FEMS MicrobiologyLetters 33 (1986) 85-88
Published by Elsevier
85
FEM 02321
Activity of the natural algicide, cyanobacterin,
on eukaryotic microorganisms
(Algae; fungi; photosynthesis; resistance)
Florence K. Gleason and Cheryl A. Baxa
Gray FreshwaterBiologicalInstitute, Universityof Minnesota, P.O. Box 100, Navarre, MN 55392, U.S.A.
Received20 August1985
Accepted13 September1985
1. SUMMARY
The natural product cyanobacterin has been
shown to be toxic to most cyanobacteria at a
concentration of approx. 5 #M. We demonstrate
here that cyanobacterin will also inhibit the growth
of most eukaryotic algae at a similar concentration. Some algae, such as Euglena gracilis, are
resistant because they are able to maintain themselves by heterotrophlc nutrition. Others, such as
Chlamydomonas reinhardtii, can apparently induce
a detoxification mechanism to maintain photosynthesis in the presence of low concentrations of
the inhibitor. Non-photosynthetic microorganisms
are not affected by cyanobacterin.
2. INTRODUCTION
The filamentous cyanobacterium Scytonema
hofmanni (UTEX2349) was reported to inhibit the
growth of other cyanobacteria and some eukaryotic
algae when the organisms were cultured together
in Petri dishes [1]. Inhibition was shown to be
caused by the production of a secondary metabolite by S. hofmanni which has been isolated and
characterized. This metabolite, named cyanobacterin, is a diaryl substituted T-lactone with a
chlorine substituent on one of the aromatic rings
[2]. Subsequent improvements in growth of S.
hofmanni and purification procedures enabled us
to extend this study using highly purified cyanobacterin. We have demonstrated that this secondary metabolite is toxic to most cyanobacteria
at a concentration of approx. 5 /~M, but has no
effect on eubacteria. Cyanobacterin has also been
shown to inhibit photosynthetic electron transport
in the unicellular cyanobacterium, Synechococcus
sp. [3]. Since the algicide acts on photosynthetic
electron transport, it might be expected to inhibit
the growth of all photosynthetic microorganisms.
However, quantitative data, especially with eukaryotic algae, were difficult to obtain using whole
cells in 2-species culture. Several algae were apparently unaffected by the proximity of S.
hofmanni [1]. We report here the effects of highly
purified cyanobacterin on cell growth of several
eukaryotic microorganisms.
3. MATERIALS AND METHODS
All cultures were axenic. Algae and Tetrahymena spp. were obtained from either the University of Texas Culture Collection (UTEX), Austin,
TX; or the American Type Culture Collection
0378-1097/86/$03.50 © 1986 Federationof EuropeanMicrobiologicalSocieties
86
(ATCC), Rockville, MD. Cultures were maintained at room temperature and algae, at constant
illumination (30 / t E / m 2 / s , photosynthetically active radiation). Toxicity tests were done on
organisms grown in liquid culture at 25-30°C with
gentle shaking. Algae were illuminated at 43-66
/~E/m2/s. Growth was monitored by measuring
the increase in optical density in a GSA-McPherson spectrophotometer or by direct counting in a
hemocytometer. Chlorophyll a was determined
spectrophotometrically after extraction with 90%
acetone [4].
Cyanobacterin was extracted from S. hofmanni
and purified as described elsewhere [2]. The algicide was dissolved in anhydrous ethyl ether or
ethanol before addition to cultures. Control cultures received an equivalent amount of solvent.
Table 1
Effect of cyanobacterin on eukaryotic microorganisms
Organisms
Source a
Medium b
Inhibition c
(4.6 ttM
cyanobacterin)
ATCC30447
UTEX89
UTEX2097
UTEX251
UTEX109
ATCC30410
UTEX175
UTEX929
ATCC30402
UTEX873
UTEX37
ATCC11460
ATCC22662
UTEX173
UTEX320
UTEX1178
MBL
C-M
Bgl 1
C-M
Bgl 1
Bgl 1
MBL
MBL
Cgl0
MBL
Bgl 1
Bgll
Bgll
MBL
Bgll
Bgl 1
+
+
+
+
+
+
+
+
+
+
+
+
UTEX755
MBL
+
UTEX753
C-M
+
ATCC30005
Tet
-
R. Crawford
Schering
Schering
Schering
Schering
Schering
Pha
-
Chlorophyta
Ankistrodesmus angustus
Chlamydomonas reinhardtii
Characium californicum
Chlorella pyrenoidosa
Chlorococcum macrostigmatum
Coelastrum proboscidium
Cosmarium botrytis
Golenkinia minutissima
Haematococcus lacustris
Pandorina morum
Pediastrum biradiatum
Scenedesmus quadricauda
Selenastrum capricornutum
Staurastrum punctulatum
Stigeoclonium pascheri
Ulothrix acuminata
Rhodophyta
Porphyridium aerugineum
Euglenophyta
Euglena gracilis Z
Protozoa
Tetrahymena pyriformis
Fungi
Phanerochaete crysosporium
Candida albicans
Saccharomyces cereoisiae
Torulopsis glabrata
Aspergillus niger
Geotrichum candidum
-
a Sources: ATCC, American Type Culture Collection, 12301 Parklawn Drive, Rockville, M D ; UTEX, University of Texas at Austin
Collection, Dept. of Botany, Austin, TX; R. Crawford, Gray Freshwater Biological Institute, University of Minnesota, Navarre,
MN; Shering, test done by Shering-Plough Corp., Kenilworth, NJ.
b Medium: MBL, Marine Biological Lab [5]; C-M, Cramer-Myers [6]; B g l l [7]; C g l 0 [8]; Tet, proteose peptone, 20 g / l , yeast extract,
0.5 g / l , Geigy iron chelate, 10 mg/1; Pha, complete m e d i u m for Phanerochaete [9].
¢ Growth in liquid m e d i u m was followed turbidometrically (A650) or by removing an aliquot and estimating cell numbers. Positive
inhibition indicates no increase in cell mass fOi" 7 days after addition of cyanobacterin as compared to a control culture to which
only solvent was added.
87
4. RESULTS AND DISCUSSION
The effects of cyanobacterin on the growth of
eukaryotic microorganisms are summarized in Table 1. Under the standardized conditions described
above, cyanobacterin was toxic to most algae at a
concentration of 4.6 #M (2 /~g/ml). As has been
shown previously for bacteria [3], non-photosynthetic microorganisms were not affected by the
algicide.
From the data presented in Table 1, it appears
that some algae are resistant to cyanobacterin.
Chlamydomonas reinhardtii was chosen for further
study. A dose-response analysis of the growth of
this organism in the presence of cyanobacterin is
illustrated in Fig. 1. All concentrations of cyanobacterin cause an immediate inhibition of cell
0.9--
o
I
I
i
0.8-
:"
"
I
oI
I
0.7_
I
0.6-
t
l
I
/
6
o
O
=®
0.5 --
,j
0.4--
I
/
0.3
/
,,
o
--
(~O, . . *
o
0,2
,,
.
I
30
o:"
•
20
d"
;"
-
,.'"
.....
X
O
O
u~
•
~
,a
.J
tu
(.I
"
~ i J ~- ~ # "
0
,.: /
A,"~ .,'0
....,,F-..~'
0.1
/
..'
.o
division. However, at concentrations up to 12 #M,
C. reinhardtii will resume growth after a lag of 3-4
days. At a concentration of 50 #M cyanobacterin
(saturation in aqueous media), inhibition of growth
is permanent. The data suggest that this organism
is as susceptible to the effects of cyanobacterin as
other algae, but that it is able to overcome this
inhibition by the latent induction of a detoxification or exclusion mechanism.
The data in Table 1 shows that cyanobacterin is
toxic to Euglena gracilis. A typical growth curve
for E. gracilis under photoautotrophic conditions
[6] is shown in Fig. 2. Addition of the algicide to a
culture of exponentially growing cells inhibits
growth immediately. The minimum inhibitory dose
is 4.6 #M. However, if cells are grown photoheterotrophically in medium containing an organic
carbon source [9], cell division will resume after
treatment with cyanobacterin at the minimum inhibitory dose. As illustrated in Fig. 3, cell numbers
will increase in the presence of 4.6 /~M cyanobacterin after a lag of 3-4 days. After 10 days,
cells remain viable in the presence of 46 /~M
cyanobacterin but do not resume growth. Presumably, in the lower concentration of inhibitor,
cells can maintain growth by heterotrophic nutri-
10
iL CYANOBACTERtN ADDED
I
I
I
I
I
I
I
2
4
6
8
10
12
14
TIME
16
(days)
Fig. 1. Effect of cyanobacterin on the growth of 'Chlamydomonas reinhardtii. Cyanobacterin, dissolved in ethyl ether,
was added at the arrow. © . . . . . . O, Control culture, ethyl
ether added; © . . . . . . O, 4.6 /~M cyanobacterin added;
O
O , 12 # M cyanobacterin added; • . . . . e, 50 /xM
cyanobacterin added, C. reinhardtii was grown photoautotrophically in C-M medium [6] and 66 # E / m 2 / s .
5
Cyanobacterin Added
I
i
~
I
I
I
3
5
7'
I0
TIME (days)
Fig. 2. Effect of cyanobacterin on the growth o f Euglena
gracilis Z. Cells were grown p h o t o a u t o t r o p h i c a ~ in C-M
medium [6] and 43/LE/m2/s. O
O, Control, ethyl ether
added; []
•, 4.6 # M cyanobacterin added; A
A,
46 # M cyanobacterin added.
88
30
l
20
o
I
I
0
x
I
10
_J
w
0
ACKNOWLEDGEMENTS
Added
3
~
I
3
5
TIME
_
I0
7
(days)
Fig. 3. Effect of cyanobacterin on the growth of Euglena
gracilis Z. Cells were grown photoheterotrophicallyin enriched
medium [10] and 43 #E/m2/s. O
O, Control, ethyl
ether added; []
D, 4.6 #M cyanobacterin added;
zx
,x, 46 #M cyanobacterin added.
o~
ov,-
x
10-
,,.I
,,.I
ILl
o
,<
,,.i
,.,.i
>"
qo
5
I
1=
Added
I
I
}
3
tion. As seen in Fig. 4, c h l o r o p h y l l a c o n t e n t p e r
cell slowly increases after the initial i n h i b i t o r y
effect of c y a n o b a c t e r i n , A t the higher c o n c e n t r a tion of algicide, cells lose all c h l o r o p h y l l a.
W e c o n c l u d e that the n a t u r a l atgicide, c y a n o bacterin, is as toxic to p h o t o s y n t h e t i c e u k a r y o t i c
m i c r o o r g a n i s m s as it is to p r o k a r y o t e s . Some of
the e u k a r y o t i c algae can survive low c o n c e n t r a tions of the i n h i b i t o r b y switching to h e t e r o t r o p h i c
m e t a b o l i s m or b y i n d u c i n g a d e t o x i f i c a t i o n m e c h a n i s m as in the case of Chlamydomonas.
~,
]
5
TIME
I
7
'r'
I0
(days)
Fig. 4. Effect of cyanobaeterin on chlorophyll a content per
cell in photoheterotrophically grown Euglena gracilis Z. Cells
were grown in enriched medium [10] and 43 #E/m2/sec.
Chlorophyll a was estimated after extraction with acetone [4].
O
O, Control, ethyl ether added; []
D, 4.6 #M
cyanobacterin added; ,',
Lx,46 #M cyanobacterin added.
This s t u d y was s u p p o r t e d b y a g r a n t f r o m the
R e s e a r c h C o r p o r a t i o n a n d the M i n n e s o t a Sea
G r a n t Institute, D O C / N A 8 3 A A - D 0 0 0 5 6 R / N P 1. T h e technical assistance of K. S u n d b e r g is gratefully a c k n o w l e d g e d .
REFERENCES
[1] Mason, C.P., Edwards, K.R., Carlson, R.E., Pignatello, J.,
Gleason, F.K. and Wood, J.M. (1982) Science 215,
400-402.
[2] Pignatello, J.J., Porwoll, J., Carlson, R.E., Xavier, A.,
Gleason, F.K. and Wood, J.M. (1983) J. Org. Chem. 48,
4035-4037.
[3] Gleason, F.K. and Paulson, J.L (1984) Arch. Microbiol.
138, 273-277.
[4] Parsons, T.R. and Strickland, J.D.H. (1963) J. Mar. Res.
21,155-163.
[5] Nichols, H.W. (1973) In Physiological Methods, Vol. I
(J.R. Stein, Ed.) pp. 7-24. Cambridge University Press,
Cambridge.
[6] Cook, J.R. (1971) Methods Enzymol. 23, 74-78.
[7] Stanier, R.Y., Kunisawa, R., Mandel, M. and CohenBazire, A. (1971) Bacteriol. Rev. 35, 171-205.
[8] Van Baalen, C. (1967) J. Phycol. 3, 154-157.
[9] Kirk, T.K., Schultz, F., Connors, W.J., Lorenz, I.F. and
Zeikus, J.G. (1978) Arch. Microbiol. 117, 277-285.
[10] Starr, R.C. (1964) Am. J. Bot. 51, 1013-1044.