Pseudomonas aeruginosa Activates Quorum Sensing, Antioxidant Enzymes and Type VI Secretion in Response to Oxidative Stress to Initiate Biofilm Formation and Wound Chronicity.

Pseudomonas aeruginosa (PA) is an opportunistic pathogen frequently isolated from cutaneous chronic wounds. How PA, in the presence of oxidative stress (OS), colonizes chronic wounds and forms a biofilm is still unknown. The purpose of this study is to investigate the changes in gene expression seen when PA is challenged with the high levels of OS present in chronic wounds. We used a biofilm-forming PA strain isolated from the chronic wounds of our murine model (RPA) and performed a qPCR to obtain gene expression patterns as RPA developed a biofilm in vitro in the presence of high levels of OS, and then compared the findings in vivo, in our mouse model of chronic wounds. We found that the planktonic bacteria under OS conditions overexpressed quorum sensing genes that are important for the bacteria to communicate with each other, antioxidant stress genes important to reduce OS in the microenvironment for survival, biofilm formation genes and virulence genes. Additionally, we performed RNAseq in vivo and identified the activation of novel genes/pathways of the Type VI Secretion System (T6SS) involved in RPA pathogenicity. In conclusion, RPA appears to survive the high OS microenvironment in chronic wounds and colonizes these wounds by turning on virulence, biofilm-forming and survival genes. These findings reveal pathways that may be promising targets for new therapies aimed at disrupting PA-containing biofilms immediately after debridement to facilitate the treatment of chronic human wounds.

Acrylate Reductase of an Anaerobic Electron Transport Chain of the Marine Bacterium Shewanella woodyi.

Many microorganisms are capable of anaerobic respiration in the absence of oxygen, by using different organic compounds as terminal acceptors in electron transport chain. We identify here an anaerobic respiratory chain protein responsible for acrylate reduction in the marine bacterium Shewanella woodyi. When the periplasmic proteins of S. woodyi were separated by ion exchange chromatography, acrylate reductase activity copurified with an ArdA protein (Swoo_0275). Heterologous expression of S. woodyi ardA gene (swoo_0275) in Shewanella oneidensis MR-1 cells did not result in the appearance in them of periplasmic acrylate reductase activity, but such activity was detected when the ardA gene was co-expressed with an ardB gene (swoo_0276). Together, these genes encode flavocytochrome c ArdAB, which is thus responsible for acrylate reduction in S. woodyi cells. ArdAB was highly specific for acrylate as substrate and reduced only methacrylate (at a 22-fold lower rate) among a series of other tested 2-enoates. In line with these findings, acrylate and methacrylate induced ardA gene expression in S. woodyi under anaerobic conditions, which was accompanied by the appearance of periplasmic acrylate reductase activity. ArdAB-linked acrylate reduction supports dimethylsulfoniopropionate-dependent anaerobic respiration in S. woodyi and, possibly, other marine bacteria.

A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

Identification of bacterial dissimilatory antimonate reductase AnrA: genes and proteins involved in antimonate respiration and resistance in Geobacter sp. strain SVR.

Geobacter sp. strain SVR uses antimonate [Sb(V)] as a terminal electron acceptor for anaerobic respiration. Here, we visualized a possible key enzyme, periplasmic Sb(V) reductase (Anr), via active staining and non-denaturing gel electrophoresis. Liquid chromatography-tandem mass spectrometry analysis revealed that a novel dimethyl sulfoxide (DMSO) reductase family protein, WP_173201954.1, is involved in Anr. This protein was closely related with AnrA, a protein suggested to be the catalytic subunit of a respiratory Sb(V) reductase in Desulfuribacillus stibiiarsenatis. The anr genes of strain SVR (anrXSRBAD) formed an operon-like structure, and their transcription was upregulated under Sb(V)-respiring conditions. The expression of anrA gene was induced by more than 1 µM of antimonite [Sb(III)]; however, arsenite [As(III)] did not induce the expression of anrA gene. Tandem mass tag-based proteomic analysis revealed that, in addition to Anr proteins, proteins in the following categories were upregulated under Sb(V)-respiring conditions: (i) Sb(III) efflux systems such as Ant and Ars; (ii) antioxidizing proteins such as ferritin, rubredoxin, and thioredoxin; (iii) protein quality control systems such as HspA, HslO, and DnaK; and (iv) DNA repair proteins such as UspA and UvrB. These results suggest that strain SVR copes with antimony stress by modulating pleiotropic processes to resist and actively metabolize antimony. To the best of our knowledge, this is the first report to demonstrate the involvement of AnrA in Sb(V) respiration at the protein level. Furthermore, this is the first example to show high expression of the Ant system proteins in the Sb(V)-respiring bacterium.IMPORTANCEAntimony (Sb) exists mainly as antimonite [Sb(III)] or antimonate [Sb(V)] in the environment, and Sb(III) is more toxic than Sb(V). Recently, microbial involvement in Sb redox reactions has received attention. Although more than 90 Sb(III)-oxidizing bacteria have been reported, information on Sb(V)-reducing bacteria is limited. Especially, the enzyme involved in dissimilatory Sb(V) reduction, or Sb(V) respiration, is unclear, despite this pathway being very important for the circulation of Sb in nature. In this study, we demonstrated that the Sb(V) reductase (Anr) of an Sb(V)-respiring bacterium (Geobacter sp. SVR) is a novel member of the dimethyl sulfoxide (DMSO) reductase family. In addition, we found that strain SVR copes with Sb stress by modulating pleiotropic processes, including the Ant and Ars systems, and upregulating the antioxidant and quality control protein levels. Considering the abundance and diversity of putative anr genes in the environment, Anr may play a significant role in global Sb cycling in both marine and terrestrial environments.

Iron limitation indirectly reduces the Escherichia coli torCAD operon expression by a reduction of molybdenum cofactor availability.

The expression of most molybdoenzymes in Escherichia coli has so far been revealed to be regulated by anaerobiosis and requires the presence of iron, based on the necessity of the transcription factor FNR to bind one [4Fe-4S] cluster. One exception is trimethylamine-N-oxide reductase encoded by the torCAD operon, which has been described to be expressed independently from FNR. In contrast to other alternative anaerobic respiratory systems, the expression of the torCAD operon was shown not to be completely repressed by the presence of dioxygen. To date, the basis for the O2-dependent expression of the torCAD operon has been related to the abundance of the transcriptional regulator IscR, which represses the transcription of torS and torT, and is more abundant under aerobic conditions than under anaerobic conditions. In this study, we reinvestigated the regulation of the torCAD operon and its dependence on the presence of iron and identified a novel regulation that depends on the presence of the bis-molybdopterin guanine dinucleotide (bis-MGD) molybdenum cofactor . We confirmed that the torCAD operon is directly regulated by the heme-containing protein TorC and is indirectly regulated by ArcA and by the availability of iron via active FNR and Fur, both regulatory proteins that influence the synthesis of the molybdenum cofactor. Furthermore, we identified a novel regulation mode of torCAD expression that is dependent on cellular levels of bis-MGD and is not used by other bis-MGD-containing enzymes like nitrate reductase.IMPORTANCEIn bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. FNR is a very important transcription factor that represents the master switch for the expression of target genes in response to anaerobiosis. Only Escherichia coli trimethylamine-N-oxide (TMAO) reductase escapes this regulation by FNR. We identified that the expression of TMAO reductase is regulated by the amount of bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor synthesized by the cell itself, representing a novel regulation pathway for the expression of an operon coding for a molybdoenzyme. Furthermore, TMAO reductase gene expression is indirectly regulated by the presence of iron, which is required for the production of the bis-MGD cofactor in the cell.

Nitrous oxide reduction by two partial denitrifying bacteria requires denitrification intermediates that cannot be respired.

Denitrification is a form of anaerobic respiration wherein nitrate (NO3-) is sequentially reduced via nitrite (NO2-), nitric oxide, and nitrous oxide (N2O) to dinitrogen gas (N2) by four reductase enzymes. Partial denitrifying bacteria possess only one or some of these four reductases and use them as independent respiratory modules. However, it is unclear if partial denitrifiers sense and respond to denitrification intermediates outside of their reductase repertoire. Here, we tested the denitrifying capabilities of two purple nonsulfur bacteria, Rhodopseudomonas palustris CGA0092 and Rhodobacter capsulatus SB1003. Each had denitrifying capabilities that matched their genome annotation; CGA0092 reduced NO2- to N2, and SB1003 reduced N2O to N2. For each bacterium, N2O reduction could be used both for electron balance during growth on electron-rich organic compounds in light and for energy transformation via respiration in darkness. However, N2O reduction required supplementation with a denitrification intermediate, including those for which there was no associated denitrification enzyme. For CGA0092, NO3- served as a stable, non-catalyzable molecule that was sufficient to activate N2O reduction. Using a ß-galactosidase reporter, we found that NO3- acted, at least in part, by stimulating N2O reductase gene expression. In SB1003, NO2- but not NO3- activated N2O reduction, but NO2- was slowly removed, likely by a promiscuous enzyme activity. Our findings reveal that partial denitrifiers can still be subject to regulation by denitrification intermediates that they cannot use.IMPORTANCEDenitrification is a form of microbial respiration wherein nitrate is converted via several nitrogen oxide intermediates into harmless dinitrogen gas. Partial denitrifying bacteria, which individually have some but not all denitrifying enzymes, can achieve complete denitrification as a community by cross-feeding nitrogen oxide intermediates. However, the last intermediate, nitrous oxide (N2O), is a potent greenhouse gas that often escapes, motivating efforts to understand and improve the efficiency of denitrification. Here, we found that at least some partial denitrifying N2O reducers can sense and respond to nitrogen oxide intermediates that they cannot otherwise use. The regulatory effects of nitrogen oxides on partial denitrifiers are thus an important consideration in understanding and applying denitrifying bacterial communities to combat greenhouse gas emissions.

Understanding wax ester synthesis in Euglena gracilis: Insights into mitochondrial anaerobic respiration.

Euglena gracilis, photosynthetic protist, has a unique ability to generate wax esters in the absence of oxygen, employing a distinctive fatty acid synthesis mechanism. Through comprehensive inhibitor assays and gene-silencing techniques, our research clearly emphasized the indispensable role of the mitochondrial anaerobic respiratory chain in this biosynthesis. We identified acyl-CoA dehydrogenase, electron transfer flavoprotein (ETF), and rhodoquinone (RQ) as central molecular components in the pathway. These findings strongly indicated a potential reversal of beta-oxidation occurring within mitochondria for fatty acid production in anaerobic conditions. Furthermore, our analysis revealed the pivotal function of nicotinamide nucleotide transhydrogenase (NNT) in efficiently managing the NADPH/NAD+ conversion essential for sustaining anaerobic metabolism. This review outlines our key findings and provides a comprehensive understanding of the molecular mechanisms that enable E. gracilis to produce wax ester anaerobically.

Pyrogenic Black Carbon Suppresses Microbial Methane Production by Serving as a Terminal Electron Acceptor.

Methane (CH4) is the second most important greenhouse gas, 27 times as potent as CO2 and responsible for >30% of the current anthropogenic warming. Globally, more than half of CH4 is produced microbially through methanogenesis. Pyrogenic black carbon possesses a considerable electron storage capacity (ESC) and can be an electron donor or acceptor for abiotic and microbial redox transformation. Using wood-derived biochar as a model black carbon, we demonstrated that air-oxidized black carbon served as an electron acceptor to support anaerobic oxidation of organic substrates, thereby suppressing CH4 production. Black carbon-respiring bacteria were immediately active and outcompeted methanogens. Significant CH4 did not form until the bioavailable electron-accepting capacity of the biochar was exhausted. An experiment with labeled acetate (13CH3COO-) yielded 1:1 13CH4 and 12CO2 without biochar and predominantly 13CO2 with biochar, indicating that biochar enabled anaerobic acetate oxidation at the expense of methanogenesis. Methanogens were enriched following acetate fermentation but only in the absence of biochar. The electron balance shows that approximately half (∼2.4 mmol/g) of biochar's ESC was utilized by the culture, corresponding to the portion of the ESC > +0.173 V (vs SHE). These results provide a mechanistic basis for quantifying the climate impact of black carbon and developing ESC-based applications to reduce CH4 emissions from biogenic sources.

The genetics of aerotolerant growth in an alphaproteobacterium with a naturally reduced genome.

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living alphaproteobacterium Zymomonas mobilis has a naturally reduced genome containing fewer than 2,000 protein-coding genes. Despite its small genome, Z. mobilis thrives in diverse conditions including the presence or absence of atmospheric oxygen. However, insufficient characterization of essential and conditionally essential genes has limited broader adoption of Z. mobilis as a model alphaproteobacterium. Here, we use genome-scale CRISPRi-seq (clustered regularly interspaced short palindromic repeats interference sequencing) to systematically identify and characterize Z. mobilis genes that are conditionally essential for aerotolerant or anaerobic growth or are generally essential across both conditions. Comparative genomics revealed that the essentiality of most "generally essential" genes was shared between Z. mobilis and other Alphaproteobacteria, validating Z. mobilis as a reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, recJ, was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F1FO ATP synthase and Rhodobacter nitrogen fixation (Rnf) respiratory complex are required for the anaerobic growth of Z. mobilis. Combining CRISPRi partial knockdowns with metabolomics and membrane potential measurements, we determined that the ATP synthase generates membrane potential that is consumed by Rnf to power downstream processes. Rnf knockdown strains accumulated isoprenoid biosynthesis intermediates, suggesting a key role for Rnf in powering essential biosynthetic reactions. Our work establishes Z. mobilis as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs Z. mobilis genome manipulation for optimized production of valuable isoprenoid-based bioproducts. IMPORTANCE The inherent complexity of biological systems is a major barrier to our understanding of cellular physiology. Bacteria with markedly fewer genes than their close relatives, or reduced genome bacteria, are promising biological models with less complexity. Reduced genome bacteria can also have superior properties for industrial use, provided the reduction does not overly restrict strain robustness. Naturally reduced genome bacteria, such as the alphaproteobacterium Zymomonas mobilis, have fewer genes but remain environmentally robust. In this study, we show that Z. mobilis is a simplified genetic model for Alphaproteobacteria, a class with important impacts on the environment, human health, and industry. We also identify genes that are only required in the absence of atmospheric oxygen, uncovering players that maintain and utilize the cellular energy state. Our findings have broad implications for the genetics of Alphaproteobacteria and industrial use of Z. mobilis to create biofuels and bioproducts.

The Campylobacter concisus BisA protein plays a dual role: oxide-dependent anaerobic respiration and periplasmic methionine sulfoxide repair.

IMPORTANCE: Campylobacter concisus is an excellent model organism to study respiration diversity, including anaerobic respiration of physiologically relevant N-/S-oxides compounds, such as biotin sulfoxide, dimethyl sulfoxide, methionine sulfoxide (MetO), nicotinamide N-oxide, and trimethylamine N-oxide. All C. concisus strains harbor at least two, often three, and up to five genes encoding for putative periplasmic Mo/W-bisPGD-containing N-/S-oxide reductases. The respective role (substrate specificity) of each enzyme was studied using a mutagenesis approach. One of the N/SOR enzymes, annotated as "BisA", was found to be essential for anaerobic respiration of both N- and S-oxides. Additional phenotypes associated with disruption of the bisA gene included increased sensitivity toward oxidative stress and elongated cell morphology. Furthermore, a biochemical approach confirmed that BisA can repair protein-bound MetO residues. Hence, we propose that BisA plays a role as a periplasmic methionine sulfoxide reductase. This is the first report of a Mo/W-bisPGD-enzyme supporting both N- or S-oxide respiration and protein-bound MetO repair in a pathogen.

Shewanella metallivivens sp. nov., a deep-sea hydrothermal vent tube worm endobiont capable of dissimilatory anaerobic metalloid oxyanion reduction.

A polyphasic taxonomic study was carried out on a Gram-stain-negative and rod-shaped strain, ER-Te-42B-LightT, isolated from the tissue of a tube worm, Riftia pachyptila, collected near a deep-sea hydrothermal vent of the Juan de Fuca Ridge in the Pacific Ocean. This bacterium was capable of performing anaerobic respiration using tellurite, tellurate, selenite and orthovanadate as terminal electron acceptors. While facultatively anaerobic, it could aerobically resist tellurite, selenite and orthovanadate up to 2000, 7000 and 10000 µg ml-1, respectively, reducing each oxide to elemental forms. Nearly complete 16S rRNA gene sequence similarity related the strain to Shewanella, with 98.8 and 98.7 % similarity to Shewanella basaltis and Shewanella algicola, respectively. The dominant fatty acids were C16 : 0 and C16 : 1. The major polar lipids were phosphatidylethanolamine and phosphatidylglycerol and MK-7 was the predominant quinone. DNA G+C content was 42.5 mol%. Computation of average nucleotide identity and digital DNA-DNA hybridization values with the closest phylogenetic neighbours of ER-Te-42B-LightT revealed genetic divergence at the species level, which was further substantiated by differences in several physiological characteristics. Based on the obtained results, this bacterium was assigned to the genus Shewanella as a new species with the name Shewanella metallivivens sp. nov., type strain ER-Te-42B-LightT (=VKM B-3580T=DSM 113370T).

Reduced chemosymbiont genome in the methane seep thyasirid and the cooperated metabolisms in the holobiont under anaerobic sediment.

Previous studies have deciphered the genomic basis of host-symbiont metabolic complementarity in vestimentiferans, bathymodioline mussels, vesicomyid clams and Alviniconcha snails, yet little is known about the chemosynthetic symbiosis in Thyasiridae-a family of Bivalvia regarded as an excellent model in chemosymbiosis research due to their wide distribution in both deep-sea and shallow-water habitats. We report the first circular thyasirid symbiont genome, named Candidatus Ruthturnera sp. Tsphm01, with a size of 1.53 Mb, 1521 coding genes and 100% completeness. Compared to its free-living relatives, Ca. Ruthturnera sp. Tsphm01 genome is reduced, lacking components for chemotaxis, citric acid cycle and de novo biosynthesis of small molecules (e.g. amino acids and cofactors), indicating it is likely an obligate intracellular symbiont. Nevertheless, the symbiont retains complete genomic components of sulphur oxidation and assimilation of inorganic carbon, and these systems were highly and actively expressed. Moreover, the symbiont appears well-adapted to anoxic environment, including capable of anaerobic respiration (i.e. reductions of DMSO and nitrate) and possession of a low oxygen-adapted type of cytochrome c oxidase. Analysis of the host transcriptome revealed its metabolic complementarity to the incomplete metabolic pathways of the symbiont and the acquisition of nutrients from the symbiont via phagocytosis and exosome. By providing the first complete genome of reduced size in a thyasirid symbiont, this study enhances our understanding of the diversity of symbiosis that has enabled bivalves to thrive in chemosynthetic habitats. The resources will be widely used in phylogenetic, geographic and evolutionary studies of chemosynthetic bacteria and bivalves.

Biosynthesis and function of microbial methylmenaquinones.

The membranous quinone/quinol pool is essential for the majority of life forms and its composition has been widely used as a biomarker in microbial taxonomy. The most abundant quinone is menaquinone (MK), which serves as an essential redox mediator in various electron transport chains of aerobic and anaerobic respiration. Several methylated derivatives of MK, designated methylmenaquinones (MMKs), have been reported to be present in members of various microbial phyla possessing either the classical MK biosynthesis pathway (Men) or the futalosine pathway (Mqn). Due to their low redox midpoint potentials, MMKs have been proposed to be specifically involved in appropriate electron transport chains of anaerobic respiration. The class C radical SAM methyltransferases MqnK, MenK and MenK2 have recently been shown to catalyse specific MK methylation reactions at position C-8 (MqnK/MenK) or C-7 (MenK2) to synthesise 8-MMK, 7-MMK and 7,8-dimethylmenaquinone (DMMK). MqnK, MenK and MenK2 from organisms such as Wolinella succinogenes, Adlercreutzia equolifaciens, Collinsella tanakaei, Ferrimonas marina and Syntrophus aciditrophicus have been functionally produced in Escherichia coli, enabling extensive quinone/quinol pool engineering of the native MK and 2-demethylmenaquinone (DMK). Cluster and phylogenetic analyses of available MK and MMK methyltransferase sequences revealed signature motifs that allowed the discrimination of MenK/MqnK/MenK2 family enzymes from other radical SAM enzymes and the identification of C-7-specific menaquinone methyltransferases of the MenK2 subfamily. It is envisaged that this knowledge will help to predict the methylation status of the menaquinone/menaquinol pool of any microbial species (or even a microbial community) from its (meta)genome.

Impacts and mechanism of biodegradable microplastics on lake sediment properties, bacterial dynamics, and greenhouse gasses emissions.

The accumulation of microplastics (MPs) in freshwater ecosystems plays a vital role in greenhouse gases (GHGs) emissions from lake sediment by altering sediment properties and microbial communities. Thus, a short-term microcosm experiment was performed to explore the effect of conventional polyethylene (PE) and biodegradable Poly (butylene-adipate-co-terephtalate) (PBAT) MPs on carbon dioxide (CO2) and methane (CH4) emissions from lake sediment and associated microbial community. The results indicated that at 1.0 % concentration, the cumulative CO2 emissions were increased by 16.8 % and the cumulative CH4 emissions were increased more than four times following the addition of biodegradable MPs compared to conventional MPs, which was due to the more dissolved organic carbon (DOC) provided by biodegradable MPs for microbial respiration. Furthermore, the cumulative CO2 and CH4 emissions significantly (p < 0.05) increased with the increasing concentrations of biodegradable MPs. Notably, the accumulation of MPs could weaken the microbial stress from requirements of energy and substrate, and increase the microbial biomass carbon (MBC) value, thus eventually improving the respiratory capacity of microbes. In addition, the biodegradable MPs significantly increased the abundance of microbes, such as Firmicutes, Myxococcota and Actinobacteriota, which were related to the function of anaerobic respiration. Overall, we concluded that the abundant DOC provided by biodegradable MPs could promote the growth of microbes in lake sediment, and they could change the structure and diversity of the microbial community, which would eventually enhance the anaerobic respiration of microbes and aggravate the GHGs emissions.

Mitochondria dysfunction and bipolar disorder: From pathology to therapy.

Bipolar disorder (BD) is one of the major psychiatric diseases in which the impairment of mitochondrial functions has been closely connected or associated with the disease pathologies. Different lines of evidence of the close connection between mitochondria dysfunction and BD were discussed with a particular focus on (1) dysregulation of energy metabolism, (2) effect of genetic variants, (3) oxidative stress, cell death and apoptosis, (4) dysregulated calcium homeostasis and electrophysiology, and (5) current as well as potential treatments targeting at restoring mitochondrial functions. Currently, pharmacological interventions generally provide limited efficacy in preventing relapses or recovery from mania or depression episodes. Thus, understanding mitochondrial pathology in BD will lead to novel agents targeting mitochondrial dysfunction and formulating new effective therapy for BD.

The Changes in Mitochondrial Morphology and Physiology Accompanying Apoptosis in Galleria mellonella (Lepidoptera) Immunocompetent Cells during Conidiobolus coronatus (Entomophthorales) Infection.

Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, their role in insects is not fully understood; thus, more indepth studies of insect cell apoptosis are necessary. The present study investigates mitochondrial involvement during Conidiobolus coronatus-induced apoptosis in Galleria mellonella hemocytes. Previous research has shown that fungal infection could induce apoptosis in insect hemocytes. Our findings indicate that mitochondria undergo several morphological and physiological changes during fungal infection, e.g., loss of mitochondrial membrane potential, megachannel formation, disturbances in intracellular respiration, increased nonrespiratory oxygen consumption in mitochondria, decreased ATP-coupled oxygen consumption and increased non-ATP-coupled oxygen consumption, decreased extracellular and intracellular oxygen consumption, and increased extracellular pH. Our findings confirm that G. mellonella immunocompetent cells demonstrate Ca2+ overload in mitochondria, translocation of cytochrome c-like protein from mitochondrial to cytosol fraction, and higher activation of caspase-9-like protein after C. coronatus infection. Most importantly, several of the changes observed in insect mitochondria are similar to those accompanying apoptosis in mammalian cells, suggesting that the process is evolutionarily conserved.

An Organismal Perspective on the Warburg Effect and Models for Proliferation Studies.

Interest in the physiology of proliferation has been generated by human proliferative diseases, i.e., cancers. A vast literature exists on the Warburg effect, which is characterized by aerobic glycolysis, diminished oxygen uptake, and lactate secretion. While these features could be rationalized via the production of biosynthetic precursors, lactate secretion does not fit this paradigm, as it wastes precursors. Forming lactate from pyruvate allows for reoxidizing cytosolic NADH, which is crucial for continued glycolysis and may allow for maintaining large pools of metabolic intermediates. Alternatively, lactate production may not be adaptive, but rather reflect metabolic constraints. A broader sampling of the physiology of proliferation, particularly in organisms that could reoxidize NADH using other pathways, may be necessary to understand the Warburg effect. The best-studied metazoans (e.g., worms, flies, and mice) may not be suitable, as they undergo limited proliferation before initiating meiosis. In contrast, some metazoans (e.g., colonial marine hydrozoans) exhibit a stage in the life cycle (the polyp stage) that only undergoes mitotic proliferation and never carries out meiosis (the medusa stage performs this). Such organisms are prime candidates for general studies of proliferation in multicellular organisms and could at least complement the short-generation models of modern biology.

Enhancing acetone production from H2 and CO2 using supplemental electron acceptors in an engineered Moorella thermoacetica.

Acetogens grow autotrophically and use hydrogen (H2) as the energy source to fix carbon dioxide (CO2). This feature can be applied to gas fermentation, contributing to a circular economy. A challenge is the gain of cellular energy from H2 oxidation, which is substantially low, especially when acetate formation coupled with ATP production is diverted to other chemicals in engineered strains. Indeed, an engineered strain of the thermophilic acetogen Moorella thermoacetica that produces acetone lost autotrophic growth on H2 and CO2. We aimed to recover autotrophic growth and enhance acetone production, in which ATP production was assumed to be a limiting factor, by supplementing with electron acceptors. Among the four selected electron acceptors, thiosulfate and dimethyl sulfoxide (DMSO) enhanced both bacterial growth and acetone titers. DMSO was the most effective and was further analyzed. We showed that DMSO supplementation enhanced intracellular ATP levels, leading to increased acetone production. Although DMSO is an organic compound, it functions as an electron acceptor, not a carbon source. Thus, supplying electron acceptors is a potential strategy to complement the low ATP production caused by metabolic engineering and to improve chemical production from H2 and CO2.

Evaluation of waterlogging tolerance and responses of protective enzymes to waterlogging stress in pumpkin.

Waterlogging caused by short and severe, or prolonged precipitation can be attributed to global warming. Pumpkin plants are drought-tolerant but not tolerate to waterlogging stress. Under frequent rain and waterlogging conditions, the production of pumpkins is of lower quality, sometimes rotten, and harvest failure occurs in severe cases. Therefore, it is of great significance to assess the waterlogging tolerance mechanism of pumpkin plants. In this study, 10 novel pumpkin varieties from Baimi series were used. The waterlogging tolerance level of pumpkin plants was evaluated by measuring waterlogging tolerance coefficient of biomass and physiological indices using waterlogging stress simulation method. The criteria to evaluate the waterlogging tolerance capacities of pumpkin plants were also explored. Using principal component and membership function analysis, waterlogging tolerance levels of the pumpkin varieties were ranked as follows: Baimi No. 10>; Baimi No. 5>; Baimi No. 1>; Baimi No. 2>; Baimi No. 3>; Baimi No. 7>; Baimi No. 9>; Baimi No. 6>; Baimi No. 4>; Baimi No. 8. Based on the results, Baimi No. 10 was identified with strong waterlogging tolerance and Baimi No. 8 with weak waterlogging tolerance. The responses of malondialdehyde (MDA), proline, key enzymes responsible for anaerobic respiration, and antioxidant enzymes to waterlogging stress were studied in pumpkin plants. The relative expression levels of related genes were determined using real-time fluorescence quantitative PCR technique. The aim of our study was to assess the waterlogging tolerance mechanism of pumpkin plants, thus laying a theoretical foundation for breeding waterlogging-tolerant varieties in the future. After flooding stress treatment, the antioxidant enzyme activities, contents of proline and alcohol dehydrogenases of Baimi No. 10 and Baimi No. 8 displayed an increase followed by a decrease. All indices of Baimi No. 10 were higher than Baimi No. 8. MDA contents gradually increased, with the content being higher in Baimi No. 8 than Baimi No. 10. The activities of pyruvate decarboxylases (PDCs) in Baimi No. 8 and Baimi No. 10 exhibited a decrease initially, followed by an increase, and then a decrease again. The PDC activity in Baimi No. 8 was generally higher than Baimi No. 10. The relative expression levels of genes encoding superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase were consistent with their corresponding enzyme activities. During the early stage of flooding stress, pumpkin plants waterlogging tolerance was improved by enhancing the expression levels of antioxidant enzyme encoding genes and increasing the antioxidant enzyme activities.

Evidence for a Putative Isoprene Reductase in Acetobacterium wieringae.

Recent discoveries of isoprene-metabolizing microorganisms suggest they might play an important role in the global isoprene budget. Under anoxic conditions, isoprene can be used as an electron acceptor and is reduced to methylbutene. This study describes the proteogenomic profiling of an isoprene-reducing bacterial culture to identify organisms and genes responsible for the isoprene hydrogenation reaction. A metagenome-assembled genome (MAG) of the most abundant (89% relative abundance) lineage in the enrichment, Acetobacterium wieringae, was obtained. Comparative proteogenomics and reverse transcription-PCR (RT-PCR) identified a putative five-gene operon from the A. wieringae MAG upregulated during isoprene reduction. The operon encodes a putative oxidoreductase, three pleiotropic nickel chaperones (2 × HypA, HypB), and one 4Fe-4S ferredoxin. The oxidoreductase is proposed as the putative isoprene reductase with a binding site for NADH, flavin adenine dinucleotide (FAD), two pairs of canonical [4Fe-4S] clusters, and a putative iron-sulfur cluster site in a Cys6-bonding environment. Well-studied Acetobacterium strains, such as A. woodii DSM 1030, A. wieringae DSM 1911, or A. malicum DSM 4132, do not encode the isoprene-regulated operon but encode, like many other bacteria, a homolog of the putative isoprene reductase (~47 to 49% amino acid sequence identity). Uncharacterized homologs of the putative isoprene reductase are observed across the Firmicutes, Spirochaetes, Tenericutes, Actinobacteria, Chloroflexi, Bacteroidetes, and Proteobacteria, suggesting the ability of biohydrogenation of unfunctionalized conjugated doubled bonds in other unsaturated hydrocarbons. IMPORTANCE Isoprene was recently shown to act as an electron acceptor for a homoacetogenic bacterium. The focus of this study is the molecular basis for isoprene reduction. By comparing a genome from our isoprene-reducing enrichment culture, dominated by Acetobacterium wieringae, with genomes of other Acetobacterium lineages that do not reduce isoprene, we shortlisted candidate genes for isoprene reduction. Using comparative proteogenomics and reverse transcription-PCR we have identified a putative five-gene operon encoding an oxidoreductase referred to as putative isoprene reductase.