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Applied and Environmental Microbiology, November 2001, p. 5069-5076, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5069-5076.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Determination of Oligopeptide Diversity within a
Natural Population of Microcystis spp. (Cyanobacteria) by
Typing Single Colonies by Matrix-Assisted Laser Desorption
Ionization-Time of Flight Mass Spectrometry
Jutta
Fastner,1,*
Marcel
Erhard,2 and
Hans
von Döhren1
Biotechnology Center and Max Vollmer
Institute, Technical University Berlin, 10587 Berlin,1 and AnagnosTec GmbH, 14943 Luckenwalde,2 Germany
Received 23 May 2001/Accepted 28 August 2001
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ABSTRACT |
Besides the most prominent peptide toxin, microcystin, the
cyanobacteria Microcystis spp. have been shown to produce a
large variety of other bioactive oligopeptides. We investigated for the
first time the oligopeptide diversity within a natural
Microcystis population by analyzing single colonies
directly with matrix-assisted laser desorption ionization-time of
flight mass spectrometry (MALDI-TOF MS). The results demonstrate a high
diversity of known cyanobacterial peptides such as microcystins,
anabaenopeptins, microginins, aeruginosins, and cyanopeptolins, but
also many unknown substances in the Microcystis colonies.
Oligopeptide patterns were mostly related to specific Microcystis taxa. Microcystis aeruginosa
(Kütz.) Kütz. colonies contained mainly microcystins,
occasionally accompanied by aeruginosins. In contrast, microcystins
were not detected in Microcystis ichthyoblabe Kütz.; instead, colonies of this species contained
anabaenopeptins and/or microginins or unknown peptides. Within a third
group, Microcystis wesenbergii (Kom.) Kom. in Kondr.,
chiefly a cyanopeptolin and an unknown peptide were found. Similar
patterns, however, were also found in colonies which could not be
identified to species level. The significance of oligopeptides as a
chemotaxonomic tool within the genus Microcystis is
discussed. It could be demonstrated that the typing of single colonies
by MALDI-TOF MS may be a valuable tool for ecological studies of the
genus Microcystis as well as in early warning of toxic
cyanobacterial blooms.
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INTRODUCTION |
Freshwater and marine cyanobacteria
are known to produce a variety of bioactive compounds, among them
potent hepatotoxins and neurotoxins (for an overview, see reference
45). Many of the toxic species of cyanobacteria tend to
massive proliferation in eutrophicated water bodies and thus have been
the cause for considerable hazards for animal and human health
(3, 23). One of the most widespread bloom-forming
cyanobacteria is the genus Microcystis, a well-known
producer of the hepatotoxic peptide microcystin (45).
Microcystins are a group of closely related cyclic heptapeptides
sharing the common structure
cyclo(D-Ala-L-X-D-MeAsp-L-Z-Adda-D-Glu-Mdha), in which MeAsp is D-erythro-
-methylaspartic acid, Mdha
is N-methyldehydroalanine, Adda is
2S,3S,8S,9S-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4E,6E-dienoic acid, and X and Z are variable L-amino acids, e.g.,
microcystin-LR (MC-LR) contains leucine (L) and arginine (R)
(5). So far, more than 60 derivatives of microcystins have
been identified, varying largely by the degree of methylation, peptide
sequence, and toxicity (for an overview, see reference
45).
The hepatotoxicity of microcystins is based on their inhibition of
protein phosphatases 1 and 2A in combination with transport into
hepatocytes via the bile acid carrier, leading to acute liver failure
due to the disruption of hepatocyte cytoskeletal components (11,
26). The widespread occurrence and acute toxicity of microcystins and their tumor-promoting properties imply the need for
identification and prediction of toxic blooms (23).
The traditional botanical code describes the genus
Microcystis as a coccal, unicellular cyanobacterium that
grows as mucilaginous colonies of irregularly arranged cells (under
natural conditions, while strain cultures usually grow as single
cells). According to this tradition, morphological criteria such as
size of the individual cells, colony morphology, and mucilage
characteristics are used for species delimitation within
Microcystis (i.e., morphospecies) (20, 21).
Microcystin-producing strains as well as strains that do not synthesize
microcystin have been reported for all species within the genus
Microcystis. However, whereas most field samples and strains
of Microcystis aeruginosa and Microcystis viridis
studied to date were found to contain microcystins (17, 47,
49-51), strains of M. wesenbergii, M. novaceckii,
and M. ichthyoblabe have only sporadically been reported to
contain microcystins (34, 38, 49).
Beside microcystins, various other linear and cyclic oligopeptides such
as aeruginosins, anabaenopeptilides, cyanopeptolins, anabaenopeptins,
and microginins are found within the genus Microcystis (31). Similar to microcystins, these peptides possess
unusual amino acids like 3-amino-6-hydroxy-2-piperidone (Ahp) in
cyanopeptolins or 2-carboxy-6-hydroxyoctahydroindol (Choi) in
aeruginosin-type molecules, and numerous structural variants also exist
within these groups (14, 29, 31). These peptides show
diverse bioactivities, frequently protease inhibition
(31).
The presence of D-amino acids, unusual amino acids, as well
as their small size suggests that the cyanobacterial oligopeptides mentioned above are synthesized nonribosomally by multifunctional enzyme complexes, generally termed peptide synthetases, a pathway studied intensively in other bacteria and fungi (1,
19). The nonribosomal synthesis of microcystins in the axenic
strain Microcystis sp. strain PCC 7806 and of
anabaenopeptilides in Anabaena sp. strain 90 was recently
demonstrated by site-directed mutagenesis and sequencing (6, 42,
46). Nonribosomal peptide synthetase genes have so far been
detected in all strains of the genus Microcystis, but genes
encoding for the so-called microcystin synthetase are usually detected
only in toxic (i.e., microcystin-containing) Microcystis
spp. (7, 35). This corresponds to the observation of
oligopeptides in all Microcystis strains investigated to
date showing various combinations of microcystins and/or other
oligopeptides such as aeruginosins, cyanopeptolins, or anabaenopeptins
(8, 27, 31).
The cooccurrence of both microcystins and other oligopeptides such as
anabaenopeptins and cyanopeptolins in natural Microcystis populations was recently demonstrated (10, 14, 36). It is well known that the species and genotype composition in natural Microcystis populations is heterogeneous, and both
microcystin- and non-microcystin-containing strains have been isolated
from the same sample (41, 48, 52). Rohrlack et al.
(41) isolated 13 Microcystis strains from Lake
Wannsee (Berlin, Germany) in 1995 which produced either microcystins or
anabaenopeptins (T. Rohrlack, M. Erhard, and M. Henning, unpublished
data). Furthermore, isolated strains may show both a different
qualitative and quantitative microcystin pattern than the original
population (41, 48). These results suggest a considerable
diversity of genotypes with different oligopeptide patterns in natural
Microcystis populations.
Our study aimed to investigate the inter- and intraspecific
oligopeptide diversity in a natural population of the genus
Microcystis. Since isolation of strains from natural
populations is likely to be selective, we recorded the oligopeptide
pattern directly in single Microcystis colonies selected
from natural populations using matrix-assisted laser desorption
ionization-time of flight mass spectrometry (MALDI-TOF MS).
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MATERIALS AND METHODS |
Sampling and isolation.
Microcystis spp. were
harvested biweekly in hypertrophic Lake Wannsee, Berlin, Germany, from
June to October 1999 with a plankton net (40-µm mesh size). The
samples were stored in the cool and dark until isolation of
Microcystis colonies the same day. The colonies were
isolated by serial dilution with tap water and micromanipulation techniques using an inverted microscope at a magnification of ×160 to
×400. Isolated colonies were washed by transferring them to several
drops of water until all other organisms were removed.
Epiphytic cyanobacteria and algae sticking in the mucilage of
Microcystis aeruginosa could not be detached (Table
1). Cell size and morphological
characteristics were recorded for each colony and species were
determined according to Komárek and Anagnostidis (21) (Table 1). Additionally, aliquots of the concentrated net samples were taken and either frozen at 
20°C and lyophilized or
fixed with formaldehyde solution and stored in the dark for detailed
cell size determination. The mean cell diameter of the species was
determined by measuring the diameters of 50 cells (10 cells per colony)
every sampling day.
Extraction and preparation of Microcystis colonies,
lyophilized strains, and field samples for MALDI-TOF MS analysis.
Single colonies were directly transferred onto a stainless steel
template, and immediately 1 µl of matrix (10 mg of
2,5-dihydroxybenzoic acid per ml in water-acetonitrile [1:1] with
0.03% trifluoroacetic acid) was added. The extraction of the
oligopeptides from the cells was achieved by the solvent fraction of
the matrix. An immediate change of the colony color from green to
brownish yellow after addition of the matrix was observed due to the
degradation of chlorophyll a by the acidic solution.
Lyophilized
Microcystis field samples, the axenic
Microcystis strains PCC 7806 and PCC 7813, and the unialgal
Microcystis strains HUB 5-2-4, HUB 5-3, and HUB 063 were
extracted with acetonitrile-ethanol-water
(1:1:1) with 0.03%
trifluoroacetic acid. Then 1 µl of the extract
was prepared for
MALDI-TOF MS analysis as described for the single
colonies.
MALDI-TOF MS analysis.
Positive ion mass spectra were
recorded from each colony and the lyophilized field samples and strains
using a MALDI-TOF mass spectrometer (Voyager DE-PRO; PerSeptive
BioSystems, Framingham, Mass.) equipped with a reflectron. For
desorption of the components, a nitrogen laser beam (
= 337 nm)
was focused on the template. The acceleration voltage was set at 20 kV.
All measurements were carried out in the delayed extraction mode,
allowing the determination of monoisotopic mass values (m/z;
mass-to-charge ratio). Analyses were performed in the positive-ion
mode, giving mainly singly protonated molecular ions
([M+H]+). Chlorophyll a degradation products
phaeophytin a and pheophorbide a with mass values
of m/z 871.57 and 593.27 [M+H]+, respectively,
were used for internal calibration. A low mass gate of 500 Da improved
measurement by filtering out the most intensive matrix ions.
After determination of monoisotopic mass values, post-source decay
(PSD) measurements for recording fragment ions were performed
directly
from the same colony on the template. The precursor ions
were selected
with a time ion selector having a mass window of
10 mass units. The
operating voltages of the reflectron were reduced
stepwise to record 12 spectral segments
sequentially.
PSD spectra of the most prominent peptides were recorded several times
over the entire sampling period from single colonies
and additionally
from the lyophilized
Microcystis samples.
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RESULTS |
Species determination.
The Microcystis population
in Lake Wannsee in 1999 consisted of several species which were present
during the entire investigation period. The majority of the isolated
colonies belonged to one of three species with distinct colonial
features and cell sizes (Table 1): M. aeruginosa
(Kützing) Kützing, M. ichthyoblabe Kützing, and M. wesenbergii (Komárek)
Komárek in Kondrateva. The other colonies isolated could not be
unequivocally determined to species level and thus were grouped as
Microcystis spp. About half of these colonies exhibited
colonial characteristics similar to those of Microcystis
ichthyoblabe but had larger cell sizes than the main phenotype
isolated from this species. The other colonies showed diverse
variations in cell size and colonial characteristics, often similar to
features described for M. flos-aquae or M. novaceckii (Komárek) Compère. The sizes of the
colonies isolated ranged between 0.2 and 4 mm, as measured at their
longest dimension.
Oligopeptides identified by MALDI-TOF MS.
Positive-ion mass
spectra were recorded from the Microcystis strains, from 258 Microcystis colonies, and from the entire
Microcystis population each time colonies were isolated
(Fig. 1 and
2). In the mass range of
m/z 500 to 1,100 Da, numerous structural variants of
microcystins, anabaenopeptins, microginins, aeruginosins, and one
cyanopeptolin were identified by means of characteristic fragment ions
obtained by PSD measurements (Table 2).
Most of these components are well known, and data about their
structures and PSD data have been published previously (9, 10,
12) (Table 2). As an example, the PSD spectrum of microcystin-LR
with characteristic fragment ions leading to an unambiguous structure
assignment is given in Fig. 3
(32). Fragment analysis assigned peptides with mass values
of m/z 726, 728, 740, 742, 749, and 751 [M+H]+
as microginin variants previously identified by means of amino acid
analysis and PSD measurements (Table 2) (U. Neumann, J. Weckesser, and
M. Erhard, unpublished data). The compound with a molecular mass of
m/z 603 [M+H]+ is probably a new variant of an
aeruginosin-type peptide, as suggested by the fragment ion of
m/z 140, indicating the presence of the unusual amino acid
Choi, which is unique to aeruginosin-type molecules (8, 28,
29).
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FIG. 1.
Positive-ion MALDI-TOF mass spectra (m/z
range 550 to 1,100 Da) of Microcystis strains PCC 7806 (axenic) (A), PCC 7813 (axenic) (B), HUB 5-3 (unialgal) (C), HUB 5-2-4 (unialgal) (D), and HUB 063 (unialgal) (E). For structure assignments,
see Table 2.
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FIG. 2.
Positive-ion MALDI-TOF mass spectra (m/z
range, 600 to 1,100 Da) of the entire Microcystis population
(A) and of colonies of M. aeruginosa (B), M. ichthyoblabe (C), and Microcystis sp. (D) in Lake
Wannsee on 24 August 1999. For structure assignments, see Table 2.
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TABLE 2.
Oligopeptides detected in Microcystis colonies
from Lake Wannsee in 1999 using MALDI-TOF MS (results from positive-ion
mass spectra and PSD measurements)
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FIG. 3.
PSD spectrum of microcystin-LR: m/z 995 [M + H]+, 861 [M 134 (Adda side
chain) + H]+, 599 [ArgAddaGlu + H]+, 375 [C11H14OGluMdha]+, 286 [ArgMeAsp + H]+, 213 [GluMdha + H]+, 155 [MdhaAla + H]+, 135 [PhCH2CH(OCH3)]+, and 70 [Leu CO + H]+.
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In addition, many unknown substances were detected in the colonies, of
which those with mass values of
m/z 619, 635, 771,
787, 804, 846, 1,007, 1,009, 1,015, and 1,021 were the most
abundant.
Inter- and intraspecific oligopeptide diversity.
Mass spectra
from the whole Microcystis population in Lake Wannsee in
1999 showed a complex mixture of different microcystins, microginins,
anabaenopeptins, and unknown components (Fig. 2A). In contrast,
isolated Microcystis strains usually have a less diverse
peptide pattern. As shown in Fig. 1, the axenic Microcystis strains PCC 7806 and PCC 7813 contain largely MC-LR and
[Asp3] MC-LR and either cyanopeptolin D or the
unknown peptide of m/z 603 [M+H]+ (Fig. 1A and
B; Table 2) (27). Microcystins are the most abundant oligopeptides in the unialgal strain HUB 5-2-4, while in HUB 5-3 an
unknown peptide of m/z 804 [M+H]+ and in HUB
063 anabaenopeptin B and F are found (Fig. 1C to E).
Similar to the clonal strains, the peptide patterns in the single
Microcystis colonies were usually less complex (Fig.
2B
to
D). Comparison of the peptide composition in the total
Microcystis population with that of single colonies suggests
that the population
is dominated by colonies with the specific
oligopeptide patterns
shown in Fig.
2B to
D.
The oligopeptide patterns in colonies of
M. aeruginosa, M. ichthyoblabe, and
M. wesenbergii revealed pronounced
differences
(Fig.
2B to D and Fig.
4). In
all but 2 of 111
M. aeruginosa colonies,
microcystins were
the chief oligopeptides detected (Fig.
2B, Fig.
4). The microcystin
profiles within this species were rather homogeneous,
with
MC-RR, MC-YR, and MC-LR being codominant in most
colonies.
Similar microcystin compositions were found both by MALDI-TOF
MS (Fig.
2A) and by high-pressure liquid chromatography (HPLC)
analysis
(data not shown) of the whole population in Lake Wannsee.
MC-RR was
detected in 79%, MC-YR in 89%, and MC-LR in 94% of all
M. aeruginosa colonies. Minor microcystins, as indicated by low
peak
intensities and small amounts in HPLC analysis, found in
M. aeruginosa were [Dha
7] MC-RR,
[Dha
7] MC-LR, [H
4] MC-YR, and MC-WR. They
were detected in 13 to 37%
of all colonies. In some colonies of
M. aeruginosa, only [Dha
7] MC-RR and
[Dha
7] MC-LR were found. In addition to microcystins,
aeruginosins
were present in some colonies, and in four colonies
anabaenopeptins
could be detected (Fig.
4). Unknown components were
detected in
many
M. aeruginosa colonies, occasionally with
molecular weights
corresponding to those of known microcystin variants.
However,
signal intensity was too low to obtain reliable PSD spectra.
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FIG. 4.
Molecular masses (m/z) oligopeptides and
unknown components in the range of m/z 500 to 1,100 Da
detected in colonies of M. aeruginosa, M. ichthyoblabe, and
M. wesenbergii. Symbols for substance classes: open
diamonds, aeruginosin; open circles, microginin; open triangles,
anabaenopeptin; open squares, microcystin; solid diamonds,
cyanopeptolin; small solid rectangles, unknown components. For
structure assignments of compounds, see Table 2.
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In order to control for the homogeneity of the colonies, eight large
colonies of
M. aeruginosa were divided into two to four
parts, and each part was analyzed separately. No difference in
the
microcystin pattern was found between the single parts (data
not
shown). Furthermore, no relationship between the presence
of epiphytic
cyanobacteria and algae in the
M. aeruginosa colonies
and
their peptide pattern was
found.
By contrast, microcystins were never detected in colonies of
M. ichthyoblabe (Fig.
2C, Fig.
4). The oligopeptide pattern within
this group was more diverse: they contained either mainly
anabaenopeptins
or microginins or both microginins and
anabaenopeptins or various
unknown peptides. Anabaenopeptins B
and F and oscillamide Y were
the most prominent anabaenopeptins, while
anabaenopeptins I and
A were less abundant. Major microginins were FR5
and FR3, while
all other variants of this substance class were less
frequent.
Aeruginosins were also detected in the colonies of
M. ichthyoblabe (Fig.
4).
M. wesenbergii colonies also did not contain microcystins.
The colonies of this species show very similar patterns, with 11
of 14 colonies containing cyanopeptolin-S together with an unknown
peptide of
m/z 635 [M+H]
+ (Fig.
4).
Oligopeptide patterns of
Microcystis colonies not identified
to species level (
Microcystis spp.) mainly fall into three
clusters,
with peptide patterns similar to those observed for
M. aeruginosa and
M. ichthyoblabe (Fig.
5). The colonies contained either
microcystins,
microginin and/or anabaenopeptin, or chiefly unknown
peptides
(Fig.
2D, Fig.
5). Aeruginosins were detected in all three
clusters.
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FIG. 5.
Molecular masses (m/z) of oligopeptides and
unknown components in the range of m/z 500 to 1,100 Da
detected in colonies of Microcystis spp. Symbols for
substance classes: open diamonds, aeruginosin; open circles,
microginin; open triangles, anabaenopeptin; open squares, microcystin;
solid diamonds, cyanopeptolin; small solid rectangles, unknown
components. For structure assignments of compounds, see Table 2.
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DISCUSSION |
MALDI-TOF MS analysis of the Microcystis populations
from Lake Wannsee in 1995 to 1999 showed a complex and persisting
mixture of microcystins, other oligopeptides, and unknown components
(8, 10) (Fig. 2A). The cooccurrence of microcystins and
cyanopeptolins in other Microcystis sp.-dominated field
samples was reported previously (15, 36). By typing single
Microcystis colonies, we could show for the first time that
the actual peptide diversity in a natural population of this genus is
substantially higher. Many of the substances detected belong to
well-known groups of cyanobacterial peptides like microcystins,
anabaenopeptins, microginins, cyanopeptolins, and aeruginosins, of
which many have been discovered in Microcystis spp.
(31). In addition, numerous unknown components have been
detected in the colonies. However, the origin of these unknown
components has yet to be investigated, since besides the observed
epiphytic cyanobacteria and algae, heterotrophic bacteria are also
known to be present in Microcystis colonies
(4).
Usually more than one type of oligopeptide was detected in the
Microcystis colonies from Lake Wannsee in 1999. With respect to known peptides, combinations of anabaenopeptins, microginins, and
aeruginosins were observed, while microcystins were found along with
aeruginosins. This correlates to the detection of aeruginosins as well
as cyanopeptolins in both toxic and nontoxic Microcystis culture strains (Fig. 1) (6, 31). Anabaenopeptins and
microginins were usually not detected together with microcystins with
the exception of four colonies containing both microcystins and
anabaenopeptins. Microcystins and anabaenopeptins were also never found
simultaneously in more than 20 Microcystis strains
investigated to date (M. Erhard, H. von Döhren, P. Jungblut, E. Dittmann, T. Börner, M. Henning, L. Rouhiainen, and K. Sivonen,
Abstracts of the 4th European Workshop on the Molecular Biology of
Cyanobacteria, p. 29, 1999). However, it must be considered that
isolation of strains is selective and may pick up only those genotypes
which are favored by the cultivation conditions. On the other hand,
although one colony can be regarded as a clone and homogeneity was
found for all of the divided colonies, a potential bias of conducting
studies with selected colonies may be the contamination of a colony
with cells of other clones.
Our data revealed a relationship between oligopeptide patterns and
certain Microcystis taxa in Lake Wannsee. Microcystins were
chiefly found in M. aeruginosa, while colonies of M. ichthyoblabe and M. wesenbergii did not contain
microcystins but did contain anabaenopeptins, microginins,
cyanopeptolins, or unknown peptides. Essentially the same results were
found for the presence of the microcystin synthetase genes in single
Microcystis colonies from Lake Wannsee in 2000 (24); microcystin synthetase genes were detected in 73%
of M. aeruginosa colonies but in only 16% of colonies assigned to M. ichthyoblabe and in 0% of M. wesenbergii colonies. The low oligopeptide diversity within
M. aeruginosa suggests that the species-related peptide
patterns observed may have been caused by the dominance of only certain
genotypes within the Microcystis species in Lake Wannsee in
1999. Restriction fragment length polymorphism of the mcyB
gene indicates the presence of five different genotypes among M. aeruginosa colonies with similar microcystin profiles in Lake
Wannsee in 2000 (24). The occurrence of various
combinations of several types of oligopeptides in M. ichthyoblabe implies a higher genotype diversity within this
species in Lake Wannsee in 1999.
M. aeruginosa is worldwide the species most often associated
with toxic water blooms and microcystin-producing strains (38, 47, 50), while the majority of M. wesenbergii and
M. ichthyoblabe strains reported in the literature did not
produce microcystin, although microcystin-producing strains are
occasionally described (34, 38, 49). Japanese strains of a
M. aeruginosa S-type (i.e., small cell size), which were
later classified as M. ichthyoblabe (51), also
rarely contained microcystins (50). Rohrlack et al.
(41) isolated 13 Microcystis strains from Lake
Wannsee in 1995, of which the strains having larger cells contained
microcystins, while those with smaller cells produced only
anabaenopeptins (T. Rohrlack, M. Erhard, and M. Henning, unpublished
data). Though these data support some relationship between
morphospecies and microcystin production, we also detected the peptide
patterns typical for M. aeruginosa and M. ichthyoblabe in colonies with colonial characteristics different
from these species (these were grouped as Microcystis spp.).
However, colony morphology and cell size, traditionally used in
taxonomic differentiation of the genus Microcystis
(20, 21), may be questionable criteria for species
distinction; both parameters have been found to be variable in
laboratory cultures and in the field (22, 39, 40). In
culture particularly some strains of M. aeruginosa, M. ichthyoblabe, and M. novaceckii have developed colony
morphologies similar to each other (39). Recent studies
investigating the taxonomy of the genus Microcystis using genetic criteria in comparison to morphological traits and microcystin production show contradictory results. 16S rRNA analysis revealed no
differences between different toxic and nontoxic strains of M. aeruginosa, M. wesenbergii, and M. viridis
(34). In contrast, sequence data for the 16S to 23S
internally transcribed spacer lead to three clusters, of which cluster
I contained both toxic and nontoxic strains of M. aeruginosa, M. novaceckii, and M. ichthyoblabe, cluster II only toxic
strains of mainly M. viridis, and cluster III only nontoxic
strains of mainly M. wesenbergii (38).
Similarly, allozyme divergence studied by Kato et al. (16)
characterized M. wesenbergii and M. viridis as
well-established species. In contrast to Otsuka et al.
(38), allozyme divergence (16) revealed a
separation of Japanese M. aeruginosa L-type strains
(equivalent to M. aeruginosa) from strains of M. aeruginosa type S (included in M. ichthyoblabe
[51]), which corresponds to our observation of a
different peptide pattern in these species. More data are needed to
determine whether or not the oligopeptide pattern may be used as a
chemotaxonomical feature to clarify the taxonomic uncertainties within
the genus Microcystis. These should include systematic
studies on the distribution of oligopeptides in combination with
morphological and molecular characterization of more strains and
original colonies from different regions.
Our study demonstrates the unequivocal identification of microcystins
and other oligopeptides in single Microcystis colonies by
employing MALDI-TOF MS. It is thus possible to directly identify the
toxic species and genotypes in natural Microcystis
populations without time-consuming and probably selective isolation
procedures. The typing of single Microcystis colonies may be
a valuable tool in early warning of toxic bloom formation, since it
enables rapid detection of whether or not a population contains
microcystin-producing genotypes in an early phase of population growth.
Furthermore, the succession of toxic and nontoxic species may be
followed and the influence of biotic and abiotic factors on genotype
succession assessed. In Lake Wannsee in 1999, M. aeruginosa
colonies showed a persisting composition of MC-RR, MC-YR, and MC-LR
over the entire sampling period from June to October. Similar
quantitative relations of microcystins were determined in the whole
Microcystis population by both HPLC and MALDI-TOF MS during
this time span. This indicates that this type of M. aeruginosa colony determined the overall microcystin pattern in 1999.
Hypotheses about possible functions of microcystins often focused on
the toxicity of microcystins. Although microcystins have been shown to
affect diverse aquatic organisms (for an overview, see reference
45), they do not necessarily seem to be produced as a
defense mechanism against zooplankton grazing. Speculations about an
inter- or intracellular function of microcystins raise the question
about substances playing a similar role in genotypes without
microcystins (37). This is supported by our observation of
various oligopeptides in the Microcystis population
investigated. The coexistence of genotypes producing either mainly
microcystins or other oligopeptides throughout the investigation period
suggests that a comprehensive understanding of their possible functions and ecological benefits requires studying oligopeptides as a group rather than focusing on microcystins.
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ACKNOWLEDGMENTS |
We thank Frank Grützner and Adam Antebi and his group (Max
Plank Institute for Molecular Genetics, Berlin, Germany) for kindly providing micromanipulation facilities. Microcystis strains
were kindly provided by Rosemarie Rippka (Institute Pasteur, Paris, France) and Manfred Henning (Humboldt University, Berlin, Germany).
This work was financially supported by funds from the EU
(ENV4-CT98-802).
| |
FOOTNOTES |
*
Corresponding author Mailing address: Federal
Environmental Agency, Corrensplatz 1, 14195 Berlin, Germany. Phone: 49 30 8903 1390. Fax: 49 30 8903 1830. E-mail:
jutta.fastner{at}uba.de.
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Applied and Environmental Microbiology, November 2001, p. 5069-5076, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5069-5076.2001
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Abstract
Introduction
