shewanella oneidensis MR-1. We took advantage of the fact that there is much less background signal produced by biological materials and support matrices in the near-infrared spectrum and thus long camera exposure times could be used. In addition, we demonstrate that a combination of probes targeting both rRNA and mRNA could be successfully employed within the same FISH assay. These results, as well as ongoing R&D improvements in NIR and infrared dyes, indicate that the FISH approach we demonstrated could be applied in certain environmental settings to monitor gene expression in mixed populations.
Coleman
James R
JR
Biological Sciences Division-Microbiology Group, 902 Battelle Blvd., Pacific Northwest National Laboratory, PO Box 999/MS P7-50, Richland, WA 99354, USA.
Culley
David E
DE
Chrisler
William B
WB
Brockman
Fred J
FJ
D015342:DNA Probes; D015870:Gene Expression; D006090:Gram-Negative Bacteria / Q000201:enzymology / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D014439:Tyramine / Q000378:metabolism*
D015342:DNA Probes; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012333:RNA, Messenger; D014439:Tyramine