Properties of partially purified photosynthetic reaction centers from scenedesmus mutant 6E and Anabaena variabilis grown in the presence of diphenylamine

When membrane fragments of Anabaena variabilis grown in the presence of diphenylamine (designated diphenylamine-Anabaena) are treated with Triton X-100 and subjected to sucrose density gradient centrifugation, a bluish-green membrane fragment enirched in P700 is obtained. This high-P700 fragment, denoted HP700, contains three P700 molecules per 100 chlorophyll a molecules and reduces NADP at a rate that is approximately nine times higher than that of HP700 fragments prepared from normally cultured Anabaena by the use of Triton X-100 following extraction with organic solvents. An HP700 fragment has also been isolated from a carotenoidless Scenedesmus mutant 6E, by the use of Triton X-100 and sucrose density gradient centrifugation.

Both HP700 fragments show the characteristic rapid absorbance changes of P700 upon illumination. The fluorescence properties of the HP700 fragments at −196° are different from those of the original membrane fragments. At −196° the long wavelength fluorescence peak is located at a shorter wavelength (724 mμ) in the diphenylamine-Anabaena HP700 fragment and is lower in intensity than that observed with the membrane fragment. Long wavelength fluorescence at −196° is low in the flurorescence spectra of the membrane fragments of Scenedesmus mutant 6E and is barely observable in the HP700 fragment. The fluorescence spectra of the HP700 fragments of both diphenylamine-Anabaena and Scenedesmus mutant 6E at −196° show a shoulder or peak at 700 nm. The data on fluorescence properties of the HP700 fragments suggest that 730 nm fluorescence does not originate from P700.     

A light-dependent oxygen-reducing system from Anabaena variabilis

Photosynthetic lamellae from Anabaena variabilis catalyze a vigorous photoreduction of O₂ to H₂O₂. The electrons come to O₂ from an artificial donor, reduced 2,3′,6-trichlorophenolindophenol, and the reaction does not require an exogenous autoxidizable substance. Evidence is presented to show that reduced 2,3′,6-trichlorophenolindophenol can donate electrons at two distinct sites. Photoreduction of O₂ is inhibited by antibodies which block the function of the ferredoxin-reducing substance. The O₂-reducing system may result from the formation of a reduced photoproduct which is more accessible to autoxidation than is the analogous product formed in higher plant chloroplasts.     

Isolation and properties of plastocyanin from Anabaena variabilis

Plastocyanin is a copper protein found in photosynethetic tissue and it exhibits the properties of a physiological redox reagent. This protein has been purified from the blue-green alga Anabaena variabilis. Plastocyanin is required for a number of partial reactions of the photosynthetic electron transfer chain. These reactions include the transfer of electrons from reduced 2,3′,6-trichlorophenolindophenol,N,N,N′,N′- tetramethyl-p-phenylenediamine and 2,3,5,6-tetramethyl-p-phenylenediamine to low potential oxidants. Reduced cytochrome c photooxidation does not appear to be dependent on plastocyanin. Cytochrome f, isolated from this alga, will partially replace plastocyanin in many of these reations. Inhibition of photosynthetic reactions by copper chelators appears to occur at some site other than the site of plastocyanin function.     

Reconstitution of electrogenic function in isolated pigment-protein complexes of Anabaena variabilis

Treatment of Anabaena variabilis membranes with lauryldimethylamine N-oxide yielded two fractions of pigment-protein complexes which were separable by gel filtration on Sepharose 6B. A green fraction was characterized which had a maximum of the chlorophyll long-wave absorption band at 678 nm and a small amount of carotenoid. In this fraction, Photosystem I activity was higher than in another (brownish-green) fraction which had a maximum of the chlorophyll absorption band at 673 nm and which was enriched in carotenoids. Similarly to isolated membranes, proteoliposomes containing pigment-protein complexes took up tetraphenylborate anions and tetraphenylphosphonium cations and were found to be capable of light-dependent membrane potential generation, when associated with a planar phospholipid membrane in the presence of reduced phenazine methosulfate upon illumination. The spatial arrangement of the pigment-protein complexes in the native and artificial membranes is discussed.     

Seasonal and interannual dynamics of polyphenols in Myriophyllum verticillatum and their allelopathic activity on Anabaena variabilis

In 4 successive years, we investigated the seasonal and interannual variability of the total polyphenolic pool and of the individual polyphenolic compounds in Myriophyllum verticillatum, as well as their allelopathic activity in a small eutrophic lake. We tested whether nutrient availability explained interannual and seasonal changes in the production of polyphenols. There were no strong interannual variations in plant tissue carbon, nitrogen and phosphorus concentrations, while total phenolic compounds (TPC) significantly differed between years, especially in apical meristems (range: 38–122 mg g⁻¹  dry weight (DW)). Seasonal patterns, with maxima between May and July, changed between years. Partially confirming the carbon-nutrient balance hypothesis sensu Bryant et al. [Bryant, J.P., Chapin III, F.S., Klein, D.R., 1983. Carbon/nutrient balance of boreal plants in relation to vertebrate herbivory. Oikos 40, 357–368], we found correlations between TPC and the C/N (carbon/nitrogen) ratio in some but not all years, especially in apical meristems. Plant tissue phosphorus content accounted also for the variability in TPC in some years. Crude extracts of apical meristems always inhibited the growth of Anabaena variabilis, used as a target cyanobacterium. Plant TPC concentration and allelopathic activity were significantly correlated in all years except in 2005. Bioassay-directed fractionation of M. verticillatum extracts coupled with LC–MS analyses of the respective fractions revealed several isomers of HHDP-di- and -tri-galloylglucose apparently responsible for the allelopathic effects. The individual active compounds revealed a more distinct seasonal pattern compared to the pool of phenolic compounds in M. verticillatum, with a clear maximum in May, the ecologically most relevant period for inhibitory effects of submerged macrophytes on phytoplankton.     

Increased rate of cyclic photophophorylation in preparations from Anabaena variabilis cells grown in the presence of diphenylamine

Cell free homogenates and membrane fractions prepared from Anabaena variabilis cells grown in the presence of diphenylamine have markedly higher activities for cyclic phosphorylation than similar preparations from normal cells. The preparations from diphenylamine-grown cells are also more active in system I mediated electron transport from reduced dichloroindophenol to oxygen or methyl viologen. The light intensity required to saturate phenazine methosulphate-supported cyclic phosphorylation, in such preparations, is higher than for preparations for normal cells.     

Photostimulation of nitrogen fixation in Anabaena cylindrica

Photostimulation of N₂ fixation in the blue-green alga Anabaena cylindrica was investigated using monochromatic light and by comparing action spectra of C₂H₂ reduction with that of photosynthetic O₂ evolution. Maximum nitrogenase activity per unit energy was measured at a wavelength which corresponds to the maximum light absorption of chlorophyll a. The action spectrum of C₂H₂ reduction indicates a primary involvement of Photosystem I in N₂ fixation by blue-green algae.     

Identification and initial utilization of a portion of the smaller plasmid of Anabaena variabilis ATCC 29413 capable of replication in Anabaena sp. strain M-131

Summary Anabaena variabilis ATCC 29413 contains two cryptic plasmids. Clones of the smaller (41 kb) plasmid, designated pRDS1, in cosmid vectors were used to construct a physical map. A clone bank of pRDS1 constructed by ligating fragments from aXhoII digest of a pRDS1 cosmid clone into a mobilizable plasmid was used to locate an origin of replication of pRDS1. Because we were unable to cureA. variabilis of pRDS1, the clone bank was transferred by conjugation to another strain ofAnabaena sp., strain M-131. A 5.3 kb fragment of pRDS1 contained all of the sequences necessary for replication inAnabaena sp. strain M-131 as judged by the ability to rescue the hybrid vector from exconjugants in unchanged form after many generations. Hybrid plasmids derived from pRDS1, one bearing genes for luciferase, were also transferred by conjugation toA. variabilis, where they appeared to recombine with pRDS1.     

High expression of Trigonopsis variabilis -amino acid oxidase in Pichia pastoris

To explore a new approach of high expression of -amino acid oxidase (DAAO) in Pichia pastoris, a gene encoding DAAO from Trigonopsis variabilis (TvDAAO gene) deleted intron was prepared by PCR amplification and cloned into the intracellular expression vector pPIC3.5K. The expression plasmid pPIC3.5K-DAAO linearized by SalI was transformed into Pichia pastoris strain GS115 (his⁻mut⁺). By means of MM and MD plates and PCR, the recombinant P. pastoris strains (his⁺mut⁺) were obtained. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant strain PD27 with the highest expression of DAAO was screened through activity assay and its high-density fermentation was carried out in a 1-l fermentor. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant cells with high expression of DAAO were screened and the high-density fermentation was carried out in a 1-l fermentor. Interestingly, the DAAO expression level reached up to 473 U/g dry cell weight in fermentation yield. Finally, 1-hexanol was used to break recombinant cells and the specific activity of DAAO was 1.46 U/mg protein in crude extraction.     

Ammonium excretion by a mutant of the nitrogen-fixing cyanobacterium Anabaena siamensis

Anabaena siamensis isolated from rice fields in Thailand is a fast growing cyanobacterium with a high nitrogen-fixing activity. Mutant strains resistant to the l-glutamate analogue, l-methionine sulfoximine (MSX) were isolated by ethyl methanesulfonate mutagenesis. A stable mutant named A. siamensis SS1, which released ammonium to the medium, was studied further. In batch cultures the rate of ammonium production peaked at the early log phase and gradually decreased until the 4th day of growth when the cultures reached a density of 90 μg chl ml⁻¹. To obtain constant release of ammonium by SS1, continuous culture experiments were performed at a cell density of 5 μg chl ml⁻¹ and the following results were obtained: (1) growth rate as the parent (μ:0·123 h⁻¹) in the presence and absence of 500 μm MSX; (2) 48% GS transferase activity when compared with the parent; (3) ammonium excretion at a rate of 8 μmol (mg chl)⁻¹ h⁻¹ as measured up to 20 generations (120 h); (4) depressed nitrogenase activity; and (5) 30% higher nitrogenase activity than that of the parent. SS1 immobilized in alginate beads (5 μg chl ml⁻¹) exhibited values of glutamine synthetase and nitrogenase activity similar to those of free cells. However, ammonium excretion at the rate of 11·61 μmol (mg chl)⁻¹ h⁻¹ was obtained only up to 20 h after loading in bioreactors, due to the fast growth of SS1 as also occurred in batch cultures.     

Nutritional control of the frequency of MNNG-induced true branching habit in Anabaena doliolum

Summary MNNG survival and mutagenesis were studied in Anabaena doliolum Bharadwaja. The survival curves of germinating spores were exponential while those of resting spores and filament-fragments were sigmoidal. Blue mutants with much higher phycocyanin contents than the parent were recovered in varying frequencies; the frequency (1.1%) was the highest with 2 mg/ml MNNG for 15 min. Some of the blue mutants sporulated normally; while others did not. A nonsporulating blue mutant (M16) showed branched filaments in liquid cultures. In nitrogen free medium. M16 had a high frequency (70%) of branched filaments during the early phase of growth. In some filaments, the branch arose from a heterocyst showing three polar nodules. There was no other difference between the parent and the mutant in cell morphology and cellular differentiation. High light intensity adversely affected phycocyanin and chlorophyll a pigments and nitrogen fixation in the mutant. Frequency of mutant branched character appears to beta factor of inorganic nitrogen nutrition.Dedicated to the memory of my revered teacher the late Prof. R. N. Singh     

Proton-ATPase activity in cells of lactobacilli grown in the presence of propionate

Lactobacillus helveticus ATCC 15009 and CRL 581, and Lact. casei LC3 were grown in a complex medium with and without 15 mmol 1^-1 of neutralized propionic acid and assayed for proton-translocating ATPase activity. The enzyme activity was higher when the medium contained fatty acid than in its absence for all strains studied. Characteristics of this increased ATPase were identical to those of the enzyme located on the membrane of normal cells. The substrate consumption rate of resting cells was increased by propionate. This effect was reverted by the specific H⁺-ATPase inhibitor N,N '-dicyclohexylcarbodiimide indicating that the increment of fermentative activity was related to the H⁺-ATPase activity. These results suggest that the amplification of H⁺-ATPase activity could be involved in the inhibition of lactobacilli growth in cultures where propionic acid is unavoidably present, such as some mixed cultures with propionibacteria.     

Polyethyleneimine-induced flocculation and flotation of cyanobacterium Anabaena flos-aquae for gas vesicle production

Gas vesicles are hollow, proteinaceous structures found in some strains of cyanobacteria. They have been used to increase the oxygen supply and improve the cultivation of shear-sensitive mammalian cells. However, the production and, especially, collection of cells and gas vesicles were laborious and ineffective. In this study we examined the use of the cationic polymer, polyethyleneimine (PEI), for improving the cell harvesting by flocculation and flotation. PEI was examined to determine the appropriate molecular weight, pH range, and dosage. The dose of 20–30 mg/l of PEI with molecular weight of 25,000 in the pH range of 6.0–8.5 was found to provide effective and efficient cell flocculation and flotation. As the PEI dose increased, the rate of flotation increased but the clearance (collection) efficacy declined slightly. The culture samples used in this study were taken from light-limiting continuous culture systems at different dilution rates (0.05–0.24 h⁻¹). Without PEI addition, the cells at dilution rates lower than 0.1 h did not float while those at higher dilution rates would float slowly. With PEI addition, the flocculated cells at the dilution rate of 0.05 h⁻¹ sank and those of higher dilution rates would float and the flotation rate increased with increasing specific growth rate. Nonetheless, PEI flocculation and flotation (or sedimentation) could be used to harvest cells at a wide range of growth states.     

不同生境对栓皮栎幼苗光合生理特性的影响

以栓皮栎天然分布的北界(北京,NP)、中心(陕西,CP)和南界(云南,SP)3个种源的实生苗为试验材料,通过在北缘(北京)和南缘(云南)的交互移植试验,探讨不同种源幼苗光合生理性状的差异及其来源。结果表明:(1)各种源在北缘生境下的潜在最大净光合速率(Pmax)、光饱和点(LSP)、羧化效率(CE)、光呼吸速率(Rp)、光化学猝灭系数(PQ)均显著高于南缘生境(P0.05);(2)南界种源(SP)则具有更高的CE、Rp、PSⅡ原初光能转换效率(F_v/F_m)和PSⅡ潜在活性(F_v/F_o)(SPCPNP);北界种源(NP)具有更高的非光化学猝灭系数(NPQ)(NPCPSP);(3)生境和种源的交互作用对最大羧化速率(A_(max))和LSP影响显著,Amax在南北缘生境下均以当地种源的最高;而LSP在北缘生境下以中心种源(CP)最高,在南缘生境下以北界种源(NP)最高。不同生境对表观量子效率(AQY)、CO2补偿点(CCP)、暗呼吸速率(Rd)以及叶绿素荧光参数如F_v/F_m、F_v/F_o以及NPQ影响不显著,其中AQY和Rd不受生境变化及其与种源的交互影响,这可能与栓皮栎自身遗传因素有关;(4)生长表现方面,栓皮栎幼苗在南、北不同生境下生长表现出明显差异,各种源在北缘生境下生长状况均明显优于南缘生境,且均以南界种源(SP)生长优势明显。     

栓皮栎幼苗对土壤干旱胁迫的生理响应

以栓皮栎一年生盆栽苗为实验材料,采用称重控水的方法,设置不同土壤水分胁迫梯度,系统分析其幼苗在不同干旱胁迫条件下的生理生化响应特征,以探索栓皮栎耐旱特性.结果显示:(1)栓皮栎幼苗叶片中3种保护酶(SOD、POD、CAT)活性在对照(CK,土壤相对含水量19.5％～21.5％)条件下保持稳定,而中度干旱(T2,9.5％～11.5％)和重度干旱(T3,5.5％～7.5％)条件下,随着胁迫时间的延长呈先增高后降低的趋势,且变化的幅度在不同胁迫强度下存在差异.(2)在整个干旱胁迫过程中,各胁迫处理叶片丙二醛(MDA)含量均呈上升趋势,不同胁迫强度的变化幅度不同;叶片中的可溶性蛋白含量和根系活力随着干旱胁迫程度的增强呈先增高后降低的趋势.(3)栓皮栎幼苗叶片的脯氨酸含量随着干旱胁迫时间的延长表现出先增加后降低的趋势;叶片叶绿素a、叶绿素b、总叶绿素含量以及叶绿素a/b值均呈逐渐降低的趋势.研究表明,栓皮栎幼苗在短期和轻度干旱胁迫下通过提高自身的保护酶活性、增加可溶性蛋白和脯氨酸含量、提高根系活力等来抵御干旱环境的伤害,从而表现出较强的耐旱特性;而在重度干旱胁迫条件下,栓皮栎幼苗自我调节能力丧失,体内代谢紊乱,导致保护酶活性、可溶性蛋白、脯氨酸含量和根系活力等下降,从而受到干旱伤害.     

Binding of metals by cell walls of Cunninghamella blakesleeana grown in the presence of copper or cobalt

Summary The binding of metals by cell walls isolated from Cunninghamella blakesleeana grown in the presence of inhibitory concentration of Cu or Co and which had altered chemical compositions was compared with the binding by control cell walls. The Co-cell walls, which had higher contents of phosphate and chitosan, bound more Cu and Co. Although the V _max for Cu and Co differed with each of the cell walls, the K _m values for the binding of Cu (6.3x10^-3 M) and Co (2.1x10^-3 M) were the same for all three types of cell walls. The cell walls also differed in their quantitative binding of various metals; control cell walls: Zn> Fe> Mn> Cd> Ca> Ni> Cu> Ag> Co> Mg; Cu-cellwalls: Zn> Fe> Mn> Cu> Ni> Cd> Ag> Ca> Co> Mg; and Co-cell walls: Fe> Zn> Cu> Mn> Cd> Ag> Ca> Ni=Co> Mg. The binding of Cu was temperature-dependent and had an optimum pH. The binding of Co was inhibited by Cu, but the binding of Cu was not inhibited by Co, and Cd totally suppressed the binding of Co but not of Cu, suggesting two binding sites on the cell walls, one exclusively for Cu and the other common to both metals but with a higher affinity for Co. The cell walls did not bind Mg. The Cu-or Co-loaded cell walls eluted with 5 mM ethylenediaminetetraacetate (EDTA) rebound these metals to the same or greater extent as the original walls, but walls eluted with 0.5 N HCl bound only 50% of that bound originally.     

Cohesive properties of axenically grown cells of the slime mould Dictyostelium discoideum

It has been found that Dictyostelium discoideum cells from the exponential growth phase of axenically grown cultures are cohesive, whereas those from stationary phase are not. These differences in cohesiveness are seen in phosphate buffer and in axenic medium. Stationary phase medium inhibits the aggregation of log phase cells; stationary phase cells inoculated into freshly prepared medium regain their cohesiveness. Stationary phase medium may contain an inhibitor of cell cohesion. pH differences between the two types of medium are not entirely responsible for loss of cohesiveness.