In
i o
modi ica ion
o
Azo obac e
ch oococcum
glu amine
syn he ase
Ma ia
C.
MUNOZ-CENTENO,
F ancisco
J.
CEJUDO
and
An onio
PANEQUE*
Ins i u o
de
Bioqu mica
Vege al
y
Fo os n esis,
Uni e sidad
de
Se illa-Consejo
Supe io
de
In es igaciones
Cien icas,
Apa ado
1113,
41080
Se illa,
Spain
A
monospeci ic
an i-(glu amine
syn he ase)
an ibody
aised
agains
glu amine
syn he ase
o
he
unicellula
cyanobac e ium
Synechocys is
sp.
s ain
PCC
6803
immuno eac ed
wi h
glu amine
syn he ase
om
he
N2- ixing
he e o ophic
bac e ium
Azo obac e
ch oococcum.
In
Wes e n-blo ing
expe imen s
his
an ibody
ecognized
a
single
p o ein
o
a
molecula
mass
o
59
kDa
co esponding
o
glu amine
syn he ase
subuni .
This
p o ein
was
in
i o-labelled
in
esponse
o
addi ion
o
ammonium,
bo h
[3H]adenine
and
H332PO4
p eincuba ion
o
he
cells
being
equally
e ec i e.
Ne e heless,
he
amoun
o
glu amine
INTRODUCTION
The
azo obac e s
a e
he e o ophic
ni ogen- ixing
ae obic
bac-
e ia
ha
can
use,
besides
molecula
ni ogen,
o he
ino ganic
ni ogen
sou ces
such
as
ni a e
o
ni i e.
Ammonium,
he
p oduc
o
he
educ ion
o
N2
and
ni a e,
con ols
in
i s
u n
he
ac i i ies
o
ni ogenase
[1-3]
and
ni a e
up ake
[4],
bo h
ene gy-
dependen
e en s.
In
Azo obac e
ch oococcum
[5]
and
A.
inelandii
[6]
ammonium
is
inco po a ed
in o
ca bon
skele ons
ia
he
glu amine
syn he ase
(GS)-glu ama e
syn hase
(GOGAT)
ou e,
which
is
hough
o
be
solely
esponsible
o
he
as-
simila ion
o
ammonium.
Since
GS,
ha
ca alyses
an
ATP-
equi ing
eac ion,
plays
a
pi o al
ole
in
ni ogen
me abolism
in
many
p oka yo es
and
euka yo es,
bo h
he
syn hesis
and
ac i i y
o
his
enzyme
ha e been
conside ed
o
be
s ic ly
egula ed
in
esponse
o
he
a ailable
ni ogen
sou ce.
Regula ion
o
GS
by
gene
exp ession,
eedback
inhibi ion,
and
co alen
modi ica ion
has
been
s udied
ex ensi ely
in
En e o-
bac e ia
[7-9].
In
Esche ichia
coli,
GS
is
a
dodecame
o
12
iden ical
subuni s,
each
o
which
can
be
egula ed
independen ly
by
e e sible
adenylyla ion
ende ing
a
less-ac i e
enzyme.
The
adenylyla ion-deadenylyla ion
eac ions
a e
ca alysed
by
adenylyl ans e ase,
he
ac i i y
o
which
is
egula ed
by
he
egula o y
p o ein
PII
which
i sel
is
egula ed
h ough
e e sible
u idylyla ion
ca ied
ou
by
an
u idylyl ans e ase.
The
la e
enzyme
esponds
o
he
a io
o
glu amine
o
a-oxoglu a a e,
shi ing
GS
o
he
mo e
adenylyla ed
s a e
as
he
a io
o
glu amine
o
a-oxoglu a a e
inc eases
and
ice
e sa
[10,1
1].
The
adenylyla ion-deadenylyla ion
sys em
is
ope a i e
in
mos
G am-
nega i e
[12-15]
bu
no
in
g am-posi i e
[16]
bac e ia,
he
me hanogen
Me hanobac e ium
i ano i
[17]
o
cyanobac e ia
[18,19].
Besides
his
co alen
modi ica ion,
GS
om
bo h
p oka yo ic
and
euka yo ic
o ganisms
has
been
desc ibed
o
be
ADP- ibosyla ed
in
i o,
and
in
some
cases
he
eac ion
was
accompanied
by
he
loss
o
GS
ac i i y
([13]
and
e e ences
ci ed
he ein).
The
in
i o
exis ence
o
his
modi ica ion
has
no
ye
been
epo ed,
howe e .
In o ma ion
on
azo obac e
GS
is
e y
sca ce.
The
enzyme
has
been
pu i ied
om
A.
inelandii
and
shown
o
con ain
AMP
syn he ase
p esen
in
A.
ch oococcum
was
independen
o
he
a ailable
ni ogen
sou ce.
Modi ied,
inac i e
glu amine
syn he ase
was
e-ac i a ed
by
ea men
wi h
snake- enom
phosphodies e ase
bu
no
by
alkaline
phospha ase.
L-
Me hionine-DL-sulphoximine,
an
inhibi o
o
glu amine
syn he ase,
p e en ed
he
enzyme
om
being
co alen ly
modi ied.
We
conclude
ha ,
in
A.
ch oococcum,
glu amine
syn he ase
is
adenylyla ed
in
esponse
o
ammonium
and
ha
o
he
modi ica ion
o
ake
place
ammonium
mus
be
me abolized.
bound
co alen ly
when
isola ed
om
ammonium-g own
cells
[20,21].
Recen ly
i
has
been
desc ibed
ha ,
in
A.
inelandii,
exp ession
o
glnA,
he
gene
encoding
glu amine
syn he ase,
is
no
con olled
by
he
a ailable
ni ogen
sou ce
[22].
He e
we
epo
ha
GS
o
A.
ch oococcum
did
c oss- eac
wi h
an ise um
aised
agains
GS
om
he
cyanobac e ium
Synechocys is
sp.
PCC
6803.
We
also
show
ha ,
in
A.
ch oococcum,
GS
is
egula ed
by
adenylyla ion-deadenylyla ion
in
esponse
o
he
addi ion
o
ammonium
o
he
g ow h
medium,
wi h
ammonium
de e mining
he
GS
ac i i y
le el
a he
han
he
enzyme
syn hesis.
Finally,
we
epo
ha
in
he
p esence
o
L-me hionine-DL-sulphoximine
(MSX),
an
inhibi o
o
GS,
adenylyla ion
o
his
enzyme
was
p e en ed.
MATERIALS
AND
METHODS
Ma e ials
Coomassie
B illian
Blue
R-250
was
om
Bio-Rad,
Richmond,
CA,
U.S.A.
Alkaline
phospha ase
and
phosphodies e ase
we e
om
Boeh inge -Mannheim,
Mannheim,
Ge many.
[3H]Adenine
(25.8
Ci/mol)
and
H332PO4
(8800
Ci/mmol)
we e
pu chased
om
du
Pon -New
England
Nuclea ,
D eieich,
Ge many.
Fo
de-
e mina ion
o
molecula
mass,
an
elec opho esis
calib a ion
ki
om
Bio-Rad
Labo a o ies,
He cules,
CA,
U.S.A.,
was
used.
All
o he
chemicals
we e
o
analy ical
g ade.
O ganism
and
g ow h
condi ions
A.
ch oococcum
A.T.C.C.
4412
( om
he
Uni e si y
o
Valencia
Collec ion,
Valencia,
Spain)
was
g own
on
ni ogen- ee
Bu k's
medium
supplemen ed
wi h
0.5
%
(w/ )
suc ose
as
he
sole
ene gy
and
ca bon
sou ce.
When
indica ed,
his
medium
was
supplemen ed
wi h
NH4C1
o
KNO3
a
he
concen a ion
speci ied
in
each
case.
G ow h
condi ions
we e
as
p e iously
desc ibed
[23].
Cell
ex ac s
Cells
we e
ha es ed
by
cen i uga ion
a
10000
g
o
10
min
a
4
°C,
washed
in
50
mM
Mops/KOH
bu X,
pH
7.5,
and
Abb e ia ions
used:
GS,
glu amine
syn he ase;
GOGAT,
glu ama e
syn hase;
MSX,
L-me hionine-DL-sulphoximine.
*
To
whom
co espondence
should
be
add essed.
641
Biochem.
J.
(1994)
298,
641-645
(P in ed
in
G ea
B i ain)
642
M.
C.
Munoz-Cen eno,
F.
J.
Cejudo
and
A.
Paneque
esuspended
a
a
concen a ion
o
1
g
o
cells/3
ml
o 50
mM
Mops/KOH
bu e ,
pH
7.5,
con aining
1
mM
DL-di hio h ei ol
and
1
mM
phenylme hanesulphonyl
luo ide.
Cells
we e
dis up ed
by
sonica ion
(20
kHz;
75
W)
o
5
min
(in
30
s
pe iods)
wi h
a
B anson
soni ie
model
B
12.
The
homogena e
was
cen i uged
a
33000
g
o
30
min
a
4
°C,
and
he
esul ing
supe na an
cons i u ed
he
cell
ex ac .
20
min
and
he
supe na an
cons i u ed
he
labelled
cell
ex ac .
Whe e
indica ed,
immunop ecipi a ion
o
labelled
p o eins
was
ca ied
ou
as
desc ibed
abo e
o
he
unlabelled
cell
ex ac .
When
gels
we e
loaded
wi h
p o eins
om
cell
ex ac s
ha
we e
no
immunop ecipi a ed,
he
GS
p o ein
was
iden i ied
by
Wes e n-blo
(immunoblo ing)
analysis.
Analy ical
me hods
Enzyme
assays
GS
ac i i y
was
de e mined
in
i o
by
he
o ma ion
o
y-
glu amylhyd oxama e
( ans e ase
assay)
in
he
p esence
o
ei he
Mn2+
(inac i e
o m)
o
Mn2+
plus
Mg2+
(ac i e
GS
o m)
[21,24].
Ni a e
educ ase
ac i i y
was
de e mined
in
i o
wi h
di hioni e- educed
Me hyl
Viologen
as
he
elec on
dono
[25].
lmmunop ecipi a ion
o
GS
p o ein
The
A. ch oococcum
GS
p o ein
was
immunop ecipi a ed
om
cell
ex ac s
wi h
an ise um
di ec ed
agains
he
Synechocys is
sp.
s ain
PCC
6803
GS
(kindly
p o ided
by
F.
J.
Flo encio,
om
his
Ins i u e)
as
ollows:
0.3
ml
aliquo s
o
ex ac s
om
A.
ch oococcum
cells
we e
mixed
wi h
inc easing
amoun s
o
he
c ude
abbi
(5
mg
o
p o ein/ml)
an ise um.
The
immuno-
p ecipi a e
was
allowed
o
o m
o e nigh
a
4
°C
and
hen
cen i uged
a
10000
g
o
10
min
a
4
'C.
The
pelle
was
dissol ed
in
40
,ul
o
sample
bu e
[26]
o
SDS/PAGE,
and
he
supe na an
sa ed
o
de e mining
he
emaining
ac i i ies
o
GS
and,
whe e
indica ed,
ni a e
educ ase.
Wes e n
blo
(immunoblo ing)
A e
SDS/PAGE,
he
p o eins
we e
ans e ed
o
ni ocellulose
shee s
as
in
[27],
using
a
mini-T ans
Blo
elec opho e ic
ans e
cell
(Bio-Rad).
To
immunode ec
p o eins,
he
ni ocellulose
il e s
we e
blocked
o e nigh
in
200
mM
NaCl
and
15
mM
T is/HCl,
pH
7.4
(T is/NaCl),
con aining
0.2
%
(w/ )
sodium
azide
and
50%
(w/ )
d ied
skimmed
milk.
An i-GS
an ibody
(1:500)
was
added,
and
he
mix u e
incuba ed
wi h
shaking
o e nigh .
The
il e s
we e
washed
ou
imes
wi h
T is/NaCl
con aining
0.05
%
( / )
Tween
20,
hen
pe oxidase-conjuga ed
an i-( abbi
IgG)
se um
(Sigma)
was
used
as
second
an ibody,
and
blo s
we e
de eloped
as
desc ibed
by
Komb ink
e
al.
[28].
In
i o
[3H]adenine-labelling
(32P-labelling)
expe imen s
A
2
ml
cul u e
was
g own
o
mid-loga i hmic
phase
[a enuance
(D560)
o
app ox.
0.5]
in
ni ogen- ee
medium.
Cells
we e
ha es ed
by
cen i uga ion
a
7000
g
o
5
min
a
oom
em-
pe a u e
and
esuspended
in
ml
o
cul u e
medium
bu e ed
wi h
50
mM
Mops/KOH,
pH
7.5,
and
wi hou
any
po assium
phospha e.
Cells
we e
incuba ed
a
30
'C
wi h
con inuous
shaking
(100
s okes
min-')
o
10
min
and
hen
supplemen ed
wi h
ei he
19
,uCi
o
[3H]adenine
o
2
,uCi
o
[32P]o ho-
phospho ic
acid.
A e
u he
incuba ion
o
2
h,
5
mM
NH4C1
was
added,
and
he
cells
we e
ha es ed
h
la e .
Sedimen a ion
was
imp o ed
by
adding
NaCl
and
EDTA
o
inal
concen a ions
o
100
mM
and
l0
mM
espec i ely.
The
cells
we e
esuspended
in
40
,u1
o
sample
bu e
[26]
o
elec opho esis
and
dis up ed
by
eezing
in
liquid
ai
ollowed
by
boiling
o
3
min
( epea ed
ou
imes
a
leas ).
The
homogena e
was
cen i uged
a
16500
g
o
P o ein
in
cell
ex ac s
was
es ima ed
by
he
me hod
o
Ma kwell
e
al.
[29]
using
BSA
as
s anda d.
SDS/PAGE
was
pe o med
acco ding
o
Laemmli
[26].
P o ein
ma ke s
we e
elec opho esed
in
pa allel.
The
p o eins
we e
s ained
wi h
Coomassie
B illian
Blue
R-250.
Fluo og ams
o
in
i o-labelled
p o eins
we e
ob ained
as
desc ibed
in
[30].
RESULTS
An ibodies
aised
agains
GS
om
Synechocys is
c oss- eac
wi h
A.
ch oococcum
GS
Addi ion
o
inc easing
amoun s
o an i-GS
an ibody
aised
agains
he
enzyme
om
he
cyanobac e ium
Synechocys is
o
an
A.
ch oococcum
cell- ee
ex ac
esul ed
in
p og essi e
dis-
appea ance
o
GS
ac i i y
(Figu e
1).
Though
no
shown,
i
could
be
asce ained
ha
he
an ise um
was
equally
e ec i e
in
he
immunop ecipi a ion
o
bo h
ac i e
and
inac i e
GS
o m.
As
illus a ed
in
Figu e
1,
no
e ec
o
he
an ibody
was
obse ed
on
ni a e
educ ase
ac i i y,
which
was
used
as
a
nega i e
con ol.
Fu he mo e,
he
analysis
o
he
immunop ecipi a ed
ma e ial
on
SDS/PAGE
e ealed
en ichmen
o
a
p o ein
o
app ox.
59
kDa
o
molecula
mass
(Figu e
2)
ha
was
no
p esen
in
he
an ise um
alone
(lane
7
in
Figu e
2).
To
ou
knowledge,
he
molecula
mass
o
A.
ch oococcum
GS
has
no
been
epo ed
p e iously.
The
A.
inelandii
GS,
howe e ,
has
been
desc ibed
as
a
dodecame
wi h
monome
o
molecula
mass
o
ei he
56.5
[21],
53
[20]
o
62
kDa,
in
he
la e
case
when
exp essed
in
Esche ichia
coli
[22].
On
he
basis
o
hese
da a
we
concluded
ha
he
p o ein
en iched
in
he
immunop ecipi a e
co esponded
o
he
GS
monome .
Taking
in o
conside a ion
ha
GS
could
be
de ec ed
on
SDS/PAGE,
we
nex
in es iga ed
whe he
o
no
his
enzyme
g
110
100io
0
90
Co(U
CD
80
70
2n
60
~
50
40
o
30
0
-10
20
30
40
50
An i-GS
(#l)
Figu e
1
Immuno i a ion
o
A.
ch oococcum
GS
Aliquo s
(300
,ul)
o
cell
ex ac
(3
mg
p o ein/ml)
om
8
mM
KNO3-g own
A.
ch oococcum
cells
we e
incuba ed
wi h
inc easing
olumes
o
he
an ibodies
(5
mg/ml)
aised
agains
pu e
GS
om
Synechococcus
sp.
s ain
PCC
6301.
The
ac i i ies
o
glu amine
syn he ase
(@)
and
ni a e
educ ase
(0)
emaining
in
supe na an s
we e
measu ed
as
desc ibed
in
he
Ma e ials
and
me hods
sec ion.
The
le el
o
100%
GS
ac i i y
co esponds
o
250
muni s/mg
o
p o ein;
100%
ni a e
educ ase
ac i i y
o
he
con ol
was
20
muni s/mg
o
p o ein.
Azo obac e
ch oococcum
glu amine
syn he ase
643
M
(kDa)
1
2
3
4
5
6
7
97.4
-
66.2
-
-
GS
40
"_
<
IgG
45
..
.
.!
-
*M
.-.-..
.-.:U
_.s.
31
-
Figu e
2
E ec
o
an l-GS
an ibody
concen a ions
on
immunop ecipl a ion
o
A.
ch oococcum
GS
Samples
desc ibed
in
Figu e
1
we e
cen i uged
and
he
co esponding
immunop ecipi a ed
p o eins
we e
elec opho esed
in
an
SDS/12%-polyac ylamide
gel.
A.
ch oococcum
GS
was
immunop ecipi a ed
wi h
0,
4,
8,
16,
30
and
40
,u
o
an i-GS
an ibodies
(lanes
1-6).
Lane
7
co esponds
o
he
an ise um
alone
(30
,ul).
Molecula
masses
(M)
o
p o ein
s anda ds
a e
indica ed.
kDa
97.4
66.2
-
45-
1
2 3
4
-
-
GS
Figu e
3
In
i o
2P
labelling
o
ammonium-inac i a ed
GS
om
A.
ch oococcum
32p
labelling
o
combined
ni ogen- ee-g own
cells
was
ca ied
ou
as
desc ibed
in
he
Ma e ials
and
me hods
sec ion.
The
cell
ex ac s
hus
ob ained
(50
jug
o
p o ein)
we e
elec opho esed
in
an
SDS/1
0%-polyac ylamide
gel
and
he
gel
subjec ed
o
au o adiog aphy.
Lanes:
1,
cell
ex ac
o
non-ammonium- ea ed
cells
(con aining
ac i e
GS);
2,
3
and
4,
cell
ex ac s
om
cells
ea ed
wi h
0.5
mM
NH4C0
o
30,
60
and
90
min
espec i ely
(con aining
inac i e
GS).
The
molecula
masses
(M)
o
p o ein
s anda ds
a e
indica ed.
was
subjec
o
co alen
modi ica ion
in
esponse
o
ammonium
in
A.
ch oococcum.
In
i o
labelling
o
A.
ch oococcum
GS
When
diazo ophically
g own
A.
ch oococcum
cells
we e
p e-
incuba ed
in
he
p esence
o
H332PO4
and
hen
subjec ed
o
an
ammonium
shock
(5
mM
NH4Cl),
he
59
kDa
p o ein
p e iously
iden i ied
as
GS
subuni
was
labelled,
his
labelling
inc easing
wi h
ime
o
incuba ion
in
he
p esence
o
ammonium
(Figu e
3).
This
esul
indica ed
ha
GS
was
modi ied
as
a
consequence
o
he
ammonium
shock
by
a
g oup
con aining
phospha e.
I
is
well
es ablished,
as
s a ed
abo e,
ha
GS
om
mos
G am-nega i e
bac e ia
is
egula ed
by
an
adenylyla ion-
deadenylyla ion
sys em
and
ha ,
in
his
case,
he
modi ied
,enzyme
is
adiolabelled
in
i o
in
he
p esence
o
32P.
To
check
whe he
his
egula o y
p ocess
is
ope a i e
in
A.
ch oococcum,
wo
app oaches
we e
made.
Fi s ,
when
cells
we e
pulse-labelled
wi h
[3H]adenine,
he
enzyme
was
adiolabelled
in
esponse
o
_0
Co
u)
E
0
Co
0
Time
(h)
Figu e
4
E ec
o
alkaline
phospha ase
and
phosphodies e ase
on
glu amine
syn he ase
Inac i a ed
by
ammonium
Diazo ophically
g own
A.
ch oococcum
cells
we e
ea ed
wi h
5
mM
NH4CI
o
1
h
o
inac i a e
GS
and
hen
used
o
ob ain he
co esponding
cell
ex ac .
The
ex ac s
we e
incuba ed
a
37
OC
wi h
30
uni s/mg
p o ein
alkaline
phospha ase
(O),
1
uni Vmg
o
p o ein
phosphodies e ase
(0)
o
wi hou
any
addi ion
(OJ),
and
he
Mg2+-dependen
in
i o
y-glu amyl ans e ase
ac i i y
(ac i e
o m)
was
de e mined
a
he
indica ed imes.
To
ollow
he
elease
o
32p
om
adiolabelled
GS
(inse ),
he
ex ac s
om
ammonium- ea ed
cells
we e
immunop ecipi a ed
a e
1
h
incuba ion
wi h
ei he
phosphodies e ase
(lane
3),
alkaline
phospha ase
(lane
4)
o
no
addi ion
(lane
2).
Lane
1
ep esen s
he
immunop ecipi a e
o
he
ex ac
om
non-ammonium-
ea ed
cells;
100%
o
GS
ac i i y
co esponds
o
300
muni s/mg
o
p o ein.
In
he
inse
M
is
molecula
mass.
ammonium
addi ion
in
a
simila
way
o
ha
desc ibed
in
Figu e
3
(see
below),
indica ing
ha he
modi ying
g oup
also
con ained
adenine.
Secondly,
ea men
o
an
enzyme
p epa a ion
om
ammonium-g own
cells,
and
hence
o
low
ac i i y,
wi h
snake-
enom
phosphodies e ase
p omo ed
he
e-ac i a ion
o
GS
ac i i y
(Figu e
4).
A
simila
ea men
wi h
alkaline
phospha ase
did
no
media e
any
e-ac i a ion
o
GS.
Analysis
by
SDS/PAGE
and
au o adiog aphy
o
he
enzyme
p epa a ion
a e
e-
ac i a ion
wi h
phosphodies e ase
and
hen
immunop ecipi a ed
demons a ed
emo al
o
he
modi ying
g oup,
as
shown
in
Figu e
4
(inse ).
Taken
oge he
hese
esul s
s ongly
suppo
ha ,
in
A.
ch oococcum,
GS
is
egula ed
by
an
ammonium-
induced
adenylyla ion-deadenylyla ion.
P e en ion
by
MSX
o
GS
modMca ion
MSX
is
an
ex ensi ely
used
inhibi o
o
GS
ac i i y.
In
A.
ch oococcum
cells
ha
had
been
p eincuba ed
wi h
MSX,
am-
monium
was
wi hou
e ec
on
ei he
ni ogenase
ac i i y
[2]
o
ni a e
up ake
[4],
he
conclusion
being
d awn
ha
ammonium
mus
be
me abolized
o
exe
i s
inhibi o y
e ec
on
hese
p ocesses.
Figu e
5
illus a es
an
expe imen
on
[3H]adenine-
labelling
o
GS
wi h
A.
ch oococcum
cells
subjec
o
an
am-
monium
shock
in
he
absence
(lane
2)
and
in
he
p esence
o
MSX
(lane
3).
In
he
con ol
ea men
(lane
1),
he
cells
we e
incuba ed
in
he
p esence
o
adiolabelled
adenine
alone.
As
can
be
seen,
cells
ea ed
wi h
ammonium
showed
e y
high
adio-
ac i e
labelling,
as
s a ed
abo e,
and
MSX
p e en ed
GS
om
being
co alen ly
modi ied.
Though
no
shown
i
was
ound
ha
MSX
emo ed
he
[3H]adenine
labelling
om
GS
when
added
once
NH4+
inhibi ion
o
GS
had
aken
place.
These
esul s
he e o e
sugges
ha
o
GS
adenylyla ion
o
ake
place
644
M.
C.
Munoz-Cen eno,
F.
J.
Cejudo
and
A.
Paneque
M
(kDa)
97.4
66.2
45
1
2 3
GS
Figu e
5
P e en ion
by
MSX
o
in
i o
[2H]adenine-labelling
o
GS
du ing
ammonium
Inac i a ion
[3H]Adenine
labelling
o
combined
ni ogen- ee-g own
cells
was
ca ied
ou
as
desc ibed
in
he
Ma e ials
and
me hods
sec ion.
The
cell
ex ac s
hus
ob ained
(50
,g
o
p o ein)
we e
elec opho esed
in
an
SDS/1
0%-polyac ylamide
gel
and
he
luo og am
was
ca ied
ou .
Lanes:
1,
ex ac
om
non-ammonium- ea ed
cells
(con aining
ac i e
GS);
lane
2,
cell
ex ac
om
bac e ia
ea ed
wi h
5
mM
NH4CI
o
60
min
(con aining
inac i e
GS);
lane
3,
cell
ex ac
om
bac e ia
ea ed
wi h
5
mM
MSX
30
min
p io
o
he
5
mM
NH4CI
addi ion.
The
molecula
masses
(M)
o
p o ein
s anda ds
a e
indica ed.
M
1
2
3
(kDa)_
1066-
80-
~-GS
49.5
-
32.5
27.5
18.5
Figu e
6
Wes e n-blo
analysis
o
A.
ch oococcum
cell
ex ac s
using
an l-GS
an ibodies
Cell
ex ac s
we e
ob ained
om
24
h-old
cells
g own
in
combined
ni ogen- ee
medium
(lane
1)
and
in
media
con aining
ei he
8
mM
KNO3
(lane
2)
o
10
mM
NH4CI
(lane
3).
Aliquo s
(60
9g
o
p o ein)
o
cell
ex ac s
we e
elec opho esed
in
an
SDS/12%-polyac ylamide
gel,
and
he
Wes e n
blo
was
ca ied
ou
as
desc ibed
in
he
Ma e ials
and
me hods
sec ion.
ammonium
has
o
be
assimila ed,
which
is
in
ag eemen
wi h
he
p oposal
ha
in
En e obac e ia
GS
adenylyla ion-
deadenylyla ion
depends
ul ima ely
on
he
glu amine/a-
oxoglu a a e
a io.
E ec
o
he
ni ogen
sou ce
on
GS
p o ein
con en
We
in es iga ed
nex
whe he ,
in
A.
ch oococcum,
he
le el
o
GS
p o ein
syn hesis
was
egula ed
by
ammonium
also.
To
check
his
possibili y
cells
ha
had
been
g own
unde
N2- ixing
condi ions,
which
i
is
known
o
exhibi
a
GS
biosyn he ic
Mg2+/Mn2+
ac i i y
a io
highe
han
in
combined
ni ogen-
con aining
medium,
indica i e
o
a
low
deg ee
o
adenylyla ion,
we e
ans e ed
o
media
wi h
ni a e
o
ammonium
as
he
ni ogen
sou ce,
o
again
o
N2- ixing
condi ions.
Cells
we e
hen
allowed
o
mul iply
o
24
h,
app ox.
10- old
he
gene a ion
ime,
and
GS
p o ein
measu ed
by
Wes e n
blo ing.
Figu e
6
depic s
ha
he
amoun
o
GS
p o ein
in
he
co esponding
cell
ex ac s
was
p ac ically
he
same
independen ly
o
he
a ailable
ni ogen
sou ce.
The e o e,
in
A.
ch oococcum,
egula ion
o
GS
ac i i y
o e ides
p o ein
syn hesis
con ol.
DISCUSSION
A
he
s uc u al
le el,
GS
om
mos
bac e ia
is
a
dodecame
wi h
iden ical
subuni s.
Elec on-mic oscopic
s udies
and
compu e ized
image
p ocessing
ha e
shown
ha
GS
is
o med
om
wo
supe imposed
hexagons,
each
composed
o
six
subuni s
a anged
adially
wi h
espec
o
he
cen al
hole
[31,32].
I
is
no
su p ising,
hen,
ha
an igenic
c oss- eac i i y
is
de ec ed
be-
ween
A.
ch oococcum
GS
and
he
an ise um
di ec ed
agains
he
enzyme
om
Synechocys is.
I
is
o
be
no ed,
howe e ,
ha
he
an ise um
c oss- eac s
less
e icien ly
wi h
A.
ch oococcum
GS
han
wi h
Calo h ix,
ano he
cyanobac e ium,
enzyme
[33].
T onick
e
al.
[34]
showed
ha
E.
coli
GS
an ise um
showed
an igenic
homology
wi h
he
GSs
o
a
numbe
o
G am-nega i e
bac e ia,
bu
no
an igenic
c oss- eac i i y
was
de ec ed
wi h
he
GSs
o
G am-posi i e
bac e ia
o
euka yo ic
o ganisms.
The
only
excep ion
o
his
was
he
c oss- eac ion
obse ed
be ween
E.
coli
GS
an ise um
and
he
G am-posi i e
S ep omyces
GS.
La e
on,
O
e
al.
[35]
held
a
di e en
iew
on
he
basis
o
he
amino
acid
composi ion,
N- e minal
sequences
and
p edic ed
con o ma ion
po en ials
o
he
GSs
om
he
ilamen ous
cyanobac e ium
Anabaena
sp.
s ain
7120,
he
G am-nega i e
bac e ium
E.
coli,
and
he
g am-posi i e
bac e ium
Bacillus
sub ilis.
Since
hey
obse ed
limi ed
sequence
simila i y
among
all
h ee
enzymes,
hey
p oposed
ha
an igenic
c oss- eac i i y
does
no
necessa ily
depend
upon
he
G am
eac ion
o
s a e
o
adenylyla ion.
The
esul s
p esen ed
he e,
showing
c oss-
eac i i y
o
A.
ch oococcum
GS
wi h
an ibody
aised
agains
he
cyanobac e ium
GS,
s ongly
suppo
his
hypo hesis,
because
he
adenylyla ion-deadenylyla ion
egula o y
sys em
is
lacking
in
Synechocys is
[19].
Ammonium
assimila ion
in
he
azo obac e s
is
hough
o
ake
place
exclusi ely
h ough
he
GS-GOGAT
ou e
[5,6],
and
inhibi ion
o
ei he
GS
o
GOGAT
has
been
widely
used
in
in es iga ions
in o
he
ammonium
egula ion
o
ni ogenase
ac i i y
o
ni a e
up ake,
as
s a ed
abo e.
On
he
basis
o
s udies
on
he
e ec
o
snake- enom
phosphodies e ase
ea men
on
he
enzyme
isola ed
om
cells
g own
on
a
ni ogen- ich
medium,
and
he
in
i o
inco po a ion
o
adioac i i y
om
[14C]ATP
in o
deadenylyla ed
enzyme
in
he
p esence
o
ei he
c ude
ex ac
om
A.
inelandii
o
adenylyl ans e ase
om
E.
coli,
i
was
concluded
ha
A.
inelandii
GS
is
egula ed
by
adenylyla ion-deadenylyla ion
[21].
Expe imen s
on
in
i o
in-
co po a ion
o
adioac i i y
om
ei he
[32P]o hophospha e
o
[3H]adenine,
bo h
componen s
o
he
modi ying
g oup,
and
he
use
o
an
an ibody
aised
agains
GS
om
a
cyanobac e ium,
allow
us
now
o
show
he
co alen
modi ica ion
o
Azo obac e
GS,
ol-
lowing
an
ammonium
shock,
by
he
combined
me hod
o
adiolabelling
and
immunop ecipi a ion.
Though
ou
esul s
canno
ule
ou
he
possibili y
ha
GS
migh
be
u he
e-
ac i a ed
by
an
enzyme
ha
emo es
ADP- ibose,
e-ac i a ion
o
modi ied
A.
ch oococcum
GS
wi h
snake- enom
phospho-
dies e ase
s ongly
sugges
ha
GS
adenylyla ion
is
o
p ime
impo ance
in
he
co alen
modi ica ion
o
GS.
As
men ioned
abo e,
an
ADP- ibosyl ans e ase
ac i i y
capable
o
modi ying
Rhodospi illum
ub um
GS
in
i o
has
been
desc ibed
ecen ly
[13].
The
in e nal
C/N
balance
plays
an
essen ial
ole
in
he
con ol
o
he
ino ganic-ni ogen
me abolism
o
mos
bac e ia.
In
his
espec ,
he
cen al
ole
pe o med
by
GS
is
unques ionable.
Azo obac e
ch oococcum
glu amine
syn he ase
645
Ac ually,
he
adenylyla ion
o
GS
is
i sel
subjec
o
such
C/N
balance,
as
shown
by
he
ac
ha
inhibi ion
o
GS
by
MSX
p e en ed
he
enzyme
om
adenylyla ion
a e
ammonium
shock.
Resul s
p esen ed
he e
sugges
ha ,
in
A.
ch oococcum,
he
cen al
ole
o
GS
in
he
con ol
o
ni ogen
me abolism
elies
only
on
modula ion
o
GS
ac i i y.
Thus
Wes e n-blo
analysis
o
c ude
ex ac s
om
cells
g own
on
di e en
ni ogen
sou ces
did
no
e eal
any
signi ican
in luence
o
he
ni ogen
sou ce
on
he
amoun
o
GS
p o ein.
In
his
connec ion
i
has
been
epo ed,
as
men ioned
abo e,
ha
he
le el
o
mRNA
o
glnA,
he
gene
coding
o
GS,
is
no
a ec ed
by
he
ni ogen
sou ce
in
A.
inelandii
ei he
[22].
By
con as ,
he
GS
p o ein
le els
in
ammonium-g own
cyanobac e ia
a e
app ox.
500%
o
hose
ound
in
cells
g own
on
ni a e
[33]
o
unde
N2- ixing
condi ions
[36].
This
wo k
was
suppo ed
by
Comisi6n
In e minis e ial
de
Ciencia
y
Tecnologia,
G an
B1091/0914
and
Plan
Andaluz
de
In es igaci6n
G oup
3156.
We
hank
J.
C.
Reyes
his
ad ice
ega ding
expe imen s
wi h
an i-GS
an ibodies.
The
sec e a ial
assis ance
o
Pepa
Pe ez
de
Le6n
is
deeply
app ecia ed.
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