Proteomic and Mutant Analysis of Hydrogenase Maturation Protein Gene hypE in Symbiotic Nitrogen Fixation of Mesorhizobium huakuii.

Hydrogenases catalyze the simple yet important redox reaction between protons and electrons and H2, thus mediating symbiotic interactions. The contribution of hydrogenase to this symbiosis and anti-oxidative damage was investigated using the M. huakuii hypE (encoding hydrogenase maturation protein) mutant. The hypE mutant grew a little faster than its parental 7653R and displayed decreased antioxidative capacity under H2O2-induced oxidative damage. Real-time quantitative PCR showed that hypE gene expression is significantly up-regulated in all the detected stages of nodule development. Although the hypE mutant can form nodules, the symbiotic ability was severely impaired, which led to an abnormal nodulation phenotype coupled to a 47% reduction in nitrogen fixation capacity. This phenotype was linked to the formation of smaller abnormal nodules containing disintegrating and prematurely senescent bacteroids. Proteomics analysis allowed a total of ninety differentially expressed proteins (fold change > 1.5 or <0.67, p < 0.05) to be identified. Of these proteins, 21 are related to stress response and virulence, 21 are involved in transporter activity, and 18 are involved in energy and nitrogen metabolism. Overall, the HypE protein is essential for symbiotic nitrogen fixation, playing independent roles in supplying energy and electrons, in bacterial detoxification, and in the control of bacteroid differentiation and senescence.

Antioxidant ability of glutaredoxins and their role in symbiotic nitrogen fixation in Rhizobium leguminosarum bv. viciae 3841.

Glutaredoxins (Grx) are redoxin family proteins that reduce disulfides and mixed disulfides between glutathione and proteins. Rhizobium leguminosarum bv. Viciae 3841 contains three genes coding for glutaredoxins: RL4289 (grxA) codes for a dithiolic glutaredoxin, RL2615 (grxB) codes for a monothiol glutaredoxin, while RL4261 (grxC) codes for a glutaredoxin-like NrdH protein. We generated mutants interrupted in one, two, or three glutaredoxin genes. These mutants had no obvious differences in growth phenotypes from the wild type RL3841. However, while a mutant of grxC did not affect the antioxidant or symbiotic capacities of R. leguminosarum, grxA-derived or grxB mutants decreased antioxidant and nitrogen fixation capacities. Furthermore, grxA mutants were severely impaired in rhizosphere colonization, and formed smaller nodules with defects of bacteroid differentiation, whereas nodules induced by grxB mutants contained abnormally thick cortices and prematurely senescent bacteroids. The grx triple mutant had the greatest defect in antioxidant and symbiotic capacities of R. leguminosarum and quantitative proteomics revealed it had 56 up-regulated and 81 down-regulated proteins relative to wildtype. Of these proteins, twenty-eight are involved in transporter activity, twenty are related to stress response and virulence, and sixteen are involved in amino acid metabolism. Overall, R. leguminosarum glutaredoxins behave as antioxidant proteins mediating root nodule symbiosis.IMPORTANCE Glutaredoxin catalyzes glutathionylation/deglutathionylation reactions, protects SH-groups from oxidation and restores functionally active thiols. Three glutaredoxins exist in R. leguminosarum and their properties were investigated in free-living bacteria and during nitrogen-fixing symbiosis. All the glutaredoxins were necessary for oxidative stress defense. Dithiol GrxA affects nodulation and nitrogen fixation of bacteroids by altering deglutathionylation reactions, monothiol GrxB is involved in symbiotic nitrogen fixation by regulating Fe-S cluster biogenesis, and GrxC may participate in symbiosis by an unknown mechanism. Proteome analysis provides clues to explain the differences between the grx triple mutant and wild-type nodules.

The Mesorhizobium huakuii transcriptional regulator AbiEi plays a critical role in nodulation and is important for bacterial stress response.

BACKGROUND: Bacterial abortive infection (Abi) systems are type IV toxin-antitoxin (TA) system, which could elicit programmed cell death and constitute a native survival strategy of pathogenic bacteria under various stress conditions. However, no rhizobial AbiE family TA system has been reported so far. Here, a M. huakuii AbiE TA system was identified and characterized. RESULTS: A mutation in M. huakuii abiEi gene, encoding an adjacent GntR-type transcriptional regulator, was generated by homologous recombination. The abiEi mutant strain grew less well in rich TY medium, and displayed increased antioxidative capacity and enhanced gentamicin resistance, indicating the abiEi operon was negatively regulated by the antitoxin AbiEi in response to the oxidative stress and a particular antibiotic. The mRNA expression of abiEi gene was significantly up-regulated during Astragalus sinicus nodule development. The abiEi mutant was severely impaired in its competitive ability in rhizosphere colonization, and was defective in nodulation with 97% reduction in nitrogen-fixing capacity. The mutant infected nodule cells contained vacuolation and a small number of abnormal bacteroids with senescence character. RNA-seq experiment revealed it had 5 up-regulated and 111 down-regulated genes relative to wild type. Of these down-regulated genes, 21 are related to symbiosis nitrogen fixation and nitrogen mechanism, 16 are involved in the electron transport chain and antioxidant responses, and 12 belong to type VI secretion system (T6SS). CONCLUSIONS: M. huakuii AbiEi behaves as a key transcriptional regulator mediating root nodule symbiosis.

Functional analysis of the ocnE gene involved in nicotine-degradation pathways in Ochrobactrum intermedium SCUEC4 and its enzymatic properties.

The SCUEC4 strain of Ochrobactrum intermedium is a newly isolated bacterium that degrades nicotine can use nicotine as the sole carbon source via a series of enzymatic catalytic processes. The mechanisms underlying nicotine degradation in this bacterium and the corresponding functional genes remain unclear. Here, we analyzed the function and biological properties of the ocnE gene involved in the nicotine-degradation pathways in strain SCUEC4. The ocnE gene was cloned by PCR with total DNA of strain SCUEC4 and used to construct the recombinant plasmid pET28a-ocnE. The overexpression of the OcnE protein was detected by SDS-PAGE analysis, and study of the function of this protein was spectrophotometrically carried out by monitoring the changes of 2,5-dihydroxypyridine. Moreover, the effects of temperature, pH, and metal ions on the biological activities of the OcnE protein were analyzed. The optimal conditions for the biological activities of OcnE, a protein of approximately 37.6 kDa, were determined to be 25 °C, pH 7.0, and 25 µmol/L Fe2+, and the suitable storage conditions for the OcnE protein were 0 °C and pH 7.0. In conclusion, the ocnE gene is responsible for the ability of 2,5-dihydroxypyridine dioxygenase. These findings will be beneficial in clarifying the mechanisms of nicotine degradation in O. intermedium SCUEC4.

Dredging mitigates cyanobacterial bloom in eutrophic Lake Nanhu: Shifts in associations between the bacterioplankton community and sediment biogeochemistry.

Cyanobacterial blooms are a worldwide environmental problem, which is partly attributed to their access to excessive nitrogen (N) and phosphorus (P). Preventing the blooms by reducing N and P from internal inputs is viewed as a challenge. To evaluate the effects of dredging on cyanobacterial abundances and bacterioplankton communities, water and sediment samples were collected from eutrophic Lake Nanhu (Wuhan, China) before dredging (2017) and after dredging (2018). After dredging, significant decreases were observed for sediment nutrients (e.g., C, N, and P sources); C-, N-, P-, and S-cycling-related enzyme activity; N- and P-cycling-related gene abundance; microbial abundance; and dramatic changes were observed in the composition of the sediment microbial community. The release rates of nutrient including nitrogen, phosphorus, and organic matter decreased after dredging, and sediment biogeochemistry was closely correlated to nutrient release rates. Additionally, our observations and analyses indicated that the abundance and diversity of the bacterioplankton community decreased significantly, the composition and interaction of the bacterioplankton community dramatically changed, and the bacterioplankton community function (e.g., N, P-cycling-related enzymes and proteins) down regulated after dredging. Water and sediment physicochemical factors explained 72.28% variation in bacterioplankton community composition, and these physicochemical factors were significantly correlated with diversity, composition, and function of bacterioplankton community. Our findings emphasized that cyanobacterial blooms in freshwater ecosystems were closely correlated with noncyanobacterial bacterioplankton that were largely conserved at the phylum level, with Proteobacteria, Actinobacteria, and Bacteroidetes as the main taxa. To our knowledge, this is the first report clarifying the mechanism of cyanobacterial blooms mitigation by dredging, via changing the association between the bacterioplankton community and sediment biogeochemistry. Our findings are of significance and indicate that dredging is effective for mitigating cyanobacterial blooms.

Glutathione is Involved in Detoxification of Peroxide and Root Nodule Symbiosis of Mesorhizobium huakuii.

Legumes interact with symbiotic rhizobia to produce nitrogen-fixation root nodules under nitrogen-limiting conditions. The contribution of glutathione (GSH) to this symbiosis and anti-oxidative damage was investigated using the M. huakuii gshB (encoding GSH synthetase) mutant. The gshB mutant grew poorly with different monosaccharides, including glucose, sucrose, fructose, maltose, or mannitol, as sole sources of carbon. The antioxidative capacity of gshB mutant was significantly decreased by these treatments with H2O2 under the lower concentrations and cumene hydroperoxide (CUOOH) under the higher concentrations, indicating that GSH plays different roles in response to organic peroxide and inorganic peroxide. The gshB mutant strain displayed no difference in catalase activity, but significantly lower levels of the peroxidase activity and the glutathione reductase activity than the wild type. The same level of catalase activity could be associated with upregulation of the transcriptional activity of the catalase genes under H2O2-induced conditions. The nodules infected by the gshB mutant were severely impaired in abnormal nodules, and showed a nodulation phenotype coupled to a 60% reduction in the nitrogen fixation capacity. A 20-fold decrease in the expression of two nitrogenase genes, nifH and nifD, is observed in the nodules induced by gshB mutant strain. The symbiotic deficiencies were linked to bacteroid early senescence.

Multiplicity of Sulfate and Molybdate Transporters and Their Role in Nitrogen Fixation in Rhizobium leguminosarum bv. viciae Rlv3841.

Rhizobium leguminosarum Rlv3841 contains at least three sulfate transporters, i.e., SulABCD, SulP1 and SulP2, and a single molybdate transporter, ModABC. SulABCD is a high-affinity transporter whose mutation prevented growth on a limiting sulfate concentration, while SulP1 and SulP2 appear to be low-affinity sulfate transporters. ModABC is the sole high-affinity molybdate transport system and is essential for growth with NO3(-) as a nitrogen source on limiting levels of molybdate (<0.25 µM). However, at 2.5 µM molybdate, a quadruple mutant with all four transporters inactivated, had the longest lag phase on NO3(-), suggesting these systems all make some contribution to molybdate transport. Growth of Rlv3841 on limiting levels of sulfate increased sulB, sulP1, modB, and sulP2 expression 313.3-, 114.7-, 6.2-, and 4.0-fold, respectively, while molybdate starvation increased only modB expression (three- to 7.5-fold). When grown in high-sulfate but not low-sulfate medium, pea plants inoculated with LMB695 (modB) reduced acetylene at only 14% of the wild-type rate, and this was not further reduced in the quadruple mutant. Overall, while modB is crucial to nitrogen fixation at limiting molybdate levels in the presence of sulfate, there is an unidentified molybdate transporter also capable of sulfate transport.

The effects of ROS in prostatic stromal cells under hypoxic environment.

OBJECTIVE: The objective of this study is to explore the effects of reactive oxygen species (ROS) under hypoxic environment in prostatic stromal cells (PSC). METHODS AND MATERIALS: To detect the expression of ROS in PSC and the tissues of benign prostatic hyperplasia (BPH) by flow cytometry; under hypoxic conditions, to observe the changes of cells growth and ROS in PSC; quantitative PCR was used to detect hypoxia inducible factor-1α (HIF-1α), androgen receptors (AR), vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) in PSC; After edaravone intervening, to examine the changes of cells growth, ROS, HIF-1α, AR, VEGF, and IL-8 under hypoxic conditions. RESULTS: The expression of ROS in tissues and cells which under hypoxic condition was significantly increased. 3% O2 promoted the proliferation. The HIF-1α, AR, VEGF, and IL-8 were upregulated under 3% O2. After edaravone intervening, ROS significantly decreased, HIF-1α and VEGF were downregulated, and cell proliferation declined. CONCLUSIONS: Hypoxia stimulates the generation of ROS, and the ROS may play a key role in BPH.

Paenibacillus enshidis sp. nov., Isolated from the Nodules of Robinia pseudoacacia L.

A Gram-positive, motile, endospore-forming, rod-shaped bacterium, designated RP-207(T), was isolated from the nodules of Robinia pseudoacacia L. plants planted in Enshi District, Hubei, PR China. Phylogenetic analyses based on the 16S rRNA gene sequence showed that the novel strain was affiliated to the genus Paenibacillus, with its closest relatives being Paenibacillus xylanilyticus XIL14(T) (95.6%), Paenibacillus peoriae DSM8320(T) (95.3%) and Paenibacillus polymyxa DSM 36(T) (95.3%). The DNA G+C content was 47.0 mol%. DNA-DNA hybridization value between strain RP-207(T) and P. xylanilyticus XIL14(T) was 40.1%. The diamino acid found in the cell wall peptidoglycan was meso-diaminopimelic. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, an unidentified amino-phospholipid and an unknown phospholipid. The predominant menaquinone was menaquinone-7 (MK-7), and the major fatty acid was anteiso-C15:0 and C16:0. On the basis of its physiological and biochemical characteristics and the level of DNA-DNA hybridization, strain RP-207(T) is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus enshidis sp. nov. is proposed. The type strain is RP-207(T) (=CCTCC AB 2013275(T) = KCTC 33519(T)).

Pretreatment of piggery wastewater by a stable constructed microbial consortium for improving the methane production.

A stable aerobic microbial consortium, established by successive subcultivation, was employed to solubilize the solid organic fraction in swine wastewater. In the 30 days' successive biological pretreatments, 30-38% of volatile solids and 19-28% total solids in raw slurry were solubilized after 10 hours at 37 °C. Meanwhile, soluble chemical oxygen demand (COD) and volatile fatty acid increased by 48%-56% and 600%-750%, respectively. Furthermore, the molecular microbial profile of the consortium in successive pretreatment was conducted by denaturing gradient gel electrophoresis (DGGE). The results indicated that bacterial species of the consortium rapidly overgrew the indigenous microbial community of raw water, and showed a stable predominance at the long-term treatment. As a consequence of biological pretreatment, pretreatment shortened digestion time by 50% and increased biogas production by 45% compared to raw water in the anaerobic process. The microbial consortium constructed herein is a potential candidate consortium for biological pretreatment of swine wastewater to enhance biogas production.

[Antioxidative function of katG gene in Rhizobium leguminosarum].

OBJECTIVE: Catalase-peroxidase KatG can protect bacteria from damage of reactive oxygen species. This study investigated the antioxidative function of catalase - peroxidase gene katG in Rhizobium leguminosarum 3841. METHODS: katG mutant strain of R. leguminosarum was constructed by homologous recombination. The wild type, katG mutant and complementary strain were challenged by oxidative stress and symbiotic ability. RESULTS: Under free - living conditions, the katG mutant exhibited no generation time extension. However, cells of the katG strain were deficient in consumption oj high concentrations of H2O2and were vulnerable after aquick exposure to H2O2. The real-time qRT-PCR results showec that katG was expressed independently of exogenous H2O2. In contrast, the katG mutant strain displayed higher expres, level of ohrB gene and lower expression level of grxC than the wild type. With regard to symbiotic capacities with Pisum sativum, the katG mutant was indistinguishable in root nodule nitrogenase activity and competition nodule ability from the wild type. However, katG gene was expressed significantly lower in bacteroids than that in free-living strains. Besides, the colonization of the pea rhizosphere by the katG mutant was impaired compared to that of the wild type. CONCLUSION: ThE deletion of katG had nosignificant effect in 3841 under the free-living and symbiosis condition but was essential ir antioxidation and colonization of the pea rhizosphere. Although katG could not be induced by H2O2, it still played acentra role in antioxidation and symbiotic nitrogen fixation by regulating the antioxidant genes such as ohrB and grxC.

Gonadotropin-positive pituitary tumors accompanied by ovarian tumors in aging female ERbeta-/- mice.

At 2 years of age, 100% (23/23) of ERbeta(-/-) female mice have developed large pituitary and ovarian tumors. The pituitary tumors are gonadotropin-positive and the ovarian tumors are sex cord (less differentiated) and granulosa cell tumors (differentiated and estrogen secreting). No male mice had pituitary tumors and no pituitary or ovarian tumors developed in ERalpha(-/-) mice or in ERalphabeta(-/-) double knockout mice. The tumors have high proliferation indices, are ERalpha-positive, ERbeta-negative, and express high levels of nuclear phospho-SMAD3. Mice with granulosa cell tumors also had hyperproliferative endometria. The cause of the pituitary tumors appeared to be excessive secretion of gonadotropin releasing hormone (GnRH) from the hypothalamus resulting from high expression of NPY. The ovarian phenotype is similar to that seen in mice where inhibin is ablated. The data indicate that ERbeta plays an important role in regulating GnRH secretion. We suggest that in the absence of ERbeta, the proliferative action of FSH/SMAD3 is unopposed and the high proliferation leads to the development of ovarian tumors. The absence of tumors in the ERalphabeta(-/-) mice suggests that tumor development requires the presence of ERalpha.

A role for epithelial-mesenchymal transition in the etiology of benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is usually described as a pathological proliferation of prostatic fibroblasts/myofibroblasts and epithelial cells. In the present study of BPH samples, we have made a morphological and immunohistochemical study of BPH prostatic sections using markers of proliferation, apoptosis, hormone receptors, and TGF-beta signaling. We found no evidence of proliferation in the stroma but in the epithelium of some ducts; 0.7% of the basal and 0.4% of luminal cells were positive for Ki67 and PCNA. Androgen receptor and estrogen receptor beta (ERbeta)1 and ERbetacx were abundant in both stromal and epithelial compartments but cells expressing ERalpha were very rare. What was very common in all BPH samples was the following: (i) regions of the ductal epithelium where the epithelial cells did not express E-cadherin, had lost their polarization, and become spindle shaped (the nuclei of these cells were strongly positive for pSmad 3 and Snail); and (ii) regions where the walls of the blood vessels were extremely thick and there was loss of endothelial layer. Loss of E-cadherin, increased pSmad 3, and high expression of Snail are all characteristic of epithelial-mesenchymal transition (EMT). We conclude that BPH is not a disease of prostatic stromal proliferation but rather of accumulation of mesenchymal-like cells derived from the prostatic epithelium and the endothelium. TGF-beta is thought to play a key role in EMT. Our data suggests that TGF-beta/Smad should be considered as targets for treatment of BPH.

Association between different concentrations of human serum albumin and 28-day mortality in intensive care patients with sepsis: A propensity score matching analysis.

Background: Human serum albumin (HSA) is a commonly used medication for the treatment of sepsis. However, there is no conclusive evidence as to whether different concentrations of HSA are associated with patient prognosis. This study aimed to evaluate the association between different concentrations of HSA and 28-day mortality in patients with sepsis. Methods: The data for this retrospective study were collected from the Medical Information Mart for Intensive Care IV database. Patients with sepsis were divided into two groups according to the concentration of HSA received: 25% and 5% HSA. The primary outcome of this study was the 28-day mortality in patients with sepsis. To ensure the robustness of our findings, we used multivariate Cox regression, propensity score matching, double-robust estimation, and inverse probability weighting models. Results: A total of 76,943 patients were screened, of whom 5,009 were enrolled. 1,258 and 3,751 patients received 25% and 5% HSA, respectively. The 28-day mortality rate was 38.2% (481/1,258) for patients in the 25% HSA group and 8.7% (325/3,751) for patients in the 5% HSA group. After propensity score matching, 1,648 patients were identified. The inverse probability weighting model suggested that 5% HSA received was associated with lower 28-day mortality (hazard ratio [HR]: 0.63, 95% confidence interval [CI]: 0.54-0.73, p < 0.001). Subgroup and sensitivity analysis confirmed the robustness of the results. Conclusion: In patients with sepsis, 5% HSA received may be associated with a lower risk of 28-day mortality than 25% HSA. Further randomized controlled trials are required to confirm this association.

Corrigendum: A GMC Oxidoreductase GmcA Is Required for Symbiotic Nitrogen Fixation in Rhizobium leguminosarum bv. viciae.

[This corrects the article DOI: 10.3389/fmicb.2020.00394.].

Corrigendum: Rhizobium leguminosarum Glutathione Peroxidase Is Essential for Oxidative Stress Resistance and Efficient Nodulation.

[This corrects the article DOI: 10.3389/fmicb.2021.627562.].

Rhizobium leguminosarum Glutathione Peroxidase Is Essential for Oxidative Stress Resistance and Efficient Nodulation.

Glutathione (GSH) plays a key role in regulating the cellular Redox Homeostasis, and appears to be essential for initiation and development of root nodules. Glutathione peroxidase (Gpx) catalyzes the reduction of H2O2 and organic hydroperoxides by oxidation of GSH to oxidized GSH (GSSG), which in turn is reduced by glutathione reductase (GR). However, it has not been determined whether the Rhizobium leguminosarum Gpx or GR is required during symbiotic interactions with pea. To characterize the role of glutathione-dependent enzymes in the symbiotic process, single and double mutants were made in gpxA (encoding glutathione peroxidase) and gshR (encoding glutathione reductase) genes. All the mutations did not affect the rhizobial growth, but they increased the sensitivity of R. leguminosarum strains to H2O2. Mutant in GpxA had no effect on intracellular GSH levels, but can increase the expression of the catalase genes. The gshR mutant can induce the formation of normal nodules, while the gpxA single and double mutants exhibited a nodulation phenotype coupled to more than 50% reduction in the nitrogen fixation capacity, these defects in nodulation were characterized by the formation of ineffective nodules. In addition, the gpxA and gshR double mutant was severely impaired in rhizosphere colonization and competition. Quantitative proteomics using the TMT labeling method was applied to study the differential expression of proteins in bacteroids isolated from pea root nodules. A total of 27 differentially expressed proteins were identified in these root bacteroids including twenty down-regulated and seven up-regulated proteins. By sorting the down-regulated proteins, eight are transporter proteins, seven are dehydrogenase, deoxygenase, oxidase, and hydrolase. Moreover, three down-regulating proteins are directly involved in nodule process.

A GMC Oxidoreductase GmcA Is Required for Symbiotic Nitrogen Fixation in Rhizobium leguminosarum bv. viciae.

GmcA is a FAD-containing enzyme belonging to the GMC (glucose-methanol-choline oxidase) family of oxidoreductases. A mutation in the Rhizobium leguminosarum gmcA gene was generated by homologous recombination. The mutation in gmcA did not affect the growth of R. leguminosarum, but it displayed decreased antioxidative capacity at H2O2 conditions higher than 5 mM. The gmcA mutant strain displayed no difference of glutathione reductase activity, but significantly lower level of the glutathione peroxidase activity than the wild type. Although the gmcA mutant was able to induce the formation of nodules, the symbiotic ability was severely impaired, which led to an abnormal nodulation phenotype coupled to a 30% reduction in the nitrogen fixation capacity. The observation on ultrastructure of 4-week pea nodules showed that the mutant bacteroids tended to start senescence earlier and accumulate poly-ß-hydroxybutyrate (PHB) granules. In addition, the gmcA mutant was severely impaired in rhizosphere colonization. Real-time quantitative PCR showed that the gmcA gene expression was significantly up-regulated in all the detected stages of nodule development, and statistically significant decreases in the expression of the redoxin genes katG, katE, and ohrB were found in gmcA mutant bacteroids. LC-MS/MS analysis quantitative proteomics techniques were employed to compare differential gmcA mutant root bacteroids in response to the wild type infection. Sixty differentially expressed proteins were identified including 33 up-regulated and 27 down-regulated proteins. By sorting the identified proteins according to metabolic function, 15 proteins were transporter protein, 12 proteins were related to stress response and virulence, and 9 proteins were related to transcription factor activity. Moreover, nine proteins related to amino acid metabolism were over-expressed.

Functional analysis of the agnH gene involved in nicotine-degradation pathways in Agrobacterium tumefaciens strain SCUEC1.

Agrobacterium tumefaciens strain SCUEC1 is a nicotine-degrading bacterium, which has been recently isolated from the tobacco waste-contaminated field soil. However, the mechanism for nicotine degradation in this strain remains unclear. Here, we analyze the function and biological properties of the agnH gene in the strain SCUEC1. The overexpression of the AgnH protein was detected by SDS-PAGE analysis, and functional insight of the AgnH protein was carried out with monitoring the changes of maleic acid into fumaric acid by high performance liquid chromatography (HPLC). Moreover, the effects of temperature, pH and metal ions on the enzymatic activities of the AgnH protein were also analyzed. The results demonstrated that the agnH gene was successfully ligated to the plasmid pET28a. The optimal condition for the enzymatic activities for the AgnH, approximately 28.0 kDa, was determined as 37 °C, pH 8.0 and 25 µM Mg2+. Conclusively, the agnH gene fulfils an important role in the conversion of maleic acid into fumaric acid involved in nicotine-degradation pathways in Agrobacterium tumefaciens strain SCUEC1.