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 (10−3–103 mm3 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

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

The genetic basis of toxin production in Cyanobacteria

The increasing incidence of mass developments of Cyanobacteria in fresh- and brackish water is a matter of growing concern due to the production of toxins that threaten human and livestock health.  The toxins that are produced by freshwater Cyanobacteria comprise hepatotoxins (cyclic peptides such as microcystins and nodularin, as well as alkaloids such as cylindrospermopsin) and neurotoxins (alkaloids such as anatoxin-a, anatoxin-a(S) and saxitoxins).  The variation in toxicity between and within species of Cyanobacteria has been recognised for a long time.  However, the toxic and non-toxic genotypes within a species cannot be discriminated under the microscope, which has been a major obstacle in identifying those factors that influence toxin production both in the laboratory and in the field.  During the last decade, major advances were achieved due to the elucidation and functional characterisation of genes, such as the gene cluster encoding the synthesis of the hepatotoxic heptapeptide, microcystin.  Genetic techniques, in particular, have been used to explore (i) the genetic basis, biosynthesis pathways, and physiological regulation of toxin (microcystin) production, (ii) gene loss processes resulting in a patchy distribution of the microcystin synthetase gene cluster among genera and species, as well as (iii) the distribution and abundance of the microcystin genes in the environment.  In recent years, experience in detecting microcystin genes directly in the field has increased enormously and robust protocols for the extraction of DNA and the subsequent detection of genes by PCR (polymerase chain reaction)- based methods are now available.  Due to the high sensitivity of PCR, it is possible to detect toxic genotypes long before a toxic cyanobacterial bloom may occur.  Consequently, waterbodies that are at risk of toxic bloom formation can be identified early on in the growing season along with environmental factors that can potentially influence the abundance of toxin producing genotypes

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

Christiansen G, Goesmann A, Kurmayer R. Elucidation of insertion elements carried on plasmids and in vitro construction of shuttle vectors from the toxic cyanobacterium planktothrix. Applied and environmental microbiology. 2014;80(16):4887-4897.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

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

Microcystin Biosynthesis in Planktothrix: Genes, Evolution, and Manipulation

Microcystins represent an extraordinarily large family of cyclic heptapeptide toxins that are nonribosomally synthesized by various cyanobacteria. Microcystins specifically inhibit the eukaryotic protein phosphatases 1 and 2A. Their outstanding variability makes them particularly useful for studies on the evolution of structure-function relationships in peptide synthetases and their genes. Analyses of microcystin synthetase genes provide valuable clues for the potential and limits of combinatorial biosynthesis. We have sequenced and analyzed 55.6 kb of the potential microcystin synthetase gene (mcy) cluster from the filamentous cyanobacterium Planktothrix agardhii CYA 126. The cluster contains genes for peptide synthetases (mcyABC), polyketide synthases (PKSs; mcyD), chimeric enzymes composed of peptide synthetase and PKS modules (mcyEG), a putative thioesterase (mcyT), a putative ABC transporter (mcyH), and a putative peptide-modifying enzyme (mcyJ). The gene content and arrangement and the sequence of specific domains in the gene products differ from those of the mcy cluster in Microcystis, a unicellular cyanobacterium. The data suggest an evolution of mcy clusters from, rather than to, genes for nodularin (a related pentapeptide) biosynthesis. Our data do not support the idea of horizontal gene transfer of complete mcy gene clusters between the genera. We have established a protocol for stable genetic transformation of Planktothrix, a genus that is characterized by multicellular filaments exhibiting continuous motility. Targeted mutation of mcyJ revealed its function as a gene coding for a O-methyltransferase. The mutant cells produce a novel microcystin variant exhibiting reduced inhibitory activity toward protein phosphatases