Single colony genetic analysis of epilithic stream algae of the genus Chamaesiphon spp.

In order to understand Chamaesiphon spp. evolution and ecological diversification, we investigated the phylogenetic differentiation of three morphospecies from field samples by means of single colony genetics. Individual colonies of three different morphospecies (C. starmachii, C. polonicus, C. geitleri,) were isolated from lotic gravel streams and their 16S rDNA nucleotide variability was analyzed. For a number of individual colonies, microscopical and ultrastructural analysis was also performed. A phylogenetic tree of all major lineages of the phylum of Cyanobacteria assigned all Chamaesiphon genotypes (1149-1176 bp) most closely with the family of Gomontiellaceae of the order Oscillatoriales. The sequences obtained from colonies assigned to C. starmachii (n = 21), C. polonicus (n = 9), and C. geitleri (n = 17) were found to reveal high average (3.5%) nucleotide diversity. No phylogenetic sub-branching in correspondence with morphology was observed suggesting that the three Chamaesiphon morphospecies did not represent monophyletic taxa. We could not attribute specific thylakoid ultrastructure to phylogenetic sub-branches; however, the observed parietally and loosely arranged thylakoids indicate that for the genus Chamaesiphon, the variability in thylakoid ultrastructure might have been underestimated. In summary, the high nucleotide diversity of the 16S rDNA gene implies phylogenetic diversity that corresponds little to morphological classification.

Evolution of Anabaenopeptin Peptide Structural Variability in the Cyanobacterium Planktothrix.

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.

Emergence of nontoxic mutants as revealed by single filament analysis in bloom-forming cyanobacteria of the genus Planktothrix.

BACKGROUND: Bloom-forming cyanobacteria cause toxic algae outbreaks in lakes and reservoirs. We aimed to explore and quantify mutation events occurring within the large mcy gene cluster (55 kbp) encoding microcystin (MC) biosynthesis that inactivate MC net production. For this purpose we developed a workflow to detect mutations in situ occurring anywhere within the large mcy gene cluster as amplified from one single filament of the red-pigmented cyanobacterium Planktothrix rubescens. From five lakes of the Alps eight hundred Planktothrix filaments were isolated and each individual filament was analyzed for mutations affecting the mcy genes. RESULTS: Mutations inactivating MC synthesis were either through an insertion element ISPlr1 or the partial deletion of mcy genes. Neutral mutations not affecting MC biosynthesis occurred within two intergenic spacer regions, either through the insertion of a Holliday-junction resolvase RusA or ISPlr1. Altogether, the insertions affected a few mcy genes only and their location was correlated with regions similar to repetitive extragenic palindromic DNA sequences (REPs). Taking all of the filaments together, the mutations leading to the inactivation of MC synthesis were more rare (0.5-6.9%), when compared with the neutral mutations (7.5-20.6%). On a spatial-temporal scale the ratio of MC synthesis-inactivating vs. neutral mutations was variable, e.g., the filament abundance carrying partial deletion of mcyD (5.2-19.4%) and/or mcyHA (0-7.3%) exceeded the abundance of neutral mutations. CONCLUSIONS: It is concluded that insertion events occurring within the Planktothrix mcy gene cluster are predictable due to their correlation with REPs. The frequency of occurrence of the REPs within the mcy gene cluster of Planktothrix relates to the rather common mutation of mcy genes in Planktothrix. Spatial-temporal variable conditions may favor the emergence of partial mcy deletion mutants in Planktothrix, in particular a higher proportion of genotypes resulting in inactivation of MC synthesis might be caused by increased ISPlr1 activity.

Structural Elucidation of the Bispecificity of A Domains as a Basis for Activating Non-natural Amino Acids.

Many biologically active peptide secondary metabolites of bacteria are produced by modular enzyme complexes, the non-ribosomal peptide synthetases. Substrate selection occurs through an adenylation (A) domain, which activates the cognate amino acid with high fidelity. The recently discovered A domain of an Anabaenopeptin synthetase from Planktothrix agardhii (ApnA A1) is capable of activating two chemically distinct amino acids (Arg and Tyr). Crystal structures of the A domain reveal how both substrates fit into to binding pocket of the enzyme. Analysis of the binding pocket led to the identification of three residues that are critical for substrate recognition. Systematic mutagenesis of these residues created A domains that were monospecific, or changed the substrate specificity to tryptophan. The non-natural amino acid 4-azidophenylalanine is also efficiently activated by a mutant A domain, thus enabling the production of diversified non-ribosomal peptides for bioorthogonal labeling.

A transcriptome derived female-specific marker from the invasive Western mosquitofish (Gambusia affinis).

Sex-specific markers are a prerequisite for understanding reproductive biology, genetic factors involved in sex differences, mechanisms of sex determination, and ultimately the evolution of sex chromosomes. The Western mosquitofish, Gambusia affinis, may be considered a model species for sex-chromosome evolution, as it displays female heterogamety (ZW/ZZ), and is also ecologically interesting as a worldwide invasive species. Here, de novo RNA-sequencing on the gonads of sexually mature G. affinis was used to identify contigs that were highly transcribed in females but not in males (i.e., transcripts with ovary-specific expression). Subsequently, 129 primer pairs spanning 79 contigs were tested by PCR to identify sex-specific transcripts. Of those primer pairs, one female-specific DNA marker was identified, Sanger sequenced and subsequently validated in 115 fish. Sequence analyses revealed a high similarity between the identified sex-specific marker and the 3´ UTR of the aminomethyl transferase (amt) gene of the closely related platyfish (Xiphophorus maculatus). This is the first time that RNA-seq has been used to successfully characterize a sex-specific marker in a fish species in the absence of a genome map. Additionally, the identified sex-specific marker represents one of only a handful of such markers in fishes.

Elucidation of insertion elements carried on plasmids and in vitro construction of shuttle vectors from the toxic cyanobacterium Planktothrix.

Several gene clusters that are responsible for toxin synthesis in bloom-forming cyanobacteria have been found to be associated with transposable elements (TEs). In particular, insertion sequence (IS) elements were shown to play a role in the inactivation or recombination of the genes responsible for cyanotoxin synthesis. Plasmids have been considered important vectors of IS element distribution to the host. In this study, we aimed to elucidate the IS elements propagated on the plasmids and the chromosome of the toxic cyanobacterium Planktothrix agardhii NIVA-CYA126/8 by means of high-throughput sequencing. In total, five plasmids (pPA5.5, pPA14, pPA50, pPA79, and pPA115, of 5, 6, 50, 79, and 120 kbp, respectively) were elucidated, and two plasmids (pPA5.5, pPA115) were found to propagate full IS element copies. Large stretches of shared DNA information between plasmids were constituted of TEs. Two plasmids (pPA5.5, pPA14) were used as candidates to engineer shuttle vectors (named pPA5.5SV and pPA14SV, respectively) in vitro by PCR amplification and the subsequent transposition of the Tn5 cat transposon containing the R6Kγ origin of replication of Escherichia coli. While pPA5.5SV was found to be fully segregated, pPA14SV consistently co-occurred with its wild-type plasmid even under the highest selective pressure. Interestingly, the Tn5 cat transposon became transferred by homologous recombination into another plasmid, pPA50. The availability of shuttle vectors is considered to be of relevance in investigating genome plasticity as a consequence of homologous recombination events. Combining the potential of high-throughput sequencing and in vitro production of shuttle vectors makes it simple to produce species-specific shuttle vectors for many cultivable prokaryotes.

Genetic variability of microcystin biosynthesis genes in Planktothrix as elucidated from samples preserved by heat desiccation during three decades.

Historic samples of phytoplankton can provide information on the abundance of the toxigenic genotypes of cyanobacteria in dependence on increased or decreased eutrophication. The analysis of a time-series from preserved phytoplankton samples by quantitative PCR (qPCR) extends observation periods considerably. The analysis of DNA from heat-desiccated samples by qPCR can be aggravated by point substitutions or the fragmentation of DNA introduced by the high temperature. In this study, we analyzed whether the heat desiccation of the cellular material of the cyanobacterium Planktothrix sp. introduced potential errors to the template DNA that is used for qPCR within (i) 16S rDNA and phycocyanin genes and (ii) the mcyA gene indicative of the incorporation of either dehydrobutyrine (Dhb) or N-methyl-dehydroalanine (Mdha) in position 7, and (ii) the mcyB gene, which is indicative of homotyrosine (Hty) in position 2 of the microcystin (MC) molecule. Due to high temperature desiccation, the deterioration of the DNA template quality was rather due to fragmentation than due to nucleotide substitutions. By using the heat-desiccated samples of Lake Zürich, Switzerland the abundance of the Dhb, Mdha and Hty genotypes was determined during three decades (1977-2008). Despite major changes in the trophic state of the lake resulting in a major increase of the total Planktothrix population density, the proportion of these genotypes encoding the synthesis of different MC congeners showed high stability. Nevertheless, a decline of the most abundant mcyA genotype indicative of the synthesis of Dhb in position 7 of the MC molecule was observed. This decline could be related to the gradual incline in the proportion of a mutant genotype carrying a 1.8kbp deletion of this gene region. The increase of this mcyA (Dhb) gene deletion mutant has been minor so far, however, and likely did not affect the overall toxicity of the population.

Putative antiparasite defensive system involving ribosomal and nonribosomal oligopeptides in cyanobacteria of the genus Planktothrix.

Parasitic chytrid fungi can inflict significant mortality on cyanobacteria but frequently fail to keep cyanobacterial dominance and bloom formation in check. Our study tested whether oligopeptide production, a common feature in many cyanobacteria, can be a defensive mechanism against chytrid parasitism. The study employed the cyanobacterial strain Planktothrix NIVA-CYA126/8 and its mutants with knockout mutations for microcystins, anabaenopeptins, and microviridins, major oligopeptide classes to be found in NIVA-CYA126/8. Four chytrid strains were used as parasite models. They are obligate parasites of Planktothrix and are unable to exploit alternative food sources. All chytrid strains were less virulent to the NIVA-CYA126/8 wild type than to at least one of its oligopeptide knockout mutants. One chytrid strain even failed to infect the wild type, while exhibiting considerable virulence to all mutants. It is therefore evident that producing microcystins, microviridins, and/or anabaenopeptins can reduce the virulence of chytrids to Planktothrix, thereby increasing the host's chance of survival. Microcystins and anabaenopeptins are nonribosomal oligopeptides, while microviridins are produced ribosomally, suggesting that Planktothrix resists chytrids by relying on metabolites that are produced via distinct biosynthetic pathways. Chytrids, on the other hand, can adapt to the oligopeptides produced by Planktothrix in different ways. This setting most likely results in an evolutionary arms race, which would probably lead to Planktothrix and chytrid population structures that closely resemble those actually found in nature. In summary, the findings of the present study suggest oligopeptide production in Planktothrix to be part of a defensive mechanism against chytrid parasitism.

Genetic variation of adenylation domains of the anabaenopeptin synthesis operon and evolution of substrate promiscuity.

Anabaenopeptins (AP) are bioactive cyclic hexapeptides synthesized nonribosomally in cyanobacteria. APs are characterized by several conserved motifs, including the ureido bond, N-methylation in position 5, and d-Lys in position 2. All other positions of the AP molecule are variable, resulting in numerous structural variants. We have identified a nonribosomal peptide synthetase (NRPS) operon from Planktothrix agardhii strain CYA126/8 consisting of five genes (apnA to apnE) encoding six NRPS modules and have confirmed its role in AP synthesis by the generation of a mutant via insertional inactivation of apnC. In order to correlate the genetic diversity among adenylation domains (A domains) with AP structure variation, we sequenced the A domains of all six NRPS modules from seven Planktothrix strains differing in the production of AP congeners. It is remarkable that single strains coproduce APs bearing either of the chemically divergent amino acids Arg and Tyr in exocyclic position 1. Since the A domain of the initiation module (the ApnA A1 domain) has been proposed to activate the amino acid incorporated into exocyclic position 1, we decided to analyze this domain both biochemically and phylogenetically. Only ApnA A1 enzymes from strains producing AP molecules containing Arg or Tyr in position 1 were found to activate these two chemically divergent amino acids in vitro. Phylogenetic analysis of apn A domain sequences revealed that strains with a promiscuous ApnA A1 domain are derived from an ancestor that activates only Arg. Surprisingly, positive selection appears to affect only three codons within the apnA A1 gene, suggesting that this remarkable promiscuity has evolved from point mutations only.

Spatial divergence in the proportions of genes encoding toxic peptide synthesis among populations of the cyanobacterium Planktothrix in European lakes.

It has been frequently reported that seasonal changes in toxin production by cyanobacteria are due to changes in the proportion of toxic/nontoxic genotypes in parallel to increases or decreases in population density during the seasonal cycle of bloom formation. In order to find out whether there is a relationship between the proportion of genes encoding toxic peptide synthesis and population density of Planktothrix spp. we compared the proportion of three gene regions that are indicative of the synthesis of the toxic heptapeptide microcystin (mcyB), and the bioactive peptides aeruginoside (aerB) and anabaenopeptin (apnC) in samples from 23 lakes of five European countries (n=153). The mcyB, aerB, and apnC genes occurred in 99%, 99%, and 97% of the samples, respectively, and on average comprised 60 ± 3%, 22 ± 2%, and 54 ± 4% of the total population, respectively. Although the populations differed widely in abundance (10(-3)-10(3) mm(3) L(-1)) no dependence of the proportion of the mcyB, aerB, and apnC genes on the density of the total population was found. In contrast populations differed significantly in their average mcyB, aerB, and apnC gene proportions, with no change between prebloom and bloom conditions. These results emphasize stable population-specific differences in mcyB, aerB, and apnC proportions that are independent from seasonal influences.

Plasticity and evolution of aeruginosin biosynthesis in cyanobacteria.

Aeruginosins are bioactive oligopeptides that are produced in high structural diversity by strains of the bloom-forming cyanobacterial genera Microcystis and Planktothrix. A hallmark of aeruginosins is the unusual Choi moiety central to the tetrapeptides, while other positions are occupied by variable moieties in individual congeners. Here we report on three aeruginosin synthetase gene clusters (aer) of Microcystis aeruginosa (strains PCC 7806, NIES-98, and NIES-843). The analysis and comparison the aer gene clusters provide the first insight into the molecular basis of biosynthetic and structural plasticity in aeruginosin pathways. Major parts of the aer gene clusters are highly similar in all strains, particularly the genes coding for the first three nonribosomal peptide synthetase (NRPS) modules except for the region coding for the second adenylation domain. However, the gene clusters differ largely in genes coding for tailoring enzymes such as halogenases and sulfotransferases, reflecting structural peculiarities in aeruginosin congeners produced by the individual strains. Significant deviations were further observed in the C-terminal NRPS modules, suggesting two distinct release mechanisms. The architecture of the gene clusters is in agreement with the particular aeruginosin variants that are produced by individual strains, the structures of two of which (aeruginosins 686 A and 686 B) were elucidated. The aer gene clusters of Microcystis and Planktothrix are proposed to originate from a common ancestor and to have evolved to their present-day diversity largely through horizontal gene transfer and recombination events.

Post-translational modification in microviridin biosynthesis.

Cyanobacteria are prolific producers of bioactive natural products that mostly belong to the nonribosomal peptide and polyketide classes. We show here how a linear precursor peptide of microviridin K, a new member of the microviridin class of peptidase inhibitors, is processed to become the mature tricyclic peptidase inhibitor. The microviridin (mvd) biosynthetic gene cluster of P. agardhii comprises six genes encoding microviridin K, an apparently unexpressed second microviridin, two RimK homologues, an acetyltransferase, and an ABC transporter. We have over-expressed three enzymes of this pathway and have demonstrated their biochemical function in vitro through chemical degradation and mass spectrometry. We show that a prepeptide undergoes post-translational modification through cross-linking by ester and amide bond formation by the RimK homologues MvdD and MvdC, respectively. In silico analysis of the mvd gene cluster suggests the potential for widespread occurrence of microviridin-like compounds in a broad range of bacteria.

Isolation and structure determination of two microcystins and sequence comparison of the McyABC adenylation domains in Planktothrix species.

Microcystins (MCs) are toxic heptapeptides found in cyanobacteria and share the common structure cyclo(-d-Ala(1)-l-X(2)-d-isoMeAsp(3)-l-Z(4)-Adda(5)-d-isoGlu(6)-Mdha(7)). The letters X and Z in the general formula above represent a wide range of l-amino acids that occupy positions 2 and 4, respectively. In general the variation in structural variants is due to the exchange of amino acids in position 7, 2, and 4. In the present work we report two homotyrosine (Hty)-containing microcystin variants, [d-Asp(3),(E)-Dhb(7)]-MC-HtyY (1) and [d-Asp(3),(E)-Dhb(7)]-MC-HtyHty (2), which were isolated from strain No80 of Planktothrix rubescens. Their structures were elucidated using amino acid analysis as well as 1D and 2D NMR techniques. The adenylation domains of McyABC involved in amino acid activation in positions 7, 2, and 4 of the microcystin molecule, respectively, were compared with corresponding genes of Planktothrix strain CYA126/8 producing [d-Asp(3),Mdha(7)]-MC-RR and [d-Asp(3),Mdha(7)]-MC-LR. While the adenylation domain comparison of McyAB between the two Planktothrix strains revealed considerable DNA recombination, the adenylation domain of McyC showed only a single amino acid substitution, which was correlated with the replacement of Arg by Hty in position 4 of the microcystin molecule.

Nontoxic strains of cyanobacteria are the result of major gene deletion events induced by a transposable element.

Blooms that are formed by cyanobacteria consist of toxic and nontoxic strains. The mechanisms that result in the occurrence of nontoxic strains are enigmatic. All the nontoxic strains of the filamentous cyanobacterium Planktothrix that were isolated from 9 European countries were found to have lost 90% of a large microcystin synthetase (mcy) gene cluster that encoded the synthesis of the toxic peptide microcystin (MC). Those strains still contain the flanking regions of the mcy gene cluster along with remnants of the transposable elements that are found in between. The majority of the strains still contain a gene coding for a distinct thioesterase type II (mcyT), which is putatively involved in MC synthesis. The insertional inactivation of mcyT in an MC-producing strain resulted in the reduction of MC synthesis by 94 +/- 2% (1 standard deviation). Nontoxic strains that occur in shallow lakes throughout Europe form a monophyletic lineage. A second lineage consists of strains that contain the mcy gene cluster but differ in their photosynthetic pigment composition, which is due to the occurrence of strains that contain phycocyanin or large amounts of phycoerythrin in addition to phycocyanin. Strains containing phycoerythrin typically occur in deep-stratified lakes. The rare occurrence of gene cluster deletion, paired with the evolutionary diversification of the lineages of strains that lost or still contain the mcy gene cluster, needs to be invoked in order to explain the absence or dominance of toxic cyanobacteria in various habitats.

Biosynthesis and structure of aeruginoside 126A and 126B, cyanobacterial peptide glycosides bearing a 2-carboxy-6-hydroxyoctahydroindole moiety.

Aeruginosins represent a group of peptide metabolites isolated from various cyanobacterial genera and from marine sponges that potently inhibit different types of serine proteases. Members of this family are characterized by the presence of a 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety. We have identified and fully sequenced a NRPS gene cluster in the genome of the cyanobacterium Planktothrix agardhii CYA126/8. Insertional mutagenesis of a NRPS component led to the discovery and structural elucidation of two glycopeptides that were designated aeruginoside 126A and aeruginoside 126B. One variant of the aglycone contains a 1-amino-2-(N-amidino-Delta(3)-pyrrolinyl)ethyl moiety at the C terminus, the other bears an agmatine residue. In silico analyses of the aeruginoside biosynthetic genes aerA-aerI as well as additional mutagenesis and feeding studies allowed the prediction of enzymatic steps leading to the formation of aeruginosides and the unusual Choi moiety.

Natural products from cyanobacteria: Exploiting a new source for drug discovery.

In the 1990s, the pharmaceutical industry shifted its focus to a combinatorial chemistry approach to fill drug-discovery pipelines; however, more recently there has been renewed interest in natural products as sources of lead compounds. Cyanobacteria are prolific producers of natural products displaying enormous chemical diversity, yet, until recently, exploitation of the genera was hampered by a number of issues related to their handling. With most of these problems now resolved, cyanobacteria have the potential to expand the variety of natural products obtained from microorganisms. The relative disregard in the past of cyanobacteria compared with other microbial sources of natural products, as well as the huge chemical diversity and biological activities of their products, recommend them as an attractive source of novel drugs for use in diverse therapeutic areas.

Transposons inactivate biosynthesis of the nonribosomal peptide microcystin in naturally occurring Planktothrix spp.

The filamentous cyanobacteria Planktothrix spp. occur in the temperate region of the Northern hemisphere. The red-pigmented Planktothrix rubescens bacteria occur in deep, physically stratified, and less eutrophic lakes. Planktothrix is a known producer of the toxic heptapeptide microcystin (MC), which is produced nonribosomally by a large enzyme complex consisting of peptide synthetases and polyketide synthases encoded by a total of nine genes (mcy genes). Planktothrix spp. differ in their cellular MC contents as well as the production of MC variants; however, the mechanisms favoring this diversity are not understood. Recently, the occurrence of Planktothrix strains containing all mcy genes but lacking MC has been reported. In this study, 29 such strains were analyzed to find out if mutations of the mcy genes lead to the inability to synthesize MC. Two deletions, spanning 400 bp (in mcyB; one strain) and 1,869 bp (in mcyHA; three strains), and three insertions (IS), spanning 1,429 bp (in mcyD; eight strains), 1,433 bp (in mcyEG; one strain), and 1,433 bp (in mcyA; one strain), were identified. Though found in different genes and different isolates and transcribed in opposite directions, IS were found to be identical and contained conserved domains assigned to transposable elements. Using mutation-specific primers, two insertions (in mcyD and mcyA) and one deletion (in mcyHA) were found regularly in populations of P. rubescens in different lakes. The results demonstrate for the first time that different mutations resulting in inactivation of MC synthesis do occur frequently and make up a stable proportion of the mcy gene pool in Planktothrix populations over several years.

The microcystin composition of the cyanobacterium Planktothrix agardhii changes toward a more toxic variant with increasing light intensity.

The cyanobacterium Planktothrix agardhii, which is dominant in many shallow eutrophic lakes, can produce hepatotoxic microcystins. Currently, more than 70 different microcystin variants have been described, which differ in toxicity. In this study, the effect of photon irradiance on the production of different microcystin variants by P. agardhii was investigated using light-limited turbidostats. Both the amount of the mRNA transcript of the mcyA gene and the total microcystin production rate increased with photon irradiance up to 60 micromol m(-2) s(-1), but they started to decrease with irradiance greater than 100 micromol m(-2) s(-1). The cellular content of total microcystin remained constant, independent of the irradiance. However, of the two main microcystin variants detected in P. agardhii, the microcystin-DeRR content decreased twofold with increased photon irradiance, whereas the microcystin-DeLR content increased threefold. Since microcystin-DeLR is considerably more toxic than microcystin-DeRR, this implies that P. agardhii becomes more toxic at high light intensities.

Genetic identification of microcystin ecotypes in toxic cyanobacteria of the genus Planktothrix.

Microcystins (MCs) are toxic heptapeptides which are synthesized by the filamentous cyanobacterium Planktothrix and other genera via non-ribosomal peptide synthesis. MCs share the common structure cyclo(-D-ala1-L-X2-D-erythro-beta-iso-aspartic acid3-L-Z4-adda5-D-Glu6-N-methyl-dehydroalanine7) [Adda; (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid], in which numerous MC variants have been reported. In general, the variation in structure is due to different amino acid residues in positions 7, 2 and 4 within the MC molecule, which are thought to be activated by the adenylation domains mcyAAd1, mcyBAd1 and mcyCAd, respectively. It was the aim of the study (i) to identify MC ecotypes that differed in the production of specific MC variants and (ii) to correlate the genetic variation within adenylation domains with the observed MC variants among 17 Planktothrix strains. Comparison of the sequences of mcyAAd1 revealed two distinctive Ad-genotypes differing in base pair composition and the insertion of an N-methyl transferase (NMT) domain. The mcyAAd1 genotype with NMT (2854 bp) correlated with N-methyl-dehydroalanine and the mcyAAd1 genotype without NMT (1692 bp) correlated with dehydrobutyrine in position 7. Within mcyBAd1, a lower genetic variation (0-4 %) and an exclusive correlation between one Ad-genotype and homotyrosine as well as another Ad-genotype and arginine in position 2 was found. The sequences of mcyCAd were found to be highly similar (0-1 % dissimilarity) and all strains contained arginine in position 4. The results on adenylation domain polymorphism do provide insights into the evolutionary origin of adenylation domains in Planktothrix and may be combined with ecological research in order to provide clues about the abundance of genetically defined MC ecotypes in nature.

Diversity of coexisting Planktothrix (cyanobacteria) chemotypes deduced by mass spectral analysis of microystins and other oligopeptides.

Cyanobacteria are reported to produce secondary metabolites of which toxic and bioactive peptides are of scientific and public interest. Many peptides are synthesized by the non-ribosomal peptide synthesis pathway and their presence is a stable feature of individual clones. We isolated 18 clonal strains of Planktothrix from a single water sample from lake Maxsee near Berlin and analyzed them by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, HPLC, and PCR for their production of peptides and the presence of microcystin synthetase genes. Microcystins could be detected in seven of the strains with considerable variability of contents and numbers of structural variants. Other known peptides like anabaenopeptins B and E/F, microviridin I, and prenylagaramide B and new variants of known peptide classes like aeruginosins and cyanopeptolins were detected in some strains while lacking in others. The 18 strains represented 15 chemotypes with respect to their peptide patterns. In contrast, all strains were morphologically very similar with respect to cell dimensions and pigmentation. Given the diversity of chemotypes among the randomly selected isolates, an immense diversity of chemotypes in the entire population can be assumed.