Diversity and biogeography of SAR11 bacteria from the Arctic Ocean.

The Arctic Ocean is relatively isolated from other oceans and consists of strongly stratified water masses with distinct histories, nutrient, temperature, and salinity characteristics, therefore providing an optimal environment to investigate local adaptation. The globally distributed SAR11 bacterial group consists of multiple ecotypes that are associated with particular marine environments, yet relatively little is known about Arctic SAR11 diversity. Here, we examined SAR11 diversity using ITS analysis and metagenome-assembled genomes (MAGs). Arctic SAR11 assemblages were comprised of the S1a, S1b, S2, and S3 clades, and structured by water mass and depth. The fresher surface layer was dominated by an ecotype (S3-derived P3.2) previously associated with Arctic and brackish water. In contrast, deeper waters of Pacific origin were dominated by the P2.3 ecotype of the S2 clade, within which we identified a novel subdivision (P2.3s1) that was rare outside the Arctic Ocean. Arctic S2-derived SAR11 MAGs were restricted to high latitudes and included MAGs related to the recently defined S2b subclade, a finding consistent with bi-polar ecotypes and Arctic endemism. These results place the stratified Arctic Ocean into the SAR11 global biogeography and have identified SAR11 lineages for future investigation of adaptive evolution in the Arctic Ocean.

Genomic evidence for the degradation of terrestrial organic matter by pelagic Arctic Ocean Chloroflexi bacteria.

The Arctic Ocean currently receives a large supply of global river discharge and terrestrial dissolved organic matter. Moreover, an increase in freshwater runoff and riverine transport of organic matter to the Arctic Ocean is a predicted consequence of thawing permafrost and increased precipitation. The fate of the terrestrial humic-rich organic material and its impact on the marine carbon cycle are largely unknown. Here, a metagenomic survey of the Canada Basin in the Western Arctic Ocean showed that pelagic Chloroflexi from the Arctic Ocean are replete with aromatic compound degradation genes, acquired in part by lateral transfer from terrestrial bacteria. Our results imply marine Chloroflexi have the capacity to use terrestrial organic matter and that their role in the carbon cycle may increase with the changing hydrological cycle.

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis.

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and metabolism. Although powerful, metagenomics alone can only elucidate functional potential. On the other hand, metaproteomics enables the description of the expressed in situ metabolism and function of a community. Here we describe a protocol for cell lysis, protein and DNA isolation, as well as peptide digestion and extraction from marine microbial cells collected on a cartridge filter unit (such as the Sterivex filter unit) and preserved in an RNA stabilization solution (like RNAlater). In mass spectrometry-based proteomics studies, the identification of peptides and proteins is performed by comparing peptide tandem mass spectra to a database of translated nucleotide sequences. Including the metagenome of a sample in the search database increases the number of peptides and proteins that can be identified from the mass spectra. Hence, in this protocol DNA is isolated from the same filter, which can be used subsequently for metagenomic analysis.

Metaproteomics of aquatic microbial communities in a deep and stratified estuary.

Here we harnessed the power of metaproteomics to assess the metabolic diversity and function of stratified aquatic microbial communities in the deep and expansive Lower St. Lawrence Estuary, located in eastern Canada. Vertical profiling of the microbial communities through the stratified water column revealed differences in metabolic lifestyles and in carbon and nitrogen processing pathways. In productive surface waters, we identified heterotrophic populations involved in the processing of high and low molecular weight organic matter from both terrestrial (e.g. cellulose and xylose) and marine (e.g. organic compatible osmolytes) sources. In the less productive deep waters, chemosynthetic production coupled to nitrification by MG-I Thaumarchaeota and Nitrospina appeared to be a dominant metabolic strategy. Similar to other studies of the coastal ocean, we identified methanol oxidation proteins originating from the common OM43 marine clade. However, we also identified a novel lineage of methanol-oxidizers specifically in the particle-rich bottom (i.e. nepheloid) layer. Membrane transport proteins assigned to the uncultivated MG-II Euryarchaeota were also specifically detected in the nepheloid layer. In total, these results revealed strong vertical structure of microbial taxa and metabolic activities, as well as the presence of specific "nepheloid" taxa that may contribute significantly to coastal ocean nutrient cycling.

Deconstructing the genetic basis of spent sulphite liquor tolerance using deep sequencing of genome-shuffled yeast.

BACKGROUND: Identifying the genetic basis of complex microbial phenotypes is currently a major barrier to our understanding of multigenic traits and our ability to rationally design biocatalysts with highly specific attributes for the biotechnology industry. Here, we demonstrate that strain evolution by meiotic recombination-based genome shuffling coupled with deep sequencing can be used to deconstruct complex phenotypes and explore the nature of multigenic traits, while providing concrete targets for strain development. RESULTS: We determined genomic variations found within Saccharomyces cerevisiae previously evolved in our laboratory by genome shuffling for tolerance to spent sulphite liquor. The representation of these variations was backtracked through parental mutant pools and cross-referenced with RNA-seq gene expression analysis to elucidate the importance of single mutations and key biological processes that play a role in our trait of interest. Our findings pinpoint novel genes and biological determinants of lignocellulosic hydrolysate inhibitor tolerance in yeast. These include the following: protein homeostasis constituents, including Ubp7p and Art5p, related to ubiquitin-mediated proteolysis; stress response transcriptional repressor, Nrg1p; and NADPH-dependent glutamate dehydrogenase, Gdh1p. Reverse engineering a prominent mutation in ubiquitin-specific protease gene UBP7 in a laboratory S. cerevisiae strain effectively increased spent sulphite liquor tolerance. CONCLUSIONS: This study advances understanding of yeast tolerance mechanisms to inhibitory substrates and biocatalyst design for a biomass-to-biofuel/biochemical industry, while providing insights into the process of mutation accumulation that occurs during genome shuffling.

Engineering microbes for plant polyketide biosynthesis.

Polyketides are an important group of secondary metabolites, many of which have important industrial applications in the food and pharmaceutical industries. Polyketides are synthesized from one of three classes of enzymes differentiated by their biochemical features and product structure: type I, type II or type III polyketide synthases (PKSs). Plant type III PKS enzymes, which will be the main focus of this review, are relatively small homodimeric proteins that catalyze iterative decarboxylative condensations of malonyl units with a CoA-linked starter molecule. This review will describe the plant type III polyketide synthetic pathway, including the synthesis of chalcones, stilbenes and curcuminoids, as well as recent work on the synthesis of these polyketides in heterologous organisms. The limitations and bottlenecks of heterologous expression as well as attempts at creating diversity through the synthesis of novel "unnatural" polyketides using type III PKSs will also be discussed. Although synthetic production of plant polyketides is still in its infancy, their potential as useful bioactive compounds makes them an extremely interesting area of study.