Ectoine accumulation in Brevibacterium epidermis

As a halotolerant bacterial species, Brevibacterium epidermis DSM 20659 can grow at relatively high salinity, tolerating up to 2 M NaCl. It synthesizes ectoine and the intracellular content increases with the medium salinity, with a maximum of 0.14 g ectoine/g CDW at 1 M NaCl. Sugar-stressed cells do not synthesize ectoine. Ectoine synthesis is also affected by the presence of external osmolytes. Added betaine is taken up and completely replaced ectoine, while L-proline is only temporarily accumulated after which ectoine is synthesized. The strain can metabolize ectoine; L-glutamate is a better carbon source for ectoine synthesis than L-aspartate.     

Operational stability of Brevibacterium imperialis CBS 489-74 nitrile hydratase

Brevibacterium imperialis CBS 489-74 was grown in broths prepared with yeast and malt extract, bacteriological peptone and 2% glucose or differently modified with the addition of Na-phosphate buffer, FeSO₄, MgSO₄ and CoCl₂. The peak production of nitrile hydratase (NHase) did not change significantly. At the stationary growth phase, the units per milliliter of broth (60 units ml⁻¹) were more important than those at the exponential growth phase.

The NHase operational stability of whole resting cells was monitored following the bioconversion of acrylonitrile to acrylamide in continuous and stirred UF-membrane reactors. The rate of inactivation was independent on buffer molarity from 25 to 75 mM and on pH from 5.8 to 7.4. Enzyme stability and activity remained unchanged in distilled water. The initial reaction rate increased from 12.8 to 23.8 g acrylamide/g dry cell/h, but NHase half-life dropped from 33 to roughly 7 h when temperature was varied from 4°C to 10°C. The addition of butyric acid up to 20 mM did not improve enzyme operational stability, and largely reduced (94%) enzyme activity. Acrylonitrile caused an irreversible damage to NHase activity. High acrylonitrile conversion (86%) was attained using 0.23 mg cells/ml in a continuously operating reactor.     

黄色短杆菌TK0303的L-亮氨酸生物合成途径分析

应用途径分析方法分析了在拟稳态时黄色短杆菌(Brevibacterium flavum)TK0303由葡萄糖发酵生产L-亮氨酸的代谢途径,确定了L-亮氨酸合成的最佳途径和最大理论产率。通过比较途径分析所获得的反应模型,确定了丙酮酸和乙酰辅酶A是L-亮氨酸合成途径的关键节点。在此基础上改变外界环境因子,强化L-亮氨酸生物合成途径中丙酮酸和乙酰辅酶A两个关键节点的代谢流,以期进一步提高L-亮氨酸产率。结果表明,经过谷氨酸以及醋酸铵的调节,代谢途径流量发生显著变化,L-亮氨酸产量有明显提高。     

Identification, characterization, and chromosomal organization of the ftsZ gene from Brevibacterium lactofermentum

The ftsZ gene was cloned from the chromosomal DNA of Brevibacterium lactofermentum by the polymerase chain reaction (PCR) using two oligonucleotides designed from two conserved regions found in most of the previously cloned and sequenced ftsZ genes from other microorganisms. ftsZ is a single-copy gene in corynebacteria and is located downstream from ftsQ and murC, indicating linkage between genes involved in peptidoglycan synthesis (mur genes) and genes involved in cell division (fts genes). The organisation of the cluster is similar to that in Streptomyces and different from those of Escherichia coli or Bacillus subtilis because ftsA is not located upstream of ftsZ. The gene was expressed in E. coli using the T7 expression system; the calculated molecular weight of the expressed protein was 50 kDa. Expression of the B. lactofermentum ftsZ gene in E. coli inhibited cell division and led to filamentation. The ftsZ gene of this organism does not complement ftsZ mutations or deletions in E. coli, when cloned on low or high-copy-number vectors. Received: 14 January 1998 / Accepted: 31 March 1998     

一株耐盐碱乳酪短杆菌G20响应盐碱胁迫的差异代谢物分析

【目的】探究耐盐碱乳酪短杆菌G20响应盐碱胁迫的代谢物组成以及代谢物合成潜力,为潜在功能分子和盐碱诱导的快速稳定响应逻辑门基因线路的挖掘提供参考。【方法】利用液相色谱-质谱联用技术(LC-MS)检测乳酪短杆菌G20盐碱环境与正常环境下4个生长时期的代谢产物。着重对富含高差异变化倍数代谢物的适应期与指数期进行分析。【结果】乳酪短杆菌G20可以在pH 10.0、9%NaCl环境中正常生长,同时环境pH值会随菌株生长逐步下降。综合正负离子2种模式,乳酪短杆菌G20在盐碱环境下各生长时期间差异代谢物数量分别为正常环境的0.69、0.75和0.81倍。盐碱胁迫诱导下适应期与指数期差异代谢物主要为苯环型化合物、有机酸及其衍生物与有机杂环类化合物。其中上调的有机酸化合物吲哚-3-乙酸、犬尿酸和葡萄糖酸指数期质谱信号强度低于适应期。菌株中可能存在的渗透保护剂有L-瓜氨酸、L-脯氨酸、N-乙酰鸟氨酸和左旋肉碱等。适应期变化倍数较大或质谱信号强度较高的差异化合物有毛果芸香碱、植物鞘氨醇和柠檬酸等,指数期有组胺、L-脯氨酸和硫胺素等。菌株差异代谢通路集中在氨基酸代谢与碳水化合物代谢。菌株代谢物中存在甜菜碱和...     

Cadmium accumulation and tolerance in Populus nigra and Salix alba

Rooted cuttings of Populus nigra L. clone Poli and Salix alba L. clone SS5 were treated for three weeks with 50 μM CdSO₄ in nutrient solution. The willow showed a far higher Cd tolerance, expressed as tolerance index (Ti), than the poplar in both roots and leaves. The root Cd content was higher in poplar than in willow, whereas in leaves the opposite was found. As a consequence, the translocation factor (Tf) revealed a greater ability of Cd transport in willow than in poplar. Cd treatment enhanced cysteine, γ-glutamylcysteine and reduced glutathione contents in roots of both species, whereas in leaves they were only enhanced in poplar. Furthermore, only poplar leaves showed an enhanced content of phytochelatins, whereas malic and citric acids rose in response to Cd only in the willow leaves. Cd treatment increased putrescine, spermidine and spermine contents in both roots and leaves of the willow, whereas in poplar only the putrescine content was enhanced in roots.     

聚球藻硅质化作用初探

硅元素是全球生地化循环的重要组成成分之一,对海洋生态系统中以浮游植物主导的初级生产力和硅碳循环具有重要的意义。普遍认为硅藻主导着全球海洋的硅循环,成为海洋硅循环和碳循环交互作用的重要桥梁。海洋单细胞聚球藻对海洋食物网和能量流具有关键启动和支撑作用,是全球碳循环中固碳过程的主要贡献者,近年又被发现其具有重要的硅质化作用,为我们提供了一个在海洋中(特别是寡营养海域),除硅藻之外,连接硅碳循环交互作用的新视角,对硅藻在全球海洋硅碳循环的绝对地位具有重要的挑战意义。面对聚球藻在大洋中如此巨大的生物量,甚至高于硅藻,有必要弄清楚其碳沉降机制以及准确的模拟其硅循环,然而关于其在海洋硅循环的研究极少,硅质化作用的吸收和储存机理以及环境调节机制也不清楚;另外,其对世界海洋硅碳循环的调节作用也未见报道。为此,通过前人对海洋单细胞聚球藻硅质化作用研究的基础上进行有针对性的探讨,可望对海洋单细胞聚球藻硅质化作用及其对硅碳循环的调控机制有一个基本的认识,为深入研究聚球藻在全球海洋硅循环中的作用提供基础。     

Metal accumulation by Halodule wrightii populations

Metal concentrations and population parameters of the seagrass Halodule wrightii were determined at three locations at Rio de Janeiro State, Brazil. The possible increase of metal availability in one of these areas, Sepetiba Bay, as a result of dredging of contaminated bottom sediments which ocurred, was evaluated by analyses of Al, Cd, Cr, Cu, Fe, Ni, Pb and Zn in root, rhizome and shoots. In addition, analyses were carried out in H. wrightii populations from non-contaminated areas located at northwestern (Cabo Frio) and southeastern (Angra do Reis) regions of Rio de Janeiro State. Concurrently, abundance and density data of the seagrass populations were obtained. It was found that concentration from Sepetiba Bay samples up to 1.6 ± 0.4 μg g⁻¹ of Cd, 12 ± 1.0 μg g⁻¹ of Cr, 27 ± 2.4 μg g⁻¹ of Pb, 291 ± 47 μg g⁻¹ of Mn, 128 ± 23 μg g⁻¹ of Zn were significantly higher than that from two other collection sites. An increase in Cd and Zn concentration was observed in H. wrightii from Sepetiba Bay indicating that metal mobilization from contaminated sediments through dredging activities were, at least in part, transferred to the biotic compartment via accumulation by the seagrass. The populations of seagrass within the region demonstrated quite substantial changes in biomass data but not in shoot or rhizome density during the study. Such changes in biomass are to be expected, as these dynamics are typical of the small, isolated monospecific populations of H. wrightii along the Rio de Janeiro coast.     

椰子脂肪酸积累及与FatB基因表达关系研究

为筛选控制椰子果实中低碳链脂肪酸积累的关键FatB基因,本研究以海南本地高种绿椰四个发育时期的果实为试验材料,参考国家标准GB/T2906-1982谷类、油料作物种子粗脂肪测定方法,用索氏提取法提取果肉脂肪酸,应用气质联用检测技术测定各发育时期脂肪酸含量及组分,同时应用qRT-PCR对椰子脂肪酸合成相关基因FatB1、FatB2及FatB3进行相对定量分析,明确各发育时期椰子果实脂肪酸积累与FatB基因表达量变化之间的关系。结果表明:(1)椰子油含9种脂肪酸成分,在椰子果实发育的早期(第6和第7个月),棕榈酸、硬脂酸和油酸的含量较高,月桂酸和肉豆蔻酸相对含量较低,通过实时荧光定量PCR发现,此时FatB1基因的相对表达量较高。(2)而在椰子果实发育的后期(第8和第9个月),棕榈酸、硬脂酸和油酸的相对含量迅速降低,月桂酸和肉豆蔻酸相对含量迅速升高,FatB2和FatB3基因的相对表达量较高,FatB1的表达量略有降低。该研究初步掌握了椰果各发育时期FatB基因表达量与脂肪酸成分间的变化趋势,该结果为将来筛选控制椰子果实中低碳链脂肪酸积累的关键FatB基因奠定基础,同时为椰子脂肪酸成分的分子改良提供理论依据。     

Regulation of 3-hydroxypropionaldehyde accumulation in Klebsiella pneumoniae by overexpression of dhaT and dhaD genes

3-Hydroxypropionaldehyde (3-HPA), an important intermediary metabolite of 1,3-propanediol (PDO) production, would be toxic to the cell growth and led to the abnormal cessation of the fermentation process. In this study, the dhaD gene encoding glycerol dehydrogenase (GDH) and dhaT gene encoding 1,3-propanediol oxidoreductase (PDOR) were overexpressed in Klebsiella pneumoniae ACCC 10082 to decrease the 3-HPA accumulation and increase the coenzyme NADH supply. By the construction of pTD plasmid, GDH and PDOR were both overexpressed and their enzyme activities were increased by 2.6- and 3.2-fold, respectively. The enzyme activity ratio of PDOR/GDHt (glycerol dehydratase) also was increased. On the other hand, NADH production was enhanced and the ratio of NADH/NAD⁺ exceeded 1 after the inducement of IPTG for the constructed strain. The two factors enhanced the transformation of 3-HPA to PDO. In the batch and fed-batch fermentation by the constructed strain, the peak of 3-HPA accumulation reduced by 52.2% and 33.3%, respectively, compared with the control. The PDO concentration and yield reached 59.2 g/L and 0.48 mol/mol, respectively. Furthermore, the fed-batch fermentation process appeared easier to be regulated. This work is considered helpful for the further understanding on the PDO metabolic mechanism of K. pneumoniae and also useful for the PDO fermentation in a large-scale bioreactor.     

Ectoine Biosynthesis in Mycobacterium smegmatis

Mycobacterium smegmatis is a commonly used mycobacterial model system. Here, we show that M. smegmatis protects itself against elevated salinity by synthesizing ectoine and hydroxyectoine and characterize the phenotype of a nonproducing mutant. This is the first analysis of M. smegmatis halotolerance and of the molecular mechanism that supports it.     

Trehalose accumulation in Baudoinia compniacensis following abiotic stress

Baudoinia compniacensis is a microfungus recently described as the principal agent of fouling known as “warehouse staining”, affecting building exteriors, fixtures and vegetation surfaces in areas proximate to distillery aging warehouses, commercial bakeries and other areas subject to low-level ethanol vapour exposure. The surfaces most affected tend to be highly exposed and undergo extreme diurnal temperature fluctuations. In previous work, we have demonstrated the existence of heat-inducible putative chaperone proteins that may also be induced by low-level exposures to ethanol vapour (e.g., <10 ppm). The present study investigated the cellular accumulation of trehalose, a disaccharide identified in some microorganisms to be important in the protection of cell components during adverse stress conditions, such as thermal stress. Following heat shock at 45 °C, we observed a 2.5-fold accumulation of trehalose relative to unheated controls maintained at 26 °C. Peak trehalose concentrations of 10 mg g⁻¹ dry wt were seen at 90 min after heat treatment, followed by a gradual return to post-treatment by 150 min. Exposure of B. compniacensis cells to ethanol resulted in a similar increased accumulation of trehalose compared to unexposed controls. These findings imply that trehalose may be important in the tolerance of this fungus to abiotic stresses, such as heat and solvent exposure, and suggest future research directions for the control and prevention of warehouse staining.     

Cell type-specific protoberberine alkaloid accumulation inThalictrum flavum

Berberine is a common benzylisoquinoline alkaloid with potent antimicrobial properties, which suggest it functions to protect some plants from pathogen challenge. Berberine was identified as the major alkaloid in meadow rue (Thalictrum flavum ssp. glaucum), a medicinal member of the Ranunculaceae, and was detected in seeds and all organs of the plant. The high level of berberine in roots, rhizomes, and older petioles is mainly responsible for the intense yellow color of these organs. In rhizomes, protoberberine alkaloids were detected throughout the pith and, to a lesser extent, the cortex, but were absent from the vascular tissues. Similarly, protoberberine alkaloids were detected in the rib parenchyma of older petioles. In roots, alkaloid accumulation was detected only in the endodermis at the onset of secondary growth. Rather than being sloughed off, the endodermis was found to undergo extensive anticlinal division leading to an expanding cellular cylinder that ultimately displaced all external tissues. Endodermal-specific protoberberine alkaloid accumulation continued throughout root development, but was extended to include 3 to 4 layers of smaller pericycle cells in the oldest roots near the base of the stem. The cell type-specific accumulation of antimicrobial alkaloids and the unusual development of the endodermis and pericycle in T. flavum roots support the putative role of berberine in plant defense.     

Ferritin accumulation under iron scarcity in Drosophila iron cells

Ferritins are highly stable, multi-subunit protein complexes with iron-binding capacities that reach 4500 iron atoms per ferritin molecule. The strict dependence of cellular physiology on an adequate supply of iron cofactors has likely been a key driving force in the evolution of ferritins as iron storage molecules. The insect intestine has long been known to contain cells that are responsive to dietary iron levels and a specialized group of “iron cells” that always accumulate iron-loaded ferritin, even when no supplementary iron is added to the diet. Here, we further characterize ferritin localization in Drosophila melanogaster larvae raised under iron-enriched and iron-depleted conditions. High dietary iron intake results in ferritin accumulation in the anterior midgut, but also in garland (wreath) cells and in pericardial cells, which together filter the circulating hemolymph. Ferritin is also abundant in the brain, where levels remain unaltered following dietary iron chelation, a treatment that depletes ferritin from the aforementioned tissues. We attribute the stability of ferritin levels in the brain to the function of the blood-brain barrier that may shield this organ from systemic iron fluctuations. Most intriguingly, our dietary manipulations demonstrably iron-depleted the iron cells without a concomitant reduction in their production of ferritin. Therefore, insect iron cells may constitute an exception from the evolutionary norm with respect to iron-dependent ferritin regulation. It will be of interest to decipher both the physiological purpose served and the mechanism employed to untie ferritin regulation from cellular iron levels in this cell type.     

Evidence for demand-regulation of ribosome accumulation in E coli

We have determined the relative concentrations of ribosomes accumulated under different growth conditions for a number of translational mutants as well as for some natural isolates of Escherichia coli. The mutants are a tRNA modification mutant (miaA), a streptomycin resistant (SmR) and a streptomycin pseudodependent (SmP) mutant as well as two ribosome ambiguity (ram) mutants. The natural isolates used in this study are known to function with submaximal ribosome kinetics. The data show that for all the ribosome mutants the concentration of ribosomes relative to that in wild type bacteria increases when the growth rate decreases. A small increase is also seen in the natural isolates. In contrast, the miaA mutant shows no increase in ribosome concentration under the same slow growth conditions. The results suggest that bacteria with kinetically impaired ribosomes can to some extent increase the number of ribosomes accumulated under poor growth conditions in order to compensate for their slower function. We use this observation to explain in part how bacteria growing in natural environments can escape the strong selection for maximized growth rates and for optimized ribosomes that are characteristic of laboratory strains.     

RAPD markers associated with quercetin accumulation in Psidium guajava

We used a random amplified polymorphic DNA (RAPD) amplification method to identify molecular markers associated with high quercetin accumulation in the leaves of Psidium guajava L. trees, selected from four different Mexican agronomic regions. We identified six polymorphic RAPD fragments of 620, 590, 370, 690, 480 and 460 bp among individuals of P. guajava. Genetic linkage disequilibrium analysis revealed that three RAPD profiles considered as DNA markers (620/590 bp, 370 bp and 480/460 bp) had a positive, direct association with quercetin content. These informative molecular markers can be used for selective identification of plants with the highest accumulation of flavonoids.     

苦草对砷的富集作用

为了探求合适的水体砷污染修复植物及砷在食物链中传递、累积的特点,以常见的沉水植物-苦草为研究对象,对受砷污染的水体进行修复,结果表明:苦草对水环境中砷的富集能在较短的时间内(3 d)达到一个较大值,到第14天,不同砷水平(2 mg/L)处理下的苦草对砷富集系数均超过200;苦草中砷浓度随处理时间及外源砷浓度的增加而增加,且与外源砷浓度之间存在极显著地正相关;苦草在不同浓度砷处理下都生长良好,对砷胁迫表现出较强的耐受性。因此,苦草对于水体的砷污染有着很好的去除效果,同时也能很好地反映出一个地区的砷污染水平。