From proteins to polysaccharides: lifestyle and genetic evolution of Coprothermobacter proteolyticus.

Microbial communities that degrade lignocellulosic biomass are typified by high levels of species- and strain-level complexity, as well as synergistic interactions between both cellulolytic and non-cellulolytic microorganisms. Coprothermobacter proteolyticus frequently dominates thermophilic, lignocellulose-degrading communities with wide geographical distribution, which is in contrast to reports that it ferments proteinaceous substrates and is incapable of polysaccharide hydrolysis. Here we deconvolute a highly efficient cellulose-degrading consortium (SEM1b) that is co-dominated by Clostridium (Ruminiclostridium) thermocellum and multiple heterogenic strains affiliated to C. proteolyticus. Metagenomic analysis of SEM1b recovered metagenome-assembled genomes (MAGs) for each constituent population, whereas in parallel two novel strains of C. proteolyticus were successfully isolated and sequenced. Annotation of all C. proteolyticus genotypes (two strains and one MAG) revealed their genetic acquisition of carbohydrate-active enzymes (CAZymes), presumably derived from horizontal gene transfer (HGT) events involving polysaccharide-degrading Firmicutes or Thermotogae-affiliated populations that are historically co-located. HGT material included a saccharolytic operon, from which a CAZyme was biochemically characterized and demonstrated hydrolysis of multiple hemicellulose polysaccharides. Finally, temporal genome-resolved metatranscriptomic analysis of SEM1b revealed expression of C. proteolyticus CAZymes at different SEM1b life stages as well as co-expression of CAZymes from multiple SEM1b populations, inferring deeper microbial interactions that are dedicated toward community degradation of cellulose and hemicellulose. We show that C. proteolyticus, a ubiquitous population, consists of closely related strains that have adapted via HGT to presumably degrade both oligo- and longer polysaccharides present in decaying plants and microbial cell walls, thus explaining its dominance in thermophilic anaerobic digesters on a global scale.

Interspecies cross-feeding orchestrates carbon degradation in the rumen ecosystem.

Because of their agricultural value, there is a great body of research dedicated to understanding the microorganisms responsible for rumen carbon degradation. However, we lack a holistic view of the microbial food web responsible for carbon processing in this ecosystem. Here, we sampled rumen-fistulated moose, allowing access to rumen microbial communities actively degrading woody plant biomass in real time. We resolved 1,193 viral contigs and 77 unique, near-complete microbial metagenome-assembled genomes, many of which lacked previous metabolic insights. Plant-derived metabolites were measured with NMR and carbohydrate microarrays to quantify the carbon nutrient landscape. Network analyses directly linked measured metabolites to expressed proteins from these unique metagenome-assembled genomes, revealing a genome-resolved three-tiered carbohydrate-fuelled trophic system. This provided a glimpse into microbial specialization into functional guilds defined by specific metabolites. To validate our proteomic inferences, the catalytic activity of a polysaccharide utilization locus from a highly connected metabolic hub genome was confirmed using heterologous gene expression. Viral detected proteins and linkages to microbial hosts demonstrated that phage are active controllers of rumen ecosystem function. Our findings elucidate the microbial and viral members, as well as their metabolic interdependencies, that support in situ carbon degradation in the rumen ecosystem.

Host-linked soil viral ecology along a permafrost thaw gradient.

Climate change threatens to release abundant carbon that is sequestered at high latitudes, but the constraints on microbial metabolisms that mediate the release of methane and carbon dioxide are poorly understood1-7. The role of viruses, which are known to affect microbial dynamics, metabolism and biogeochemistry in the oceans8-10, remains largely unexplored in soil. Here, we aimed to investigate how viruses influence microbial ecology and carbon metabolism in peatland soils along a permafrost thaw gradient in Sweden. We recovered 1,907 viral populations (genomes and large genome fragments) from 197 bulk soil and size-fractionated metagenomes, 58% of which were detected in metatranscriptomes and presumed to be active. In silico predictions linked 35% of the viruses to microbial host populations, highlighting likely viral predators of key carbon-cycling microorganisms, including methanogens and methanotrophs. Lineage-specific virus/host ratios varied, suggesting that viral infection dynamics may differentially impact microbial responses to a changing climate. Virus-encoded glycoside hydrolases, including an endomannanase with confirmed functional activity, indicated that viruses influence complex carbon degradation and that viral abundances were significant predictors of methane dynamics. These findings suggest that viruses may impact ecosystem function in climate-critical, terrestrial habitats and identify multiple potential viral contributions to soil carbon cycling.

Non-Invasive Analysis of Recombinant mRNA Stability in Escherichia coli by a Combination of Transcriptional Inducer Wash-Out and qRT-PCR.

mRNA stability is one among many parameters that can potentially affect the level of recombinant gene expression in bacteria. Blocking of the entire prokaryotic transcription machinery by addition of rifampicin is commonly used in protocols for analysis of mRNA stability. Here we show that such treatment can be effectively replaced by a simple, non-invasive method based on removal of the relevant transcriptional inducers and that the mRNA decay can then be followed by qRT-PCR. To establish the methodology we first used the m-toluate-inducible XylS/Pm expression cassette as a model system and analyzed several examples of DNA modifications causing gene expression stimulation in Escherichia coli. The new method allowed us to clearly discriminate whether an improvement in mRNA stability contributes to observed increases in transcript amounts for each individual case. To support the experimental data a simple mathematical fitting model was developed to calculate relative decay rates. We extended the relevance of the method by demonstrating its application also for an IPTG-inducible expression cassette (LacI/Ptac) and by analyzing features of the bacteriophage T7-based expression system. The results suggest that the methodology is useful in elucidating factors controlling mRNA stability as well as other specific features of inducible expression systems. Moreover, as expression systems based on diffusible inducers are almost universally available, the concept can be most likely used to measure mRNA decay for any gene in any cell type that is heavily used in molecular biology research.