Partial Purification and Characterization of an Inducible Indole-3-Acetyl-L-Aspartic Acid Hydrolase from Enterobacter agglomerans

Indole-3-acetyl-amino acid conjugate hydrolases are believed to be important in the regulation of indole-3-acetic acid (IAA) metabolism in plants and therefore have potential uses for the alteration of plant IAA metabolism. To isolate bacterial strains exhibiting significant indole-3-acetyl-aspartate (IAA-Asp) hydrolase activity, a sewage sludge inoculation was cultured under conditions in which IAA-Asp served as the sole source of carbon and nitrogen. One isolate, Enterobacter agglomerans, showed hydrolase activity inducible by IAA-L-Asp or N-acetyl-L-Asp but not by IAA, (NH4)2SO4, urea, or indoleacetamide. Among a total of 17 IAA conjugates tested as potential substrates, the enzyme had an exclusively high substrate specificity for IAA-L-Asp. Substrate concentration curves and Lineweaver-Burk plots of the kinetic data showed a Michaelis constant value for IAA-L-Asp of 13.5 mM. The optimal pH for this enzyme was between 8.0 and 8.5. In extraction buffer containing 0.8 mM Mg2+ the hydrolase activity was inhibited to 80% by 1 mM dithiothreitol and to 60% by 1 mm CuSO4; the activity was increased by 40% with 1 mM MnSO4. However, in extraction buffer with no trace elements, the hydrolase activity was inhibited to 50% by either 1 mM dithiothreitol or 1% Triton X-100 (Sigma). These results suggest that disulfide bonding might be essential for enzyme activity. Purification of the hydrolase by hydroxyapatite and TSK-phenyl (HP-Genenchem, South San Francisco, CA) preparative high-performance liquid chromatography yielded a major 45-kD polypeptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Purification and Partial Characterization of Arginine Decarboxylase from Brassica campestris

Arginine decarboxylase (EC 4.1.1.19) has been purified and characterized from Brassica campestris cv B-9. The enzyme was purified 1120 fold and the recovery was 9%. The mol wt of the enzyme determined by gel filtration was 240 kD with identical subunits of 60 kD. The pH and temperature optima for the enzyme were 8.0 and 30°C respectively. The Km was 0.31mM. Polyamines inhibited the enzyme activity significantly. Immunodiffusion with ADC-specific antibodies showed cross reactivity against purified ADC from Brassica.     

Purification and Characterization of Aromatic L-Amino Acid Decarboxylase from Rat Kidney and Monoclonal Antibody to the Enzyme

Aromatic L-amino acid decarboxylase was purified from rat kidney to homogeneity, as judged by polyacrylamide gel electrophoresis, in the presence and absence of sodium dodecyl sulfate (SDS). The final preparation showed an activity of 3,4-dihydroxyphenylalanine (dopa) decarboxylation of approximately 11,000 nmol/min/mg of protein at 37 degrees C. The purified enzyme also catalyzed the decarboxylation of 5-hydroxytryptophan, tyrosine, tryptophan, and phenylalanine. The enzyme appeared to be composed of two identical subunits, each possessing a molecular weight of 48,000. The isoelectric point of the enzyme was estimated to be 6.7 in the presence of 8 M urea and 5.60-5.85 in its absence. To examine the identity of aromatic L-amino acid decarboxylase from various tissues, a monoclonal antibody directed against the enzyme from rat kidney was prepared. Immunotitration and analysis by antibody-affinity chromatography followed by SDS-polyacrylamide gel electrophoresis revealed that the enzymes from the striatum, adrenal medulla, pineal gland, liver, and kidney were indistinguishable with respect to immunological cross-reactivity and molecular size.     

猪脑谷氨酸脱羧酶的分离纯化以及生物学活性鉴定

L-谷氨酸脱羧酶是γ-氨基丁酸合成的关键限速酶,广泛的存在于脊椎动物神经细胞以及β-胰腺细胞,是胰岛素依赖型糖尿病(IDDM)病人以及僵硬综合症(SMS)病人血清的关键抗原。运用sephamryl S-200以及DEAEsepharose可以从猪脑中分离纯化出谷氨酸脱羧酶。纯化的GAD在变性条件下电泳,经考马斯亮蓝R250染色以及Western-Blot鉴定主要有两条带,分子量分别为67kD和44kD。根据L-谷氨酸脱羧酶能够分解谷氨酸产生γ-氨基丁酸和CO2的特性,通过测定产物γ-氨基丁酸推断酶活。以上实验结果表明从猪脑中分离纯化到的是具有生物学活性以及免疫原性的谷氨酸脱羧酶,可进一步改良为IDDM检测试剂盒,用于IDDM的预防和预测。     

Purification and Characterization of Glutamate Decarboxylase from Lactobadllus bvevis IFO 12005

Glutamate decarboxylase (GAD) [EC 4.1.1.15] was purified from a cell-free extract of Lactobadllus brevis TFO 12005 by chromatographies on Sephadex G-100, DEAE-Sepharose CL-6B, and Mono Q. About 9 mg of purified GAD was obtained from 90.2 g of wet cells. The purified preparation showed a single protein band on SDS-PAGE. The molecular weights of purified GAD by SDS-PAGE and gel filtration on Superdex 200 were 60,000 and 120,000, respectively, indicating that GAD from L. brevis exists as a dimer. The N-terminal amino acid sequence of the purified GAD was NH₂-Met-Asn-Lys-Asn-Asp-Gln-Glu-Gln-Thr-. The optimum pH and temperature of GAD were at pH 4.2 and at 30°C. The GAD activity was increased by the addition of sulfate ions in a dose-dependent manner. The order of effect was as follows: ammonium sulfate?>?sodium sulfate?>?magnesium sulfate, indicating that the increase of hydrophobic interaction between subunits causes the increase of GAD activity. The purified GAD reacted only with l-glutamic acid as a substrate and the K_m, k_cat, and k_cat/K_m values were 9.3 mm, 6.5 s^?1, and 7 × 10² m^?1 s^?1, respectively.     

Genome sequence of Pantoea agglomerans strain IG1

Pantoea agglomerans is a gram-negative bacterium that grows symbiotically with various plants. Here we report the 4.8-Mb genome sequence of P. agglomerans strain IG1. The lipopolysaccharides derived from P. agglomerans IG1 have been shown to be effective in the prevention of various diseases, such as bacterial or viral infection, lifestyle-related diseases. This genome sequence represents a substantial step toward the elucidation of pathways for production of lipopolysaccharides.     

Inhibition of morphine dependence by a lipopolysaccharide from Pantoea agglomerans.

A lipopolysaccharide from Pantoea agglomerans (LPSp) was purified and examined for relief of morphine dependence by observing its inhibition of the jumping of mice on naloxone-precipitate withdrawal. Administration of LPSp either intravenously or intradermally showed marked inhibition of the jumping. Beta-endorphin in mouse serum and brain tissue were recognized to be in synchrony with the time course of the relief. Administration of TNF-alpha gave similar effect, suggesting that LPSp induces a cytokine cascade to produce endogenous TNF followed by ACTH/beta-LPH gene products and beta-endorphin. The effect of LPSp was better than that of LPS from E. coli or Bordetella pertussis, and thus is considered to be applicable for clinical use.     

Virulence mechanisms and host specificity of gall-forming Pantoea agglomerans

Pantoea agglomerans has been transformed from a commensal bacterium associated with many plants into a host-specific gall-forming pathogen by acquiring a plasmid-borne pathogenicity island. This pathogenicity island harbors the hrp/hrc gene cluster, in addition to genes encoding type III effector proteins, biosynthesis of the phytohormones indole-3-acetic acid and cytokinin, multiple diverse insertion sequences and pseudogenes. This review describes a unique model for understanding the emergence of new pathogens or new pathogenic variants, offering an insight into the function of type III effectors in host specificity and the evolution of a pathogen into pathovars. It also addresses the primary role of type III effectors in gall initiation as compared with a secondary role of phytohormones secreted by the pathogen.     

林生山黧豆谷氨酸脱羧酶的分离纯化及部分性质的研究

以林生山黧豆为材料,利用硫酸按分段盐析,丙酮沉淀,DEAE-SepharoseFF离子交换柱层析,SephacrylS300凝胶过滤柱层析及FPLC-MonoQ柱层析技术,以聚酰胺薄膜层析荧光定量法为酶活力检测手段,分离纯化了谷氨酸脱羧酶,达到电泳银染纯．纯化后的林生山黧豆谷氨酸脱羧酶活力达375．09U·mp-1,纯化倍数38．2倍,经SDS-PAGE测定,其亚基分子量为70kD,经梯度PAGE确定,天然分子量为140kD,表明该酶是由两个亚基组成的二聚体．酶学研究表明,纯化的林生山黧豆谷氨酸脱羧酶的最适pH值为5．4,对谷氨酸的Km值为1．62×10-3mol·L-1,酶的最适温度为40℃,酶特异性地使谷氨酸脱羧,不能使天门冬氨酸等其它氨基酸脱羧．     

Characterization of a small cryptic plasmid from endophytic Pantoea agglomerans and its use in the construction of an expression vector

A circular cryptic plasmid named pPAGA (2,734 bp) was isolated from Pantoea agglomerans strain EGE6 (an endophytic bacterial isolate from eucalyptus). Sequence analysis revealed that the plasmid has a G+C content of 51% and contains four potential ORFs, 238(A), 250(B), 131(C), and 129(D) amino acids in length without homology to known proteins. The shuttle vector pLGM1 was constructed by combining the pPAGA plasmid with pGFPmut3.0 (which harbors a gene encoding green fluorescent protein, GFP), and the resulting construct was used to over-express GFP in E. coli and P. agglomerans cells. GFP production was used to monitor the colonization of strain EGE6gfp in various plant tissues by fluorescence microscopy. Analysis of EGE6gfp colonization showed that 14 days after inoculation, the strain occupied the inner tissue of Eucalyptus grandis roots, preferentially colonizing the xylem vessels of the host plants.     

Purification and Characterization of Arginine Decarboxylase from Soybean (Glycine max) Hypocotyls

Arginine decarboxylase (EC 4.1.1.19 [EC] ) was purified from soybean,Glycine max, hypocotyls by a procedure which includes ammoniumsulfate fractionation, acetone precipitation, gel filtrationchromatography, and affinity chromatography. Using this procedure,ADC was purified to one band in non-denaturing PAGE. The purifiedADC has an M_r of 240 kDa based on gel filtration chromatographyand is a trimer of identical subunits which has an estimatedM_r of 74 kDa based on SDS-PAGE. ADC is active between 30 and50°C and has a K_m value of 46.1 µM. ADC is very sensitiveto agmatine or putrescine but not to spermidine or spermine.In the presence of 0.5 mM agmatine (or putrescine), the enzymeactivity was inhibited by 70%. However, at the same concentrationof spermidine (or spermine), the enzyme activity was inhibitedby only 10–20%. (Received April 2, 1997; Accepted August 18, 1997)     

Effects of a lipopolysaccharide from Pantoea agglomerans on the cocaine-induced place preference

A lipopolysaccharide from Pantoea agglomerans (LPSp) was purified, and its effect on the cocaine-induced place preference was examined in rats. Cocaine (4 mg/kg, i.p.) produced a significant place preference. Administration of LPSp (5 – 1000 μg/kg, i.p.) alone resulted in neither preference nor aversion for either the drug- or saline-associated place. However, pretreatment with LPSp (500 and 1000 μg/kg, i.p.) abolished the place preference that had been induced by cocaine. Furthermore, treatment with LPSp (500 μg/kg, i.p.) abolished cocaine (20 mg/kg, i.p.)-induced locomotor enhancement in mice. These results suggest that while LPSp itself may possess neither reinforcing nor locomotor enhancing effects, it blocks both the reinforcing and the locomotor enhancing effects of cocaine. Therefore, LPSp might be useful in pharmacotherapy for prevention of recurrent cocaine abuse.     

Partial Purification and Characterization of A Novel Histidine Decarboxylase from Enterobacter aerogenes DL-1

Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca²⁺ and Mn²⁺ showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver–Burk plot showed that K _m and V _max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.     

Purification and partial characterization of azoreductase from Enterobacter agglomerans

Azoreductase, an enzyme catalyzing the reductive cleavage of the azo bond of methyl red (MR) and related dyes, was purified to electrophoretic homogeneity from Enterobacter agglomerans. This bacterial strain, isolated from dye-contaminated sludge, has a higher ability to grow, under aerobic conditions, on culture medium containing 100mg/L of MR. The enzyme was purified approximately 90-fold with 20% yield by ammonium sulfate precipitation, followed by three steps of column chromatography (gel-filtration, anion-exchange, and dye-affinity). The purified enzyme is a monomer with a molecular weight of 28,000 Da. The maximal azoreductase activity was observed at pH 7.0 and at 35 degrees C. This activity was NADH dependent. The K(m) values for both NADH and MR were 58.9 and 29.4 microM, respectively. The maximal velocity (V(max)) was 9.2 micromol of NADH min(-1)mg(-1). The purified enzyme is inhibited by several metal ions including Fe(2+) and Cd(2+).     

Isolation of endophytic diazotroph Pantoea agglomerans and nondiazotroph Enterobacter asburiae from sweetpotato stem in Japan

AIMS: To isolate and identify diazotrophic endophytes in the stem of Japanese sweetpotato cv. Koganesengan. METHODS AND RESULTS: Surface-sterilized and thinly sliced (1-2 mm) sweetpotato stem samples were incubated in test tubes with semi-solid modified Rennie (MR) medium. The test tubes were assayed for acetylene reduction activity (ARA) 5 days after incubation at 30 degrees C. Twelve isolates were obtained from MR plates inoculated with a loop of semi-solid MR medium from ARA+ tubes. However, ARA test showed that only nine isolates were diazotrophic and three were nondiazotrophic strains. Using the API 20E diagnostic kit, four diazotrophic isolates were identified as strains of Pantoea spp. and five isolates as Klebsiella spp. The nondiazotrophic bacteria were strains of Enterobacter spp. A diazotrophic isolate Pantoea sp. MY1 and nondiazotrophic isolate Enterobacter sp. MY2 were identified to the species level by full sequence analysis of 16S rRNA gene. The results showed that MY1 had 99.2% similarity to Pantoea agglomerans ATCC 27155 and MY2 had 99.5% similarity to Enterobacter asburiae ATCC 35953. CONCLUSION: The stem of sweetpotato cv. Koganesengan was colonized by diazotrophic endophyte P. agglomerans and nondiazotrophic endophyte E. asburiae. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is an essential step toward understanding the ecology and interaction between endophytic bacteria and sweetpotato.     

Purification of L-Glutamate Decarboxylase from Monkey Brain

Glutamate decarboxylase (GAD) is an enzyme that synthesizes γ-aminobutyrate (GABA), a major inhibitory neurotransmitter in the central nervous system. Post-translational modification of GAD, such as N-terminal blockage, phosphorylation-dephosphorylation, and palmitoylation, is an important factor in the biological activity of GAD. In order to address the significance of post-translational events on GAD, we thought it crucial to obtain a non-recombinant form of GAD. In this study, we attempted to isolate GAD protein from the monkey brain, a model animal close to the human that has not been studied. Monkey brain was homogenized, fractionated with ammonium sulphate, and applied to a series of chromatographic steps, including hydrophobic, ion-exchange, and gel filtration. Purified GAD showed a single band on SDS–PAGE, and the enzyme was found to have a molecular weight of 61,000 and exhibited 1,100 nmol/min/mg of specific activity. It had an optimal pH of 7 and optimal thermal stability at 40 °C.     

Colonization dynamics of Pantoea agglomerans in the wheat root habitat

Plants are colonized by microbial communities that have diverse implications for plant development and health. The establishment of a stable plant–bacteria interaction depends on a continuous coexistence over generations. Transmission via the seed is considered as the main route for vertical inheritance of plant-associated bacteria. Nonetheless, the ecological principles that govern the plant colonization by seed endophytes remain understudied. Here we quantify the contribution of arrival time and colonization history to bacterial colonization of the wheat root. Establishing a common seed endophyte, Pantoea agglomerans, and wheat as a model system enabled us to document bacterial colonization of the plant roots during the early stages of germination. Using our system, we estimate the carrying capacity of the wheat roots as 10⁸ cells g⁻¹, which is robust among individual plants and over time. Competitions in planta reveal a significant advantage of early incoming colonizers over late-incoming colonizers. Priming for the wheat environment had little effect on the colonizer success. Our experiments thus provide empirical data on the root colonization dynamics of a seed endophyte. The persistence of seed endophyte bacteria with the plant population over generations may contribute to the stable transmission that is one route for the evolution of a stable host-associated lifestyle.     

水稻内生优势成团泛菌GFP标记菌株的性质与标记丢失动力学

为研究内生细菌对宿主植物侵染定殖的机理和其共生生物学作用 ,对水稻内生优势成团泛菌 (Pantoeaagglomerans)YS19与绿色荧光蛋白 (GFP)标记的YS19B ::gfp菌株的生长动力学进行了比较研究 ,探讨了成团泛菌YS19B ::gfp的标记稳定性和荧光性质 .标记菌株与野生型菌株相比 ,最大比生长速率和最大生物量仅减小 12 4 %和 6 % ,代时延长 14 0 % .成团泛菌YS19B ::gfp在指数期连续传代培养 10 0代后 ,GFP标记的保持率为 89 1% ,建立了标记菌株在有标记丢失存在时的生长动力学模型 :dX+ dt =μ+ (1-p)X+ ,解析出细胞分裂时标记丢失的概率p =9 75 6× 10 -7,确定了方程的模型参数 .标记菌株的荧光光谱在激发波长为 4 0 0nm时 ,最大发射波长为 5 0 8nm ,与供体菌株完全相同 .在LB培养基上生长时 ,成团泛菌YS19B ::gfp的GFP产生时间在指数期末期到稳定期较快 ,并于培养至 2 0h时达到最高 ,同时单位菌体生物量的荧光强度也达到最大 .结果说明 ,在GFP标记后成团泛菌YS19B ::gfp的生长仅受到较小影响 ,不致对成团泛菌的生理活动造成大的改变 ,同时由于该菌对宿主的侵染能力比其它内生细菌要强得多 ,因而该菌对植物的侵染活性影响也较小 ,该菌仍然可以保持其内生优势地位 .该标记的稳定性比较高 ,荧光产生正常 ,很适     

The structure of the O-specific polysaccharide of the lipopolysaccharide from Pantoea agglomerans strain FL1

A neutral O-specific polysaccharide consisting of d-rhamnose was obtained by mild acid hydrolysis of the lipopolysaccharide of the plant pathogenic bacterium Pantoea agglomerans strain FL1, a common epiphyte of many plant species, and associated with Pseudomonas savastanoi pv. savastanoi in young and apparently intact olive knots. By means of compositional and methylation analyses, and NMR spectroscopy, the chemical repeating unit of the polymer was identified as a linear tetrasaccharide of the structure: