JOURNAL OF BACTERIOLOGY, July 1999, p.

40894097
0021-9193/99/$04.0010
Copyright 1999, American Society for Microbiology. All Rights Reserved.

Vol. 181, No. 13

Nonribosomal Peptide Synthesis and Toxigenicity

of Cyanobacteria
BRETT A. NEILAN,1,2* ELKE DITTMANN,2 LEO ROUHIAINEN,3 R. AMANDA BASS,1
RNER2
VERENA SCHAUB,2 KAARINA SIVONEN,3 AND THOMAS BO
School of Microbiology and Immunology, The University of New South Wales, Sydney 2052, New South Wales,
Australia1; Institute for Biology (Genetics), Humboldt University of Berlin, D-10115 Berlin, Germany2; and
Department of Applied Chemistry and Microbiology, University of Helsinki, FIN-00014 Helsinki, Finland3
Received 14 December 1998/Accepted 21 April 1999

Nonribosomal peptide synthesis is achieved in prokaryotes and lower eukaryotes by the thiotemplate
function of large, modular enzyme complexes known collectively as peptide synthetases. These and other multifunctional enzyme complexes, such as polyketide synthases, are of interest due to their use in unnatural-product or combinatorial biosynthesis (R. McDaniel, S. Ebert-Khosla, D. A. Hopwood, and C. Khosla, Science 262:
15461557, 1993; T. Stachelhaus, A. Schneider, and M. A. Marahiel, Science 269:6972, 1995). Most nonribosomal peptides from microorganisms are classified as secondary metabolites; that is, they rarely have a role
in primary metabolism, growth, or reproduction but have evolved to somehow benefit the producing organisms.
Cyanobacteria produce a myriad array of secondary metabolites, including alkaloids, polyketides, and nonribosomal peptides, some of which are potent toxins. This paper addresses the molecular genetic basis of nonribosomal peptide synthesis in diverse species of cyanobacteria. Amplification of peptide synthetase genes was
achieved by use of degenerate primers directed to conserved functional motifs of these modular enzyme complexes. Specific detection of the gene cluster encoding the biosynthetic pathway of the cyanobacterial toxin
microcystin was shown for both cultured and uncultured samples. Blot hybridizations, DNA amplifications,
sequencing, and evolutionary analysis revealed a broad distribution of peptide synthetase gene orthologues in
cyanobacteria. The results demonstrate a molecular approach to assessing preexpression microbial functional
diversity in uncultured cyanobacteria. The nonribosomal peptide biosynthetic pathways detected may lead to
the discovery and engineering of novel antibiotics, immunosuppressants, or antiviral agents.
ation, hydroxylation, epimerization, peptide sequence, and toxicity have been identified (26, 29). These peptides are potent
inhibitors of eukaryotic protein phosphatases type 1 and 2A,
with inhibition being dependent on particular structural variations (1), including the substitution of two variable L-amino
acids and the methylation of aspartate (b-iso-Asp) and dehydroalanine (Fig. 1A). The modified b-amino acid (Adda in Fig.
1A), which is also found in the hepatotoxic pentapeptides
nodularin and motuporin, is conserved in all known toxic microcystins. Microcystins and related cyclic peptides are carried
into hepatocytes via the bile acid transport system, where hyperphosphorylation of microfilaments, including cytokeratins,
is the primary toxic effect (33). Microcystins may also activate
phospholipase A2 and cyclooxygenase in hepatocytes, while in
macrophages they induce tumor necrosis factor alpha and interleukin 1. These functions, together with hyperphosphorylation of DNA, have implicated microcystins as agents promoting hepatocellular carcinoma (8).
Like other small peptides which contain unusual amino acids, microcystins are synthesized nonribosomally (2, 4). We
have recently identified peptide synthetase genes in cyanobacteria of the genera Microcystis and Anabaena (3, 19). The
characteristic modular structure of the peptide synthetase
genes and particular conserved sequence motifs seen in other
bacteria and fungi were also found in these cyanobacterial
genes (Fig. 1B) (3). The insertional inactivation of a peptide
synthetase gene from the hepatotoxic strain Microcystis aeruginosa PCC7806 resulted in transformation to the nontoxic state
and a loss of microcystins, demonstrating that this gene, called
mcyB, encodes a microcystin synthetase (4).
To date, peptide synthetase genes have been isolated from
two cyanobacterial species, while a microcystin synthetase gene

Not all proteins are synthesized on the ribosome. Small

polypeptides, with fewer than about 50 amino acids, can be
assembled by peptide synthetases just as other compounds,
such as fatty acids, are linked by other synthases. The products
of microbial nonribosomal peptide synthesis include the immunosuppressant cyclosporine and antibiotics such as gramicidin S, tyrocidin A, and surfactins (for a review, see reference
13). A modular structure of peptide synthetases has been
shown to be responsible for the sequential and amino-acidspecific elongation of peptide chains (17). The specific combination of modules and various functional domains within the
peptide synthetase determines the structure and hence the
activity of the peptide product.
The Cyanobacteria, as determined on the basis of several
molecular phylogenies, comprise a single and coherent group
of prokaryotes (35). Commonly, these bacteria proliferate in
eutrophic marine and freshwater habitats, resulting in the formation of water blooms. Cyanobacteria represent a relatively
unexplored and potentially rich source of bioactive secondary
metabolites (6, 20, 21). Of these bioactive compounds, the toxins
produced by certain planktonic species of cyanobacteria have
been particularly well studied. These oxygenic phototrophs are
the only known producers of the hepatotoxic microcystins, and
several morphologically and physiologically diverse genera
have been shown to synthesize these compounds (11, 14, 30).
Microcystins are cyclic heptapeptides (Fig. 1A). Sixty-five
isoforms of these compounds which vary by degree of methyl* Corresponding author. Mailing address: School of Microbiology
and Immunology, The University of New South Wales, Sydney 2052,
NSW, Australia. Phone: 612 9385 3235. Fax: 612 9385 1591. E-mail: b
.neilan@unsw.edu.au.
4089

4090

J. BACTERIOL.

NEILAN ET AL.

FIG. 1. (A) General structure of microcystins, cyanobacterial heptapeptide hepatotoxins, showing the most frequently found variations. X and Z, variable L-amino
acids L-leucine and L-arginine, respectively; R1 and R2, H (demethylmicrocystins) and CH3, respectively; D-MeAsp, D-erythro-b-methylaspartic acid; Adda,
(2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid; Mdha, N-methyldehydroalanine (Dha, dehydroalanine). (B) Diagram depicting the
gene encoding a single peptide synthetase module. The functional domains of a type I peptide synthetase module include condensation (shaded box), amino-acidspecific acyladenylation (open box), thioesterification (hatching), and possibly epimerization (cross-hatching). A type II module would contain an N-methyltransferase
domain between the acyladenylation and thioesterification regions. Peptide synthase conserved motifs (13) are shown as roman numerals (I to VI) (for a more detailed
description of functional domains in peptide synthetases, see reference 17). Arrows indicate the relative positions of degenerate cyanobacterial PS-PCR primers (at
motifs I and V; MTR2) and MS-PCR primers.

has been isolated from only one of several microcystin-producing genera. The present study describes the detection and
characterization of microcystin and peptide synthetase genes in
a genetically diverse range of microcystin-producing and nontoxic cyanobacterial species. Oligonucleotide primers which
amplify DNA from both the conserved sequence motifs of
peptide synthetase genes and specific sequences of microcystin
synthetase genes in cyanobacteria were designed (Table 1).
These PCR products were used as hybridization probes and/or
directly sequenced. Correlations were made between the pres-

ence of peptide synthetase genes and the production of

microcystins by hepatotoxic cyanobacteria. The data presented provide initial indications of the distribution of microcystin
synthetase and other peptide synthetase genes in the phylum
Cyanobacteria and the possible mechanism underlying their
transmission. In addition, these gene-targeting procedures enable the isolation and characterization of sequences from novel
peptide synthetase modules with potentially diverse biosynthetic activities. Isolation and culturing of the many microorganisms
which may produce nonribosomal peptides are not required.

TABLE 1. Peptide synthetase gene consensus and specific PCR primers

Primer (annealing)
temp

PS-PCR (52C)
MTF2
MTR
MS-PCR (50C)
FAA
RAA

Degeneracy (fold)

Tm (C)

Product (bp)

Oligonucleotide primer sequence, from 59339 (conserved peptide motif)

512
192

53
51

;1,000
;1,000

GCNGG(C/T)GG(C/T)GCNTA(C/T)GTNCC(AGGAYVP, core motif I)

CCNCG(AGT)AT(TC)TTNAC(T/C)TG(QVKIRG, core motif V)

1
1

50
50

758
758

CTATGTTATTTATACATCAGG
CTCAGCTTAACTTGATTATC

VOL. 181, 1999

NONRIBOSOMAL PEPTIDE SYNTHESIS OF CYANOBACTERIA

MATERIALS AND METHODS

Cyanobacterial strains and culturing. Cyanobacterial strains with the designations AWT, HUB, NIES, PCC, and UNSW were obtained from the culture
collections of Australian Water Technologies (AWT; Sydney, Australia), Humboldt University (HUB; Berlin, Germany), the National Institute for Environmental Studies (NIES; Tsukuba, Japan), the Institut Pasteur (PCC; Paris,
France), and The University of New South Wales (UNSW; Sydney, Australia),
respectively (Table 2). The remaining strains were from a culture collection of
one of the authors (K. Sivonen, University of Helsinki). Strains were grown in
JM, BG-11, or Z8 (Helsinki strains only) medium at 20 6 2C and under
continuous illumination of 25 mmol/m2/s as detailed earlier (16, 25). Nitrogenfixing species were grown in nitrogen-free medium (25), and Nodularia species
were grown in salt-containing medium (16). Strains obtained from culture collections were axenic.
Measurement of strain toxicity and chemical analyses of microcystins. Detection of microcystins by mouse bioassay, high-pressure liquid chromatography
(HPLC), commercially available enzyme-linked immunosorbent assay (ELISA)
(EnviroGard Microcystins Plate Kit; Strategic Diagnostics), and protein phosphatase inhibition was performed according to previously published procedures.
Briefly, the mouse bioassay was performed by intraperitoneal injection of aqueous cell suspensions, measurement of the 50% lethal dose, and histological
observation of hepatic hemorrhage (5). For HPLC analyses, microcystin was
extracted from a log-phase culture with methanol. Unquantified measurement of
microcystin content was performed with a LiChroCART RP18 column (15).
Protein phosphatase type 2A inhibition activity was determined with frozenthawed cell extracts and standardized for total protein and dry cell mass (1, 4).
For many of the strains listed in Table 2, at least one microcystin has been
isolated and the structure has been identified (26, 29).
DNA extraction, amplification, sequencing, and probe hybridization. Total
genomic DNA was extracted by standard methods commonly used for cyanobacteria and plants (10, 22). Alternatively, a PCR template was prepared by rapid
cell lysis in the presence of a cation-exchange resin and nonionic detergents (24).
PCR annealing step temperatures are shown in Table 1 along with peptide
synthetase gene-directed oligonucleotide primer sequences. In capillary or
200-ml tubes, the PCR thermal cycling protocol included an initial denaturation
at 94C for 2 min, followed by 35 cycles at 93C for 10 s, at the annealing
temperature (Table 1) for 20 s, and at 72C for 1 min. Amplification reaction
components were as previously described (23), and incubations were performed
with an FTS-1S capillary thermocycler (Corbett Research, Sydney, Australia) or
a PE2400 apparatus (Perkin-Elmer Cetus Corporation, Emeryville, Calif.).
Amplified DNA was purified from surplus reaction components and sequenced directly by standard automated fluorescence techniques (19, 23).
Larger, contiguous fragments of the M. aeruginosa PCC7806 and M. aeruginosa
HUB524 microcystin synthetase genes (accession no. U97078 and Z28338, respectively) were also isolated from phage and plasmid gene libraries by PCR
probe hybridization. A method of semidegenerate, long PCR was developed to
extend sequence information flanking that obtained from the gene libraries. This
procedure used the highly redundant primers MTF2 and MTR (Table 1) directed to the conserved motifs of known peptide synthetase genes combined with
primers specific for the Microcystis genome (19). By modifying and using longPCR protocols, we found it possible to amplify regions of DNA encoding entire
modules of the synthetase. In this way, sequences flanking the known microcystin
synthetase gene could be extended to unknown conserved regions of the peptide
synthetase gene. This method is termed module jumping. Sequencing of these
PCR fragments was performed with 100 pmol of the degenerate primer and
automated protocols. DNA sequences of the peptide and microcystin synthetase
genes were aligned with the program Pileup (Genetics Computer Group) and the
multiple-sequence-alignment tool Clustal W. Manual confirmation of the sequence alignment was also performed. Phylogenetic analyses are described in the
corresponding figure legends.
Cyanobacterial genomic DNA (about 100 ng) was dot blotted onto nylon
membranes (Hybond N1; Amersham, Little Chalfont, United Kingdom) by a
previously described protocol (27). A 2.0-kb clone and a 2.4-kb clone corresponding to different modules of a peptide synthetase gene from Anabaena sp.
strain 90, as well as a 1.15-kb amplified clone of the mcyB gene of strain HUB524
(4), were used as probes. Probe labeling, DNA hybridization, which was performed at 60C, and membrane washing were done by standard procedures (27).

RESULTS AND DISCUSSION

Detection of peptide synthetase genes by dot blot hybridization. In order to compare the distributions of two peptide
synthetase genes derived from M. aeruginosa HUB524 (mcyB)
(4) and Anabaena sp. strain 90, DNAs from hepatotoxic, neurotoxic, and nontoxic strains belonging to various species of
Anabaena, Nostoc, Microcystis, Nodularia, Oscillatoria, and
Aphanizomenon were simultaneously probed in dot blot experiments (Fig. 2A). The mcyB fragment recently identified as a

4091

microcystin synthetase gene was shown to hybridize, with variable signal intensity, to DNAs from all hepatotoxic strains and
to DNAs from three nontoxic strains (Microcystis sp. strain 130,
Aphanizomenon sp. strain 202, and Oscillatoria sp. strain 2)
(Table 2 and Fig. 2B). This probe did not hybridize to DNAs
from neurotoxic strains and other nontoxic strains (Table 2).
The two Anabaena sp. strain 90 peptide synthetase gene fragments gave strong signals with all hepatotoxic Anabaena strains
but showed insignificant hybridization to most other microcystin-producing species (Fig. 2C). These probes did, however,
cross-hybridize to Nostoc sp. strain 152, Nodularia sp. strain
HEM, nontoxic Anabaena sp. strain 299 (data not shown), and
(weakly) Nodularia sp. strain NSOR12. Therefore, we have
evidence for two distinct peptide synthetase genes showing
different distributions among the cyanobacterial strains tested.
These results reflect the observation that nonribosomal peptide and microcystin contents vary among cyanobacterial species (26). The mcyB hybridization data revealed the presence
of similar genes across all genera investigated. It remains to be
determined whether all positive hybridization signals corresponded to microcystin synthetase genes. To obtain more detailed information on the distribution of peptide synthetase
genes among toxic and bloom-forming cyanobacteria, several
selected strains were further investigated by DNA amplification and sequencing.
Design of amplification primers for peptide synthetase
genes in cyanobacteria. A comparison of peptide synthetases
and other adenylate-forming enzymes from various prokaryotes and eukaryotes revealed the presence of highly conserved functional domains (13, 17) (Fig. 1B). Degenerate PCR
primers were directed to these conserved sequence motifs (Table 1). The design of these primers was based on conserved
peptide motifs in other bacteria and fungi. Back-translation
(reverse translation from protein to DNA) of these consensus
motifs was sensitive to the codon bias of cyanobacterial genes
in general (7). Specifically, sequence motifs I and V of the
adenylate-forming domain of known peptide synthetase modules were used as target sequences (Fig. 1B). These functional
domains are only weakly conserved in non-peptide synthetase
adenylate-forming enzymes (34). Degenerate primers were
used to detect genes encoding peptide synthetases (degenerate
peptide synthetase PCR [PS-PCR]).
The mcyB gene of M. aeruginosa PCC7608 (4) and its orthologue in M. aeruginosa HUB524 (19) were aligned in order to
design PCR primers specific for peptide synthetase genes involved in microcystin biosynthesis (microcystin synthetase
PCR [MS-PCR]). These primers were directed to regions
within the peptide synthetase module which were shown not to
be part of the series of conserved functional motifs (17). The
resulting PCR product did, however, contain a conserved core
sequence (core motif II) which was used to align DNA sequences for further phylogenetic analyses (Fig. 1B). Specific
amplification primers (for MS-PCR) based on the characterized peptide synthetase gene sequence of M. aeruginosa (4)
were designed and directed to the region between conserved
peptide synthetase motifs I and III (Fig. 1B and Table 1). The
priming sites for MS-PCR did not have a sequence identical in
the two Microcystis strains, PCC7806 and HUB524, and were
selected to enable amplification of a microcystin synthetase
fragment from a broad range of microcystin-producing cyanobacteria.
Detection of peptide synthetase genes in cyanobacteria by
PCR. All DNA samples were checked for integrity in a cyanobacterium-specific 16S ribosomal DNA amplification reaction prior to use in PS-PCR and MS-PCR (23). The degenerate
PCR (PS-PCR) was used to identify cyanobacterial strains

4092

NEILAN ET AL.

J. BACTERIOL.

TABLE 2. Cyanobacterial strain toxicities and amplification and hybridization of DNA involved in the biosynthesis of microcystins and other
nonribosomal peptides
Strain

Anabaena circinalis AWT006

Anabaena cylindrica NIES19
Anabaena flos-aquae CYA83/1
Anabaena sp. strain 90
Anabaena sp. strain 202A1
Anabaena sp. strain 66A
Anabaena sp. strain 186
Anabaena sp. strain 54
Anabaena sp. strain 79B
Anabaena sp. strain 123
Anabaena sp. strain 14
Anabaena sp. strain 277
Anabaena sp. strain 302
Anabaena sp. strain 299A
Aphanizomenon sp. strain 3
Aphanizomenon sp. strain 301
Aphanizomenon sp. strain TR18
Aphanizomenon sp. strain 202
Aphanizomenon flos-aquae NIES81
Cylindrospermopsis raciborskii
AWT205
Lyngbya sp. strain AWT211
Microcystis aeruginosa PCC7806
Microcystis aeruginosa HUB524
Microcystis aeruginosa PCC7820
Microcystis wesenbergii NIES107
Microcystis viridis NIES102
Microcystis sp. strain UNSWCP1
Microcystis sp. strain AWT139
Microcystis sp. strain 199
Microcystis sp. strain 98
Microcystis sp. strain 205
Microcystis aeruginosa PCC7005
Microcystis aeruginosa NIES99
Microcystis elabens NIES42
Microcystis sp. strain HUB53
Microcystis sp. strain 269
Microcystis sp. strain 130
Nodularia spumigena PCC73104
Nodularia spumigena HEM
Nodularia spumigena BY1
Nodularia spumigena NSO
Nodularia sphaerocarpa HKVV
Nostoc sp. strain 152
Nostoc commune NIES24
Nostoc punctiforme PCC73120
Nostoc sp. strain 268
Nostoc sp. strain PCC7120
Oscillatoria agardhii 195
Oscillatoria agardhii 18R
Oscillatoria agardhii 223
Oscillatoria agardhii 49
Oscillatoria agardhii 97
Oscillatoria agardhii 126
Oscillatoria agardhii 128
Oscillatoria agardhii NIES204
Oscillatoria agardhii 2
Plectonema sp. strain UNSW901700
Pseudanabaena sp. strain AWT300
Synechococcus sp. strain AWT400
a

Toxinsa

PS-PCRb

MS-PCRb

Australia
England
Norway
Finland
Finland
Finland
Finland
Finland
Finland
Finland
Finland
Finland
Finland
Finland
Finland
Baltic Sea
Baltic Sea
Finland
Japan
Australia

S
nt
M
M
M
M
M
A
A
A
A
nt
nt
nt
A
nt
nt
nt
nt
C

1
1
1
1
1
ND
ND
ND
ND
ND
ND
1
1
1
ND
ND
ND
1
1
1

Australia
The Netherlands
Germany
Scotland
Japan
Japan
Australia
Australia
Finland
Finland
Finland
United States
Japan
Japan
Germany
Finland
Finland
Canada
Baltic Sea
Baltic Sea
Australia
Baltic Sea
Finland
Japan
Australia
Russia
United States
Finland
Finland
Finland
Finland
Finland
Finland
Finland
Japan
Finland
United States
Australia
Australia

nt
M
M
M
M
M
M
M
M
M
M
nt
nt
nt
nt
nt
nt
Nd
N
N
N
nt
M
nt
nt
nt
nt
M
M
M
M
M
M
M
M
nt
nt
nt
nt

1
1
1
1
1
1
1
1
ND
ND
ND
1
1
1
1
ND
ND
1
1
1
1
1
1
1
1
1
1
1
1
1
ND
ND
ND
ND
1
ND
1
1
2

Origin

Probesc
HUB

ANA

2
2
1
1
1
ND
ND
ND
ND
ND
ND
2
2
2
ND
ND
ND
2
2
2

ND
ND
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
1
ND
ND

ND
ND
1
1
1
1
1
2
2
2
2
2
2
1
2
2
2
6
ND
ND

1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
1
2
1
1
1
1
2
1
2
2
2
2
1
1
1
ND
ND
ND
ND
1
2
2
2
2

ND
1
1
ND
ND
ND
ND
ND
1
1
1
ND
ND
ND
ND
2
1
ND
1
1
1
2
1
ND
ND
2
2
1
1
6
1
1
1
1
ND
1
ND
ND
ND

ND
2
2
ND
ND
ND
ND
ND
2
2
6
ND
ND
ND
ND
2
6
ND
1
2
1
2
1
ND
ND
2
2
6
6
2
2
2
2
2
ND
6
ND
ND
ND

S, paralytic shellfish toxins (e.g., saxitoxins); nt, nontoxic; M, microcystins; A, anatoxin-a; C, cylindrospermopsin; N, nodularin.
1, positive; 2, negative; ND, not determined.
HUB, peptide synthetase gene probe amplified from M. aeruginosa HUB524; ANA, two amplified probes from Anabaena sp. strain 90.
d
N. spumigena PCC73104 mildly inhibits protein phosphatase 2A activity; however, the structure of this inhibitory compound has not been characterized.
b
c

VOL. 181, 1999

NONRIBOSOMAL PEPTIDE SYNTHESIS OF CYANOBACTERIA

4093

FIG. 3. Ethidium bromide-stained 1.5% agaroseTris-acetate-EDTA electrophoresis gels showing peptide synthetase gene amplification products from
various toxic and nontoxic cyanobacteria (Table 2). (A) Amplification products
obtained with the degenerate PS-PCR. Lanes 1 to 13, PCR fragments from the
cultured cyanobacteria Anabaena sp. strain 90, A. circinalis AWT006, Aphanizomenon sp. strain 202, C. raciborskii AWT205, Lyngbya sp. strain AWT211, M.
aeruginosa PCC7806, M. aeruginosa PCC7005, M. elabens NIES42, N. spumigena
PCC73104, Nostoc sp. strain 152, Nostoc sp. strain 203, Oscillatoria sp. strain 195,
and O. agardhii NIES204, respectively. The marker lane (M) contains fX174
digested with HaeIII, the top four bands being 1,358, 1,078, 872, and 603 bp. (B)
Specific amplification of the microcystin synthetase gene by use of the MS-PCR
described in the text. Lanes 1 to 13 are the same as in panel A. (C) Results of the
MS-PCR for uncultured cyanobacterial bloom samples containing the genera
Oscillatoria (lane 1), Nodularia (lane 2), and Microcystis (lanes 3 to 6). The
marker lane (M) contains SPP-1 digested with EcoRI, the six bottom bands being
1,390, 1,160, 980, 720, 480, and 360 bp.

FIG. 2. Schematic representation of cyanobacterial DNAs on a template dot

blot filter and results of hybridization and autoradiography. (A) Abbreviations
for cyanobacterial genera are as follows: Anab, Anabaena; Nos, Nostoc; Mic,
Microcystis; Osc, Oscillatoria; Nod, Nodularia; and Aph, Aphanizomenon. Hepatotoxic (H), neurotoxic (N), and nontoxic () strains were investigated. (B)
Hybridization pattern of the cyanobacterial dot blot filter with a 1.157-kb fragment of the mcyB gene of M. aeruginosa HUB524 as the probe. (C) Hybridization
pattern of the dot blot filter with a 2.0-kb fragment of a peptide synthetase gene
from Anabaena sp. strain 90 as the probe.

which contained significant DNA sequence similarity to the

adenylate-forming domains of known peptide synthetase genes
(13, 28, 34). With the described amplification reaction, putative
peptide synthetase genes were detected in strains of the cyanobacterial genera Anabaena, Aphanizomenon, Cylindrosper-

mopsis, Microcystis, Nodularia, Nostoc, Oscillatoria, Plectonema, and Pseudanabaena. However, similar degenerate PCR
products were not observed for the Synechococcus strain analyzed (Table 2). The results presented in Table 2 were consistent with a larger body of data generated for several other
strains of the species listed (data not shown).
Several members of the genera Anabaena, Aphanizomenon,
and Nostoc were tested for the presence of cyclic peptide toxins
and sequences homologous to those of peptide synthetase
genes (Fig. 3A). No strains of nonhepatotoxic Anabaena representing the species Anabaena circinalis, A. cylindrica, and A.
flos-aquae were shown to possess a microcystin synthetase gene
orthologue by the described MS-PCR or probe hybridization.
However, these species were shown to contain peptide synthetase gene orthologues. Of the 15 Anabaena strains examined and listed in Table 2, 7 were microcystin producers; the
other strains were either nontoxic or produced alkaloid neurotoxins (anatoxins or saxitoxins). These seven strains were

4094

NEILAN ET AL.

identified by amplification of the MS-PCR product and by

positive probe hybridization (Fig. 2B and 3B).
The closely related genus Nostoc, which was represented by
both microcystin-producing and nontoxic strains possessed
peptide synthetase gene homologues in toxic and nontoxic
types and microcystin synthetase gene homologues selectively
in only the hepatotoxic strain. Neurotoxic and nontoxic strains
of Aphanizomenon possessed peptide synthetase but not microcystin synthetase gene orthologues. This result correlates
with the lack of microcystin-based toxicity observed to date for
the genus Aphanizomenon (Fig. 2 and 3). MS-PCR of nonmicrocystin-producing strains of cyanobacteria from these
three genera did not reveal an amplification product.
The other filamentous and heterocyst-forming cyanobacteria used in this study, Nodularia and Cylindrospermopsis, also
showed gene detection experiment results which were congruent with toxin analyses. Members of the genus Nodularia are
responsible for the synthesis of nodularin, a cyclic pentapeptide, similar in structure to microcystin and likewise hepatotoxic due to its potent inhibition of eukaryotic protein phosphatases. PS-PCR and MS-PCR products were amplified from
all strains of Nodularia spumigena. These strains were also
shown to produce nodularin, as detected by HPLC, or to inhibit protein phosphatase 2A, as in the case of N. spumigena
PCC73104. The Nodularia strains used in this study were isolated from distinct geographic regions, implying a high degree
of gene conservation or a cosmopolitan distribution of hepatotoxic strains of this brackish-water cyanobacterium (31). No
amplification of the MS-PCR fragment correlated with the lack
of microcystin-based toxicity in Cylindrospermopsis raciborskii.
This cyanobacterium is capable of synthesizing a hepatotoxic
alkaloid known as cylindrospermopsin. The presence of PSPCR products for the strains studied may be related to the
presence of peptide synthetase genes of unknown function.
PS-PCR revealed that all strains of Microcystis studied possess one or more peptide synthetase loci. It has been shown
that strains of Microcystis, irrespective of their ability to produce microcystin, as determined by chemical analyses and bioassays, contain DNA sequences with significant identity to
known peptide synthetase genes (3). The data presented here
support these findings and describe a rapid method for the
determination of potentially hepatotoxic Microcystis based on
the amplification of the MS-PCR template. Samples of uncultured cyanobacterial blooms containing hepatotoxic Microcystis, Nodularia, or Oscillatoria strains were all detected by
MS-PCR (Fig. 3C). Both the degenerate and the specific amplification products were generated coordinately only for
strains which were shown to produce microcystin or otherwise
inhibit eukaryotic protein phosphatases (Fig. 3 and Table 2).
Moreover, we sequenced the products obtained by MS-PCR
from M. aeruginosa strains and consistently found significant
identity to the mcyB gene of strain PCC7806 (see below). It has
recently been shown that a single gene cluster inclusive of
mcyB is responsible for the synthesis of all microcystin isoforms
in M. aeruginosa PCC7806 and that all protein phosphatase
inhibition activity is due to cellular microcystin content (4).
The amplification of an MS-PCR product for nonhepatotoxic Microcystis sp. strain 269 was not supported by the
HUB524 microcystin synthetase gene probe hybridization (Fig.
2A). All other nontoxic strains of this genus did not show
amplification of the microcystin-specific DNA sequence (Table
2). Strain 269 may contain a genome sequence compatible with
the MS-PCR primers but not the dot blot probe. In addition, it
is also possible that toxin levels in this strain are below detection limits or that the altered expression of peptide synthetase
genes in laboratory cultures has inhibited microcystin biosyn-

J. BACTERIOL.

thesis by this strain (36). It is also possible that mutational

inactivation of the microcystin synthetase gene in regions other
than the fragments assayed resulted in the observed differences
among MS-PCR, probe hybridizations, and toxicity tests.
All the Oscillatoria agardhii strains produced detectable levels of microcystin, except for one, Oscillatoria sp. strain 2. This
strain also cross-hybridized with the HUB524 peptide synthetase probe but was found to be nontoxigenic by MS-PCR.
MS-PCR experiments and/or probe hybridizations indicated
that these strains, including strain 2, contained orthologous
toxin biosynthesis genes (Fig. 2B and 3B).
Of the remaining cyanobacterial groups to be tested with the
described molecular methods, the filamentous and non-heterocyst-forming genera Lyngbya, Pseudanabaena, and Plectonema
(Table 2) and several stromatolite-associated (benthic) cyanobacteria (data not shown) showed genomic orthology to the
conserved peptide synthetase loci but not to the microcystin
synthetase gene. The sole Synechococcus isolate examined did
not possess peptide synthetase or microcystin synthetase (Table 2). Similarly, there was a lack of peptide synthetase orthologues in the genome of Synechocystis sp. strain PCC6803 (12).
These results indicate that a broad range of cyanobacteria are
capable of nonribosomal peptide synthesis.
Sequence analysis and evolutionary relationships between
the putative microcystin synthetase gene and other peptide
synthetase genes in cyanobacteria. The specific MS-PCR products from 12 hepatotoxic cyanobacterial strains (representing
five genera and seven species) and four PS-PCR products
(representing peptide synthetase modules of unknown function) from nontoxic M. aeruginosa HUB53 (three modules)
and NIES99 (one module) were purified and sequenced. Approximately 700 bp of the putative microcystin synthetase gene
was determined for each strain. Furthermore, we included the
respective most similar sequence from another module of the
microcystin synthetase gene, mcyC, that had been determined
during a study aimed at identifying the entire microcystin synthetase gene cluster of M. aeruginosa PCC7806 (33a). DNA
sequences of the microcystin synthetase gene and other peptide synthetase gene orthologues were aligned with each other,
and the pairwise (Jukes-Cantor) genetic distances were calculated. These distances were represented in a phylogenetic analysis (Fig. 4A). Differences between the peptide synthetase gene
sequences were surprisingly large and reflected relatively low
sequence similarity between the highly conserved and functional domain motifs of each peptide synthetase module (Fig.
4B). The unicellular cyanobacteria of the genus Microcystis had
a second cluster of MS-PCR sequences which was delineated
from the PS-PCR sequences of the nontoxic Microcystis strains.
We concluded from this observation that, for the Microcystis
species studied, MS-PCR specifically amplified a sequence of
the mcyB gene and thus may be used to identify potential
microcystin producers in these species.
However, the situation appears more complicated for strains
of Anabaena, Nodularia, Nostoc, and Oscillatoria. For these
strains, MS-PCR amplified sequences were clearly different
from the sequences amplified for Microcystis strains. Moreover,
some of these sequences (N. spumigena BY1, Nostoc sp. strain
152, and O. agardhii 195) clustered more closely with the MSPCR products from Microcystis strains, whereas other sequences (Anabaena sp. strain 90 and N. spumigena HEM and
PCC73104) were more related to the PS-PCR products from
nontoxic M. aeruginosa strains. Since the sequences between
different modules (having different functions) and of different
peptide synthetase genes (again, having different functions)
were compared (Fig. 4B), no phylogenetic relationships can be
concluded from the PS-PCR sequences. The above-mentioned

FIG. 4. (A) Phylogenetic affiliations in a defined region of peptide synthetase genes from strains of the cyanobacterial genera Anabaena, Microcystis, Oscillatoria,
Nodularia, and Nostoc. M. aeruginosa HUB53 is nonhepatotoxic and contains a peptide synthetase with paralogous modules designated M, S, and U (27a). Comparisons
were based on partial sequence data from MS-PCR products (hepatotoxic strains in Table 2) and PS-PCR products (nontoxic strains). The phenogram was
reconstructed from a pairwise distance matrix by use of the neighbor-joining method. Hepatotoxic and nontoxic clusters of Microcystis strains are enclosed by square
brackets. (B) Evolutionary relationships among peptide synthetases of microcystin-producing cyanobacteria, nontoxic cyanobacteria, and other bacteria and fungi.
Translated MS-PCR and PS-PCR DNA sequences of cyanobacteria were compared to sequences of similar database peptides. Divergence between amino acid
sequences was calculated by use of PAM-Dayhoff matrix, and the tree was constructed by use of the neighbor-joining method obtained from the PHYLIP suite of
programs. Peptide synthetases, including various modules of the same multienzyme complex, are as follows: GrsA and GrsB, gramicidin A and B synthetases (with
modules A to C), respectively, from Bacillus brevis; SrfA, SrfB, and SrfC, surfactin A, B, and C synthetases, respectively, from B. subtilis; TycA, tyrocidin A synthetase
from Brevibacillus brevis; EntF, enterobactin synthetase component F from Escherichia coli; Hts1, HC toxin synthetase 1 (modules A to D) from Cochliobolus carbonum;
and SimA, cyclosporin A synthetase from Tolypocladium niveum. McyB and McyC are involved in microcystin biosynthesis in the M. aeruginosa strains indicated (Table
2). M. aeruginosa strains HUB53 and NIES99 are nontoxic and possess putative peptide synthetases of unknown function. The scale for each tree indicates inferred
evolutionary distances. Bootstrap values greater than 50% which were derived from 100 resampling events of the aligned sequence data and which support the tree
topologies are also shown.

4095

4096

NEILAN ET AL.

cluster of sequences from different cyanobacterial genera that

possibly represents the mcyB gene (Fig. 4A), however, reflects
organismic phylogenetic relationships, as inferred by 16S
rRNA gene sequences (9, 23, 35). More information on the
sequence and organization of peptide synthetase genes in cyanobacteria and of the microcystin synthetase gene in particular
is required before final conclusions can be made regarding the
evolution and phylogeny of microcystin biosynthesis.
The present study has shown, for the first time, that microcystin synthetase gene orthologues are present not only in all
toxic strains of the genus Microcystis but also in microcystinproducing strains of the genera Anabaena, Oscillatoria, and
Nostoc. Nodularin-producing cyanobacteria of the genus
Nodularia also appear to possess a microcystin synthetase gene
orthologue and therefore a similar biosynthetic pathway for
toxin production. We have also shown that strains of other
toxic and nontoxic cyanobacterial genera, such as Anabaena,
Aphanizomenon, Cylindrospermopsis, Lyngbya, Nodularia, Nostoc, Oscillatoria, Plectonema, and Pseudanabaena contain genes
for similar peptide synthetase complexes of unknown function.
Due to this broad intergeneric distribution of integrated enzyme systems, therefore, cyanobacteria provide a rich and
novel source of many uncharacterized amino-acid-activating
and -modifying peptide synthetase modules (18, 28, 32). This
study reveals a molecular approach to the discovery of novel
bioactive compound synthetic pathways in uncultured cyanobacteria and probably other microorganisms. The specific
PCR (MS-PCR) was applicable to the rapid and sensitive
detection of toxigenic strains of Microcystis. This method
could also be used to identify microcystin-producing strains of
other cyanobacterial genera. Further characterization (including insertional inactivation) of various microcystin synthetase
gene orthologues from Nodularia, Anabaena, Nostoc, and Oscillatoria will enable the design of specific PCRs for the detection of potential hepatoxin producers in each of these genera.
This strategy will provide a procedure for detecting toxic genotypes prior to the production of toxins by relevant cyanobacterial species.
ACKNOWLEDGMENTS
Additional strains used in this study were kindly supplied by Sue
Blackburn (CSIRO) and Boris Gromov (University of St. Petersburg).
The anonymous reviewers are thanked for their contributions to the
manuscript.
This work was funded in Australia by the Australian Research
Council, Australian Water Technologies, and the Co-operative Research Center for Water Quality and Treatment; in Germany by the
German Research Foundation, DFG (grant Bo 1045/13-3), and the
European Commission (grant BIO 4 CT96-0256); and in Finland by
the University of Helsinki and the Academy of Finland. B.A.N. was
supported by fellowships from the Alexander von Humboldt Foundation and the Australian Research Council. K.S. is a senior research
scientist of the Academy of Finland.
REFERENCES
1. An, J., and W. W. Carmichael. 1994. Use of a colorimetric protein phosphatase inhibition assay and enzyme linked immunosorbent assay for the study
of microcystins and nodularins. Toxicon 32:14951507.
2. Arment, A. R., and W. W. Carmichael. 1995. Evidence that microcystin is a
thiotemplate product. J. Phycol. 32:591597.
3. Dittmann, E., K. Meiner, and T. Borner. 1996. Conserved sequences of
peptide synthetase genes in the cyanobacterium Microcystis aeruginosa. Phycologia 35:6267.
4. Dittmann, E., B. A. Neilan, M. Erhard, H. von Do
hren, and T. Bo
rner. 1997.
Insertional mutagenesis of a peptide synthetase gene which is responsible for
hepatotoxin production in the cyanobacterium Microcystis PCC7806. Mol.
Microbiol. 26:779787.

J. BACTERIOL.
5. Eloff, J. N. 1981. Autecological studies on Microcystis, p. 7196. In W. W.
Carmichael (ed.), The water environmentalgal toxins and health. Plenum
Press, New York, N.Y.
6. Erhard, M., H. von Dohren, and P. Junblut. 1997. Rapid typing and elucidation of new secondary metabolites of intact cyanobacteria using MALDITOF mass spectrometry. Nat. Biotechnol. 15:906909.
7. Fay, P., and C. van Baalen. 1987. The cyanobacteria. Elsevier/North-Holland
Publishing Co., Amsterdam, The Netherlands.
8. Fujiki, H. 1992. Is the inhibition of protein phosphatase 1 and 2A activities
a general mechanism of tumor promotion in human cancer development?
Mol. Carcinog. 5:9194.
9. Giovanonni, S. J., S. Turner, G. J. Olsen, S. Barns, D. J. Lane, and N. R.
Pace. 1988. Evolutionary relationships among cyanobacteria and green chloroplasts. J. Bacteriol. 170:35843592.
10. Golden, J. W., C. D. Carrasco, M. E. Mulligan, G. J. Schneider, and R.
Haselkorn. 1988. Deletion of a 55-kilobase-pair DNA element from the
chromosome during heterocyst differentiation of Anabaena sp. strain PCC
7120. J. Bacteriol. 170:50345041.
11. Honkanen, R. E., F. R. Caplan, K. K. Baker, C. L. Baldwin, S. C. Bobzin,
C. M. Bolis, G. M. Cabrera, L. A. Johnson, J. H. Jung, L. K. Larsen, I. A.
Levine, R. E. Moore, C. S. Nelson, G. M. L. Patterson, K. D. Tschappat,
G. D. Tuang, A. L. Boynton, A. R. Arment, J. An, W. W. Carmichael, K. D.
Rodland, B. E. Magun, and R. A. Lewin. 1995. Protein phosphatase inhibitory activity in extracts of cultured blue-green algae (Cyanophyta). J. Phycol.
31:478486.
12. Kaneko, T., A. Tanaka, S. Sato, H. Kotani, T. Sazuka, N. Miyajima, M.
Sugiura, and S. Tabata. 1995. Sequence analysis of the genome of the
unicellular cyanobacterium Synechocystis sp. PCC6803. I. Sequence features
in the 1 Mb region from map position 64% to 92% of the genome. DNA Res.
2:153166.
13. Kleinkauf, H., and H. von Do
hren. 1996. A non-ribosomal system of peptide
biosynthesis. Eur. J. Biochem. 236:335351.
14. Krishnamurthy, T., W. W. Carmichael, and E. W. Sarver. 1986. Toxic peptides from freshwater cyanobacteria (blue-green algae). I. Isolation, purification and characterisation of peptides from Microcystis aeruginosa and
Anabaena flos-aquae. Toxicon 24:865873.
15. Lawton, L. A., C. E. Edward, and G. A. Codd. 1994. Extraction and high
performance liquid chromatographic method for the determination of microcystins in raw and treated waters. Analyst 119:1525.
16. Lehtima
ki, J., P. Moisander, K. Sivonen, and K. Kononen. 1997. Growth,
nitrogen fixation, and nodularin production by two Baltic Sea cyanobacteria.
Appl. Environ. Microbiol. 63:16471656.
17. Marahiel, M. A., T. Stachelhaus, and H. D. Mootz. 1997. Modular peptide
synthetases involved in non-ribosomal peptide synthesis. Mol. Gen. Genet.
97:26512673.
18. McDaniel, R., S. Ebert-Khosla, D. A. Hopwood, and C. Khosla. 1993. Engineered biosynthesis of novel polyketides. Science 262:15461557.
19. Meiner, K., E. Dittmann, and T. Bo
rner. 1996. Toxic and non-toxic strains
of the cyanobacterium Microcystis aeruginosa contain sequences homologous
to peptide synthetase genes. FEMS Microbiol. Lett. 135:295303.
20. Moore, R. E. 1996. Cyclic peptides and depsipeptides from cyanobacteria: a
review. J. Ind. Microbiol. 16:134143.
21. Namikoshi, M., and K. L. Rinehart. 1996. Bioactive compounds produced by
cyanobacteria. J. Ind. Microbiol. 17:373384.
22. Neilan, B. A. 1995. Identification and phylogenetic analysis of toxigenic
cyanobacteria by multiplex randomly amplified polymorphic DNA PCR.
Appl. Environ. Microbiol. 61:22862291.
23. Neilan, B. A., D. Jacobs, T. Del Dot, L. Blackall, P. R. Hawkins, P. T. Cox,
and A. E. Goodman. 1997. Ribosomal RNA sequences and evolutionary
relationships among the toxigenic cyanobacteria of genus Microcystis. Int. J.
Syst. Bacteriol. 47:693697.
24. Neilan, B. A., D. Jacobs, and A. E. Goodman. 1995. Genetic diversity and
phylogeny of toxic cyanobacteria determined by DNA polymorphisms within
the phycocyanin locus. Appl. Environ. Microbiol. 61:38753883.
25. Rapala, J., K. Sivonen, C. Lyra, and S. I. Niemela
. 1997. Variation of
microcystins, cyanobacterial hepatotoxins, in Anabaena spp. as a function of
growth stimuli. Appl. Environ. Microbiol. 64:22062212.
26. Rinehart, K. L., M. Namikoshi, and B. W. Choi. 1994. Structure and biosynthesis of toxins from blue-green algae (cyanobacteria). J. Appl. Phycol.
6:159176.
27. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a
laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.
27a.Schaub, V. Unpublished results.
28. Schneider, A., T. Stachelhaus, and M. A. Marahiel. 1998. Targeted alteration of the substrate specificity of peptide synthetases by rational module
swapping. Mol. Gen. Genet. 257:308318.
29. Sivonen, K. 1996. Cyanobacterial toxins and toxin production. Phycologia
35:1224.
30. Sivonen, K., W. W. Carmichael, M. Namikoshi, K. L. Rinehart, A. M.
Dahlem, and S. I. Niemela
. 1990. Isolation and characterization of hepato-

VOL. 181, 1999

NONRIBOSOMAL PEPTIDE SYNTHESIS OF CYANOBACTERIA

toxic microcystin homologs from Nostoc sp. strain 152. Appl. Environ. Microbiol. 56:26502657.
31. Sivonen, K., K. Kononen, W. W. Carmichael, A. M. Dahlem, K. L. Rinehart,
J. Kiviranta, and S. I. Niemela
. 1989. Occurrence of the hepatotoxic cyanobacterium Nodularia spumigena in the Baltic Sea and structure of the
toxin. Appl. Environ. Microbiol. 55:19901995.
32. Stachelhaus, T., A. Schneider, and M. A. Marahiel. 1995. Rational design of
peptide antibiotics by targeted replacement of bacterial and fungal domains.
Science 269:6972.
33. Theiss, W. C., W. W. Carmichael, J. Wyman, and R. Bruner. 1988. Blood
pressure and hepatocellular effects of the cyclic heptapeptide toxin produced

4097

by the freshwater cyanobacterium (blue-green alga) Microcystis aeruginosa

strain PCC7820. Toxicon 26:603613.
33a.Tillett, D. Unpublished data.
34. Turgay, K., and M. A. Marahiel. 1994. A general approach for identifying
and cloning peptide synthetase genes. Peptide Res. 7:238241.
35. Turner, S. 1998. Molecular systematics of oxygenic photosynthetic bacteria.
Plant Syst. Evol. 11:1352.
36. Watanabe, M. F., and S. Oishi. 1985. Effects of environmental factors on
toxicity of a cyanobacterium (Microcystis aeruginosa) under culture conditions. Appl. Environ. Microbiol. 49:13421344.