Effects of salinity and betaine addition on anaerobic granular sludge properties and microbial community succession patterns in organic saline wastewater.

In this study, the effects of different salinity gradients and addition of compatible solutes on anaerobic treated effluent water qualities, sludge characteristics and microbial communities were investigated. The increase in salinity resulted in a decrease in particle size of the granular sludge, which was concentrated in the range of 0.5-1.0 mm. The content of EPS (extracellular polymeric substances) in the granular sludge gradually increased with increasing salinity and the addition of betaine (a typical compatible solute). Meanwhile, the microbial community structure was significantly affected by salinity, with high salinity reducing the diversity of bacteria. At higher salinity, Patescibacteria and Proteobacteria gradually became the dominant phylum, with relative abundance increasing to 13.53% and 12.16% at 20 g/L salinity. Desulfobacterota and its subordinate Desulfovibrio, which secrete EPS in large quantities, dominated significantly after betaine addition.Their relative abundance reached 13.65% and 7.86% at phylum level and genus level. The effect of these changes on the treated effluent was shown as the average chemical oxygen demand (COD) removal rate decreased from 82.10% to 79.71%, 78.01%, 68.51% and 64.55% when the salinity gradually increased from 2 g/L to 6, 10, 16 and 20 g/L. At the salinity of 20 g/L, average COD removal increased to 71.65% by the addition of 2 mmol/L betaine. The gradient elevated salinity and the exogenous addition of betaine played an important role in achieving stability of the anaerobic system in a highly saline environment, which provided a feasible strategy for anaerobic treatment of organic saline wastewater.

Gut microbiota metabolites and risk of major adverse cardiovascular events and death: A systematic review and meta-analysis.

BACKGROUND: Gut microbial metabolites such as trimethylamine N-oxide (TMAO) and its precursors, namely betaine, L-carnitine, and choline, have been implicated as risk factors for cardiovascular events and mortality development. Therefore, we aim to perform a systematic review and meta-analysis to assess the validity of these associations. METHODS: MEDLINE and Scopus were queried from their inception to August 2023 to identify studies that quantified estimates of the associations of TMAO with the development of major adverse cardiovascular events (MACE) or death. A random-effects meta-analysis was conducted to pool unadjusted or multivariable-adjusted hazard ratios (HR) and their 95% confidence intervals. The primary endpoint was the risk of MACE and all-cause death. RESULTS: 30 prospective observational studies (n = 48 968) were included in the analysis. Elevated TMAO levels were associated with a significantly greater risk of MACE and all-cause death compared to low TMAO levels (HR: 1.41, 95% CI 1.2-1.54, P < .00001, I2 = 43%) and (HR: 1.55, 95% CI 1.37-1.75, P < .00001, I2 = 46%), respectively. Furthermore, high levels of either L-carnitine or choline were found to significantly increase the risk of MACE. However, no significant difference was seen in MACE in either high or low levels of betaine. CONCLUSION: Elevated concentrations of TMAO were associated with increased risks of MACE and all-cause mortality. High levels of L-carnitine/choline were also significantly associated with an increased risk of MACE. However, no significant difference was found between high or low levels of betaine for the outcome of MACE.

Glycine betaine: A multifaceted protectant against salt stress in Indian mustard through ionic homeostasis, ROS scavenging and osmotic regulation.

Salt stress is a prevalent environmental issue that disrupts the redox balance and metabolic processes in plants, leading to reduced crop growth and productivity. Currently, over 6.74 million hectares in India are salt-affected, and about 75% of this land lies in states that are the major cultivators of edible oilseed crops (rapeseed-mustard). Therefore, this study focused on the efficacy of glycine betaine (GB) supplementation in mitigating the detrimental effects of salt stress in Brassica juncea L. (Indian mustard) plants. Indian mustard plants were subjected to salt stress [0, 50, 100, and 150 mM sodium chloride] 20 days after sowing (DAS), while a foliar spray of 20 mM GB was applied to the foliage at 50 and 70 DAS. The data showed that salt stress substantially reduced growth, photosynthetic rate, membrane stability, and yield by significantly increasing lipid peroxidation, ion toxicity, cell death, electrolyte leakage, and reactive oxygen species accumulation that triggered oxidative stress. Supplementation with 20 mM GB provided tolerance to plants against salt-induced toxicity since it substantially increased growth, biomass, water content, nutrient uptake, and photosynthetic efficiency. Additionally, GB enhances the accumulation of osmolytes, enhances the antioxidant defence system, improves ionic balance, and enhances cell viability. Taken together, the obtained data provides deeper insights into the beneficial effect of the exogenous GB application that could have biotechnological uses to enhance crop stress tolerance in challenging environments.

Synergistic effects of melatonin and glycine betaine on seed germination, seedling growth, and biochemical attributes of maize under salinity stress.

Salinity stress represents a major threat to crop production by inhibiting seed germination, growth of seedlings, and final yield and, therefore, to the social and economic prosperity of developing countries. Recently, plant growth-promoting substances have been widely used as a chemical strategy for improving plant resilience towards abiotic stresses. This study aimed to determine whether melatonin (MT) and glycine betaine (GB) alone or in combination could alleviate the salinity-induced impacts on seed germination and growth of maize seedlings. Increasing NaCl concentration from 100 to 200 mM declined seed germination rate (4.6-37.7%), germination potential (24.5-46.7%), radical length (7.7-40.0%), plumule length (2.2-35.6%), seedling fresh (1.7-41.3%) and dry weight (23.0-56.1%) compared to control (CN) plants. However, MT and GB treatments lessened the adverse effects of 100 and 150 mM NaCl and enhanced germination comparable to control plants. In addition, results from the pot experiments show that 200 mM NaCl stress disrupted the osmotic balance and persuaded oxidative stress, presented by higher electrolyte leakage, hydrogen peroxide, superoxide radicals, and malondialdehyde compared to control plants. However, compared to the NaCl treatment, NaCl+MT+GB treatment decreased the accumulation of malondialdehyde (24.2-42.1%), hydrogen peroxide (36.2-44.0%), and superoxide radicals (20.1-50.9%) by up-regulating the activity of superoxide dismutase (28.4-51.2%), catalase (82.2-111.5%), ascorbate peroxidase (40.3-59.2%), and peroxidase (62.2-117.9%), and by enhancing osmolytes accumulation, thereby reducing NaCl-induced oxidative damages. Based on these findings, the application of MT+GB is an efficient chemical strategy for improving seed germination and growth of seedlings by improving the physiological and biochemical attributes of maize under 200 mM NaCl stress.

Structure and mechanism of the osmoregulated choline transporter BetT.

The choline-glycine betaine pathway plays an important role in bacterial survival in hyperosmotic environments. Osmotic activation of the choline transporter BetT promotes the uptake of external choline for synthesizing the osmoprotective glycine betaine. Here, we report the cryo-electron microscopy structures of Pseudomonas syringae BetT in the apo and choline-bound states. Our structure shows that BetT forms a domain-swapped trimer with the C-terminal domain (CTD) of one protomer interacting with the transmembrane domain (TMD) of a neighboring protomer. The substrate choline is bound within a tryptophan prism at the central part of TMD. Together with functional characterization, our results suggest that in Pseudomonas species, including the plant pathogen P. syringae and the human pathogen Pseudomonas aeruginosa, BetT is locked at a low-activity state through CTD-mediated autoinhibition in the absence of osmotic stress, and its hyperosmotic activation involves the release of this autoinhibition.

Metabolic gene function discovery platform GeneMAP identifies SLC25A48 as necessary for mitochondrial choline import.

Organisms maintain metabolic homeostasis through the combined functions of small-molecule transporters and enzymes. While many metabolic components have been well established, a substantial number remains without identified physiological substrates. To bridge this gap, we have leveraged large-scale plasma metabolome genome-wide association studies (GWAS) to develop a multiomic Gene-Metabolite Association Prediction (GeneMAP) discovery platform. GeneMAP can generate accurate predictions and even pinpoint genes that are distant from the variants implicated by GWAS. In particular, our analysis identified solute carrier family 25 member 48 (SLC25A48) as a genetic determinant of plasma choline levels. Mechanistically, SLC25A48 loss strongly impairs mitochondrial choline import and synthesis of its downstream metabolite betaine. Integrative rare variant and polygenic score analyses in UK Biobank provide strong evidence that the SLC25A48 causal effects on human disease may in part be mediated by the effects of choline. Altogether, our study provides a discovery platform for metabolic gene function and proposes SLC25A48 as a mitochondrial choline transporter.

Structural insights into the molecular effects of the anthelmintics monepantel and betaine on the Caenorhabditis elegans acetylcholine receptor ACR-23.

Anthelmintics are drugs used for controlling pathogenic helminths in animals and plants. The natural compound betaine and the recently developed synthetic compound monepantel are both anthelmintics that target the acetylcholine receptor ACR-23 and its homologs in nematodes. Here, we present cryo-electron microscopy structures of ACR-23 in apo, betaine-bound, and betaine- and monepantel-bound states. We show that ACR-23 forms a homo-pentameric channel, similar to some other pentameric ligand-gated ion channels (pLGICs). While betaine molecules are bound to the classical neurotransmitter sites in the inter-subunit interfaces in the extracellular domain, monepantel molecules are bound to allosteric sites formed in the inter-subunit interfaces in the transmembrane domain of the receptor. Although the pore remains closed in betaine-bound state, monepantel binding results in an open channel by wedging into the cleft between the transmembrane domains of two neighboring subunits, which causes dilation of the ion conduction pore. By combining structural analyses with site-directed mutagenesis, electrophysiology and in vivo locomotion assays, we provide insights into the mechanism of action of the anthelmintics monepantel and betaine.

Betaine and I-LG may have a predictive value for ATB: A causal study in a large European population.

PURPOSE: To analyze the causal relationship between 486 human serum metabolites and the active tuberculosis (ATB) in European population. METHODS: In this study, the causal relationship between human serum metabolites and the ATB was analyzed by integrating the genome-wide association study (GWAS). The 486 human serum metabolites were used as the exposure variable, three different ATB GWAS databases in the European population were set as outcome variables, and single nucleotide polymorphisms were used as instrumental variables for Mendelian Randomization. The inverse variance weighting was estimated causality, the MR-Egger intercept to estimate horizontal pleiotropy, and the combined effects of metabolites were also considered in the meta-analysis. Furthermore, the web-based MetaboAnalyst 6.0 was engaged for enrichment pathway analysis, while R (version 4.3.2) software and Review Manager 5.3 were employed for statistical analysis. RESULTS: A total of 21, 17, and 19 metabolites strongly associated with ATB were found in the three databases after preliminary screening (P < 0.05). The intersecting metabolites across these databases included tryptophan, betaine, 1-linoleoylglycerol (1-monolinolein) (1-LG), 1-eicosatrienoylglycerophosphocholine, and oleoylcarnitine. Among them, betaine (I2 = 24%, P = 0.27) and 1-LG (I2 = 0%, P = 0.62) showed the lowest heterogeneity among the different ATB databases. In addition, the metabolic pathways of phosphatidylethanolamine biosynthesis (P = 0.0068), methionine metabolism (P = 0.0089), betaine metabolism (P = 0.0205) and oxidation of branched-chain fatty acids (P = 0.0309) were also associated with ATB. CONCLUSION: Betaine and 1-LG may be biomarkers or auxiliary diagnostic tools for ATB. They may provide new guidance for medical practice in the early diagnosis and surveillance of ATB. In addition, by interfering with phosphatidylethanolamine biosynthesis, methionine metabolism, betaine metabolism, oxidation of branched-chain fatty acids, and other pathways, it is helpful to develop new anti-tuberculosis drugs and explore the virulence or pathogenesis of ATB at a deeper level, providing an effective reference for future studies.

Dysregulation of hepatic one-carbon metabolism in classical homocystinuria: Implications of redox-sensitive DHFR repression and tetrahydrofolate depletion for pathogenesis and treatment.

Cystathionine beta-synthase-deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. HCU can be treated by using betaine to lower tissue and plasma levels of homocysteine (Hcy). Here, we show that mice with severely elevated Hcy and potentially deficient in the folate species tetrahydrofolate (THF) exhibit a very limited response to betaine indicating that THF plays a critical role in treatment efficacy. Analysis of a mouse model of HCU revealed a 10-fold increase in hepatic levels of 5-methyl -THF and a 30-fold accumulation of formiminoglutamic acid, consistent with a paucity of THF. Neither of these metabolite accumulations were reversed or ameliorated by betaine treatment. Hepatic expression of the THF-generating enzyme dihydrofolate reductase (DHFR) was significantly repressed in HCU mice and expression was not increased by betaine treatment but appears to be sensitive to cellular redox status. Expression of the DHFR reaction partner thymidylate synthase was also repressed and metabolomic analysis detected widespread alteration of hepatic histidine and glutamine metabolism. Many individuals with HCU exhibit endothelial dysfunction. DHFR plays a key role in nitric oxide (NO) generation due to its role in regenerating oxidized tetrahydrobiopterin, and we observed a significant decrease in plasma NOx (NO2 + NO3) levels in HCU mice. Additional impairment of NO generation may also come from the HCU-mediated induction of the 20-hydroxyeicosatetraenoic acid generating cytochrome CYP4A. Collectively, our data shows that HCU induces dysfunctional one-carbon metabolism with the potential to both impair betaine treatment and contribute to multiple aspects of pathogenesis in this disease.

Metabolome analysis revealed the critical role of betaine for arsenobetaine biosynthesis in the marine medaka (Oryzias melastigma).

Arsenobetaine (AsB), a non-toxic arsenic (As) compound found in marine fish, structurally resembles betaine (GB), a common methyl donor in organisms. This study investigates the potential role of GB in AsB synthesis in marine medaka (Oryzias melastigma) using metabolomic analysis. Dietary exposure to arsenate (As(V)) and varying GB concentrations (0.05% and 0.1% in diets) increased total As and AsB bioaccumulation, particularly in marine medaka muscle. Metabolomic analysis revealed that GB played a crucial role in promoting up-regulation in methylthioadenosine (MTA) by modulating the methionine cycle and down-regulation in glutathione (GSH) by modulating the glutathione cycle. Methionine metabolism and GSH, potentially binding again to exogenous GB, could synchronously produce more non-toxic AsB. Combining verification experiments of differential metabolites of Escherichia coli in vitro, GB, GSH, S-adenosylmethionine (SAM), and arsenocholine (AsC) entered methionine and glutathione metabolism pathways to generate more AsB. These findings underscore the GB's crucial regulatory role in modulating the synthesis of AsB. This study provides vital insights into the interplay between the structural analogs GB and AsB, offering specific strategies to enhance the detoxification mechanisms of marine fish in As-contaminated environments.

Effects of guanidino acetic acid and betaine supplementation on growth, dietary nutrient digestion and intestinal creatine metabolism in sheep.

BACKGROUND: The intestine of young ruminants is in the developmental stage and has weaker resistance to the changes of external environment. Improving intestinal health is vital to promoting growth of young ruminants. This study investigated effects of guanidino acetic acid (GAA) and rumen-protected betaine (RPB) supplementation on growth, dietary nutrient digestion and GAA metabolism in the small intestine of sheep. METHODS: Eighteen healthy Kazakh rams (27.46 ± 0.10 kg of body weight and 3-month old) were categorized into control, test group I and test group II, which were fed a basal diet, 1500 mg/kg GAA and 1500 mg/kg GAA + 600 mg/kg RPB, respectively. RESULTS: Compared with control group, test group II had increased (p < 0.05) average daily gain, plasma creatine level, ether extract (EE) and phosphorus digestibility on day 30. On day 60, the EE apparent digestibility, jugular venous plasma GAA, GAA content in the duodenal mucosa and GAA content in the jejunal and ileal mucosa of test group II were higher (p < 0.05) than other groups. Transcriptome analysis revealed that the differentially expressed genes (DEGs) involved in the duodenal pathways of oxidative phosphorylation and non-alcoholic fatty liver disease were significantly altered in test group II versus test group I (p < 0.05). Moreover, in the jejunum, the MAPK signalling pathway, complement and coagulation cascade and B-cell receptor signalling pathway were significantly enriched, with ATPase, solute carrier transporter protein, DHFR, SI, GCK, ACACA and FASN being the significantly DEGs (p < 0.05). CONCLUSION: Dietary supplementation of RPB on top of GAA in sheep diets may promote sheep growth and development by improving the body's energy, amino acid, glucose and lipid metabolism capacity.

Enhanced catabolism of glycine betaine and derivatives provides improved osmotic stress protection in Methylorubrum extorquens PA1.

Integration of metabolites into the overall metabolic network of a cell requires careful coordination dependent upon the ultimate usage of the metabolite. Different stoichiometric needs, and thus pathway fluxes, must exist for compounds destined for diverse uses, such as carbon sources, nitrogen sources, or stress-protective agents. Herein, we expand upon our previous work that highlighted the nature of glycine betaine (GB) metabolism in Methylobacteria to examine the utilization of GB-derivative compounds dimethylglycine (DMG) and sarcosine into Methylorubrum extorquens in different metabolic capacities, including as sole nitrogen and/or carbon sources. We isolated gain-of-function mutations that allowed M. extorquens PA1 to utilize dimethylglycine as a carbon source and dimethylglycine and sarcosine as nitrogen source. Characterization of mutants demonstrated selection for variants of the AraC-like regulator Mext_3735 that confer constitutive expression of the GB metabolic gene cluster, allowing direct utilization of the downstream GB derivatives. Finally, among the distinct isolates examined, we found that catabolism of the osmoprotectant used for selection (GB or dimethylglycine) enhanced osmotic stress resistance provided in the presence of that particular osmolyte. Thus, access to the carbon and nitrogen and osmoprotective effects of GB and DMG are made readily accessible through adaptive mutations. In M. extorquens PA1, the limitations to exploiting this group of compounds appear to exist predominantly at the levels of gene regulation and functional activity, rather than being constrained by transport or toxicity.IMPORTANCEOsmotic stress is a common challenge for bacteria colonizing the phyllosphere, where glycine betaine (GB) can be found as a prevalent osmoprotectant. Though Methylorubrum extorquens PA1 cannot use GB or its demethylation products, dimethylglycine (DMG) and sarcosine, as a sole carbon source, utilization is highly selectable via single nucleotide changes for both GB and DMG growth. The innate inability to use these compounds is due to limited flux through steps in the pathway and regulatory constraints. Herein, the characterization of the transcriptional regulator, Mext_3735 (GbdR), expands our understanding of the various roles in which GB derivatives can be used in M. extorquens PA1. Interestingly, increased catabolism of GB and derivatives does not interfere with, but rather improves, the ability of cells to thrive under increased salt stress conditions, suggesting that metabolic flux improves stress tolerance rather than providing a distinct tension between uses.

Osmotic stress response of the coral and oyster pathogen Vibrio coralliilyticus: acquisition of catabolism gene clusters for the compatible solute and signaling molecule myo-inositol.

Marine bacteria experience fluctuations in osmolarity that they must adapt to, and most bacteria respond to high osmolarity by accumulating compatible solutes also known as osmolytes. The osmotic stress response and compatible solutes used by the coral and oyster pathogen Vibrio coralliilyticus were unknown. In this study, we showed that to alleviate osmotic stress V. coralliilyticus biosynthesized glycine betaine (GB) and transported into the cell choline, GB, ectoine, dimethylglycine, and dimethylsulfoniopropionate, but not myo-inositol. Myo-inositol is a stress protectant and a signaling molecule that is biosynthesized and used by algae. Bioinformatics identified myo-inositol (iol) catabolism clusters in V. coralliilyticus and other Vibrio, Photobacterium, Grimontia, and Enterovibrio species. Growth pattern analysis demonstrated that V. coralliilyticus utilized myo-inositol as a sole carbon source, with a short lag time of 3 h. An iolG deletion mutant, which encodes an inositol dehydrogenase, was unable to grow on myo-inositol. Within the iol clusters were an MFS-type (iolT1) and an ABC-type (iolXYZ) transporter and analyses showed that both transported myo-inositol. IolG and IolA phylogeny among Vibrionaceae species showed different evolutionary histories indicating multiple acquisition events. Outside of Vibrionaceae, IolG was most closely related to IolG from a small group of Aeromonas fish and human pathogens and Providencia species. However, IolG from hypervirulent A. hydrophila strains clustered with IolG from Enterobacter, and divergently from Pectobacterium, Brenneria, and Dickeya plant pathogens. The iol cluster was also present within Aliiroseovarius, Burkholderia, Endozoicomonas, Halomonas, Labrenzia, Marinomonas, Marinobacterium, Cobetia, Pantoea, and Pseudomonas, of which many species were associated with marine flora and fauna.IMPORTANCEHost associated bacteria such as Vibrio coralliilyticus encounter competition for nutrients and have evolved metabolic strategies to better compete for food. Emerging studies show that myo-inositol is exchanged in the coral-algae symbiosis, is likely involved in signaling, but is also an osmolyte in algae. The bacterial consumption of myo-inositol could contribute to a breakdown of the coral-algae symbiosis during thermal stress or disrupt the coral microbiome. Phylogenetic analyses showed that the evolutionary history of myo-inositol metabolism is complex, acquired multiple times in Vibrio, but acquired once in many bacterial plant pathogens. Further analysis also showed that a conserved iol cluster is prevalent among many marine species (commensals, mutualists, and pathogens) associated with marine flora and fauna, algae, sponges, corals, molluscs, crustaceans, and fish.

Unveiling the crucial role of betaine: modulation of GABA homeostasis via SLC6A1 transporter (GAT1).

Betaine is an endogenous osmolyte that exhibits therapeutic potential by mitigating various neurological disorders. However, the underlying cellular and molecular mechanisms responsible for its neuroprotective effects remain puzzling.In this study, we describe a possible mechanism behind the positive impact of betaine in preserving neurons from excitotoxicity. Here we demonstrate that betaine at low concentration modulates the GABA uptake by GAT1 (slc6a1), the predominant GABA transporter in the central nervous system. This modulation occurs through the temporal inhibition of the transporter, wherein prolonged occupancy by betaine impedes the swift transition of the transporter to the inward conformation. Importantly, the modulatory effect of betaine on GAT1 is reversible, as the blocking of GAT1 disappears with increased extracellular GABA. Using electrophysiology, mass spectroscopy, radiolabelled cellular assay, and molecular dynamics simulation we demonstrate that betaine has a dual role in GAT1: at mM concentration acts as a slow substrate, and at µM as a temporal blocker of GABA, when it is below its K0.5. Given this unique modulatory characteristic and lack of any harmful side effects, betaine emerges as a promising neuromodulator of the inhibitory pathways improving GABA homeostasis via GAT1, thereby conferring neuroprotection against excitotoxicity.

The Role of One-Carbon Metabolism and Methyl Donors in Medically Assisted Reproduction: A Narrative Review of the Literature.

One-carbon (1-C) metabolic deficiency impairs homeostasis, driving disease development, including infertility. It is of importance to summarize the current evidence regarding the clinical utility of 1-C metabolism-related biomolecules and methyl donors, namely, folate, betaine, choline, vitamin B12, homocysteine (Hcy), and zinc, as potential biomarkers, dietary supplements, and culture media supplements in the context of medically assisted reproduction (MAR). A narrative review of the literature was conducted in the PubMed/Medline database. Diet, ageing, and the endocrine milieu of individuals affect both 1-C metabolism and fertility status. In vitro fertilization (IVF) techniques, and culture conditions in particular, have a direct impact on 1-C metabolic activity in gametes and embryos. Critical analysis indicated that zinc supplementation in cryopreservation media may be a promising approach to reducing oxidative damage, while female serum homocysteine levels may be employed as a possible biomarker for predicting IVF outcomes. Nonetheless, the level of evidence is low, and future studies are needed to verify these data. One-carbon metabolism-related processes, including redox defense and epigenetic regulation, may be compromised in IVF-derived embryos. The study of 1-C metabolism may lead the way towards improving MAR efficiency and safety and ensuring the lifelong health of MAR infants.

Osmolyte-producing microbial biostimulants regulate the growth of Arachis hypogaea L. under drought stress.

Globally, drought stress poses a significant threat to crop productivity. Improving the drought tolerance of crops with microbial biostimulants is a sustainable strategy to meet a growing population's demands. This research aimed to elucidate microbial biostimulants' (Plant Growth Promoting Rhizobacteria) role in alleviating drought stress in oil-seed crops. In total, 15 bacterial isolates were selected for drought tolerance and screened for plant growth-promoting (PGP) attributes like phosphate solubilization and production of indole-3-acetic acid, siderophore, hydrogen cyanide, ammonia, and exopolysaccharide. This research describes two PGPR strains: Acinetobacter calcoaceticus AC06 and Bacillus amyloliquefaciens BA01. The present study demonstrated that these strains (AC06 and BA01) produced abundant osmolytes under osmotic stress, including proline (2.21 and 1.75 µg ml- 1), salicylic acid (18.59 and 14.21 µg ml- 1), trehalose (28.35 and 22.74 µg mg- 1 FW) and glycine betaine (11.35 and 7.74 mg g- 1) respectively. AC06 and BA01 strains were further evaluated for their multifunctional performance by inoculating in Arachis hypogaea L. (Groundnut) under mild and severe drought regimes (60 and 40% Field Capacity). Inoculation with microbial biostimulants displayed distinct osmotic-adjustment abilities of the groundnut, such as growth parameters, plant biomass, photosynthetic pigments, relative water content, proline, and soluble sugar in respective to control during drought. On the other hand, plant sensitivity indexes such as electrolyte leakage and malondialdehyde (MDA) contents were decreased as well as cooperatively conferred plant drought tolerance by induced alterations in stress indicators such as catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD). Thus, Acinetobacter sp. AC06 and Bacillus sp. BA01 can be considered as osmolyte producing microbial biostimulants to simultaneously induce osmotic tolerance and metabolic changes in groundnuts under drought stress.

The origin of betaine in mouse oocytes and preimplantation embryos†.

Betaine has important roles in preimplantation mouse embryos, including as an organic osmolyte that functions in cell volume regulation in the early preimplantation stages and as a donor to the methyl pool in blastocysts. The origin of betaine in oocytes and embryos was largely unknown. Here, we found that betaine was present from the earliest stage of growing oocytes. Neither growing oocytes nor early preantral follicles could take up betaine, but antral follicles were able to transport betaine and supply the enclosed oocyte. Betaine is synthesized by choline dehydrogenase, and female mice lacking Chdh did not have detectable betaine in their oocytes or early embryos. Supplementing betaine in their drinking water restored betaine in the oocyte only when supplied during the final stages of antral follicle development but not earlier in folliculogenesis. Together with the transport results, this implies that betaine can only be exogenously supplied during the final stages of oocyte growth. Previous work showed that the amount of betaine in the oocyte increases sharply during meiotic maturation due to upregulated activity of choline dehydrogenase within the oocyte. This betaine present in mature eggs was retained after fertilization until the morula stage. There was no apparent role for betaine uptake via the SIT1 (SLC6A20) betaine transporter that is active at the 1- and 2-cell stages. Instead, betaine was apparently retained because its major route of efflux, the volume-sensitive organic osmolyte - anion channel, remained inactive, even though it is expressed and capable of being activated by a cell volume increase.

Total replacement of soybean meal with alternative plant-based ingredients and a combination of feed additives in broiler diets from 1 day of age during the whole growing period.

The capacity of combinations of feed enzymes, natural betaine and a probiotic, combined with alternative plant-based ingredients, to totally replace soybean meal (SBM) in a broiler diet was evaluated. Day-old Ross 308 males (2,574) were assigned to 9 treatments (13 pens/treatment, 22 birds/pen) in a completely randomized design. All diets were pelleted and fed ad libitum in 4 phases: starter, grower, finisher 1, finisher 2 (0-10, 10-21, 21-35, and 35-42 d of age, respectively). Treatments included: 1) control diet containing SBM (SBM control), supplemented with phytase (PhyG), at 2,000, 1,500, 1000 and 1,000 FTU/kg in each phase and xylanase (X) at 750 U/kg, [crude protein (CP): 23.5%, 22.0%, 20.2% and 19.3% in each phase]; 2) to 5), alternative (ALT), SBM-free diets, containing the same CP level as the control ("CP high"), supplemented with PhyG as in the control, protease (P, 800 U/kg) and in 2) xylanase (750 U/kg) (ALT+PhyG+P+X), 3) xylanase-ß-glucanase (XB, 1,200 U/kg and 152 U/kg) (Alt+PhyG+P+XB), 4) XB plus betaine (800 g/ton) (ALT+PhyG+P+XB+Bet), and 5) XB plus a probiotic [150,000 colony forming units (CFU)/g] (ALT+PhyG+P+XB+Prob); 6) to 9) as treatments 2) to 5) but with CP reduced by -2.0 to -1.5% points vs. control ('CP low'). Final (d 42) BW and overall (d 0-42) feed conversion ratio (FCR) of birds fed the SBM control exceeded breeder objectives (+3.8% and -1.9%, respectively). Overall FCR was reduced and d 42 BW increased in birds fed "low" vs. "high" CP (P < 0.01). Overall FCR and feed intake were not different in ALT+PhyG+XB+P+Bet and ALT+PhyG+XB+P+Prob vs. the control, whereas final BW was reduced (P < 0.05) in all ALT treatments but close to breeder objectives (98.3%) in ALT+PhyG+XB+P+Prob. Feed costs of this treatment were similar to the control. Total replacement of SBM with alternative plant-based ingredients in a CP-low diet supplemented with hydrolytic enzymes and probiotics can achieve growth performance outcomes close to commercial breeder objectives.

The substrate-binding domains of the osmoregulatory ABC importer OpuA transiently interact.

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism.

Choline Dehydrogenase Contributes to Salt Tolerance in Dunaliella through Betaine Synthesis.

In Dunaliella tertiolecta, a microalga renowned for its extraordinary tolerance to high salinity levels up to 4.5 M NaCl, the mechanisms underlying its stress response have largely remained a mystery. In a groundbreaking discovery, this study identifies a choline dehydrogenase enzyme, termed DtCHDH, capable of converting choline to betaine aldehyde. Remarkably, this is the first identification of such an enzyme not just in D. tertiolecta but across the entire Chlorophyta. A 3D model of DtCHDH was constructed, and molecular docking with choline was performed, revealing a potential binding site for the substrate. The enzyme was heterologously expressed in E. coli Rosetta (DE3) and subsequently purified, achieving enzyme activity of 672.2 U/mg. To elucidate the role of DtCHDH in the salt tolerance of D. tertiolecta, RNAi was employed to knock down DtCHDH gene expression. The results indicated that the Ri-12 strain exhibited compromised growth under both high and low salt conditions, along with consistent levels of DtCHDH gene expression and betaine content. Additionally, fatty acid analysis indicated that DtCHDH might also be a FAPs enzyme, catalyzing reactions with decarboxylase activity. This study not only illuminates the role of choline metabolism in D. tertiolecta's adaptation to high salinity but also identifies a novel target for enhancing the NaCl tolerance of microalgae in biotechnological applications.