Activity-resolved microbial community profiling using rpoB gene and transcript sequencing

Deciphering which members of a microbial community are biologically active remains a profound challenge in microbial ecology. While the 16S rRNA gene serves as the primary marker for bacterial community examination, it fails to definitively differentiate active cells from those in dormant or deceased states. Consequently, supplementary phylogenetic markers with transcript abundance patterns closely mirroring cellular activity are imperative.

In this study, researchers meticulously examined 80 bacterial protein-coding marker genes and identified rpoB, the gene encoding the beta subunit of bacterial RNA polymerase, as the most suitable candidate. A novel primer pair (1528F 2041R) was crafted from a meticulously curated database of 305,274 unique rpoB sequences and subsequently validated for quantitative PCR and amplicon sequencing of DNA and RNA templates.

The rpoB qPCR assay exhibited a detection limit two orders of magnitude lower than the established 16S rRNA assay, rendering the latter's limit of detection inconclusive due to the absence of a template control. Across soil and sediment communities, rpoB successfully replicated community composition akin to 16S rRNA yet provided a quantitative activity signal, as evidenced by rpoB cDNA:DNA ratios correlating significantly with taxon-level transcript abundance (R squared values ranging from 0.22 to 0.29, p values less than 0.5).

Furthermore, in a biological activated carbon biofilter experiment, rpoB transcript abundance mirrored the decline in dissolved organic carbon removal rates over a 72-hour period (correlation coefficients ranging from 0.84 to 0.99), whereas 16S rRNA transcripts proved inconsequential (correlation coefficients ranging from -0.4 to 0.98).

These findings firmly establish rpoB as a quantitatively robust, activity-responsive adjunct to 16S rRNA, enabling a more accurate linkage between community composition and ecosystem processes.