Potential Pathogenicity Determinants Identified from Structural Proteomics of SARS-CoV and SARS-CoV-2.

Despite SARS-CoV and SARS-CoV-2 being equipped with highly similar protein arsenals, the corresponding zoonoses have spread among humans at extremely different rates. The specific characteristics of these viruses that led to such distinct outcomes remain unclear. Here, we apply proteome-wide comparative structural analysis aiming to identify the unique molecular elements in the SARS-CoV-2 proteome that may explain the differing consequences. By combining protein modeling and molecular dynamics simulations, we suggest nonconservative substitutions in functional regions of the spike glycoprotein (S), nsp1, and nsp3 that are contributing to differences in virulence. Particularly, we explain why the substitutions at the receptor-binding domain of S affect the structure-dynamics behavior in complexes with putative host receptors. Conservation of functional protein regions within the two taxa is also noteworthy. We suggest that the highly conserved main protease, nsp5, of SARS-CoV and SARS-CoV-2 is part of their mechanism of circumventing the host interferon antiviral response. Overall, most substitutions occur on the protein surfaces and may be modulating their antigenic properties and interactions with other macromolecules. Our results imply that the striking difference in the pervasiveness of SARS-CoV-2 and SARS-CoV among humans seems to significantly derive from molecular features that modulate the efficiency of viral particles in entering the host cells and blocking the host immune response.

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.

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.

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 of a gutless marine worm and its symbiotic microbial community reveal unusual pathways for carbon and energy use.

Low nutrient and energy availability has led to the evolution of numerous strategies for overcoming these limitations, of which symbiotic associations represent a key mechanism. Particularly striking are the associations between chemosynthetic bacteria and marine animals that thrive in nutrient-poor environments such as the deep sea because the symbionts allow their hosts to grow on inorganic energy and carbon sources such as sulfide and CO(2). Remarkably little is known about the physiological strategies that enable chemosynthetic symbioses to colonize oligotrophic environments. In this study, we used metaproteomics and metabolomics to investigate the intricate network of metabolic interactions in the chemosynthetic association between Olavius algarvensis, a gutless marine worm, and its bacterial symbionts. We propose previously undescribed pathways for coping with energy and nutrient limitation, some of which may be widespread in both free-living and symbiotic bacteria. These pathways include (i) a pathway for symbiont assimilation of the host waste products acetate, propionate, succinate and malate; (ii) the potential use of carbon monoxide as an energy source, a substrate previously not known to play a role in marine invertebrate symbioses; (iii) the potential use of hydrogen as an energy source; (iv) the strong expression of high-affinity uptake transporters; and (v) as yet undescribed energy-efficient steps in CO(2) fixation and sulfate reduction. The high expression of proteins involved in pathways for energy and carbon uptake and conservation in the O. algarvensis symbiosis indicates that the oligotrophic nature of its environment exerted a strong selective pressure in shaping these associations.

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.

Discovery and annotation of small proteins using genomics, proteomics, and computational approaches.

Small proteins (10-200 amino acids [aa] in length) encoded by short open reading frames (sORF) play important regulatory roles in various biological processes, including tumor progression, stress response, flowering, and hormone signaling. However, ab initio discovery of small proteins has been relatively overlooked. Recent advances in deep transcriptome sequencing make it possible to efficiently identify sORFs at the genome level. In this study, we obtained ~2.6 million expressed sequence tag (EST) reads from Populus deltoides leaf transcriptome and reconstructed full-length transcripts from the EST sequences. We identified an initial set of 12,852 sORFs encoding proteins of 10-200 aa in length. Three computational approaches were then used to enrich for bona fide protein-coding sORFs from the initial sORF set: (1) coding-potential prediction, (2) evolutionary conservation between P. deltoides and other plant species, and (3) gene family clustering within P. deltoides. As a result, a high-confidence sORF candidate set containing 1469 genes was obtained. Analysis of the protein domains, non-protein-coding RNA motifs, sequence length distribution, and protein mass spectrometry data supported this high-confidence sORF set. In the high-confidence sORF candidate set, known protein domains were identified in 1282 genes (higher-confidence sORF candidate set), out of which 611 genes, designated as highest-confidence candidate sORF set, were supported by proteomics data. Of the 611 highest-confidence candidate sORF genes, 56 were new to the current Populus genome annotation. This study not only demonstrates that there are potential sORF candidates to be annotated in sequenced genomes, but also presents an efficient strategy for discovery of sORFs in species with no genome annotation yet available.

Niche of harmful alga Aureococcus anophagefferens revealed through ecogenomics.

Harmful algal blooms (HABs) cause significant economic and ecological damage worldwide. Despite considerable efforts, a comprehensive understanding of the factors that promote these blooms has been lacking, because the biochemical pathways that facilitate their dominance relative to other phytoplankton within specific environments have not been identified. Here, biogeochemical measurements showed that the harmful alga Aureococcus anophagefferens outcompeted co-occurring phytoplankton in estuaries with elevated levels of dissolved organic matter and turbidity and low levels of dissolved inorganic nitrogen. We subsequently sequenced the genome of A. anophagefferens and compared its gene complement with those of six competing phytoplankton species identified through metaproteomics. Using an ecogenomic approach, we specifically focused on gene sets that may facilitate dominance within the environmental conditions present during blooms. A. anophagefferens possesses a larger genome (56 Mbp) and has more genes involved in light harvesting, organic carbon and nitrogen use, and encoding selenium- and metal-requiring enzymes than competing phytoplankton. Genes for the synthesis of microbial deterrents likely permit the proliferation of this species, with reduced mortality losses during blooms. Collectively, these findings suggest that anthropogenic activities resulting in elevated levels of turbidity, organic matter, and metals have opened a niche within coastal ecosystems that ideally suits the unique genetic capacity of A. anophagefferens and thus, has facilitated the proliferation of this and potentially other HABs.

Discovery and Characterization of Fluopipamine, a Putative Cellulose Synthase 1 Antagonist within Arabidopsis.

Herbicide-resistant weeds are increasingly a problem in crop fields when exposed to similar chemistry over time. To avoid future yield losses, identifying herbicidal chemistry needs to be accelerated. We screened 50,000 small molecules using a liquid-handling robot and light microscopy focusing on pre-emergent herbicides in the family of cellulose biosynthesis inhibitors. Through phenotypic, chemical, genetic, and in silico methods we uncovered 6-{[4-(2-fluorophenyl)-1-piperazinyl]methyl}-N-(2-methoxy-5-methylphenyl)-1,3,5-triazine-2,4-diamine (fluopipamine). Symptomologies support fluopipamine as a putative antagonist of cellulose synthase enzyme 1 (CESA1) from Arabidopsis (Arabidopsis thaliana). Ectopic lignification, inhibition of etiolation, phenotypes including loss of anisotropic cellular expansion, swollen roots, and live cell imaging link fluopipamine to cellulose biosynthesis inhibition. Radiolabeled glucose incorporation of cellulose decreased in short-duration experiments when seedlings were incubated in fluopipamine. To elucidate the mechanism, ethylmethanesulfonate mutagenized M2 seedlings were screened for fluopipamine resistance. Two loci of genetic resistance were linked to CESA1. In silico docking of fluopipamine, quinoxyphen, and flupoxam against various CESA1 mutations suggests that an alternative binding site at the interface between CESA proteins is necessary to preserve cellulose polymerization in compound presence. These data uncovered potential fundamental mechanisms of cellulose biosynthesis in plants along with feasible leads for herbicidal uses.

Environmental proteomics reveals early microbial community responses to biostimulation at a uranium- and nitrate-contaminated site.

High-performance MS instrumentation coupled with improved protein extraction techniques enables metaproteomics to identify active members of soil and groundwater microbial communities. Metaproteomics workflows were applied to study the initial responses (i.e. 4 days post treatment) of the indigenous aquifer microbiota to biostimulation with emulsified vegetable oil (EVO) at a uranium-contaminated site. Members of the Betaproteobacteria (i.e. Dechloromonas, Ralstonia, Rhodoferax, Polaromonas, Delftia, Chromobacterium) and the Firmicutes dominated the biostimulated aquifer community. Proteome characterization revealed distinct differences between the microbial biomass collected from groundwater influenced by biostimulation and groundwater collected upgradient of the EVO injection points. In particular, proteins involved in ammonium assimilation, EVO degradation, and polyhydroxybutyrate granule formation were prominent following biostimulation. Interestingly, the atypical NosZ of Dechloromonas spp. was highly abundant, suggesting active nitrous oxide (N2 O) respiration. c-Type cytochromes were barely detected, as was citrate synthase, a biomarker for hexavalent uranium reduction activity, suggesting that uranium reduction has not commenced 4 days post EVO amendment. Environmental metaproteomics identified microbial community responses to biostimulation and elucidated active pathways demonstrating the value of this technique as a monitoring tool and for complementing nucleic acid-based approaches.

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.

Insights into the structure and metabolic function of microbes that shape pelagic iron-rich aggregates ("iron snow").

Microbial ferrous iron [Fe(II)] oxidation leads to the formation of iron-rich macroscopic aggregates ("iron snow") at the redoxcline in a stratified lignite mine lake in east-central Germany. We aimed to identify the abundant Fe-oxidizing and Fe-reducing microorganisms likely to be involved in the formation and transformation of iron snow present in the redoxcline in two basins of the lake that differ in their pH values. Nucleic acid- and lipid-stained microbial cells of various morphologies detected by confocal laser scanning microscopy were homogeneously distributed in all iron snow samples. The dominant iron mineral appeared to be schwertmannite, with shorter needles in the northern than in the central basin samples. Total bacterial 16S rRNA gene copies ranged from 5.0 × 10(8) copies g (dry weight)(-1) in the acidic central lake basin (pH 3.3) to 4.0 × 10(10) copies g (dry weight)(-1) in the less acidic (pH 5.9) northern basin. Total RNA-based quantitative PCR assigned up to 61% of metabolically active microbial communities to Fe-oxidizing- and Fe-reducing-related bacteria, indicating that iron metabolism was an important metabolic strategy. Molecular identification of abundant groups suggested that iron snow surfaces were formed by chemoautotrophic iron oxidizers, such as Acidimicrobium, Ferrovum, Acidithiobacillus, Thiobacillus, and Chlorobium, in the redoxcline and were rapidly colonized by heterotrophic iron reducers, such as Acidiphilium, Albidiferax-like, and Geobacter-like groups. Metaproteomics yielded 283 different proteins from northern basin iron snow samples, and protein identification provided a glimpse into some of their in situ metabolic processes, such as primary production (CO2 fixation), respiration, motility, and survival strategies.

Strain-resolved community proteomics reveals recombining genomes of acidophilic bacteria.

Microbes comprise the majority of extant organisms, yet much remains to be learned about the nature and driving forces of microbial diversification. Our understanding of how microorganisms adapt and evolve can be advanced by genome-wide documentation of the patterns of genetic exchange, particularly if analyses target coexisting members of natural communities. Here we use community genomic data sets to identify, with strain specificity, expressed proteins from the dominant member of a genomically uncharacterized, natural, acidophilic biofilm. Proteomics results reveal a genome shaped by recombination involving chromosomal regions of tens to hundreds of kilobases long that are derived from two closely related bacterial populations. Inter-population genetic exchange was confirmed by multilocus sequence typing of isolates and of uncultivated natural consortia. The findings suggest that exchange of large blocks of gene variants is crucial for the adaptation to specific ecological niches within the very acidic, metal-rich environment. Mass-spectrometry-based discrimination of expressed protein products that differ by as little as a single amino acid enables us to distinguish the behaviour of closely related coexisting organisms. This is important, given that microorganisms grouped together as a single species may have quite distinct roles in natural systems and their interactions might be key to ecosystem optimization. Because proteomic data simultaneously convey information about genome type and activity, strain-resolved community proteomics is an important complement to cultivation-independent genomic (metagenomic) analysis of microorganisms in the natural environment.

Exploring the role of plant lysin motif receptor-like kinases in regulating plant-microbe interactions in the bioenergy crop Populus.

For plants, distinguishing between mutualistic and pathogenic microbes is a matter of survival. All microbes contain microbe-associated molecular patterns (MAMPs) that are perceived by plant pattern recognition receptors (PRRs). Lysin motif receptor-like kinases (LysM-RLKs) are PRRs attuned for binding and triggering a response to specific MAMPs, including chitin oligomers (COs) in fungi, lipo-chitooligosaccharides (LCOs), which are produced by mycorrhizal fungi and nitrogen-fixing rhizobial bacteria, and peptidoglycan in bacteria. The identification and characterization of LysM-RLKs in candidate bioenergy crops including Populus are limited compared to other model plant species, thus inhibiting our ability to both understand and engineer microbe-mediated gains in plant productivity. As such, we performed a sequence analysis of LysM-RLKs in the Populus genome and predicted their function based on phylogenetic analysis with known LysM-RLKs. Then, using predictive models, molecular dynamics simulations, and comparative structural analysis with previously characterized CO and LCO plant receptors, we identified probable ligand-binding sites in Populus LysM-RLKs. Using several machine learning models, we predicted remarkably consistent binding affinity rankings of Populus proteins to CO. In addition, we used a modified Random Walk with Restart network-topology based approach to identify a subset of Populus LysM-RLKs that are functionally related and propose a corresponding signal transduction cascade. Our findings provide the first look into the role of LysM-RLKs in Populus-microbe interactions and establish a crucial jumping-off point for future research efforts to understand specificity and redundancy in microbial perception mechanisms.

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.

Defining the boundaries and characterizing the landscape of functional genome expression in vascular tissues of Populus using shotgun proteomics.

Current state-of-the-art experimental and computational proteomic approaches were integrated to obtain a comprehensive protein profile of Populus vascular tissue. This featured: (1) a large sample set consisting of two genotypes grown under normal and tension stress conditions, (2) bioinformatics clustering to effectively handle gene duplication, and (3) an informatics approach to track and identify single amino acid polymorphisms (SAAPs). By applying a clustering algorithm to the Populus database, the number of protein entries decreased from 64,689 proteins to a total of 43,069 protein groups, thereby reducing 7505 identified proteins to a total of 4226 protein groups, in which 2016 were singletons. This reduction implies that ∼50% of the measured proteins shared extensive sequence homology. Using conservative search criteria, we were able to identify 1354 peptides containing a SAAP and 201 peptides that become tryptic due to a K or R substitution. These newly identified peptides correspond to 502 proteins, including 97 previously unidentified proteins. In total, the integration of deep proteome measurements on an extensive sample set with protein clustering and peptide sequence variants provided an exceptional level of proteome characterization for Populus, allowing us to spatially resolve the vascular tissue proteome.

Heterotrophic archaea contribute to carbon cycling in low-pH, suboxic biofilm communities.

Archaea are widely distributed and yet are most often not the most abundant members of microbial communities. Here, we document a transition from Bacteria- to Archaea-dominated communities in microbial biofilms sampled from the Richmond Mine acid mine drainage (AMD) system (∼pH 1.0, ∼38°C) and in laboratory-cultivated biofilms. This transition occurs when chemoautotrophic microbial communities that develop at the air-solution interface sink to the sediment-solution interface and degrade under microaerobic and anaerobic conditions. The archaea identified in these sunken biofilms are from the class Thermoplasmata, and in some cases, the highly divergent ARMAN nanoarchaeal lineage. In several of the sunken biofilms, nanoarchaea comprise 10 to 25% of the community, based on fluorescent in situ hybridization and metagenomic analyses. Comparative community proteomic analyses show a persistence of bacterial proteins in sunken biofilms, but there is clear evidence for amino acid modifications due to acid hydrolysis. Given the low representation of bacterial cells in sunken biofilms based on microscopy, we infer that hydrolysis reflects proteins derived from lysed cells. For archaea, we detected ∼2,400 distinct proteins, including a subset involved in proteolysis and peptide uptake. Laboratory cultivation experiments using complex carbon substrates demonstrated anaerobic enrichment of Ferroplasma and Aplasma coupled to the reduction of ferric iron. These findings indicate dominance of acidophilic archaea in degrading biofilms and suggest that they play roles in anaerobic nutrient cycling at low pH.

Global transcriptomic and proteomic responses of Dehalococcoides ethenogenes strain 195 to fixed nitrogen limitation.

Bacteria of the genus Dehalococcoides play an important role in the reductive dechlorination of chlorinated ethenes. A systems-level approach was taken in this study to examine the global transcriptomic and proteomic responses of exponentially growing cells of Dehalococcoides ethenogenes strain 195 to fixed nitrogen limitation (FNL), as dechlorination activity and cell yield both decrease during FNL. As expected, the nitrogen-fixing (nif) genes were differentially upregulated in the transcriptome and proteome of strain 195 during FNL. Aside from the nif operon, a putative methylglyoxal synthase-encoding gene (DET1576), the product of which is predicted to catalyze the formation of the toxic electrophile methylglyoxal and is implicated in the uncoupling of anabolism from catabolism in bacteria, was strongly upregulated in the transcriptome and could potentially play a role in the observed growth inhibition during FNL. Carbon catabolism genes were generally downregulated in response to FNL, and a number of transporters were differentially regulated in response to nitrogen limitation, with some playing apparent roles in nitrogen acquisition, while others were associated with general stress responses. A number of genes related to the functions of nucleotide synthesis, replication, transcription, translation, and posttranslational modifications were also differentially expressed. One gene coding for a putative reductive dehalogenase (DET1545) and a number of genes coding for oxidoreductases, which have implications in energy generation and redox reactions, were also differentially regulated. Interestingly, most of the genes within the multiple integrated elements were not differentially expressed. Overall, this study elucidates the molecular responses of strain 195 to FNL and identifies differentially expressed genes that are potential biomarkers to evaluate environmental cellular nitrogen status.

Shotgun proteomic monitoring of Clostridium acetobutylicum during stationary phase of butanol fermentation using xylose and comparison with the exponential phase.

Economically viable production of solvents through acetone-butanol-ethanol (ABE) fermentation requires a detailed understanding of Clostridium acetobutylicum. This study focuses on the proteomic profiling of C. acetobutylicum ATCC 824 from the stationary phase of ABE fermentation using xylose and compares with the exponential growth by shotgun proteomics approach. Comparative proteomic analysis revealed 22.9% of the C. acetobutylicum genome and 18.6% was found to be common in both exponential and stationary phases. The proteomic profile of C. acetobutylicum changed during the ABE fermentation such that 17 proteins were significantly differentially expressed between the two phases. Specifically, the expression of five proteins namely, CAC2873, CAP0164, CAP0165, CAC3298, and CAC1742 involved in the solvent production pathway were found to be significantly lower in the stationary phase compared to the exponential growth. Similarly, the expression of fucose isomerase (CAC2610), xylulose kinase (CAC2612), and a putative uncharacterized protein (CAC2611) involved in the xylose utilization pathway were also significantly lower in the stationary phase. These findings provide an insight into the metabolic behavior of C. acetobutylicum between different phases of ABE fermentation using xylose.

Evaluating the performance of random forest and iterative random forest based methods when applied to gene expression data.

Gene-to-gene networks, such as Gene Regulatory Networks (GRN) and Predictive Expression Networks (PEN) capture relationships between genes and are beneficial for use in downstream biological analyses. There exists multiple network inference tools to produce these gene-to-gene networks from matrices of gene expression data. Random Forest-Leave One Out Prediction (RF-LOOP) is a method that has been shown to be efficient at producing these gene-to-gene networks, frequently known as GEne Network Inference with Ensemble of trees (GENIE3). Random Forest can be replaced in this process by iterative Random Forest (iRF), which performs variable selection and boosting. Here we validate that iterative Random Forest-Leave One Out Prediction (iRF-LOOP) produces higher quality networks than GENIE3 (RF-LOOP). We use both synthetic and empirical networks from the Dialogue for Reverse Engineering Assessment and Methods (DREAM) Challenges by Sage Bionetworks, as well as two additional empirical networks created from Arabidopsis thaliana and Populus trichocarpa expression data.