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Vol.
45,
No.
4
APPLIED
AND
ENVIRONMENTAL
MICROBIOLOGY,
Apr.
1983,
p.
1351-1359
0099-2240/83/041351-O9S02.0O/0
Copyright
0
1983,
American
Society
for
Microbiology
Regulation
of
Expression
of
Nitrate
and
Dinitrogen
Assimilation
by
Anabaena
Species
JOHN
C.
MEEKS,*
KEITH
L.
WYCOFF,t
JOHN
S.
CHAPMAN,
AND
CAROL
S.
ENDERLIN
Department
of
Bacteriology,
University
of
California,
Davis,
California
95616
Received
18
August
1982/Accepted
14
January
1983
Anabaena
sp.
strain
7120
appeared
more
responsive
to
nitrogen
control
than
A.
cylindrica.
Growth
in
the
presence
of
nitrate
strongly
repressed
the
differentiation
of
heterocysts
and
fixation
of
dinitrogen
in
Anabaena
sp.
strain
7120,
but
only
weakly
in
A.
cylindrica.
Nitrate
assimilation
by
ammonium-grown
cultures
was
strongly
repressed
in
Anabaena
sp.
strain
7120,
but
less
so
in
A.
cylindrica.
The
repressive
effect
of
nitrate
on
dinitrogen
assimilation
in
Anabaena
sp.
strain
7120,
compared
to
A.
cylindnca,
did
not
correlate
with
a
greater
rate
of
nitrate
transport,
reduction
to
ammonium,
assimilation
into
amino
acids,
or
growth.
Although
both
species
grew
at
similar
rates
with
dinitrogen,
A.
cylindrica
grew
faster
with
nitrate,
incorporated
more
'3NO3-
into
amino
acids,
and
assimilated
(transported)
nitrate
at
the
same
rate
as
Anabaena
sp.
strain
7120.
Full
expression
of
nitrate
assimilation
in
the
two
species
occurred
within
2.5
h
(10
to
14%
of
their
generation
times)
after
transfer
to
nitrate
medium.
The
induction
and
continued
expression
of
nitrate
assimilation
was
dependent
on
protein
synthesis.
The
half-
saturation
constants
for
nitrate
assimilation
and
for
nitrate
and
ammonium
repression
of
dinitrogen
assimilation
have
ecological
significance
with
respect
to
nitrogen-dependent
growth
and
competitiveness
of
the
two
Anabaena
species.
The
studies
of
Fogg
(7)
demonstrating
that
ammonium
represses
heterocyst
formation
and
dinitrogen
fixation
by
Anabaena
species
have
been
confirmed
by
numerous
studies
on
a
varie-
ty
of
cyanobacteria
(c.f.
for
reviews
references
8,
10,
and
13).
The
single
reported
exception
comes
from
observation
of
a
recently
isolated
marine
Anabaena
species
in
which
nitrate
but
not
ammonium
prevents
formation
of
hetero-
cysts
and
fixation
of
dinitrogen
(4).
Nitrate
has
been
reported
to
have
variable
and
transitory
inhibitory
effects
on
heterocyst
formation
and
dinitrogen
fixation
by
other
species
(10,
31).
In
a
preliminary
survey
of
freshwater
cyano-
bacteria,
including
two
strains
of
A.
cylindrica
(the
Wolk
and
Fogg
strains),
A.
variabilis,
A.
doliolum,
A.
azollae,
Nostoc
muscorum,
and
Anabaenopsis
circularis,
we
too
observed
that
growth
in
the
presence
of
nitrate
has
a
variable
inhibitory
effect
on
heterocyst
differentiation;
only
in
Anabaena
sp.
strain
7120
(formerly
Nos-
toc
muscorum)
(10)
was
heterocyst
differentia-
tion
consistently
repressed
by
5
mM
nitrate.
Ammonium
represses
the
synthesis
of
nitrate
reductase
by
cyanobacteria
(11,
12,
15,
19,
26).
The
effects
of
combined
nitrogen
on
enzyme
activities
(including
the
nitrate
and
aerobic
dini-
t
Present
address:
Department
of
Botany,
Harvard
Univer-
sity,
Cambridge,
MA
02138.
trogen
assimilatory
systems)
may
reflect
a
form
of
nitrogen
control
similar
to
that
observed
in
heterotrophic
bacteria
(18)
and
fungi
(3).
In
cyanobacteria,
exogenous
ammonium
per
se
does
not
appear
to
be
the
primary
regulatory
signal,
at
least
for
the
synthesis
of
nitrogenase
and
differentiation
of
heterocysts
(27).
Rather,
glutamine
or
a
metabolic
derivative
of
it
has
been
implicated
(10,
24,
30),
as
it
also
has
been
implicated
in
the
control
of
nitrate
metabolism
in
Neurospora
species
(22).
To
gain
further
insight
into
the
regulation
of
nitrogen
metabolism
in
cyanobacteria,
we
com-
pared
nitrate
assimilation
by
Anabaena
sp.
strain
7120
and
A.
cylindrica
(Wolk
strain).
We
chose
A.
cylindrica
as
the
representative
of
cyanobacteria
in
which
nitrate
is
a
weak
repres-
sor
because
considerable
information
has
al-
ready
been
accumulated
on
nitrogen
metabolism
in
this
species
(16).
In
this
report
we
compare
these
two
species
with
respect
to
the
effects
of
nitrate
and
ammonium
on
growth,
nitrogenase
activity,
and
heterocyst
formation.
We
also
compared
the
effects
of
various
nitrogen
sources
on
nitrate
assimilation.
MATERLALS
AND
METHODS
Culture
of
cyanobacteria.
Axenic
cultures
of
Ana-
baena
sp.
strain
7120
(ATCC
27893)
and
A.
cylindrica
Lemm
(ATCC
29414)
were
obtained
from
the
Ameri-
can
Type
Culture
Collection,
Rockville,
Md.
1351
APPL.
ENVIRON.
MICROBIOL.
Cyanobacteria
were
initially
grown
in
1-
or
4-liter
Erlenmeyer
flasks
fitted
with
sparging
tubes
and
inlet
and
outlet
ports
and
mixed
by
magnetic
stirring,
illuminated
from
the
side
with
cool
white
fluorescence
lamps
at
7.5
W/m2,
and
maintained
as
semicontinuous
cultures
by
daily
removal
of
cells
and
spent
medium
and
addition
of
fresh
medium.
The
basal
medium
was
an
eightfold
dilution
of
that
described
by
Allen
and
Arnon
(2).
For
growth
on
nitrate
or
ammonium,
the
basal
medium
was
supplemented
with
5
to
20
mM
NO3-
(equimolar
Na+
and
K+
salts
in
all
cases
of
supplementation
or
addition
of
N03)
or
2.5
mM
NH4Cl
plus,
as
buffer
in
all
cases,
5
mM
sodium
N-
tris(hydroxymethyl)methyl-2-aminoethanesulfonic
acid
(TES),
pH
7.2
to
7.5.
In
experiments
to
determine
the
immediate
effect
of
high
exogenous
N03-
or
NH4'
concentrations
on
nitrogenase
activity,
the
experimen-
tal
and
control
semicontinuous
cultures
were
operated
in
a
batch
mode
after
addition
of
the
nitrogen
source.
In
all
other
cases,
cells
were
harvested
aseptically
from
the
semicontinuous
cultures,
concentrated
by
centrifugation,
and
suspended
in
fresh
medium
to
the
cell
concentration
noted.
For
growth
experiments,
the
cyanobacteria
were
subcultured
at
room
temperature
in
test
tubes
(2
by
15
cm)
fitted
with
side
arms
and
holding
15
ml
of
medium,
with
8.5
W/m2
of
fluorescent
illumination
directed
toward
the
sides
of
the
tubes.
The
cultures
were
mixed
by
sparging
with
membrane-filtered
(0.20-,um
pore
size;
Millipore
Corp.,
Bedford,
Mass.)
humidified
gas-
es
at
a
flow
rate
of
ca.
400
ml/min.
In
the
case
of
N2-
supported
growth,
the
cultures
were
sparged
with
air
with
or
without
1%
CO2.
In
the
cases
of
N03-
and
NH4+-supported
growth,
cultures
were
sparged
with
air
with
or
without
1%
CO2
or
with
argon-oxygen-
carbon
dioxide,
80:19:1
(vol/vol/vol),
obtained
by
mix-
ing
cylinder
gases
in
a
rotameter
(model
7471;
Mathe-
son,
Newark,
Calif.).
Growth
was
monitored
by
changes
in
light
scattering
(measured
at
750
nm
in
a
Bausch
&
Lomb
Spectronic
20)
or
chlorophyll
a
(Chl
a)
content.
For
Chl
a
determinations,
1.0-ml
subsam-
ples
were
harvested
at
1,000
x
g
for
5
min,
extracted
with
90%o
(vol/vol)
methanol,
and
quantitated
at
663
nm,
assuming
a
specific
absorption
coefficient
of
78.74
(17).
To
determine
the
long-term
(up
to
7
days
of
culture)
effects
of
micromolar
concentrations
of
N03
and
NH4'
on
acetylene
reduction
and
heterocyst
forma-
tion,
we
suspended
the
harvested
semicontinuous
cul-
ture
cells
in
basal
medium
and
inoculated
it
to
an
initial
concentration
of
approximately
5
x
104
cells/ml
into
1
liter
of
basal
medium
containing
TES-buffered
nitro-
gen
supplements
in
2-liter
Erlenmeyer
flasks.
The
cultures
were
incubated
at
room
temperature
on
an
orbital
shaker
under
8.4
W/m2
of
fluorescent
illumina-
tion.
The
rates
of
acetylene
reduction
and
heterocyst
frequencies
were
monitored
at
8-
to
48-h
intervals
by
centrifuging
400
ml
of
culture
and
suspending
the
pellets
in
5
ml
of
culture
supernatant.
Duplicate
2-ml
subsamples
were
used
to
determine
the
rate
of
acety-
lene
reduction,
and
the
remainder
was
used
for
Chl
a
concentration
and
heterocyst
frequency
(400
to
700
total
cells
counted)
determinations.
To
avoid
time
delays
and
additional
experimental
manipulation
before
exposure
to
13N,
the
cyanobac-
teria
in
semicontinuous
culture
were
first
harvested
by
centrifugation
at
500
x
g
for
5
min,
washed
twice
with
basal
medium
lacking
combined
nitrogen,
suspended
to
2.5
,ug
of
Chl
a
per
ml,
and
incubated
under
growth
conditions
for
2
to
3
h
near
the
site
of
13N
generation.
The
suspensions
were
then
concentrated
10-fold
by
centrifugation
and
immediately
exposed
to
"NO3-.
In
some
experiments,
L-methionine-DL-sulfoximine
(MSX;
Sigma
Chemical
Co.,
St.
Louis,
Mo.)
was
added
to
the
concentrated
cell
suspension
(1.0
mM
after
dilution
with
13N03-).
Enzyme
and
protein
assay.
Nitrogenase
activity
was
assayed
by
whole
cell
reduction
of
acetylene
to
ethyl-
ene;
2
ml
of
cyanobacterial
suspension
was
incubated
in
6.4-ml
vials
containing
5%
(vol/vol)
acetylene
in
air
and
at
30°C
under
tungsten
illumination.
Ethylene
and
acetylene
were
determined
on
a
0.1-ml
subsample
of
the
vial
atmosphere
on
a
Varian
model
940
flame
ionization
detector
gas
chromatograph
(Varian
Asso-
ciates,
Palo
Alto,
Calif.)
employing
a
column
(½/8
in.
by
6
ft.
[ca.
3.2
mm
by
1.8
ml)
of
Porapak
R
(Alltech
Associates,
Arlington
Heights,
Ill.).
Protein
was
assayed
by
the
method
of
Lowry
et
al.
(13),
using
bovine
serum
albumin
as
standard.
Cells
were
lysed
by
cavitation
for
150
s
per
ml
of
suspension
(model
W-225R
sonicator,
equipped
with
a
microtip;
Heat
Systems
Inc.,
Plainview,
N.Y.),
and
protein
was
determined
in
the
supernatant
fraction
after
centrifu-
gation
at
1,000
x
g
for
5
min.
Nitrate
uptake.
Semicontinuous
cultures
grown
with
TES-buffered
1
to
5
mM
N03-,
0.5
to
1.0
mM
N02-,
1
mM
NH4',
or
N2
were
harvested
aseptically,
washed
twice
with
basal
medium
lacking
combined
nitrogen,
and
suspended
to
a
final
concentration
of
approxi-
mately
5
x
107
cells/ml.
One
milliliter
of
concentrated
suspension
was
added
to
49
ml
of
basal
medium
supplemented
with
5
to
250
,uM
N03-
in
125-ml
flasks.
The
suspensions
were
incubated
on
an
orbital
shaker
(100
rpm)
at
room
temperature
under
fluorescent
illu-
mination.
Subsamples
(2.0
ml)
were
withdrawn
at
various
times
from
1
to
12
h
and
centrifuged
at
1,000
x
g
for
5
min.
The
pellets
were
analyzed
for
Chl
a
content.
Supernatant
fractions
(10
to
50
p.I)
were
analyzed
for
nitrate
and
nitrite
by
separation
on
a
high-
performance
liquid
chromatography
(HPLC)
strong
anion-exchange
column
(Partisil-10
SAX;
Whatman
Inc.,
Clifton,
N.J.);
nitrate
and
nitrite
were
eluted
with
30
mM
phosphate
buffer,
pH
3.0,
and
monitored
at
210
nm,
and
the
concentrations
were
determined
from
peak
heights
with
a
standard
curve
(28).
Labeling
with
13N03-.
Details
of
the
target
system
for
the
generation
of
'3N
at
the
Crocker
Nuclear
Laboratory
on
the
University
of
California,
Davis
campus
and
the
purification
of
13N03-
have been
given
previously
(5,
16,
21).
The
13N03-
used
in
this
study
had
a
greater
than
99.99%
radiochemical
purity
and
1
to
4
mCi
of
13N
per
ml.
Labeling
with
13N03-
was
done
under
air
in
15-ml
conical
centrifuge
tubes
containing
0.25
ml
of
cyano-
bacterial
suspension
and
0.25
ml
of
purified
13N03-
solution.
The
samples
were
incubated
at
room
tem-
perature
on
an
orbital
shaker
(200
rpm)
in
front
of
a
cool
white
fluorescent
lamp
(ca.
600
ft-c
[ca.
7,056
lx]).
The
reactions
were
terminated
after
incubations
of
5
to
900
s
by
the
addition
of
4
volumes
of
100%
methanol.
The
cells
were
extracted
by
mixing
on
a
micromixer
for
1
min,
and
the
cellular
debris
was
removed
by
centrifugation
at
1,000
x
g
for
1
min.
The
methanolic
extracts
were
processed
either
for
amino
acid
separa-
1352
MEEKS
ET
AL.
NITRATE
ASSIMILATION
BY
ANABAENA
SPECIES
1353
tion
by
high-voltage
electrophoresis
and
quantitation
by
integration
of
peaks
in
radioscans
or
for
13NH4+
determination
by
vacuum
distillation
and
quantitation
by
scintillation
spectroscopy
as
previously
described
(16).
RESULTS
Effects
of
inorganic
nitrogen
sources
on
growth,
nitrogenase
activity,
and
heterocyst
formation.
Both
Anabaena
sp.
strain
7120
and
A.
cylindrica
grew
faster
and
produced
a
lower
frequency
of
heterocysts
when
cultured
with
ammonium
(in
the
presence
or
absence
of
dinitrogen)
as
com-
pared
with
dinitrogen
and
nitrate
(Table
1).
However,
the
two
species
differed
significantly
with
respect
to
their
response
to
dinitrogen
or
nitrate
as
nitrogen
sources.
With
nitrate,
A.
cylindrica
grew
almost
as
rapidly
as
it
did
with
ammonium
and
slightly
more
rapidly
than
it
did
with
dinitrogen,
but
it
produced
only
about
30%
fewer
heterocysts.
Repeated
subculturing
of
this
strain
in
5
mM
nitrate
caused,
at
most,
a
50o
reduction
in
heterocyst
frequency.
In
contrast,
Anabaena
sp.
strain
7120
grew
at
about
the
same
rate
in
5
mM
nitrate
or
with
dinitrogen,
but
the
frequency
of
heterocysts
was
reduced
approxi-
mately
85%
within
7
days
of
nitrate
culture
and
was
essentially
0
after
two
to
three
subcultures.
In
both
Anabaena
species
there
was
an
imme-
diate
linear
decline
in
nitrogenase
activity
upon
the
addition
of
either
5
mM
nitrate
or
2.5
mM
ammonium
to
dinitrogen-grown
cultures
(Fig.
1).
When
ammonium
was
added
to
cultures
of
TABLE
1.
Growth
rates
and
heterocyst
frequencies
of
Anabaena
sp.
strain
7120
and
A.
cylindrica
cultured
with
dinitrogen,
nitrate,
and
ammoniuma
Nirgn
Doubling
Heterocyst
Species
souren
time
frequency
Spce
irogen
(h)
±
SE
(%)
±
SE
Anabaena
sp.
N2
21.5
±
1.0
8.4
±
0.3
strain
7102
NO3
21.1
±
1.7
1.3
±
0.2
NH4+
18.8
±
0.5
0
A.
cylindrica
N2
18.2
1.0
6.3
±
0.5
N03
15.0
±
1.6 4.3
±
0.3
NH4+
14.3
±
0.3
0.1
±
0.1
a
Growth
was
measured
by
changes
in
light
scatter-
ing
at
750
mm
or
by
Chl
a
content.
In
the
experiments
with
combined
nitrogen,
the
atmosphere
was
air
with
or
without
CO2
or
argon-oxygen-carbon
dioxide,
80:19:1
(vol/vol/vol),
and
at
N03
and
NH4+
concen-
trations
of
5
and
2.5
mM,
respectively.
There
were
no
significant
differences
between
cultures
grown
with
combined
nitrogen
under
air
or
argon-oxygen-carbon
dioxide.
In
the
case
of
N2,
the
atmosphere
was
air
with
or
without
1%
CO2.
Buffer
in
all
cases
was
5
mM
TES,
pH
7.5.
Each
value
is
the
mean
±
standard
error
of
the
mean
of
four
to
six
separate
experiments.
8
0
0C
20
1'
60-n*t
4,V
lo
20
30
10
20
30
Time
(h)
FIG.
1.
Effect
of
added
nitrate
and
ammonium
to
dinitrogen-growing
cultures
of
Anabaena
sp.
strain
7120
(A)
and
A.
cylindrica
(B)
on
the
activity
of
nitrogenase
(acetylene
reduction)
as
a
function
of
time
after
the
additions.
Experiments
were
conducted
on
semicontinuous
cultures
converted
to
batch
culture
conditions
at
the
time
of
addition
of
5
mM
nitrate
or
2.5
mM
ammonium.
Open
symbols
refer
to
nitrate
additions
and
closed
symbols
to
ammonium
additions.
Each
point
is
the
mean
±
standard
error
of
the
mean
of
three
to
five
experiments.
Control
rates
of
acetylene
reduction
were
9.95
±
0.7
and
20.20
±
1.5
nmol
per
mg
of
protein
per
min
for
Anabaena
sp.
strain
7120
and
A.
cylindrica,
respectively.
either
species
or
nitrate
to
cultures
of
Anabaena
sp.
strain
7120,
nitrogenase
activity
declined
until
little
or
no
activity
could
be
detected
after
48
to
56
h.
The
time
course
of
decline
in
nitro-
genase
activity
was
similar
to
that
observed
in
other
cyanobacterial
strains
(1,
27).
Less
than
24
h
after
the
addition
of
nitrate
to
A.
cylindrica,
nitrogenase
activity
appeared
to
stabilize
at
40
to
45%
of
the
rate
of
dinitrogen-grown
cultures
and
thereafter
declined
only
slowly
(Fig.
1).
By
using
low-cell-density,
large-volume
cul-
ture
conditions,
we
were
able
to
extend
observa-
tions
on
the
effect
of
exogenous
nitrate
and
ammonium
concentrations
on
nitrogenase
activ-
ity
and
heterocyst
formation
to
micromolar
lev-
els.
The
results
of
these
experiments
(Fig.
2
and
summarized
in
Table
2)
showed
that
the
half-
saturation
constants
(Ki)
for
inhibition
of
acety-
lene
reduction
and
heterocyst
formation
were
in
the
very
low
micromolar
range.
The
values
were
also
reasonably
similar
for
both
species
with
respect
to
the
specific
nitrogen
source
and
pa-
rameter
analyzed.
Nevertheless,
there
were
dis-
tinct
differences
between
nitrogen
sources
in
terms
of
acetylene
reduction,
as
well
as
inhibi-
tion
of
acetylene
reduction
compared
with
het-
erocyst
formation.
Ammonium
inhibited
expres-
sion
of
nitrogenase
activity
at
three-
to
ninefold
lower
concentrations
than
did
nitrate.
The
Ki
values
for
acetylene
reduction
were
also
lower
VOL.
45,
1983
1354
MEEKS
ET
AL.
100
80
60
40
Z
20
u
0
c
0
.0
c
100
C
0-
@
80
0
60
40
20
A.
*
a
/
6-
V
'I-
L
r,
B.
0
50
100
1
50
200
250
Nitrate
or
Ammonium
(pAM)
A--b
250,
2500
FIG.
2.
Effect
of
various
nitrate
or
ammonium
con-
centrations
on
heterocyst
formation
and
acetylene
reduction
by
Anabaena
sp.
strain
7120
(A)
and
A.
cylindrica
(B).
Open
symbols
refer
to
nitrate
and
closed
symbols
to
ammonium:
0
and
*,
acetylene
reduction;
A
and
A,
heterocyst
frequency.
The
points
represent
the
means
of
four
to
six
experiments.
Con-
trol
rates
of
acetylene
reduction
were
0.21
±
0.03
and
0.47
±
0.06
(mean
±
standard
error
of
the
mean
of
15
experiments)
,umol
of
C2H2
reduced
per
mg
of
Chl
a
per
min
for
Anabaena
sp.
strain
7120
and
A.
cylin-
drica,
respectively.
Heterocyst
frequencies
in
the
ab-
sence
of
combined
nitrogen
were
7.8
±
0.2
and
5.5
±
0.1
(16
determinations)
for
Anabaena
sp.
strain
7120
and
A.
cylindrica,
respectively.
Mean
Chl
a
concentra-
tion
at
the
start
of
the
experiments
was
1.5
ng/ml,
increasing
to
a
maximum
of
335
ng/ml
within
6
to
7
days.
with
either
nitrogen
source
than
they
were
for
heterocyst
formation.
The
maximal
inhibition
of
acetylene
reduction
and
heterocyst
formation
was
different
in
the
two
species
(Fig.
1
and
2
and
Tables
1
and
2).
Exogenous
ammonium
inhibited
the
expression
of
both
in
the
two
species
by
greater
than
97%.
However,
nitrate
caused
only
69
and
36%
inhibi-
tion
of
acetylene
reduction
activity
and
hetero-
cyst
formation,
respectively,
in
A.
cylindrica.
Maximal
inhibition
of
acetylene
reduction
and
heterocyst
formation
in
A.
cylindrica
occurred
between
25
and
100
,M
and
did
not
increase
at
higher
nitrate
concentrations.
In
contrast,
higher
nitrate
concentrations
increased
inhibition
of
heterocyst
formation
in
Anabaena
sp.
strain
7120.
This
increased
inhibition
in
Anabaena
sp.
strain
7120
was
not
a
direct
function
of time
to
dilute
preexisting
heterocysts
since
both
species
grew
at
their
respective
rates
at
all
nitrate
con-
centrations
above
50
,uM.
Double
reciprocal
plots
of
nitrate
concentration
versus
inhibition
of
heterocyst
formation
in
Anabaena
sp.
strain
7120
showed
a
departure
from
linearity
above
250
,uM,
which
could
imply
a
concentration
dependence
on
nitrate
transport
or
utilization.
Kinetics
of
nitrate
assimilation.
The
effect
of
nitrate
concentration
on
nitrate
assimilation
by
Anabaena
sp.
strain
7120
and
A.
cylindrica
was
determined
by
HPLC
analysis
of
nitrate
disap-
pearance
from
the
growth
medium
(Fig.
3).
Nitrate
assimilation
(including
transport,
reduc-
tion,
and
incorporation
into
organic
metabolites)
by
both
species
showed
saturation
kinetics
be-
tween
50
and
250
,uM
nitrate
(Fig.
3).
There
were
no
significant
changes
in
the
rate
of
assimilation
by
either
species
at
concentrations
up
to
2.5
mM
(data
not
shown).
Thus,
the
Anabaena
species
appear
to
have
one
nitrate
assimilatory
(trans-
port)
system,
compared
with
two
in
Klebsiella
pneumoniae
(29).
The
calculated
Vma,
values
were
26.2
and
27.5
,umoles
per
mg
of
Chl
a
per
h
and
the
apparent
K,
values
were
29.7
and
26.2
,uM
for
Anabaena
sp.
strain
7120
and
A.
cylin-
drica,
respectively.
The
Vmax
values
are
approx-
imately
threefold
greater
than
those
previously
calculated
for
a
Nostoc
species
and
an
unrelated
Anabaena
species
(6).
The
Ks
values
are
similar
to
those
reported
for
other
cyanobacteria
(33).
Our
initial
experiments
examining
13NO3-
accu-
mulation
in
filtered cells
resulted
in
curves
with
shapes
similar
to
those
in
Fig.
3.
However,
the
13NO3-
experiments
were
difficult
to
quantitate
due
to
variable
counting
efficiencies
of
the
de-
tectors
available.
To
determine
whether
differences
in
the
re-
duction
of
nitrate
to
nitrite
and
to
ammonium
caused
the
differential
effect
of
nitrate
repres-
sion,
we
attempted
to
quantitate
the
intracellular
concentrations
and
ratios
of
these
ions
by
modi-
fications
of
the
transport
assay
of
Thayer
and
Huffaker
(29).
Cells
were
exposed
to
13NO3-
and
then
centrifuged
at
15,000
x
g
through
mixtures
of
silicone
oils
and
into
1.0
M
perchlo-
ric
acid.
The
perchloric
acid
lysate
was
subject-
ed
to
HPLC
analysis
for
13N03-,
13NO2-,
and
13NH4+
plus
organic
compounds.
Due
to
exten-
sive
transfer
of
exogenous
13NO3-
through
the
silicone
oils
in
the
presence
of
Anabaena
fila-
ments,
the
results
of
these
experiments
were
inconclusive.
Mild
cavitation
of
the
Anabaena
cultures
to
yield
10-
to
15-cell
filaments
did
not
r-
APPL.
ENVIRON.
MICROBIOL.
A---./
A-M
NITRATE
ASSIMILATION
BY
ANABAENA
SPECIES
1355
TABLE
2.
Kinetic
constants
of
nitrate
and
ammonium
repression
of
acetylene
reduction
and
heterocyst
formation
in
Anabaena
sp.
strain
7120
and
A.
cylindricaa
Acetylene
reduction
Heterocyst
formation
Species
Nitrogen
K
Maximum
%
Maximum
%
source
KLM)
inhibition
rb
K
inhibition
(ILM)
(6
day)
(6
day)
Anabaena
sp.
NO3-
2.3
94.0
0.91
6.2
51.0 0.92
strain
7120
NH4+
0.25
98.0
0.78
7.6
99.9
0.99
A.
cylindrica
NO3-
2.6
69.0
0.83
9.5
36.0
0.99
NH4+
0.74
99.0
0.92
3.2
97.0
0.97
a
Kinetic
parameters
were
calculated
from
the
data
in
Fig.
2.
b
The
correlation
coefficients
(r)
were
determined
from
linear
regression
analysis.
reduce
the
amount
of
13NO3-
contamination
in
the
perchloric
acid
lysate.
Nevertheless,
nitrite
did
not
accumulate
in
media
as
it
does
in
a
"leaky"
nitrite
reductase
mutant
of
a
unicellular
cyanobacterium
(25).
The
lower
limits
of
detection
in
the
nonradioactive
HPLC
system
were
near
50
pmol
of
nitrite
and
200
pmol
of
nitrate
in
a
50-Jl
injection
volume
(28).
More
than
50
pmol
of
nitrite
did
not
accu-
mulate
in
the
growth
medium
of
either
Ana-
I
co
-
C0
E
0
-9-
0
4
0
0-
z
E
10
30
20
10
50
loo
150
200
250
Nitrate
(,IlM)
FIG.
3.
Effect
of
various
nitrate
concentrations
on
the
rates
of
nitrate
assimilation
by
Anabaena
sp.
strain
7120
(A)
and
A.
cylindrica
(B).
The
data
presented
are
means
+
standard
errors
of
the
means
of
8
to
14
experiments
with
cells
of
both
species
previously
grown
in
the
presence
of
1
to
5
mM
nitrate,
0.5
to
1
mM
nitrite,
1
mM
ammonium,
or
dinitrogen
gas
as
the
nitrogen
source.
After
2
to
4
h
of
incubation
in
the
respective
nitrate
concentration,
the
rates
of
nitrate
assimilation
were
determined
by
monitoring
nitrate
disappearance
from
growth
medium,
using
HPLC
as
described
in
the
text
(see
also
Fig.
4).
baena
sp.
strain
7120
or
A.
cylindrica
at
any
external
nitrate
concentration
after
any
incuba-
tion
period.
In
parallel
experiments
with
'3NO3-
plus
50
,uM
14NO3-
as
carrier,
no
13N02-
above
that
in
the
original
13N
solution
(less
than
0.01%)
could
be
detected
in
the
medium
of
either
spe-
cies
after
120
s
of
incubation.
Substantial
amounts
of
ammonium
also
did
not
accumulate
within
the
cells
of
either
species.
Less
than
0.1%
(lower
limit
of
detection)
of
the
no-carrier-added
13NO3-
(4
to
40
nM
total
nitrate)
could
be
distilled
as
13NH3
from
methanolic
extracts
after
900
s
of
incubation.
However,
when
cultures
were
incubated
in
the
additional
presence
of
1
mM
MSX
(an
inhibitor
of
glutamine
synthetase),
11.0
and
10.2%
of
the
13NO3-
distilled
as
13NH3
from
extracts
of
Anabaena
sp.
strain
7120
and
A.
cylindrica,
respectively.
Effect
of
nitrogen
sources
for
growth
on
nitrate
assmilation.
The
assimilation
of
nitrate
by
dini-
trogen-fixing
(11,
12,
20)
and
non-dinitrogen-
fixing
cyanobacteria
(6,
15,
26)
is
reported
to
be
lower
in
the
presence
of
ammonium.
We
verified
this
observation
in
Anabaena
sp.
strain
7120
and
A.
cylindrica
and
also
showed
that
the
presence
of
ammonium
did
not
completely
repress
nitrate
assimilation
in
either
species
(Fig.
4
and
Table
3).
A
low
level
of
nitrate
disappearance
could
be
detected
by
HPLC
analysis
of
ammonium-sup-
plemented
growth
medium
(Fig.
4),
and
signifi-
cant
amounts
of
13NO3-
were
incorporated
into
organic
metabolites
by
ammonium-grown
cells
of
both
species
(Table
3).
The
recovery
from
ammonium
repression
of
nitrate
assimilation
oc-
curred
within
2.5
h
after
transfer
to
ammonium-
minus,
nitrate-plus
growth
conditions
(Fig.
4).
Full
expression
of
nitrate
assimilation
after
transfer
from
ammonium
was
dependent
on
new
protein
synthesis
as
shown
by
chloramphenicol
treatment
in
A.
cylindrica
(Fig.
4).
Treatment
with
chloramphenicol
also
reduced
the
rate
of
nitrate
assimilation
within
2
h
of
exposure
in
nitrate-grown
cells
(Fig.
4),
implying
a
rapid
turnover
rate
of
an
essential
assimilatory
pro-
A.
B.
4t
VOL.
45,
1983
30~
20~
1356
MEEKS
ET
AL.
Time
(h)
FIG.
4.
Initial
time
course
of
the
assimilation
of
100
,M
nitrate
by
suspensions
of
Anabaena
sp.
strain
7120
(A)
and
A.
cylindrica
(B)
previously
grown
in
the
presence
of
1
mM
nitrate
(0),
0.5
mM
nitrite
(V),
1
mM
ammonium
(A),
or
nitrogen
gas
(O).
Symbols:
A,
nitrate
assimilation
by
suspensions
incubated
in
the
additional
presence
of
1
mM
ammonium;
0,
nitrate
assimilation
by
nitrate-grown
A.
cylindrica
in
the
presence
of
50
,ug
of
chloramphenicol
per
ml
added
at
time
zero;
*,
nitrate
assimilation
by
ammonium-
grown
cells
of
A.
cylindrica
in
the
presence
of
50
,ug
of
chloramphenicol
per
ml
added
at
time
zero.
The
data
points
reflect
means
of
two
to
four
individual
experi-
ments.
The
rates
of
nitrate
assimilation
were
deter-
mined by
monitoring
disappearance
of
nitrate
from
the
growth
medium,
using
HPLC
as
described
in
the
text.
tein.
We
have
not
attempted
to
identify
the
protein(s)
required
for
continued
expression
by
nitrate-grown
cells
or
for
induction
in
ammoni-
um-grown
cells.
Dinitrogen-grown
cells
showed
a
higher
level
of
nitrate
assimilation
into
organic
metabolites
than
did
ammonium-grown
cells
of
both
species
(Table
3).
The
time
course
of
full
expression
of
nitrate
assimilation
by
dinitrogen-grown
cells
when
transferred
to
nitrate
medium
was
similar
to
that
of
ammonium-grown
cells
(Fig.
4).
There
were
differences
between
Anabaena
sp.
strain
7120
and
A.
cylindrica
in
the
effect
of
nitrogen
sources
for
growth
on
nitrate
assimila-
tion.
For
example,
growth
on
nitrite
appeared
to
support
full
expression
of
nitrate
assimilation
in
Anabaena
sp.
strain
7120
but
not
in
A.
cylindrica
(Fig.
4).
Compared
to
A.
cylindrica,
growth
of
Anabaena
sp.
strain
7120
in
ammonium
resulted
in
much
greater
repression
of
nitrate
assimila-
tion
(Fig.
4
and
Table
3).
The
differential
effect
of
ammonium
on
nitrate
assimilation
was
similar
to
the
effect
of
nitrate
on
acetylene
reduction
and
heterocyst
formation
(Fig.
2).
DISCUSSION
The
two
dinitrogen-fixing
Anabaena
species
used
in
this
study
differed
in
their
physiological
responses
to
exogenous
combined
nitrogen.
In
general,
Anabaena
sp.
strain
7120
was
distinctly
more
responsive
to
nitrogen
control
(18)
than
was
A.
cylindrica.
For
example,
growth
in
the
presence
of
nitrate
ultimately
caused
complete
repression
of
heterocyst
formation
and
dinitro-
gen
fixation
(the
aerobic
dinitrogen
assimilatory
system)
in
Anabaena
sp.
strain
7120,
but
only
approximately
50
and
70%
reductions
in
these
activities,
respectively,
in
A.
cylindrica
(Tables
1
and
2
and
Fig.
1
and
2).
We
expected
the
repressive
effect
of
nitrate
on
the
dinitrogen
assimilatory
system
of
Anabaena
sp.
strain
7120,
in
comparison
with
A.
cylindrica,
to
corre-
late
with:
(i)
a
greater
rate
of
nitrate
transport;
(ii)
more
incorporation
of
13NO3-
into
amino
TABLE
3.
Principal
radioactive
constituents
observed
after
900
s
of
assimilation
of
'3N03-
by
Anabaena
sp.
strain
7120
and
A.
cylindrica
cultured
with
dinitrogen,
nitrate,
and
ammonium
Nitrogen
"3N
found
in
compound
(%
13N
added)a
Species
source
for
Total
amino
growth
Asp
+
Glub
Gln
Cit
+
Alac
Arg
acids
Anabaena
sp.
NH4+
0.01
±
0.006
0.05
+
0.03
0.001
+
0.001
0.06
±
0.04
strain
7120
N2
0.79
±
0.35
0.29
t
0.08
0.10
t
0.06 1.19
t
0.47
N03
2.59
t
0.51
0.35
t
0.11
0.09
±
0.04
0.08
±
0.04
3.09
±
0.63
A.
cylindrica
NH4+
2.35
t
1.48
0.50
±
0.18
0.21
±
0.13
0.016
±
0.008
3.08
±
1.73
N2
4.23
±
0.46
0.51
±
0.24
0.21
±
0.07
0.06
±
0.016
4.79
±
0.48
NO3
3.25
±
1.18
3.77
±
0.65
1.31
±
0.78
0.51
±
0.28
8.88
±
2.77
a
Values
are
means
+
standard
errors
of
the
mean
of
two
to
six
experiments
and
were
determined
by
integration
and
time
correction
of
peaks
of
radioactivity
from
13N
after
electrophoresis
at
pH
9.2,
in
comparison
with
l3N03-
radioactivity
added
as
determined
by
scintillation
spectroscopy.
Asp,
aspartate;
Glu,
glutamate;
Gln,
glutamine;
Cit,
citrulline;
Ala,
alanine;
Arg,
arginine.
b
In
A.
cylindrica,
the
Asp
+
Glu
peak
was
approximately
50%o
Asp
in
N03--grown
cultures
and
approximately
25%
Asp
in
N2-
and
NH4+-grown
cultures.
In
Anabaena
sp.
strain
7120,
the
Asp
+
Glu
peak
was
approximately
33%
Asp
in
NO3
-grown
cultures
and
not
observed
in
N2-
or
NH4+-grown
cultures.
c
In
all
cases,
the
peak
was
primarily
Cit
with
a
variable
small
shoulder
of
Ala.
APPL.
ENVIRON.
MICROBIOL.
NITRATE
ASSIMILATION
BY
ANABAENA
SPECIES
1357
acids;
and
(iii)
an
increase
in
the
growth
rate
relative
to
dinitrogen
and
comparable
to
that
supported
by
ammonium.
These
correlations
were
not
observed;
in
fact,
they
were
generally
reversed.
Based
on
the
kinetics
of
preliminary
short-
term
13NO3-
accumulation
experiments,
we
previously
suggested
that
the
rates
of
nitrate
transport
differed
in
the
two
species
(1;
J.
C.
Meeks,
Third
International
Symposium
on
Pho-
tosynthetic
Prokaryotes,
Oxford,
1979,
abstract
D-15).
However,
with
the
data
presented
here,
differences
in
rates
of
nitrate
uptake
or
assimila-
tion
cannot
explain
the
differential
effect
of
nitrate
on
the
aerobic
dinitrogen
assimilatory
system.
Nitrate
disappeared
from
the
growth
medium
of
both
species
at
the
same
rate
(Fig.
3),
although
under
identical
culture
conditions
the
rates
of
acetylene
reduction
and
numbers
of
heterocysts
formed
differed
markedly.
When
ammonium
assimilation
was
inhibited
by
MSX,
the
two
species
accumulated
nearly
equal
amounts
of
13NH4+
from
13NO3-.
Thus,
their
rates
of
reduction
of
nitrite
to
ammonium
were
also
quite
similar.
The
fact
that
detectable
amounts
of
13N03-
derived
13NH4'
did
not
accumulate
in
the
absence
of
MSX
implies
that
ammonium
was
rapidly
assimilated
by
both
spe-
cies.
These
observations
suggest
that
dinitrogen
fixation
could
have
contributed
to
the
growth
of
A.
cylindrica
in
the
presence
of
nitrate.
The
growth
rate
of
A.
cylindrica
was
the
same
in
the
presence
of
nitrate
and
the
presence
or
absence
of
dinitrogen
(Table
1).
The
rates
of
nitrate
assimilation
were
measured
under
air;
thus,
it
is
possible
that
assimilation
was
greater
when
there
was
no
contribution
of
fixed
nitrogen
by
nitrogenase.
However,
the
relative
amount
of
13NO3-
in-
corporated
into
methanol-extractable
amino
ac-
ids
differed;
Anabaena
sp.
strain
7120
accumu-
lated
significantly
less
than
A.
cylindrica.
These
results
imply
that
a
significant
fraction
of
nitrate-
derived
ammonium
was
converted
into
metabo-
lites
in
Anabaena
sp.
strain
7120,
including
polypeptides
that
were
not
available
for
growth.
In
fact,
nitrate
did
not
stimulate
growth
of
Anabaena
sp.
strain
7120
relative
to
dinitrogen
(Table
1).
Since
the
addition
of
1%
CO2
and
5
mM
fructose
or
glucose
and
higher
light
intensi-
ties
did
not
increase
the
nitrate-dependent
growth
rate,
availability
of
nitrogen
was
proba-
bly
more
rate
limiting
than
carbon
in
Anabaena
sp.
strain
7120.
In
A.
cylindrica,
nitrate
support-
ed
nearly
as
rapid
a
growth
rate
as
ammonium.
Assuming
a
nitrogen
content
of
7
to
10%
and
a
Chl
a
content
of
1%
of
the
dry weight
(32),
the
maximal
rates
of
nitrate
assimilation
(Fig.
3)
predict
a
generation
time
of
13
to
18
h
in
nitrate-
supplemented
medium;
i.e.,
a
generation
time
similar
to
that
observed
by
A.
cylindrica
(Table
1).
Thus,
nitrate
assimilation
and
growth
appear
to
be
closely
coupled
in
A.
cylindrica,
but
not
in
Anabaena
sp.
strain
7120.
The
nitrate
assimilatory
systems
of
Anabaena
sp.
strain
7120
and
A.
cylindrica
showed
three
general
levels
of
expression:
repressed
(growth
with
ammonium),
constitutive
(growth
with
dini-
trogen),
and
induced
(growth
with
nitrate).
There
were
differences
between
Anabaena
sp.
strain
7120
and
A.
cylindrica
in
their
degree
of
repression
by
ammonium
(Table
3).
Nitrate
as-
similation
by
ammonium-grown
cells
of
A.
cylin-
drica
was
about
65%
that
of
dinitrogen-grown
cells.
A
similar
small
difference
in
nitrate
reduc-
tase
activity
between
dinitrogen
and
ammonium-
grown
Nostoc
species
and
an
unrelated
Ana-
baena
species
was
reported
by
Herrero
et
al.
(12).
In
that
study,
growth
in
the
presence
of
nitrate
increased
the
rate
of
nitrate
reductase
activity
about
3.8-fold
relative
to
dinitrogen-
and
ammonium-grown
cells.
Nitrate-grown
A.
cylin-
drica
showed
similar
increases
of
1.9-
to
2.8-fold
relative
to
dinitrogen-
and
ammonium-grown
cells,
respectively
(Table
3).
However,
ammoni-
um-grown
Anabaena
sp.
strain
7120
had
only
5%
of
the
rate
of
13N03-
incorporation
by
dinitrogen-grown
cells,
and
there
was
an
ap-
proximate
50-fold
difference
between
nitrate-
and
ammonium-grown
cells.
In
this
respect,
Anabaena
sp.
strain
7120
is
similar
to
the
unicel-
lular
non-dinitrogen-
fixing
cyanobacterium
An-
acystis
nidulans
(12).
Nevertheless,
both
of
the
dinitrogen-fixing
species
used
here
and
those
used
by
Herrero
et
al.
(12)
differ
from
A.
nidu-
lans
in
that
growth
on
nitrate
gave
higher
rates
of
nitrate
reductase
activity
and
assimilation.
Nitrate
reductase
activity
in
A.
nidulans
was
fully
expressed
in
the
absence
of
both
ammoni-
um
and
nitrate
(12).
Full
expression
of
nitrate
assimilation
in
Ana-
baena
sp.
strain
7120
and
A.
cylindrica
was
attained
within
2
to
2.5
h
after
transfer
from
repressed
or
constitutive
growth
conditions
to
nitrate
medium
(Fig.
4).
A
comparable
time
span
was
plotted
with
nitrate
uptake
in
ammonium-to-
nitrate
step-down
experiments
with
N.
mus-
corum
(23).
The
time
span
for
full
expression
of
the
nitrate
assimilatory
system
in
Anabaena
sp.
strain
7120
and
A.
cylindrica
corresponds
to
less
than
10
to
14%
of
their
generation times.
This
is
a
relatively
short
time
frame
for
complete
syn-
thesis
and
assembly
of
a
complex
assimilatory
system.
A
comparable
time
span
with
respect
to
generation
time
was
observed
in
A.
nidulans
for
full
expression
of
nitrate
reductase
activity
dur-
ing
transitions
from
ammonium
to
nitrate
or
no
combined
nitrogen
(12).
In
A.
cylindrica
(Fig.
4)
and
A.
nidulans
(12),
induction
of
the
activities
did
not
occur
in
the
presence
of
chlorampheni-
VOL.
45,
1983
APPL.
ENVIRON.
MICROBIOL.
col,
and
the
fully
induced
activity
in
A.
cylin-
drica
declined
after
2
h
of
incubation.
These
data
indicate
de
novo
and
continued
synthesis
of
one
or
more
essential
proteins.
However,
the
pro-
tein(s)
responsible
has
not
been
identified.
Thus,
one
cannot
unequivocally
state
whether
an
inac-
tive
or
less
active
form
of
nitrate
reductase
or
of
the
nitrate
assimilatory
system
was
present
in
these
cyanobacteria
as
has
been
suggested for
eucaryotic
cells
(9).
The
regulatory
signal(s)
for
repressed
or
in-
duced
expression
of
the
nitrate
or
aerobic
dini-
trogen
assimilatory
systems
in
cyanobacteria
is
unknown.
The
effect
of
MSX
on
reversal
of
short-term
ammonium
inhibition
of
nitrate
up-
take
(6)
and
long-term
ammonium
repression
of
nitrate
reductase
activity
(12)
is
identical
to
its
effect
on
the
aerobic
dinitrogen
assimilatory
system
(27).
Thus,
inhibition
or
repression
of
both
nitrogen
assimilatory
systems
in
cyanobac-
teria
appears
to
be
dependent
on
incorporation
of
exogenous
or
nitrate-derived
ammonium
into
glutamine.
Whether
a
nitrogen
regulatory
com-
pound
is
glutamine
or
a
derivative
of
it
remains
speculative.
The
kinetics
of
13NO3-
assimilation
into
amino
acids
by
Anabaena
sp.
strain
7120
and
A.
cylindrica
showed
no
significant
varia-
tions
in
the
rates
or
amounts
of
glutamine
formed
relative
to
other
amino
acids
that
indi-
cate
a
direct
role
for
glutamine
in
control
of
the
aerobic
dinitrogen
assimilatory
system
(16).
The
kinetics
of
nitrate
and
ammonium
inhibi-
tion
of
the
aerobic
dinitrogen
assimilatory
sys-
tem
indicate
that
the
effective
concentrations
are
ecologically
significant
(Ki
values
of
0.25
to
9.5
,uM;
Table
2).
Concentrations
of
0.3
to
5
F.M
nitrate
and
0.7
to
25
,uM
ammonium
are
com-
monly
detected
in
mesotrophic
to
eutrophic
al-
kaline
freshwater
lakes
(14)
where
these
orga-
nisms
proliferate
(8).
The
kinetics
of
nitrate
assimilation
also
indicate
that
both
species
could
complete
for
the
low
environmental
concentra-
tions
of
nitrate.
The
K,
values
of
26
to
30
,uM
are
within
the
same
range
as
those
determined
for
the
non-dinitrogen-fixing
dominant
planktonic
cyanobacterium,
Oscillatoria
agardhii
(33),
with
which
they
would
compete.
ACKNOWLEDGMENTS
This
work
was
supported
in
part
by
the
California
Agricul-
tural
Experiment
Station,
under
project
number
CAD*-3620-
H,
and
National
Science
Foundation
grant
PCM
79-04136.
We
thank
K.
Krohn
and
N.
J.
Parks
for
assistance
in
the
general
13N
experimentation,
Joe
Chasco
for
HPLC
analysis
of
13N
solutions,
Jim
Thayer
for
HPLC
analysis
of
both
13N
and
nonradioactive
nitrate
and
nitrite
solutions,
and
J.
L.
Ingraham
and
S.
G.
Kustu
for
their
comments
during
prepara-
tion
of
the
manuscript.
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VOL.
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1983
