Transfer of nitrate reductase genes of the cyanobacterium Nostoc muscorum into Rhizobium japonicum

Transformation of Rhizobium japonicum CB1809 was studied using DNA from the cyanobacterium Nostoc muscorum ATCC 27893. A spontaneous nitrate reductase deficient (Nar-) mutant (NR-6) of R. japonicum CB1809 was isolated with a frequency of 8.4 X 10(-7). Streptomycin (Sm) and Neomycin (Neo) resistance markers were introduced into strain NR-6, and the resulting strain was designated NR-6 SmR NeoR. Experiments with cyanobacterial DNA and live cells of strain NR-6 SmR NeoR indicated transformation of nitrate reductase (nar) genes of N. muscorum into this strain. This conclusion was supported by the reversion frequency of strain NR-6 SmR NeoR to Nar+ and the transformation frequency when recipient cells were exposed to N. muscorum DNA (with heat-treated DNA as control). Comparisons of growth, nitrate uptake, assimilatory nitrate reductase activity and nodulation of parent CB1809, NR-6 SmR NeoR and five transformant clones (Nar+) suggest that there may be considerable homology between the nar genes of R. japonicum CB1809 and N. muscorum.     

Isolation and characterization of chlorate-resistant mutants of the blue-green alga Nosoc muscorum.

Spontaneous chlorate-resistant (Clr) mutants of three classes were isolated from Nostoc muscorum under three different selective conditions. A Clr-N2 class of mutants lacked nitrate reductase and showed nitrate inhibition of nitrogen fixation. A Clr-NO3 group of het+ nif- mutants formed heterocysts, but lacked nitrogen fixation and active nitrogenase enzyme. The Clr-NO2 class included those mutants deficient in both active nitrogenase and nitrate reductase, as they were unable to grow at the expense of molecular nitrogen or with nitrate nitrogen. The results suggest a common genetic determinant of active nitrogenase and nitrate reductase in the blue-green alga N. muscorum.     

Actinorhodin production by Streptomyces coelicolor A3(2) in iron-restricted media

Production of the polyketide antibiotic actinorhodin by Streptomyces coelicolor A3(2) was investigated using a defined medium with or without iron supplementation. Iron limitation was found to enhance the intracellular production and export of the pigmented antibiotic. The effect of iron deficiency was particularly pronounced when the bacterium was grown with nitrate instead of ammonium. Analysis of the excreted pigment led to the identification of the lactone form of actinorhodin, gamma-actinorhodin.     

Global gene expression profiles of Bacillus subtilis grown under anaerobic conditions

Bacillus subtilis can grow under anaerobic conditions, either with nitrate or nitrite as the electron acceptor or by fermentation. A DNA microarray containing 4,020 genes from this organism was constructed to explore anaerobic gene expression patterns on a genomic scale. When mRNA levels of aerobic and anaerobic cultures during exponential growth were compared, several hundred genes were observed to be induced or repressed under anaerobic conditions. These genes are involved in a variety of cell functions, including carbon metabolism, electron transport, iron uptake, antibiotic production, and stress response. Among the highly induced genes are not only those responsible for nitrate respiration and fermentation but also those of unknown function. Certain groups of genes were specifically regulated during anaerobic growth on nitrite, while others were primarily affected during fermentative growth, indicating a complex regulatory circuitry of anaerobic metabolism.     

pH Changes Associated with Iron-Stress Response

When Fe-inefficient T3238fer and Fe-efficient T3238FER tomatoes were supplied iron, and nitrogen as nitrate, they increased the pH of the nutrient culture. When they were supplied nitrogen as ammonium, they decreased the pH. When Fe supply was limited, Fe-stress response developed in T3238FER that opposed the usual nitrate response and decreased, rather than increased, the pH. A “reductant” which reduced Fe³⁺ to Fe²⁺ was released from the roots of these plants and lowered the pH; and there was a tremendous increase in the uptake of Fe. T3238fer did not produce “reductant” in response to Fe-stress; the pH increased, and the plants developed Fe-deficiency when nitrogen was supplied as nitrate. Nitrogen nutrition and iron-stress response are important factors associated with iron chlorosis in plants. Release of hydrogen ions from roots of Fe-stressed plants is caused by more than response to imbalanced uptake of cations and anions.     

Cloning and sequencing of the pheU gene for tRNAPhe of Thermus thermophilus HB8, and genomic mapping of the pheU and pheST genes

Abstract The effect of the iron content of the medium on the yield of the peptide antibiotic microcin 25 was examined; synthesis was optimal in minimal media and was reduced by adding iron. Escherichia coli AY25, the wild-type producer of the antibiotic, showed a 95% decrease in microcin yield when grown in minimal medium containing 10 μM iron (high iron) as compared to 0.2 μM (low iron). Addition of chelators to Luria broth elicited microcin production, and there was a complete reversal of the effect of the chelators by adding iron. Studies with Escherichia coli mutants deficient in iron-regulated proteins ( fur ) suggested that factors other than Fur could mediate iron regulation of microsin synthesis.     

Evaluation of critical nutritional parameters and their significance in the production of rhamnolipid biosurfactants from Pseudomonas aeruginosa BS‐161R

Eleven biosurfactant producing bacteria were isolated from different petroleum‐contaminated soil and sludge samples. Among these 11 isolates, two were identified as promising, as they reduced the surface tension of culture medium to values below 27 mN m^?1. Besides biosurfactant production property, they exhibited good flocculating activity. Microbacterium sp. was identified as a new addition to the list of biosurfactant and bioflocculant‐producers. Optimization of various conditions for rhamnolipid production was carried out for one of the promising isolate, Pseudomonas aeruginosa BS‐161R. Bioglycerol (2.5%), as a cheap renewable carbon source, attained better rhamnolipid yield, while sodium nitrate appeared to be the preferable nitrogen source. The optimum carbon to nitrogen (C/N) and carbon to iron (C/Fe) ratios achieved were 15 and 28,350, respectively, which favored rhamnolipid production. Physical parameters like pH, temperature, and agitation speed also affected the production of rhamnolipids. Results from shake flask optimization indicated that the concentration of bioglycerol, sodium nitrate, and iron were the most significant factors affecting rhamnolipid production, which was supported by the results of central composite rotatable design. After optimization of the culture conditions, the production of rhamnolipids increased by ninefold from 0.369 to 3.312 g L^?1. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Anthracene biodegradation and surface activity by an iron-stimulated Pseudomonas sp

Iron may enhance polycyclic aromatic hydrocarbons (PAHs) degradation directly by increasing the activity of the enzymes involved in the aerobic biodegradation pathways for hydrocarbons, and indirectly by increasing the PAHs bioavailability due to the stimulation of biosurfactant production. In the present work, the PAH anthracene was used in order to study the effect of different forms and concentrations of iron on its biodegradation and surfactant production by Pseudomonas spp. isolates from a 14-years old petrochemical sludge landfarm site. Among the iron forms, iron nitrate was chosen based on its high solubility and effect on the increase in the growth of the isolate. Iron concentration of 0.1mM was selected as the limit between deficiency and toxicity for isolates growth and anthracene degradation. After 48 days Pseudomonas citronellolis isolate 222A degraded 72% of anthracene related to iron stimulation and surface tension decrease, indicating surfactant production. Pseudomonas aeruginosa isolate 332C was iron-stimulated but did not reduce surface tension while P. aeruginosa isolate 312A exhibited a noniron and surfactant dependence to degrade 72% of anthracene. Isolate 222A showed a direct dependence on iron to stimulate surfactant activity, which probably increased anthracene bioavailability. To our knowledge, this is the first report about the iron effect on anthracene degradation and surfactant production by a Pseudomonas sp. Based on the iron requirement and surfactant activity, the Pseudomonas isolates may be useful for bioremediation of PAHs.     

EPR spectra of photosystem I and other iron protein components in intact cells of cyanobacteria.

Electron paramagnetic resonance (EPR) spectra were recorded of whole filaments of the cyanobacteria Nostoc muscorum and Anabaena cylindrica. Signals due to manganese were removed by freezing and thawing the cells in EDTA. EPR spectra were assigned on the basis of their g values, linewidths, temperature dependence and response to dithionite and light treatments. The principal components identified were: (i) rhombic Fe3+ (signal at g = 4.3), probably a soluble storage form of iron; (ii) iron-sulfur centers A and B of Photosystem I; (iii) the photochemical electron acceptor 'X' of Photosystem I; this component was also observed for the first time in isolated heterocysts; (iv) soluble ferredoxin which was present at a concentration of 1 molecule per 140 +/- 20 chlorophyll molecules; (v) a membrane-bound iron-sulfur protein (g = 1.92). A signal g = 6 in the oxidized state was probably due to an unidentified heme compound. During deprivation of iron the rhombic Fe3+, centers A, B and X of Photosystem I, and soluble ferredoxin were all observed to decrease.     

基于响应面法对一株好氧反硝化菌脱氮效能优化

【目的】水体富营养化是当今我国水环境面临的重大水域环境问题,氮素超标排放是主要的引发因素之一。好氧反硝化菌构建同步硝化反硝化工艺比传统脱氮工艺优势更大。获得高效的好氧反硝化菌株并通过生长因子优化使脱氮效率达到最高。【方法】经过序批式生物反应器(Sequencing batch reactor,SBR)的定向驯化,筛选获得高效好氧反硝化菌株,采用响应面法优化好氧反硝化过程影响总氮去除效率的关键因子(碳氮、溶解氧、pH、温度)。【结果】从运行稳定的SBR反应器中定向筛选高效好氧反硝化菌株Pseudomonas T13,采用响应面法对碳氮比、pH和溶解氧关键因子综合优化获得在18 h内最高硝酸盐去除率95%,总氮去除率90%。该菌株的高效反硝化效果的适宜温度范围为25?30 °C;最适pH为中性偏碱;适宜的COD/NO3?-N为4:1以上;最佳溶解氧浓度在2.5 mg/L。【结论】从长期稳定运行的SBR反应器中筛选获得一株高效好氧反硝化菌Pseudomonas T13,硝酸盐还原酶比例占脱氮酶基因的30%以上,通过运行条件优化获得硝氮去除率达到90%以上,对强化废水脱氮工艺具有良好应用价值。     

Assessment of Salinity-Induced Antioxidative Defense System of Diazotrophic Cyanobacterium Nostoc muscorum

The present study examines the salinity-induced oxidative damage and differential response of enzymatic and non-enzymatic antioxidants of Nostoc muscorum. As compared to carotenoid content which showed induction the chlorophyll and phycocyanin contents were inhibited after salt stress. Acceleration of lipid peroxidation and peroxide production suggested onset of oxidative damage. The activities of all studied enzymatic antioxidants were significantly increased by salt stress with maximum induction of superoxide dismutase (154.8% at 200 mM NaCl treatment). Interestingly under severe stress condition (250 mM NaCl) ascorbate peroxidase seems to be more crucial than catalase for peroxide scavenging. Among the studied non-enzymatic antioxidants alpha-tocopherol was induced maximally (56.0%), however, ascorbate and reduced glutathione were increased by only 8.9% after 250 mM NaCl treatment as compared to control cells. Therefore, salinity was found to induce antioxidative defense system of N. muscorum.     

Medium optimization for antifungal active substances production from a newly isolated Paenibacillus sp. using response surface methodology

Statistics based experimental designs were used to optimize the medium for antifungal active substances production from a newly isolated Paenibacillus polymyxa Cp-S316 in shaker flask cultivation. The medium components having significant effect on the production were first identified using a fractional factorial design. Then steepest ascent method was employed to approach the experimental design space, followed by an application of response surface methodology for further optimization. A quadratic model was found to fit the antifungal active substances production. Response surface analysis revealed that the optimum values of the tested variables for the production of active substances were 12.3 (g/l) lactose, 17.5 (g/l) peptone, 0.4 (g/l) sodium nitrate, 4.5 (g/l) magnesium sulfate and 100 (g/l) potato. A production of 4687.71mug/ml, which was in agreement with the prediction, was observed in verification experiment. In comparison to the production of basal medium, 3.05-fold increase had been obtained.     

Inorganic nitrogen regulation of glutamate uptake in the cyanobacterium Nostoc muscorum

In Nostoc muscorum (Anabaena ATCC 27893) glutamate was not metabolised as a fixed nitrogen source, rather it functioned as an inhibitor of growth. The latter effect was nitrogen source specific and occurred in N₂-fixing cultures but not in cultures assimilating nitrate or ammonium. NO₃^--grown cultures lacked heterocysts and nitrogenase activity and showed a nearly 50% reduction in glutamate uptake rates, as well as in the final extent of glutamate taken up, compared to N₂-fixing or nitrogen-limited control cultures. NH₄⁺-grown cultures showed a similar response, except that the reduction in glutamate uptake rates and the final exten of glutamate taken up was over 80%. The present results suggest a relation between nitrate/ammounium nitrogen-dependent inhibition of glutamate uptake, probably via repression of the glutamate transport system, and glutamate toxicity.     

INFLUENCE OF IRON LIMITATION AND NITROGEN SOURCE ON GROWTH AND SIDEROPHORE PRODUCTION BY CYANOBACTERIA1

To characterize the mobilization and uptake of iron by cyanobacteria, 14 species were screened for ability to scavenge iron in a competitive system. The cyanobacteria exhibited a range of growth responses to iron limitation which could be separated into three groups, and a representative species from each group was chosen for further study. Effects of iron-limitation on growth and siderophore production of Anacystis nidulans R2, Anabaena variabilis ATCC 29413, and Plectonema boryanum UTEX 581 were determined. Both A. nidulans R2 and A. variabilis showed a reduced rate of growth with decreased available iron concentration (PFe 17–19). Growth rates increased with further reduction in the level of available iron (pFe 20 to pFe 21). The increase in growth rate occurred at the same available iron concentration as the initiation of extracellular siderophore production. In contrast, the growth of P. boryanum decreased with decreasing available iron levels. No siderophore production was detected from P. boryanum cultures. The growth kinetics of siderophore-producing species differ from traditional nutrient-limited growth kinetics and clearly reflect the presence of a high affinity, siderophore-mediated iron transport system in A. nidulans R2 and A. variabilis. Iron-limited growth kinetics more similar to traditional nutrient-limited growth kinetics were found in P. boryanum. The available nitrogen source influenced amount of siderophore produced and concentration of available iron which induced siderophore production. Siderophores were produced at high iron concentrations (pFe 18) when A. variablilis cultures were grown in the absence of combined nitrogen source. When nitrate was supplied to the culture, iron concentrations had to be reduced to pFe 20 before siderophores were produced. Cells grown on nitrogen also produced greater than two times the amount of siderophore compared with nitrate grown cells. This may be indicative of an increased demand for iron by nitrogen fixing A. variabilis Cultures.     

Process optimization for poly-beta-hydroxybutyrate production in a nitrogen fixing cyanobacterium, Nostoc muscorum using response surface methodology

A five-level-four-factor central composite rotary design was employed to find out the interactive effects of four variables, viz. concentrations of acetate, glucose and K2HPO4, and dark incubation period on poly-beta-hydroxybutyrate (PHB) production in a N2-fixing cyanobacterium, Nostoc muscorum. Acetate, glucose and dark incubation period exhibited positive impacts on PHB yield. Using response surface methodology (RSM), a second order polynomial equation was obtained by multiple regression analysis. A yield of 45.6% of dry cell weight (dcw) was achieved at reduced level of nutrients, i.e. 0.17% acetate, 0.16% glucose and 5 mg l(-1) K2HPO4 at a dark incubation period of 95 h as compared to 41.6% PHB yield in 0.4% acetate, 0.4% glucose and 40 mg l(-1) K2HPO4 at a dark incubation period of 168 h under single factor optimization strategy.     

Optimization of medium components for increased production of C-phycocyanin from Phormidium ceylanicum and its purification by single step process

Phycocyanin is a major protein produced by cyanobacteria, but very few phycocyanin-producing strains have been reported. In the present study, response surface methodology (RSM) involving a central composite design for four factors was successfully employed to optimize medium components for increased production of phycocyanin from Phormidium ceylanicum. The production of phycocyanin and interactions between sodium nitrate, calcium chloride, trace metal mix and citric acid stock were investigated and modeled. Under optimized condition P. ceylanicum was able to give 2.3-fold increase in phycocyanin production in comparison to commonly used BG 11 medium in 32 days. We have demonstrated the extraction, purification and characterization of C-phycocyanin using novel method based on filtration and single step chromatography. The protein was extracted by repeated freeze-thaw cycles and the crude extract was filtered and concentrated in stirred ultrafiltration cell (UFC). The UFC concentrate was then subjected to a single ion exchange chromatographic step. A purity ratio of 4.15 was achieved from a starting value of 1.05. The recovery efficiency of C-phycocyanin from crude extract was 63.50%. The purity was checked by electrophoresis and UV-Vis spectroscopy.     

Galactomyces geotrichum Y25产脂肪酶条件的优化

应用响应面法对Galactomyces geotrichumY25液体发酵产脂肪酶的条件进行了优化。首先采用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出黄豆粉、玉米浆和发酵时间3个对产酶影响显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面设计对显著因素进行优化,得出黄豆粉、玉米浆最佳质量分数分别为2.51%、2.12%,最佳发酵时间101.95 h。优化后液体发酵液中脂肪酶活力提高到34.65 U/mL,比初始酶活力9.6 U/mL提高了3.61倍。表明响应面法可显著优化Galactomyces geotrichumY25液体发酵产脂肪酶条件。     

Statistical optimization for tannase production from <Emphasis Type="Italic">Aspergillus niger</Emphasis> under submerged fermentation

Statistically based experimental design was employed for the optimization of fermentation conditions for maximum production of enzyme tannase from Aspergillus niger. Central composite rotatable design (CCRD) falling under response surface methodology (RSM) was used. Based on the results of ‘one-at-a-time’ approach in submerged fermentation, the most influencing factors for tannase production from A. niger were concentrations of tannic acid and sodium nitrate, agitation rate and incubation period. Hence, to achieve the maximum yield of tannase, interaction of these factors was studied at optimum production pH of 5.0 by RSM. The optimum values of parameters obtained through RSM were 5% tannic acid, 0.8% sodium nitrate, 5.0 pH, 5 × 10⁷ spores/50mL inoculum density, 150 rpm agitation and incubation period of 48 h which resulted in production of 19.7 UmL⁻¹ of the enzyme. This activity was almost double as compared to the amount obtained by ‘one-at-a-time’ approach (9.8 UmL⁻¹).     

应用响应面法优化发酵培养基提高达托霉素产量

[背景]达托霉素来自玫瑰孢链霉菌NRRL 11379的发酵产物,是重要的临床用抗生素.其原始产生菌发酵周期长,影响达托霉素的生产效率.本实验室前期在天蓝色链霉菌中重构了达托霉素的生物合成途径,有效地缩短了发酵周期,但重组菌株K10中达托霉素发酵产量很低,制约了后续的研究和开发.[目的]利用响应面法优化产达托霉素的重组菌...