Light-harvesting adaptations of planktonic phototrophic micro-organisms to different light quality conditions

The effects of light spectral distribution on the composition of phototrophic microbial communities were analyzed in three metalimnetic levels (relative depth positions) of 41 lakes. Principal Component Analysis was used to compare light quality conditions reaching the populations of phototrophic micro-organisms containing different photosynthetic pigments. Results allowed to identify the optimal light quality conditions for the selection of each microbial group at their respective levels. Two general light-harvesting adaptations were defined, according to the wavebands that could be related to the selection of these microbial groups. The micro-organisms adapted to use red and near-infrared light – eukaryotic phytoplankton, Chloronema spp. and green-coloured Chlorobiaceae – predominated at shallow depths (specially in waters containing high gilvin contents) using their respective Q_y absorption bands. The micro-organisms adapted to green-yellow light – phycoerythrin-containing cyanobacteria, Chromatiaceae and brown-coloured Chlorobiaceae – were dominant in deep metalimnetic communities. Laboratory experiments with cultures of Chlorobium limicola and C. phaeobacteroides growing under different light quality conditions showed that the green-coloured species had higher photosynthetic activity under red light, while the brown-coloured species was more active under green light. These results demonstrated that physiological differences between micro-organisms with different light-harvesting adaptations are responsible of their selection under different light quality conditions. This selection is experimented by Chlorobiaceae (as it was previously indicated by other investigators) at the deepest positions of the metalimnetic communities (level 3), but also by Chromatiaceae and Chloronema spp. at level 2 and by the eukaryotic phytoplankton and cyanobacteria at level 1.     

沼泽红假单胞菌光合色素的分离、组成分析与光稳定性

【目的】探求光对不产氧光合细菌类胡萝卜素(Car)和细菌叶绿素a(BChl a)稳定性的影响规律。【方法】以沼泽红假单胞菌CQV97为材料,采用硅胶柱层析和HPLC方法进行Car和BChl a组分的纯化和成分分析,采用吸收光谱法研究Car和BChl a组分的光稳定性。【结果】在Car和BChl a组分分离过程中,Car组分回收率高且稳定,而BChl a回收率波动性较大。Car组分中含有6种螺菌黄质系Car和极少量(0.25%)的细菌脱镁叶绿素a。BChl a组分中包含BChl aGG、BChl aDHGG、BChl aTHGG和BChl aP4种成分。Car和BChl a组分在黑暗条件下非常稳定。2 000 lx白炽灯、日光灯和自然光照射时,Car在70 min内非常稳定,但对紫外光敏感,半衰期为11.15 min,BChl a组分对白炽灯、日光灯、自然光和紫外灯的光降解速率常数(min–1)分别为0.169 8、0.028 9、0.213 9和0.026 4,半衰期(min)分别为4.47、29.68、4.20和26.19。【结论】一步硅胶柱层析可同时得到Car和BChl a纯组分。Car对白光相对稳定,对紫外光不稳定。BChl光稳定性很差,分离过程中短期见光是导致BChl a回收率波动性较大的原因,光降解过程中产生了相对稳定的中间产物。该研究结果为光合色素的精制、功能研究和应用提供了理论依据。     

Separation of asymmetrical hybrid hemoglobins by anaerobic cation-exchange high-performance liquid chromatography

Asymmetrical hybrid hemoglobins formed from mixtures of two structurally different hemoglobins were found to be readily separated by cation-exchange high-performance liquid chromatography under anaerobic conditions. When oxyhemoglobins A and S were mixed and deoxygenated, the resulting HPLC chromatogram showed three peaks. The distribution of the three components follow the binomial expansion a² + 2 ab + b² = 1, where a and b are the initial fractions of parent hemoglobins. The middle peak was collected in a test tube saturated with CO gas and reanalyzed under the same experimental conditions. This middle component gave two peaks of equal areas with retention times identical to those of the CO-form of the parent hemoglobins without the appearance of the hybrid hemoglobin band. No intermediate peak was observed in solutions of mixtures of liganded hemoglobins under aerobic conditions. Hybrid hemoglobins AC and SC were also formed when oxyhemoglobins A and C, S and C were mixed, respectively. The separation and the identification of hemoglobins and hybrid hemoglobin employing cation-exchange HPLC can be achieved within 30 min by gradient elution. In addition, the ability to isolate hybrid hemoglobins may be a valuable tool for the study of physical and chemical properties of hybrid hemoglobins.     

Phototrophic growth and chlorosome formation in Chloroflexus aurantiacus under conditions of carotenoid deficiency

Chloroflexus aurantiacus was grown phototrophically in the presence of 60 µg of 2-hydroxybiphenyl (HBP) per ml, which inhibited the formation of colored carotenoids up to about 90%, while bacteriochlorophyll (BChl) c formation and phototrophic growth were not significantly influenced. When the HBP concentration was increased, the latter two processes became inhibited, yet, no further inhibition of carotenoid formation was possible. The typical shape of chlorosomes was not affected after growth in the presence of 60 µg of HBP per ml. However, the chlorosome dimensions were slightly decreased. Chlorosome preparations from carotenoid-deficient cultures lacked significant amounts of colored carotenoids and exhibited lower buoyant densities than chlorosomes from uninhibited control cultures. As compared on a BChl c basis, the relative contents of the two larger chlorosomal polypeptides of 10.8 and 15.5 kDa (apparent M_r= 11000 and 18000) were not affected by HBP, while formation of the 5.7 kDa (M_r= 3700) polypeptide was inhibited by 50%. The data suggest that, in C. aurantiacus, colored carotenoids are largely dispensable for phototrophic growth and chlorosome formation. Moreover, chlorosome formation and BChl c incorporation into chlorosomes in the absence of about half of the regular amount of the 5.7 kDa polypeptide excludes a constant relationship between these parameters.     

Separation of polyprenol and dolichol by monolithic silica capillary column chromatography

We attempted an analysis of naturally occurring polyprenol and dolichol using a monolithic silica capillary column in HPLC. First, the separation of the polyprenol mixture alone was performed using a 250 x 0.2 mm inner diameter (ID) octadecylsilyl (ODS)-monolithic silica capillary column. The resolution of the separation between octadecaprenol (prenol 18) and nonadecaprenol (prenol 19) exceeded by >or=2-fold the level recorded when using a conventional ODS-silica particle-packed column (250 x 4.6 mm ID) under the same elution conditions. Next, the mixture of the prenol type (polyprenol) and dolichol type (dihydropolyprenol) was subjected to this capillary HPLC system, and the separation of each homolog was successfully achieved. During the analysis of polyprenol fraction derived from Eucommia ulmoides leaves, dolichols were found as a single peak, including all-trans-polyprenol and cis-polyprenol previously identified. This sensitive high-resolution system is very useful for the analysis of compounds that are structurally close to polyprenols and dolichols and that have a low content.     

峡谷型喀斯特不同生态系统的土壤微生物数量及生物量特征

采用样地调查与室内分析相结合的方法,研究了峡谷型喀斯特水田、旱地、草地、灌丛、人工林、次生林6种生态系统不同深度土壤微生物数量、微生物生物量特征及其分形关系。结果表明:峡谷型喀斯特不同生态系统的土壤微生物数量及组成不同,微生物数量均以次生林最高,旱地最低,其组成数量均为细菌放线菌真菌,细菌是土壤微生物的主要类群,数量多达26.66×105—71.64×105cfu/g,占全部微生物比例为87.00%—95.50%,其次为放线菌数量,为1.45×105—3.78×105cfu/g,所占比例为4.21%—12.39%,真菌数量最小,为0.07×105—0.23×105cfu/g,所占比例仅为0.24%—0.61%,不足1%。不同生态系统土壤微生物生物量碳(MBC)、氮(MBN)、磷(MBP)的含量不同,次生林MBC与MBN最高,人工林MBP最高,旱地MBC最低,草地MBN与MBP最低;各生态系统均为MBCMBNMBP。不同生态系统的MBC/SOC、MBN/TN、MBP/TP分别为0.44%—0.97%、2.13%—3.13%、1.46%—2.13%,差异不显著;MBC/MBN在3.06—6.54之间,其中次生林极显著高于其他生态系统,其他生态系统差异不显著。不同生态系统土壤微生物数量及生物量均随土层加深而减少,且具有良好分形关系,均达到了极显著水平(P0.01)。探讨土壤微生物活性为提高石灰土土壤肥力、促进喀斯特植被迅速恢复提供依据。     

Superior resolution of gamma-crystallins from microdissected eye lens by cation-exchange high-performance liquid chromatography

A novel procedure is presented for the rapid quantitative analysis of eye lens gamma-crystallins and beta s-crystallin by ion-exchange high-performance liquid chromatography on Synchropak CM300. At least six different gamma-crystallin gene products can be resolved from the soluble fraction of calf lens extract. This method is applicable to the analysis of microsections from individual lenses, and can be used to rapidly characterize spatial variations in gamma-crystallin composition which occur with aging and cataractogenesis.     

Characteristics and role of the exocellular polysaccharides produced by five cyanobacteria isolated from phototrophic biofilms growing on stone monuments

Three coccoid and two filamentous cyanobacterial strains were isolated from phototrophic biofilms exposed to intense solar radiation on lithic surfaces of the Parasurameswar Temple and Khandagiri caves, located in Orissa State, India. Based on to their morphological features, the three coccoid strains were assigned to the genera Gloeocapsosis and Gloeocapsa, while the two filamentous strains were assigned to the genera Leptolyngbya and Plectonema. Eleven to 12 neutral and acidic sugars were detected in the slime secreted by the five strains. The secretions showed a high affinity for bivalent metal cations, suggesting their ability to actively contribute to weakening the mineral substrata. The secretion of protective pigments in the polysaccharide layers, namely mycosporine amino acid-like substances (MAAs) and scytonemins, under exposure to UV radiation showed how the acclimation response contributes to the persistence of cyanobacteria on exposed lithoid surfaces in tropical areas.     

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF PHYTOPLANKTON PIGMENTS USING A POLYMERIC REVERSED-PHASE C18 COLUMN1

A high-performance liquid chromatographic (HPLC) method is described that allows improved resolution of several chemotaxonomically significant phytoplankton pigments. The protocol, which employs two pumps and a modified Mantoura and Llewellyn (1983) solvent system, can be easily adapted for many HPLC systems currently in use. The most unique aspect of the method is the use of a polymeric C₁₈ reversed phase HPLC column (VYDAC 201TP). In comparison to the monomeric C₁₈ columns typically used in the characterization of phytoplankton pigments, polymeric C₁₈ columns offer superior selectivity for structurally similar compounds. The protocol was evaluated for the ability to resolve most of the phytoplankton pigments of diagnostic importance using algal cultures from nine classes. Pigment pairs that were resolved by the method include a) lutein and zeaxanthin, b) neoxanthin and 19′-hexanoyloxyfucoxanthin, and c) α-carotene and β-carotene, and partial resolution of chlorophyll c₁ and chlorophyll c₂.     

Spectrochromatography of photosynthetic pigments as a fingerprinting technique for microbial phototrophs

Abstract: The combination of chromatographic separation using reverse-phase high-performance liquid chromatography and continuous monitoring of the column eluate using a diode-array spectrophotometer allowed qualitative and quantitative pigment profiling of extracts of photosynthetic material in a single run. Carotenoids and the spectrally distinct types of chlorophyll and bacteriochlorophyll can be unambiguously identified even when imperfectly separated on the column. The resulting spectrochromatogram is a fingerprint useful for the rapid characterization of pure cultures or mixed populations. We have developed software to allow recording, manipulation, and presentation of the resulting spectrochromatograms and present results from photosynthetic microbes in pure and mixed cultures. We describe a number of approaches to the presentation of the resulting data in a readily comprehensible form.     

Intermediary purine-metabolizing enzymes from the cytosol of Dictyostelium discoideum monitored by high-performance liquid chromatography

The use of high-performance liquid chromatography to identify and quantitate five purine-metabolizing enzymes from a partially purified subcellular fraction of the eucaryotic microorganism Dictyostelium discoideum is described. All HPLC separations were carried out in an isocratic manner using reverse-phase C18 as the stationary phase. The mobile phase consisted of a phosphate buffer with either methanol or acetonitrile as cosolvent, and optimal separation conditions were attained by varying the organic concentration or the pH of the buffer or by employing paired-ion chromatographic techniques. Substrates and products were detected at either 254 nm for the purines or 295 nm for the formycin analogs. An adenosine kinase activity was identified, and it was demonstrated that formycin A (FoA) could be substituted for adenosine as the phosphate acceptor, yielding FoAMP as the product. With FoA as the substrate an apparent Km of 18.2 microM and an apparent Vmax of 32.4 mmol min-1 mg-1 were observed for the activity. A purine-nucleoside phosphorylase activity was found to cleave adenosine to adenine and ribosylphosphate. FoA was not found to be a substrate for this activity due to the unusual formycin C-glycosyl bond which was not hydrolyzed by enzymes or chemically with either HCl or NaOH. An adenylate deaminase activity was found to be present in the cytosolic S-100 of cells harvested during the onset of development, and this deaminase activity was greatly stimulated by ATP. With FoAMP as the substrate, an apparent Km of 236 microM and Vmax of 2.78 mumol min-1 mg-1 were observed. The deamination of FoAMP could be inhibited by the addition of the natural substrate AMP. An apparent Ki value of 136 microM was determined from initial rate data. An adenylosuccinate synthetase activity was observed to have a Km value for GTP, IMP, and aspartic acid of 23, 34, and 714 microM, respectively. The formycin analog FoIMP was not a substrate with this activity but was a competitive inhibitor of IMP. Finally hypoxanthine-guanine phosphoribosyltransferase was found to have Km and Vmax values for hypoxanthine of 55.5 microM and 34.3 nmol-1 min-1 mg-1. When guanine was used as the substrate, the rate of nucleotide formation was 50% that with hypoxanthine as the substrate. The advantages of using HPLC to examine the interconnecting activities of a multienzyme complex in subcellular fractions are discussed, including the increased sensitivity obtained by using formycin analogs in the assay procedures.(ABSTRACT TRUNCATED AT 400 WORDS)     

玛珥湖好氧不产氧光合细菌pufM基因DNA和mRNA的定量及多样性分析

【目的】湖光岩玛珥湖是一类特殊的火山口湖,它完全封闭,地质年代久远,尚未受人类活动的剧烈影响,孕育着丰富而特殊的微生物种群。好氧不产氧光合细菌(AAPB)是以其在有氧情况下能行使光合功能而定义的一类专性异养细菌,其生理生态特征独特,进化年代久远,在水生生态系统的上层水体中广泛分布。目前,AAPB在玛珥湖水体中是否有分布仍是未知。【方法】构建和比较夏季湖水1 m、5 m、12 m三个水层的总DNA和总RNA的AAPB光合中心合成的关键基因pufM的6个克隆文库,并结合定量PCR技术,分析了不同水层AAPB的分布、系统发育多样性及其在总细菌中的比重。【结果】6个文库覆盖率和稀释曲线显示样本初步揭示了各水层优势AAPB类群的多样性。BLAST核苷酸同源性介于80%93%;多样性指数表明,湖光岩表层和底层多样性相当,中间层最低,总RNA的多样性高于总DNA。系统发育分析结果表明,OTU21 24所含的序列(占总序列的49.43%)与β-变形细菌的进化距离最接近,是湖光岩玛珥湖的优势AAPB菌群。定量PCR结果显示1 m水层中AAPB在总细菌中的比重最高,可达38.06%;而5 m水层中AAPB所占的比重最低,仅为0.85%;12 m为9.54%。【结论】湖光岩玛珥湖孕育着丰富而多样的AAPB类群。     

Purification and characterization of reaction centers from the obligate aerobic phototrophic bacteria Erythrobacter litoralis,Erythromonas ursincola and Sandaracinobacter sibiricus

Reaction centers (RC) from the species Erythrobacter (Eb.) litoralis, Erythromonas (Em.) ursincola and Sandaracinobacter (S.) sibiricus have been purified by LDAO treatment of light-harvesting-reaction center complexes and DEAE chromatography. The content and overall organisation of the RCs' chromophores, determined by linear dichroism (LD) and absorption spectroscopy, are similar to those isolated from anaerobic photosynthetic bacteria. The redox properties of the primary electron donor are pH-independent and very similar to those determined for anaerobic photosynthetic bacteria with midpoint potential values equal to 445 (± 10), 475 and 510 mV for Eb. litoralis, S. sibiricus and Em. ursincola, respectively. The RC purified from Eb. litoralis does not contain bound cytochrome (cyt), whereas RCs isolated from S. sibiricus and Em. ursincola possess a tetraheme cyt c. Each of these tetraheme cyts contains two high potential hemes and two low potential hemes. Their redox properties are very similar, with midpoint potentials equal to 385 (± 10), 305, 40, -40 mV for Em. ursincola and 355, 285, 30, -48 mV for S. sibiricus. At physiological pH, the midpoint potential of the primary electron acceptor (Q_A) varies with a slope of -60 mV/pH unit. The reduced form of Q_A presents pK values of 9, 9.8, 10.5 for S. sibiricus, Em. ursincola and Eb. litoralis, respectively. The main difference observed between RCs isolated from anaerobic photosynthetic and from obligate aerobic bacteria is the _Emvalues of Q_A which are 65 to 120 mV higher in the last case. This difference is proposed to be a major reason for the inability of these species to grow under anaerobic photosynthetic conditions.     

二维液相色谱法在羊草地上部总蛋白分离中的应用

取8周龄羊草的地上部分,用三氯乙酸-丙酮法沉淀总蛋白,沉淀裂解后将缓冲液置换为起始缓冲液,进行第一维色谱聚焦分离。将第一维分离收集的pH值为8.5至4.0之间的组分分别进行第二维无孔硅胶反相高效液相色谱分离,利用ProteoVue软件获得羊草植株总蛋白pI/UV图谱,即羊草植株总蛋白质表达谱。文中对二维液相色谱法分离羊草蛋白质进行了方法学的研究,在第二维分离中尝试用3种不同的洗脱梯度条件进行分离,优化二维液相色谱分离条件并与传统凝胶双向电泳进行了比较,另外还对二维液相色谱的重现性和准确性进行了检验。实验建立了利用二维液相色谱分离羊草总蛋白的技术方法。     

Measurement of stable isotopic enrichment and concentration of long-chain fatty acyl-carnitines in tissue by HPLC-MS

We have developed a new method for the simultaneous measurements of stable isotopic tracer enrichments and concentrations of individual long-chain fatty acyl-carnitines in muscle tissue using ion-pairing high-performance liquid chromatography-electrospray ionization quadrupole mass spectrometry in the selected ion monitoring (SIM) mode. Long-chain fatty acyl-carnitines were extracted from frozen muscle tissue samples by acetonitrile/methanol. Baseline separation was achieved by reverse-phase HPLC in the presence of the volatile ion-pairing reagent heptafluorobutyric acid. The SIM capability of a single quadrupole mass analyzer allows further separation of the ions of interest from the sample matrixes, providing very clean total and selected ion chromatograms that can be used to calculate the stable isotopic tracer enrichment and concentration of long-chain fatty acyl-carnitines in a single analysis. The combination of these two separation techniques greatly simplifies the sample preparation procedure and increases the detection sensitivity. Applying this protocol to biological muscle samples proves it to be a very sensitive, accurate, and precise analytical tool.     

A sensitive method for quantification of aceticlastic methanogens and estimation of total methanogenic cells in natural environments based on an analysis of ether-linked glycerolipids

Abstract A highly sensitive method for the quantification of methanogens in anaerobic digestor sludges was developed, based on an analysis of ether-linked glycerolipids. Core lipids were prepared from total lipids by HF treatment and mild methanolysis, and these core lipids were quantified as the corresponding 9-anthroyl derivatives by high-performance liquid chromatography with fluorescence detection. The amounts, in terms of cell carbon content, of Methanosaeta and Methanosarcina were proportional to the amounts of α-hydroxyarchaeol and β-hydroxyarchaeol, respectively. Moreover, the total amount of core lipids was well correlated with the cell mass of aceticlastic and H₂/CO₂-consuming methanogens. The limit of detection for Methanosaeta concilii was 17 ng of cell carbon when the signal/noise ratio was 3. This method allowed us to quantitate aceticlastic methanogens with high accuracy and to make a rough estimate of total methanogenic cells without any interference by the multifarious impurities that are present in anaerobic sludges. These results suggest that the present method will be a useful tool for investigations of methanogenic ecosystems.     

Rat liver homogenate (S9)-mediated binding of benzo[a]pyren to DNA in V79 cells,V79 cell nuclei and aqueous solution

The role of the target cell in determining the structures and the amounts of hydrocarbon-DNA adducts formed after hydrocarbon activation by an exogenous metabolic ativation system was investigated by exposing intact cells of the Chinese hamster lung cell line V79, V79 cell nuclei and calf thymus DNA to benzo[a]pyrene (B[a]P) in the presenceof a rat liver homogenate activation system (S9). The DNA was isolated, enzymatically degraded to deoxyribonucleosides and the B[a]P-deoxyribonucleoside adducts analyzed by high-performance liquid chromatography. Two major adducts were present in all samples; one formed by reaction of r-7, t-8-dihydroxy-t-9, 10-epoxy-7, 8, 9, 10-tetrahydro-B[a]P (anti-B[a]PDE) with the 2-amino group of deoxyguanosine, the other formed by reaction of a metabolite of 9-hydroxybenzo[a]pyrene (9-OH-B[a]P) with an unidentified deoxyribonucleoside. The ratios of the anti-B[a]PDE-DNA adduct to the 9-OH-B[a]P-DNA adduct were: calf thymus DNA, 3 to 1: DNA from V79 nuclei, 8 to 1; DNA from intact V79 cells, 11 to 1. Similar several-fold increases in the proportion of anti-B[a]PDE-DNA adducts in V79 cells over those in calf thymus DNA were observed for a dose range of 1–10 μg B[a]P per ml. The relative extent of binding of the activated metabolite of 9-OH-B[a]P to DNA was also much lower in intact V79 cells than in calf thymus DNA after exposure to 9-OH-B[a]P in the presence of the S9 activation system.These results demonstrate that the relative abilities of various reactive bbenzo[a]pyrene metabolites formed by an exogenous activation system to reach DNA differ substantially. Therefore, assessment of the biological activity of hydrocarbons in mutation assays using exogenous activation systems must take into account not only the amounts of different reactive hydrocarbon metabolites formed but also the relative abilities of these metabolites to reach the DNA of the target cell.     

Differentiation of endopeptidases and aminopeptidases by high-performance liquid chromatography of reaction products from chromogenic peptide p-nitroanilines as substrates

A method for differentiating endopeptidases and aminopeptidases on the basis of substrate specificity is presented. Various synthetic chromogenic substrates, succinyl-(Ala)3-p-nitroaniline, succinyl-(Ala)2-p-nitroaniline, (Ala)3-p-nitroaniline, and (Ala)2-p-nitroaniline, were incubated with various peptidases and the incubation mixtures were directly analyzed by high-performance liquid chromatography to determine the splitting patterns of these substrates by the enzymes. The substrates and hydrolyzed products containing the chromophore were separated on a reverse-phase column under isocratic conditions, and the chromophore was specifically detected in the effluent fractions by absorbance measurement at 314 nm. Endopeptidases, leucine aminopeptidase, and dipeptidyl aminopeptidase showed different patterns of cleavage of the substrates. This simple and rapid high-performance liquid chromatographic procedure is suitable for identifying the above activities in different fractions obtained during separation and purification studies. The same approach was applied to the simultaneous determination of three types of endopeptidase activities in rat tissues based on the ability of the enzymes to hydrolyze different sites in succinyl-(Ala)3-p-nitroaniline.