Transcriptome-Stable Isotope Probing Provides Targeted Functional and Taxonomic Insights Into Microaerobic Pollutant-Degrading Aquifer Microbiota.

While most studies using RNA-stable isotope probing (SIP) to date have focused on ribosomal RNA, the detection of 13 C-labeled mRNA has rarely been demonstrated. This approach could alleviate some of the major caveats of current non-target environmental "omics." Here, we demonstrate the feasibility of total RNA-SIP in an experiment where hydrocarbon-degrading microbes from a BTEX-contaminated aquifer were studied in microcosms with 13 C-labeled toluene under microoxic conditions. From the total sequencing reads (∼30 mio. reads per density-resolved RNA fraction), an average of 1.2% of reads per sample were identified as non-rRNA, including mRNA. Members of the Rhodocyclaceae (including those related to Quatrionicoccus spp.) were most abundant and enriched in 13 C-rRNA, while well-known aerobic degraders such as Pseudomonas spp. remained unlabeled. Transcripts related to cell motility, secondary metabolite formation and xenobiotics degradation were highly labeled with 13 C. mRNA of phenol hydroxylase genes were highly labeled and abundant, while other transcripts of toluene-activation were not detected. Clear labeling of catechol 2,3-dioxygenase transcripts supported previous findings that some of these extradiol dioxygenases were adapted to low oxygen concentrations. We introduce a novel combination of total RNA-SIP with calculation of transcript-specific enrichment factors (EFs) in 13 C-RNA, enabling a targeted approach to process-relevant gene expression in complex microbiomes.