Integration of absolute multi-omics reveals translational and metabolic interplay in mixed-kingdom microbiomes

Microbiology is founded on well-known model organisms. For example, the majority of our fundamental knowledge regarding the quantitative levels of DNA, RNA, and protein backdates to keystone pure culture-based studies. Nowadays, meta-omic approaches allow us to directly access the molecules that constitute microbes and microbial communities, however due to a lack of absolute measurements, many original culture-derived “microbiology statutes” have not been updated or adapted to more complex microbiome settings. Within a cellulose-degrading and methanogenic consortium, we temporally measured genome-centric absolute RNA and protein levels per gene, and obtained a protein-to-RNA ratio of 102-104 for bacterial populations, whereas Archaeal RNA/protein dynamics (103-105: Methanothermobacter thermoautotrophicus) were more comparable to Eukaryotic representatives humans and yeast. The linearity between transcriptome and proteome had a population-specific change over time, highlighting a minimal subset of four functional carriers (cellulose degrader, fermenter, syntrophic acetate-oxidizer and methanogen) that coordinated their respective metabolisms, cumulating in the overarching community phenotype of converting polysaccharides to methane. 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.

Analysis of a hybrid TATA box binding protein originating from mesophilic and thermophilic donor organisms

The TATA Box Binding Protein (TBP) is a 20 kD protein that is essential and universally conserved in eucarya and archaea. Especially among archaea, organisms can be found that live below 0°C as well as organisms that grow above 100°C. The archaeal TBPs show a high sequence identity and a similar structure consisting of α-helices andβ-sheets that are arranged in a saddle-shape 2-symmetric fold. In previous studies, we have characterized the thermal stability of thermophilic and mesophilic archaeal TBPs by infrared spectroscopy and showed the correlation between the transition temperature (Tm) and the optimal growth temperature (OGT) of the respective donor organism. In this study, a “new” mutant TBP has been constructed, produced, purified and analyzed for a deeper understanding of the molecular mechanisms of thermoadaptation. Theβ-sheet part of the mutant consists of the TBP fromMethanothermobacter thermoautotrophicus(OGT 65°C, MtTBP65) whose α-helices have been exchanged by those ofMethanosarcina mazei(OGT 37°C, MmTBP37). The Hybrid-TBP irreversibly aggregates after thermal unfolding just like MmTBP37 and MtTBP65, but theTm lies between that of MmTBP37 and MtTBP65 indicating that the interaction between the α-helical andβ-sheet part of the TBP is crucial for the thermal stability. The temperature stability is probably encoded in the variable α-helices that interact with the highly conserved and DNA bindingβ-sheets.

Interactions between RNase P protein subunits in Archaea

A yeast two-hybrid system was used to identify protein–protein interactions between the ribonuclease P (RNase P) protein subunits Mth11p, Mth687p, Mth688p and Mth1618p from the archaeonMethanothermobacter thermoautotrophicus. Clear interactions between Mth688p and Mth687p, and between Mth1618p and Mth11p, were confirmed byHIS3andLacZreporter expression. Weaker interactions of Mth687p and Mth688p with Mth11p, and Mth11p with itself, are also suggested. These interactions resemble, and confirm, those previously seen among the homologs of these proteins in the more complex yeast RNase P holoenzyme.

Differential Expression of Methanogenesis Genes of Methanothermobacter thermoautotrophicus (Formerly Methanobacterium thermoautotrophicum) in Pure Culture and in Cocultures with Fatty Acid-Oxidizing Syntrophs

ABSTRACT The expression of genes involved in methanogenesis in a thermophilic hydrogen-utilizing methanogen, Methanothermobacter thermoautotrophicus strain TM, was investigated both in a pure culture sufficiently supplied with H2 plus CO2 and in a coculture with an acetate-oxidizing hydrogen-producing bacterium, Thermacetogenium phaeum strain PB, in which hydrogen partial pressure was constantly kept very low (20 to 80 Pa). Northern blot analysis indicated that only the mcr gene, which encodes methyl coenzyme M reductase I (MRI), catalyzing the final step of methanogenesis, was expressed in the coculture, whereas mcr and mrt, which encodes methyl coenzyme M reductase II (MRII), the isofunctional enzyme of MRI, were expressed at the early to late stage of growth in the pure culture. In contrast to these two genes, two isofunctional genes (mtd and mth) for N 5,N 10-methylene-tetrahydromethanopterin dehydrogenase, which catalyzes the fourth step of methanogenesis, and two hydrogenase genes (frh and mvh) were expressed both in a pure culture and in a coculture at the early and late stages of growth. The same expression pattern was observed for Methanothermobacter thermoautotrophicus strain ΔH cocultured with a thermophilic butyrate-oxidizing syntroph, Syntrophothermus lipocalidus strain TGB-C1. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole proteins of M. thermoautotrophicus strain TM obtained from a pure culture and a coculture with the acetate-oxidizing syntroph and subsequent N-terminal amino acid sequence analysis confirmed that MRI and MRII were produced in the pure culture, while only MRI was produced in the coculture. These results indicate that under syntrophic growth conditions, the methanogen preferentially utilizes MRI but not MRII. Considering that hydrogenotrophic methanogens are strictly dependent for growth on hydrogen-producing fermentative microbes in the natural environment and that the hydrogen supply occurs constantly at very low concentrations compared with the supply in pure cultures in the laboratory, the results suggest that MRI is an enzyme primarily functioning in natural methanogenic ecosystems.