RESUMO
Biological Nitrification Inhibition (BNI) of Brachiaria humidicola has been mainly attributed to the root-exuded fusicoccane-type diterpene brachialactone. We hypothesized, however, that according to the high diversity of fusicoccanes described for plants and microorganisms, BNI of B. humidicola is caused by an assemblage of bioactive fusicoccanes. B. humidicola root exudates were collected hydroponically and compounds isolated by semi-preparative HPLC. Chemical structures were revealed by spectroscopic techniques, including HRMS as well as 1D and 2D NMR. Nitrification inhibiting (NI) potential of isolated compounds was evaluated by a Nitrosomonas europaea based bioassay. Besides the previously described brachialactone (1), root exudates contained 3-epi-brachialactone (2), the C3-epimer of 1 (m/z 334), as well as 16-hydroxy-3-epi-brachialactone (3) with an additional hydroxyl group at C16 (m/z 350) and 3,18-epoxy-9-hydroxy-4,7-seco-brachialactone (4), which is a ring opened brachialactone derivative with a 3,18 epoxide ring and a hydroxyl group at C9 (m/z 332). The 3-epi-brachialactone (2) showed highest NI activity (ED50 ~ 20 µg mL-1, ED80 ~ 40 µg mL-1), followed by compound 4 with intermediate (ED50 ~ 40 µg mL-1), brachialactone (1) with low and compound 3 without activity. In coherence with previous reports on fusicoccanes, stereochemistry at C3 was of high relevance for the biological activity (NI potential) of brachialactones.
Assuntos
Brachiaria/química , Lactonas/química , Nitrificação , Exsudatos de Plantas/química , Nitrosomonas europaea , Raízes de PlantasRESUMO
Fusion proteins play an important role in the production of recombinant proteins in Escherichia coli. They are mostly used for cytoplasmic expression since they can be designed to increase the solubility of the target protein, which then can be easily purified via affinity chromatography. In contrast, fusion proteins are not usually included in construct designs for periplasmic production. Instead, a signal sequence is inserted for protein transport into the periplasm and a C-terminal his-tag added for subsequent purification. Our research group has proposed the small metal-binding protein (SmbP) isolated from the periplasm of Nitrosomonas europaea as a new fusion protein to express recombinant proteins in the cytoplasm or periplasm of E. coli. SmbP also allows purification via immobilized metal affinity chromatography using Ni(II) ions. Recently, we have optimized the periplasmic production of proteins tagged with SmbP by exchanging its native signal peptide with one taken from pectate lyase B (PelB), substantially increasing the amount of protein produced. In this work, we have expressed and purified soluble bioactive human growth hormone (hGH) tagged with PelB-SmbP and obtained the highest periplasmic production reported for this protein so far. Its activity, tested on Nb2-11 cells, was equivalent to commercial growth hormone at 50 ng·mL-1 . Therefore, we strongly recommend the use of PelB-SmbP as a protein tag for the expression and purification of hGH or other possible target proteins in the periplasm of E. coli.
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hormônio do Crescimento Humano/biossíntese , Engenharia Metabólica/métodos , Metaloproteínas/genética , Nitrosomonas europaea/metabolismo , Periplasma/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Transporte/genética , Hormônio do Crescimento Humano/genética , Humanos , Polissacarídeo-Liases/química , Sinais Direcionadores de Proteínas , Transporte ProteicoRESUMO
We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.
Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Nitrosomonas europaea/genética , Periplasma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cobre , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nitrosomonas europaea/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Periplasma/química , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Proteína Vermelha FluorescenteRESUMO
Nitrosomonas europaea is a chemolithotroph that obtains energy through the oxidation of ammonia to hydroxylamine while assimilates atmospheric CO2 to cover the cell carbon demands for growth. This microorganism plays a relevant role in the nitrogen biogeochemical cycle on Earth but its carbon metabolism remains poorly characterized. Based on sequence homology, we identified two genes (cbbG and gabD) coding for redox enzymes in N. europaea. Cloning and expression of the genes in Escherichia coli, allowed the production of recombinant enzymes purified to determine their biochemical properties. The protein CbbG is a glyceraldehyde-3-phosphate (Ga3P) dehydrogenase (Ga3PDHase) catalyzing the reversible oxidation of Ga3P to 1,3-bis-phospho-glycerate (1,3bisPGA), using specifically NAD+/NADH as cofactor. CbbG showed â¼6-fold higher Km value for 1,3bisPGA but â¼5-fold higher kcat for the oxidation of Ga3P. The protein GabD irreversibly oxidizes Ga3P to 3Pglycerate using NAD+ or NADP+, thus resembling a non-phosphorylating Ga3PDHase. However, the enzyme showed â¼6-fold higher Km value and three orders of magnitude higher catalytic efficiency with succinate semialdehyde (SSA) and NADP+. Indeed, the GabD protein identity corresponds to an SSA dehydrogenase (SSADHase). CbbG seems to be the only Ga3PDHase present in N. europaea; which would be involved in reducing triose-P during autotrophic carbon fixation. Otherwise, in cells grown under conditions deprived of ammonia and oxygen, the enzyme could catalyze the glycolytic step of Ga3P oxidation producing NADH. As an SSADHase, GabD would physiologically act producing succinate and preferentially NADPH over NADH; thus being part of an alternative pathway of the tricarboxylic acid cycle converting α-ketoglutarate to succinate. The properties determined for these enzymes contribute to better identify metabolic steps in CO2 assimilation, glycolysis and the tricarboxylic acid cycle in N. europaea. Results are discussed in the framework of metabolic pathways that launch biosynthetic intermediates relevant in the microorganism to develop and fulfill its role in nature.
Assuntos
Proteínas de Bactérias , Carbono/metabolismo , Gliceraldeído 3-Fosfato/metabolismo , Nitrosomonas europaea , Oxirredutases , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dióxido de Carbono/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Glicólise/fisiologia , Nitrosomonas europaea/enzimologia , Nitrosomonas europaea/genética , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismoRESUMO
Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E. coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E. coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9 kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli.
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Expressão Gênica , Nitrosomonas europaea/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte , Escherichia coli/química , Escherichia coli/metabolismo , Metais/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Nitrifying bacteria are highly susceptible to silver nanoparticles (AgNPs). However, the effect of sublethal exposure to AgNPs after their release of nitrogenous compounds of environmental concern (e.g., the greenhouse gas nitrous oxide [N2 O] and the common water pollutant nitrite [NO2 -]) has not been systematically investigated. The present study reports the effect of AgNPs (and potentially released silver ions [Ag(+) ]) on NO2 - and N2 O production by Nitrosomonas europaea, and on the transcription of the associated genes. The release of NO2 - was more negatively affected than the production of N2 O. For example, exposure to AgNPs at 0.075 mg/L temporarily enhanced N2 O production (by 12%) without affecting nitrite release, whereas higher AgNP concentrations (>0.25 mg/L) inhibited NO2 - release (by >12%) but not N2 O production. Transcriptomic analyses corroborated these trends; AgNPs at 0.075 mg/L increased the expression of the nitric oxide reductase gene (norQ) associated with N2 O production (by 5.3-fold to 12.8-fold), whereas both 0.075 mg/L of Ag(+) and 0.75 mg/L of AgNPs down-regulated the ammonia monooxygenase gene (amoA2; by 0.08-fold to 0.15-fold and 0.32-fold to 0.64-fold, respectively), the nitrite reductase gene (nirK; by 0.01-fold to 0.02-fold and 0.22-fold to 0.44-fold, respectively), and norQ (by 0.11-fold to 0.15-fold and 0.32-fold to 0.57-fold, respectively). These results suggest that AgNP release to sewage treatment plants and land application of AgNP-containing biosolids should be minimized because of their potential temporary stimulation of N2 O release and interference with nitrification. Environ Toxicol Chem 2015;34:2231-2235. © 2015 SETAC.
Assuntos
Nanopartículas Metálicas/química , Nitrosomonas europaea/metabolismo , Óxido Nitroso/metabolismo , Prata/química , Cromatografia Gasosa , Nanopartículas Metálicas/toxicidade , Nitrificação/efeitos dos fármacos , Nitritos/análise , Nitritos/metabolismo , Nitrosomonas europaea/efeitos dos fármacos , Nitrosomonas europaea/enzimologia , Óxido Nitroso/análise , Oxirredutases/genética , Oxirredutases/metabolismo , Transcrição GênicaRESUMO
Nitrosomonas europaea is a chemolithoautotroph that obtains energy by oxidizing ammonia in the presence of oxygen and fixes CO(2) via the Benson-Calvin cycle. Despite its environmental and evolutionary importance, very little is known about the regulation and metabolism of glycogen, a source of carbon and energy storage. Here, we cloned and heterologously expressed the genes coding for two major putative enzymes of the glycogen synthetic pathway in N. europaea, ADP-glucose pyrophosphorylase and glycogen synthase. In other bacteria, ADP-glucose pyrophosphorylase catalyzes the regulatory step of the synthetic pathway and glycogen synthase elongates the polymer. In starch synthesis in plants, homologous enzymes play similar roles. We purified to homogeneity the recombinant ADP-glucose pyrophosphorylase from N. europaea and characterized its kinetic, regulatory, and oligomeric properties. The enzyme was allosterically activated by pyruvate, oxaloacetate, and phosphoenolpyruvate and inhibited by AMP. It had a broad thermal and pH stability and used different divalent metal ions as cofactors. Depending on the cofactor, the enzyme was able to accept different nucleotides and sugar phosphates as alternative substrates. However, characterization of the recombinant glycogen synthase showed that only ADP-Glc elongates the polysaccharide, indicating that ATP and glucose-1-phosphate are the physiological substrates of the ADP-glucose pyrophosphorylase. The distinctive properties with respect to selectivity for substrates and activators of the ADP-glucose pyrophosphorylase were in good agreement with the metabolic routes operating in N. europaea, indicating an evolutionary adaptation. These unique properties place the enzyme in a category of its own within the family, highlighting the unique regulation in these organisms.