Complete Genome Sequence of Pseudomonas aeruginosa K34-7, a Carbapenem-Resistant Isolate of the High-Risk Sequence Type 233.

Carbapenem-resistant Pseudomonas aeruginosa is defined as a "critical" priority pathogen for the development of new antibiotics. Here we report the complete genome sequence of an extensively drug-resistant, Verona integron-encoded metallo-ß-lactamase-expressing isolate belonging to the high-risk sequence type 233.

Whole-genome sequencing of mutants with increased resistance against the two-peptide bacteriocin plantaricin JK reveals a putative receptor and potential docking site.

By whole-genome sequencing of resistant mutants, a putative receptor for plantaricin JK, a two-peptide bacteriocin produced by some Lactobacillus plantarum strains, was identified in Lactobacillus plantarum NCFB 965 and Weissella viridescens NCFB 1655. The receptors of the two species had 66% identical amino acid sequences and belong to the amino acid-polyamine-organocation (APC) transporter protein family. The resistant mutants contained point mutations in the protein-encoding gene resulting in either premature stop codons, leading to truncated versions of the protein, or single amino acid substitutions. The secondary structure of the W. viridescens protein was predicted to contain 12 transmembrane (TM) helices, a core structure shared by most members of the APC protein family. The single amino acid substitutions that resulted in resistant strains were located in a confined region of the protein that consists of TM helix 10, which is predicted to be part of an inner membrane pore, and an extracellular loop between TM helix 11 and 12. By use of template-based modeling a 3D structure model of the protein was obtained, which visualizes this mutational hotspot region and further strengthen the hypothesis that it represents a docking site for plantaricin JK.

Complete Genome Sequence of a Multidrug-Resistant, blaNDM-1-Expressing Klebsiella pneumoniae K66-45 Clinical Isolate from Norway.

Multidrug-resistant Klebsiella pneumoniae is a major cause of hospital-acquired infections. Here, we report the complete genome sequence of the multidrug-resistant, blaNDM-1-positive strain K. pneumoniae K66-45, isolated from a hospitalized Norwegian patient.

Single Cell Transcriptomics, Mega-Phylogeny, and the Genetic Basis of Morphological Innovations in Rhizaria.

The innovation of the eukaryote cytoskeleton enabled phagocytosis, intracellular transport, and cytokinesis, and is largely responsible for the diversity of morphologies among eukaryotes. Still, the relationship between phenotypic innovations in the cytoskeleton and their underlying genotype is poorly understood. To explore the genetic mechanism of morphological evolution of the eukaryotic cytoskeleton, we provide the first single cell transcriptomes from uncultured, free-living unicellular eukaryotes: the polycystine radiolarian Lithomelissa setosa (Nassellaria) and Sticholonche zanclea (Taxopodida). A phylogenomic approach using 255 genes finds Radiolaria and Foraminifera as separate monophyletic groups (together as Retaria), while Cercozoa is shown to be paraphyletic where Endomyxa is sister to Retaria. Analysis of the genetic components of the cytoskeleton and mapping of the evolution of these on the revised phylogeny of Rhizaria reveal lineage-specific gene duplications and neofunctionalization of α and ß tubulin in Retaria, actin in Retaria and Endomyxa, and Arp2/3 complex genes in Chlorarachniophyta. We show how genetic innovations have shaped cytoskeletal structures in Rhizaria, and how single cell transcriptomics can be applied for resolving deep phylogenies and studying gene evolution in uncultured protist species.

A putative amino acid transporter determines sensitivity to the two-peptide bacteriocin plantaricin JK.

Lactobacillus plantarum produces a number of antimicrobial peptides (bacteriocins) that mostly target closely related bacteria. Although bacteriocins are important for the ecology of these bacteria, very little is known about how the peptides target sensitive cells. In this work, a putative membrane protein receptor of the two-peptide bacteriocin plantaricin JK was identified by comparing Illumina sequence reads from plantaricin JK-resistant mutants to a crude assembly of the sensitive wild-type Weissella viridescens genome using the polymorphism discovery tool VAAL. Ten resistant mutants harbored altogether seven independent mutations in a gene encoding an APC superfamily protein with 12 transmembrane helices. The APC superfamily transporter thus is likely to serve as a target for plantaricin JK on sensitive cells.

Sensitivity to the two-peptide bacteriocin lactococcin G is dependent on UppP, an enzyme involved in cell-wall synthesis.

Most bacterially produced antimicrobial peptides (bacteriocins) are thought to kill target cells by a receptor-mediated mechanism. However, for most bacteriocins the receptor is unknown. For instance, no target receptor has been identified for the two-peptide bacteriocins (class IIb), whose activity requires the combined action of two individual peptides. To identify the receptor for the class IIb bacteriocin lactococcin G, which targets strains of Lactococcus lactis, we generated 12 lactococcin G-resistant mutants and performed whole-genome sequencing to identify mutations causing the resistant phenotype. Remarkably, all had a mutation in or near the gene uppP (bacA), encoding an undecaprenyl pyrophosphate phosphatase; a membrane protein involved in peptidoglycan synthesis. Nine mutants had stop codons or frameshifts in the uppP gene, two had point mutations in putative regulatory regions and one caused an amino acid substitution in UppP. To verify the receptor function of UppP, it was shown that growth of non-sensitive Streptococcus pneumoniae could be inhibited by lactococcin G when L. lactis uppP was expressed in this bacterium. Furthermore, we show that the related class IIb bacteriocin enterocin 1071 also uses UppP as receptor. The approach used here should be broadly applicable to identify receptors for other bacteriocins as well.

A Zn-dependent metallopeptidase is responsible for sensitivity to LsbB, a class II leaderless bacteriocin of Lactococcus lactis subsp. lactis BGMN1-5.

Lactococcus lactis subsp. lactis BGMN1-5 produces a leaderless class II bacteriocin called LsbB. To identify the receptor for LsbB, a cosmid library of the LsbB-sensitive strain BGMN1-596 was constructed. About 150 cosmid clones were individually isolated and transferred to LsbB-resistant mutants of BGMN1-596. Cosmid pAZILcos/MN2, carrying a 40-kb insert, was found to restore LsbB sensitivity in LsbB-resistant mutants. Further subcloning revealed that a 1.9-kb fragment, containing only one open reading frame, was sufficient to restore sensitivity. The fragment contains the gene yvjB coding for a Zn-dependent membrane-bound metallopeptidase, suggesting that this gene may serve as the receptor for LsbB. Further support for this notion derives from several independent experiments: (i) whole-genome sequencing confirmed that all LsbB-resistant mutants contain mutations in yvjB; (ii) disruption of yvjB by direct gene knockout rendered sensitive strains BGMN1-596 and IL1403 resistant to LsbB; and (iii) most compellingly, heterologous expression of yvjB in naturally resistant strains of other species, such as Lactobacillus paracasei and Enterococcus faecalis, also rendered them sensitive to the bacteriocin. To our knowledge, this is the first time a membrane-bound peptidase gene has been shown to be involved in bacteriocin sensitivity in target cells. We also demonstrated a novel successful approach for identifying bacteriocin receptors.

Gene flow, recombination, and selection in cyanobacteria: population structure of geographically related Planktothrix freshwater strains.

Several Planktothrix strains, each producing a distinct oligopeptide profile, have been shown to coexist within Lake Steinsfjorden (Norway). Using nonribosomal peptide synthetase (NRPS) genes as markers, it has been shown that the Planktothrix community comprises distinct genetic variants displaying differences in bloom dynamics, suggesting a Planktothrix subpopulation structure. Here, we investigate the Planktothrix variants inhabiting four lakes in southeast of Norway utilizing both NRPS and non-NRPS genes. Phylogenetic analyses showed similar topologies for both NRPS and non-NRPS genes, and the lakes appear to have similar structuring of Planktothrix genetic variants. The structure of distinct variants was also supported by very low genetic diversity within variants compared to the between-variant diversity. Incongruent topologies and split decomposition revealed recombination events between Planktothrix variants. In several strains the gene variants seem to be a result of recombination. Both NRPS and non-NRPS genes are dominated by purifying selection; however, sites subjected to positive selection were also detected. The presence of similar and well-separated Planktothrix variants with low internal genetic diversity indicates gene flow within Planktothrix populations. Further, the low genetic diversity found between lakes (similar range as within lakes) indicates gene flow also between Planktothrix populations and suggests recent, or recurrent, dispersals. Our data also indicate that recombination has resulted in new genetic variants. Stability within variants and the development of new variants are likely to be influenced by selection patterns and within-variant homologous recombination.

Metagenomic and geochemical characterization of pockmarked sediments overlaying the Troll petroleum reservoir in the North Sea.

BACKGROUND: Pockmarks (depressions in the seabed) have been discovered throughout the world's oceans and are often related to hydrocarbon seepage. Although high concentrations of pockmarks are present in the seabed overlaying the Troll oil and gas reservoir in the northern North Sea, geological surveys have not detected hydrocarbon seepage in this area at the present time. In this study we have used metagenomics to characterize the prokaryotic communities inhabiting the surface sediments in the Troll area in relation to geochemical parameters, particularly related to hydrocarbon presence. We also investigated the possibility of increased potential for methane oxidation related to the pockmarks. Five metagenomes from pockmarks and plain seabed sediments were sequenced by pyrosequencing (Roche/454) technology. In addition, two metagenomes from seabed sediments geologically unlikely to be influenced by hydrocarbon seepage (the Oslofjord) were included. The taxonomic distribution and metabolic potential of the metagenomes were analyzed by multivariate analysis and statistical comparisons to reveal variation within and between the two sampling areas. RESULTS: The main difference identified between the two sampling areas was an overabundance of predominantly autotrophic nitrifiers, especially Nitrosopumilus, and oligotrophic marine Gammaproteobacteria in the Troll metagenomes compared to the Oslofjord. Increased potential for degradation of hydrocarbons, especially aromatic hydrocarbons, was detected in two of the Troll samples: one pockmark sample and one from the plain seabed. Although presence of methanotrophic organisms was indicated in all samples, no overabundance in pockmark samples compared to the Oslofjord samples supports no, or only low level, methane seepage in the Troll pockmarks at the present time. CONCLUSIONS: Given the relatively low content of total organic carbon and great depths of hydrocarbon containing sediments in the Troll area, it is possible that at least part of the carbon source available for the predominantly autotrophic nitrifiers thriving in this area originates from sequential prokaryotic degradation and oxidation of hydrocarbons to CO2. By turning CO2 back into organic carbon this subcommunity could play an important environmental role in these dark oligotrophic sediments. The oxidation of ammonia to nitrite and nitrate in this process could further increase the supply of terminal electron acceptors for hydrocarbon degradation.

Radiolaria associated with large diversity of marine alveolates.

We have isolated cells of unculturable radiolarians from marine coastal waters. Individual cells were subjected to single cell whole genome amplification (SCWGA) and gene-targeted PCR. Using this approach we recover a surprisingly large diversity of sequences related to the enigmatic marine alveolate groups 1 and 2 (MALV I and MALV II) that most likely represent intracellular symbionts or parasites of the radiolarian cells. 18S rDNA phylogeny of the MALV sequences reveals 4 distinct clades of radiolarian associates here named Radiolarian Associated Sequences (RAS) 1-4. One clade of both phaeodarian and radiolarian associates and one clade of only phaeodarian associates are also identified. The MALV sequences cluster according to host type, i.e. sequences from associates identified in radiolarians, fish, copepods, ciliates or dinoflagellates are not intermixed but separated into distinct clades. This implies several independent colonizations of host lineages and links a large diversity of MALV to radiolarian-associated species. This demonstrates that radiolarians may be an important reservoir for MALV, making them a key group for understanding the impact of intracellular symbionts on the marine ecosystem. This study shows that applying SCWGA on unculturable cells is a promising approach to study the vast diversity and interactions of intracellular eukaryote organisms.

A metagenomic study of methanotrophic microorganisms in Coal Oil Point seep sediments.

BACKGROUND: Methane oxidizing prokaryotes in marine sediments are believed to function as a methane filter reducing the oceanic contribution to the global methane emission. In the anoxic parts of the sediments, oxidation of methane is accomplished by anaerobic methanotrophic archaea (ANME) living in syntrophy with sulphate reducing bacteria. This anaerobic oxidation of methane is assumed to be a coupling of reversed methanogenesis and dissimilatory sulphate reduction. Where oxygen is available aerobic methanotrophs take part in methane oxidation. In this study, we used metagenomics to characterize the taxonomic and metabolic potential for methane oxidation at the Tonya seep in the Coal Oil Point area, California. Two metagenomes from different sediment depth horizons (0-4 cm and 10-15 cm below sea floor) were sequenced by 454 technology. The metagenomes were analysed to characterize the distribution of aerobic and anaerobic methanotrophic taxa at the two sediment depths. To gain insight into the metabolic potential the metagenomes were searched for marker genes associated with methane oxidation. RESULTS: Blast searches followed by taxonomic binning in MEGAN revealed aerobic methanotrophs of the genus Methylococcus to be overrepresented in the 0-4 cm metagenome compared to the 10-15 cm metagenome. In the 10-15 cm metagenome, ANME of the ANME-1 clade, were identified as the most abundant methanotrophic taxon with 8.6% of the reads. Searches for particulate methane monooxygenase (pmoA) and methyl-coenzyme M reductase (mcrA), marker genes for aerobic and anaerobic oxidation of methane respectively, identified pmoA in the 0-4 cm metagenome as Methylococcaceae related. The mcrA reads from the 10-15 cm horizon were all classified as originating from the ANME-1 clade. CONCLUSIONS: Most of the taxa detected were present in both metagenomes and differences in community structure and corresponding metabolic potential between the two samples were mainly due to abundance differences. The results suggests that the Tonya Seep sediment is a robust methane filter, where taxa presently dominating this process could be replaced by less abundant methanotrophic taxa in case of changed environmental conditions.

Radiolaria divided into Polycystina and Spasmaria in combined 18S and 28S rDNA phylogeny.

Radiolarians are marine planktonic protists that belong to the eukaryote supergroup Rhizaria together with Foraminifera and Cercozoa. Radiolaria has traditionally been divided into four main groups based on morphological characters; i.e. Polycystina, Acantharia, Nassellaria and Phaeodaria. But recent 18S rDNA phylogenies have shown that Phaeodaria belongs within Cerocozoa, and that the previously heliozoan group Taxopodida should be included in Radiolaria. 18S rDNA phylogenies have not yet resolved the sister relationship between the main Radiolaria groups, but nevertheless suggests that Spumellaria, and thereby also Polycystina, are polyphyletic. Very few sequences other than 18S rDNA have so far been generated from radiolarian cells, mostly due to the fact that Radiolaria has been impossible to cultivate and single cell PCR has been hampered by low success rate. Here we have therefore investigated the mutual evolutionary relationship of the main radiolarian groups by using the novel approach of combining single cell whole genome amplification with targeted PCR amplification of the 18S and 28S rDNA genes. Combined 18S and 28S phylogeny of sequences obtained from single cells shows that Radiolaria is divided into two main lineages: Polycystina (Spumellaria+Nassellaria) and Spasmaria (Acantharia+Taxopodida). Further we show with high support that Foraminifera groups within Radiolaria supporting the Retaria hypothesis.

A genome-wide analysis of nonribosomal peptide synthetase gene clusters and their peptides in a Planktothrix rubescens strain.

BACKGROUND: Cyanobacteria often produce several different oligopeptides, with unknown biological functions, by nonribosomal peptide synthetases (NRPS). Although some cyanobacterial NRPS gene cluster types are well described, the entire NRPS genomic content within a single cyanobacterial strain has never been investigated. Here we have combined a genome-wide analysis using massive parallel pyrosequencing ("454") and mass spectrometry screening of oligopeptides produced in the strain Planktothrix rubescens NIVA CYA 98 in order to identify all putative gene clusters for oligopeptides. RESULTS: Thirteen types of oligopeptides were uncovered by mass spectrometry (MS) analyses. Microcystin, cyanopeptolin and aeruginosin synthetases, highly similar to already characterized NRPS, were present in the genome. Two novel NRPS gene clusters were associated with production of anabaenopeptins and microginins, respectively. Sequence-depth of the genome and real-time PCR data revealed three copies of the microginin gene cluster. Since NRPS gene cluster candidates for microviridin and oscillatorin synthesis could not be found, putative (gene encoded) precursor peptide sequences to microviridin and oscillatorin were found in the genes mdnA and oscA, respectively. The genes flanking the microviridin and oscillatorin precursor genes encode putative modifying enzymes of the precursor oligopeptides. We therefore propose ribosomal pathways involving modifications and cyclisation for microviridin and oscillatorin. The microviridin, anabaenopeptin and cyanopeptolin gene clusters are situated in close proximity to each other, constituting an oligopeptide island. CONCLUSION: Altogether seven nonribosomal peptide synthetase (NRPS) gene clusters and two gene clusters putatively encoding ribosomal oligopeptide biosynthetic pathways were revealed. Our results demonstrate that whole genome shotgun sequencing combined with MS-directed determination of oligopeptides successfully can identify NRPS gene clusters and the corresponding oligopeptides. The analyses suggest independent evolution of all NRPS gene clusters as functional units. Our data indicate that the Planktothrix genome displays evolution of dual pathways (NRPS and ribosomal) for production of oligopeptides in order to maximize the diversity of oligopeptides with similar but functional discrete bioactivities.

Expression of genes involved in GABAergic neurotransmission in anoxic crucian carp brain (Carassius carassius).

The crucian carp, Carassius carassius, survives days to months without oxygen, depending on temperature. In the anoxic crucian carp brain, increased GABAergic inhibition, mediated by increased extracellular levels of GABA, has been shown to suppress electric activity and ATP consumption. To investigate an involvement of gene expression in this response, we utilized real-time RT-PCR to test the effect of 1 and 7 days anoxia (8 degrees C) on the expression of 22 genes, including nine GABA(A) receptor subunits (alpha(1-6), beta(2), delta, and gamma(2)), three GABA(B) receptor subunits (G(B)1a-1b and G(B)2), three enzymes involved in GABA metabolism (GAD65 and GAD67, GABAT), four GABA transporters (GAT1, 2a-b and 3), two GABA(A) receptor-associated proteins (GABARAP 1 and 2), and the K(+)/Cl(-) cotransporter KCC2. While the expression of GABA(A) receptor subunits was dominated by alpha(4)-, alpha(6)-, and delta-subunits, all of which are located to extrasynaptic sites in mammalian brains and respond to elevations in extracellular levels of GABA by showing tonic activity patterns, the expression of GABA transporters was dominated by GAT2 (a and b) and GAT3, which also show extrasynaptic location in mammals. These expression patterns differ from those observed in mammals and may be a prerequisite for GABAergic inhibition of anoxic metabolic rate in crucian carp. Furthermore, while the expression of the majority of the genes was largely unaltered by anoxia, the expression of GAT2 and GAT3 decreased to 20%. This suggests impairment of GABA transport, which could be a mechanism behind the accumulation of extracellular GABA and the increased GABAergic inhibition.

Evidence for positive selection acting on microcystin synthetase adenylation domains in three cyanobacterial genera.

BACKGROUND: Cyanobacteria produce a wealth of secondary metabolites, including the group of small cyclic heptapeptide hepatotoxins that constitutes the microcystin family. The enzyme complex that directs the biosynthesis of microcystin is encoded in a single large gene cluster (mcy). mcy genes have a widespread distribution among cyanobacteria and are likely to have an ancient origin. The notable diversity within some of the Mcy modules is generated through various recombination events including horizontal gene transfer. RESULTS: A comparative analysis of the adenylation domains from the first module of McyB (McyB1) and McyC in the microcystin synthetase complex was performed on a large number of microcystin-producing strains from the Anabaena, Microcystis and Planktothrix genera. We found no decisive evidence for recombination between strains from different genera. However, we detected frequent recombination events in the mcyB and mcyC genes between strains within the same genus. Frequent interdomain recombination events were also observed between mcyB and mcyC sequences in Anabaena and Microcystis. Recombination and mutation rate ratios suggest that the diversification of mcyB and mcyC genes is driven by recombination events as well as point mutations in all three genera. Sequence analysis suggests that generally the adenylation domains of the first domain of McyB and McyC are under purifying selection. However, we found clear evidence for positive selection acting on a number of amino acid residues within these adenylation domains. These include residues important for active site selectivity of the adenylation domain, strongly suggesting selection for novel microcystin variants. CONCLUSION: We provide the first clear evidence for positive selection acting on amino acid residues involved directly in the recognition and activation of amino acids incorporated into microcystin, indicating that the microcystin complement of a given strain may influence the ability of a particular strain to interact with its environment.

Recombination and selectional forces in cyanopeptolin NRPS operons from highly similar, but geographically remote Planktothrix strains.

BACKGROUND: Cyanopeptolins are nonribosomally produced heptapetides showing a highly variable composition. The cyanopeptolin synthetase operon has previously been investigated in three strains from the genera Microcystis, Planktothrix and Anabaena. Cyanopeptolins are displaying protease inhibitor activity, but the biological function(s) is (are) unknown. Cyanopeptolin gene cluster variability and biological functions of the peptide variants are likely to be interconnected. RESULTS: We have investigated two cyanopeptolin gene clusters from highly similar, but geographically remote strains of the same genus. Sequencing of a nonribosomal peptide synthetase (NRPS) cyanopeptolin gene cluster from the Japanese strain Planktothrix NIES 205 (205-oci), showed the 30 kb gene cluster to be highly similar to the oci gene cluster previously described in Planktothrix NIVA CYA 116, isolated in Norway. Both operons contained seven NRPS modules, a sulfotransferase (S) and a glyceric acid loading (GA)-domain. Sequence analyses showed a high degree of conservation, except for the presence of an epimerase domain in NIES 205 and the regions around the epimerase, showing high substitution rates and Ka/Ks values above 1. The two strains produce almost identical cyanopeptolins, cyanopeptolin-1138 and oscillapeptin E respectively, but with slight differences regarding the production of minor cyanopeptolin variants. These variants may be the result of relaxed adenylation (A)-domain specificity in the nonribosomal enzyme complex. Other genetic markers (16S rRNA, ntcA and the phycocyanin cpcBA spacer) were identical, supporting that these geographically separated Planktothrix strains are closely related. CONCLUSION: A horizontal gene transfer event resulting in exchange of a whole module-encoding region was observed. Nucleotide statistics indicate that both purifying selection and positive selection forces are operating on the gene cluster. The positive selection forces are acting within and around the epimerase insertion while purifying selection conserves the remaining (major) part of the gene cluster. The presence of an epimerase in the gene cluster is in line with the D-configuration of Htyr, determined experimentally in oscillapeptin E in a previous study.

Expression of genes involved in excitatory neurotransmission in anoxic crucian carp (Carassius carassius) brain.

The crucian carp, Carassius carassius, survives months without oxygen. During anoxia it needs to keep energy expenditure low, particularly in the brain, with its high rate of ATP use related to neuronal activity. This could be accomplished by reducing neuronal excitability through altered expression of genes involved in excitatory neurotransmission. Through cloning and the use of a recently developed real-time RT-PCR approach, with an external RNA control for normalization, we investigated the effect of 1 and 7 days of anoxia (12 degrees C) on the expression of 29 genes, including 8 3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits, 6 N-methyl-d-aspartate (NMDA) receptor subunits, 7 voltage-gated sodium and calcium channels, 4 glutamate transporters, and 4 genes involved in NMDA receptor-mediated neuroplasticity. The subunits of the majority of the gene families had expression profiles similar to those observed in the mammalian brain and showed remarkably stable expression during anoxia. This suggests that the genes may have similar functions in crucian carp and mammals, and that the excitatory abilities of the crucian carp brain are retained during anoxia. Although the data generally argue against profound neural depression ("channel arrest"), NMDA receptor subunit (NR) expression showed features that could mediate reduced neural excitability. Primarily, the NR2 subunit expression, which was dominated by NR2B and NR2D, resembled that seen in hypoxia-tolerant neonatal rats, and decreased anoxic expression of NR1, NR2C, and NR3A indicated reduced numbers of functional NMDA receptors. We also report the full-length sequence of crucian carp NR1 mRNA and a novel NR1 splice cassette introducing an N-glycosylation site into the extracellular S1S2 domain.

The mosaic structure of the mcyABC operon in Microcystis.

An extensive study of the mcyABC genes and regions flanking the mcy gene cluster was performed in naturally occurring Microcystis strains. Lack of methylation in strains producing only desmethyl(7)-microcystin was found to be associated with point mutations in substrate-binding sequence motifs of the N-methyltransferase (NMT) domain in McyA. Multiple recombination events giving rise to 'phylogenetic mosaics' were detected within the NMT-domain-encoding mcyA sequences and the adenylation (A) domain sequences of mcyB and mcyC. Recombination leading to exchanges between the mcyB and mcyC regions encoding A domains in modules McyB1 and McyC was also detected. A previously reported replacement of the A domain in McyB1 was found to involve the region between the conserved motifs A3 and A8/A9. In all microcystin-producing strains the mcy gene cluster was flanked by the genes uma1 and dnaN. Clear indications of recombination, an insertion element and footprints of IS elements were found in the dnaN-mcyJ intergenic region. Among the non-microcystin producers, uma1 and dnaN were linked in some, but not all strains. Most non-producing strains lacked all mcy genes, while one strain possessed a partially deleted mcy operon. Our results show that frequent horizontal gene transfer events in addition to point mutations and insertions/deletions contribute to variation in the mcy gene cluster.

Natural occurrence of microcystin synthetase deletion mutants capable of producing microcystins in strains of the genus Anabaena (Cyanobacteria).

Microcystins form a large family of small cyclic heptapeptides harbouring extensive modifications in amino acid residue composition and functional group chemistry. These peptide hepatotoxins contain a range of non-proteinogenic amino acids and unusual peptide bonds, and are typically N-methylated. They are synthesized on large enzyme complexes consisting of non-ribosomal peptide synthetases and polyketide synthases in a variety of distantly related cyanobacterial genera. Here we report a 1236 bp in-frame deletion mutation in the mcyA gene of the microcystin biosynthetic pathway in nine strains of the genus Anabaena. The deletion removed almost the entire N-methyltransferase (NMT) domain. Strains of Anabaena carrying the in-frame deletion mutation incorporated mainly dehydroalanine (Dha) into the microcystins they produce while strains with full-length mcyA genes incorporated mainly N-methyldehydroalanine (Mdha). Interestingly, the strains of Anabaena lacking the NMT domain also incorporated elevated amounts of L-Ser, the precursor of Mdha and Dha, into the microcystin they produced relative to strains carrying functional NMT domains. We provide evidence for the in-frame deletion of the NMT domain without the co-conversion of the flanking adenylation domain. Our results demonstrate a further example of the strategies employed by cyanobacteria in the biosynthesis of microcystin variants.