Elsevier

Peptides

Volume 27, Issue 9, September 2006, Pages 2090-2103
Peptides

Detection and identification of oligopeptides in Microcystis (cyanobacteria) colonies: Toward an understanding of metabolic diversity

https://doi.org/10.1016/j.peptides.2006.03.014Get rights and content

Abstract

Cyanobacteria and particularly Microcystis sp. (Chroococcales) are known to produce a multitude of peptide metabolites. Here we report on the mass spectral analysis of cyanobacterial peptides in individual colonies of Microcystis sp. collected in a drinking water reservoir. A total number of more than 90 cyanopeptides could be detected, 61 of which could be identified either as known peptides or new structural variants of known peptide classes. For 18 new peptides flat structures are proposed. New congeners differed from known ones mainly in chlorination (aeruginosins), methylation (microginins), or amino acid sequences (cyanopeptolins). The high number of peptides and especially the new peptides underline the capability of Microcystis strains as producers of a high diversity of potentially bioactive compounds.

Introduction

Cyanobacteria of the genus Microcystis are notorious for their mass developments in eutrophied inland and brackish waters. In the majority of Microcystis blooms hepatotoxic peptides of the microcystin class can be detected, thus creating a potential health hazard by respective blooms when the infested water is used as source of drinking water or accidentally taken up during recreational activities. Microcystins have been studied intensively during the last two decades with emphasis on genetics [12], physiology [77], biochemistry [52], toxicology [10], and ecology [73], to name some aspects and studies. One major outcome of these studies was that microcystins are synthesized constitutively thus resulting in fairly stable cell quota but only by strains that possess the respective peptide synthetase gene cluster. The gene cluster coding for the non-ribosomal peptide synthetase (NRPS), mcyA-I [69], has a size of some 60 kbp and seems to be distributed among Microcystis clones independently of the phylogeny based on housekeeping genes such as the phycocyanin operon [50]. In individual clones a single gene cluster can be responsible for the formation of a multitude of congeners diverging in amino acid composition (e.g. Mcyst-LR and Mcyst-RR) and methylation [45].

Besides microcystins, cyanobacteria, and especially Microcystis, can produce a high number of oligopeptides that are presumably synthesized by NRPS biosynthetical pathways [7]. Many peptide structures of Microcystis and cyanobacteria in general can be classified in types with shared structural properties like microcystins, cyanopeptolins, and aeruginosins. For a number of peptides bioactivity has been reported but respective studies were driven by pharmacological interests and the resulting data can shed only little light on the function of the peptides in cyanobacterial physiology and ecology [24], [30], [46], [56]. In fact, no consistent hypothesis has been developed so far to explain the high structural variability and patchy distribution of cyanopeptides. This is partly due to the still very limited knowledge on the occurrence of individual peptides and peptide classes in environmental samples. The diversity of peptide chemotypes has been reported previously [17]. Mass spectral analyzes of Microcystis colonies and strains showed that new structural variants of known peptide classes are frequently encountered [9], [74]. In peptide classes for which several congeners have been described, these differ either by exchanges of amino acids or by modifications like chlorination, methylation, or glycosilation.

Several analytical methods have been applied for chemotaxonomic characterization of cyanobacteria [32], [58], based on fatty acid compositions, for example [40]. Likewise, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been applied for the rapid typing of micro-organisms [11], [22]. In studies on cyanobacteria, MALDI-TOF MS proved to be a very efficient tool to detect oligopeptides in very small samples and to elucidate flat structures of new congeners without laborious cultivation and isolation procedures [14], [39]. Post-source-decay (PSD) fragmentation supported by collision-induced-dissociation (CID) has been studied for many different peptides and fragmentation schemes have been developed allowing a reliable reconstruction of amino acid sequences [21], [66], [71]. Structure elucidation by MALDI-TOF MS is facilitated when respective fragment patterns of similar compounds are available for comparison [15], [79]. Partial structures or fragments with near identical masses can sometimes not be distinguished and for a full structure elucidation NMR-techniques would then be necessary. This required, however, the isolation of the compounds of interest in the milligram range—and thus in amounts in which many structural variants will probably never be available.

The aim of the present project was to follow changes in peptide chemotype composition during the summer season; a report on the chemotype dynamics will be published elsewhere. Here we report on the detection of cyanopeptides in single colonies of Microcystis by MALDI-TOF MS and the identification of known and new structural variants. The present report is focused on the structural variability of peptides in a Microcystis population and on the frequency of individual peptides and peptide classes.

Section snippets

Experimental

Microcystis colonies originated from Brno reservoir near Brno (Czech Republic) and from Brilicky pond in Trebon (Czech Republic). Colonies were collected with a plankton net from the first 2 m of the water column at a central location of Brno reservoir. From Brilicky pond a water sample was taken from the shore and passed through a plankton net. The net samples were stored cool (<8 °C) upon return to the laboratory within less than 4 h where the isolation of individual colonies was performed

Results

More than 150 distinct mass signals in a range of 450–2000 Da were recorded in the Microcystis colonies, i.e. with a signal to noise ratio in excess of 10, at least three isotopic peaks, and purity after peak deisotoping. Of these, only a minor part could be directly assigned to known peptides as they have been detected previously on multiple occasions. A larger number of low-intensity mass signals likely also represented peptidic compounds but was excluded from the further analysis for

Discussion

In 850 Microcystis colonies analyzed for the present study a high number of peptide metabolites could be detected and identified. Identification could be achieved either by comparison of actual fragment spectra to spectra obtained from standard material or by calculation of theoretical fragment spectra allowing an error margin of maximum 0.5 Da. Although MALDI-TOF MS does not enable the elucidation of full structures including chirality it allows a fast identification of congeners of known

Acknowledgements

This study was supported by the EU-research project PEPCY (Bioactive Peptides in Cyanobacteria) and by the Grant Agency of the Czech Republic (project nr. 206/03/1215). We thank Hana Slovackova for support in collecting and isolating of Microcystis colonies and Marcel Erhard, Jutta Fastner, and Jürgen Weckesser for providing reference mass spectra and other supportive information. Keishi Ishida, Shmuel Carmeli, and Rosmarie Rippka generously provided reference peptides and/or reference strain

References (79)

  • K. Ishida et al.

    Kawaguchipeptin A, a novel cyclic undecapeptide from the cyanobacterium Microcystis aeruginosa (NIES-88)

    Tetrahedron

    (1996)
  • K. Ishida et al.

    Aeruginoguanidines 98-A - 98C: cytotoxic unusual peptides from the cyanobacterium Microcystis aeruginosa

    Tetrahedron

    (2002)
  • K. Ishida et al.

    Aeruginosins, protease inhibitors from the cyanobacterium Microcystis aeruginosa

    Tetrahedron

    (1999)
  • M. Kansiz et al.

    Fourier transform infrared microspectroscopy and chemometrics as a tool for the discrimination of cyanobacterial strains

    Phytochemistry

    (1999)
  • J. Kiviranta et al.

    Structure determination and toxicity of a new microcystin from Microcystis aeruginosa strain 205

    Toxicon

    (1992)
  • S. Kodani et al.

    Five new cyanobacterial peptides from water bloom materials of lake Teganuma (Japan)

    FEMS Microbiol Lett

    (1999)
  • T. Kusumi et al.

    a toxin from the cyanobacterium (blue-green alga) Microcystis viridis

    Tetrahedron Lett

    (1987)
  • R.H. Li et al.

    Fatty acid profiles and their chemotaxonomy in planktonic species of Anabaena (Cyanobacteria) with straight trichomes

    Phytochemistry

    (2001)
  • U. Matern et al.

    Binding structure of elastase inhibitor scyptolin A

    Chem Biol

    (2003)
  • H. Matsuda et al.

    Aeruginosins 102-A and B, new thrombin inhibitors from the cyanobacterium Microcystis viridis (NIES-102)

    Tetrahedron

    (1996)
  • M. Murakami et al.

    Aeruginosins 98-A and B, trypsin inhibitors from the blue-green alga Microcystis aeruginosa (NIES-98)

    Tetrahedron Lett

    (1995)
  • M. Murakami et al.

    A cyclic peptide, anabaenopeptin B, from the cyanobacterium Oscillatoria agardhii

    Phytochemistry

    (1997)
  • U. Neumann et al.

    Co-occurrence of non-toxic (Cyanopeptolin) and toxic (Microcystin) peptides in a bloom of Microcystis sp from a Chilean Lake

    Syst Appl Microbiol

    (2000)
  • T. Okino et al.

    Microginin, an angiotensin-converting enzyme inhibitor from the blue-green alga Microcystis aeruginosa

    Tetrahedron Lett

    (1993)
  • T. Okino et al.

    Micropeptins A and B, plasmin and trypsin inhibitors from the blue green alga Microcystis aeruginosa

    Tetrahedron Lett

    (1993)
  • V. Reshef et al.

    Protease inhibitors from a water bloom of the cyanobacterium Microcystis aeruginosa

    Tetrahedron

    (2001)
  • I. Romano et al.

    Lipid profile: a useful chemotaxonomic marker for classification of a new cyanobacterium in Spirulina genus

    Phytochemistry

    (2000)
  • T. Sano et al.

    A chymotrypsin inhibitor from toxic Oscillatoria agardhii

    Tetrahedron Lett

    (1995)
  • T. Sano et al.

    Leucine aminopeptidase M inhibitors, cyanostatins A and B, isolated from cyanobacterial water blooms in Scotland

    Phytochemistry

    (2005)
  • K. Sivonen et al.

    Isolation and structures of five microcystins from a russian Microcystis aeruginosa strain CALU 972

    Toxicon

    (1992)
  • D. Tillett et al.

    Structural organization of microcystin biosynthesis in Microcystis aeruginosa Pcc7806: an integrated peptide-poliketide synthetase system

    Chem Biol

    (2000)
  • L. Via-Ordorika et al.

    Distribution of microcystin-producing and non-microcystin-producing Microcystis sp in European freshwater bodies: detection of microcystins and microcystin genes in individual colonies

    Syst Appl Microbiol

    (2004)
  • B. Bister et al.

    Cyanopeptolin 963A, a chymotrypsin inhibitor of Microcystis PCC 7806

    J Nat Prod

    (2004)
  • D.P. Botes et al.

    Structural studies on cyanoginosins-LR, YR, YA, and YM, peptide toxins from Microcystis aeruginosa

    J Chem Soc Perkin Trans

    (1985)
  • A.K. Brunberg et al.

    Recruitment of Microcystis (cyanophyceae) from lake sediments: the importance of littoral inocula

    J Phycol

    (2003)
  • Cadel-Six S, Welker M, Dauga C, Tandeau de Marsac N. Putative halogenase genes in two cyanopeptide clusters of...
  • G. Christiansen et al.

    Nonribosomal peptide synthetase genes occur in most cyanobacterial genera as evidenced by their distribution in axenic strains of the PCC

    Arch Microbiol

    (2001)
  • G. Christiansen et al.

    Microcystin biosynthesis in Planktothrix: genes, evolution, and manipulation

    J Bacteriol

    (2003)
  • O. Czarnecki et al.

    Identification of peptide metabolites of Microcystis (Cyanobacteria) that inhibit trypsin-like activity in plankonic herbivorous Daphnia (Cladocera)

    Environ Microbiol

    (2006)
  • Cited by (124)

    View all citing articles on Scopus
    View full text