Interaction of the general transcription factor TnrA with
the PII-like protein GlnK and glutamine synthetase in
Bacillus subtilis
Airat Kayumov
1,2
, Annette Heinrich
3
, Kseniya Fedorova
2
, Olga Ilinskaya
2
and Karl Forchhammer
3
1 Kazan State University of Architecture and Engineering, Russia
2 Kazan Federal University, Department of Microbiology, Russia
3 Interfaculty Institute of Microbiology and Infection Medicine, Eberhard-Karls-Universita
¨
tTu
¨
bingen, Germany
Keywords
Bacillus subtilis; GlnK; glutamine synthetase;
nitrogen regulation; PII protein; transcription
factor TnrA
Correspondence
K. Forchhammer, Interfaculty Institute of
Microbiology and Infection Medicine,
Eberhard-Karls-Universita
¨
tTu
¨

ferentially to adenylate nucleotide levels, with ATP weakening interactions
with both partners.
Structured digital abstract
l
tnrA binds to glnK by surface plasmon resonance (View interaction)
l
GS binds to tnrA by pull down (View interaction)
l
tnrA binds to glnK by pull down (View interaction)
l
tnrA binds to GS by pull down (View interaction)
l
GS physically interacts with tnrA by anti bait coimmunoprecipitation (View interaction)
l
glnK binds to tnrA by pull down (View interaction)
l
glnK physically interacts with tnrA by anti bait coimmunoprecipitation (View interaction)
l
tnrA physically interacts with GS by anti bait coimmunoprecipitation (View interaction)
l
tnrA physically interacts with glnK by anti bait coimmunoprecipitation (View interaction)
l
tnrA binds to tnrA by cross-linking study (View interaction)
l
tnrA binds to GS by surface plasmon resonance (View interaction)
Abbreviations
FC, flow cell; GlnK-ST, Strep-tag II-tagged variant of GlnK; GS, glutamine synthetase; GS-ST, Strep-tag II-tagged variant of glutamine
synthetase; ITC, isothermal titration calorimetry; NAGK, N-acetyl-
L-glutamate kinase; SPR, surface plasmon resonance.
FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS 1779

was recently found. When B. subtilis cells were grown
with a poor nitrogen source such as nitrate, TnrA was
found to be almost completely associated with the cell
membrane via the ammonium uptake proteins AmtB
and GlnK, originally termed NrgA and NrgB, respec-
tively [11,12]. AmtB is a homotrimeric transmembrane
ammonium transporter that is active under nitrogen-
limited conditions [13]. GlnK consists of three 12-kDa
monomers, and is a small regulatory protein that
belongs to the PII protein family. GlnK homologs
bind to AmtB and regulate its activity, depending on
the cellular nitrogen status [14]. Like other GlnK pro-
teins, B. subtilis GlnK was shown to bind to the mem-
brane in an AmtB-dependent manner [11,12].
Furthermore, B. subtilis GlnK exhibits the unique fea-
ture of lacking a response to 2-oxoglutarate, but seem-
ing to primarily respond to ATP. Depending on the
ATP levels, B. subtilis GlnK was shown in vitro to be
soluble or membrane-bound: 4 mm ATP caused almost
full solubilization of GlnK [12]. In wild-type B. subtilis,
TnrA was shown to bind specifically to the membrane-
bound AmtB–GlnK complex, but not to soluble, ATP-
saturated GlnK. TnrA-dependent expression of the
nrgAB (amtBglnK) promoter was shown to be reduced
in a GlnK-deficient strain under conditions of ammo-
nium-limited growth [11], indicating that GlnK could
be involved in fine-tuning TnrA-dependent gene
expression. Furthermore, the cellular levels of TnrA
are modulated by proteolysis [15]. After shifting of
nitrate-grown cells to a medium containing no usable

detergent-containing buffer and elution of antibody-
bound protein, the samples were separated by
SDS ⁄ PAGE and analyzed by immunoblotting.
In agreement with earlier data [12], GlnK was copre-
cipitated with TnrA from crude extracts of nitrate-
grown wild-type cells, when antibodies against TnrA
were used for immunoprecipitation (Fig. 1A). When
the cells were shifted to nitrate-deprived medium prior
to extraction of the proteins, much less TnrA was
immunoprecipitated, and, in consequence, less GlnK
Interaction of TnrA with GlnK and GS A. Kayumov et al.
1780 FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS
was detected, as TnrA is degraded by proteolysis fol-
lowing the shift to nitrate-deprived medium [15]. By
contrast, in the AmtB-deficient mutant strain, the same
amount of TnrA was immunoprecipitated and the
same amount of GlnK was copurified with TnrA in
both nitrogen regimes (Fig. 1A, lanes I and II). It
should be noted that GlnK is present only as soluble
protein in the AmtB-deficient mutant, whereas, in
wild-type cells, it is predominantly bound to the trans-
membrane AmtB channel, and only AmtB-bound
GlnK was able to interact with TnrA [11,12,15]. In the
TnrA immunoprecipitate of GlnK-deficient cells, again
no effect was observed on the recovery of TnrA fol-
lowing nitrate deprivation, in agreement with the lack
of TnrA degradation in this strain. GS was copurified
with TnrA and the recovery of GS was independent of
whether the cells were nitrate-grown or shifted to
nitrate-deprived medium. By contrast, no GS was co-

interactions.
Figure 2A shows a response difference sensorgram
(FC2 – FC1) of interactions of GlnK with immobilized
TnrA. For this analysis, an analyte concentration of
40 nm GlnK (trimer) was used. Binding of GlnK
was not observed when another His-tagged protein
(His
6
-NtcA from S. elongatus) was immobilized on the
sensor chip (not shown), revealing that the observed
binding was specific for TnrA. The GlnK–TnrA com-
plex appeared to be quite stable, as revealed by the
very slow dissociation of the complex following the
injection phase (Fig. 2A). In the course of the mea-
surements, we found that 2 mm ATP (in the absence
of Mg
2+
) led to rapid dissociation of the GlnK–TnrA
complex (see below), which was subsequently used to
regenerate the TnrA-coated chip surface. The dissociat-
ing effect of 2 mm ATP on the GlnK–TnrA complex is
shown in Fig. 2A. Immediately after application of
2mm ATP to the preformed GlnK–TnrA complex,
rapid dissociation was observed, reaching the basal
levels of resonance units (GlnK free surface) within
seconds. To test the effects of various molecules on the
interaction of TnrA with GlnK, 40 nm GlnK (trimer)
Fig. 1. Coimmunoprecipitation of TnrA, GlnK and GS. Immunopre-
cipitation experiments were performed with either TnrA-specific
(A), GlnK-specific (B) or GS-specific (C) antibodies. Cells were

with the effector molecules
ATP and 2-oxoglutarate. As shown in Fig. 2B, MgCl
2
or MnCl
2
alone did not affect TnrA binding to GlnK.
However, Mg
2+
and Mn
2+
gradually relieved the
inhibitory effect of ATP on the GlnK–TnrA inter-
action, so that, in the presence of 1 mm Mg
2+
or
Mn
2+
, ATP at 2 mm was not fully inhibitory, and
2mm Mg
2+
or Mn
2+
restored more than 50% of the
GlnK–TnrA interaction in the presence of 2 mm ATP.
On the other hand, 2-oxoglutarate did not influence
the GlnK–TnrA interaction, either alone, in the
absence of divalent metals, or in combination with
ATP and Mg
2+
or Mn

The graph shows the response difference between FC 2 (His
6
-
TnrA) and FC 1 (His
6
-NAGK). (A) ATP effect on dissociation of the
GlnK–TnrA complex. First, GlnK (40 n
M trimers) was injected onto
the His
6
-TnrA surface. After 50 s of washing with HBS buffer,
25 lL of 2 mK ATP was injected (indicated by the arrow), which
removed the GlnK bound to the His
6
-TnrA surface within a few sec-
onds. (B) Binding of GlnK to TnrA in the presence of different Mg
2+
or Mn
2+
concentrations with or without 2 mM ATP and 1 mM 2-oxo-
glutarate (2-OG) present, as indicated. GlnK in pure HBS buffer
served as a control (set as 100% binding).
Interaction of TnrA with GlnK and GS A. Kayumov et al.
1782 FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS
1–3 mm had only a small effect on the GlnK–TnrA
interaction, except for ADP, which was moderately
inhibitory, although less so than ATP (Fig. 3B). Taken
together, these measurements, although performed
under rather artificial conditions, indicate that the
GlnK–TnrA complex could be stable in vivo in the

The results of the immunoprecipitation experiments
revealed a constitutively present GS–TnrA complex in
the GlnK-deficient cells transferred to nitrate-deprived
conditions, as well as in nitrate-grown cells. Under
these conditions, GS is supposed to be in an active
state, whereas only feedback-inhibited GS was previ-
ously reported to bind TnrA [9,18]. To test whether,
indeed, non-feedback-inhibited GS can also bind
TnrA, a Strep II-tagged variant of GS (GS-ST) was
overproduced in E. coli BL21, purified to apparent
electrophoretic homogeneity [12], and used in BIAcore
analysis on immobilized His
6
-tagged TnrA immobilized
on a chelating nitrilotriacetic acid sensor chip. NAGK
from S. elongatus was bound to the reference cell as a
control for nonspecific interactions.
The response difference sensogram (FC2 – FC1) in
Fig. 5A shows the binding of non-feedback-inhibited
GS to immobilized TnrA. The GS–TnrA complex was
quite stable under the conditions used: almost no com-
plex dissociation appeared after the injection phase. In
contrast to what was found for GlnK, no efficient
effector molecule was found to remove GS from the
His
6
-TnrA surface. ATp at 10 mm caused only partial
release of GS from TnrA (Fig. 5A).
The effects of various metabolites on the GS–TnrA
interaction were also investigated. GS at 40 nm was

intracellular proteolysis
Previously, it had been reported that the DNA-binding
domain of TnrA is located on its N-terminus, whereas
the C-terminus is responsible for GS binding [10]. Six
amino acids required for this interaction on the C-termi-
nus were identified (Met96, Leu97, Gln100, Leu101,
Ala103, and Phe105) (Fig. S2). A previous study
showed [19] that the TnrA-dependent nrg and nasB pro-
moters were constitutively expressed when seven or 20
amino acids were deleted from the C-terminus of TnrA,
whereas deletion of 34 amino acids from the C-terminus
resulted in a TnrA null mutation phenotype. This
implied that the TnrA signal transduction domain is
most likely located at the C-terminus. In nitrate-grown
cells, TnrA is almost completely membrane-bound via
GlnK [12]. We have speculated that GlnK may also
interact with the C-terminus of TnrA, and may play a
role in the regulation of TnrA activity and its proteo-
lysis [15]. To test this assumption, various truncations
of TnrA (lacking six, 20 and 35 amino acids from the
C-terminus) were constructed and overproduced in
E. coli (Fig. S2). Glutaraldehyde crosslinking assays
revealed that all proteins were in a dimeric state, con-
firming that the C-terminus is not required for dimeriza-
tion (Fig. S3) [10,19]. Interactions of the truncated
TnrA proteins with GlnK and with GS were determined
by pulldown and SPR analysis, as described above
(Fig. 6). As expected, the C-terminus of TnrA was abso-
lutely required for GS binding: deletion of even six
amino acids abolished this interaction (Fig. 6A,B).

-TnrA surface. After 180 s of washing with HBS buffer,
25 lLof10m
M ATP was injected (indicated by the arrow), which
partially removed the GS bound to the His
6
-TnrA surface. (B, C)
Effects of AMP, glutamine and ATP on GS binding to TnrA. GS
was preincubated with effector molecules at the concentrations
indicated, and injected onto the His
6
-TnrA surface. GS incubated in
pure HBS buffer served as a control.
Interaction of TnrA with GlnK and GS A. Kayumov et al.
1784 FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS
region located between 20 and 35 amino acids from
the C-terminus of TnrA is required for protease recog-
nition and, at the same time, overlaps with the GlnK
recognition site (see above). This finding agrees with the
assumption that binding of GS or GlnK protects TnrA
from proteolytic degradation [15], as these proteins
would shed the recognition site for proteolytic degrada-
tion. As soon as GlnK or GS dissociate from TnrA, the
C-terminus becomes accessible to proteolysis.
Discussion
TnrA, a major transcription factor in B. subtilis for the
control of nitrogen assimilation, is active under nitro-
gen-limited conditions and is membrane-bound via the
AmtB–GlnK complex [6,12]. Its activity was shown to
be regulated by complex formation with feedback-
inhibited GS, and in the absence of a nitrogen source

6
-NAGK served as a control
in FC 1. (B) Pulldown analysis of GS binding to TnrAwt, TnrA6,
TnrA20, and TnrA35 (see Experimental procedures for details). (C)
Pulldown analysis of GlnK binding to TnrAwt, TnrA6, TnrA20, and
TnrA35. TnrA (dimer) at 10 n
M was premixed with 10 nM GS (12-
mer) or 10 n
M GlnK (trimer), and incubated in buffer B at 20 °C for
30 min. The protein mix was loaded onto an Ni
2+
–nitrilotriacetic
acid Sepharose column to affinity-purify TnrA (I) or Strep-Tactin
Sepharose to affinity-purify GS or GlnK (II), after the columns had
been washed with buffer B. Proteins were eluted with 250 m
M
imidazole (I) or with 2.5 mM destiobiotin (II), and the eluates were
analyzed by western blot with TnrA-specific, GlnK-specific and GS-
specific antibodies, as indicated on the left.
A. Kayumov et al. Interaction of TnrA with GlnK and GS
FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS 1785
of TnrA shields the recognition site for proteolytic deg-
radation of TnrA (Figs 6 and 7).
In the AmtB-deficient strain, GlnK is located in the
cytoplasm and constitutively binds TnrA. Previously,
the AmtB-deficient strain (with constitutive GlnK–
TnrA binding) was shown to display high levels of tran-
scription from the TnrA-dependent nrgAB promoter
under ammonia-limited conditions (ammonium at low
pH) [11]. This suggests that TnrA bound to GlnK is still

assays [9,10,18]. Constitutive binding of GS to TnrA
in the GlnK-deficient strain provides an explanation
for the so-far elusive observation that TnrA-dependent
transcription from the nrgAB promoter is impaired in
a GlnK-deficeint strain growing under ammonia-
limited conditions (ammonium at low pH) [11], as GS
binding was shown to depress the transcriptional activ-
ity of TnrA [9,10,18].
Taken together, the results from this investigation
provide indications of the physiological role of the
GlnK–TnrA interaction, which has previously been
unclear. In the GlnK-bound state, TnrA is protected
from proteolysis without affecting its ability to induce
gene expression. When TnrA dissociates from the
AmtB–GlnK complex (after a shift to nitrate-deprived
conditions), it becomes rapidly degraded. Under these
conditions, GS should be in a highly active, non-
feedback-inhibited state, which has reduced affinity
for TnrA, Therefore, TnrA could be preferentially rec-
ognized by a protease as an idle protein and
degraded, as has been proposed for many proteins in
B. subtilis [21]. When, however, TnrA is complexed by
GS before nitrate downshift, as is the case in the
GlnK-deficient mutant, it remains bound and is pro-
tected from proteolysis.
Experimental procedures
Bacterial strains and growth conditions
The B. subtilis strains used in this study – strain 168 (wild
type), the AmtB-deficient strain GP 254, and the GlnK-
deficient mutant GP 253 – have been described previously

(Roche Diagnostics, Mannheim, Germany).
Coupling antibodies to Protein A Sepharose
One hundred milligrams of Protein A Sepharose beads (GE
Healthcare, Munich, Germany) were incubated for 2 h at
24 °C in 0.5 mL of NaCl ⁄ P
i
(4.3 mm Na
2
HPO
4
, 1.8 mm
KH
2
PO
4
, 137 mm NaCl, 2.7 mm KCl, pH 8.0). The beads
were harvested by short centrifugation (11 500 g,30s,
Interaction of TnrA with GlnK and GS A. Kayumov et al.
1786 FEBS Journal 278 (2011) 1779–1789 ª 2011 The Authors Journal compilation ª 2011 FEBS
4 °C), and incubated with 0.5 mL of antiserum for 1 h at
24 °C with gentle shaking. After being washed times with
5 mL of 0.2 m Na
3
BO
3
(pH 9.0), the Sepharose beads were
resuspended in 5 mL of 0.2 m Na
3
BO
3

trifugation (15 000 g, 10 min, 4 °C) to remove debris and
unbroken cells, the samples, containing 3 mg of total
protein, were diluted with detergent-containing buffer
[NET buffer I: 50 mm Tris ⁄ HCl, pH 7.0, 150 mm NaCl,
0.1% (v ⁄ v) nonionic detergent Nonidet P-40, 1 mm EDTA]
to a total volume of 1.5 mL, and following a 15-min incuba-
tion at 24 °C, the sample was briefly centrifuged (16 000 g,
30 s) to remove debris. To this extract, 100 lL of a suspen-
sion of Protein A Sepharose beads with coupled antibodies
was added. After a 3-h incubation at 4 °C, Sepharose beads
were harvested by centrifugation (16 000 g,30s,4°C), and
the sediment was washed twice with NET buffer I, once with
NET buffer II (NET buffer I with 500 mm NaCl), and once
with buffer IP [10 mm Tris ⁄ HCl, pH 7.5, 0.1% (v ⁄ v) Noni-
det P-40]. The bound proteins were eluted from Protein A
Sepharose by 10 consecutive additions of 50 lL each of
buffer IE (100 mm glycine, pH 2.4), and the elutions were
pooled and analyzed by immunoblot analysis with TnrA-
specific, GlnK-specific and GS-specific antibodies.
BIAcore SPR detection
SPR experiments were performed with a BIAcore X biosen-
sor system (Biacore AB, Uppsala, Sweden). To immobilize
His
6
-TnrA on the nitrilotriacetic acid biosensor surface,
Ni
2+
was first bound to the nitrilotriacetic acid surfaces of
both flow chambers through injection of 10 lLofa5mm
NiSO

. Subsequently, the
chip was loaded again with Ni
2+
and His
6
-TnrA or His
6
-
NAGK as described above. This procedure was performed
when the performance of analyte binding to the His
6
-TnrA
surface started to decrease.
ITC
ITC experiments were performed on a VP-ITC microcalo-
rimeter (MicroCal, LCC, New York, USA) in 10 mm
Hepes ⁄ NaOH, 50 mm KCl and 100 mm NaCl (pH 7.4) at
20 °C [24]. For determination of ATP, ADP, AMP and
GTP binding isotherms for wild-type GlnK, 25 lm protein
(trimer concentration) was titrated with 2 mm ATP, 2 mm
ADP, 2 mm AMP, or 2 mm GTP, respectively. The ligand
(5 lL) was injected 35 times into the 1.4285-mL cell with
stirring at 350 r.p.m. The binding isotherms were calculated
from received data, and fitted to a three-site binding model
with MicroCal origin software (Northampton, MA, USA).
Construction of mutant tnrA genes
All DNA manipulations were performed by standard meth-
ods as described in [23]. Mutant tnrA genes were amplified
with pfu polymerase from chromosomal DNA of B. subtil-
is 168. Briefly, the tnrA gene coding for the protein with dele-

2+
–nitrilotriacetic acid Sepharose (Qiagen,
Hilden, Germany) or Strep-Tactin Sepharose (IBA, Go
¨
ttin-
gen, Germany) equilibrated with 10 column volumes
(10 · 0.2 mL) of buffer B, with subsequent washing four
times with five volumes of the same buffer. Proteins were
eluted with buffer E (buffer B supplemented with 250 mm
imidazole from Ni
2+
–nitrilotriacetic acid Sepharose or
2.5 mm destiobiotin from the Strep-Tactin column). The
samples were collected and analyzed by western blot with
TnrA-specific, GlnK-specific and GS-specific antibodies.
Acknowledgements
J. Stu
¨
lke (Go
¨
ttingen) is gratefully acknowledged for
providing B. subtilis strains. This work was supported
by DFG grant Fo195, the Russian–German program
‘Michail Lomonosov’ A ⁄ 08 ⁄ 75091, and the Ministry of
Education and Science of the Russian Federation (gov-
ernment contract No. P2573 from 25 November 2009).
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Supporting information
The following supplementary material is available:
Fig. S1. Isothermal titration calorimetry of (A) ADP,
(B) AMP and (C) GTP binding to GlnK.
Fig. S2. TnrA C-terminal truncations.
Fig. S3. Crosslinking analysis of truncated TnrA
proteins.
Doc. S1. Purification of His
6
-tagged TnrA proteins,
purification of GlnK-ST and GS-ST, and glutaralde-
hyde crosslinking assays.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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should be addressed to the authors.
A. Kayumov et al. Interaction of TnrA with GlnK and GS