The monofunctional CO dehydrogenase CooS is essential for growth of Thermoanaerobacter kivui on carbon monoxide

Thermoanaerobacter kivui is a thermophilic acetogen that can grow on carbon monoxide as sole carbon and energy source. To identify the gene(s) involved in CO oxidation, the genome sequence was analyzed. Two genes potentially encoding CO dehydrogenases were identified. One, cooS, potentially encodes a monofunctional CO dehydrogenase, whereas another, acsA, potentially encodes the CODH component of the CODH/ACS complex. Both genes were cloned, a His-tag encoding sequence was added, and the proteins were produced from a plasmid in T. kivui. His-AcsA copurified by affinity chromatography with AcsB, the acetyl-CoA synthase of the CO dehydrogenase/acetyl CoA synthase complex. His-CooS copurified with CooF1, a small iron-sulfur center containing protein likely involved in electron transport. Both protein complexes had CO:ferredoxin oxidoreductase as well as CO:methyl viologen oxidoreductase activity, but the activity of CooSF1 was 15-times and 231-times lower, respectively. To underline the importance of CooS, the gene was deleted in the CO-adapted strain. Interestingly, the ∆cooS deletion mutant did not grow on CO anymore. These experiments clearly demonstrated that CooS is essential for growth of T. kivui on CO. This is in line with the hypothesis that CooS is the CO-oxidizing enzyme in cells growing on CO.

Transcriptome Reveals the Dynamic Response Mechanism of Pearl Millet Roots under Drought Stress

Drought is a major threat to global agricultural production that limits the growth, development and survival rate of plants, leading to tremendous losses in yield. Pearl millet (Cenchrus americanus (L.) Morrone) has an excellent drought tolerance, and is an ideal plant material for studying the drought resistance of cereal crops. The roots are crucial organs of plants that experience drought stress, and the roots can sense and respond to such conditions. In this study, we explored the mechanism of drought tolerance of pearl millet by comparing transcriptomic data under normal conditions and drought treatment at four time points (24 h, 48 h, 96 h, and 144 h) in the roots during the seedling stage. A total of 1297, 2814, 7401, and 14,480 differentially expressed genes (DEGs) were found at 24 h, 48 h, 96 h, and 144 h, respectively. Based on Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analyses, we found that many DEGs participated in plant hormone-related signaling pathways and the “oxidoreductase activity” pathway. These results should provide a theoretical basis to enhance drought resistance in other plant species.

Vaccinium virgatum Aiton Leaves Extract Suppressed Lipid Accumulation and Uric Acid Production in 3T3-L1 Adipocytes

Blueberry (Vaccinium virgatum Aiton; Kinisato 35 Gou) leaves have recently attracted increasing attention as a useful material for the prevention of lifestyle diseases. Here, we examined the effects of the hot water extract of blueberry leaves (BLEx) on lipogenesis and uric acid production in 3T3-L1 adipocytes. The results showed that BLEx suppressed lipid accumulation and the mRNA expression of differentiation markers in 3T3-L1 adipocytes. A fractionation study showed that the highly polymerized proanthocyanidin-rich fraction was responsible for this effect. Upon maturation to adipocytes, 3T3-L1 cells produced uric acid and tumor necrosis factor-α, and hypoxia stimulated the production of uric acid and xanthine oxidoreductase activity. BLEx suppressed the production of uric acid under these conditions. Although BLEx inhibited the enzymatic activity of xanthine oxidase, this activity was observed in several fractions containing catechin, epicatechin, chlorogenic acid, rutin, and low molecular weight proanthocyanidins. Taken together, these results indicate that BLEx contains various compounds with the ability to suppress lipid accumulation and uric acid production in adipocytes.

Genome-Wide Identification and Functional Characterization of GATA Transcription Factor Gene Family in Alternaria alternata

In the present study, we identified six GATA transcription factors (AaAreA, AaAreB, AaLreA, AaLreB, AaNsdD, and AaSreA) and characterized their functions in response to environmental stress and virulence in the tangerine pathotype of Alternaria alternata. The targeted gene knockout of each of the GATA-coding genes decreased the growth to varying degrees. The mutation of AaAreA, AaAreB, AaLreB, or AaNsdD decreased the conidiation. All the GATA transcription factors were found to be required for tolerance to cumyl hydroperoxide and tert-butyl-hydroperoxide (oxidants) and Congo red (a cell-wall-destructing agent). Pathogenicity assays assessed on detached citrus leaves revealed that mutations of AaAreA, AaLreA, AaLreB, or AaNsdD significantly decreased the fungal virulence. A comparative transcriptome analysis between the ∆AreA mutant and the wild-type strain revealed that the inactivation of AaAreA led to alterations in the expression of genes involved in a number of biological processes, including oxidoreductase activity, amino acid metabolism, and secondary metabolite biogenesis. Taken together, our findings revealed that GATA-coding genes play diverse roles in response to environmental stress and are important regulators involved in fungal development, conidiation, ROS detoxification, as well as pathogenesis. This study, for the first time, systemically underlines the critical role of GATA transcription factors in response to environmental stress and virulence in A. alternata.

Gender Differences in the Impact of Plasma Xanthine Oxidoreductase Activity on Coronary Artery Spasm

Xanthine oxidoreductase (XOR) is the rate-limiting enzyme in uric acid (UA) production that plays a pivotal role in generating oxidative stress. Gender differences in the impact of plasma XOR activity on coronary artery spasm (CAS) remain unclear. We investigated plasma XOR activity in 132 patients suspected of having CAS (male, n = 78; female, n = 54) and who underwent an intracoronary acetylcholine provocation test. Plasma XOR activity was significantly lower in female patients compared with male patients. CAS was provoked in 36 male patients and 17 female patients, and both had significantly higher plasma XOR activity than those without. Multivariate logistic regression analysis showed that this activity was independently associated with the incidence of CAS in both sexes after adjusting for confounding factors. The optimal cut-off values for predicting CAS were lower in female patients than in male patients. Multivariate analysis demonstrated that female patients with high XOR activity exhibited a higher incidence of CAS than male patients. Plasma XOR activity was an independent predictor of the incidence of CAS in both sexes. The impact of plasma XOR activity on CAS was stronger in female patients than in male patients.

MSMEG_3955 from Mycobacterium smegmatis is a FMN bounded homotrimeric NAD(P)H:Flavin mononucleotide (FMN) oxidoreductase

Background Tuberculosis (TB) remains an important public health problem since it is the major cause of elevated morbidity and mortality globally. Previous works have shown that Mycobacterium tuberculosis (Mtb); the prime causative agent of the deadly disease has dormancy survival regulator (DosR) regulon, a two-component regulatory system which controls the transcription of more than 50 genes. However, the structure and detailed functions of these DosR regulated genes are largely undetermined. Out of many DosR regulon genes, Rv3131 gets up regulated in hypoxic conditions and was believed to encode for a nitroreductase flavoprotein. The utilization of mycobacteria-specific model systems has greatly added to our understanding of the molecular mechanisms involved in the life cycle and pathogenesis of Mtb. Results In this study the non-pathogenic mycobacterial model organism Mycobacterium smegmatis (Msmeg) was used to reveal the structure and function of MSMEG_3955; which is a homologue of Rv3131 from Mtb. Using chromatography and spectroscopy techniques it was revealed that cofactor flavin mononucleotide (FMN) was bound to flavoprotein MSMEG_3955. Consistent with the homology modelling predictions, Circular Dichroism (CD) analysis indicated that the MSMEG_3955 is composed of 39.3% α-helix and 24.9% β-pleated sheets. In contrast to the current notions, the enzymatic assays performed in the present study revealed that MSMEG_3955 was not capable of reducing nitro substrates but showed NADPH dependent FMN oxidoreductase activity. Also, gel permeation chromatography, dynamic light scattering and native acidic gels showed that MSMEG_3955 exists as a homotrimer. Furthermore, the presence of NADPH dependent FMN oxidoreductase and homotrimeric existence could be an alternative function of the protein to help the bacteria survive in dormant state or may be involved in other biochemical pathways. Conclusion MSMEG_3955 is a FMN bound flavoprotein, which exits as a trimer under in vitro conditions. There is no disulphide linkages in between the three protomers of the homotrimer MSMEG_3955. It has a NADPH dependent FMN oxidoreductase activity.

Genome-Wide Identification of Long Non-Coding RNAs and Their Potential Functions in Poplar Growth and Phenylalanine Biosynthesis

Poplar is an important bioenergy tree species. lncRNAs play important roles in various biological regulatory processes, and their expression pattern is more tissue-specific than mRNAs. In this study, P. deltoides “Danhong” (Pd) and P. simonii “Tongliao1” (Ps) with different growth rates and wood quality were used as experimental materials, and the transcriptomes of their shoot apical meristem, xylem, and phloem were sequenced. Furthermore, high-throughput RNA sequencing analysis revealed that the expression patterns of genes and lncRNAs are different between the two genotypes. 6,355 lncRNAs were identified. Based on target prediction, lncRNAs and target genes were involved in ADP binding, oxidoreductase activity, phenylpropanoid biosynthesis, and cyanoamino acid metabolism. The DElncRNAs in two poplars were co-expressed with transcription factors and structural genes of lignin and flavonoid pathways. In addition, we found the potential target lncRNAs of miRNA. This result provides basic evidence for a better understanding of the regulatory role of lncRNAs in regulating phenylalanine molecular pathways and wood formation.

Transcriptomic Analysis of Stropharia Rugosoannulata Reveals Potential Carbohydrate Metabolism and Cold Resistance Mechanisms Under Low-Temperature Stress

Low temperature is an important environmental factor that restricts the growth of Stropharia rugosoannulata; however, the molecular mechanisms underlying S. rugosoannulata responses to low-temperature stress are largely unknown. In this study, we performed a transcriptome analysis of a high-sensitivity strain (DQ-1) and low-sensitivity strain (DQ-3) under low-temperature stress. The liquid hyphae of S. rugosoannulata treated at 25°C and 10°C were analyzed by RNA-Seq, and a total of 9499 differentially expressed genes (DEGs) were identified. GO and KEGG enrichment analyses showed that these genes were enriched in “xenobiotic biodegradation and metabolism”, “carbohydrate metabolism”, “lipid metabolism” and “oxidoreductase activity”. Further research found that carbohydrate enzyme (AA, GH, CE, and GT) genes were downregulated more significantly in DQ-1 than DQ-3 and several cellulase activities were also reduced to a greater extent. Moreover, the CAT1, CAT2, GR, and POD genes and more heat shock protein genes (HSP20, HSP78 and sHSP) were upregulated in the two strains after low-temperature stress, and the GPX gene and more heat shock protein genes were upregulated in DQ-3. In addition, the enzyme activity and qRT–PCR results showed trends similar to those of the RNA-Seq results. This result indicates that low-temperature stress reduces the expression of different AA, GH, CE, and GT enzyme genes and reduces the secretion of cellulase, thereby reducing the carbohydrate metabolism process and mycelial growth of S. rugosoannulata. Moreover, the expression levels of different types of antioxidant enzymes and heat shock proteins are also crucial for S. rugosoannulata to resist low-temperature stress. In short, this study will provide a basis for further research on important signaling pathways, gene functions and variety breeding of S. rugosoannulata related to low-temperature stress.

The flavin transferase ApbE flavinylates the ferredoxin:NAD+-oxidoreductase Rnf required for N2 fixation in Azotobacter vinelandii

Azotobacter vinelandii, the model microbe in nitrogen fixation studies, uses the ferredoxin:NAD+-oxidoreductase Rnf to regenerate ferredoxin (flavodoxin) acting as an electron donor for nitrogenase. However, the relative contribution of Rnf into nitrogenase functioning is unknown because this bacterium contains another ferredoxin reductase, FixABCX. Furthermore, Rnf is flavinylated in the cell, but the importance and pathway of this modification reaction also remain largely unknown. We have constructed A. vinelandii cells with impaired activities of FixABCX and/or putative flavin transferase ApbE. The ApbE-deficient mutant could not produce covalently flavinylated membrane proteins and demonstrated a markedly decreased flavodoxin:NAD+ oxidoreductase activity and significant growth defect under diazotrophic conditions. The double ΔFix/ΔApbE mutation abolished the flavodoxin:NAD+ oxidoreductase activity and the ability of A. vinelandii to grow in the absence of fixed nitrogen source. ApbE flavinylated a truncated RnfG subunit of Rnf1 by forming a phosphoester bond between FMN and a threonine residue. These findings indicate that Rnf (presumably its Rnf1 form) is the major ferredoxin-reducing enzyme in the nitrogen fixation system and that the activity of Rnf depends on its covalent flavinylation by the flavin transferase ApbE.

Structure of mycobacterial CIII2CIV2 respiratory supercomplex bound to the tuberculosis drug candidate telacebec (Q203)

The imidazopyridine telacebec, also known as Q203, is one of only a few new classes of compounds in more than fifty years with demonstrated antituberculosis activity in humans. Telacebec inhibits the mycobacterial respiratory supercomplex composed of complexes III and IV (CIII2CIV2). In mycobacterial electron transport chains, CIII2CIV2 replaces canonical CIII and CIV, transferring electrons from the intermediate carrier menaquinol to the final acceptor, molecular oxygen, while simultaneously transferring protons across the inner membrane to power ATP synthesis. We show that telacebec inhibits the menaquinol:oxygen oxidoreductase activity of purified Mycobacterium smegmatis CIII2CIV2 at concentrations similar to those needed to inhibit electron transfer in mycobacterial membranes and Mycobacterium tuberculosis growth in culture. We then used electron cryomicroscopy (cryoEM) to determine structures of CIII2CIV2 both in the presence and absence of telacebec. The structures suggest that telacebec prevents menaquinol oxidation by blocking two different menaquinol binding modes to prevent CIII2CIV2 activity.