Anneliese Ernst

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.

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.

Mechanisms of dominance : coexistence of picocyanobacterial genotypes in a freshwater ecosystem

Abstract The autotrophic picoplankton of the pelagic zone of the mesotrophic Lake Constance is dominated by phycoerythrin-rich unicellular cyanobacteria phylogenetically related to the marine Synechococcus and Prochlorococcus cluster. In Lake Constance, the abundance of picocyanobacteria shows a recurrent pattern of seasonal variations. Evidence of diverse subpopulations was obtained by electron-microscopic examination of natural water samples and isolated strains that unveiled different surface structures of picocyanobacteria. Further evidence was obtained by DNA analysis of 26 clonal isolates representing 12 different genotypes. Variations in light and nutrient supply revealed distinct abilities of the genetically different strains to cope with these stress situations. Furthermore, cultured heterotrophic nanoflagellates exhibited differential feeding preferences for certain Synechococcus strains. The findings imply that growth and loss rates of the natural cyanobacterial community may be influenced by its genetic composition. Phylogenetic analyses of isolated strains indicated that the physiological diversification of pelagic Synechococcus spp. has occurred during a recent adaptive radiation. An example for genetic mechanisms underlying physiological diversification is indicated by mobile DNA elements found in a Synechocystis strain also isolated from the pelagic zone of Lake Constance. The observations suggest that dominance of Synechococcus spp. was achieved by evolutionary adaptation and coexistence of numerous genotypes generating a physiologically highly diversified population.

Genetic diversity and distribution of periphytic Synechococcus spp. in biofilms and picoplankton of Lake Constance

In various water depths of the littoral zone of Lake Constance (Bodensee) cyanobacteria of the Synechococcus-type were isolated from biofilms (periphyton) on three natural substrates and an artificial one (unglazed tiles). From one tile three strains of phycoerythrin (PE)-rich Synechococcus spp. were isolated, the first examples of these organisms in the epibenthos. Phylogenetic inference based on the 16S-23S rRNA intergenic spacer (ITS-1) assigned all periphytic isolates to two clusters of the picophytoplankton clade (evolutionary lineage VI of cyanobacteria). The sequence divergence in the ITS-1 was used to design specific PCR primers to allow direct, culture-independent detection and quantification of isolated Synechococcus strains in natural periphytic and pelagic samples. Denaturing gradient gel electrophoresis (DGGE) analysis revealed depth-related differences of Synechococcus spp. distribution on tiles placed in the littoral zone. Synechococcus genotypes were observed which occurred in both the periphyton (on tiles) and in the pelagic picoplankton. A strain with one of these genotypes, Synechococcus sp. BO 8805, was isolated from the pelagic zone in 1988. Its genotype was found on tiles that had been exposed at different water depths in the littoral zone in spring and autumn of the year 2000. Quantitative analysis with a genotype-specific TaqMan probe and real-time Taq nuclease assays (TNA) confirmed its presence in the pelagic zone, although appearance of this and related genotypes was highly irregular and exhibited strong differences between consecutive years. Our results show that the ability to form significant subpopulations in pelagic and periphytic communities exists in three out of four phylogenetic clusters of Synechococcus spp. in Lake Constance. This versatility may be a key feature in the ubiquity of the evolutionary lineage VI of cyanobacteria.

Glycogen Accumulation and the Induction of Nitrogenase Activity in the Heterocyst-forming Cyanobacterium Anabaena variabilis

Summary: Anabaena variabilis (ATCC 29413) grown in batch cultures for 2 d exhibited rapid glycogen synthesis upon dilution of the cell suspension. The cellular polysaccharide concentration attained a maximum after 9-12 h growth in fresh medium and decreased thereafter. The growth rate, the rate of glycogen accumulation and the maximum amount of glycogen increased when light intensity was increased. Accumulation occurred with both N2 and ammonia as N-sources. In cultures grown in the presence of ammonia, the increase in the cellular C-fraction upon dilution caused neither induction of nitrogenase nor heterocyst formation. However, ammonia depletion during subsequent growth gave rise to a second accumulation of glycogen followed by differentiation. Thus, in this phototroph, glycogen is synthesized without subsequent differentiation following an increase in average irradiation as long as the N-supply is maintained. Glycogen synthesis following N-depletion, however, is accompanied by induction of nitrogenase.

Phosphorylation and nitrogenase activity in isolated heterocysts from Anabaena variabilis (ATCC 29413)

Adenylate-pool composition, energy charge, and nitrogenase activity were examined in isolated heterocysts from Anabaena variabilis (ATCC 29413). ATP formation was detected as a light- or oxygen-induced increase in ATP concentration. No cofactors or substrates had to be added for photophosphorylation to occur, whereas oxidative phosphorylation was dependent on hydrogen and oxygen (Knallgas reaction). The increase in ATP concentration was reflected by a decrease in AMP concentration, accompanied by small changes in ADP levels. Thus, a regulation of the adenylate pool by a myokinase (adenylate kinase) has to be assumed. Upon dark-light transitions, the energy charge in heterocysts increased from values below 0.4 to values approaching 0.8. High energy-charge values, reached in the light only, allowed for high rates of acetylene reduction in the presence of hydrogen. The increase in the energy charge in the dark to approx. 0.64 by addition of oxygen (5% (vv) in the presence of hydrogen) resulted in low nitrogenase activities, generally not exceeding 1–3% of the light-induced rates. In the dark, oxygen concentrations above 10% were inhibitory to both ATP formation and acetylene reduction. Increasing light intensities led to a steep increase in energy charge followed by an increase in nitrogenase activity. Plotting enzyme activity versus energy charge, a nonlinear, asymptotic relationship was observed.

Increase and Stabilization of Photoproduction of Hydrogen in Nostoc muscorum by Photosynthetic Electron Transport Inhibitors

Abstract Nittrogen-fixing cells of Nostoc muscorum grown under nitrogen or in the presence of nitrate exhibit substantial light-induced hydrogen production for over 15 hours in the presence of electron transport inhibitors. Rates attain levels of 12 μmol H2 evolved/ml packed cell volume and hour. The ATP-dependent nitrogenase, not a hydrogenase, is responsible for hydrogen production. This is indicated by poor sensitivity to CO and inhibition of the reaction by uncouplers, acetylene, and N2 . An active uptake hydrogenase minimizes light-induced H2 production. Although nitrogenase activity is somewhat decreased by several photosynthetic electron transport inhibitors, hydrogen production is markedly increased. This is due to lowering the partial pressure of oxygen in the cell, preventing oxidative hydrogen consumption.

Induction and modification of dinitrogenase reductase in the unicellular cyanobacterium Synechocystis BO 8402

Oxygen is an important regulatory factor of nitrogenase induced in a unicellular cyanobacterium, Synechocystis BO 8402, during nitrogen starvation. Synthesis of the enzyme is limited by the efficiency of the cells to remove oxygen by respiration, supported by hydrogenases and, in the light, by inhibition of photosynthesis. With a polyclonal antibody against dinitrogenase reductase (the Fe protein of nitrogenase) a single polypeptide is detected, indicative of an active dimeric enzyme in dense cell suspensions. Inhibition of nitrogenase by addition of oxygen is accompanied by the appearance of a second polypeptide of the Fe protein having a 1.5 kDa higher molecular weight. This disappears upon removal of oxygen from the gas phase while nitrogenase activity is restored. No protein synthesis is required indicating that a fraction of the existing polypeptides is reversibly modified in response to oxygen. After induction of nitrogenase activity in dilute culture suspensions, both forms of the Fe-protein are found in variable amounts possibly due to oxygen contamination during the experiment.