Phthalates Boost Natural Transformation of Extracellular Antibiotic Resistance Genes through Enhancing Bacterial Motility and DNA Environmental Persistence.

The environmental dissemination of extracellular antibiotic resistance genes (eARGs) in wastewater and natural water bodies has aroused growing ecological concerns. The coexisting chemical pollutants in water are known to markedly affect the eARGs transfer behaviors of the environmental microbial community, but the detailed interactions and specific impacts remain elusive so far. Here, we revealed a concentration-dependent impact of dimethyl phthalate (DMP) and several other types of phthalate esters (common water pollutants released from plastics) on the natural transformation of eARGs. The DMP exposure at an environmentally relevant concentration (10 µg/L) resulted in a 4.8-times raised transformation frequency of Acinetobacter baylyi but severely suppressed the transformation at a high concentration (1000 µg/L). The promotion by low-concentration DMP was attributed to multiple mechanisms, including increased bacterial mobility and membrane permeability to facilitate eARGs uptake and improved resistance of the DMP-bounded eARGs (via noncovalent interaction) to enzymatic degradation (with suppressed DNase activity). Similar promoting effects of DMP on the eARGs transformation were also found in real wastewater and biofilm systems. In contrast, higher-concentration DMP suppressed the eARGs transformation by disrupting the DNA structure. Our findings highlight a potentially underestimated eARGs spreading in aquatic environments due to the impacts of coexisting chemical pollutants and deepen our understanding of the risks of biological-chemical combined pollution in wastewater and environmental water bodies.

Optimization strategies for CO2 biological fixation.

Global sustainable development faces a significant challenge in effectively utilizing CO2. Meanwhile, CO2 biological fixation offers a promising solution. CO2 has the highest oxidation state (+4 valence state), whereas typical multi­carbon chemicals have lower valence states. The Gibbs free energy (ΔG) changes of CO2 reductive reactions are generally positive and this renders it necessary to input different forms of energy. Although biological carbon fixation processes are friendly to operate, the thermodynamic obstacles must be overcome. To make this reaction occur favorably and efficiently, diverse strategies to enhance CO2 biological fixation efficiency have been proposed by numerous researchers. This article reviews recent advances in optimizing CO2 biological fixation and intends to provide new insights into achieving efficient biological utilization of CO2. It first outlines the thermodynamic characteristics of diverse carbon fixation reactions and proposes optimization directions for CO2 biological fixation. A comprehensive overview of the catalytic mechanisms, optimization strategies, and challenges encountered by common carbon-fixing enzymes is then provided. Subsequently, potential routes for improving the efficiency of biological carbon fixation are discussed, including the ATP supply, reducing power supply, energy supply, reactor design, and carbon enrichment system modules. In addition, effective artificial carbon fixation pathways were summarized and analyzed. Finally, prospects are made for the research direction of continuously improving the efficiency of biological carbon fixation.

Ca2+ pushes and pulls energetics to maintain ATP balance in atrial cells: computational insights.

To maintain atrial function, ATP supply-to-demand matching must be tightly controlled. Ca2+ can modulate both energy consumption and production. In light of evidence suggesting that Ca2+ affects energetics through "push" (activating metabolite flux and enzymes in the Krebs cycle to push the redox flux) and "pull" (acting directly on ATP synthase and driving the redox flux through the electron transport chain and increasing ATP production) pathways, we investigated whether both pathways are necessary to maintain atrial ATP supply-to-demand matching. Rabbit right atrial cells were electrically stimulated at different rates, and oxygen consumption and flavoprotein fluorescence were measured. To gain mechanistic insight into the regulators of ATP supply-to-demand matching in atrial cells, models of atrial electrophysiology, Ca2+ cycling and force were integrated with a model of mitochondrial Ca2+ and a modified model of mitochondrial energy metabolism. The experimental results showed that oxygen consumption increased in response to increases in the electrical stimulation rate. The model reproduced these findings and predicted that the increase in oxygen consumption is associated with metabolic homeostasis. The model predicted that Ca2+ must act both in "push" and "pull" pathways to increase oxygen consumption. In contrast to ventricular trabeculae, no rapid time-dependent changes in mitochondrial flavoprotein fluorescence were measured upon an abrupt change in workload. The model reproduced these findings and predicted that the maintenance of metabolic homeostasis is due to the effects of Ca2+ on ATP production. Taken together, this work provides evidence of Ca2+ "push" and "pull" activity to maintain metabolic homeostasis in atrial cells.

Oligodendrocyte-derived transcellular signaling regulates axonal energy metabolism.

The unique morphology and functionality of central nervous system (CNS) neurons necessitate specialized mechanisms to maintain energy metabolism throughout long axons and extensive terminals. Oligodendrocytes (OLs) enwrap CNS axons with myelin sheaths in a multilamellar fashion. Apart from their well-established function in action potential propagation, OLs also provide intercellular metabolic support to axons by transferring energy metabolites and delivering exosomes consisting of proteins, lipids, and RNAs. OL-derived metabolic support is crucial for the maintenance of axonal integrity; its dysfunction has emerged as an important player in neurological disorders that are associated with axonal energy deficits and degeneration. In this review, we discuss recent advances in how these transcellular signaling pathways maintain axonal energy metabolism in health and neurological disorders.

Mitochondrial Dysfunction in Cardiac Arrhythmias.

Electrophysiological and structural disruptions in cardiac arrhythmias are closely related to mitochondrial dysfunction. Mitochondria are an organelle generating ATP, thereby satisfying the energy demand of the incessant electrical activity in the heart. In arrhythmias, the homeostatic supply-demand relationship is impaired, which is often accompanied by progressive mitochondrial dysfunction leading to reduced ATP production and elevated reactive oxidative species generation. Furthermore, ion homeostasis, membrane excitability, and cardiac structure can be disrupted through pathological changes in gap junctions and inflammatory signaling, which results in impaired cardiac electrical homeostasis. Herein, we review the electrical and molecular mechanisms of cardiac arrhythmias, with a particular focus on mitochondrial dysfunction in ionic regulation and gap junction action. We provide an update on inherited and acquired mitochondrial dysfunction to explore the pathophysiology of different types of arrhythmias. In addition, we highlight the role of mitochondria in bradyarrhythmia, including sinus node dysfunction and atrioventricular node dysfunction. Finally, we discuss how confounding factors, such as aging, gut microbiome, cardiac reperfusion injury, and electrical stimulation, modulate mitochondrial function and cause tachyarrhythmia.

Estimation of energy pathway fluxes in cancer cells - Beyond the Warburg effect.

Glycolytic and respiratory fluxes were analyzed in cancer and non-cancer cells. The steady-state fluxes in energy metabolism were used to estimate the contributions of aerobic glycolytic and oxidative phosphorylation (OxPhos) pathways to the cellular ATP supply. The rate of lactate production - corrected for the fraction generated by glutaminolysis - is proposed as the appropriate way to estimate glycolytic flux. In general, the glycolytic rates estimated for cancer cells are higher than those found in non-cancer cells, as originally observed by Otto Warburg. The rate of basal or endogenous cellular O2 consumption corrected for non-ATP synthesizing O2 consumption, measured after inhibition by oligomycin (a specific, potent and permeable ATP synthase inhibitor), has been proposed as the appropriate way to estimate mitochondrial ATP synthesis-linked O2 flux or net OxPhos flux in living cells. Detecting non-negligible oligomycin-sensitive O2 consumption rates in cancer cells has revealed that the mitochondrial function is not impaired, as claimed by the Warburg effect. Furthermore, when calculating the relative contributions to cellular ATP supply, under a variety of environmental conditions and for different types of cancer cells, it was found that OxPhos pathway was the main ATP provider over glycolysis. Hence, OxPhos pathway targeting can be successfully used to block in cancer cells ATP-dependent processes such as migration. These observations may guide the re-design of novel targeted therapies.

Total Cellular ATP Production Changes With Primary Substrate in MCF7 Breast Cancer Cells.

Cancer growth is predicted to require substantial rates of substrate catabolism and ATP turnover to drive unrestricted biosynthesis and cell growth. While substrate limitation can dramatically alter cell behavior, the effects of substrate limitation on total cellular ATP production rate is poorly understood. Here, we show that MCF7 breast cancer cells, given different combinations of the common cell culture substrates glucose, glutamine, and pyruvate, display ATP production rates 1.6-fold higher than when cells are limited to each individual substrate. This increase occurred mainly through faster oxidative ATP production, with little to no increase in glycolytic ATP production. In comparison, non-transformed C2C12 myoblast cells show no change in ATP production rate when substrates are limited. In MCF7 cells, glutamine allows unexpected access to oxidative capacity that pyruvate, also a strictly oxidized substrate, does not. Pyruvate, when added with other exogenous substrates, increases substrate-driven oxidative ATP production, by increasing both ATP supply and demand. Overall, we find that MCF7 cells are highly flexible with respect to maintaining total cellular ATP production under different substrate-limited conditions, over an acute (within minutes) timeframe that is unlikely to result from more protracted (hours or more) transcription-driven changes to metabolic enzyme expression. The near-identical ATP production rates maintained by MCF7 and C2C12 cells given single substrates reveal a potential difficulty in using substrate limitation to selectively starve cancer cells of ATP. In contrast, the higher ATP production rate conferred by mixed substrates in MCF7 cells remains a potentially exploitable difference.

Regulation of intracellular ATP supply and its application in industrial biotechnology.

Efficient cell factories are the core of industrial biotechnology. In recent years, synthetic biology develops rapidly, and more and more modified microbial cell factories are employed in industrial biotechnology. ATP plays vital roles in biosynthesis, metabolism regulation, and cellular maintenance. Regulating cellular ATP supply can effectively modify cellular metabolism. This paper presents a review of recent studies on the regulation of the intracellular ATP supply and its application in industrial biotechnology. Detailed strategies for regulating the ATP supply and the resulting impact on bioproduction are introduced. It is observed that regulating the cellular ATP supply can provide great possibilities for making microbial cells into efficient factories. Future perspectives for further understanding the function of ATP are also discussed.

[Enhanced production of bacitracin via energy metabolism engineering in Bacillus licheniformis DW2].

Bacitracin is a broad-spectrum cyclic peptide antibiotic, and mainly produced by Bacillus. Energy metabolism plays as a critical role in high-level production of target metabolites. In this study, Bacillus licheniformis DW2, an industrial strain for bacitracin production, was served as the original strain. First, our results confirmed that elimination of cytochrome bd oxidase branch via deleting gene cydB benefited bacitracin synthesis. Bacitracin titer and ATP content were increased by 10.97% and 22.96%, compared with those of original strain, respectively. Then, strengthening cytochrome aa3 oxidase branch via overexpressing gene qoxA was conducive to bacitracin production. Bacitracin titer and ATP content were increased by 18.97% and 34.00%, respectively. In addition, strengthening ADP synthesis supply is also proven as an effective strategy to promote intracellular ATP accumulation, overexpression of adenosine kinase DcK and adenylate kinase AdK could all improve bacitracin titers, among which, dck overexpression strain showed the better performance, and bacitracin titer was increased by 16.78%. Based on the above individual methods, a method of combining the deletion of gene cydB and overexpression of genes qoxA, dck were used to enhance ATP content of cells to 39.54 nmol/L, increased by 49.32% compared to original strain, and bacitracin titer produced by the final strain DW2-CQD (DW2ΔcydB::qoxA::dck) was 954.25 U/mL, increased by 21.66%. The bacitracin titer produced per cell was 2.11 U/CFU, increased by 11.05%. Collectively, this study demonstrates that improving ATP content was an efficient strategy to improve bacitracin production, and a promising strain B. licheniformis DW2-CQD was attained for industrial production of bacitracin.

Enhanced production of bacitracin via energy metabolism engineering in Bacillus licheniformis DW2 / 生物工程学报

Bacitracin is a broad-spectrum cyclic peptide antibiotic, and mainly produced by Bacillus. Energy metabolism plays as a critical role in high-level production of target metabolites. In this study, Bacillus licheniformis DW2, an industrial strain for bacitracin production, was served as the original strain. First, our results confirmed that elimination of cytochrome bd oxidase branch via deleting gene cydB benefited bacitracin synthesis. Bacitracin titer and ATP content were increased by 10.97% and 22.96%, compared with those of original strain, respectively. Then, strengthening cytochrome aa3 oxidase branch via overexpressing gene qoxA was conducive to bacitracin production. Bacitracin titer and ATP content were increased by 18.97% and 34.00%, respectively. In addition, strengthening ADP synthesis supply is also proven as an effective strategy to promote intracellular ATP accumulation, overexpression of adenosine kinase DcK and adenylate kinase AdK could all improve bacitracin titers, among which, dck overexpression strain showed the better performance, and bacitracin titer was increased by 16.78%. Based on the above individual methods, a method of combining the deletion of gene cydB and overexpression of genes qoxA, dck were used to enhance ATP content of cells to 39.54 nmol/L, increased by 49.32% compared to original strain, and bacitracin titer produced by the final strain DW2-CQD (DW2ΔcydB::qoxA::dck) was 954.25 U/mL, increased by 21.66%. The bacitracin titer produced per cell was 2.11 U/CFU, increased by 11.05%. Collectively, this study demonstrates that improving ATP content was an efficient strategy to improve bacitracin production, and a promising strain B. licheniformis DW2-CQD was attained for industrial production of bacitracin.

Ultrahigh-Pressure Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry-Based Metabolomics Reveal the Mechanism of Methyl Jasmonate in Delaying the Deterioration of Agaricus bisporus.

Conquering rapid postripeness and deterioration of Agaricus bisporus is quite challenging. We previously observed that methyl jasmonate (MeJA) pretreatment postponed the deterioration of A. bisporus, but the mechanism is unknown. Here, a nontargeted metabolomics analysis by ultrahigh-pressure liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) revealed that MeJA increased the synthesis of malate by inhibiting the decomposition of fumarate and cis-aconitate. MeJA maintained energy supply by enhancing ATP content and energy charge level and improving hexokinase and glucose-6-phosphate dehydrogenase activities as well. These results promoted ATP supply by maintaining glycolysis, the TCA cycle, and the pentose phosphate pathway. In addition, we revealed that the delayed deterioration was attributed to MeJA treatment which stimulated the energy status of A. bisporus by reducing the respiration rate and nutrient decomposition, thus maintaining energy production. Our results provide a new insight into the role of MeJA treatment in delaying deterioration of A. bisporus through ATP production and supply.

Enhanced production of poly-γ-glutamic acid by improving ATP supply in metabolically engineered Bacillus licheniformis.

Poly-γ-glutamic acid (γ-PGA) is an important multifunctional biopolymer with various applications, for which adenosine triphosphate (ATP) supply plays a vital role in biosynthesis. In this study, the enhancement of γ-PGA production was attempted through various approaches of improving ATP supply in the engineered strains of Bacillus licheniformis. The first approach is to engineer respiration chain branches of B. licheniformis, elimination of cytochrome bd oxidase branch reduced the maintenance coefficient, leading to a 19.27% increase of γ-PGA yield. The second approach is to introduce Vitreoscilla hemoglobin (VHB) into recombinant B. licheniformis, led to a 13.32% increase of γ-PGA yield. In the third approach, the genes purB and adK in ATP-biosynthetic pathway were respectively overexpressed, with the AdK overexpressed strain increased γ-PGA yield by 14.69%. Our study also confirmed that the respiratory nitrate reductase, NarGHIJ, is responsible for the conversion of nitrate to nitrite, and assimilatory nitrate reductase NasBC is for conversion of nitrite to ammonia. Both NarGHIJ and NasBC were positively regulated by the two-component system ResD-ResE, and overexpression of NarG, NasC, and ResD also improved the ATP supply and the consequent γ-PGA yield. Based on the above individual methods, a method of combining the deletion of cydBC gene and overexpression of genes vgB, adK, and resD were used to enhance ATP content of the cells to 3.53 µmol/g of DCW, the mutant WX-BCVAR with this enhancement produced 43.81 g/L of γ-PGA, a 38.64% improvement compared to wild-type strain WX-02. Collectively, our results demonstrate that improving ATP content in B. licheniformis is an efficient strategy to improve γ-PGA production.

Enhanced Production of Poly-γ-glutamic acid by Overexpression of the Global Anaerobic Regulator Fnr in Bacillus licheniformis WX-02.

Poly-γ-glutamic acid is a multi-functional biopolymer with various applications. ATP supply plays an important role in poly-γ-glutamic acid (γ-PGA) synthesis. Global anaerobic regulator Fnr plays a key role in anaerobic adaptation and nitrate respiration, which might affect ATP generation during γ-PGA synthesis. In this study, we have improved γ-PGA production by overexpression of Fnr in Bacillus licheniformis WX-02. First, the gene fnr was knocked out in WX-02, and the γ-PGA yields have no significant differences between WX-02 and the fnr-deficient strain WXΔfnr in the medium without nitrate (BFC medium). However, the γ-PGA yield of 8.95 g/L, which was produced by WXΔfnr in the medium with nitrate addition (BFCN medium), decreased by 74% compared to WX-02 (34.53 g/L). Then, the fnr complementation strain WXΔfnr/pHY-fnr restored the γ-PGA synthesis capability, and γ-PGA yield was increased by 13% in the Fnr overexpression strain WX/pHY-fnr (39.96 g/L) in BFCN medium, compared to WX/pHY300 (35.41 g/L). Furthermore, the transcriptional levels of narK, narG, and hmp were increased by 5.41-, 4.93-, and 3.93-fold in WX/pHY-fnr, respectively, which led to the increases of nitrate consumption rate and ATP supply for γ-PGA synthesis. Collectively, Fnr affects γ-PGA synthesis mainly through manipulating the expression level of nitrate metabolism, and this study provides a novel strategy to improve γ-PGA production by overexpression of Fnr.

Mitochondrial Aspects of Synaptic Dysfunction in Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by brain deposition of amyloid plaques and tau neurofibrillary tangles along with steady cognitive decline. Synaptic damage, an early pathological event, correlates strongly with cognitive deficits and memory loss. Mitochondria are essential organelles for synaptic function. Neurons utilize specialized mechanisms to drive mitochondrial trafficking to synapses in which mitochondria buffer Ca2+ and serve as local energy sources by supplying ATP to sustain neurotransmitter release. Mitochondrial abnormalities are one of the earliest and prominent features in AD patient brains. Amyloid-ß (Aß) and tau both trigger mitochondrial alterations. Accumulating evidence suggests that mitochondrial perturbation acts as a key factor that is involved in synaptic failure and degeneration in AD. The importance of mitochondria in supporting synaptic function has made them a promising target of new therapeutic strategies for AD. Here, we review the molecular mechanisms regulating mitochondrial function at synapses, highlight recent findings on the disturbance of mitochondrial dynamics and transport in AD, and discuss how these alterations impact synaptic vesicle release and thus contribute to synaptic pathology associated with AD.

Improvement of the intracellular environment for enhancing l-arginine production of Corynebacterium glutamicum by inactivation of H2O2-forming flavin reductases and optimization of ATP supply.

l-arginine, a semi essential amino acid, is an important amino acid in food flavoring and pharmaceutical industries. Its production by microbial fermentation is gaining more and more attention. In previous work, we obtained a new l-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through mutation breeding. In this work, we enhanced l-arginine production through improvement of the intracellular environment. First, two NAD(P)H-dependent H2O2-forming flavin reductases Frd181 (encoded by frd1 gene) and Frd188 (encoded by frd2) in C. glutamicum were identified for the first time. Next, the roles of Frd181 and Frd188 in C. glutamicum were studied by overexpression and deletion of the encoding genes, and the results showed that the inactivation of Frd181 and Frd188 was beneficial for cell growth and l-arginine production, owing to the decreased H2O2 synthesis and intracellular reactive oxygen species (ROS) level, and increased intracellular NADH and ATP levels. Then, the ATP level was further increased by deletion of noxA (encoding NADH oxidase) and amn (encoding AMP nucleosidase), and overexpression of pgk (encoding 3-phosphoglycerate kinase) and pyk (encoding pyruvate kinase), and the l-arginine production and yield from glucose were significantly increased. In fed-batch fermentation, the l-arginine production and yield from glucose of the final strain reached 57.3g/L and 0.326g/g, respectively, which were 49.2% and 34.2% higher than those of the parent strain, respectively. ROS and ATP are important elements of the intracellular environment, and l-arginine biosynthesis requires a large amount of ATP. For the first time, we enhanced l-arginine production and yield from glucose through reducing the H2O2 synthesis and increasing the ATP supply.

The mechanism of improved pullulan production by nitrogen limitation in batch culture of Aureobasidium pullulans.

Batch culture of Aureobasidium pullulans CCTCC M 2012259 for pullulan production at different concentrations of ammonium sulfate and yeast extract was investigated. Increased pullulan production was obtained under nitrogen-limiting conditions, as compared to that without nitrogen limitation. The mechanism of nitrogen limitation favoring to pullulan overproduction was revealed by determining the activity as well as gene expression of key enzymes, and energy supply for pullulan biosynthesis. Results indicated that nitrogen limitation increased the activities of α-phosphoglucose mutase and glucosyltransferase, up-regulated the transcriptional levels of pgm1 and fks genes, and supplied more ATP intracellularly, which were propitious to further pullulan biosynthesis. The economic analysis of batch pullulan production indicated that nitrogen limitation could reduce more than one third of the cost of raw materials when glucose was supplemented to a total concentration of 70 g/L. This study also helps to understand the mechanism of other polysaccharide overproduction by nitrogen limitation.

In vivo identification of the steps that control energy metabolism and survival of Entamoeba histolytica.

The steps that control the Entamoeba histolytica glycolytic flux were here identified by elasticity analysis, an experimental approach of metabolic control analysis. The concentrations of glycolytic metabolites were gradually varied in live trophozoites by (a) feeding with different glucose concentrations and (b) inhibiting the final pathway steps; in parallel, the changes in the pathway flux were determined. From the metabolite concentration-flux relationship, the elasticity coefficients of individual or groups of pathway reactions were determined and used to calculate their respective degrees of control on the glycolytic flux (flux control coefficients). The results indicated that the pathway flux was mainly controlled (72-86%) by the glucose transport/hexokinase/glycogen degradation group of reactions and by bifunctional aldehyde-alcohol dehydrogenase (ADHE; 18%). Further, inhibition of the first pathway reactions with 2-deoxyglucose (2DOG) decreased the glycolytic flux and ATP content by 75% and 50%, respectively. Cell viability was also decreased by 2DOG (25%) and more potently (50%) by 2DOG plus the ADHE inhibitor tetraethylthiuram disulfide (disulfiram). Biosate as an alternative carbon (amino acid) source was unable to replace glucose for ATP supply, which indicated that glucose was the main nutrient for amoebal ATP synthesis and survival. These results indicated that glycolysis in the parasite is mainly controlled by the initial pathway reactions and that their inhibition can decrease the parasite energy load and survival.

Organelle biogenesis and interorganellar connections: Better in contact than in isolation.

Membrane contact sites (MCSs) allow the exchange of molecules and information between organelles, even when their membranes cannot fuse directly. In recent years, a number of functions have been attributed to these contacts, highlighting their critical role in cell homeostasis. Although inter-organellar connections typically involve the endoplasmic reticulum (ER), we recently reported the presence of a novel MCSs between melanosomes and mitochondria. Melanosome-mitochondrion contacts appear mediated by fibrillar bridges resembling the protein tethers linking mitochondria and the ER, both for their ultrastructural features and the involvement of Mitofusin 2. The frequency of these connections correlates spatially and timely with melanosome biogenesis, suggesting a functional link between the 2 processes and in general that organelle biogenesis in the secretory pathway requires interorganellar crosstalks at multiple steps. Here, we summarize the different functions attributed to MCSs, and discuss their possible relevance for the newly identified melanosome-mitochondrion liaison.