APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2017
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IF 3.807
Wenjun Yu,Rongshui Wang,Haiyan Huang,Huijun Xie,Shuning Wang
Agrobacterium tumefaciens S33 can grow with nicotine as the sole source of carbon, nitrogen, and energy via a novel hybrid of the pyridine pathway and the pyrrolidine pathway. Characterization of the enzymes involved in the hybrid pathway is important for understanding its biochemical mechanism. Here, we report that the molybdenum-containing nicotine dehydrogenase (NdhAB), which catalyzes the initial step of nicotine degradation, is located in the periplasm of strain S33, while the 6-hydroxynicotine oxidase and 6-hydroxypseudooxynicoine oxidase are in the cytoplasm. This is consistent with the fact that NdhA has a Tat signal peptide. Interestingly, an open reading frame (ORF) adjacent to the ndhAB gene was verified to encode a copper-containing electron carrier, pseudoazurin (Paz), which has a signal peptide typical of bacterial Paz proteins. Both were transported into the periplasm after being produced in the cytoplasm. We purified NdhAB from the periplasmic fraction of strain S33 and found that with Paz as the physiological electron acceptor, NdhAB catalyzed the hydroxylation of nicotine at a specific rate of 110.52 ± 8.09 μmol · min−1 · mg of protein−1, where the oxygen atom in the hydroxyl group of the product 6-hydroxynicotine was derived from H2O. The apparent Km values for nicotine and Paz were 1.64 ± 0.07 μM and 3.61 ± 0.23 μM, respectively. NAD(P)+, O2, and ferredoxin could not serve as electron acceptors. Disruption of the paz gene disabled the strain for nicotine degradation, indicating that Paz is required for nicotine catabolism in the strain. These findings help our understanding of electron transfer during nicotine degradation in bacteria.IMPORTANCE Nicotine is a toxic and addictive N-heterocyclic aromatic alkaloid produced in tobacco. Its catabolism in organisms and degradation in tobacco wastes have become major concerns for human health and the environment. Bacteria usually decompose nicotine using the classical strategy of hydroxylating the pyridine ring with the help of activated oxygen by nicotine dehydrogenase, which binds one molybdopterin, two [2Fe2S] clusters, and usually one flavin adenine dinucleotide (FAD) as well. However, the physiological electron acceptor for the reaction is still unknown. In this study, we found that the two-component nicotine dehydrogenase from Agrobacterium tumefaciens S33, naturally lacking an FAD-binding domain, is located in the periplasmic space and uses a copper-containing electron carrier, pseudoazurin, as its physiological electron acceptor. We report here the role of pseudoazurin in a reaction catalyzed by a molybdopterin-containing hydroxylase occurring in the periplasmic space. These results provide new biochemical knowledge on microbial degradation of N-heterocyclic aromatic compounds.
研究领域:
微生物学
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生物化学
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环境科学
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分子生物学
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PubMed
高影响因子
JOURNAL OF APPLIED MICROBIOLOGY
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2022
·Vol. 133
·期 5
·3161-3175
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IF 4.059
Pan Deng,Wang Xing‐Yun,Zhou Jin‐Wei,Yang Liang,Khan Abbas,Wei Dong‐Qing,Li Jun‐Jian,Jia Ai‐Qun
AimsIn the current study the anti‐virulence and anti‐biofilm activities of the cinnamic acid derivative, 3‐methoxycinnamic acid, was investigated against Agrobacterium tumefaciens.Methods and ResultsBased on the disc diffusion test and β‐galactosidase activity assay, 3‐methoxycinnamic acid was shown to interfere with the quorum sensing (QS) system of A. tumefaciens. Crystal violet staining assay, phenol‐sulfuric acid method, Bradford protein assay and confocal laser scanning microscopy (CLSM) revealed that the biofilm formation of A. tumefaciens was inhibited after the treatment of 3‐methoxycinnamic acid. Employing high‐performance liquid chromatography (HPLC) analysis of culture supernatant revealed that the production of 3‐oxo‐octanoylhomoserine lactone (3‐oxo‐C8‐HSL) decreased concentration‐dependently after treatment with 3‐methoxycinnamic acid. Swimming and chemotaxis assays also indicated that 3‐methoxycinnamic acid had a good effect on reducing the motility and chemotaxis of A. tumefaciens. In addition, the RT‐qPCR, molecular docking and simulations further demonstrated that 3‐methoxycinnamic acid could competitively inhibit the binding of 3‐oxo‐C8‐HSL to TraR and down‐regulate virulence‐related genes.Conclusions3‐Methoxycinnamic acid is proved to have good anti‐virulence and anti‐biofilm activities against A. tumefaciens.Significance and Impact of the StudyThis is the first study that investigates the anti‐virulence and anti‐biofilm activities of 3‐methoxycinnamic acid against A. tumefaciens. With its potential QS‐related virulence and biofilm inhibitory activities, 3‐methoxycinnamic acid is expected to be developed as a potent pesticide or adjuvant for the prevention and treatment of crown gall caused by A. tumefaciens.
研究领域:
微生物学
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植物病理学
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药理学
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PubMed
高影响因子
MOLECULAR BREEDING
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2026
·Vol. 46
·期 3
·20
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IF 3.6
Lin Fan,Ma Hongping,Chen Jianhui,Liang Shuang,Wu Zhijun,Fan Haili,Chen Shenglong,Kong Xiangqi,Zhang Tong,Hu Hongyan,Wang Hong
Genetic transformation has been a crucial technology for plant molecular biology research and molecular design breeding, particularly in modern agriculture, and currently gene function validation is mostly conducted on model plants, which severely limits the development of non-model plants such as medicinal plants. Polygonum cuspidatum Sieb. et Zucc. plays a significant role in the treatment of diabetes, cancer, respiratory and cardiovascular diseases, however, its optimal regeneration and gene transformation system is still not available. Here, we at first establish an efficient regeneration system for P. cuspidatum as: stems are the most suitable explants for callus induction and regeneration of buds, and N1 medium (MS medium with 1.0 mg/L 1-naphthylacetic acid (NAA), 0.1 mg/L kinetin (KT)) is the most suitable regeneration medium. Then based on this regeneration system, an Agrobacterium tumefaciens -mediated genetic transformation system via callus regeneration (CR) and organ regeneration (OR) is established and optimized as: the stems are immersed in the A. tumefaciens resuspension medium (1/4 MS medium added with 0.02% Silwet L-77) and vacuum infiltrated for 10 min, the optimal co-culture time is 5 days, and the most suitable screening antibiotic is 100 mg/L hygromycin (Hyg). In addition, the practicality of our introduced transformation system is confirmed through the experimental verification of the reporter gene DsRED and two candidate genes: stilbene synthase ( PcSTS) and MYB transcription factor ( PcMYB62) . In summary, our established system can not only efficiently regenerate and genetically transform P. cuspidatum , a typical non-model but highly valuable plant, but it could also serve as a reference for the construction of other non-model plant transformation systems.
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高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2019
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IF 3.571
Ruiqi Bao,Ning Gao,Jing Lv,Chaofan Ji,Huipeng Liang,Shengjie Li,Chenxu Yu,Zhenyu Wang,Xinping Lin
Nine transformants of Rhodosporidium toruloides with significant changes in the carotenoid profile were obtained by Agrobacterium tumefaciens-mediated transformation, including a white, three red, and four yellow mutants. A red mutant A1-15-BRQ that showed a high torularhodin production was selected for culture condition optimization. Results indicated that the torularhodin yield was boosted with glucose as the carbon source, at a carbon/nitrogen ratio of 22, a loading volume of 75 mL, and 28 °C. The torularhodin yield of 21.3 mg/L consisting of 94.4% total carotenoids was obtained by Box–Behnken design experiments. The torularhodin yield was 17.0-fold higher than that of the wild type, with time shortened from 9 to 3 days. This study reports an effective strategy for improving torularhodin production and provides a candidate R. toruloides strain for highly selective production of torularhodin.
研究领域:
微生物学
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生物技术
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代谢工程
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高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2019
·
IF 4.077
Rongshui Wang,Jihong Yi,Jinmeng Shang,Wenjun Yu,Zhifeng Li,Haiyan Huang,Huijun Xie,Shuning Wang
Agrobacterium tumefaciens S33 degrades nicotine via a novel hybrid of the pyridine and the pyrrolidine pathways. The hybrid pathway consists of at least six steps involved in oxidoreductive reactions before the N-heterocycle can be broken down. Collectively, the six steps allow electron transfer from nicotine and its intermediates to the final acceptor O2 via the electron transport chain (ETC). 6-Hydroxypseudooxynicotine oxidase, renamed 6-hydroxypseudooxynicotine dehydrogenase in this study, has been characterized as catalyzing the fourth step using the artificial electron acceptor 2,6-dichlorophenolindophenol. Here, we used biochemical, genetic, and liquid chromatography-mass spectrometry (LC-MS) analyses to determine that 6-hydroxypseudooxynicotine dehydrogenase utilizes the electron transfer flavoprotein (EtfAB) as the physiological electron acceptor to catalyze the dehydrogenation of pseudooxynicotine, an analogue of the true substrate 6-hydroxypseudooxynicotine, in vivo, into 3-succinoyl-semialdehyde-pyridine. NAD(P)+, O2, and ferredoxin could not function as electron acceptors. The oxygen atom in the aldehyde group of the product 3-succinoyl-semialdehyde-pyridine was verified to be derived from H2O. Disruption of the etfAB genes in the nicotine-degrading gene cluster decreased the growth rate of A. tumefaciens S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine, an intermediate downstream of the hybrid pathway, indicating the requirement of EtfAB for efficient nicotine degradation. The electrons were found to be further transferred from the reduced EtfAB to coenzyme Q by the catalysis of electron transfer flavoprotein:ubiquinone oxidoreductase. These results aid in an in-depth understanding of the electron transfer process and energy metabolism involved in the nicotine oxidation and provide novel insights into nicotine catabolism in bacteria.IMPORTANCE Nicotine has been studied as a model for toxic N-heterocyclic aromatic compounds. Microorganisms can catabolize nicotine via various pathways and conserve energy from its oxidation. Although several oxidoreductases have been characterized to participate in nicotine degradation, the electron transfer involved in these processes is poorly understood. In this study, we found that 6-hydroxypseudooxynicotine dehydrogenase, a key enzyme in the hybrid pyridine and pyrrolidine pathway for nicotine degradation in Agrobacterium tumefaciens S33, utilizes EtfAB as a physiological electron acceptor. Catalyzed by the membrane-associated electron transfer flavoprotein:ubiquinone oxidoreductase, the electrons are transferred from the reduced EtfAB to coenzyme Q, which then could enter into the classic ETC. Thus, the route for electron transport from the substrate to O2 could be constructed, by which ATP can be further sythesized via chemiosmosis to support the baterial growth. These findings provide new knowledge regarding the catabolism of N-heterocyclic aromatic compounds in microorganisms.
研究领域:
微生物学
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生物化学
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分子生物学
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环境科学
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高影响因子
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
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2021
·Vol. 69
·期 4
·1428-1437
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IF 2.431
Di Liu,Linbo Gou,Yajun Bai,Tai-Ping Fan,Xiaohui Zheng,Yujie Cai
In this study, the 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) was modified by site-directed mutagenesis. And we further obtained a saturation mutant library in which the residue 197 was mutated. A single-point mutation converted the wild enzyme that originally had no catalytic activity in reduction of ethyl 4-chloroacetoacetate (COBE) into an enzyme with catalytic activity. The results of enzyme activity assays showed that the seven variants could asymmetrically reduce COBE to ethyl ( S )-4-chloro-3-hydroxybutyrate (( S )-CHBE) with NADH as coenzyme. In the library, the variant E197N showed higher catalytic efficiency than others. The E197N was optimally active at pH 6.0 and 40°C, and the catalytic efficiency ( k cat / K m ) for COBE was 51.36 s –1 ·mM –1 . This study showed that the substrate specificity of AtQR could be changed through site-directed mutagenesis at the residue 197.
研究领域:
生物化学
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酶学
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生物催化
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PubMed
高影响因子
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
·
2020
·Vol. 68
·期 1
·71-81
·
IF 1.638
Zeping Sun,Jing Lv,Chaofan Ji,Huipeng Liang,Shengjie Li,Zhaoxia Yang,Wenhuan Xu,Sufang Zhang,Xinping Lin
Rhodosporidium toruloides has been reported as a potential biotechnological microorganism to produce carotenoids. The most commonly used molecular and genetic manipulation methods based on Agrobacterium -mediated transformation (ATMT). However, this method was of relatively lower transformation efficiency. In this study, we optimized the ATMT method for R. toruloides on account of the promoter on T-DNA, the ratio of A. tumefaciens to R. toruloides NP11, acetosyringone concentration, cocultivation temperature and time, and a transformation efficiency of 2,369 cells per 10 5 recipient cells was obtained and was 24 times as that of the previous report. With this optimized method, four redder mutants and four yellower mutants were selected out with torularhodin and β-carotene production preference, respectively. The highest torularhodin production was 1,638.15 µg/g dry cell weight in A1-13. The yellower mutants were found to divert the metabolic flux from torularhodin and torulene to γ-carotene and β-carotene, and the proportion of γ-carotene and β-carotene were all over 92%. TAIL-PCR was carried out to found T-DNA insertion in these mutants, and insertion hotspot was found. RT-qPCR results showed that CTA 1 genes in these mutants were closely related to the synthesis of total carotenoids, especially torularhodin, and was a potenial metabolic engineering site in the future.
研究领域:
微生物学
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分子生物学
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生物技术
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PubMed
高影响因子
Frontiers in Microbiology
·
2021
·
IF 5.64
Li Jingxin,Zhang Yuxiao,Wang Xing,Walk Seth T.,Wang Gejiao
Antimony (Sb)-resistant bacteria have potential applications in the remediation of Sb-contaminated sites. However, the effect of Sb(III) exposure on whole-cell metabolic change has not been studied. Herein, we combined untargeted metabolomics with a previous proteomics dataset and confirmatory gene transcription analysis to identify metabolic responses to Sb(III) exposure in Agrobacterium tumefaciens GW4. Dynamic changes in metabolism between control and Sb(III)-exposed groups were clearly shown. KEGG pathway analysis suggested that with Sb(III) exposure: (1) the branching pathway of gluconeogenesis is down-regulated, resulting in the up-regulation of pentose phosphate pathway to provide precursors of anabolism and NADPH; (2) glycerophospholipid and arachidonic acid metabolisms are down-regulated, resulting in more acetyl-CoA entry into the TCA cycle and increased capacity to produce energy and macromolecular synthesis; (3) nucleotide and fatty acid synthesis pathways are all increased perhaps to protect cells from DNA and lipid peroxidation; (4) nicotinate metabolism increases which likely leads to increased production of co-enzymes (e.g., NAD+ and NADP+) for the maintenance of cellular redox and Sb(III) oxidation. Expectedly, the total NADP+/NADPH content, total glutathione, and reduced glutathione contents were all increased after Sb(III) exposure in strain GW4, which contribute to maintaining the reduced state of the cytoplasm. Our results provide novel information regarding global bacterial responses to Sb(III) exposure from a single gene level to the entire metabolome and provide specific hypotheses regarding the metabolic change to be addressed in future research.
研究领域:
微生物学
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环境科学
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分子生物学
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代谢组学
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PubMed
高影响因子
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