Genome sequence of Agrobacterium tumefaciens strain F2, a bioflocculant-producing bacterium

Agrobacterium tumefaciens F2 is an efficient bioflocculant-producing bacterium. But the genes related to the metabolic pathway of bioflocculant biosynthesis in strain F2 are unknown. We present the draft genome of A. tumefaciens F2. It could provide further insight into the biosynthetic mechanism of polysaccharide-like bioflocculant in strain F2.     

Further characterization of Renibacterium salmoninarum extracellular products.

Renibacterium salmoninarum, the agent of bacterial kidney disease in salmonids, releases high concentrations of extracellular protein in tissues of infected fish. The extracellular protein consists almost entirely of a 57-kDa protein and derivatives of degradation and aggregation of the same molecule. The 57-kDa protein and its derivatives were fractionated into defined ranges of molecular mass. Separated fractions continued to produce degradation and aggregation products. One-dimensional electrophoretic separation of extracellular protein revealed a number of proteolytically active bands from > 100 to approximately 18 kDa associated with various 57-kDa protein derivatives in the different molecular mass fractions. Two-dimensional separation of extracellular protein showed that continued degradation and aggregation, similar both in location and behavior to some of the 57-kDa protein derivatives, was also displayed by the proteolytically active bands after their separation. Effects of reducing agents and sulfhydryl group proteinase inhibitors indicated a common mechanism for the proteolytically active polypeptides characteristic of a thiol proteinase. The results suggested that the 57-kDa protein and some of its derivatives undergo autolytic cleavage, releasing a proteolytically active polypeptide(s) of at least 18 kDa. Soluble polysaccharide-like material also was detected in extracellular products and tissue from infected fish. Antiserum to the polysaccharide-like material cross-reacted with O-polysaccharide of the fish pathogen Aeromonas salmonicida, suggesting some structural similarity between these polysaccharides. The polysaccharide and the proteolytic activity associated with the 57-kDa protein derivatives should be investigated with respect to the pathogenesis of R. salmoninarum infections.     

Manure and peat based iron-organo complexes

Earlier studies have shown that some naturally occuring organic materials can be used as an Fe source for plants. The aim of this research was to study enrichment procedures that would result in complex formation in common, low cost organic materials and to determine the maximum attainable Fe enrichment levels.Three organic materials-farmyard manure (FYM), poultry manure (PM) and Huleh Valley peat (PE) were studied for their characteristics as Fe, Mn and Zn carriers for plant uptake. Various enrichment procedures were investigated. These studies have shown that the enrichment level depends on the metal, pH and the water soluble fraction (WSF) of the organic materials. Maximum enrichment levels (at pH=3.5) were measured after the excess of inorganic salts was removed by leaching. These levels were 5.8–6.6% for Fe, 3.0–3.4% for Mn and 6.0–6.3% for Zn. An infrared spectrum of the Fe enriched WSF showed that most of the ligands in the complex formed are polysaccharides or polysaccharide-like compounds.     

Consumer value‐based edible insect market segmentation [edible insect market segmentation]

Edible insect food products have been launched and in the market since 2011. Since then the number of companies that produce edible insect food product has increased greatly. However, the guidance of how these firms should effectively target which consumer market is unclear. The goal of this study is to provide information about the US consumer market that could potentially increase edible insect food product sales. This study uses consumer value as a segmenting axle and provides the cluster analysis results. From this analysis, academics and industry practitioners can have better insights about edible insect food product consumer profiles.     

Identification of the protein encoded by rodC, a cell division gene from Bacillus subtilis

The rodC1 mutation of Bacillus subtilis is a temperature-sensitive marker which affects the orientation of the plane of cell division. We have cloned the rodC gene and have localized the site of the rodC1 lesion. To identify the rodC gene product, we have subjected several plasmid clones containing B. subtilis chromosomal DNA from the rodC region to maxicell analysis in Escherichia coli. A 68 kiloDalton protein has been identified as the rodC gene product. This is the initial cloning of a cell division gene and the identification of its product from B. subtilis. The rodC gene has also been implicated as being directly associated with the synthesis of glycerol teichoic acid.     

Biochemical and immunological characterization of the cell surface of the fish pathogenic bacterium Aeromonas salmonicida

The identification of lipopolysaccharide as periodic acid-Schiff positive material, present in the membrane fraction of the fish pathogenic Gram-negative bacterium Aeromonas salmonicida, analyzed by SDS-polyacrylamide gel electrophoresis, is shown. Such analysis has revealed several periodic acid-Schiff positive bands and many membrane proteins among which a pathogenicity-related Mr 54000 protein as a constituent of an additional surface layer outside the outer membrane (Evenberg et al., (1982) Biochim. Biophys. Acta 684, 241-248). The latter protein, designated as additional cell envelope protein or ACE protein, has been purified and characterized in our laboratory (Evenberg and Lugtenberg, (1982) Biochim. Biophys. Acta 684, 249-254). Most strains produce both high and low molecular weight lipopolysaccharide species, presumably corresponding with the presence and (virtual) absence, respectively, of an O-antigenic chain. The property to produce high molecular weight lipopolysaccharide can be lost upon subculturing in laboratory growth media and such is greatly enhanced by the prior loss of the ability to produce ACE protein. Lipopolysaccharide and ACE protein were identified as the major antigens. A new polysaccharide-like antigen, designated as PS-antigen, was detected. Moreover, immunological indications for the presence of a lipoprotein in A. salmonicida are described. The surface localization of the antigens was determined by testing whether preadsorption of antisera by intact cells decreased the binding of IgG to these antigens, or decreased the ability of the sera to agglutinate cells. According to these criteria lipopolysaccharide, ACE protein and PS-antigen are the major surface-located antigens. Material cross-reactive with lipopolysaccharide, ACE protein and PS-antigen has been found in a large number of strains. Several lines of evidence indicate the presence of interactions between ACE protein and lipopolysaccharide. Based on these results a molecular model of the cell envelope of virulent A. salmonicida is presented.     

Kinetic analysis of the michaelis-menten mechanism in which the substrate and the product are unstable

• 1.1. A kinetic analysis of the Michaelis-Menten mechanism for the case in which both the substrate and the product are unstable, either spontaneously or as the result of the addition of a reagent, has been made.
• 2.2. The explicit time course equations of the immediate product and the species into which it subsequently is transformed have been derived under the conditions of rapid equilibrium and limiting substrate concentration.
• 3.3. The validity of these equations has been checked using numerical simulations.
• 4.4. The kinetic data analysis which we suggest is based on the time progress curves of the product or, in the case in which the product accumulation cannot be monitored experimentally, on the time progress curve of the species into which the immediate product is transformed.
• 5.5. This analysis allows the determination of the rate and the equilibrium constants if adequate experimental results are available.
• 6.6. We have chosen a numerical example, with which we illustrate the procedure of the kinetic data analysis by simulating some curves with assumed experimental errors.

     

High‐Resolution Insight into Materials Criticality: Quantifying Risk for By‐Product Metals from Primary Production

Many advanced energy and environmentally relevant technologies rely on metals that have been identified as critical, or whose availability may be limited. Several of these elements are produced mostly as by‐products of mining other base metals (carriers). This by‐product dependence has been proposed as a significant supply‐risk indicator by the materials criticality community. This article provides new quantitative evidence that, in several cases, by‐product metals’ availability may not be directly limited by carrier supply. We perform an assessment based on characteristics essential to by‐product metals, including physical concentration, market value of metals, and extraction technology efficiency. We analyze 40 carrier/by‐product pairs and identify five ‘high‐by‐product’ pairs. We assess the supply responsiveness of these metals. Our analysis suggests that rather than limited primary production of carrier, lack of incentive for improving recovery efficiency may limit availability of the by‐product. This behavior is found in the zinc‐indium and copper‐selenium systems. For germanium, on the other hand, we instead propose influence from the by‐product market itself leading to price inelasticity of supply. As a complement to other quantitative methods developed for material systems, such as material flow analysis, we provide an essential technoeconomic analysis of the by‐product metals problem by employing cluster analysis and econometric modeling. This approach provides insight into supply‐risk mitigation strategies related to extraction efficiency and supply‐chain structure.     

Cloning, mapping and gene product identification of rhaT from Escherichia coli K12

Rhamnose utilization requires the function of a specific rhamnose transport system. Rhamnose transport mutants have been isolated and characterized. The structural gene, rhaT, encoding the rhamnose permease has been cloned from Escherichia coli. rhaT has been mapped in the rha locus (87.7 min) by analysis of cotransduction with glpK and other rha markers. The precise location of the gene has been determined by complementation analysis of rhamnose transport mutants transformed with recombinant plasmids containing different fragments of the cloned region. Gene order (counterclockwise) is established as glpK . . . rhaT-rhaR-rhaS-rhaB-rhaA-rhaD. The gene product has been identified by expression of rhaT in a T7 RNA polymerase/promoter system. This 23 kDa protein has been assigned to the rhaT product and has been shown to be located in the cell membrane.     

后基因组时代的真菌天然产物发现

真菌产生的次级代谢产物是新药发现的重要来源之一,然而近年来传统的真菌天然产物发现方法在大量真菌基因组测序完成的时代遇到了很大的挑战。如何利用这些基因组数据来发现真菌中新的天然产物已成为后基因组时代天然产物发现的研究重点和热点。本综述先后介绍了真菌天然产物的类型及其相应基因簇和骨架酶的特征,基于基因组挖掘技术发展而来的天然产物发现新策略,以及利用合成生物学理念和技术在真菌天然产物发现中的应用现状,最后展望了后基因组时代中的天然产物发现研究前沿及基因组数据在后基因组时代对真菌天然产物发现的应用前景。     

Dimers of nicotinamide adenine dinucleotide: New evidence for the structure and the involvement in an enzymatic redox process

The composition and the structure of the product from the known electrochemical dimerization of the NAD⁺ have been conclusively demonstrated. A detailed analysis of the ¹H and ¹³C nmr spectra has in fact led to the conclusion that the product contains three diastereoisomeric dimers of the 4,4′-tetrahydrobipyridyl type. Furthermore, the cytoplasmic fraction obtained from a standard mitochondrial preparation of rat liver has been shown to catalyze the oxygen uptake by the dimers. A 1 : 1 molar ratio of the reagents in the redox process is indicated by manometric data on oxygen uptake complemented by spectrophotometric analysis of the oxidized substrates, suggesting that H₂O₂ is the reduction product. NAD⁺ was identified as the oxidation product by an enzymatic method.     

Feedforward inhibition in biosynthetic pathways: inhibition of the aminoacyl-tRNA synthetase by the penultimate product.

Inhibition of the aminoacyl-tRNA synthetase by the penultimate product of a pathway for the biosynthesis of an amino acid has been reported for several pathways in many different types of organisms. A regulatory role for this mechanism often has been suggested, although there is some conflicting experimental evidence. The significance of such feedforward inhibition is examined here by mathematical analysis. The techniques that have proved successful in the analysis of control by feedback inhibition—showing that the nearly universal pattern of end-product inhibition is an optimal design—indicate that feedforward inhibition by the penultimate product does not contribute significantly to the functional effectiveness of regulation at the level of enzymatic activity. Feedforward inhibition by the penultimate product may have no physiological role, or it may be involved in differential signalling of intra- and extracellular changes and/or in directing the metabolic flow in branched pathways. These possibilities are discussed in light of analytical results presented in this paper and published experimental evidence.     

西双版纳不同热带生态系统土壤有机质的光谱学特性

 为了探讨西双版纳地区土地利用变化对土壤有机质含量及其化学组成的影响,选取了相邻的次生林、耕种6年的农田和定植3年的橡胶园样地,对其0～5 cm和5～20 cm表层土壤中有机质含量、胡敏酸的光谱学特性进行了分析。研究结果表明,次生林转变为农田之后,0～5 cm和5～20 cm 表层土壤有机质含量分别降低33.6%和23.7%;而次生林转变为橡胶园,分别降低28.6%和27.6%。胡敏酸可见-红外光谱结果显示,次生林转变为农田和橡胶园后,0～20 cm表层土壤E4/E6显著降低,这表明胡敏酸化学组成当中芳香族结构增加。傅立叶变换红外光谱结果同样表明,土地利用变化影响土壤有机质的化学组成。次生林转变为农田和橡胶园后,胡敏酸中羧基和酚基结构比例降低,而脂肪族、芳香族和多聚糖比例增加。     

Vector correlations in the F + HO → HF + O reaction and its isotopic variant

The F + H(D)O → HF(DF) + O reactions have been studied using quasi-classical trajectory (QCT) calculation method, based on the three different potential energy surfaces (PESs) of Gomez-Carrasco et al. (J Chem Phys 2004, 121:4605; J Chem Phys 2005, 123:114310; Chem Phys Lett 2007, 435:188). Facilitated with the analysis of the QCT results, the pictures for product scattering and product polarizations have been presented to investigate the vector correlations in the two reactions, with effects of isotope substitution and electronic state as well as collision energy being revealed at a chemical stereodynamical level.     

Detection of multiple β-casein (CASB) alleles by amplification created restriction sites (ACRS)

Direct sequencing of polymerase chain reaction (PCR) amplified DNA has been used to detect the DNA sequences for bovine beta-casein (CASB) A3 and B variants. Based on these sequences we have designed primers which create allele-specific restriction sites in the PCR product. Restriction analysis of PCR product generated in one reaction enable us to identify the A1, A2, A3 and B alleles of CASB rapidly without the use of radioactivity.     

Benzidine dihydrochloride as a chromogen for single- and double-label light and electron microscopic immunocytochemical studies

Although very sensitive chromogens have been adapted for localization of horseradish peroxidase in anterograde and retrograde tracing studies, they have not been successfully applied in immunocytochemical studies. This report describes a protocol which uses benzidine dihydrochloride (BDHC) as the chromogen for light (LM) and electron microscopic (EM) immunocytochemical studies. The protocol is comparable to that used for tetramethylbenzidine, except that the pH of the reaction is above 6.0. At the LM level, the BDHC reaction product is bluish-green and crystalline. Both the color and form of the product are readily distinguished from the reddish-brown DAB reaction product. LM double-labeling studies are therefore feasible. The use of BDHC also increases significantly the sensitivity of the immunoreaction. Higher fixative concentrations can be used, less detergent is necessary, and higher primary antibody dilutions are possible. By osmicating at 45 degrees C in an s-collidine buffer it is possible to preserve the soluble BDHC reaction product for EM analysis. Immunoreactive cells are particularly well labeled with this new protocol. The BDHC crystals are easily detected at the EM level and can be distinguished from flocculent DAB reaction product. This feature makes EM double-labeling studies possible.     

Kinetic analysis of enzyme systems with suicide substrate in the presence of a reversible, uncompetitive inhibitor.

We present a general kinetic analysis of enzyme catalyzed reactions evolving according to a Michaelis-Menten mechanism, in which an uncompetitive, reversible inhibitor acts. Simultaneously, enzyme inactivation is induced by an unstable suicide substrate, i.e. it is a Michaelis-Menten mechanism with double inhibition: one originating from the substrate and another originating from the reversible inhibitor. Rapid equilibrium of the reversible reaction steps involved is assumed and the time course equations for the reaction product have been derived under the assumption of limiting enzyme. The goodness of the analytical solutions has been tested by comparison with simulated curves obtained by numerical integration. A kinetic data analysis to determine the corresponding kinetic parameters from the time progress curve of the product is suggested.     

合成生物学应用产品开发现状与趋势

目的：从产品开发角度分析全球合成生物学发展现状和趋势。方法：在伍德罗·威尔逊国际学者中心的合成生物学产品和应用清单（synthetic biology products and applications inventory）的数据基础上,对全球合成生物学产品的开发状态、市场应用和发展前景等进行补充检索和分析。结果：至2015年,全球至少已有81家企业（或研究机构）的116种合成生物学产品得到了市场应用开发,主要开发者为美国企业（或研究机构）,产品主要集中于化学和医药领域。结论：合成生物学实现了从生物学分析向生物学合成的范式转移,其产品开发将给一系列的行业带来深刻的变革。