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Appl Environ Microbiol, July 1998, p. 2361-2366, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Growth on Urea Can Trigger Death and Peroxidation
of the Cyanobacterium Synechococcus sp. Strain PCC
7002
Toshio
Sakamoto,
Victoria B.
Delgaizo, and
Donald A.
Bryant*
Department of Biochemistry and Molecular
Biology, The Pennsylvania State University, University Park,
Pennsylvania 16802
Received 6 March 1998/Accepted 2 May 1998
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ABSTRACT |
Laboratory conditions have been identified that cause the rapid
death of cultures of cyanobacteria producing urease. Once the death
phase had initiated in the stationary growth phase, cells were rapidly
bleached of all pigmentation. Null mutations in the ureC
gene, encoding the alpha subunit of urease, were constructed, and these
mutants were no longer sensitive to growth in the presence of urea.
High levels of peroxides, including lipid peroxides, were detected in
the bleaching cells. Exogenously added polyunsaturated fatty acids
triggered a similar death response. Vitamin E suppressed the formation
of peroxides and delayed the onset of cell bleaching. The results
suggest that these cyanobacterial cells undergo a metabolic imbalance
that ultimately leads to oxidative stress and lipid peroxide formation.
These observations may provide insights into the mechanism of sudden
cyanobacterial bloom disappearance in nature.
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INTRODUCTION |
Cyanobacteria are morphologically
and physiologically diverse eubacterial phototrophs which perform
oxygen-evolving photosynthesis. Certain cyanobacteria, such as species
of Anabaena, Microcystis, Trichodesmium, and Synechococcus, are capable of
forming massive blooms (26). In the spring and summer, when
nutrients are abundant, blooms occur as the water temperature rises.
Numerous field investigators have reported that cyanobacterial blooms
sometimes disappear suddenly from lake or sea surfaces and that such
disappearances can occur as rapidly as overnight (1, 21,
23). Three hypotheses have been proposed to explain the fates of
such cyanobacterial blooms: (i) the cyanobacterial cells are lysed by
virus infection; (ii) the cells are consumed by predators; and (iii)
the bloom at the water surface is dispersed by mixing with bottom
layers due to storms or tidal changes (21). However, no
satisfying explanation for the large-scale disappearance or death of
cyanobacterial blooms has yet been established. Considering the
potential threat to both animals and humans posed by toxins produced by
bloom-forming cyanobacteria (22), the ability to trigger
such disappearances could have both economic and human health-related
significance. In this study, we discovered growth conditions that
promote catastrophic cell death during the stationary growth phase of
both marine and freshwater cyanobacteria. On the basis of our
laboratory observations, we propose a novel explanation for the
massive, rapid die-off of cyanobacteria in natural blooms.
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MATERIALS AND METHODS |
Chemicals.
Oleic acid, linoleic acid, and 
-linolenic acid
were kindly provided by John H. Golbeck (The Pennsylvania State
University). The purity of these free fatty acids, which were sealed
under nitrogen in ampoules and had been analyzed at the Hormel
Institute, University of Minnesota, was >99%. The sodium salt of
oleic acid (O-7501), the sodium salt of linoleic acid (L-8134),
ricinoleic acid (R-7257), cis-12-octadecenoic acid (O-8881),
linoleladic acid (trans-9,
trans-12-octadecadienoic acid; L-2126), (±)--tocopherol (vitamin E; T-3251), butylated hydroxytoluene (BHT; B-1378), methyl viologen (paraquat dichloride; M-2254), and xylenol orange (X-0127) were purchased from Sigma Chemical Co. (St. Louis, Mo.).
Organisms and culture conditions.
A laboratory wild-type
strain of Synechococcus sp. strain PCC 7002, designated
strain PR6000, was originally obtained from S. E. Stevens, Jr.,
and has been maintained at The Pennsylvania State University. Mutant
strain PR6080 of Synechococcus sp. strain PCC 7002, lacking
polyunsaturated fatty acids, was constructed by interposon mutagenesis
(20). A DNA fragment encoding the aminoglycoside
5'-phosphotransferase gene (aphII) of Tn5 was
inserted into a unique restriction site within the cloned
desA gene, encoding acyl-lipid 
12 desaturase of
Synechococcus sp. strain PCC 7000. Cells were grown
photoautotrophically in medium A+ containing 1 mg of
NaNO3 ml
1 (24) or medium A-U
containing 50 mM urea (19) under constant illumination from
cool-white fluorescent lamps (250 microeinsteins m2
s1) with aeration from 1% (vol/vol) CO2 in
air at 38°C. For the selection of kanamycin-resistant mutants,
kanamycin (50 µg ml1) was added to the medium. When
appropriate, 5 µM NiSO4 was added to the growth medium.
Liquid cultures (25 ml) were incubated in glass culture tubes (22 by
175 mm). Cell growth was monitored by determining optical density at
550 nm (OD550) with a Spectronic 20 spectrophotometer
(Milton Roy, Rochester, N.Y.). A cell suspension from an
exponential-phase culture grown at 38°C in medium A+ at a
light intensity of 250 microeinsteins m2 s1
with an OD550 of 1.0 contained 3.4 ± 0.3 µg of
chlorophyll ml1 and (1.0 ± 0.2) × 108
cells ml1 as determined by microscopic count
(19). Colony-forming activity was measured by plating cells
on medium A+ solidified with 1.5% (wt/vol) Difco Bacto
Agar. Colonies were counted after 10 days of growth under constant
illumination.
The freshwater cyanobacteria Synechococcus sp. strain PCC
6301, Synechocystis sp. strain PCC 6803, and
Anabaena sp. strain PCC 7120 were obtained from the Pasteur
Culture Collection and grown under 120 microeinsteins m2
s1 in medium B-HEPES (3). When appropriate,
sodium nitrate was replaced with urea as a nitrogen source.
Cloning and insertional mutagenesis of the ureC gene
in Synechococcus sp. strain PCC 7002.
A 4.5-kb
HindIII fragment was isolated from a genomic library of
Synechococcus sp. strain PCC 7002 by cross-hybridization with a 1.0-kb portion of the ureC gene of
Synechocystis sp. strain PCC 6803. The screening probe was
amplified by PCR using genomic DNA from Synechocystis sp.
strain PCC 6803 as the template and the following specific primers:
forward, 5'-GGC AAT GGC AGT CAA CC; reverse, 5'-ATC CCG TTT GGT TAA CT
(10). The ureC gene was insertionally inactivated
by insertion of a 1.4-kb KpnI fragment derived from plasmid
pRL170 (4) and containing the aphII gene of
Tn5 into a unique KpnI site within the
ureC coding region. Wild-type cells of
Synechococcus sp. strain PCC 7002 (strain PR6000) were
transformed with plasmid DNA containing the interrupted gene essentially described by Stevens and Porter (25).
Kanamycin-resistant transformants were selected on plates with medium
A+ supplemented with 50 µg of kanamycin
ml1. The ureC mutants were designated strain
PR6091, in which the transcription orientation of the aphII
gene is the opposite of that of ureC, and strain PR6092, in
which the transcription orientation of the aphII gene is the
same as that of the ureC gene (Fig.
1).
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FIG. 1.
Physical map of the 4.5-kb HindIII
fragment encoding the ureC gene of Synechococcus
sp. strain PCC 7002. Arrows indicate the direction of transcription.
The aphII gene, which encodes aminoglycoside
3'-phosphotransferase II and confers resistance to kanamycin, was
inserted into the unique KpnI site of the ureC
gene in both orientations. The ureC mutants were designated
strain PR6091, in which the transcription orientation of the
aphII gene is the opposite of that of ureC, and
strain PR6092, in which the transcription orientation of the
aphII gene is the same as that of the ureC
gene.
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Detection of ammonium.
The concentration of ammonium ions in
the culture medium was determined by using the Nessler reagent
(Aldrich, Milwaukee, Wis.). The culture medium was diluted 1,000-fold,
and 1/20 volume of Nessler reagent was added. The
A395 was immediately measured, and the ammonium
concentration was determined from a standard curve of 25 to 200 µM
ammonium chloride (12).
Detection of hydroperoxides.
The level of hydroperoxides in
intact cyanobacterial cells was measured by the ferrous
oxidation-xylenol orange method that was originally developed for
detection of hydrogen peroxide (H2O2) in the
nanomolar range and that has been applied for the detection of lipid
hydroperoxides in yeast cells (2). Cells were collected by
centrifugation, and the pellet was suspended in 0.8 ml of methanol containing 0.01% (wt/vol) BHT. After addition of 0.1 ml of reagent A
[2.5 mM ammonium ferrous(II) sulfate; 0.25 M sulfuric acid] and 0.1 ml of reagent B (40 mM BHT; 1.25 mM xylenol orange in methanol),
samples were quickly centrifuged to remove cell debris. The
A560 of the supernatant was measured
immediately. The amount of hydroperoxides was calculated from an
apparent extinction coefficient (E560, 4.3 × 104 M1 cm1) (7,
8).
Polyunsaturated fatty acid sensitivity.
Cells of
desA mutant strain PR6080 (20) were grown
overnight in medium A-U containing 50 mM urea and 50 µg of kanamycin ml1 under 250 microeinsteins of light m2
s1 and 1% CO2 conditions at 38°C. The cell
suspension was diluted with fresh medium A-U to produce an
OD550 of 1.0. Free fatty acids in methanol (25 µl) were
added to the cell suspension (25 ml) to produce a final calculated
concentration of 0.5 mM. Fatty acid sodium salts were dissolved in
water and added to the cell suspensions to produce a final
concentration of 0.5 mM. Changes in cell coloration were observed after
overnight incubation under standard growth conditions.
Paraquat sensitivity.
Wild-type cells of
Synechococcus sp. strain PCC 7002 were grown
photoautotrophically in medium A+ under standard growth
conditions at 38°C overnight. Freshwater strains
Synechococcus sp. strain PCC 6301, Synechocystis
sp. strain PCC 6803, and Anabaena sp. strain PCC 7120 were
grown in medium B-HEPES. The cell suspension was diluted in the same
medium to an OD550 of 0.5. Paraquat solutions in water (25 µl) were added to 25 ml of the cell suspension to final
concentrations of 1 µM to 0.5 mM. After overnight incubation under
standard growth conditions, visible changes in cell coloration were
determined.
Nucleotide sequence accession number.
The complete
nucleotide sequence of the 4.5-kb HindIII fragment
encoding the ureC gene of strain PCC 7002 was determined and has been submitted to GenBank (accession no. AF035751).
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RESULTS |
Death and peroxidation of cells grown with urea as the N
source.
Under our standard laboratory growth conditions, the
wild-type PR6000 strain of the unicellular marine cyanobacterium
Synechococcus sp. strain PCC 7002 is grown in medium
A+ containing nitrate as the nitrogen source. Figure
2A shows a typical growth curve for this
organism under these conditions. When grown at 38°C at 250 microeinsteins m2 s1, wild-type strain
PR6000 had a doubling time of about 4 h. During the mid- to
late-exponential growth phase (OD550, 4 to 16; Fig. 2A),
the colony-forming activity of cultures was constant at (4.4 ± 0.4) × 107 colonies ml1
(OD550)1 (n = 6). After 5 days of incubation, the OD550 typically reached 21 ± 2 (n = 11) and cell growth stopped. Although little or
no change in the OD550 was observed, the colony-forming
activity of cultures in stationary phase decreased but was still
greater than 6 × 106 colonies ml1
(OD550)1 (n = 4).
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FIG. 2.
Growth curves of Synechococcus sp. strain PCC
7002 in medium A+ (A) and in medium A-U (B). In medium A-U,
cells entered the death phase after growth in culture for 72 h.
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When strain PR6000 cells were grown in medium A-U containing 50 mM urea
as the N source, the doubling time decreased to 3.5
h under
otherwise standard growth conditions (Fig.
2B). The colony-forming
activity of such cultures during the mid- to late-exponential
growth
phase (OD
550, 3 to 6) was (6.5 ± 0.7) × 10
7 colonies ml
1
(OD
550)
1 (
n = 4). After the
OD
550 reached 10 ± 2 (
n = 18), cell
growth
ceased. In stationary phase, the color of the culture changed
from the normal blue-green to yellowish green, and this change
consistently occurred 1 to 3 days after the culture reached stationary
phase (Fig.
3). This chlorosis only
occurred at OD
550 values of
greater than ~7.5 after
cultures had entered stationary phase.
Once the bleaching process
became visibly detectable, no colony-forming
activity could be detected
and the cells were rapidly (within
18 h) bleached of all
pigmentation.
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FIG. 3.
Bleaching of pigmentation of Synechococcus
sp. strain PCC 7002 during stationary phase. Wild-type cells of
Synechococcus sp. strain PCC 7002 (strain PR6000) were grown
in medium A+ (lane A, control) and in medium A-U (lane U).
3d, cells 3 days after initial inoculation, in the stationary growth
phase. 4d, 4 days after initial inoculation, in the initial stages of
bleaching or the death phase. 5d, completely bleached cells 5 days
after initial inoculation, at the end of the death phase.
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When the concentration of urea in medium A-U was increased to 100 to
200 mM, the chlorosis and death phase occurred more quickly
as the urea
concentration increased. For
Synechococcus sp. strain
PCC
7002, the death phase in stationary phase was not initiated
when the
concentration of urea in medium A-U was reduced to 25
mM or less. At 5 mM urea, the cell color also changed from the
normal blue-green to a
chlorotic yellowish green. However, this
change of cell color was not
due to the initiation of cell death
but rather was induced by nitrogen
limitation. The chlorotic cells
obtained in stationary phase after
growth on medium containing
5 mM urea could recover normal cell
coloration and growth when
the cells were transferred into fresh
A
+ medium (nitrate as the N source). No ammonium was
detected in
the culture medium in stationary phase when the initial
concentration
of urea was 5 mM. When the initial concentration of urea
in medium
A-U was more than 5 mM, ammonium ions (
1 mM) could be
detected
in the growth medium in the stationary growth phase (data not
shown). However, the death phase was not triggered by the presence
of
ammonium ions in the medium during stationary phase, since
addition of
up to 100 mM ammonium chloride to stationary-phase
cells grown in
medium A
+ did not cause cell bleaching or entrance into the
death phase.
Interestingly, the death phase for
Synechococcus sp. strain PCC
7002 also required elevated
CO
2 conditions (1% [vol/vol] CO
2 in
air).
Neither bleaching nor cell death occurred when cells were
bubbled with
air levels of CO
2 (0.03%) and grown in medium A-U
(50 mM
urea). When 5 µM NiSO
4 was added to medium A-U, the death
phase occurred more quickly. Since nickel and carbon dioxide are
components of the active site of urease (
15), the elevated
CO
2 and Ni
2+ could enhance the urease activity
in cyanobacterial cells and
thereby trigger the death phase.
In solution, hydrolysis of urea results in a net increase in pH.
However, the external pH of cultures after cell death had
occurred in
the urea-containing medium was essentially unchanged
at pHs 7.8 to 8.3. Thus, a shift of pH to one that would promote
cell death in the
external medium is not the cause of cell death
during growth on urea.
On the other hand, cells were killed during
growth on NH
4Cl
if adequate buffering was not provided because
of acidification of the
external medium resulting from ammonia
utilization (data not shown).
When cells were grown on 5 mM NH
4Cl
in medium A, the pH of
the medium decreased to less than 5.0 and
cells were killed and
bleached. This acidification of the medium
during growth on
NH
4Cl was easily remedied by increasing the buffering
capacity of the growth medium by addition of HEPES-NaOH to the
medium.
When growth medium containing 5 mM NH
4Cl was buffered
with
12.5 to 200 mM HEPES-NaOH (pH 7.5), the pH of the growth
medium was
maintained above neutrality and no cell death occurred.
To test for possible lethal changes in the composition of medium A-U
after cell death, fresh cells grown in medium A
+ were
inoculated into the supernatant of culture medium A-U after
the debris
from dead cells had been removed by centrifugation.
Wild-type cells
grew in this conditioned medium under standard
growth conditions, and
rapid cell death was not observed. When
cell debris was collected by
centrifugation and added to fresh,
mid-exponential-phase cultures in
medium A
+, cell death did not occur. These results
demonstrate that nutrients
were not limiting in conditioned medium A-U
and that no toxic
compound(s) capable of causing rapid cell death
remained in the
culture medium or cell debris after cell death had
occurred. These
observations are consistent with the notion that the
death phase
is initiated by an intracellular event or process.
Similar results were obtained with the freshwater cyanobacteria
Synechocystis sp. strain PCC 6803 and
Anabaena
sp. strain
PCC 7120, two strains that can also synthesize urease and
utilize
this compound as the sole nitrogen source for growth (
10,
11,
13).
Anabaena sp. strain PCC 7120 was much more
sensitive to
urea and was killed when 5 mM urea was added to the growth
medium,
irrespective of the CO
2 level supplied.
Synechocystis sp. strain
PCC 6803 exhibited intermediate
sensitivity to urea and was killed
when at least 25 mM urea was added
to the growth medium. Interestingly,
Synechococcus sp.
strain PCC 6301, which cannot utilize urea as
the sole nitrogen source
and does not appear to synthesize urease
(
11), was not
killed by urea concentrations of up to 200 mM,
and no growth inhibition
was detectable when growth media were
supplemented with urea at
concentrations of up to 100 mM. These
results suggested that a direct
or indirect product of urease
activity causes cell death.
Urease deletion mutant.
To test directly the involvement of
urease in the causation of cell death during growth on urea, urease
null mutants were constructed by insertional inactivation of the
ureC gene, encoding the subunit of urease, in
Synechococcus sp. strain PCC 7002. Both ureC
disruption mutants, strains PR6091 and PR6092 (Fig. 1), did not grow at
all on urea-containing medium. This indicates that the ureC
gene product is essential for cell growth on urea as the sole nitrogen
source and that these mutants had lost all urease activity. When these
ureC disruption mutants were grown in medium A+
supplemented with 100 mM urea and 5 µM NiSO4, the mutant
cells did not die, even after prolonged incubation in stationary phase, although wild-type cells were killed by this growth medium (Fig. 4). These results clearly demonstrate
that hydrolysis of urea by urease is required for the urea-mediated
cell death phenomenon.
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FIG. 4.
Resistance of ureC mutants to urea in
stationary phase. Wild-type cells of Synechococcus sp.
strain PCC 7002 (strain PR6000) (lane A, control) and cells of
ureC mutant strains PR6091 (lane B) and PR6092 (lane C) were
grown in medium A+ supplemented with 100 mM urea and 5 µM
NiSO4 for 5 days. The mutant cells did not die and were not
bleached, even after prolonged incubation (up to 11 days), although
wild-type cells were killed and bleached after 3 days when grown in
this medium.
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Lipid peroxide formation during the death phase.
It is
generally believed that membrane lipids are major targets for cellular
damage induced by oxygen radicals (9). The photosynthetic or
thylakoid membranes in many cyanobacteria contain high levels of
polyunsaturated fatty acids (16) which are easily oxidized
to form lipid peroxides (9). Since the bleached cultures of
Synechococcus sp. strain PCC 7002 in medium A-U had a strong odor resembling that of mushrooms (determined empirically by
questioning laboratory members), it was thus reasoned that the death
conditions were causing the formation of volatile products, the
so-called "green notes" (6), derived from oxidative
degradation of polyunsaturated fatty acids. Therefore, the levels of
lipid peroxides of whole cells grown in medium A-U during the death
phase were measured. Peroxide levels increased dramatically as
chlorosis increased. In cells for which the onset of chlorosis or
bleaching was first detected (Fig. 3, 4d, U), peroxide levels were
about 0.4 ± 0.1 nmol ml1
(OD550)1 (this level increased to 2.2 ± 0.7 nmol ml1 [OD550]1) in cells
that had undergone 15 to 18 h of bleaching (Fig. 3, 5d, U).
Peroxide levels were below the detection limit (0.1 nmol ml1 [OD550]1) in
stationary-phase cells grown in medium A+ or in
exponential-phase cells grown in medium A-U.
When 0.5 mM vitamin E, an antioxidant and a scavenger of radicals, was
added to wild-type cells in medium A-U at 24-h intervals
after 3 days
of growth, the onset of the death phase was delayed
by 24 to 36 h.
However, once the death phase had been initiated,
addition of vitamin E
did not stop the progressive cell bleaching,
which led to complete
death in a manner similar to that observed
in the absence of vitamin E. Daily addition of vitamin E was required
to observe a significant delay
in the onset of the death phase.
Rapid cell death triggered by polyunsaturated fatty acids.
The
results presented above suggest that growth in the presence of a high
concentration of urea ultimately leads to the formation of toxic lipid
peroxides and that all cellular pigmentation is rapidly destroyed by
oxidative reactions under these conditions. To investigate the toxicity
of fatty acids for cyanobacterial cells, we initially tested the
effects of various fatty acids by using mutant strain PR6080, which
contains no polyunsaturated fatty acids (20). Oleic acid
[18:1 (9)], its sodium salt, cis-12-octadecenoic acid [18:1 (12)], and
linoleladic acid [trans-9, trans-12-octadecenoic
acid; 18:2 (trans-9, trans-12)] all had little or no effect on the cyanobacterial cells, and no obvious changes
were observed in cultures after overnight incubation with these fatty
acids (data not shown). Linoleic acid [18:2 (9, 12)],
linoleic acid-sodium salt -linolenic acid [18:3 (9, 12,
15)], and ricinoleic acid [18:1 (9, 12-OH)] triggered rapid
cell death. Within 18 h of the addition of these fatty acids to
cultures, no surviving cells could be detected and all pigmentation was
completely bleached (Fig. 5). Hence,
fatty acids with a high propensity to form lipid peroxides, linoleic
acid and its derivatives (9), triggered cell death and
peroxidation while fatty acids with a low propensity to form lipid
peroxides did not. Cell death and bleaching occurred after addition of
polyunsaturated fatty acids whether the cells were incubated under
light or dark conditions. However, the cell suspension remained blue
after overnight incubation in the dark with linoleic acid 18:2 (9,
12) (data not shown). This result suggests that phycobiliproteins
were less susceptible to bleaching under these conditions than
chlorophyll. Wild-type Synechococcus sp. strains PCC 7002 and PCC 6301, Synechocystis sp. strain PCC 6803, and
Anabaena sp. strain PCC 7120 were also sensitive to 18:2 but
not 18:1 fatty acids.
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FIG. 5.
Effects of polyunsaturated fatty acids on cell death and
pigment oxidation. Cells of desA mutant strain PR6080
containing no polyunsaturated fatty acids (14) were grown at
38°C in medium A-U with 1% (vol/vol) CO2 in air, and the
culture (OD550, ~1.0) was divided into five equal
aliquots. The aliquots were incubated under the same growth conditions
overnight with no addition (lane 1), with 0.1% (vol/vol) methanol
(lane 2), with 18:1 (9) fatty acid (lane 3), with 18:2
(9, 12) fatty acid (lane 4), or with 18:3 (9, 12,
15) fatty acid (lane 5). The calculated final concentration of
free fatty acids was 0.5 mM. Cells in lanes 4 and 5 were killed and
completely bleached of all pigmentation.
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Figure
6 shows the levels of peroxides
induced by addition of free fatty acids to cells of
desA
mutant strain PR6080. The
formation of peroxides, especially lipid
peroxides, correlates
precisely with cell death and pigment bleaching.
For example,
oleic acid [18:1 (
9)] did not cause cell
death and did not
induce peroxide formation, whereas linoleic acid
[18:2 (
9,
12)] triggered cell death and promoted formation
of peroxides.
Interestingly, vitamin E suppressed the formation of
peroxides.
These results strongly suggest that peroxidation of
polyunsaturated
fatty acids is involved in the killing phenomenon
triggered by
polyunsaturated fatty acids.
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FIG. 6.
Effect of free fatty acids on cellular peroxide
formation. Cells of desA mutant strain PR6080 containing no
polyunsaturated fatty acids (6) were grown at 38°C in
medium A-U on 1% (vol/vol) CO2 in air, and the culture
(OD550, ~1.0) was divided into five equal aliquots.
Peroxide levels were determined as described in Materials and Methods
after 20 h (column 1) or 40 h (column 2) of incubation with
0.1% (vol/vol) methanol (the solvent for the fatty acid additions),
18:1 (9), 18:2 (9, 12) with or without vitamin E,
or vitamin E alone, as indicated. The calculated fatty acid
concentration was 0.5 mM. N.D., none detected.
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To investigate the possibility that toxic compounds were accumulating
in the growth medium, linoleic acid [18:2 (
9,
12)]
was
added to a culture of
desA mutant strain PR6080, which was
isolated by a dialysis bag having a pore size capable of passing
compounds with molecular weights of 6,000 to 8,000. When linoleic
acid
was added to cells inside the bag, only the cells inside
the bag were
killed. Correspondingly, addition of linoleic acid
to the cells outside
the bag killed only the cells outside the
dialysis bag (Fig.
7). These results demonstrate that direct
contact
between cells and the free fatty acids is required for
initiation
of cell death by linoleic acid. Any toxic compound, should
one
exist, must have a nominal molecular weight greater than
approximately
8,000. Considering that the added fatty acids formed
visible,
floating droplets on the surface of the medium, these results
suggest that direct contact with the fatty acids is required to
initiate cell death.
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FIG. 7.
Dialysis bag experiment. Cells of desA mutant
strain PR6080 containing no polyunsaturated fatty acids (6)
were grown at 38°C in medium A-U (150 ml in a glass tube [34 by 295 mm]) with 1% (vol/vol) CO2 in air (OD550,
approximately 1), and 25 ml of the culture was placed into a dialysis
bag (17.5-mm diameter; molecular weight cutoff, 6,000 to 8,000).
Linoleic acid [18:2 (9, 12); 150 µl of a 0.5 M solution
in methanol] was added to the cell culture inside the dialysis bag (A)
or to that outside the bag (B). The calculated final concentration of
free fatty acids was 0.5 mM, but lipid droplets could be seen floating
on the surface of the culture medium. After 2 to 3 days of incubation
under standard culture conditions, cells in contact with the fatty
acid-containing solution were killed and bleached. The dialysis bags
were taken from the culture tubes and photographed.
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Paraquat sensitivity.
Paraquat, also called methyl viologen,
is a herbicide that can be reduced through a one-electron transfer from
reduced Fe-S centers associated with photosystem I, resulting in the
formation of a stable cation radical. The rapid, toxic effect of
paraquat on plants is due to subsequent reduction of oxygen, forming
the superoxide radical anion (4a). To investigate the
possible involvement of superoxide during the initiation of the death
phase, the effect of paraquat on Synechococcus sp. strain
PCC 7002 was tested. Addition of 0.5 mM paraquat to cultures led to
complete cell death after overnight incubation under standard growth
conditions. Cell growth was inhibited, but cells retained their
blue-green coloration when the paraquat concentration was reduced to
100 to 200 µM. Little or no visible effect of paraquat was observed
at concentrations below 10 µM. These results show that the elevation
of superoxide anions, which is produced by addition of paraquat to
cells, can also mimic the initiation of rapid cell death and the
complete bleaching of cells which accompanies cell death during growth on urea or in the presence of polyunsaturated fatty acids. Similar results were also obtained with Synechocystis sp. strain PCC
6803, Synechococcus sp. strain PCC 6301, and
Anabaena sp. strain PCC 7120, although each of these strains
was much more sensitive to paraquat than was Synechococcus
sp. strain PCC 7002. Synechocystis sp. strain PCC 6803 and
Synechococcus sp. strain PCC 6301 were killed by 10 µM
paraquat, while Anabaena sp. strain PCC 7120 was killed by 1 µM paraquat.
| |
DISCUSSION |
The studies presented here demonstrate that rapid cell death of
urease-producing cyanobacteria occurs under specific growth conditions
in the laboratory. The cell death and oxidation of pigmentation
observed during the stationary growth phase in the presence of urea
could be mimicked by addition of either polyunsaturated fatty acids or
paraquat to the growth medium
treatments that induce oxidative stress.
Vitamin E suppressed the bleaching and delayed the onset of the death
phase. These results strongly implicate the peroxidation of lipids in
the cell death and pigment oxidation phenomenon that we have observed.
Paraquat causes an increase of superoxide radicals by short-circuiting
photosynthetic electron transport by capturing electrons from the
reduced Fe-S centers of photosystem I, thereby forming superoxide;
superoxide radicals, in turn, can attack membrane lipids to form lipid
peroxides (4a, 9). It remains to be determined in further
studies what the primary event is that triggers the catastrophic cell
death phenomenon in cyanobacterial cultures grown with urea as the N
source. However, once the increase in cellular levels of superoxide
radical or hydrogen peroxide has commenced and the formation of lipid
peroxides is detectable, cells lose all pigmentation within 18 h.
This can be explained by a peroxidative chain reaction of all
intracellular materials, including membrane lipids. These reactions
must occur inside the cyanobacterial cells in the postexponential
growth phase, because the medium in which cells had been killed did not contain compounds capable of initiating the death phase.
Although they did not characterize it in detail, Gorham et al.
(5) noted that urea promoted the death of the toxic,
bloom-forming cyanobacterium Anabaena flos-aquae. It was
also previously reported that growth of Synechococcus sp.
strain PCC 7002 began to decline before depletion of the urea in the
medium, although this observation was not characterized further
(18). In our studies, the concentration of ammonium ions in
the growth medium increased to significant levels (1 mM) in the
midexponential phase when cells were cultured in medium A-U containing
50 mM urea. However, high concentrations of ammonium ions alone did not
induce the peroxidative death phenomenon because when stationary-phase
cells grown in medium A+ (the nitrogen source was nitrate)
were transferred to a medium containing 100 mM NH4Cl at the
same cell density, they retained their blue-green color. Since
Synechococcus sp. strain PCC 6301, a strain that cannot use
urea as the sole nitrogen source and apparently does not synthesize
urease, was never observed to undergo urea-induced death (at
concentrations of up to 200 mM) and Synechococcus sp. strain
PCC 7002 ureC mutants were not killed when grown in the
presence of urea, we conclude that urease plays the critical role in
potentiating this phenomenon.
Urea is initially hydrolyzed by urease to form ammonia and carbamate,
which subsequently decomposes to carbonic acid and ammonia, resulting
in an increase in intracellular pH (14, 15). The increased
level of ammonium ions (1 mM) in the medium in the midexponential
phase of growth on urea suggested that hydrolysis of urea exceeded the
consumption of ammonia by cells. Hydrolysis of urea presumably
continues in the postexponential phase. Although the internal pH could
increase as urea passively diffuses into the cells and is hydrolyzed,
passive loss of NH3 from the cells could actually cause the
intracellular pH to decrease significantly. In either case, a pH
imbalance inside the postexponential-phase cells could either lead to
cell death directly or impair the oxidative protection mechanism(s).
Alternatively, such an imbalance might affect electron transport
components that would lead to an increase in active oxygen species in
the cells. However, it remains to be discovered precisely what causes
cell death and leads to the formation of the oxidizing radicals and
peroxides.
The phenomenon reported here may provide novel insights into the
mechanism of the sudden disappearance of cyanobacterial blooms in
nature (1, 21, 23). Considering the rapidity of bloom disappearance in the field, the majority of the research community seemingly believes that viruses cause lysis of the cells, and thus,
viral infections have been extensively studied (17, 21). Our
studies suggest that peroxidative chain reactions could also account
for rapid bloom disappearance phenomena and that such chain reactions
might be triggered by nutrient stress conditions in stationary-phase
cells within the bloom. A metabolic imbalance could lead to a change in
intracellular pH or the formation of active oxygen species;
additionally, suppression or inactivation of the normal oxidative
protection mechanism(s) might occur and thus increase intracellular
active oxygen species. Alternatively, cell lysis in blooms could
release polyunsaturated fatty acids that could, in turn, promote the
formation of peroxides. In either case, catastrophic cell death would
occur and the algal bloom would be bleached by an increase in
intracellular peroxides due to a peroxidative chain reaction. The
ability to promote such processes artificially could lead to better
mechanisms for the control of toxic, bloom-forming cyanobacteria.
| |
ACKNOWLEDGMENTS |
This work was supported by USPHS grant GM-31625 to D.A.B.
T.S. was the recipient of a postdoctoral fellowship from the Yamada Foundation (Osaka, Japan) in 1995.
We thank Tanja Gruber, Kaori Inoue, Elena Vassilieva (Penn State
University), and Masayuki Ohmori (Tokyo University) for their helpful
suggestions and Veronica L. Stirewalt for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: S-234 Frear
Building, Dept. of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802. Phone: (814) 865-1992. Fax: (814) 863-7024. E-mail: dab14{at}psu.edu.
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Abstract
Introduction
