Multi-Omics Analysis of Lipid Metabolism for a Marine Probiotic Meyerozyma guilliermondii GXDK6 Under High NaCl Stress

Investigating microbial lipid regulation contributes to understanding the lipid-dependent signal transduction process of cells and helps to improve the sensitivity of microorganisms to environmental factors by interfering with lipid metabolism, thus beneficial for constructing advanced cell factories of novel molecular drugs. Integrated omics technology was used to systematically reveal the lipid metabolism mechanism of a marine Meyerozyma guilliermondii GXDK6 under high NaCl stress and test the sensitivity of GXDK6 to antibiotics when its lipid metabolism transformed. The omics data showed that when GXDK6 perceived 10% NaCl stress, the expression of AYR1 and NADPH-dependent 1-acyldihydroxyacetone phosphate reductase was inhibited, which weaken the budding and proliferation of cell membranes. This finding was further validated by decreased 64.39% of OD600 under 10% NaCl stress when compared with salt-free stress. In addition, salt stress promoted a large intracellular accumulation of glycerol, which was also verified by exogenous addition of glycerol. Moreover, NaCl stress remarkably inhibited the expression of drug target proteins (such as lanosterol 14-alpha demethylase), thereby increasing sensitivity to fluconazole. This study provided new insights into the molecular mechanism involved in the regulation of lipid metabolism in Meyerozyma guilliermondii strain and contributed to developing new methods to improve the effectiveness of killing fungi with lower antibiotics.

Epimerization of Deoxynivalenol by the Devosia Strain A6-243 Assisted by Pyrroloquinoline Quinone

Deoxynivalenol (DON) is a secondary metabolite produced by several Fusarium species that is hazardous to humans and animals after entering food chains. In this study, by adding cofactors, the Devosia strain A6-243 is identified as the DON-transforming bacteria from a bacterial consortium with the ability to biotransform DON of Pseudomonas sp. B6-24 and Devosia strain A6-243, and its effect on the biotransformation process of DON is studied. The Devosia strain A6-243 completely biotransformed 100 μg/mL of DON with the assistance of the exogenous addition of PQQ (pyrroloquinoline quinone) within 48 h and produced non-toxic 3-epi-DON (3-epi-deoxynivalenol), while Pseudomonas sp. B6-24 was not able to biotransform DON, but it had the ability to generate PQQ. Moreover, the Devosia strain A6-243 not only degraded DON, but also exhibited the ability to degrade 3-keto-DON (3-keto-deoxynivalenol) with the same product 3-epi-DON, indicating that DON epimerization by the Devosia strain A6-243 is a two-step enzymatic reaction. The most suitable conditions for the biodegradation process of the Devosia strain A6-243 were a temperature of 16–37 °C and pH 7.0–10, with 15–30 μM PQQ. In addition, the Devosia strain A6-243 was found to completely remove DON (6.7 μg/g) from DON-contaminated wheat. The results presented a reference for screening microorganisms with the ability of biotransform DON and laid a foundation for the development of enzymes for the detoxification of mycotoxins in grain and its products.

Pseudomonas response regulators produced in an E. coli heterologous expression host exhibit host-derived post-translational phosphorylation

In this report, we systematically characterize 32 response regulators (RRs) from a metal tolerant groundwater isolate, Pseudomonas stutzeri RCH2 to assess the impact of host-derived post-translational phosphorylation. As observed by distinct shifted bands in a phos-tag gel, 12 of the 24 detected RRs show homogenous mixtures of phosphorylated proteins or heterogenous mixtures of unphosphorylated and phosphorylated proteins. By evaluating the phosphorylation state of CzcR and CopR II under varying assay parameters, we found that changes to pH and exogenous addition of phospho-donors (e.g. acetyl phosphate) have little to no effect on phosphorylation state. By applying protein production conditions that decrease the pool of intracellular acetyl-phosphate in E. coli, we found a reduction in the phosphorylated population of CopR II when magnesium was added to the media, but observed no change in phosphorylated population when CopR II is expressed in E. coli BL21 (DE3) ∆pta, a mutant with a metabolic disruption to the acetyl-phosphate pathway. Therefore, the specific mechanism of post-translational phosphorylation of RRs in E. coli remains obscure. These findings show the importance of characterizing the phosphorylations state of proteins when heterologously expressed, since their biochemical and physiological properties are dependent on post-translational modification.

Effect of arginine, putrescine and spermidine on the polyamine, proline and chlorophyll content of tobacco (Nicotiana tabacum L.)

Polyamines, such as spermidine (Spd) spermine (Spm) and their direct precursor, the diamine putrescine (Put) are vital and essential aliphatic amines which are also present in plants. Although ethylene and polyamines are also involved in fruit ripening, the genes coding them must also take part in other biosynthetic pathways. In the ethylene and polyamines play an important role in development of salt stress tolerance, and in responses for biotic and abiotic stresses. Exogenous application of all three main polyamines (Put, Spd, Spm) increase salt tolerance of plants, but, accordingly to previous experiments, spermidine has the main effect on the enhancement of salt tolerance. Nicotiana tabacum L. plants were grown in vitro on MS medium, the treatments were as follows: arginine (150 mg l-1), putrescine (10 mg l-1), spermidine (10 mg l-1). Proline, chlorophyll a, b and polyamine contents were measured. The obtained results show that the arginine decarboxylase and the spermidine synthase genes involved in polyamine metabolism, cannot be enhanced by exogenous addition of their precursor molecules. On the contrary, the spermine synthase gene has a positive effect to the lower-class forms of polyamines.

The Effect of Exogenous Oxytetracycline on High-Temperature Anaerobic Digestion of Elements in Swine Wastewater

Tetracycline antibiotics (TCs) are a common type of antibiotic found in swine wastewater. Oxytetracycline (OTC) is a significant type of TC. This study mainly examined the influence of OTC on high-temperature anaerobic digestion by adding OTC to collections of swine wastewater at different times during the digestion process. The results showed that high-temperature anaerobic digestion was suitable for the removal of TCs, with an 87% OTC removal efficiency achieved by day 20. Additionally, OTC added from external sources was found to inhibit the chlortetracycline degradation process and affect the first-order degradation kinetic model of TCs. Complexation reactions were the main ways in which OTC affected the heavy metal content of the water. The exogenous addition of OTC was found to inhibit the activity of some digester microbial strains, reduce the proportion of dominant strains, such as MBA03, and kill certain specific strains. This performance alteration was most obvious when OTC was added in the middle of the reaction.

Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm

In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed prior to cryopreservation, thereby exposing sperm cells to further oxidative stress. The exogenous addition of antioxidants to the freezing medium can reduce the detrimental effects caused by ROS generation. Therefore, the aim of this study was to evaluate how the addition of different reduced glutathione (GSH) concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) to fresh sperm affect their cryotolerance. Total and progressive motility, kinematic parameters and motile sperm subpopulations were significantly (p < 0.05) different from the control in treatments containing 8 mM and 10 mM GSH, but not at lower concentrations. Plasma and acrosome membrane integrity, mitochondrial membrane potential (MMP) and intracellular superoxide levels (O2−) were not affected (p > 0.05) by any GSH concentration. Interestingly, however, the addition of 8 mM or 10 mM GSH reduced (p < 0.05) the percentages of viable sperm with high overall ROS levels compared to the control. In conclusion, frozen-thawed donkey sperm are able to tolerate high GSH concentrations, which differs from what has been observed in other species. This antioxidant capacity suggests that ROS could be important during post-AI and that the impact of using exogenous antioxidants like GSH to improve the sperm resilience to freeze-thawing is limited in this species.

Completely dechlorinating of trichloroethene by a Dehalococcoides mccartyi-containing microbial consortium in the absence of cobalamin

Completely dechlorinating of trichloroethene (TCE) by Dehalococcoides mccartyi (D.mccartyi) is catalyzed by reductive dehalogenases (RDases) which possess cobalamin as the crucial cofactor, whereas virtually all pure D.mccartyi strains isolated thus far are corrinoid auxotrophs. Exogenous addition of commercially available cobalamin for real TCE-contaminated site decontamination is deemed to be unrealistic. In this study, TCE reduction by a D.mccartyi-containing microbial consortium utilizing biosynthetic cobalamin generated by interior corrinoid-producing organisms within this mixed consortia was studied. The results confirmed that subcultures with exogenous cobalamin omitting from the medium apparently were impervious and enabled to successively metabolize TCE to non-chlorinated ethene. The 2-bromoethanesulfonate and ampicillin resistance tests results suggested that bacteria (particularly certain ampicillin-sensitive ones) rather than methanogenic archaea within this microbial consortium were responsible for biosynthesizing cobalamin. Moreover, relative stable Ɛ-carbon values of TCE among treatments in disregard of whether exogenous cobalamin or selective inhibitors were existed in the medium also speculated that cobalamin biosynthesized by these organisms was enable to uptake and utilize by D.mccartyi for RDases synthesis and eventually participated in TCE reduction. Finally, the Illumina MiSeq sequencing analysis indicated that Desulfitobacterium and Acetobacterium in this microbial consortium probably both were in charge of de novo cobalamin biosynthesis to fulfillment the requirements of D.mccartyi for TCE metabolism.

Industrial Heap Bioleaching of Copper Sulfide Ore Started with Only Water Irrigation

Sulfuric acid solution containing ferric iron is the extractant for industrial heap bioleaching of copper sulfides. To start a heap bioleaching plant, sulfuric acid is usually added to the irrigation solution to maintain adequate acidity (pH 1.0–2.0) for copper dissolution. An industrial practice of heap bioleaching of secondary copper sulfide ore that began with only water irrigation without the addition of sulfuric acid was successfully implemented and introduced in this manuscript. The mineral composition and their behavior related to the production and consumption of sulfuric acid during the bioleaching in heaps was analyzed. This indicated the possibility of self-generating of sulfuric acid in heaps without exogenous addition. After proving by batches of laboratory tests, industrial measures were implemented to promote the sulfide mineral oxidation in heaps throughout the acidifying stages, from a pH of 7.0 to 1.0, thus sulfuric acid and iron was produced especially by pyrite oxidation. After acidifying of the heaps, adapted microbial consortium was inoculated and established in a leaching system. The launch of the bioleaching heap and finally the production expansion were realized without the addition of sulfuric acid, showing great efficiency under low operation costs.

Effect of Foliar Spraying of Organic and Inorganic Selenium Fertilizers On Selenium Accumulation and Speciation During Different Stages of Rice Growth

Aims Most crops are supplemented with selenium (Se) through the exogenous addition of inorganic selenium fertilizer. There is a lack of in-depth research on organic selenium fertilizers. Meanwhile, the dosage range between human selenium deficiency and selenium toxicity is narrow, so the selenium content of agricultural products needs to be controlled within a reasonable interval. Methods W e analyzed and compared the Se accumulation and speciation in rice during three different growth stages (late tillering stage, initiate heading stage, and full heading stage) using three selenium fertilizers, selenite, fermented Se, and potassium Selenocyanoacetate (Se-AAF) via the foliar application. Results The selenium content in rice sprayed with organic selenium fertilizer was controlled in the relatively safe range and met the human selenium supplement requirement compared to the sprayed sodium selenite, which was too high of a dose. The percentage of organic Se and protein Se in brown rice was found to be similar in all three Se fertilizers. The highest organic selenium content of 91.57% was found in the grain of rice at the full heading stage by spraying Se-AAF. The main Se species in the grain was selenomethionine (SeMet), which reached 80% of the total selenium. Se-methyl selenocysteine(SeMeCys) was found only in Se-AAF. The grain quality showed that all three Se fertilizers increased the consistency of gelatinization. Conclusions Appropriately delaying the spraying time and selecting organic Se fertilizer as the Se source can help to produce green and safe selenium-rich rice.

Human endothelial cells and fibroblasts express and produce the coagulation proteins necessary for thrombin generation

In a previous study, we reported that human endothelial cells (ECs) express and produce their own coagulation factors (F) that can activate cell surface FX without the additions of external proteins or phospholipids. We now describe experiments that detail the expression and production in ECs and fibroblasts of the clotting proteins necessary for formation of active prothrombinase (FV–FX) complexes to produce thrombin on EC and fibroblast surfaces. EC and fibroblast thrombin generation was identified by measuring: thrombin activity; thrombin–antithrombin complexes; and the prothrombin fragment 1.2 (PF1.2), which is produced by the prothrombinase cleavage of prothrombin (FII) to thrombin. In ECs, the prothrombinase complex uses surface-attached FV and γ-carboxyl-glutamate residues of FX and FII to attach to EC surfaces. FV is also on fibroblast surfaces; however, lower fibroblast expression of the gene for γ-glutamyl carboxylase (GGCX) results in production of vitamin K-dependent coagulation proteins (FII and FX) with reduced surface binding. This is evident by the minimal surface binding of PF1.2, following FII activation, of fibroblasts compared to ECs. We conclude that human ECs and fibroblasts both generate thrombin without exogenous addition of coagulation proteins or phospholipids. The two cell types assemble distinct forms of prothrombinase to generate thrombin.