Integration of absolute multi-omics reveals dynamic protein-to-RNA ratios and metabolic interplay within mixed-domain microbiomes.

While the field of microbiology has adapted to the study of complex microbiomes via modern meta-omics techniques, we have not updated our basic knowledge regarding the quantitative levels of DNA, RNA and protein molecules within a microbial cell, which ultimately control cellular function. Here we report the temporal measurements of absolute RNA and protein levels per gene within a mixed bacterial-archaeal consortium. Our analysis of this data reveals an absolute protein-to-RNA ratio of 102-104 for bacterial populations and 103-105 for an archaeon, which is more comparable to Eukaryotic representatives' humans and yeast. Furthermore, we use the linearity between the metaproteome and metatranscriptome over time to identify core functional guilds, hence using a fundamental biological feature (i.e., RNA/protein levels) to highlight phenotypical complementarity. Our findings show that upgrading multi-omic toolkits with traditional absolute measurements unlocks the scaling of core biological questions to dynamic and complex microbiomes, creating a deeper insight into inter-organismal relationships that drive the greater community function.

16S rDNA directed PCR primers and detection of methanogens in the bovine rumen.

AIMS: To assess the diversity of ruminal methanogens in a grazing cow, and develop PCR primers targeting the predominant methanogens. METHODS AND RESULTS: DNA was extracted from rumen contents collected from a cow grazing pasture. Archaeal 16S rRNA genes were amplified by PCR using two pairs of archaea-specific primers, and clone libraries prepared. Selected clones were sequenced. Phylogenetic analysis revealed that for one primer pair, most sequences clustered with Methanobrevibacter spp. whereas with the other primer pair most clustered with Methanosphaera stadtmanae. One sequence belonged to the Crenarcheota. PCR primers were designed to detect Msp. stadtmanae and differentiate between Mbb. ruminantium and Mbb. smithii and successfully tested. CONCLUSIONS: The ruminal methanogens included Mbb. ruminantium, Mbb. smithii, Mbb. thaueri and methanogens similar to Msp.stadtmanae. The study showed that apparent methanogen diversity can be affected by selectivity from the archaea-specific primers used to create clone libraries. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed a greater diversity of ruminal methanogens in grazing cows than previously recognized. It also shows the need for care in interpreting methanogen diversity using PCR-based analyses. The new PCR primers will enable more information to be obtained on Msp. stadtmanae and Methanobrevibacter spp. in the rumen.