Amino acid treatment enhances protein recovery from sediment and soils for metaproteomic studies.

Characterization of microbial protein expression provides information necessary to better understand the unique biological pathways that occur within soil microbial communities that contribute to atmospheric CO2 levels and the earth's changing climate. A significant challenge in studying the soil microbial community proteome is the initial dissociation of bacterial proteins from the complex mixture of particles found in natural soil. The differential extraction of intact bacterial cells limits the characterization of the complete representation of a microbial community. However, in situ lysis of bacterial cells in soil can lead to potentially high levels of protein adsorption to soil particles. Here, we investigated various amino acids for their ability to block soil protein adsorption sites prior to in situ lysis of bacterial cells, as well as their compatibility with both tryptic digestion and mass spectrometric analysis. The treatments were tested by adding proteins from lysed Escherichia coli cells to representative treated and untreated soil samples. The results show that it is possible to significantly increase protein identifications through blockage of binding sites on a variety of soil and sediment textures; use of an optimized desorption buffer further increases the number of identifications.

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.

New roles in hemicellulosic sugar fermentation for the uncultivated Bacteroidetes family BS11.

Ruminants have co-evolved with their gastrointestinal microbial communities that digest plant materials to provide energy for the host. Some arctic and boreal ruminants have already shown to be vulnerable to dietary shifts caused by changing climate, yet we know little about the metabolic capacity of the ruminant microbiome in these animals. Here, we use meta-omics approaches to sample rumen fluid microbial communities from Alaskan moose foraging along a seasonal lignocellulose gradient. Winter diets with increased hemicellulose and lignin strongly enriched for BS11, a Bacteroidetes family lacking cultivated or genomically sampled representatives. We show that BS11 are cosmopolitan host-associated bacteria prevalent in gastrointestinal tracts of ruminants and other mammals. Metagenomic reconstruction yielded the first four BS11 genomes; phylogenetically resolving two genera within this previously taxonomically undefined family. Genome-enabled metabolic analyses uncovered multiple pathways for fermenting hemicellulose monomeric sugars to short-chain fatty acids (SCFA), metabolites vital for ruminant energy. Active hemicellulosic sugar fermentation and SCFA production was validated by shotgun proteomics and rumen metabolites, illuminating the role BS11 have in carbon transformations within the rumen. Our results also highlight the currently unknown metabolic potential residing in the rumen that may be vital for sustaining host energy in response to a changing vegetative environment.

Proteomic detection of non-annotated protein-coding genes in Pseudomonas fluorescens Pf0-1.

Genome sequences are annotated by computational prediction of coding sequences, followed by similarity searches such as BLAST, which provide a layer of possible functional information. While the existence of processes such as alternative splicing complicates matters for eukaryote genomes, the view of bacterial genomes as a linear series of closely spaced genes leads to the assumption that computational annotations that predict such arrangements completely describe the coding capacity of bacterial genomes. We undertook a proteomic study to identify proteins expressed by Pseudomonas fluorescens Pf0-1 from genes that were not predicted during the genome annotation. Mapping peptides to the Pf0-1 genome sequence identified sixteen non-annotated protein-coding regions, of which nine were antisense to predicted genes, six were intergenic, and one read in the same direction as an annotated gene but in a different frame. The expression of all but one of the newly discovered genes was verified by RT-PCR. Few clues as to the function of the new genes were gleaned from informatic analyses, but potential orthologs in other Pseudomonas genomes were identified for eight of the new genes. The 16 newly identified genes improve the quality of the Pf0-1 genome annotation, and the detection of antisense protein-coding genes indicates the under-appreciated complexity of bacterial genome organization.