FEMS Microbiology Ecology 37 (2001) 39^43
www.fems-microbiology.org
Consequences of impaired microcystin production for
light-dependent growth and pigmentation of
Microcystis aeruginosa PCC 7806
Karina Hesse *, Elke Dittmann, Thomas Bo«rner
Institut fu«r Biologie, Humboldt-Universita«t, Unter den Linden 6, D-10099 Berlin, Germany
Received 17 November 2000; received in revised form 31 May 2001; accepted 31 May 2001
First published online 22 June 2001
Abstract
Microcystis aeruginosa PCC 7806 produces potent inhibitors of eukaryotic protein phosphatases called microcystins, whose function to
the organism is presently unknown. Mutants with impaired microcystin biosynthesis should provide a useful tool for investigations of
microcystin function. This study has focussed on the comparison of growth and pigment content of strain PCC 7806 and its mcyB3 mutant
deficient in microcystin biosynthesis, under semicontinuous culture conditions. Both wild-type and mutant are characterised by a very low
light demand and low-maximum specific growth rates in comparison to other Microcystis strains studied. While growth of wild-type and
mutant were similar under different light conditions, the mutant cells showed significantly higher specific absorbances in the range of
photosynthetic active radiance 420^700 nm, under light-limiting conditions. The mutant cells possess lower contents of chlorophyll a,
L-carotene, zeaxanthin and echinenone under light limitation and of myxoxanthophyll under saturated light conditions. Though
microcystins are clearly not essential for growth, the observed effects of the mutation are a first indication of their involvement in
intracellular processes. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Microcystin-de¢cient mutant; Microcystin function; Chlorophyll; Carotenoid; Microcystis aeruginosa
1. Introduction
Cyanobacteria are known to produce a variety of bioactive substances including antibiotics, cytotoxins, neurotoxins and hepatotoxins as well as substances with antiviral and anti-algal e¡ects [1,2]. Scienti¢c interests have
focussed especially on Microcystis aeruginosa, due to its
frequent occurrence in toxic blooms world-wide and the
related health hazard for humans and livestock [3]. Most
of the risk can be attributed to a family of small cyclic
heptapeptides, called microcystins. Microcystins are specific inhibitors of eukaryotic protein phosphatases 1 and 2A
and are primarily responsible for damage of blood-vessels
in the liver of vertebrates leading to an acute haemorrhetic
shock, i.e. they act as hepatotoxins [2,4]. Microcystins are
produced by a number of bloom-forming species of cya-
* Corresponding author. Tel. : +49 (30) 2093 6533 ;
Fax: +49 (30) 2093 6530.
E-mail address : [email protected] (K. Hesse).
nobacteria, but their function for the producing organisms
remains obscure. An important role of microcystins in
cellular processes of cyanobacteria has been discussed previously in relation to the compound's ability to chelate
metal ions and thus function as siderophores, possibly
regulating the amount of free Fe2‡ in the cell [5]. A role
for microcystin as a feeding deterrent against zooplankton
or other grazers has also been suggested [6,7]. Overall, the
role of microcystin is still highly disputed. Even its common classi¢cation as a secondary metabolite has come
under scrutiny, due to a linear correlation calculated between microcystin production and cell division rates [8].
Recently a gene cluster (mcy genes) was identi¢ed encoding non-ribosomal peptide synthetases, polyketide synthases and other enzymes involved in microcystin biosynthesis in M. aeruginosa [9,10]. Mutants unable to produce
microcystins were generated by insertional mutagenesis of
some of the mcy genes. These knock-out mutants (mcyB3 ,
MT), still producing cyanopeptolines (the second group of
small peptides detected in the wild-type (WT) strain [11])
o¡er the possibility to ¢nd out if the loss of microcystin
has phenotypic consequences for the cyanobacterial cells
0168-6496 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 1 4 2 - 8
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K. Hesse et al. / FEMS Microbiology Ecology 37 (2001) 39^43
and may thus provide a clue to the function of microcystins for the cyanobacterial cell. Transcription of mcy genes
is positively regulated by light in M. aeruginosa [12,13]
suggesting a function of microcystins in light-dependent
processes. Therefore, in a ¢rst attempt to elucidate a possible role of microcystins, we compared growth and pigment composition of the WT strain M. aeruginosa PCC
7806, a microcystin producer, and the MT mutant, lacking
microcystins [11], under di¡erent light conditions.
2. Materials and methods
2.1. Cyanobacterial strains and cultivation
The microcystin-producing M. aeruginosa strain
PCC7806 was kindly provided by R. Rippka (Institute
Pasteur, Paris, France). The mutant used in this study,
unable to produce microcystins, was previously generated
by insertion of a chloramphenicol resistance gene cassette
into mcyB [11]. MT and WT were maintained on BG11
agar medium, the MT being usually kept in medium containing 5 Wg ml31 chloramphenicol. For all comparative
experiments during this study, chloramphenicol was omitted from the medium to exclude an in£uence of the antibiotic on physiological processes and morphological characteristics. The persistence of the mutation, as indicated by
the lack of toxin, was checked during and after culturing
by HPLC [14,15].
Cells were grown in 100 ml M IV/2 medium [16] in 500ml Erlenmeyer £asks, shaken in a water bath at 20³C and
illuminated in a 12-h/12-h light^dark cycle with £uorescent
tubes (Lampi warmwhite LT 36 W, Germany) in a light
intensity gradient between 4 and 110 WE m32 s31 : to
maintain nutrient saturated conditions cultures were
grown semicontinuously (turbidostat principle), diluted
every 2^3 days with MIV/2 nutrient solution.
2.2. Biovolume determination
A photometrical measurement of culture density was
performed with double-frame technique [17] in order to
facilitate the biovolume detection procedure for culture
control. In this double-frame method priority is given to
light attenuation by scattering which is caused by strain
speci¢c morphological characteristics. Optical density
(light extinction), when measured using this technique is
less in£uenced by cellular pigmentation. The obtained attenuation signals of the culture suspensions of both the
WT and the MT strain at 436 nm (Photometer 1101 M,
Eppendorf Gera«tebau, Netheler+Hinz GmbH, Hamburg,
Germany) were calibrated to their real biovolumes, which
were determined microscopically both by measuring diameters of the spherical cells calculated as mean cell volume,
and by cell counting. The data sets of both variants were
tested for their normal distribution. Signi¢cance of di¡er-
ences between WT and MT was tested by comparing the
slopes of the biovolume vs light extinction curves using a
Student's t-test and hypothesis-test (STATGRAPHICS0
Plus 3.0, Statistical Graphics Corp., USA).
2.3. Pigment extraction
Cells were harvested as 20-ml samples from the cultures
maintained at exponential growth phase on three di¡erent
days at de¢ned biovolumes. The cells were ¢ltered onto
glass ¢bre ¢lters (Whatman GF/C) in two aliquots (10 ml
each). Subsequently, the ¢lters were freeze-dried and
stored at 320³C. For extraction, ¢lters were ground in a
mortar with 2 ml of cold acetone (90%), sonicated in an
ice bath for 10 min and centrifuged at 8000 rpm for 10
min (4³C). The sediments were re-extracted with 1 ml acetone (100%) in the same way. Collected extracts were
mixed with IPR-solution (15 mg ml31 tetrabutylammonium acetate in 77 mg ml31 ammonium acetate solution)
and ¢ltered through 0.2-Wm syringe-¢lters into autosampler tubes. Chromatography was carried out with a binary
gradient system in a Waters 600E (Waters, Milford, USA)
gradient module (eluent 1: methanol/water/IPR ^
80:10:10, eluent 2: methanol/acetone ^ 80:20). Peaks
were recorded at 440 nm. A detailed description of the
pigment extraction method and analysis is given in Woitke
et al. [16].
Phycocyanin, which is water soluble, can not be extracted by the method described above and hence is not
detected by HPLC. However it has a distinct absorption
maximum at 625 nm. The intensity of the photometric
signal (extent of the peak) indicates the amount of phycocyanin in the cell. Hence, cytophotometric in vivo measurements complemented the pigment analysis performed
with HPLC. Prior to measurement, samples were pressurised to collapse the gas vesicles and avoid otherwise disturbing optical interferences. This was achieved by placing
the cell suspension, bubble-free, in a glass syringe, sealing
the needle with a rubber stopper and pressurising by 2^3
hits with a rubber mallet on the syringe pistol until light
microscopy showed cells without gas vesicles. Cytophotometric absorbance spectra from 420 to 750 nm were recorded for at least 10 single cells of both a light-limited
and a nearly light-saturated culture of WT and MT (Leitz
DMRB with MPV-SP, Leitz, Wetzlar, Germany). Each
spectrum was normalised by the actual cell diameter to
obtain comparable speci¢c absorbance values. For comparison of WT and MT data, the speci¢c absorbances at
characteristic pigment absorption maxima (435 nm ^ short
wave maximum of chlorophyll a (chl a), 490 nm ^ carotenoids, 625 nm ^ phycocyanin, 675 nm ^ long-wave maximum of chl a) and the ratio E625 /E675 (representing the
proportion of phycocyanin in relation to chl a) were determined. In addition, the speci¢c total absorbance of the
cells in the range of photosynthetic active radiance (420^
700 nm) was determined.
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41
3. Results and discussion
Table 1
Model parameters of estimated light^growth curves (Fig. 2)
3.1. Growth under di¡erent light conditions
Parameter
31
In this study, microcystin producing PCC 7806 (WT)
and its microcystin-de¢cient mutant (MT) were compared
with respect to light intensity-dependent growth and pigment contents under di¡erent light conditions. We did not
observe signi¢cant di¡erences between the light and
growth curves (Fig. 1) and the maximum speci¢c growth
rate (Table 1) of MT and WT. Thus, the lack of microcystins in MT had no e¡ect on the growth under the tested
light conditions. However, the low-maximum speci¢c
growth rates measured for both variants di¡erentiate
them from other strains of M. aeruginosa [18,19]. Similarly, light saturation intensities (Isat : light intensity at
W = 0.95 Wm ) of approximately 32 WE m32 s31 for the
WT, and 38 WE m32 s31 for the MT were low compared
to other Microcystis strains that require higher intensities
(single cell strains 50 WE m32 s31 , colony-forming strains
80 WE m32 s31 , [18]) to reach light saturation conditions.
3.2. Biovolume vs absorption
We could not detect signi¢cant di¡erences in the size of
cells between WT and MT. Surprisingly, WT and MT
variants exhibited signi¢cant di¡erences in the biovolume
vs light extinction curves (Fig. 2). At the same biovolume
a 30% higher optical signal was measured from the WT
culture compared to the MT.
Light re£ection, refraction and absorption are the components that constitute light attenuation of culture solutions during photometric biovolume measurements. While
light absorption by pigments (chl a at 436 nm) represents
only a minor proportion of this measurement and light
Fig. 1. Light intensity-dependent growth of PCC 7806 WT and MT,
means (n = 25) and con¢dence limits; Values of ¢lled symbols were
tested with Student's t-test (P = 0.05) for signi¢cant di¡erences at comparable light supply. Grey ¢lled symbols ^ tested values without signi¢cant di¡erences; black ¢lled symbols ^ tested values with signi¢cant difference; open symbols ^ not tested ; Light^growth curves were estimated
by non-linear regression to a modi¢ed Mitscherlich model [17]
(W = Wm (13exp(3ln2(I3i0 )/(KI 3i0 )).
Wm (day )
i0 (WE m32 s31 )
KI (WE m32 s31 )
Isat (WE m32 s31 )
WT
MT
a
0.211 (0.205^0.217)
1.85 (0.73^2.97)
8.79 (8.16^9.42)
31.8
0.208 (0.202^0.214)
0.61 (30.43^1.70)
9.44 (8.60^10.28)
37.7
Wm : maximum speci¢c growth rate; i0 : minimum light demand; KI : half
saturation constant; Isat : saturating light supply, calculated at W = 0.95
Wm
a
Standard deviations
re£ection is mainly in£uenced by cellular surface structure,
refraction is determined by the internal organisation of
membrane systems, the nature and size of inclusions and
the occurrence of gas vesicles within the cell. The doubleframe technique for measuring photometric signals, used
in this study, is based on light attenuation as a consequence of light refraction (scattering). Therefore, the signi¢cantly lower photometric absorbance signals observed
in MT cultures in comparison to the WT, are an indication of internal structural di¡erences in MT cells.
3.3. Pigment composition
Structural changes in the MT are also suggested by the
signi¢cantly higher speci¢c absorbance values (for individual pigments), but lower chl a and carotenoid contents
(Fig. 3, Table 2). To investigate possible adaptive responses to di¡erent light intensities, the pigment contents
of WT and MT cells were analysed by HPLC. Clear separation of peaks was seen for chl a, the carotenoids Lcarotene (L-car) and zeaxanthin (zea), the ketocarotenoid
echinenone (ech), and the carotenoid-glycoside myxoxanthophyll (myx). The detected crocetindial (cro) represents
an arti¢cial degradation product of L-car or zea due to
oxidative splitting by the carotene oxygenase enzyme complex [20]. Due to the highly variable occurrence of cro in
the samples, the sum of L-car, zea and cro was used to
compare WT and MT.
The observed cellular pigment concentrations re£ect
adaptation of the pigment apparatus to light intensities
between 4 and 110 WE m32 s31 . Both the WT and the
Fig. 2. Light extinction vs biovolume ratios of WT and MT cultures
with linear regression lines.
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K. Hesse et al. / FEMS Microbiology Ecology 37 (2001) 39^43
Table 2
Cytophotometrically determined values of speci¢c absorbances of light limited and nearly saturated WT and MT cells
Speci¢c absorbance (Wm31 )
WT (5.3 WE m32 31 )
MT (4.0 WE m32 s31 )
E435
E490
E625
E675
Total speci¢c absorbance (420^700 nm)
E625 /E675
0.055a
0.017a
0.037a
0.041a
7.1a
0.89a
0.063a
0.023a
0.053a
0.055a
10.1a
0.97a
WT (34.6 WE m32 s31 )
MT (31.0 WE m32 s31 )
0.042
0.018
0.027
0.031
5.9
0.86
0.040
0.018
0.026
0.031
5.8
0.85
E435
E490
E625
E675
total speci¢c absorbance (420^700 nm)
E625 /E675
a
Values are signi¢cantly di¡erent, tested with Student's t-test at a level of P 6 0.05
MT adapted to light by altering their intracellular concentrations of chl a and carotenoids. Chl a, L-car+zea+cro
and ech decreased with increasing light intensity, especially
in the low light range, while high irradiance resulted in
higher myx concentrations. When normalised to chl a,
all pigment ratios increased with increasing light supply,
largely re£ecting a much greater reduction in chl a relative
to accessory carotenoids. The carotenoid/chl a ratios were
identical for both variants.
While the adaptation to di¡erent light intensities was
shown to be the same in MT and WT, the pigment concentrations revealed di¡erences between the two variants
independent of light intensities (Fig. 3). The cellular concentrations (means) of all pigments were approximately
20% lower in the MT compared to the WT. Signi¢cant
di¡erences were found at light limited conditions for chl
a and ech, and at light saturation for myx.
The cytophotometrically determined absorbance data
are displayed in Table 2. Under nearly light saturated
conditions, there were no signi¢cant di¡erences in the speci¢c absorbances, the ratio of E625 /E675 and total speci¢c
absorbances between 420 and 700 nm. However, the cytophotometric in vivo absorbance spectra revealed a signi¢cantly higher PC content (E625 /E675 ) relative to chl a in the
Fig. 3. Light intensity-dependent pigment contents (A, B, C and D) and pigment/chl a-ratios (E, F and G) of PCC 7806 WT and MT, means (n = 6)
and standard deviations ; Values of ¢lled symbols were tested with Student's t-test (P = 0.05) for signi¢cant di¡erences at comparable light supply. Grey
¢lled symbols ^ tested values without signi¢cant di¡erences ; black ¢lled symbols ^ tested values with signi¢cant di¡erence; open symbols ^ not tested;
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K. Hesse et al. / FEMS Microbiology Ecology 37 (2001) 39^43
MT cells under light limitation. PC is the main component
of the phycobilisomes in M. aeruginosa, which act as lightharvesting systems and transfer collected light energy to
the photosynthetic reaction centres, primarily to PS II [21].
The lower chl a content, compared to the WT, and the
simultaneously increasing ratio of PC to chl a, exhibited
under light limited conditions in MT cells, point toward a
changed PS I/PS II ratio. In cyanobacteria light adaptation mechanisms are guided by variations in the PS I/PS II
ratio [22]. Therefore, it can be speculated that microcystins
could play a role in light adaptation processes. The lightregulated transcription of the microcystin synthetase gene
cluster [13] and the subcellular localisation of microcystins
on thylakoid membranes [23] support such assumptions.
In this case, presumably, compounds with similar chemical
structure (i.e. cyanopeptolines, microginins [24]) serve similar functions in non-microcystin-containing WT strains.
On the other hand our observations could be explained
by structural changes in the cellular membrane system
caused by the lack of membrane bound microcystin.
Then the assumed change in the PS I/PS II ratio could
be a consequence of the lack of space for the photosynthetic reaction centres and the expanded antennae assembly under light-de¢cient conditions.
It is likely that more information will be gained, when
the second cyclic peptide present in PCC 7806 cyanopeptoline is absent also.
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
Acknowledgements
[16]
We thank Marion Dewender and Christa-Dora Martin
for excellent technical assistance and Melanie Kaebernick
for critically reading of the manuscript. This study was
supported by grants from the Humboldt-University, Berlin, and from the Deutsche Forschungsgemeinschaft,
Bonn, to T.B.
[17]
[18]
[19]
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