Dataset Information

Application of real-time PCR to estimate toxin production by the cyanobacterium Planktothrix sp.

ABSTRACT: Quantitative real-time PCR methods are increasingly being applied for the enumeration of toxic cyanobacteria in the environment. However, to justify the use of real-time PCR quantification as a monitoring tool, significant correlations between genotype abundance and actual toxin concentrations are required. In the present study, we aimed to explain the concentrations of three structural variants of the hepatotoxin microcystin (MC) produced by the filamentous cyanobacterium Planktothrix sp., [Asp, butyric acid (Dhb)]-microcystin-RR (where RR means two arginines), [Asp, methyl-dehydro-alanine (Mdha)]-microcystin-RR, and [Asp, Dhb]-microcystin-homotyrosine-arginine (HtyR), by the abundance of the microcystin genotypes encoding their synthesis. Three genotypes of microcystin-producing cyanobacteria (denoted the Dhb, Mdha, and Hty genotypes) in 12 lakes of the Alps in Austria, Germany, and Switzerland from 2005 to 2007 were quantified by means of real-time PCR. Their absolute and relative abundances were related to the concentration of the microcystin structural variants in aliquots determined by high-performance liquid chromatography (HPLC). The total microcystin concentrations varied from 0 to 6.2 microg liter(-1) (mean +/- standard error [SE] of 0.6 +/- 0.1 microg liter(-1)) among the samples, in turn resulting in an average microcystin content in Planktothrix of 3.1 +/- 0.7 microg mm(-3) biovolume. Over a wide range of the population density (0.001 to 3.6 mm(3) liter(-1) Planktothrix biovolume), the Dhb genotype and [Asp, Dhb]-MC-RR were most abundant, while the Hty genotype and MC-HtyR were found to be in the lowest proportion only. In general, there was a significant linear relationship between the abundance/proportion of specific microcystin genotypes and the concentration/proportion of the respective microcystin structural variants on a logarithmic scale. We conclude that estimating the abundance of specific microcystin genotypes by quantitative real-time PCR is useful for predicting the concentration of microcystin variants in water.

SUBMITTER: Ostermaier V

PROVIDER: S-EPMC2876456 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Application of real-time PCR to estimate toxin production by the cyanobacterium Planktothrix sp.

Ostermaier Veronika V Kurmayer Rainer R

Applied and environmental microbiology 20100402 11

Quantitative real-time PCR methods are increasingly being applied for the enumeration of toxic cyanobacteria in the environment. However, to justify the use of real-time PCR quantification as a monitoring tool, significant correlations between genotype abundance and actual toxin concentrations are required. In the present study, we aimed to explain the concentrations of three structural variants of the hepatotoxin microcystin (MC) produced by the filamentous cyanobacterium Planktothrix sp., [Asp ...[more]

PMID: 20363794

Similar Datasets

Project description:The production of biofuels in photosynthetic microalgae and cyanobacteria is considered a promising alternative to the generation of fuels from fossil resources. However, to be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria to forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain. Here we report the results of a monitoring experiment, in which the transcriptome-wide response to continuous ethanol production in the unicellular model cyanobacterium Synechocystis sp. PCC6803 was examined using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) EtOH d-1 ± 0.002 and 0.0303% (v/v) d-1 ± 0.002 were obtained over 18 consecutive days, measuring biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation in the producer strains and the development of a bleaching phenotype. Absorption spectroscopy indicated in particular a down-regulation of light harvesting capacity. Microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an about 4 fold reduction in cpcB (sll1577) and an about 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional operon discoordination in the cpcBA operon leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, Western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype. We conclude that the changes in gene expression upon induction of long term ethanol production in Synechocystis sp. PCC6803 are highly specific. in particular we did not observe a comprehensive stress response contributing to a complex phenotype as might have been expected.

Project description:The production of biofuels in photosynthetic microalgae and cyanobacteria is considered a promising alternative to the generation of fuels from fossil resources. However, to be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria to forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain. Here we report the results of a monitoring experiment, in which the transcriptome-wide response to continuous ethanol production in the unicellular model cyanobacterium Synechocystis sp. PCC6803 was examined using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) EtOH d-1 M-BM-1 0.002 and 0.0303% (v/v) d-1 M-BM-1 0.002 were obtained over 18 consecutive days, measuring biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation in the producer strains and the development of a bleaching phenotype. Absorption spectroscopy indicated in particular a down-regulation of light harvesting capacity. Microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an about 4 fold reduction in cpcB (sll1577) and an about 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional operon discoordination in the cpcBA operon leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, Western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype. We conclude that the changes in gene expression upon induction of long term ethanol production in Synechocystis sp. PCC6803 are highly specific. in particular we did not observe a comprehensive stress response contributing to a complex phenotype as might have been expected. Gene expression of Synechocystis sp. PCC 6803 WT (#621) and the isogenic ethanol producing strain #309 was monitored at 4, 7, 11 and 18 days after induction of ethanol production by copper depletion. Each condition was sampled in biological duplicates

Project description:Cyanobacteria are frequently involved in the formation of harmful algal blooms wherein, apart from the toxic microcystins, other groups of bioactive peptides are abundant as well, such as anabaenopeptins (APs). The APs are synthesized nonribosomally as cyclic hexapeptides with various amino acids at the exocyclic position. We investigated the presence and recombination of the AP synthesis gene cluster (apnA-E) through comparing 125 strains of the bloom-forming cyanobacterium Planktothrix spp., which were isolated from numerous shallow and deep water habitats in the temperate and tropical climatic zone. Ten ecologically divergent strains were purified and genome sequenced to compare their entire apnA-E gene cluster. In order to quantify apn gene distribution patterns, all the strains were investigated by PCR amplification of 2 kbp portions of the entire apn gene cluster without interruption. Within the 11 strains assigned to P. pseudagardhii, P. mougeotii, or P. tepida (Lineage 3), neither apnA-E genes nor remnants were observed. Within the P. agardhii/P. rubescens strains from shallow waters (Lineage 1, 52 strains), strains both carrying and lacking apn genes occurred, while among the strains lacking the apnA-E genes, the presence of the 5'end flanking region indicated a gene cluster deletion. Among the strains of the more derived deep water ecotype (Lineage 2, 62 strains), apnA-E genes were always present. A high similarity of apn genes of the genus Planktothrix when compared with strains of the genus Microcystis suggested its horizontal gene transfer during the speciation of P. agardhii/P. rubescens. Genetic analysis of the first (A1-) domain of the apnA gene, encoding synthesis of the exocyclic position of the AP molecule, revealed four genotype groups that corresponded with substrate activation. Groups of genotypes were either related to Arginine only, the coproduction of Arginine and Tyrosine or Arginine and Lysine, or even the coproduction of Arginine, Tyrosine, and Lysine in the exocyclic position of the AP-molecule. The increased structural diversity resulted from the evolution of apnA A1 genotypes through a small number of positively selected point mutations that occurred repeatedly and independently from phylogenetic association.

Project description:Cyanobacteria are phototrophic prokaryotes that can convert inorganic carbon as CO2 into organic carbon compounds at the expense of light energy. In addition, they need only a few inorganic nutrients and can be cultivated in high densities using non-arable land and seawater. This features qualified cyanobacteria as attractive organisms for the production of third generation biofuels as part of the development of future CO2-neutral energy production. Synechocystis sp. PCC 6803 represents one of the most widely used cyanobacterial model strains. On the basis of its available genome sequence and genetic tools, many strains of Synechocystis have been generated that produce different biotechnological products. Efficient isoprene production is an attractive goal, since this compound represents not only an energy-rich biofuel but is also used as chemical feedstock. Here, we report on our attempts to generate isoprene-producing strains of Synechocystis. The cDNA of a codon-optimized plant isoprene synthase (IspS) was cloned under the control of different Synechocystis promoters, which ensure strong constitutive or light-regulated ispS expression. The expression of the ispS gene was quantified by qPCR, whereas the amount of isoprene was quantified using GC-MS. Incubation of our strains at different salt conditions had marked impact on the isoprene production rates. Under low salt conditions, a good correlation was found between ispS expression and isoprene production rate. However, the cultivation of isoprene production strains under salt-supplemented conditions decreased isoprene production despite the fact that ispS expression was salt-stimulated. The characterization of the metabolome of isoprene producing strains indicated that isoprene production might be limited by insufficient precursor levels. Our isoprene production rates under low salt conditions were 2 - 6.5times higher compared to the previous report of Lindberg et al. (2010). These results can be used to guide future attempts establishing the isoprene production with cyanobacterial host systems.

Dataset Information

Application of real-time PCR to estimate toxin production by the cyanobacterium Planktothrix sp.

Publications

Application of real-time PCR to estimate toxin production by the cyanobacterium Planktothrix sp.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets