Peter Böger

PCR Bias in Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial Communities

ABSTRACT Succession of ecotypes, physiologically diverse strains with negligible rRNA sequence divergence, may explain the dominance of small, red-pigmented (phycoerythrin-rich) cyanobacteria in the autotrophic picoplankton of deep lakes (C. Postius and A. Ernst, Arch. Microbiol. 172:69–75, 1999). In order to test this hypothesis, it is necessary to determine the abundance of specific ecotypes or genotypes in a mixed background of phylogenetically similar organisms. In this study, we examined the performance of Taq nuclease assays (TNAs), PCR-based assays in which the amount of an amplicon is monitored by hydrolysis of a labeled oligonucleotide (TaqMan probe) when hybridized to the amplicon. High accuracy and a 7-order detection range made the real-time TNA superior to the corresponding end point technique. However, in samples containing mixtures of homologous target sequences, quantification can be biased due to limited specificity of PCR primers and probe oligonucleotides and due to accumulation of amplicons that are not detected by the TaqMan probe. A decrease in reaction efficiency, which can be recognized by direct monitoring of amplification, provides experimental evidence for the presence of such a problem and emphasizes the need for real-time technology in quantitative PCR. Use of specific primers and probes and control of amplification efficiency allow correct quantification of target DNA in the presence of an up to 104-fold excess of phylogenetically similar DNA and of an up to 107-fold excess of dissimilar DNA.

Rewetting of drought-resistant blue-green algae: Time course of water uptake and reappearance of respiration, photosynthesis, and nitrogen fixation

SummaryThe response of the terrestrial blue-green algae Nostoc flagelliforme, Nostoc commune, and Nostoc spec. to water uptake has been investigated after a drought period of approximately 2 years. Rapid half-times of rewetting (0.6, 3.3, and 15.5 min, respectively) are found. The surfaceto-mass ratio of the three species is inversely correlated to the speed of water uptake and loss. The ecological relevance of these different time courses is discussed.Respiration starts immediately after a 30-min rewetting period, whereas photosynthetic oxygen evolution reaches its maximum activity after 6 and 8 h with N. commune and N. flagelliforme, respectively. In the dark, recovery of oxygen uptake by N. commune is somewhat impaired, while slightly stimulated with N. flagelliforme. With both species, recovery of photosynthesis is inhibited by darkness.Using colonies kept dry for two years, nitrogenase activity of N. commune attains its maximum 120 to 150 h after rewetting, while only 50 h were needed with algal mats kept dry for two days.Thus, after a 2-year drought period, the physiological sequence of reactivation is respiration—photosynthesis—nitrogen fixation. Respiration and photosynthesis precede growth and are exhibited by existing vegetative cells, whereas recovery of nitrogen fixation is dependent on newly differentiated heterocysts.

Glycogen content and nitrogenase activity in Anabaena variabilis

Nitrogenase (=acetylene-reducing activity) was followed during photoautotrophic growth of Anabaena variabilis (ATCC 29413). When cell density increased during growth, (1) inhibition of light-dependent activity by DCMU, an inhibitor of photosynthesis, increased, and (2) nitrogenase activity in the dark decreased. Addition of fructose stabilized dark activity and alleviated the DCMU effect in cultures of high cell density.The resistance of nitrogenase towards oxygen inactivation decreased after transfer of autotrophically grown cells into the dark at subsequent stages of increasing culture density. The inactivation was prevented by addition of fructose. Recovery of acetylene-reducing activity in the light, and in the dark with fructose present, was suppressed by ammonia or chloramphenicol. In the light, also DCMU abolished recovery.To prove whether the observed effects were related to a lack of photosynthetic storage products, glycogen of filaments was extracted and assayed enzymatically. The glycogen content of cells was highest 10 h after inoculation, while light-dependent nitrogenase activity was at its maximum about 24 h after inoculation. Glycogen decreased markedly as growth proceeded and dropped sharply when the cells were transferred to darkness. Thus, when C-supply (by photosynthesis or added fructose) was not effective, the glycogen content of filaments determined the activity of nitrogenase and its stability against oxygen. In cells lacking glycogen, nitrogenase activity recovered only when carbohydrates were supplied by exogenously added fructose or by photosynthesis.

The biotechnological potential and design of novel carotenoids by gene combination in Escherichia coli

Carotenoids are antioxidants with considerable pharmaceutical potential. More than 600 carotenoid structures are known but their availability is limited owing to practical difficulties associated with chemical synthesis and isolation from microorganisms or plant tissue. To overcome some of these problems, heterologous expression of carotenoid genes in Escherichia coli can be used for the synthesis of rare derivatives or even of novel carotenoids. Novel and rare carotenoids can be obtained by combining carotenoid genes from different host species in E. coli.

Towards the primary target of chloroacetamides –new findings pave the way

This review reports on research of the last ten years to find the primary target enzyme for chloroacetamides. As could be shown first with the green alga Scenedesmus, the formation of very-long-chain fatty acids (VLCFAs) is severely impaired. Subsequently, in short-term experiments, labelled malonate or stearate could be incorporated into leaf discs of cucumber, barley or leek seedlings. While the formation of ‘normal’ long-chain fatty acids (up to C18) was not influenced, phytotoxic chloroacetamides strongly inhibited the synthesis of VLCFAs of C20, 22 and 24, with I50 values of 10–100 nM. Inhibition depends on the amide structure and on stereospecificity. Also cafenstrole or recently developed tetrazolinones and phosphosulfonates were found active to inhibit fatty-acid elongation. Subsequently, a cell-free elongase assay was developed using a microsomal preparation from leek seedlings (Allium porrum L), [14C]malonyl-CoA and C18, 20, or C22 acyl-CoA primer substrates. All elongation steps were strongly affected by those phytotoxic herbicides which were also active in vivo. The inhibitors form a tight-binding complex with the condensing elongase enzyme system which develops with time and lowers the I50 values markedly. Apparently, a nucleophilic attack of the inhibitor takes place at the specific target enzyme. Acyl-CoA elongation inhibition is correlated with growth inhibition of the intact cell. Due to the low I50 values and the specific inhibition, we assume that impaired VLCFA-formation is the primary phytotoxic impact of chloroacetamides and functionally related structures. © 2000 Society of Chemical Industry

Copper-induced exchange of plastocyanin and cytochrome c-533 in cultures of Anabaena variabilis and Plectonema boryanum

Abstract Anabaena variabilis and Plectonema boryanum exhibited copper-induced plastocyanin formation accompanied by a replacement of thylakoidal cytochrome c-533. This could be shown by optical absorption and EPR spectroscopy. The membrane-bound cytochrome φ-557 was not influenced. KCN treatment of spheroplasts only affected the electron transport (with either water or reduced diaminodurene as electron donor) of plastocyanin-containing membranes, whereas little influence was observed with thylakoids in which the copper protein was replaced by cytochrome c-533.

Distribution of plastocyanin and soluble plastidic cytochrome c in various classes of algae

Several eukaryotic algae belonging to the main taxonomic classes have been cultured autotrophically in liquid medium supplemented with or depleted of copper to assay their ability to form plastocyanin or exchange it against plastidic cytochrome c-553. Most Chlorophyceae are able to substitute cytochrome c-533 for plastocyanin with some exceptions like Haematococcus or Dunaliella, which can only synthesize plastocyanin. Also within the Chlorella group, about half of the 28 strains assayed cannot synthesize cytochrome c-553 under copper deficiency. Species of Chrysophyceae, Xanthophyceae, and Rhodophyceae, on the other hand, cannot synthesize plastocyanin even when a comparatively high copper concentration (10μM) is available.Serological cross-reactions of various plastocyanincontaining Chlorella homogenates against an antibody towards Scenedesmus plastocyanin exhibit a pattern which cannot be taxonomically used at the moment.Including previous data on blue-green algae, it appears that, in the course of evolution, cytochrome c-553 dominates in the older species. In the Chlorophyceae, it is mutually exchangeable against plastocyanin which becomes the only electron donor to P700 in higher plants.

Quantitative Tracing, by Taq Nuclease Assays, of a Synechococcus Ecotype in a Highly Diversified Natural Population

ABSTRACT Quantitative Taq nuclease assays (TNAs) (TaqMan PCR), nested PCR in combination with denaturing gradient gel electrophoresis (DGGE), and epifluorescence microscopy were used to analyze the autotrophic picoplankton (APP) of Lake Constance. Microscopic analysis revealed dominance of phycoerythrin (PE)-rich Synechococcus spp. in the pelagic zone of this lake. Cells passing a 3-μm-pore-size filter were collected during the growth period of the years 1999 and 2000. The diversity of PE-rich Synechococcus spp. was examined using DGGE to analyze GC-clamped amplicons of a noncoding section of the 16S-23S intergenic spacer in the ribosomal operon. In both years, genotypes represented by three closely related PE-rich Synechococcus strains of our culture collection dominated the population, while other isolates were traced sporadically or were not detected in their original habitat by this method. For TNAs, primer-probe combinations for two taxonomic levels were used, one to quantify genomes of all known Synechococcus-type cyanobacteria in the APP of Lake Constance and one to enumerate genomes of a single ecotype represented by the PE-rich isolate Synechococcus sp. strain BO 8807. During the growth period, genome numbers of known Synechococcus spp. varied by 2 orders of magnitude (2.9 × 103 to 3.1 × 105 genomes per ml). The ecotype Synechococcus sp. strain BO 8807 was detected in every sample at concentrations between 1.6 × 101 and 1.3 × 104 genomes per ml, contributing 0.02 to 5.7% of the quantified cyanobacterial picoplankton. Although the quantitative approach taken in this study has disclosed several shortcomings in the sampling and detection methods, this study demonstrated for the first time the extensive internal dynamics that lie beneath the seemingly arbitrary variations of a population of microbial photoautotrophs in the pelagic habitat.

Copper Deficiency and Toxicity in Scenedesmus

Summary The microalga Scenedesmus acutus can accumulate high amounts of copper ions. Depending on the concentration in the medium, an enrichment of up to 1000 fold within the cells is possible. For optimum growth a copper supply of 0.1 to 1 µM is necessary. Growth is reduced to 50% of the normal rate and chlorophyll is bleached when the medium is depleted of copper. Copper concentrations exceeding 10 µM are toxic. However, toxicity is temporary only and the algae can adapt to up to 50 µM copper ions within 24 h. During this transient period, chlorophyll is broken down and lipids are decomposed by oxidative processes. By following ethane and ethylene production, lipid peroxidation could be correlated with decrease of photosynthetic oxygen evolution and growth.

In vitro studies on pathways and regulation of electron transport to nitrogenase with a cell-free extract from heterocysts of Anabaena variabilis

A cell-free preparation of heterocysts from Anabaena variabilis showed high nitrogenase activities with several physiological electron donors, dependent on addition of an ATP-generating system. Light-induced acetylene reduction with the artificial electron donor to photosystem I, diaminodurol, exhibited the same light saturation as with hydrogen as donor. Inhibitors of electron flow through plastoquinone affected light-induced, hydrogen- or NADH-dependent nitrogenase activity in a similar way. Several uncoupling agents were without effect, indicating that energized membranes are not a prerequisite for nitrogen fixation. We conclude that NADH or hydrogen deliver electrons to nitrogenase via photosystem I and ferredoxin, feeding in at the plastoquinone site.In the light, addition of NADP induced a lag in H2- or NADH-supported acetylene reduction apparently by competing with nitrogenase for electrons at the reducing side of photosystem I. Time reversal of this inibition reflects a regulation of photosystem I-dependent nitrogenase activity by the NADPH/NADP ratio in the cell. This was directly demonstrated by differently adjusted NADPH/NADP ratios.NADPH donates electrons to nitrogenase in the dark and in the light, the light reaction being DBMIB-sensitive. NADPH-supported acetylene reduction was inhibited by NADP. This inhibition was not reversed with time, pointing to an involvement of ferredoxin: NADP oxidoreductase (EC 1.18.1.2) in this pathway. Apparently, in the dark, this enzyme is able to directly reduce ferredoxin, whereas in the light electrons from NADPH first have to pass through photosystem I before reducing ferredoxin, hence nitrogenase.Intermediates of glycolysis, like glucose-6-phosphate, fructose-1,6-bisphosphate, and dihydroxyacetone phosphate supported nitrogenase activity in the dark, each with catalytic amounts of both NAD and NADP as equally effective cofactors.We conclude that in heterocysts electrons for nitrogen fixation are essentially supplied by dark reactions, mainly by glycolysis. NADH (and hydrogen) contribute electrons via photosystem I in the light, whereas the NADPH/NADP ratio regulates linear and cyclic electron flow at the reducing side of photosystem I to provide a ratio of ATP/electrons most effective for nitrogenase.