Isolation from Ochrobactrum anthropi of a Novel Class II 5-Enopyruvylshikimate-3-Phosphate Synthase with High Tolerance to Glyphosate

Applying the genomic library construction process and colony screening, a novel aroA gene encoding 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified, cloned, and overexpressed, and the enzyme was purified to homogeneity. Furthermore, site-directed mutagenesis was employed to assess the role of single amino acid residues in glyphosate resistance.The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19) is the sixth enzyme in the shikimate pathway, which is essential for the synthesis of aromatic amino acids and many aromatic metabolites in plants, fungi, and microorganisms (2, 11, 16), including apicomplexan parasites (22). It converts shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) to 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate. Interest in the characterization of EPSPS has increased significantly since the enzyme was identified as the primary target of the broad-spectrum, nonselective herbicide glyphosate [N-(phosphonomethyl)glycine] (25). Glyphosate is a competitive inhibitor with respect to PEP and binds adjacent to S3P in the active site of EPSPS, thereby mimicking an intermediate state of the ternary enzyme-substrate complex (23).Two classes of EPSPS, class I and II enzymes, sharing less than 30% amino acid similarity have been reported (9). Class I includes those found in plants and bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium, whose catalytic activity is inhibited at low micromolar concentrations of glyphosate (8). Class II EPSPS, found in Pseudomonas sp. strain PG2982, Agrobacterium tumefaciens strain CP4, Streptococcus pneumoniae, and Staphylococcus aureus, was distinguished by its ability to sustain efficient catalysis in the presence of high glyphosate concentrations (6, 9).Although a large number of AroA enzymes (EPSPS) have been cloned, identified, and tested as glyphosate resistant, only AroA variants derived from the A. tumefaciens strain CP4 have been successfully used commercially (9). To find a new enzyme similar to that of the AroA_{A. tumefaciens CP4}, in this study a highly glyphosate-tolerant strain from the rhizosphere of rice in a field where glyphosate is frequently used has been selected and identified on M9 minimal medium containing 200 mM glyphosate, and its 16S rRNA gene sequence confirmed that this strain was strongly related to Ochrobactrum anthropi (99.9%). Additionally, the aroA_{O. anthropi} gene was isolated and kinetic characteristics of the Ochrobactrum anthropi strain EPSP synthase were determined in this study.     

不同氮素水平下施硫对高产小麦碳氮运转和产量的影响

在大田栽培条件下,以2个不同穗型的高产冬小麦品种为试验材料,在施240 kg·hm-2(N240)和330 kg·hm-2(N330)纯氮水平下,分别施纯硫60 kg·hm-2(S60)和0 kg·hm-2(S0),研究了施硫对小麦不同器官碳氮运转及其对籽粒产量和蛋白质产量的影响.结果显示:(1)2个供氮水平下,施硫(S60)均比对照(S0)增加了2个小麦品种的叶片、茎、鞘、颖壳和穗轴等营养器官花前贮藏干物质、氮素的运转量和运转率以及总运转量和总运转率,提高了转运干物质、氮素对籽粒重和籽粒氮素的贡献率,极显著提高了籽粒和蛋白质产量.(2)施硫对豫农949品种籽粒和蛋白质产量提高幅度显著高于兰考矮早八;与对照(S0)相比,豫农949的N240S60和N330S60处理使籽粒产量分别增加12.74%和16.41%,蛋白质产量分别增加16.84%和16.14%.结果表明,中氮和高氮水平下施硫均可明显促进高产小麦植株的C-N运转,提高植株对氮的吸收利用和碳物质的积累,从而增加籽粒产量,但不同品种间的施硫效应存在差异.     

Genomic Features of Lactococcus lactis IO-1, a Lactic Acid Bacterium That Utilizes Xylose and Produces High Levels of L-Lactic Acid

Lactococcus lactis IO-1 (JCM7638) produces L-lactic acid predominantly when grown at high xylose concentrations, and its utilization is highly desired in the green plastics industry. Therefore it is worthwhile studying its genomic traits. In this study, we focused on (i) genes of possible horizontal transfer derivation (prophages, the nisin-sucrose transposon, and several restriction-modification systems), and (ii) genes for the synthetic pathways of amino acids and vitamins in the IO-1 genome. In view of the results of this analysis, we consider their meanings in strain IO-1.     

A Novel Replication-Competent Vaccinia Vector MVTT Is Superior to MVA for Inducing High Levels of Neutralizing Antibody via Mucosal Vaccination

Mucosal vaccination offers great advantage for inducing protective immune response to prevent viral transmission and dissemination. Here, we report our findings of a head-to-head comparison of two viral vectors modified vaccinia Ankara (MVA) and a novel replication-competent modified vaccinia Tian Tan (MVTT) for inducing neutralizing antibodies (Nabs) via intramuscular and mucosal vaccinations in mice. MVTT is an attenuated variant of the wild-type VTT, which was historically used as a smallpox vaccine for millions of Chinese people. The spike glycoprotein (S) of SARS-CoV was used as the test antigen after the S gene was constructed in the identical genomic location of two vectors to generate vaccine candidates MVTT-S and MVA-S. Using identical doses, MVTT-S induced lower levels (∼2-3-fold) of anti- SARS-CoV neutralizing antibodies (Nabs) than MVA-S through intramuscular inoculation. MVTT-S, however, was capable of inducing consistently 20-to-100-fold higher levels of Nabs than MVA-S when inoculated via either intranasal or intraoral routes. These levels of MVTT-S-induced Nab responses were substantially (∼10-fold) higher than that induced via the intramuscular route in the same experiments. Moreover, pre-exposure to the wild-type VTT via intranasal or intraoral route impaired the Nab response via the same routes of MVTT-S vaccination probably due to the pre-existing anti-VTT Nab response. The efficacy of intranasal or intraoral vaccination, however, was still 20-to-50-fold better than intramuscular inoculation despite the subcutaneous pre-exposure to wild-type VTT. Our data have implications for people who maintain low levels of anti-VTT Nabs after historical smallpox vaccination. MVTT is therefore an attractive live viral vector for mucosal vaccination.     

A Novel High Biosorbent of Pb-resistant Bacterium Isolate for the Removal of Hazardous Lead from Alkaline Soil and Water: Biosorption Isotherms In Vivo and Bioremediation Strategy

A novel Pb-resistant bacterium was isolated from aged lead-contaminated alkaline soils, and was identified as Bacillus megaterium via the MIDI protocol. The biosorption isotherms and kinetics of Pb(II) associated with B. megaterium in vivo in the alkaline environment were investigated at the first time. All the batch experiments of biosorption demonstrate that the B. megaterium uptake of lead is pH-dependent, exothermic (ΔH° = ?5224.86 KJ mol^?1), spontaneous, and fits well with the Langmuir isotherm, resulting in different kinetics under different examination temperatures. The maximum biosorption capacity is 503.86 mg g^?1 at optimum conditions, which is much better in comparison to the biosorbent reported at the acidic condition in the literature. The Fourier-transform Infrared spectroscopic analysis of lead-loaded biomass confirms that the biosorption between B. megaterium and lead is the chemical adsorption in vivo. A site test indicates that B. megaterium really increases mobility and bioavailability of lead in Pb-contaminated alkaline soil in terms of chemical fractionation in vivo, which will potentially increase its uptake by hyperaccumulated plants in alkaline soils in arid or semi-arid areas of NW, China. Therefore, the novel isolate of B. megaterium with the highest adsorption capacity is a new promising biosorbent for the lead removal in alkaline water and soil.     

从富含淀粉的水稻胚乳组织中提取功能总RNA 和克隆wee1基因片段的方法研究

水稻胚乳中含有大量的淀粉等物质,用传统的方法提取总RNA难度很大．改良了一种SDS／苯酚法．可以从水稻胚乳中提取高质量的总RNA,解决了RNA易降解、易被污染以及由于总RNA与淀粉等物质共沉淀所造成的低产量等问题．通过分光光度计测量OD值以及变性胶电泳,可以检测出所提取的总RNA质量较高,从1.5g水稻胚乳中可提取到800μg左右的总RNA．提取的总RNA已成功用于RT-PCR克隆目的基因．