A Novel Antibacterial Component and the Mechanisms of an Amaranthus tricolor Leaf Ethyl Acetate Extract against Acidovorax avenae subsp. citrulli

The aim of the present investigation was to determine the active ingredients in Amaranthus tricolor L. leaves and develop a biological pesticide. Organic solvent extraction, column chromatography, liquid chromatography, ODS-C18 reverse elution, Sephadex LH-20 gel filtration, H spectrum, and C spectrum were used to isolate the pure product for an assessment of the agricultural activity and bacteriostatic mechanisms. The results showed that the activity of the crude extract following carbon powder filtration was 1.63-fold that of the non-filtered extract. Further isolation was performed to obtain two pure products, namely, hydroxybenzoic acid (HBA) and benzo[b]furan-2-carboxaldehyde (BFC), and their molecular formulas and molecular weights were C7H6O3 and 138.12, and C9H6O2 and 146.12, respectively. Our study is the first to determine that HBA has bacteriostatic activity (MIC 125 μg/mL) and is also the first to isolate BFC from A. tricolor. The ultrastructure observation results showed that HBA caused the bacteria to become shriveled, distorted, and deformed, as well as exhibit uneven surfaces. After HBA treatment, 70 differentially expressed metabolites were detected in the bacteria, of which 9 were downregulated and 61 were upregulated. The differentially expressed metabolites were mainly strigolactones, organic acids and derivatives, fatty acids, benzene and substituted benzene derivatives, amino acids and associated metabolites, and alcohols and amines. Among all of the downregulated differentially expressed metabolites, MEDP1280 was the most critical, as it participates in many physiological and biochemical processes. The enrichment analysis showed that the differentially expressed metabolites mainly participate in tyrosine metabolism, biosynthesis of amino acids, cysteine and methionine metabolism, and arginine and proline metabolism. Additionally, HBA was found to disrupt cell membrane permeability and integrity, causing the leakage of substances and apoptosis. The physiological and biochemical test results showed that HBA could increase the pyruvate levels in bacteria but could decrease the activities of respiratory enzymes (malate dehydrogenase (MDH) and NADH oxidase) and antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX)). Inverse molecular docking was used to study the binding between HBA and respiratory and antioxidant enzymes. The results showed that HBA could bind to MDH, NADH oxidase, SOD, and GSH-PX, suggesting that these enzymes may be the effector targets of HBA. Conclusion: The optimal active ingredient in A. tricolor that can inhibit Acidovorax avenae subsp. citrulli was identified as HBA. HBA mainly disrupts the cell membrane, damages the metabolic system, and inhibits respiration and antioxidant enzyme activity to control bacterial growth. These results provide a reference for the further development of biological pesticides.

NADH inhibition of SIRT1 links energy state to transcription during time-restricted feeding

In mammals, circadian rhythms are entrained to the light cycle and drive daily oscillations in levels of NAD+, a cosubstrate of the class III histone deacetylase sirtuin 1 (SIRT1) that associates with clock transcription factors. Although NAD+ also participates in redox reactions, the extent to which NAD(H) couples nutrient state with circadian transcriptional cycles remains unknown. Here we show that nocturnal animals subjected to time-restricted feeding of a calorie-restricted diet (TRF-CR) only during night-time display reduced body temperature and elevated hepatic NADH during daytime. Genetic uncoupling of nutrient state from NADH redox state through transduction of the water-forming NADH oxidase from Lactobacillus brevis (LbNOX) increases daytime body temperature and blood and liver acyl-carnitines. LbNOX expression in TRF-CR mice induces oxidative gene networks controlled by brain and muscle Arnt-like protein 1 (BMAL1) and peroxisome proliferator-activated receptor alpha (PPARα) and suppresses amino acid catabolic pathways. Enzymatic analyses reveal that NADH inhibits SIRT1 in vitro, corresponding with reduced deacetylation of SIRT1 substrates during TRF-CR in vivo. Remarkably, Sirt1 liver nullizygous animals subjected to TRF-CR display persistent hypothermia even when NADH is oxidized by LbNOX. Our findings reveal that the hepatic NADH cycle links nutrient state to whole-body energetics through the rhythmic regulation of SIRT1.

Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment

Background Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. Results The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L−1 and 0.47 g L−1 9-OH-4-HP from 1 g L−1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L−1 from 5 g L−1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. Conclusion KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis

Cellular respiration is essential for multiple bacterial pathogens and a validated antibiotic target. In addition to driving oxidative phosphorylation, bacterial respiration has a variety of ancillary functions that obscure its contribution to pathogenesis. We find here that the intracellular pathogen Listeria monocytogenes encodes two respiratory pathways which are partially functionally redundant and indispensable for pathogenesis. Loss of respiration decreased NAD+ regeneration, but this could be specifically reversed by heterologous expression of a water-forming NADH oxidase (NOX). NOX expression fully rescued intracellular growth defects and increased L. monocytogenes loads >1,000-fold in a mouse infection model. Consistent with NAD+ regeneration maintaining L. monocytogenes viability and enabling immune evasion, a respiration-deficient strain exhibited elevated bacteriolysis within the host cytosol and NOX rescued this phenotype. These studies show that NAD+ regeneration, rather than oxidative phosphorylation, represents the primary role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis.

Efficient 2,3-Butanediol/Acetoin Production Using Whole-Cell Biocatalyst with a New Nadh/Nad(+) Regeneration System

An auto-inducing expression system was developed that could express target genes in S. marcescens MG1. Using this system, MG1 was constructed as a whole-cell biocatalyst to produce 2,3-butanediol/acetoin. Formate dehydrogenase (FDH) and 2,3-butanediol dehydrogenase were expressed together to build an NADH regeneration system to transform diacetyl to 2,3-butanediol. After fermentation, the extract of recombinant S. marcescens MG1ABC (pETDuet-bdhA-fdh) showed 2,3-BDH activity of 57.8 U/mg and FDH activity of 0.5 U/mg. And 27.95 g/L of 2,3-BD was achieved with a productivity of 4.66 g/Lh using engineered S. marcescens MG1(Pswnb+pETDuet-bdhA-fdh) after 6 h incubation. Next, to produce 2,3-butanediol from acetoin, NADH oxidase and 2,3-butanediol dehydrogenase from Bacillus subtilis were co-expressed to obtain a NAD+ regeneration system. After fermentation, the recombinant strain S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC) showed AR activity of 212.4 U/mg and NOX activity of 150.1 U/mg. We obtained 44.9 g/L of acetoin with a productivity of 3.74 g/Lh using S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC). This work confirmed that S. marcescens could be designed as a whole-cell biocatalyst for 2,3-butanediol and acetoin production.

Tangeretin and its Derivatives: An Integrative Bioinformatic Study of Obesity and Related Immunodeficiency

Obesity is a complex and major public health concern known to exacerbate many diseases. There are increasing evidences stating the obese people due to adiposity are getting more susceptible to immune deficiency disorders. Tangeretin is a key member of flavonoids reported to have many favourable biological activities. In search of novel leads in ameliorating obesity and related immunodeficiency, the present study is aimed at the in silico evaluation of tangeretin derivatives to assess their biological role. Initially tangeretin derivatives are designed by molecular manipulation approach.Drug likeness and bioactivity score prediction was done using Molinspiration web tool. Swiss ADME prediction and toxicological predictions were performed. In silico Molecular Docking studies were performed by employing a flexible ligand docking approach using Schrodinger on the protein targets namely leptin, Fat mass and obesity associated protein (FTO), Pancreatic lipase, Peroxisome proliferated receptor (PPARɣ) and NADH oxidase. Further the electronic parameters were computed for the best fitted ligands by DFT analysis. The evaluation of results was made based on Glide (Schrodinger) dock score. Out of 18 screened compounds, some of them showed the best docking scores with the targets when compared with the standard (Lovastatin). Particularly the two ligands (L-13 and L-8) showed the best binding score with all five targets. Moreover, DFT analysis carried out for the tangeretin and best fitted ligands (L13 and L8) substantiated the other in silico studies. These findings probably provide excellent lead candidates for the development of therapeutic drugs in combating obesity and related immune deficiency.

Efficient 1-Hydroxy-2-Butanone Production from 1,2-Butanediol by Whole Cells of Engineered E. coli

1-Hydroxy-2-butanone (HB) is a key intermediate for anti-tuberculosis pharmaceutical ethambutol. Commercially available HB is primarily obtained by the oxidation of 1,2-butanediol (1,2-BD) using chemical catalysts. In present study, seven enzymes including diol dehydrogenases, secondary alcohol dehydrogenases and glycerol dehydrogenase were chosen to evaluate their abilities in the conversion of 1,2-BD to HB. The results showed that (2R, 3R)- and (2S, 3S)-butanediol dehydrogenase (BDH) from Serratia sp. T241 could efficiently transform (R)- and (S)-1,2-BD into HB respectively. Furthermore, two biocatalysts co-expressing (2R, 3R)-/(2S, 3S)-BDH, NADH oxidase and hemoglobin protein in Escherichia coli were developed to convert 1,2-BD mixture into HB, and the transformation conditions were optimized. Maximum HB yield of 341.35 and 188.80 mM could be achieved from 440 mM (R)-1,2-BD and 360 mM (S)-1,2-BD by E. coli (pET-rrbdh-nox-vgb) and E. coli (pET-ssbdh-nox-vgb) under the optimized conditions. In addition, two biocatalysts showed the ability in chiral resolution of 1,2-BD isomers, and 135.68 mM (S)-1,2-BD and 112.43 mM (R)-1,2-BD with the purity of 100 % could be obtained from 300 and 200 mM 1,2-BD mixture by E. coli (pET-rrbdh-nox-vgb) and E. coli (pET-ssbdh-nox-vgb), respectively. These results provided potential application for HB production from 1,2-BD mixture and chiral resolution of (R)-1,2-BD and (S)-1,2-BD.

The Effect of Benzyl Isothiocyanate On The Expression of Genes Encoding NADH Oxidase And Fibronectin-Binding Protein In Oral Streptococcal Biofilms

Background: Recent studies have shown that antibiotic treatment results in up- or down regulation of several virulence-associated genes. The genes encoding NADH oxidase (nox) and fibronectin-binding protein (fbp) are known to play important roles in biofilms of some oral bacterial species. The objective was to study the effect of benzyl isothiocyanate (BITC), an antimicrobial agent from Miswak plant, on the expression of nox and fbp genes in some oral streptococci.Methods: Bacterial strains were grown as biofilms in brucella broth. The crystal violet stained biofilms were quantified by optical density measurements at 590 nm. The biofilms were treated with an antimicrobial agent (BITC) for 2 h and fold change in mRNA expression of nox and fbp genes in BITC treated selected oral streptococci was measured by comparative ∆∆Ct method on Real-Time PCR machine.Results: The highest amount of biofilm mass was produced by A. defectiva, followed by S. gordonii, S. mutans, G. elegans and G. adiacens. Upon treatment with BITC, S. gordonii biofilms showed highest mRNA expression for both fbp and nox genes. Mean (SE) folds increase in the expression of nox mRNA: S. gordonii 2 (0.30), followed by S. mutans 1.25 (0.18), A. defectiva 1.03 (0.09), G. adiacens 0.7 (0.03). Similarly for fbp, folds increase in mRNA expression was: S. gordonii 2.65 (0.03), followed by A. defectiva 2.09 (0.60), G. elegans 1.61 (0.40), S. mutans 1.57 (0.20), and G. adiacens 0.58 (0.06). G. elegans mRNA levels for nox were extremely low (0.006-fold). Conclusion: BITC treatment of the biofilms caused an upregulation of biofilm-associated genes fbp and nox genes in most of the tested species suggesting the significance of these genes in biofilm lifestyle of these oral bacteria. Increased expression of nox and fbp genes in the biofilm lifestyle of these species needs further investigation to understand if it contributes to antimicrobial resistance.