Integrated Application of Multiomics Strategies Provides Insights Into the Environmental Hypoxia Response in Pelteobagrus vachelli Muscle

Increasing pressures on aquatic ecosystems because of pollutants, nutrient enrichment, and global warming have severely depleted oxygen concentrations. This sudden and significant lack of oxygen has resulted in persistent increases in fish mortality rates. Revealing the molecular mechanism of fish hypoxia adaptation will help researchers to find markers for hypoxia induced by environmental stress. Here, we used a multiomics approach to identify several hypoxia-associated miRNAs, mRNAs, proteins, and metabolites involved in diverse biological pathways in the muscles of Pelteobagrus vachelli. Our findings revealed significant hypoxia-associated changes in muscles over 4 h of hypoxia exposure and discrete tissue-specific patterns. We have previously reported that P. vachelli livers exhibit increased anaerobic glycolysis, heme synthesis, erythropoiesis, and inhibit apoptosis when exposed to hypoxia for 4 h. However, the opposite was observed in muscles. According to our comprehensive analysis, fishes show an acute response to hypoxia, including activation of catabolic pathways to generate more energy, reduction of biosynthesis to decrease energy consumption, and shifting from aerobic to anaerobic metabolic contributions. Also, we found that hypoxia induced muscle dysfunction by impairing mitochondrial function, activating inflammasomes, and apoptosis. The hypoxia-induced mitochondrial dysfunction enhanced oxidative stress, apoptosis, and further triggered interleukin-1β production via inflammasome activation. In turn, interleukin-1β further impaired mitochondrial function or apoptosis by suppressing downstream mitochondrial biosynthesis–related proteins, thus resulting in a vicious cycle of inflammasome activation and mitochondrial dysfunction. Our findings contribute meaningful insights into the molecular mechanisms of hypoxia, and the methods and study design can be utilized across different fish species.     

The effects of changing climate on faunal depth distributions determine winners and losers

Changing climate is predicted to impact all depths of the global oceans, yet projections of range shifts in marine faunal distributions in response to changing climate seldom evaluate potential shifts in depth distribution. Marine ectotherms' thermal tolerance is limited by their ability to maintain aerobic metabolism (oxygen‐ and capacity‐limited tolerance), and is functionally associated with their hypoxia tolerance. Shallow‐water (<200 m depth) marine invertebrates and fishes demonstrate limited tolerance of increasing hydrostatic pressure (pressure exerted by the overlying mass of water), and hyperbaric (increased pressure) tolerance is proposed to depend on the ability to maintain aerobic metabolism, too. Here, we report significant correlation between the hypoxia thresholds and the hyperbaric thresholds of taxonomic groups of shallow‐water fauna, suggesting that pressure tolerance is indeed oxygen limited. Consequently, it appears that the combined effects of temperature, pressure and oxygen concentration constrain the fundamental ecological niches (FENs) of marine invertebrates and fishes. Including depth in a conceptual model of oxygen‐ and capacity‐limited FENs' responses to ocean warming and deoxygenation confirms previous predictions made based solely on consideration of the latitudinal effects of ocean warming (e.g. Cheung et al., 2009), that polar taxa are most vulnerable to the effects of climate change, with Arctic fauna experiencing the greatest FEN contraction. In contrast, the inclusion of depth in the conceptual model reveals for the first time that temperate fauna as well as tropical fauna may experience substantial FEN expansion with ocean warming and deoxygenation, rather than FEN maintenance or contraction suggested by solely considering latitudinal range shifts.     

Comparative proteomic analysis of biofilm and planktonic cells of Lactobacillus plantarum DB200

This study investigated the relative abundance of extracellular and cell wall associated proteins (exoproteome), cytoplasmic proteins (proteome), and related phenotypic traits of Lactobacillus plantarum grown under planktonic and biofilm conditions. Lactobacillus plantarum DB200 was preliminarily selected due to its ability to form biofilms and to adhere to Caco2 cells. As shown by fluorescence microscope analysis, biofilm cells became longer and autoaggregated at higher levels than planktonic cells. The molar ratio between glucose consumed and lactate synthesised was markedly decreased under biofilm compared to planktonic conditions. DIGE analysis showed a differential exoproteome (115 protein spots) and proteome (44) between planktonic and biofilm L. plantarum DB200 cells. Proteins up‐ or downregulated by at least twofold (p < 0.05) were found to belong mainly to the following functional categories: cell wall and catabolic process, cell cycle and adhesion, transport, glycolysis and carbohydrate metabolism, exopolysaccharide metabolism, amino acid and protein metabolisms, fatty acid and lipid biosynthesis, purine and nucleotide metabolism, stress response, oxidation/reduction process, and energy metabolism. Many of the above proteins showed moonlighting behavior. In accordance with the high expression levels of stress proteins (e.g., DnaK, GroEL, ClpP, GroES, and catalase), biofilm cells demonstrated enhanced survival under conditions of environmental stress.     

Comprehensive proteome profiling of the Fe(III)‐reducing myxobacterium Anaeromyxobacter dehalogenans 2CP‐C during growth with fumarate and ferric citrate

Anaeromyxobacter dehalogenans is a microaerophilic member of the delta‐proteobacteria which is able to utilize a wide range of electron acceptors, including halogenated phenols, U(VI), Fe(III), nitrate, nitrite, oxygen and fumarate. To date, the knowledge regarding general metabolic activities of this ecologically relevant bacterium is limited. Here, we present a first systematic 2‐D reference map of the soluble A. dehalogenans proteome in order to provide a sound basis for further proteomic studies as well as to gain first global insights into the metabolic activities of this bacterium. Using a combination of 2‐DE and MALDI‐TOF‐MS, a total of 720 proteins spots were identified, representing 559 unique protein species. Using the proteome data, altogether 50 metabolic pathways were found to be expressed during growth with fumarate as primary electron acceptor. An analysis of the pathways revealed an extensive display of enzymes involved in the catabolism and anabolism of a variety of amino acids, including the unexpected fermentation of lysine to butyrate. Moreover, using the reference gel as basis, a semi‐quantitative analysis of protein expression changes of A. dehalogenans during growth with ferric citrate as electron acceptor was conducted. The adaptation to Fe(III) reducing conditions involved the expression changes of a total of 239 proteins. The results suggest that the adaptation to Fe(III) reductive conditions involves an increase in metabolic flux through the tricarboxylic acid cycle, which is fueled by an increased catabolism of amino acids.     

Metabolic adaptation of Chlamydia trachomatis to mammalian host cells

Metabolic adaptation is a key feature for the virulence of pathogenic intracellular bacteria. Nevertheless, little is known about the pathways in adapting the bacterial metabolism to multiple carbon sources available from the host cell. To analyze the metabolic adaptation of the obligate intracellular human pathogen Chlamydia trachomatis, we labeled infected HeLa or Caco‐2 cells with ¹³C‐marked glucose, glutamine, malate or a mix of amino acids as tracers. Comparative GC‐MS‐based isotopologue analysis of protein‐derived amino acids from the host cell and the bacterial fraction showed that C. trachomatis efficiently imported amino acids from the host cell for protein biosynthesis. FT‐ICR‐MS analyses also demonstrated that label from exogenous ¹³C‐glucose was efficiently shuffled into chlamydial lipopolysaccharide probably via glucose 6‐phosphate of the host cell. Minor fractions of bacterial Ala, Asp, and Glu were made de novo probably using dicarboxylates from the citrate cycle of the host cell. Indeed, exogenous ¹³C‐malate was efficiently taken up by C. trachomatis and metabolized into fumarate and succinate when the bacteria were kept in axenic medium containing the malate tracer. Together, the data indicate co‐substrate usage of intracellular C. trachomatis in a stream‐lined bipartite metabolism with host cell‐supplied amino acids for protein biosynthesis, host cell‐provided glucose 6‐phosphate for cell wall biosynthesis, and, to some extent, one or more host cell‐derived dicarboxylates, e.g. malate, feeding the partial TCA cycle of the bacterium. The latter flux could also support the biosynthesis of meso‐2,6‐diaminopimelate required for the formation of chlamydial peptidoglycan.     

Differential Proteomic Analysis of Dimethylnitrosamine (DMN)‐Induced Liver Fibrosis

Liver fibrosis is a common pathological feature of many chronic liver diseases. To characterize the entire panorama of proteome changes in dimethylnitrosamine (DMN)‐induced liver fibrosis, isobaric tags for relative and absolute quantitation (iTRAQ)‐based differential proteomic analysis is performed with DMN‐induced liver fibrosis rats. A total of 4155 confidently identified proteins are found, with 365 proteins showing significant changes (fold changes of >1.5 or < 0.67, p < 0.05). In metabolic activation, proteins assigned to drug metabolism enzymes (e.g., CYP2D1) change, suggesting that the liver protection mechanism is activated to relieve DMN toxicity. In addition, the altered proteins of immune response and oxidative stress may activate hepatic stellate cells. Glucose metabolism disorder in DMN model rats is demonstrated by a decrease in key enzymes (e.g., ACSL1) in fatty acid metabolism, a tricabolic acid cycle‐related enzyme (SDH), glycogenolysis enzyme, and gluconeogenesis enzymes (PC, PCKGC) and by an increase in glycolysis enzymes (e.g., HXK1). Meanwhile, alterations in iron and calcium ion homeostasis proteins are observed. Our results also show that mitochondrial dysfunction may be involved in DMN hepatotoxicity. In conclusion, these altered liver proteins in the DMN model and control rats provide data for understanding the functional mechanism of liver fibrosis.     

Evolutionarily Conserved Optimization of Amino Acid Biosynthesis

The “cognate bias hypothesis” states that early in evolutionary history the biosynthetic enzymes for amino acid x gradually lost residues of x, thereby reducing the threshold for deleterious effects of x scarcity. The resulting reduction in cognate amino acid composition of the enzymes comprising a particular amino acid biosynthetic pathway is predicted to confer a selective growth advantage on cells. Bioinformatic evidence from protein-sequence data of two bacterial species previously demonstrated reduced cognate bias in amino acid biosynthetic pathways. Here we show that cognate bias in amino acid biosynthesis is present in the other domains of life—Archaebacteria and Eukaryota. We also observe evolutionarily conserved underrepresentations (e.g., glycine in methionine biosynthesis) and overrepresentations (e.g., tryptophan in asparagine biosynthesis) of amino acids in noncognate biosynthetic pathways, which can be explained by secondary amino acid metabolism. Additionally, we experimentally validate the cognate bias hypothesis using the yeast Saccharomyces cerevisiae. Specifically, we show that the degree to which growth declines following amino acid deprivation is negatively correlated with the degree to which an amino acid is underrepresented in the enzymes that comprise its cognate biosynthetic pathway. Moreover, we demonstrate that cognate fold representation is more predictive of growth advantage than a host of other potential growth-limiting factors, including an amino acid’s metabolic cost or its intracellular concentration and compartmental distribution. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Reviewing Editor: Dr. Niles Lehman Ethan O. Perlstein and Benjamin L. de Bivort contributed equally to this work.     

High‐coverage proteomics reveals methionine auxotrophy in Deinococcus radiodurans

Deinococcus radiodurans is a robust bacterium best known for its capacity to resist to radiation. In this study, the SDS‐PAGE coupled with high‐precision LC‐MS/MS was used to study the D. radiodurans proteome. A total of 1951 proteins were identified which covers 63.18% protein‐coding genes. Comparison of the identified proteins to the key enzymes in amino acid biosyntheses from KEGG database showed the methionine biosynthesis module is incomplete while other amino acid biosynthesis modules are complete, which indicated methionine auxotrophy in D. radiodurans. The subsequent amino acid‐auxotrophic screening has verified methionine instead of other amino acids is essential for the growth of D. radiodurans. With molecular evolutionary genetic analysis, we found the divergence in methionine biosynthesis during the evolution of the common ancestor of bacteria. We also found D. radiodurans lost the power of synthesizing methionine because of the missing metA and metX in two types of methionine biosyntheses. For the first time, this study used high‐coverage proteome analysis to identify D. radiodurans amino acid auxotrophy, which provides the important reference for the development of quantitative proteomics analysis using stable isotope labeling in metabolomics of D. radiodurans and in‐depth analysis of the molecular mechanism of radiation resistance.     

System level analysis of cacao seed ripening reveals a sequential interplay of primary and secondary metabolism leading to polyphenol accumulation and preparation of stress resistance

Theobroma cacao and its popular product, chocolate, are attracting attention due to potential health benefits including antioxidative effects by polyphenols, anti‐depressant effects by high serotonin levels, inhibition of platelet aggregation and prevention of obesity‐dependent insulin resistance. The development of cacao seeds during fruit ripening is the most crucial process for the accumulation of these compounds. In this study, we analyzed the primary and the secondary metabolome as well as the proteome during Theobroma cacao cv. Forastero seed development by applying an integrative extraction protocol. The combination of multivariate statistics and mathematical modelling revealed a complex consecutive coordination of primary and secondary metabolism and corresponding pathways. Tricarboxylic acid (TCA) cycle and aromatic amino acid metabolism dominated during the early developmental stages (stages 1 and 2; cell division and expansion phase). This was accompanied with a significant shift of proteins from phenylpropanoid metabolism to flavonoid biosynthesis. At stage 3 (reserve accumulation phase), metabolism of sucrose switched from hydrolysis into raffinose synthesis. Lipids as well as proteins involved in lipid metabolism increased whereas amino acids and N‐phenylpropenoyl amino acids decreased. Purine alkaloids, polyphenols, and raffinose as well as proteins involved in abiotic and biotic stress accumulated at stage 4 (maturation phase) endowing cacao seeds the characteristic astringent taste and resistance to stress. In summary, metabolic key points of cacao seed development comprise the sequential coordination of primary metabolites, phenylpropanoid, N‐phenylpropenoyl amino acid, serotonin, lipid and polyphenol metabolism thereby covering the major compound classes involved in cacao aroma and health benefits.     

Expression of a Brassica Isopropylmalate Synthase Gene in Arabidopsis Perturbs Both Glucosinolate and Amino Acid Metabolism

Isopropylmalate synthase (IPMS) is a key enzyme in the biosynthesis of the essential amino acid leucine, and thus primary metabolism. In Arabidopsis, the functionally similar enzyme, methythiolalkylmalate synthase (MAM), is an important enzyme in the elongation of methionine prior to glucosinolate (GSL) biosynthesis, as part of secondary metabolism. We describe the cloning of an IPMS gene from Brassica, BatIMS, and its functional characterisation by heterologous expression in E. coli and Arabidopsis. Over expression of BatIMS in Arabidopsis resulted in plants with an aberrant phenotype, reminiscent of mutants in GSL biosynthesis. Metabolite analyses showed that these plants had both perturbed amino acid metabolism and enhanced levels of GSLs. Microarray profiling showed that BatIMS over expression caused up regulation of the genes for methionine-derived GSL biosynthesis, and down regulation of genes involved in leucine catabolism, in addition to perturbed expression of genes involved in auxin and ethylene metabolism. The results illustrate the cross talk that can occur between primary and secondary metabolism within transgenic plants. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Biotechnological production of polyamines by bacteria: recent achievements and future perspectives

In Bacteria, the pathways of polyamine biosynthesis start with the amino acids l-lysine, l-ornithine, l-arginine, or l-aspartic acid. Some of these polyamines are of special interest due to their use in the production of engineering plastics (e.g., polyamides) or as curing agents in polymer applications. At present, the polyamines for industrial use are mainly synthesized on chemical routes. However, since a commercial market for polyamines as well as an industry for the fermentative production of amino acid exist, and since bacterial strains overproducing the polyamine precursors l-lysine, l-ornithine, and l-arginine are known, it was envisioned to engineer these amino acid-producing strains for polyamine production. Only recently, researchers have investigated the potential of amino acid-producing strains of Corynebacterium glutamicum and Escherichia coli for polyamine production. This mini-review illustrates the current knowledge of polyamine metabolism in Bacteria, including anabolism, catabolism, uptake, and excretion. The recent advances in engineering the industrial model bacteria C. glutamicum and E. coli for efficient production of the most promising polyamines, putrescine (1,4-diaminobutane), and cadaverine (1,5-diaminopentane), are discussed in more detail.     

Strain engineering for high‐level 5‐aminolevulinic acid production in Escherichia coli

Herein, we report the development of a microbial bioprocess for high‐level production of 5‐aminolevulinic acid (5‐ALA), a valuable non‐proteinogenic amino acid with multiple applications in medical, agricultural, and food industries, using Escherichia coli as a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5‐ALA biosynthesis in E. coli. To reduce, but not to abolish, the carbon flux toward essential tetrapyrrole/porphyrin biosynthesis, we applied clustered regularly interspersed short palindromic repeats interference (CRISPRi) to repress hemB expression, leading to extracellular 5‐ALA accumulation. We then applied metabolic engineering strategies to direct more dissimilated carbon flux toward the key precursor of succinyl‐CoA for enhanced 5‐ALA biosynthesis. Using these engineered E. coli strains for bioreactor cultivation, we successfully demonstrated high‐level 5‐ALA biosynthesis from glycerol (~30 g L^?1) under both microaerobic and aerobic conditions, achieving up to 5.95 g L^?1 (36.9% of the theoretical maximum yield) and 6.93 g L^?1 (50.9% of the theoretical maximum yield) 5‐ALA, respectively. This study represents one of the most effective bio‐based production of 5‐ALA from a structurally unrelated carbon to date, highlighting the importance of integrated strain engineering and bioprocessing strategies to enhance bio‐based production.     

A proteomic analysis of rice seed germination as affected by high temperature and ABA treatment

Seed germination is a critical phase in the plant life cycle, but the specific events associated with seed germination are still not fully understood. In this study, we used two‐dimensional gel electrophoresis followed by mass spectrometry to investigate the changes in the proteome during imbibition of Oryza sativa seeds at optimal temperature with or without abscisic acid (ABA) and high temperature (germination thermoinhibition) to further identify and quantify key proteins required for seed germination. A total of 121 protein spots showed a significant change in abundance (1.5‐fold increase/decrease) during germination under all conditions. Among these proteins, we found seven proteins specifically associated with seed germination including glycosyl hydrolases family 38 protein, granule‐bound starch synthase 1, Os03g0842900 (putative steroleosin‐B), N‐carbamoylputrescine amidase, spermidine synthase 1, tubulin α‐1 chain and glutelin type‐A; and a total of 20 imbibition response proteins involved in energy metabolism, cell growth, cell defense and storage proteins. High temperature inhibited seed germination by decreasing the abundance of proteins involved in methionine metabolism, amino acid biosynthesis, energy metabolism, reserve degradation, protein folding and stress responses. ABA treatment inhibited germination and decreased the abundance of proteins associated with methionine metabolism, energy production and cell division. Our results show that changes in many biological processes including energy metabolism, protein synthesis and cell defense and rescue occurred as a result of all treatments, while enzymes involved in methionine metabolism and weakening of cell wall specifically accumulated when the seeds germinated at the optimal temperature.     

Physiological refugia: swamps,hypoxia tolerance and maintenance of fish diversity in the Lake Victoria region

In Lake Nabugabo, Uganda, a satellite of Lake Victoria, approximately 50% of the indigenous fishes disappeared from the open waters subsequent to the establishment of the introduced predatory Nile perch, Lates niloticus. This pattern is similar to the faunal loss experienced in the much larger Lake Victoria. Several of these species persisted in wetland refugia (e.g. ecotonal wetlands, swamp lagoons); however, deep swamp refugia (habitats lying well within the dense interior of fringing wetlands), are available only to a subset of the basin fauna with extreme tolerance to hypoxia. Although air-breathers are common in deep swamp refugia; we also documented a surprisingly high richness and abundance of non-air-breathing fishes. We describe several mechanisms that may facilitate survival in deep swamp refugia including high hemoglobin concentration, high hematocrit, large gill surface area and a low critical oxygen tension (P(c)). In addition, swamp-dwelling fishes showed lower PO(2) thresholds for onset of aquatic surface respiration than the lake-dwelling fishes. This suggests higher tolerance to hypoxia in the swamp fishes because they are able to withstand a lower oxygen tension before approaching the surface. We suggest that physiological refugia may be important in modulating the impact of Nile perch and indigenous fishes in the Lake Nabugabo region; this highlights the need to evaluate relative tolerance of introduced predators and indigenous prey to environmental stressors.     

Arsenic hyperaccumulation induces metabolic reprogramming in Pityrogramma calomelanos to reduce oxidative stress

Arsenic (As) pollution is a major environmental concern due to its worldwide distribution and high toxicity to organisms. The fern Pityrogramma calomelanos is one of the few plant species known to be able to hyperaccumulate As, although the mechanisms involved are largely unknown. This study aimed to investigate the metabolic adjustments involved in the As‐tolerance of P. calomelanos. For this purpose, ferns with five to seven fronds were exposed to a series of As concentrations. Young fronds were used for biochemical analysis and metabolite profiling using gas chromatography–mass spectrometry. As treatment increased the total concentration of proteins and soluble phenols, enhanced peroxidase activities, and promoted disturbances in nitrogen and carbon metabolism. The reduction of the glucose pool was one of the striking responses to As. Remarkable changes in amino acids levels were observed in As‐treated plants, including those related to biosynthesis of glutathione and phenols, osmoregulation and two photorespiratory intermediates. In addition, increases in polyamines levels and antioxidant enzyme activities were observed. In summary, this study indicates that P. calomelanos tolerates high concentration of As due to its capacity to upregulate biosynthesis of amino acids and antioxidants, without greatly disturbing central carbon metabolism. At extremely high As concentrations, however, this protective mechanism fails to block reactive oxygen species production, leading to lipid peroxidation and leaf necrosis.