Impact of Dietary Resistant Starch on the Human Gut Microbiome, Metaproteome, and Metabolome.

Diet can influence the composition of the human microbiome, and yet relatively few dietary ingredients have been systematically investigated with respect to their impact on the functional potential of the microbiome. Dietary resistant starch (RS) has been shown to have health benefits, but we lack a mechanistic understanding of the metabolic processes that occur in the gut during digestion of RS. Here, we collected samples during a dietary crossover study with diets containing large or small amounts of RS. We determined the impact of RS on the gut microbiome and metabolic pathways in the gut, using a combination of "omics" approaches, including 16S rRNA gene sequencing, metaproteomics, and metabolomics. This multiomics approach captured changes in the abundance of specific bacterial species, proteins, and metabolites after a diet high in resistant starch (HRS), providing key insights into the influence of dietary interventions on the gut microbiome. The combined data showed that a high-RS diet caused an increase in the ratio of Firmicutes to Bacteroidetes, including increases in relative abundances of some specific members of the Firmicutes and concurrent increases in enzymatic pathways and metabolites involved in lipid metabolism in the gut.IMPORTANCE This work was undertaken to obtain a mechanistic understanding of the complex interplay between diet and the microorganisms residing in the intestine. Although it is known that gut microbes play a key role in digestion of the food that we consume, the specific contributions of different microorganisms are not well understood. In addition, the metabolic pathways and resultant products of metabolism during digestion are highly complex. To address these knowledge gaps, we used a combination of molecular approaches to determine the identities of the microorganisms in the gut during digestion of dietary starch as well as the metabolic pathways that they carry out. Together, these data provide a more complete picture of the function of the gut microbiome in digestion, including links between an RS diet and lipid metabolism and novel linkages between specific gut microbes and their metabolites and proteins produced in the gut.

Comparative Omics and Trait Analyses of Marine Pseudoalteromonas Phages Advance the Phage OTU Concept.

Viruses influence the ecology and evolutionary trajectory of microbial communities. Yet our understanding of their roles in ecosystems is limited by the paucity of model systems available for hypothesis generation and testing. Further, virology is limited by the lack of a broadly accepted conceptual framework to classify viral diversity into evolutionary and ecologically cohesive units. Here, we introduce genomes, structural proteomes, and quantitative host range data for eight Pseudoalteromonas phages isolated from Helgoland (North Sea, Germany) and use these data to advance a genome-based viral operational taxonomic unit (OTU) definition. These viruses represent five new genera and inform 498 unaffiliated or unannotated protein clusters (PCs) from global virus metagenomes. In a comparison of previously sequenced Pseudoalteromonas phage isolates (n = 7) and predicted prophages (n = 31), the eight phages are unique. They share a genus with only one other isolate, Pseudoalteromonas podophage RIO-1 (East Sea, South Korea) and two Pseudoalteromonas prophages. Mass-spectrometry of purified viral particles identified 12-20 structural proteins per phage. When combined with 3-D structural predictions, these data led to the functional characterization of five previously unidentified major capsid proteins. Protein functional predictions revealed mechanisms for hijacking host metabolism and resources. Further, they uncovered a hybrid sipho-myovirus that encodes genes for Mu-like infection rarely described in ocean systems. Finally, we used these data to evaluate a recently introduced definition for virus populations that requires members of the same population to have >95% average nucleotide identity across at least 80% of their genes. Using physiological traits and genomics, we proposed a conceptual model for a viral OTU definition that captures evolutionarily cohesive and ecologically distinct units. In this trait-based framework, sensitive hosts are considered viral niches, while host ranges and infection efficiencies are tracked as viral traits. Quantitative host range assays revealed conserved traits within virus OTUs that break down between OTUs, suggesting the defined units capture niche and fitness differentiation. Together these analyses provide a foundation for model system-based hypothesis testing that will improve our understanding of marine copiotrophs, as well as phage-host interactions on the ocean particles and aggregates where Pseudoalteromonas thrive.

Illuminating structural proteins in viral "dark matter" with metaproteomics.

Viruses are ecologically important, yet environmental virology is limited by dominance of unannotated genomic sequences representing taxonomic and functional "viral dark matter." Although recent analytical advances are rapidly improving taxonomic annotations, identifying functional dark matter remains problematic. Here, we apply paired metaproteomics and dsDNA-targeted metagenomics to identify 1,875 virion-associated proteins from the ocean. Over one-half of these proteins were newly functionally annotated and represent abundant and widespread viral metagenome-derived protein clusters (PCs). One primarily unannotated PC dominated the dataset, but structural modeling and genomic context identified this PC as a previously unidentified capsid protein from multiple uncultivated tailed virus families. Furthermore, four of the five most abundant PCs in the metaproteome represent capsid proteins containing the HK97-like protein fold previously found in many viruses that infect all three domains of life. The dominance of these proteins within our dataset, as well as their global distribution throughout the world's oceans and seas, supports prior hypotheses that this HK97-like protein fold is the most abundant biological structure on Earth. Together, these culture-independent analyses improve virion-associated protein annotations, facilitate the investigation of proteins within natural viral communities, and offer a high-throughput means of illuminating functional viral dark matter.

Tissue-specific transcriptomics and proteomics of a filarial nematode and its Wolbachia endosymbiont.

BACKGROUND: Filarial nematodes cause debilitating human diseases. While treatable, recent evidence suggests drug resistance is developing, necessitating the development of novel targets and new treatment options. Although transcriptomic and proteomic studies around the nematode life cycle have greatly enhanced our knowledge, whole organism approaches have not provided spatial resolution of gene expression, which can be gained by examining individual tissues. Generally, due to their small size, tissue dissection of human-infecting filarial nematodes remains extremely challenging. However, canine heartworm disease is caused by a closely related and much larger filarial nematode, Dirofilaria immitis. As with many other filarial nematodes, D. immitis contains Wolbachia, an obligate bacterial endosymbiont present in the hypodermis and developing oocytes within the uterus. Here, we describe the first concurrent tissue-specific transcriptomic and proteomic profiling of a filarial nematode (D. immitis) and its Wolbachia (wDi) in order to better understand tissue functions and identify tissue-specific antigens that may be used for the development of new diagnostic and therapeutic tools. METHODS: Adult D. immitis worms were dissected into female body wall (FBW), female uterus (FU), female intestine (FI), female head (FH), male body wall (MBW), male testis (MT), male intestine (MI), male head (MH) and 10.1186/s12864-015-2083-2 male spicule (MS) and used to prepare transcriptomic and proteomic libraries. RESULTS: Transcriptomic and proteomic analysis of several D. immitis tissues identified many biological functions enriched within certain tissues. Hierarchical clustering of the D. immitis tissue transcriptomes, along with the recently published whole-worm adult male and female D. immitis transcriptomes, revealed that the whole-worm transcriptome is typically dominated by transcripts originating from reproductive tissue. The uterus appeared to have the most variable transcriptome, possibly due to age. Although many functions are shared between the reproductive tissues, the most significant differences in gene expression were observed between the uterus and testis. Interestingly, wDi gene expression in the male and female body wall is fairly similar, yet slightly different to that of Wolbachia gene expression in the uterus. Proteomic methods verified 32 % of the predicted D. immitis proteome, including over 700 hypothetical proteins of D. immitis. Of note, hypothetical proteins were among some of the most abundant Wolbachia proteins identified, which may fulfill some important yet still uncharacterized biological function. CONCLUSIONS: The spatial resolution gained from this parallel transcriptomic and proteomic analysis adds to our understanding of filarial biology and serves as a resource with which to develop future therapeutic strategies against filarial nematodes and their Wolbachia endosymbionts.

Environmental Effects of Nanoceria on Seed Production of Common Bean (Phaseolus vulgaris): A Proteomic Analysis.

The rapidly growing literature on the response of edible plants to nanoceria has provided evidence of its uptake and bioaccumulation, which delineates a possible route of entry into the food chain. However, little is known about how the residing organic matter in soil may affect the bioavailability and resulting impacts of nanoceria on plants. Here, we examined the effect of nanoceria exposure (62.5-500 mg/kg) on kidney bean (Phaseolus vulgaris) productivity and seed quality as a function of soil organic matter content. Cerium accumulation in the seeds produced from plants in organic matter enriched soil showed a dose-dependent increase, unlike in low organic matter soil treatments. Seeds obtained upon nanoceria exposure in soils with higher organic matter were more susceptible to changes in nutrient quality. A quantitative proteomic analysis of the seeds produced upon nanoceria exposure provided evidence for upregulation of stress-related proteins at 62.5 and 125 mg/kg nanoceria treatments. Although the plants did not exhibit overt toxicity, the major seed proteins primarily associated with nutrient storage (phaseolin) and carbohydrate metabolism (lectins) were significantly down-regulated in a dose dependent manner upon nanoceria exposure. This study thus suggests that nanoceria exposures may negatively affect the nutritional quality of kidney beans at the cellular and molecular level. More confirmatory studies with nanoceria along different species using alternative and orthogonal "omic" tools are currently under active investigation, which will enable the identification of biomarkers of exposure and susceptibility.

Multi-omics of permafrost, active layer and thermokarst bog soil microbiomes.

Over 20% of Earth's terrestrial surface is underlain by permafrost with vast stores of carbon that, once thawed, may represent the largest future transfer of carbon from the biosphere to the atmosphere. This process is largely dependent on microbial responses, but we know little about microbial activity in intact, let alone in thawing, permafrost. Molecular approaches have recently revealed the identities and functional gene composition of microorganisms in some permafrost soils and a rapid shift in functional gene composition during short-term thaw experiments. However, the fate of permafrost carbon depends on climatic, hydrological and microbial responses to thaw at decadal scales. Here we use the combination of several molecular 'omics' approaches to determine the phylogenetic composition of the microbial communities, including several draft genomes of novel species, their functional potential and activity in soils representing different states of thaw: intact permafrost, seasonally thawed active layer and thermokarst bog. The multi-omics strategy reveals a good correlation of process rates to omics data for dominant processes, such as methanogenesis in the bog, as well as novel survival strategies for potentially active microbes in permafrost.

Life-style and genome structure of marine Pseudoalteromonas siphovirus B8b isolated from the northwestern Mediterranean Sea.

Marine viruses (phages) alter bacterial diversity and evolution with impacts on marine biogeochemical cycles, and yet few well-developed model systems limit opportunities for hypothesis testing. Here we isolate phage B8b from the Mediterranean Sea using Pseudoalteromonas sp. QC-44 as a host and characterize it using myriad techniques. Morphologically, phage B8b was classified as a member of the Siphoviridae family. One-step growth analyses showed that this siphovirus had a latent period of 70 min and released 172 new viral particles per cell. Host range analysis against 89 bacterial host strains revealed that phage B8b infected 3 Pseudoalteromonas strains (52 tested, >99.9% 16S rRNA gene nucleotide identity) and 1 non-Pseudoaltermonas strain belonging to Alteromonas sp. (37 strains from 6 genera tested), which helps bound the phylogenetic distance possible in a phage-mediated horizontal gene transfer event. The Pseudoalteromonas phage B8b genome size was 42.7 kb, with clear structural and replication modules where the former were delineated leveraging identification of 16 structural genes by virion structural proteomics, only 4 of which had any similarity to known structural proteins. In nature, this phage was common in coastal marine environments in both photic and aphotic layers (found in 26.5% of available viral metagenomes), but not abundant in any sample (average per sample abundance was 0.65% of the reads). Together these data improve our understanding of siphoviruses in nature, and provide foundational information for a new 'rare virosphere' phage-host model system.

Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are an important class of glycoproteins that are tethered to the surface of mammalian cells via the lipid GPI. GPI-APs have been implicated in many important cellular functions including cell adhesion, cell signaling, and immune regulation. Proteomic identification of mammalian GPI-APs en masse has been limited technically by poor sensitivity for these low abundance proteins and the use of methods that destroy cell integrity. Here, we present methodology that permits identification of GPI-APs liberated directly from the surface of intact mammalian cells through exploitation of their appended glycans to enrich for these proteins ahead of LC-MS/MS analyses. We validate our approach in HeLa cells, identifying a greater number of GPI-APs from intact cells than has been previously identified from isolated HeLa membranes and a lipid raft preparation. We further apply our approach to define the cohort of endogenous GPI-APs that populate the distinct apical and basolateral membrane surfaces of polarized epithelial cell monolayers. Our approach provides a new method to achieve greater sensitivity in the identification of low abundance GPI-APs from the surface of live cells and the nondestructive nature of the method provides new opportunities for the temporal or spatial analysis of cellular GPI-AP expression and dynamics.

Colonization state influences the hemocyte proteome in a beneficial squid-Vibrio symbiosis.

The squid Euprymna scolopes and the luminescent bacterium Vibrio fischeri form a highly specific beneficial light organ symbiosis. Not only does the host have to select V. fischeri from the environment, but it must also prevent subsequent colonization by non-symbiotic microorganisms. Host macrophage-like hemocytes are believed to play a role in mediating the symbiosis with V. fischeri. Previous studies have shown that the colonization state of the light organ influences the host's hemocyte response to the symbiont. To further understand the molecular mechanisms behind this process, we used two quantitative mass-spectrometry-based proteomic techniques, isobaric tags for relative and absolute quantification (iTRAQ) and label-free spectral counting, to compare and quantify the adult hemocyte proteomes from colonized (sym) and uncolonized (antibiotic-treated/cured) squid. Overall, iTRAQ allowed for the quantification of 1,024 proteins with two or more peptides. Thirty-seven unique proteins were determined to be significantly different between sym and cured hemocytes (p value < 0.05), with 20 more abundant proteins and 17 less abundant in sym hemocytes. The label-free approach resulted in 1,241 proteins that were identified in all replicates. Of 185 unique proteins present at significantly different amounts in sym hemocytes (as determined by spectral counting), 92 were more abundant and 93 were less abundant. Comparisons between iTRAQ and spectral counting revealed that 30 of the 37 proteins quantified via iTRAQ exhibited trends similar to those identified by the label-free method. Both proteomic techniques mutually identified 16 proteins that were significantly different between the two groups of hemocytes (p value < 0.05). The presence of V. fischeri in the host light organ influenced the abundance of proteins associated with the cytoskeleton, adhesion, lysosomes, proteolysis, and the innate immune response. These data provide evidence that colonization by V. fischeri alters the hemocyte proteome and reveals proteins that may be important for maintaining host-symbiont specificity.

Discovery of a novel methanogen prevalent in thawing permafrost.

Thawing permafrost promotes microbial degradation of cryo-sequestered and new carbon leading to the biogenic production of methane, creating a positive feedback to climate change. Here we determine microbial community composition along a permafrost thaw gradient in northern Sweden. Partially thawed sites were frequently dominated by a single archaeal phylotype, Candidatus 'Methanoflorens stordalenmirensis' gen. nov. sp. nov., belonging to the uncultivated lineage 'Rice Cluster II' (Candidatus 'Methanoflorentaceae' fam. nov.). Metagenomic sequencing led to the recovery of its near-complete genome, revealing the genes necessary for hydrogenotrophic methanogenesis. These genes are highly expressed and methane carbon isotope data are consistent with hydrogenotrophic production of methane in the partially thawed site. In addition to permafrost wetlands, 'Methanoflorentaceae' are widespread in high methane-flux habitats suggesting that this lineage is both prevalent and a major contributor to global methane production. In thawing permafrost, Candidatus 'M. stordalenmirensis' appears to be a key mediator of methane-based positive feedback to climate warming.

Twelve previously unknown phage genera are ubiquitous in global oceans.

Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in "unknowns" dominating culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four well-known viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria. Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage-host systems for experimental hypothesis testing.

Metaproteomics reveals abundant transposase expression in mutualistic endosymbionts.

Transposases, enzymes that catalyze the movement of mobile genetic elements, are the most abundant genes in nature. While many bacteria encode an abundance of transposases in their genomes, the current paradigm is that the expression of transposase genes is tightly regulated and generally low due to its severe mutagenic effects. In the current study, we detected the highest number of transposase proteins ever reported in bacteria, in symbionts of the gutless marine worm Olavius algarvensis with metaproteomics. At least 26 different transposases from 12 different families were detected, and genomic and proteomic analyses suggest that many of these are active. This high expression of transposases indicates that the mechanisms for their tight regulation have been disabled or no longer exist.

New group in the Leptospirillum clade: cultivation-independent community genomics, proteomics, and transcriptomics of the new species "Leptospirillum group IV UBA BS".

Leptospirillum spp. are widespread members of acidophilic microbial communities that catalyze ferrous iron oxidation, thereby increasing sulfide mineral dissolution rates. These bacteria play important roles in environmental acidification and are harnessed for bioleaching-based metal recovery. Known members of the Leptospirillum clade of the Nitrospira phylum are Leptospirillum ferrooxidans (group I), Leptospirillum ferriphilum and "Leptospirillum rubarum" (group II), and Leptospirillum ferrodiazotrophum (group III). In the Richmond Mine acid mine drainage (AMD) system, biofilm formation is initiated by L. rubarum; L. ferrodiazotrophum appears in later developmental stages. Here we used community metagenomic data from unusual, thick floating biofilms to identify distinguishing metabolic traits in a rare and uncultivated community member, the new species "Leptospirillum group IV UBA BS." These biofilms typically also contain a variety of Archaea, Actinobacteria, and a few other Leptospirillum spp. The Leptospirillum group IV UBA BS species shares 98% 16S rRNA sequence identity and 70% average amino acid identity between orthologs with its closest relative, L. ferrodiazotrophum. The presence of nitrogen fixation and reverse tricarboxylic acid (TCA) cycle proteins suggest an autotrophic metabolism similar to that of L. ferrodiazotrophum, while hydrogenase proteins suggest anaerobic metabolism. Community transcriptomic and proteomic analyses demonstrate expression of a multicopper oxidase unique to this species, as well as hydrogenases and core metabolic genes. Results suggest that the Leptospirillum group IV UBA BS species might play important roles in carbon fixation, nitrogen fixation, hydrogen metabolism, and iron oxidation in some acidic environments.

The two-faced T cell epitope: examining the host-microbe interface with JanusMatrix.

Advances in the field of T cell immunology have contributed to the understanding that cross-reactivity is an intrinsic characteristic of the T cell receptor (TCR), and that each TCR can potentially interact with many different T cell epitopes. To better define the potential for TCR cross-reactivity between epitopes derived from the human genome, the human microbiome, and human pathogens, we developed a new immunoinformatics tool, JanusMatrix, that represents an extension of the validated T cell epitope mapping tool, EpiMatrix. Initial explorations, summarized in this synopsis, have uncovered what appear to be important differences in the TCR cross-reactivity of selected regulatory and effector T cell epitopes with other epitopes in the human genome, human microbiome, and selected human pathogens. In addition to exploring the T cell epitope relationships between human self, commensal and pathogen, JanusMatrix may also be useful to explore some aspects of heterologous immunity and to examine T cell epitope relatedness between pathogens to which humans are exposed (Dengue serotypes, or HCV and Influenza, for example). In Hand-Foot-Mouth disease (HFMD) for example, extensive enterovirus and human microbiome cross-reactivity (and limited cross-reactivity with the human genome) seemingly predicts immunodominance. In contrast, more extensive cross-reactivity with proteins contained in the human genome as compared to the human microbiome was observed for selected Treg epitopes. While it may be impossible to predict all immune response influences, the availability of sequence data from the human genome, the human microbiome, and an array of human pathogens and vaccines has made computationally-driven exploration of the effects of T cell epitope cross-reactivity now possible. This is the first description of JanusMatrix, an algorithm that assesses TCR cross-reactivity that may contribute to a means of predicting the phenotype of T cells responding to selected T cell epitopes. Whether used for explorations of T cell phenotype or for evaluating cross-conservation between related viral strains at the TCR face of viral epitopes, further JanusMatrix studies may contribute to developing safer, more effective vaccines.

Biostimulation induces syntrophic interactions that impact C, S and N cycling in a sediment microbial community.

Stimulation of subsurface microorganisms to induce reductive immobilization of metals is a promising approach for bioremediation, yet the overall microbial community response is typically poorly understood. Here we used proteogenomics to test the hypothesis that excess input of acetate activates complex community functioning and syntrophic interactions among autotrophs and heterotrophs. A flow-through sediment column was incubated in a groundwater well of an acetate-amended aquifer and recovered during microbial sulfate reduction. De novo reconstruction of community sequences yielded near-complete genomes of Desulfobacter (Deltaproteobacteria), Sulfurovum- and Sulfurimonas-like Epsilonproteobacteria and Bacteroidetes. Partial genomes were obtained for Clostridiales (Firmicutes) and Desulfuromonadales-like Deltaproteobacteria. The majority of proteins identified by mass spectrometry corresponded to Desulfobacter-like species, and demonstrate the role of this organism in sulfate reduction (Dsr and APS), nitrogen fixation and acetate oxidation to CO2 during amendment. Results indicate less abundant Desulfuromonadales, and possibly Bacteroidetes, also actively contributed to CO2 production via the tricarboxylic acid (TCA) cycle. Proteomic data indicate that sulfide was partially re-oxidized by Epsilonproteobacteria through nitrate-dependent sulfide oxidation (using Nap, Nir, Nos, SQR and Sox), with CO2 fixed using the reverse TCA cycle. We infer that high acetate concentrations, aimed at stimulating anaerobic heterotrophy, led to the co-enrichment of, and carbon fixation in Epsilonproteobacteria. Results give an insight into ecosystem behavior following addition of simple organic carbon to the subsurface, and demonstrate a range of biological processes and community interactions were stimulated.

Integrated metagenomics/metaproteomics reveals human host-microbiota signatures of Crohn's disease.

Crohn's disease (CD) is an inflammatory bowel disease of complex etiology, although dysbiosis of the gut microbiota has been implicated in chronic immune-mediated inflammation associated with CD. Here we combined shotgun metagenomic and metaproteomic approaches to identify potential functional signatures of CD in stool samples from six twin pairs that were either healthy, or that had CD in the ileum (ICD) or colon (CCD). Integration of these omics approaches revealed several genes, proteins, and pathways that primarily differentiated ICD from healthy subjects, including depletion of many proteins in ICD. In addition, the ICD phenotype was associated with alterations in bacterial carbohydrate metabolism, bacterial-host interactions, as well as human host-secreted enzymes. This eco-systems biology approach underscores the link between the gut microbiota and functional alterations in the pathophysiology of Crohn's disease and aids in identification of novel diagnostic targets and disease specific biomarkers.

Coupling a detergent lysis/cleanup methodology with intact protein fractionation for enhanced proteome characterization.

The expanding use of surfactants for proteome sample preparations has prompted the need to systematically optimize the application and removal of these MS-deleterious agents prior to proteome measurements. Here we compare four detergent cleanup methods (trichloroacetic acid (TCA) precipitation, chloroform/methanol/water (CMW) extraction, a commercial detergent removal spin column method (DRS) and filter-aided sample preparation (FASP)) to provide efficiency benchmarks with respect to protein, peptide, and spectral identifications in each case. Our results show that for protein-limited samples, FASP outperforms the other three cleanup methods, while at high protein amounts, all the methods are comparable. This information was used to investigate and contrast molecular weight-based fractionated with unfractionated lysates from three increasingly complex samples ( Escherichia coli K-12, a five microbial isolate mixture, and a natural microbial community groundwater sample), all of which were prepared with an SDS-FASP approach. The additional fractionation step enhanced the number of protein identifications by 8% to 25% over the unfractionated approach across the three samples.

Fermentation, hydrogen, and sulfur metabolism in multiple uncultivated bacterial phyla.

BD1-5, OP11, and OD1 bacteria have been widely detected in anaerobic environments, but their metabolisms remain unclear owing to lack of cultivated representatives and minimal genomic sampling. We uncovered metabolic characteristics for members of these phyla, and a new lineage, PER, via cultivation-independent recovery of 49 partial to near-complete genomes from an acetate-amended aquifer. All organisms were nonrespiring anaerobes predicted to ferment. Three augment fermentation with archaeal-like hybrid type II/III ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) that couples adenosine monophosphate salvage with CO(2) fixation, a pathway not previously described in Bacteria. Members of OD1 reduce sulfur and may pump protons using archaeal-type hydrogenases. For six organisms, the UGA stop codon is translated as tryptophan. All bacteria studied here may play previously unrecognized roles in hydrogen production, sulfur cycling, and fermentation of refractory sedimentary carbon.

Phage-induced expression of CRISPR-associated proteins is revealed by shotgun proteomics in Streptococcus thermophilus.

The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas) proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response in S. thermophilus DGCC7710 infected with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infection and across various time points using two-dimensional liquid chromatography tandem mass spectrometry. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance following infection, including the signature Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection.

The temporal analysis of yeast exponential phase using shotgun proteomics as a fermentation monitoring technique.

System biology and bioprocess technology can be better understood using shotgun proteomics as a monitoring system during the fermentation. We demonstrated a shotgun proteomic method to monitor the temporal yeast proteome in early, middle and late exponential phases. Our study identified a total of 1389 proteins combining all 2D-LC-MS/MS runs. The temporal Saccharomyces cerevisiae proteome was enriched with proteolysis, radical detoxification, translation, one-carbon metabolism, glycolysis and TCA cycle. Heat shock proteins and proteins associated with oxidative stress response were found throughout the exponential phase. The most abundant proteins observed were translation elongation factors, ribosomal proteins, chaperones and glycolytic enzymes. The high abundance of the H-protein of the glycine decarboxylase complex (Gcv3p) indicated the availability of glycine in the environment. We observed differentially expressed proteins and the induced proteins at mid-exponential phase were involved in ribosome biogenesis, mitochondria DNA binding/replication and transcriptional activator. Induction of tryptophan synthase (Trp5p) indicated the abundance of tryptophan during the fermentation. As fermentation progressed toward late exponential phase, a decrease in cell proliferation was implied from the repression of ribosomal proteins, transcription coactivators, methionine aminopeptidase and translation-associated proteins.