Comparative Protein Expression in Different Strains of the Bloom-forming Cyanobacterium Microcystis aeruginosa*

Toxin production in algal blooms presents a significant problem for the water industry. Of particular concern is microcystin, a potent hepatotoxin produced by the unicellular freshwater species Microcystis aeruginosa. In this study, the proteomes of six toxic and nontoxic strains of M. aeruginosa were analyzed to gain further knowledge in elucidating the role of microcystin production in this microorganism. This represents the first comparative proteomic study in a cyanobacterial species. A large diversity in the protein expression profiles of each strain was observed, with a significant proportion of the identified proteins appearing to be strain-specific. In total, 475 proteins were identified reproducibly and of these, 82 comprised the core proteome of M. aeruginosa. The expression of several hypothetical and unknown proteins, including four possible operons was confirmed. Surprisingly, no proteins were found to be produced only by toxic or nontoxic strains. Quantitative proteome analysis using the label-free normalized spectrum abundance factor approach revealed nine proteins that were differentially expressed between toxic and nontoxic strains. These proteins participate in carbon-nitrogen metabolism and redox balance maintenance and point to an involvement of the global nitrogen regulator NtcA in toxicity. In addition, the switching of a previously inactive toxin-producing strain to microcystin synthesis is reported.

[1]  B. Neilan,et al.  NtcA from Microcystis aeruginosa PCC 7806 Is Autoregulatory and Binds to the Microcystin Promoter , 2010, Applied and Environmental Microbiology.

[2]  A. M. Plominsky,et al.  Optimization of 2D‐PAGE protocols for proteomic analysis of two nonaxenic toxin‐producing freshwater cyanobacteria: Cylindrospermopsis raciborskii and Raphidiopsis sp. , 2009, Letters in applied microbiology.

[3]  B. Kan,et al.  Proteins involved in difference of sorbitol fermentation rates of the toxigenic and nontoxigenic Vibrio cholerae El Tor strains revealed by comparative proteome analysis , 2009, BMC Microbiology.

[4]  M. A. De la Rosa,et al.  Proteomic analyses of the response of cyanobacteria to different stress conditions , 2009, FEBS letters.

[5]  M. Desvaux,et al.  Insight into the core and variant exoproteomes of Listeria monocytogenes species by comparative subproteomic analysis , 2009, Proteomics.

[6]  M. E. Silva-Stenico,et al.  Microcystin production by a freshwater spring cyanobacterium of the genus Fischerella. , 2009, Toxicon : official journal of the International Society on Toxinology.

[7]  T. Kieselbach,et al.  Disulphide proteomes and interactions with thioredoxin on the track towards understanding redox regulation in chloroplasts and cyanobacteria. , 2009, Journal of proteomics.

[8]  J. Humbert,et al.  Competition between microcystin- and non-microcystin-producing Planktothrix agardhii (cyanobacteria) strains under different environmental conditions. , 2008, Environmental microbiology.

[9]  S. Burgess,et al.  Comparative Proteomic Analysis of Listeria monocytogenes Strains F2365 and EGD , 2008, Applied and Environmental Microbiology.

[10]  Ning Li,et al.  Association between the availability of environmental resources and the atomic composition of organismal proteomes: evidence from Prochlorococcus strains living at different depths. , 2008, Biochemical and biophysical research communications.

[11]  M. Baker,et al.  A combination of immobilised pH gradients improves membrane proteomics. , 2008, Journal of proteome research.

[12]  M. Bes,et al.  Exploring the interaction of microcystin-LR with proteins and DNA. , 2008, Toxicology in vitro : an international journal published in association with BIBRA.

[13]  M. Bes,et al.  Iron availability affects mcyD expression and microcystin-LR synthesis in Microcystis aeruginosa PCC7806. , 2008, Environmental microbiology.

[14]  A. Muro-Pastor,et al.  Role of Two NtcA-Binding Sites in the Complex ntcA Gene Promoter of the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120 , 2008, Journal of bacteriology.

[15]  B. Neilan,et al.  Unnatural production of natural products: heterologous expression and combinatorial biosynthesis of cyanobacterial-derived compounds , 2008, Journal and proceedings of the Royal Society of New South Wales.

[16]  Sang Yup Lee,et al.  Comparison of the extracellular proteomes of Escherichia coli B and K‐12 strains during high cell density cultivation , 2008, Proteomics.

[17]  P. Schürmann,et al.  The ferredoxin/thioredoxin system of oxygenic photosynthesis. , 2008, Antioxidants & redox signaling.

[18]  H. Lüthi,et al.  Topology and enhanced toxicity of bound microcystins in Microcystis PCC 7806. , 2008, Toxicon : official journal of the International Society on Toxinology.

[19]  Peter Lindblad,et al.  Quantitative shotgun proteomics of enriched heterocysts from Nostoc sp. PCC 7120 using 8-plex isobaric peptide tags. , 2008, Journal of proteome research.

[20]  T. Yeates,et al.  Atomic-Level Models of the Bacterial Carboxysome Shell , 2008, Science.

[21]  S. Tabata,et al.  Complete Genomic Structure of the Bloom-forming Toxic Cyanobacterium Microcystis aeruginosa NIES-843 , 2008, DNA research : an international journal for rapid publication of reports on genes and genomes.

[22]  Marc S. Lavine,et al.  A Robotic Future , 2007, Science.

[23]  T. Downing,et al.  Optimization of laboratory scale production and purification of microcystin-LR from pure cultures of Microcystis aeruginosa , 2007 .

[24]  M. Franklin,et al.  Strain-specific proteome responses of Pseudomonas aeruginosa to biofilm-associated growth and to calcium. , 2007, Microbiology.

[25]  L. Breci,et al.  Proteomic analysis of shade-avoidance response in tomato leaves. , 2007, Journal of agricultural and food chemistry.

[26]  Eva-Mari Aro,et al.  Cyanobacterial NDH-1 complexes: multiplicity in function and subunit composition. , 2007, Physiologia plantarum.

[27]  P. Wright,et al.  2‐DE proteomic analysis of the model cyanobacterium Anabaena variabilis , 2007, Electrophoresis.

[28]  A. Kaplan,et al.  Towards clarification of the biological role of microcystins, a family of cyanobacterial toxins. , 2007, Environmental microbiology.

[29]  J. Huisman,et al.  Competition for Light between Toxic and Nontoxic Strains of the Harmful Cyanobacterium Microcystis , 2007, Applied and Environmental Microbiology.

[30]  T. Ogawa,et al.  Cyanobacterial NADPH dehydrogenase complexes , 2007, Photosynthesis Research.

[31]  Peter Lindblad,et al.  An iTRAQ-based quantitative analysis to elaborate the proteomic response of Nostoc sp. PCC 7120 under N2 fixing conditions. , 2007, Journal of proteome research.

[32]  J. Meeks,et al.  A soluble 3D LC/MS/MS proteome of the filamentous cyanobacterium Nostoc punctiforme. , 2006, Journal of proteome research.

[33]  Luis López-Maury,et al.  The diversity and complexity of the cyanobacterial thioredoxin systems , 2006, Photosynthesis Research.

[34]  Michael K. Coleman,et al.  Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae. , 2006, Journal of proteome research.

[35]  K. Forchhammer,et al.  Interaction network in cyanobacterial nitrogen regulation: PipX, a protein that interacts in a 2‐oxoglutarate dependent manner with PII and NtcA , 2006, Molecular microbiology.

[36]  T. Smith,et al.  Atomic structure of a nitrate-binding protein crucial for photosynthetic productivity. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Kenneth F Reardon,et al.  Shotgun proteomics of cyanobacteria--applications of experimental and data-mining techniques. , 2006, Briefings in functional genomics & proteomics.

[38]  Nobuyuki Takatani,et al.  Effects of PII deficiency on expression of the genes involved in ammonium utilization in the cyanobacterium Synechocystis sp. Strain PCC 6803. , 2006, Plant & cell physiology.

[39]  K. Williams,et al.  Improved 2‐DE of microorganisms after acidic extraction , 2006, Electrophoresis.

[40]  W. Carmichael,et al.  Sublethal exposure from microcystins to renal insufficiency patients in Rio de Janeiro, Brazil , 2006, Environmental toxicology.

[41]  S. Gerbersdorf.  An advanced technique for immuno-labelling of microcystins in cryosectioned cells of Microcystis aeruginosa PCC 7806 (cyanobacteria): implementations of an experiment with varying light scenarios and culture densities. , 2006, Toxicon : official journal of the International Society on Toxinology.

[42]  E. Dittmann,et al.  A mannan binding lectin is involved in cell–cell attachment in a toxic strain of Microcystis aeruginosa , 2006, Molecular microbiology.

[43]  P. Babica,et al.  EXPLORING THE NATURAL ROLE OF MICROCYSTINS—A REVIEW OF EFFECTS ON PHOTOAUTOTROPHIC ORGANISMS 1 , 2006 .

[44]  R. Srivastava,et al.  Proteomic studies of the thylakoid membrane of Synechocystis sp. PCC 6803 , 2005, Proteomics.

[45]  Alan E. Wilson,et al.  Genetic Variation of the Bloom-Forming Cyanobacterium Microcystis aeruginosa within and among Lakes: Implications for Harmful Algal Blooms , 2005, Applied and Environmental Microbiology.

[46]  Takashi Yoshida,et al.  Coordination of DNA replication and cell division in cyanobacteria Microcystis aeruginosa. , 2005, FEMS microbiology letters.

[47]  Ying Xu,et al.  Comparative genomics analysis of NtcA regulons in cyanobacteria: regulation of nitrogen assimilation and its coupling to photosynthesis , 2005, Nucleic acids research.

[48]  J. Huisman,et al.  The Microcystin Composition of the Cyanobacterium Planktothrix agardhii Changes toward a More Toxic Variant with Increasing Light Intensity , 2005, Applied and Environmental Microbiology.

[49]  Martin Phillips,et al.  Protein Structures Forming the Shell of Primitive Bacterial Organelles , 2005, Science.

[50]  G. Codd,et al.  Immunogold localisation of microcystins in cryosectioned cells of Microcystis. , 2005, Journal of structural biology.

[51]  A. Kaplan,et al.  Ecological implications of the emergence of non-toxic subcultures from toxic Microcystis strains. , 2005, Environmental microbiology.

[52]  T. Börner,et al.  Abundance of active and inactive microcystin genotypes in populations of the toxic cyanobacterium Planktothrix spp. , 2004, Environmental microbiology.

[53]  M. Hippler,et al.  Subunit Composition of NDH-1 Complexes of Synechocystis sp. PCC 6803 , 2004, Journal of Biological Chemistry.

[54]  Robertson Craig,et al.  TANDEM: matching proteins with tandem mass spectra. , 2004, Bioinformatics.

[55]  K. Forchhammer,et al.  Global carbon/nitrogen control by PII signal transduction in cyanobacteria: from signals to targets. , 2004, FEMS microbiology reviews.

[56]  Christiane Bouchier,et al.  The Gas Vesicle Gene Cluster from Microcystis aeruginosa and DNA Rearrangements That Lead to Loss of Cell Buoyancy , 2004, Journal of bacteriology.

[57]  J. Vaitomaa,et al.  Phylogenetic evidence for the early evolution of microcystin synthesis. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[58]  V. Paakkarinen,et al.  Towards Functional Proteomics of Membrane Protein Complexes in Synechocystis sp. PCC 68031 , 2004, Plant Physiology.

[59]  R. Beavis,et al.  A method for reducing the time required to match protein sequences with tandem mass spectra. , 2003, Rapid communications in mass spectrometry : RCM.

[60]  Kjetill S. Jakobsen,et al.  Natural Variation in the Microcystin Synthetase Operon mcyABC and Impact on Microcystin Production in Microcystis Strains , 2003, Journal of bacteriology.

[61]  K. Forchhammer,et al.  Signal Transduction Protein PII Is Required for NtcA-Regulated Gene Expression during Nitrogen Deprivation in the Cyanobacterium Synechococcus elongatus Strain PCC 7942 , 2003, Journal of bacteriology.

[62]  L. R. Mur,et al.  Effects of Light on the Microcystin Content of Microcystis Strain PCC 7806 , 2003, Applied and Environmental Microbiology.

[63]  M. Badger,et al.  CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversity and evolution. , 2003, Journal of experimental botany.

[64]  J. Vaitomaa,et al.  Effect of Nitrogen and Phosphorus on Growth of Toxic and Nontoxic Microcystis Strains and on Intracellular Microcystin Concentrations , 2002, Microbial Ecology.

[65]  E. Dittmann,et al.  Altered expression of two light-dependent genes in a microcystin-lacking mutant of Microcystis aeruginosa PCC 7806. , 2001, Microbiology.

[66]  T. Rohrlack,et al.  A spontaneous mutant of microcystin biosynthesis: genetic characterization and effect on Daphnia. , 2001, Environmental microbiology.

[67]  S. Shibata,et al.  A proposal for the unification of five species of the cyanobacterial genus Microcystis Kützing ex Lemmermann 1907 under the rules of the Bacteriological Code. , 2001, International journal of systematic and evolutionary microbiology.

[68]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[69]  B. Neilan,et al.  Ecological and molecular investigations of cyanotoxin production. , 2001, FEMS microbiology ecology.

[70]  M. Rodríguez-Buey,et al.  Changes in carboxysome structure and grouping and in photosynthetic affinity for inorganic carbon in Anabaena strain PCC 7119 (Cyanophyta) in response to modification of CO2 and Na+ supply. , 2001, Plant & cell physiology.

[71]  G. Peltier,et al.  The gene encoding the NdhH subunit of type 1 NAD(P)H dehydrogenase is essential to survival of Synechocystis PCC6803 , 2000, FEBS letters.

[72]  E. Dittmann,et al.  Light and the Transcriptional Response of the Microcystin Biosynthesis Gene Cluster , 2000, Applied and Environmental Microbiology.

[73]  F. Florencio,et al.  Electron transport controls transcription of the thioredoxin gene (trxA) in the cyanobacterium Synechocystis sp. PCC 6803 , 2000, Plant Molecular Biology.

[74]  H. Oh,et al.  Microcystin Production by Microcystis aeruginosa in a Phosphorus-Limited Chemostat , 2000, Applied and Environmental Microbiology.

[75]  R. Jeanjean,et al.  Protein PII regulates both inorganic carbon and nitrate uptake and is modified by a redox signal in Synechocystis PCC 6803 , 1999, FEBS letters.

[76]  W. Carmichael,et al.  Using an enzyme linked immunosorbent assay (ELISA) and a protein phosphatase inhibition assay (PPIA) for the detection of microcystins and nodularins. , 1999, Natural toxins.

[77]  G. Gadd,et al.  Binding of copper and zinc to three cyanobacterial microcystins quantified by differential pulse polarography , 1997 .

[78]  B. Sykes,et al.  A Molecular Basis for Different Interactions of Marine Toxins with Protein Phosphatase-1 , 1997, The Journal of Biological Chemistry.

[79]  H. Utkilen,et al.  Iron-stimulated toxin production in Microcystis aeruginosa , 1995, Applied and environmental microbiology.

[80]  T. Börner,et al.  Plasmids in toxic and nontoxic strains of the cyanobacteriumMicrocystis aeruginosa , 1988, Current Microbiology.

[81]  I. Falconer,et al.  Clinical and Pathological Changes in Sheep Experimentally Poisoned by the Blue-Green Alga Microcystis aeruginosa , 1984, Veterinary pathology.

[82]  J. Noirel,et al.  Quantitative overview of N2 fixation in Nostoc punctiforme ATCC 29133 through cellular enrichments and iTRAQ shotgun proteomics. , 2009, Journal of proteome research.

[83]  Andrew C. Tolonen,et al.  UvA-DARE ( Digital Academic Repository ) Highly plastic genome of Microcystis aeruginosa PCC 7806 , a ubiquitous toxic freshwater cyanobacterium , 2008 .

[84]  J. Reyes,et al.  Ammonium assimilation in cyanobacteria , 2004, Photosynthesis Research.

[85]  Joshua E. Elias,et al.  Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. , 2003, Journal of proteome research.

[86]  S. Pflugmacher.  Possible allelopathic effects of cyanotoxins, with reference to microcystin‐LR, in aquatic ecosystems , 2002, Environmental toxicology.

[87]  O. Ohara,et al.  Towards a proteome project of cyanobacterium Synechocystis sp. strain PCC6803: Linking 130 protein spots with their respective genes , 1997, Electrophoresis.

[88]  L. Lawton,et al.  Extraction and high-performance liquid chromatographic method for the determination of microcystins in raw and treated waters. , 1994, The Analyst.