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Original article
Azospirillum brasilense PII proteins GlnB and GlnZ do notform heterotrimers and GlnB shows a unique trimericuridylylation pattern
Juliana Inaba, Luciano F. Huergo, Ana C. Bonatto, Leda S. Chubatsu, Rose A. Monteiro,M. Berenice Steffens, Giseli Klassen, Liu U. Rigo, Fabio O. Pedrosa, Emanuel M. Souza*
Department of Biochemistry and Molecular Biology, Universidade Federal do Parana, Francisco H. dos Santos s/n,
CP 19046, Curitiba, PR, Brazil
a r t i c l e i n f o
Article history:
Received 17 March 2008
Received in revised form
1 August 2008
Accepted 22 August 2008
Published online 1 October 2008
Keywords:
Azospirillum brasilense
GlnB
GlnZ
PII-like protein,
Nitrogen fixation
* Corresponding author. Tel.: þ55 41 3361 16E-mail address: souzaem@ufpr.br (E.M. S
1164-5563/$ – see front matter ª 2008 Elsevidoi:10.1016/j.ejsobi.2008.08.006
a b s t r a c t
In many organisms, nitrogen metabolism is co-ordinated by a class of highly conserved
proteins from the PII family. In Gram-negative bacteria PII proteins are trimers that can be
covalently modified by uridylylation according to the cellular nitrogen status. Several
prokaryotes have more than one gene that code for PII proteins. In Escherichia coli it was
shown that the two PII proteins (GlnB and GlnK) can form heterotrimers and it was sug-
gested that heterotrimerization of PII proteins could be widespread in Bacteria. The
nitrogen-fixing plant-associative bacteria Azospirillum brasilense code for two PII proteins,
GlnB and GlnZ. The expression of glnB and glnZ genes are induced under nitrogen fixing
conditions and these proteins control both the expression and the activity of the nitroge-
nase enzyme. Here we show that unlike E. coli PII proteins, A. brasilense GlnB and GlnZ, do
not form heterotrimers in vitro. Our data also suggest that A. brasilense GlnB shows a unique
uridylylation pattern.
ª 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction This organism can only fix nitrogen under microaerobic
The associative nitrogen-fixing bacteria Azospirillum brasilense
has attracted considerable attention due to its capacity of
colonize the roots and enhance the growth and yield of several
economically important crops [8,27]. Inoculants of Azospir-
illum spp. have been used in many part of the world for crops
such as rice and maize. The understanding of its nitrogen
metabolism and the fine tuning of its regulatory network is
essential for genetically engineering strains with improved
agronomic efficiency.
67; fax: þ55 41 3266 2042.ouza).er Masson SAS. All rights
conditions and when fixed nitrogen is limiting. Nitrogenase
expression and activity in A. brasilense are controlled by the PII
proteins GlnB and GlnZ [12,13,18,24,25]. In response to
nitrogen levels, these proteins are reversibly modified by the
bifunctional enzyme GlnD. In vitro assays showed that GlnB
and GlnZ are uridylylated by GlnD in the presence of ATP and
2-oxoglutarate. The presence of glutamine inhibits uridylyla-
tion and stimulates deuridylylation of both GlnB and GlnZ [3].
The GlnB protein controls the activity of the transcriptional
activator NtrC through its partner NtrB [13] and is required for
reserved.
e u r o p e a n j o u r n a l o f s o i l b i o l o g y 4 5 ( 2 0 0 9 ) 9 4 – 9 9 95
the activity of NifA, the transcriptional activator of the nif
genes [2]. The GlnZ protein cannot substitute GlnB in these
functions [13]. GlnB and GlnZ also play specific roles in the
control of the nitrogenase activity through the reversible ADP-
ribosylation of dinitrogenase reductase (NifH) [19,24,25].
Under nitrogen fixing conditions GlnZ-UMP3 interacts with
the enzyme DraG (dinitrogenase reductase ADP-ribosyl-
glycohydrolase), which activates nitrogenase by removing the
ADP-ribosyl group from NifH [19]. After an ammonium shock,
the deuridylylated form of GlnB interacts with the enzyme
DraT (dinitrogenase reductase ADP-ribosyl-transferase),
which inactivates nitrogenase by ADP-ribosylation of NifH.
Under these conditions, GlnZ is deuridylylated and the GlnZ–
DraG complex associates with the membrane protein AmtB,
inhibiting nitrogenase activation by DraG [17,18].
The PII family comprises highly conserved signal trans-
duction proteins. These proteins are found in Archaea, Bacteria
and Eukarya and control the activity of transcriptional activa-
tors, key metabolic enzymes and membrane transporters
through direct protein–protein interactions [16,26]. In Proteo-
bacteria, PII proteins undergo reversible uridylylation by the bi-
functional enzyme GlnD [16]. The structural models of some PII
proteins have been determined. The PII proteins from Escher-
ichia coli (GlnB and GlnK) are homotrimers that form a compact
barrel with a central and three lateral clefts [10,30]. Each
subunit extends a flexible region known as the T-loop, which
contains the uridylylation site, a highly conserved tyrosine
residue (Tyr51). Uridylylation of E. coli PII proteins requires ATP
and 2-oxoglutarate and these proteins have a binding site for
each effector [6,22]. The ATP binding sites are located in the
lateral clefts between the PII subunits [30,31]. The 2-oxogluta-
rate binding site of GlnB proteins from E. coli and Herbaspirillum
seropedicae seems to be also in the lateral cleft [9,21]. Recent
data have suggested that PII proteins can also bind ADP and
AMP [20,23,29,32]. The ability of PII proteins to interact and thus
regulate their targets depends on the PII uridylylation status
and the effectors bound.
E. coli PII proteins (GlnB and GlnK) can form heterotrimers
both in vivo and in vitro [15,28]. The formation of heterotrimers
between these proteins seems to promote fine regulation of PII
target activities such as glutamine synthetase [28]. It was
suggested that heterotrimerization could be widespread in
Bacteria. As both PII proteins coded by A. brasilense (GlnB and
GlnZ) are induced under nitrogen limiting conditions [12,17],
we decided to verify if PII heterotrimerization could also occur
in this organism. Here we show that A. brasilense PII proteins,
GlnB and GlnZ, do not form heterotrimers in vitro. The data
also suggest that A. brasilense GlnB trimer is only stable when
completely unmodified or when fully uridylylated.
2. Material and methods
2.1. Expression and purification of A. brasilense GlnB,GlnZ and His-GlnD proteins
The A. brasilense GlnB, GlnZ and His-GlnD (N-terminal 6xHis
tagged version of the GlnD enzyme) proteins were expressed
in E. coli BL 21 from plasmids bearing the T7 promoter and
purified as described previously [3].
2.2. In vitro uridylylation of GlnB and GlnZ andpurification of fully uridylylated forms
The uridylylation reactions were performed as described
previously [3]. The products of the reactions were analyzed by
native gel electrophoresis as described [3] and MALDI-TOF
spectrometry (see below). Samples were collected at 0, 5, 10, 20,
30 and 60 min and the reaction stopped by addition of 1 mM
EDTA and 5 ml of native electrophoresis sample buffer. PII-UMP
was purified after uridylylation by removing GlnD-His with
HisMagnetics beads (Promega Co., Madison, WI) and the system
was dialyzed against 50 mM Tris–HCl pH 7.5, 0.1 M KCl, 20%
glycerol overnight at 4 �C to remove ATP and 2-oxoglutarate.
2.3. Heterotrimer formation of PII
Purified GlnB, GlnB-UMP3, GlnZ and GlnZ-UMP3 proteins were
mixed (final concentration of 0.5 mg/ml) at the indicated ratios
in a buffer containing 100 mM Tris–HCl pH 7.5, 100 mM KCl
and 25 mM MgCl2. When indicated, samples were boiled for
5 min and kept on ice for 5 min. The samples were analyzed by
native gel electrophoresis [3].
2.4. Native polyacrylamide gel electrophoresis (PAGE)
Samples were mixed with 5 ml of loading buffer (62.5 mM Tris–
HCl pH 6.8, 10% glycerol, 0.01% bromophenol blue) and sepa-
rated in a non-denaturing polyacrylamide gel electrophoresis
(10%) as described [14].
2.5. MALDI-TOF analysis
The protein bands separated by native gel electrophoresis
were excised, distained with a solution containing 50% of
acetonitrile (ACN) and ammoniumbicarbonate 25 mM. Samples
were dehydrated in acetonitrile 100%, lyophilized in a Speed-
Vac, and digested with porcine trypsin (10 mg/ml) (Promega Co.,
Madison, WI) overnight at 37 �C. The peptides were extracted
using 30 ml of ACN 50% and trifluoroacetic acid (TFA) 5% for
10 min in an ultrasonic water bath. The supernatant was
collected, lyophilized in a SpeedVac and mixed with 2 ml TFA
0.1% and 1 ml of matrix (a-cyano 4-hydroxycinnamic acid). The
samples were spotted on a MALDI plate (Bruker Daltonics) and
the mass spectra obtained in a MALDI-TOF–MS Autoflex spec-
trometer (Bruker Daltonics, Bremen, Germany) using a positive
reflector mode, accelerating voltage of 20 kV, delay time of
150 ns and acquisition mass-range 800–3200 Da. The spectra
were analyzed by the FlexAnalysis 2.0 software (Bruker Dal-
tonics, Bremen, Germany) and Mascot Program.
3. Results and discussion
3.1. In vitro GlnB uridylylation
The PII proteins are found in solution as trimers. The uridy-
lylation of A. brasilense GlnZ by GlnD (Utase/UR) generates
trimers containing one, two or three subunits linked to UMP
that can be resolved in a non-denaturing gel electrophoresis:
as uridylylation increases the protein mobility due to the
e u r o p e a n j o u r n a l o f s o i l b i o l o g y 4 5 ( 2 0 0 9 ) 9 4 – 9 996
addition of negatively charged groups [3,4]. We observed that,
differently from the A. brasilense GlnZ, only two forms of GlnB
were resolved under native page after an in vitro uridylylation
assay (Fig. 1I). Unmodified GlnB (time zero) produces a single
band and five minutes after starting the reaction, about 30% of
GlnB was modified and migrated faster then unmodified
protein. Ninety percent of uridylylated form was present at
60 min. No intermediate bands corresponding to GlnB-UMP1
and GlnB-UMP2 were observed (Fig. 1I). A similar result was
reported by Araujo et al. (2008) [3]. The bands corresponding to
unmodified GlnB trimer at time 0 and uridylylated GlnB trimer
at 5, 10 and 60 min were then excised from the gel (Fig. 1I),
digested with trypsin and analyzed by Maldi-Tof spectrometry
0 5 10A
B
Reaction
GlnB
GlnB-UMP
m/z
GlnB 48-56
I
II
C
Fig. 1 – (I) Uridylylation of A. brasilense (Ab) GlnB protein. The re
(2 mM), UTP (1 mM), purified GlnB (10 mM) and Ab GlnD (200 nM
analyzed by MALDI-TOF spectrometry. (II) The A–D spectra are
uridylylated and uridylylated forms of the GlnB48–56 peptide con
indicated by arrows. Peak m/z 1287.8950 corresponds to residue
residues 59–72; and peaks 1237.9770 and 1543.8520 to residues
to investigate the presence of hybrid trimers not resolved in
the native gel electrophoresis. A peak with m/z ratio of
1237.977 obtained after digestion of the band corresponding to
the unmodified GlnB trimer at 0 min matched with the frag-
ment containing the residues 48–58 of unmodified GlnB (m/z
1237.6463) that contains the uridylylation site. This peak was
substituted at times 5, 10 and 60 min by a peak with m/z ratio
of 1543.852 that corresponded to the same fragment attached
to a UMP residue (mass difference of 306 Da) (Fig. 1II). These
data confirm previous results [3,19], which showed that GlnB
uridylylation and deuridylylation, in vivo and in vitro, respec-
tively, seem to occur without the formation of hybrid forms
(GlnB-UMP1 and GlnB-UMP2).
20 30 60
D
time (minute)
A
B
C
D
GlnB 48-56
-UMP
GlnB 48-56
-UMP
GlnB 48-56
-UMP
action system contained ATP (100 mM), 2-oxoglutarate
). The bands A–D were excised digested by trypsin and
from excised bands A, B, C and D of panel I. The non-
taining residues 48–56 of GlnB (m/z difference of 306) are
s 91–101 of GlnB; 1365.9790 to residues 73–85; 1646.0780 to
48–56 non-uridylylated and uridylylated, respectively.
e u r o p e a n j o u r n a l o f s o i l b i o l o g y 4 5 ( 2 0 0 9 ) 9 4 – 9 9 97
The uridylylation of each subunit of the trimeric E. coli GlnB
is independent of each other as partially uridylylated trimers
are observed in native PAGE [5]. During the A. brasilense GlnZ
time course uridylylation reaction there is also the formation
of the intermediates, GlnZ-UMP1 and GlnZ-UMP2, which could
be resolved by native gel electrophoresis [3,4]. The results
obtained with the A. brasilense GlnB are different and may be
explained by two possibilities: (1) the A. brasilense GlnB uri-
dylylation is a cooperative process and therefore, once the
first UMP is added to the trimer the addition of the second and
third UMP groups are so fast that we cannot detect the inter-
mediates (GlnB-UMP1 and GlnB-UMP2) under our assay
condition; and (2) the partially uridylylated trimers are not
stable and thus are not readily observed in vitro.
3.2. In vitro heterotrimer formation of A. brasilense PII
proteins
To investigate the formation of GlnB/GlnZ heterotrimers in A.
brasilense the purified GlnB, GlnZ, GlnB-UMP3 and GlnZ-UMP3
were subjected to a denaturation/renaturation reaction as
described in Section 2 and loaded on a native gel electropho-
resis. Studies involving denaturation/renaturation of GlnB
and GlnK proteins were previously reported in E. coli using
urea as denaturing agent [22,28]. The treatment was efficient
to promote hybrid formation, but the renatured protein had
decreased activity [21].
To allow hybrid trimer formation without changing PII
activity we tested a temperature induced denaturation/rena-
turation treatment as described in Section 2. The denatur-
ation/renaturation treatment did not remove the UMP moiety
from GlnB or GlnZ, nor altered the uridylylation profile of both
PII proteins (Fig. 2I and results not shown).
I
Z Z* Z/Z* Z Z* Z/Z*
Room temperature Boiled 5 minutes
II
Boiled 5 minutes
1 2 3 4 5 6
B B* Z Z* B/ZB/B*(1:2)
Fig. 2 – The purified proteins (final concentration 0.5 mg/ml) were
by native gel electrophoresis (10%). When indicated, the sample
electrophoresis. The symbols indicate: B: A. brasilense (Ab) GlnB
At room temperature GlnZ and GlnZ-UMP3 are found as
homogeneous bands as observed by native PAGE (Fig. 2I). When
both proteins (GlnZ and GlnZ-UMP3) were mixed at 1:1 ratio and
kept at room temperature the mono- and di-uridylylated hybrid
trimers were not observed: as expected only the two bands cor-
responding to the homotrimers fully uridylylated and fully
unmodified were detected (Fig. 2I). When GlnZ and GlnZ-UMP3
were mixed (at 1:1 ratio), boiled for 5 min and allowed to renature
on ice for 5 min, new oligomers corresponding to the mono- and
di-uridylylated forms were found in native gel electrophoresis.
These results were expected since the thermic treatment
disrupts the quaternary and tertiary structures of both GlnZ and
GlnZ-UMP3; once the mix is cooled the proteins would renature
randomly leading to the formation of trimers containing 0, 1, 2
and3uridylylated monomers.Theaffinityof interactionbetween
a non-uridylylated GlnZ monomer with a GlnZ-UMP monomer or
with other non-uridylylated GlnZ monomers seems to be similar,
since the most abundant forms of GlnZ observed after thermic
treatment of GlnZ:GlnZ-UMP3 mixture (1:1 ratio) are the mono-
and di-uridylylated forms (Fig. 2II).
In contrast, when purified GlnB and GlnB-UMP3 were mixed,
boiled and allowed to renature, mono- and di-uridylylated
forms were not observed; only two bands corresponding to free
or fully uridylylated GlnB were observed (lanes 3 and 9, Fig. 2II).
When non-uridylylated GlnB and GlnZ (lane 6, Fig. 2II), non-
uridylylated GlnB and GlnZ-UMP-3 (lane 7, Fig. 2II) or non-
uridylylated GlnZ and GlnB-UMP3 (lane 8, Fig. 2II) were mixed
we did not observed formation of any new band, suggesting that
heterotrimerization between these group of proteins did not
occur under our assay condition. Since it is very unlikely that
the thermic treatment did not promote the complete denatur-
ation of these proteins, these results indicate that the A. brasi-
lense GlnB and GlnZ proteins do not form stable heterotrimers in
GlnZ
GlnZ-UMP3
GlnZ-UMP1
GlnZ-UMP2
7 8 9
B/Z* Z/B*
GlnB
GlnB-UMP3
GlnZ
GlnZ-UMP3
B/B*
mixed in a 1:1 (or 1:2, when indicated) ratio and separated
s were boiled 5 min and kept on ice before native gel
; B*: Ab GlnB-UMP3; Z: Ab GlnZ; Z*: Ab GlnZ-UMP3.
e u r o p e a n j o u r n a l o f s o i l b i o l o g y 4 5 ( 2 0 0 9 ) 9 4 – 9 998
vitro, confirming in vivo data [19]. Furthermore our data suggest
that non-uridylylated monomer of GlnB does not form stable
complexes with uridylylated GlnB monomers, thus not allowing
partially uridylylated trimer formation. This could explain why
the mono- and di-uridylylated forms are not observed upon
uridylylation of GlnB in vivo [19] and in vitro ([3] and Fig. 1).
Previous studies in E. coli involving a GlnB D47–53 mutant have
shown that GlnB is found as homotrimer mono-, di- and tri-
uridylylated and the presence of a single T-loop subunit is suffi-
cient to promote PII interaction with their receptors. Hybrid
trimers were able to regulate NtrB activity, promoting NtrC
dephosphorylation, and GS activity by the adenylyltransferase
(ATase) [21]. Van Heeswijk et al. (2000) [28] showed that GlnB
forms heterotrimer with GlnK both in vivo and in vitro, and sug-
gested that heterotrimers GlnK/GlnB are involved in fine tuning
of the regulation of glutamine synthetase by GlnB [28]. The het-
erotrimers had intermediate activity compared with the
homotrimers of GlnB or GlnK. In E. coli both GlnB and GlnK
seem to have several overlapping functions including control of
NtrB [6], ATase [28], AmtB [11]. It was suggested that the distinct
functions of GlnB and GlnK were correlated with the timing of
expression and levels of accumulation of the two proteins [7].
On the other hand, despite the high degree of sequence simi-
larity (81%), the A. brasilense PII proteins GlnB and GlnZ seems to
have evolved to perform very specific functions that usually do
not overlap. Most of the A. brasilense PII targets reported so far
including NifA, NtrB, DraT and DraG are specifically regulated
by only one of the proteins (GlnB or GlnZ) [1,13,18]. The only
target shared by these two proteins seems to be the ammonia
channel AmtB [20], but the effect of either protein on AmtB
activity is not yet known. Thus, the functional segregation
between A. brasilense GlnB and GlnZ seems to exclude a regu-
latory role of GlnB/GlnZ heterotrimer formation.
In conclusion, here we have reported evidence that the A.
brasilense PII proteins GlnB and GlnZ do not form heterotrimers
in vitro and that GlnB shows a unique pattern of trimer uri-
dylylation. Since PII proteins have a central role in the control
of nitrogen metabolism in Bacteria our results may be relevant
for the goal of obtaining new genetically improved strains for
use as inoculant for crop plants. Further structural analysis
will provide the molecular basis of the differences observed
between GlnB and GlnZ trimer formation.
Acknowledgments
We are grateful to Daniela F. Seixas for helping with the
MALDI-TOF analysis, Valter de Baura, Roseli Prado and Julieta
Pie are thanked for their technical support. This work was
supported by CNPq (Instituto do Milenio) and Fundacao
Araucaria.
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