Eukaryotic Protein Phosphatases Research Articles

Identification of a homoarginine biosynthetic gene from a microcystin biosynthetic pathway in Fischerella sp. PCC 9339

Microcystins (MCs) are a family of chemically diverse toxins produced by numerous distantly related cyanobacteria. They are potent inhibitors of eukaryotic protein phosphatases 1 and 2A and are responsible for the toxicosis and death of wild and domestic animals around the world. Microcystins are synthesized on large enzyme complexes comprised of peptide synthetases, polyketide synthases, and additional modifying enzymes. Bioinformatic analysis identified the presence of an additional uncharacterized enzyme in the microcystin (mcy) biosynthetic gene cluster in Fischerella sp. PCC 9339, which we named McyK, that lacked a clearly defined role in the biosynthesis of microcystin. Further bioinformatic analysis suggested that McyK belongs to the inosamine-phosphate amidinotransferase family and could be involved in synthesizing homo amino acids. Quadrupole time-of-flight tandem mass spectrometry (Q-TOFMS/MS) analysis confirmed that Fischerella sp. PCC 9339 produces MC-Leucine2-Homoarginine4(MC-LHar) and [Aspartic acid3]MC-Leucine2-Homoarginine4 ([Asp3]MC-LHar) as the dominant chemical variants. We hypothesized that the McyK enzyme might be involved in the production of microcystin variants containing homoarginine (Har) in the strain. Heterologous expression of a codon-optimized mcyK gene in Escherichia coli confirmed that McyK is responsible for the synthesis of L-Har. These results confirm the production of MC-LHar, a novel microcystin chemical variant [Asp3]MC-LHar, and a new microcystin biosynthetic enzyme involved in supply of the rare homo-amino acid Har to the microcystin biosynthetic pathway in Fischerella sp. PCC 9339. This study provides new insights into the logic underpinning the biosynthesis of microcystin chemical variants and broadens our knowledge of structural diversity of the microcystin family of toxins.

Protein phosphatases and their targets: Comprehending the interactions in plant signaling pathways.

Protein phosphorylation is a vital reversible post-translational modification. This process is established by two classes of enzymes: protein kinases and protein phosphatases. Protein kinases phosphorylate proteins while protein phosphatases dephosphorylate phosphorylated proteins, thus, functioning as 'critical regulators' in signaling pathways. The eukaryotic protein phosphatases are classified as phosphoprotein phosphatases (PPP), metallo-dependent protein phosphatases (PPM), protein tyrosine (Tyr) phosphatases (PTP), and aspartate (Asp)-dependent phosphatases. The PPP and PPM families are serine (Ser)/threonine (Thr) specific phosphatases (STPs) that dephosphorylate Ser and Thr residues. The PTP family dephosphorylates Tyr residues while dual-specificity phosphatases (DsPTPs/DSPs) dephosphorylate Ser, Thr, and Tyr residues. The composition of these enzymes as well as their substrate specificity are important determinants of their functional significance in a number of cellular processes and stress responses. Their role in animal systems is well-understood and characterized. The functional characterization of protein phosphatases has been extensively covered in plants, although the comprehension of their mechanistic basis is an ongoing pursuit. The nature of their interactions with other key players in the signaling process is vital to our understanding. The substrates or targets determine their potential as well as magnitude of the impact they have on signaling pathways. In this article, we exclusively overview the various substrates of protein phosphatases in plant signaling pathways, which are a critical determinant of the outcome of various developmental and stress stimuli.

Comprehensive and functional analyses reveal the genomic diversity and potential toxicity of Microcystis

Microcystis is a cyanobacteria that is widely distributed across the world. It has attracted great attention because it produces the hepatotoxin microcystin (MC) that can inhibit eukaryotic protein phosphatases and pose a great risk to animal and human health. Due to the high diversity of morphospecies and genomes, it is still difficult to classify Microcystis species. In this study, we investigated the pangenome of 23 Microcystis strains to detect the genetic diversity and evolutionary dynamics. Microcystis revealed an open pangenome containing 22,009 gene families and exhibited different functional constraints. The core-genome phylogenetic analysis accurately differentiated the toxic and nontoxic strains and could be used as a taxonomic standard at the genetic level. We also investigated the functions of HGT events, of which were mostly conferred from cyanobacteria and closely related species. In order to detect the potential toxicity of Microcystis, we searched and characterized MC biosynthetic gene clusters and other secondary metabolite gene clusters. Our work provides insights into the genetic diversity, evolutionary dynamics, and potential toxicity of Microcystis, which could benefit the species classification and development of new methods for drinking water quality control and management of bloom formation in the future.

Protein Phosphatase 2A Catalytic Subunit PP2A-1 Enhances Rice Resistance to Sheath Blight Disease.

Rice (Oryza sativa) production is damaged to a great extent by sheath blight disease (ShB). However, the defense mechanism in rice against this disease is largely unknown. Previous transcriptome analysis identified a significantly induced eukaryotic protein phosphatase 2A catalytic subunit 1 (PP2A-1) after the inoculation of Rhizoctonia solani. Five genes encoding PP2A exist in rice genome, and these five genes are ubiquitously expressed in different tissues and stages. Inoculation of R. solani showed that the genome edited pp2a-1 mutants using the CRISPR/Cas9 were more susceptible to ShB than the wild-type control, but other PP2A gene mutants exhibited similar response to ShB compared to wild-type plants. In parallel, PP2A-1 expression level was higher in the activation tagging line, and PP2A-1 overexpression inhibited plant height and promoted the resistance to ShB. PP2A-1-GFP was localized in the cytoplasm and nucleus. In addition, R. solani-dependent induction kinetics of pathogen-related genes PBZ1 and PR1b was lower in pp2a-1 mutants but higher in PP2A-1 activation line compared to those in the wild-type. In conclusion, our analysis shows that PP2A-1 is a member of protein phosphatase, which regulates rice resistance to ShB. This result broadens the understanding of the defense mechanism against ShB and provides a potential target for rice breeding for disease resistance.

Plant protein phosphatases: What do we know about their mechanism of action?

Protein phosphorylation is a major reversible post-translational modification. Protein phosphatases function as 'critical regulators' in signaling networks through dephosphorylation of proteins, which have been phosphorylated by protein kinases. A large understanding of their working has been sourced from animal systems rather than the plant or the prokaryotic systems. The eukaryotic protein phosphatases include phosphoprotein phosphatases (PPP), metallo-dependent protein phosphatases (PPM), protein tyrosine (Tyr) phosphatases (PTP), and aspartate (Asp)-dependent phosphatases. The PPP and PPM families are serine(Ser)/threonine(Thr)-specific phosphatases (STPs), while PTP family is Tyr specific. Dual-specificity phosphatases (DsPTPs/DSPs) dephosphorylate Ser, Thr, and Tyr residues. PTPs lack sequence homology with STPs, indicating a difference in catalytic mechanisms, while the PPP and PPM families share a similar structural fold indicating a common catalytic mechanism. The catalytic cysteine (Cys) residue in the conserved HCX5 R active site motif of the PTPs acts as a nucleophile during hydrolysis. The PPP members require metal ions, which coordinate the phosphate group of the substrate, followed by a nucleophilic attack by a water molecule and hydrolysis. The variable holoenzyme assembly of protein phosphatase(s) and the overlap with other post-translational modifications like acetylation and ubiquitination add to their complexity. Though their functional characterization is extensively reported in plants, the mechanistic nature of their action is still being explored by researchers. In this review, we exclusively overview the plant protein phosphatases with an emphasis on their mechanistic action as well as structural characteristics.

The Biosynthesis of Rare Homo-Amino Acid Containing Variants of Microcystin by a Benthic Cyanobacterium.

Microcystins are a family of chemically diverse hepatotoxins produced by distantly related cyanobacteria and are potent inhibitors of eukaryotic protein phosphatases 1 and 2A. Here we provide evidence for the biosynthesis of rare variants of microcystin that contain a selection of homo-amino acids by the benthic cyanobacterium Phormidium sp. LP904c. This strain produces at least 16 microcystin chemical variants many of which contain homophenylalanine or homotyrosine. We retrieved the complete 54.2 kb microcystin (mcy) gene cluster from a draft genome assembly. Analysis of the substrate specificity of McyB1 and McyC adenylation domain binding pockets revealed divergent substrate specificity sequences, which could explain the activation of homo-amino acids which were present in 31% of the microcystins detected and included variants such as MC-LHty, MC-HphHty, MC-LHph and MC-HphHph. The mcy gene cluster did not encode enzymes for the synthesis of homo-amino acids but may instead activate homo-amino acids produced during the synthesis of anabaenopeptins. We observed the loss of microcystin during cultivation of a closely related strain, Phormidium sp. DVL1003c. This study increases the knowledge of benthic cyanobacterial strains that produce microcystin variants and broadens the structural diversity of known microcystins.

IEKPD 2.0: an update with rich annotations for eukaryotic protein kinases, protein phosphatases and proteins containing phosphoprotein-binding domains.

Here, we described the updated database iEKPD 2.0 (http://iekpd.biocuckoo.org) for eukaryotic protein kinases (PKs), protein phosphatases (PPs) and proteins containing phosphoprotein-binding domains (PPBDs), which are key molecules responsible for phosphorylation-dependent signalling networks and participate in the regulation of almost all biological processes and pathways. In total, iEKPD 2.0 contained 197 348 phosphorylation regulators, including 109 912 PKs, 23 294 PPs and 68 748 PPBD-containing proteins in 164 eukaryotic species. In particular, we provided rich annotations for the regulators of eight model organisms, especially humans, by compiling and integrating the knowledge from 100 widely used public databases that cover 13 aspects, including cancer mutations, genetic variations, disease-associated information, mRNA expression, DNA & RNA elements, DNA methylation, molecular interactions, drug-target relations, protein 3D structures, post-translational modifications, protein expressions/proteomics, subcellular localizations and protein functional annotations. Compared with our previously developed EKPD 1.0 (∼0.5 GB), iEKPD 2.0 contains ∼99.8 GB of data with an ∼200-fold increase in data volume. We anticipate that iEKPD 2.0 represents a more useful resource for further study of phosphorylation regulators.

Positive selection analysis highlights key positions in plant PP2A regulatory subunits

ABSTRACTA versatile hub for cellular control, the eukaryotic protein phosphatase 2A (PP2A) enzyme family is thought to achieve specificity through combinatorial complexity. Phylogenetic analysis has revealed that expansion of PP2A gene families resulted from whole genome duplications followed by non-random gene loss, and selection analysis suggests that retention of B56/PPP2R5 gene family members after genome duplication events was driven by functional diversification. Here we identify the sites at which positive selection is detected in the plant B56 gene family, and discuss the significance of selection at these positions in the context of PP2A holoenzyme structure. The pattern of positive selection observed in the B11 subclade is distinctive, and suggests selective pressure on interactions with substrates and the enzymatic core.

PLATINUM SENSITIVE 2 LIKE impacts growth, root morphology, seed set, and stress responses.

Eukaryotic protein phosphatase 4 (PP4) is a PP2A-type protein phosphatase that is part of a conserved complex with regulatory factors PSY2 and PP4R2. Various lines of Arabidopsis thaliana with mutated PP4 subunit genes were constructed to study the so far completely unknown functions of PP4 in plants. Mutants with knocked out putative functional homolog of the PSY2 LIKE (PSY2L) gene were dwarf and bushy, while plants with knocked out PP4R2 LIKE (PP4R2L) looked very similar to WT. The psy2l seedlings had short roots with disorganized morphology and impaired meristem. Seedling growth was sensitive to the genotoxin cisplatin. Global transcript analysis (RNA-seq) of seedlings and rosette leaves revealed several groups of genes, shared between both types of tissues, strongly influenced by knocked out PSY2L. Receptor kinases, CRINKLY3 and WAG1, important for growth and development, were down-regulated 3–7 times. EUKARYOTIC ELONGATION FACTOR5A1 was down-regulated 4–6 fold. Analysis of hormone sensitive genes indicated that abscisic acid levels were high, while auxin, cytokinin and gibberellic acid levels were low in psy2l. Expression of specific transcription factors involved in regulation of anthocyanin synthesis were strongly elevated, e.g. the master regulator PAP1, and intriguingly TT8, which is otherwise mainly expressed in seeds. The psy2l mutants accumulated anthocyanins under conditions where WT did not, pointing to PSY2L as a possible upstream negative regulator of PAP1 and TT8. Expression of the sugar-phosphate transporter GPT2, important for cellular sugar and phosphate homeostasis, was enhanced 7–8 times. Several DNA damage response genes, including the cell cycle inhibitor gene WEE1, were up-regulated in psy2l. The activation of DNA repair signaling genes, in combination with phenotypic traits showing aberrant root meristem and sensitivity to the genotoxic cisplatin, substantiate the involvement of Arabidopsis PSY2L in maintenance of genome integrity.

EKPD: a hierarchical database of eukaryotic protein kinases and protein phosphatases

We present here EKPD (http://ekpd.biocuckoo.org), a hierarchical database of eukaryotic protein kinases (PKs) and protein phosphatases (PPs), the key molecules responsible for the reversible phosphorylation of proteins that are involved in almost all aspects of biological processes. As extensive experimental and computational efforts have been carried out to identify PKs and PPs, an integrative resource with detailed classification and annotation information would be of great value for both experimentalists and computational biologists. In this work, we first collected 1855 PKs and 347 PPs from the scientific literature and various public databases. Based on previously established rationales, we classified all of the known PKs and PPs into a hierarchical structure with three levels, i.e. group, family and individual PK/PP. There are 10 groups with 149 families for the PKs and 10 groups with 33 families for the PPs. We constructed 139 and 27 Hidden Markov Model profiles for PK and PP families, respectively. Then we systematically characterized ∼50 000 PKs and >10 000 PPs in eukaryotes. In addition, >500 PKs and >400 PPs were computationally identified by ortholog search. Finally, the online service of the EKPD database was implemented in PHP + MySQL + JavaScript.

Microcystins as novel leads against OATP1B3-expressing tumors

Microcystins are cyclic heptapeptides produced by cyanobacteria. They potently inhibit the eukaryotic protein phosphatase families PP1 and PP2A. They are able to display toxicity only after transporter-mediated uptake by the cell. This uptake is mediated by the organic anion transporting polypeptides OATP1B1, OATP1B3, and OATP1A2. Liver cells express OATP1B1 and OATP1B3 transporters, which results in a pronounced liver toxicity of microcystins. However, OATP1B3 is also expressed in a significant percentage of hepatocellular carcinoma cells as well as gastrointestinal, lung, breast and colon tumors. Interestingly, compared to OATP1B1, OATP1B3 is found only in low abundance in liver cells. Thus selectivity that favors OATP1B3 over OATP1B1 should lead to a decreased hepatic clearance and toxicity, and an increased uptake in OATP1B3-expressing tumors, thus creating a therapeutic window for the respective compound.

A high-throughput screen to identify novel calcineurin inhibitors

Calcineurin is a eukaryotic protein phosphatase important for many signalling and developmental processes in cells. Inhibitors of this enzyme are used clinically and there is interest in identifying novel inhibitors for therapeutic applications. This report describes a high-throughput assay that can be used to screen natural or chemical libraries of compounds to identify new calcineurin inhibitors. The microtitre plate assay is based on a yeast reporter strain and was validated with known inhibitors and tested in a pilot screen of bacterial extracts.

ICP34.5 Protein of Herpes Simplex Virus Facilitates the Initiation of Protein Translation by Bridging Eukaryotic Initiation Factor 2α (eIF2α) and Protein Phosphatase 1

The ICP34.5 protein of herpes simplex virus type 1 is a neurovirulence factor that plays critical roles in viral replication and anti-host responses. One of its functions is to recruit protein phosphatase 1 (PP1) that leads to the dephosphorylation of the α subunit of translation initiation factor eIF2 (eIF2α), which is inactivated by infection-induced phosphorylation. As PP1 is a protein phosphatase with a wide range of substrates, the question remains to be answered how ICP34.5 directs PP1 to specifically dephosphorylate eIF2α. Here we report that ICP34.5 not only binds PP1 but also associates with eIF2α by in vitro and in vivo assays. The binding site of eIF2α is identified at amino acids 233-248 of ICP34.5, which falls in the highly homologous region with human gene growth arrest and DNA damage 34. The interaction between ICP34.5 and eIF2α is independent of the phosphorylation status of eIF2α at serine 51. Deletion mutation of this region results in the failure of dephosphorylation of eIF2α by PP1 and, consequently, interrupts viral protein synthesis and replication. Our data illustrated that the binding between viral protein ICP34.5 and the host eIF2α is crucial for the specific dephosphorylation of eIF2α by PP1. We propose that herpes simplex virus protein ICP34.5 bridges PP1 and eIF2α via their binding motifs and thereby facilitates the protein synthesis and viral replication.

Structure of the bifunctional isocitrate dehydrogenase kinase/phosphatase

The Escherichia coli isocitrate dehydrogenase kinase/phosphatase (AceK) is a unique bifunctional enzyme that phosphorylates or dephosphorylates isocitrate dehydrogenase (ICDH) in response to environmental changes, resulting in the inactivation or, respectively, activation of ICDH. ICDH inactivation short-circuits the Krebs cycle by enabling the glyoxlate bypass. It was the discovery of AceK and ICDH that established the existence of protein phosphorylation regulation in prokaryotes. As a 65-kDa protein, AceK is significantly larger than typical eukaryotic protein kinases. Apart from the ATP-binding motif, AceK does not share sequence homology with any eukaryotic protein kinase or phosphatase. Most intriguingly, AceK possesses the two opposing activities of protein kinase and phosphatase within one protein, and specifically recognizes only intact ICDH. Additionally, AceK has strong ATPase activity. It has been shown that AceK kinase, phosphatase and ATPase activities reside at the same site, although the molecular basis of such multifunctionality and its regulation remains completely unknown. Here we report the structures of AceK and its complex with ICDH. The AceK structure reveals a eukaryotic protein-kinase-like domain containing ATP and a regulatory domain with a novel fold. As an AceK phosphatase activator and kinase inhibitor, AMP is found to bind in an allosteric site between the two AceK domains. An AMP-mediated conformational change exposes and shields ATP, acting as a switch between AceK kinase and phosphatase activities, and ICDH-binding induces further conformational change for AceK activation. The substrate recognition loop of AceK binds to the ICDH dimer, allowing higher-order substrate recognition and interaction, and inducing critical conformational change at the phosphorylation site of ICDH.

Identification of a Small Molecule Inhibitor of the Human DNA Repair Enzyme Polynucleotide Kinase/Phosphatase

Human polynucleotide kinase/phosphatase (hPNKP) is a 57.1-kDa enzyme that phosphorylates DNA 5'-termini and dephosphorylates DNA 3'-termini. hPNKP is involved in both single- and double-strand break repair, and cells depleted of hPNKP show a marked sensitivity to ionizing radiation. Therefore, small molecule inhibitors of hPNKP should potentially increase the sensitivity of human tumors to gamma-radiation. To identify small molecule inhibitors of hPNKP, we modified a novel fluorescence-based assay to measure the phosphatase activity of the protein, and screened a diverse library of over 200 polysubstituted piperidines. We identified five compounds that significantly inhibited hPNKP phosphatase activity. Further analysis revealed that one of these compounds, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3), was the most effective, with an IC50 of 0.06 micromol/L. When tested for its specificity, A12B4C3 displayed no inhibition of two well-known eukaryotic protein phosphatases, calcineurin and protein phosphatase-1, or APTX, another human DNA 3'-phosphatase, and only limited inhibition of the related PNKP from Schizosaccharomyces pombe. At a nontoxic dose (1 micromol/L), A12B4C3 enhanced the radiosensitivity of human A549 lung carcinoma and MDA-MB-231 breast adenocarcinoma cells by a factor of two, which was almost identical to the increased sensitivity resulting from shRNA-mediated depletion of hPNKP. Importantly, A12B4C3 failed to increase the radiosensitivity of the hPNKP-depleted cells, implicating hPNKP as the principal cellular target of A12B4C3 responsible for increasing the response to radiation. A12B4C3 is thus a useful reagent for probing hPNKP cellular function and will serve as the lead compound for further development of PNKP-targeting drugs.

The sole Serine/Threonine protein kinase and its cognate phosphatase from Aquifex aeolicus targets Pyrimidine biosynthesis

Serine/Threonine kinases participate in complex, interacting signaling pathways in eukaryotes, prokaryotes, and archae. While most organisms contain many different kinases, the extreme hyperthermophile, Aquifex aeolicus encodes a single hypothetical Ser/Thr kinase. A gene homologous to eukaryotic protein phosphatases overlaps the kinase gene by a single base pair. The putative kinase, AaSTPK and phosphatase, AaPPM, were cloned and expressed in E. coli, purified to homogeneity and found to be functional. AaSTPK is a 34-kDa monomer that can use MgATP, MnATP, or MnGTP as co-substrates, although MgATP appears to be the preferred substrate. AaSTPK was autophosphorylated on a threonine residue and was dephosphorylated by AaPPM. AaPPM phosphatase is homologous to the PPM sub-family of Ser/Thr phosphatases and was stimulated by MnCl2 and CoCl2 but not MgCl2. AaSTPK also phosphorylated one threonine residue on the carbamoyl phosphate synthetase, CPS.A subunit. Carbamoyl phosphate synthetase reconstituted with phosphorylated CPS.A had unaltered catalytic activity but allosteric inhibition by UMP and activation by the arginine intermediate, ornithine, were both appreciably attenuated. These changes in allosteric regulation would be expected to activate pyrimidine biosynthesis by releasing the constraints imposed on carbamoyl phosphate synthetase activity by UMP and uncoupling the regulation of pyrimidine and arginine biosynthesis. CPS.A was also dephosphorylated by AaPPM. Aquifex aeolicus occupies the lowest branch on the prokaryotic phylogenetic tree. The Thr/Ser kinase, its cognate phosphatase and a protein substrate may be elements of a simple signaling pathway, perhaps the most primitive example of this mode of regulation described thus far.

Screening and characterization of microbial inhibitors against eukaryotic protein phosphatases (PP1 and PP2A)

To identify novel microbial inhibitors of protein phosphatase 1 (PP1). 750 actinomycetes and 408 microfungi were isolated from Sabah forest soils and screened for production of potential PP1 inhibitors using an in vivo screening system, in which candidate inhibitors were identified through mimicking the properties of PP1-deficient yeast cells. Acetone extracts of two fungi, H9318 (Penicillium) and H9978 (non-Penicillium) identified in this way showed inhibitory activity towards both mammalian PP1 and PP2A in an in vitro phosphatase assay, while extract from H7520 (Streptomyces) inhibited PP2A but not PP1. Consistently, using a drug-induced haploinsufficiency test, strains with either reduced PP1 or PP2A function were hypersensitive to H9318 and H9978 extracts whereas only the latter strain showed hypersensitivity to H7250 extract. H9318 extract was fractionated using RP-HPLC into two active peaks (S1 and S2). A yeast strain with reduced PP1 function showed hypersensitivity to fraction S2 whereas a strain with reduced PP2A function was hypersensitive to fraction S1. However, S1 and S2 inhibited both PP1 and PP2A activities to a similar extent. Three candidate PP inhibitors have been identified. Further development may generate useful research tools and ultimately therapeutic agents.

High crustacean toxicity of microcystin congeners does not correlate with high protein phosphatase inhibitory activity

Microcystins are strong toxins and efficient inhibitors of eukaryotic protein phosphatases. To determine structure related properties of six different microcystin congeners, we applied standardized inhibition assays for the protein phosphatases 1 and 2A, and an acute toxicity assay with Thamnocephalus platyurus. Protein phosphatase inhibition and acute toxicity did not correlate with each other. While the inhibition of the protein phosphatases 1 and 2A was much weaker for [ d-Asp 3,( E)-Dhb 7]microcystin-RR than for the other congeners, the toxicity was one of the highest. [ d-Asp 3]microcystin-LR exhibited only small differences to microcystin-LR. The data show that mechanisms other than the inhibition of protein phosphatases, such as uptake, transport, detoxification or other target sites may have a strong modulating effect on the toxicity of a microcystin congener for a particular animal. Structural changes can offset or even reverse the specific toxicity of microcystin congeners.

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.

Altered expression of two light-dependent genes in a microcystin-lacking mutant of Microcystis aeruginosa PCC 7806.

Microcystin is a potent inhibitor of eukaryotic protein phosphatases and has been implicated in causing hepatotoxicity to humans and animals worldwide. It is produced primarily by the bloom-forming cyanobacterium Microcystis aeruginosa, although the function of the peptide in this micro-organism is unknown. In this study, a microcystin-related protein, MrpA, was identified using a microcystin-lacking mutant of M. aeruginosa, PCC 7806. Comparative two-dimensional protein electrophoresis showed that MrpA was strongly expressed in wild-type PCC 7806, but was not detectable in the mcyB mutant. MrpA showed similarity to the RhiA protein from Rhizobium leguminosarum, which is encoded by the rhiABC operon and controlled by quorum-sensing mediators. Sequencing of mrpA flanking regions in M. aeruginosa PCC 7806 revealed the presence of a rhiB homologue, mrpB, directly downstream of mrpA. Northern blot analyses of mrpA expression in cells exposed to different light conditions revealed a rapid decline of transcription under high light conditions. Most striking was a strong increase in transcript levels from cultures irradiated with blue light. The mrpA transcription level was strongly reduced in two independent microcystin-lacking mutants under all light conditions investigated.