the PII-like protein GlnK and glutamine synthetase inBacillus subtilis Airat Kayumov1,2, Annette Heinrich3, Kseniya Fedorova2, Olga Ilinskaya2and Karl Forchhammer3 1 Kazan State Universi
Trang 1the PII-like protein GlnK and glutamine synthetase in
Bacillus subtilis
Airat Kayumov1,2, Annette Heinrich3, Kseniya Fedorova2, Olga Ilinskaya2and Karl Forchhammer3
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¨t Tu¨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¨t Tu¨bingen, Auf der
Morgenstelle 28, D-72076 Tu¨bingen,
Germany
Fax: +49 7071295843
Tel: +49 70712972096
E-mail: karl.forchhammer@uni-tuebingen.de
(Received 26 January 2011, revised
10 March 2011, accepted 14 March 2011)
doi:10.1111/j.1742-4658.2011.08102.x
TnrA is a master transcription factor regulating nitrogen metabolism in Bacillus subtilisunder conditions of nitrogen limitation When the preferred nitrogen source is in excess, feedback-inhibited glutamine synthetase (GS) has been shown to bind TnrA and disable its activity In cells grown with
an energetically unfavorable nitrogen source such as nitrate, TnrA is fully membrane-bound via a complex of AmtB and GlnK, which are the trans-membrane ammonium transporter and its cognate regulator, respectively, originally termed NrgA and NrgB The complete removal of nitrate from the medium leads to rapid degradation of TnrA in wild-type cells In con-trast, in AmtB-deficient or GlnK-deficient strains, TnrA is neither mem-brane-bound nor degraded in response to nitrate depletion Here, we show that TnrA forms either a stable soluble complex with GlnK in the absence
of AmtB, or constitutively binds to GS in the absence of GlnK In vitro, the TnrA C-terminus is responsible for interactions with either GS or GlnK, and this region appears also to mediate proteolysis, suggesting that binding of GlnK or GS protects TnrA from degradation Surface plasmon resonance detection assays have demonstrated that GS binds to TnrA not only in its feedback-inhibited form, but also in its non-feedback-inhibited form, although less efficiently TnrA binding to GlnK or GS responds dif-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.
Trang 2Spore-forming bacteria of the genus Bacillus have a
variety of regulatory responses to changes in the
environment TnrA, a major transcription factor
in Bacillus subtilis under nitrogen-limited conditions
(conditions in which the nitrogen source becomes
growth-limiting), controls gene expression in response
to nitrogen availability During nitrogen-limited
growth, TnrA serves either as an activator or a
repres-sor of genes involved in nitrogen assimilation TnrA
activates its own gene [1], the nitrate and nitrite
utiliza-tion genes [2], the nrgAB (amtBglnK) operon
(ammo-nium transport) [3], and some other target promoters
[1,4,5] TnrA is a negative regulator of glnA and gltAB,
encoding the ammonium assimilatory enzymes
gluta-mine synthetase (GS) and glutamate synthase,
respec-tively [6–8] TnrA belongs to the MerR family of
transcription factors, and is present as a homodimer of
two 12-kDa subunits [1] The signal for its activation
remains unclear [1,6,8,9] Several lines of evidence
indi-cate that GS acts as a sensor of nitrogen availability in
B subtilis [1,9] The feedback-inhibited form of GS
binds tightly to TnrA, preventing its binding to DNA,
with the most effective feedback inhibitors of GS being
glutamine and AMP [9] Mutations in TnrA that result
in constitutive expression of the TnrA-activated amtB
promoter all lie within the C-terminal region of TnrA,
and impair the interaction between GS and TnrA
[9,10]
Another mechanism for controlling TnrA activity
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 Dependseem-ing 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 nitrogen source, TnrA is released from the membrane and, concomitantly, it is degraded within 15 min by proteolysis By contrast, no degradation of TnrA was observed during this kind of shift experiment in B sub-tilis AmtB-deficient and GlnK-deficient strains, despite TnrA being soluble in these cells [12,15] To gain dee-per insights in the involvement of proteolysis in modu-lating TnrA-dependent gene expression, we aimed to elucidate why TnrA is resistant to proteolysis in the GlnK-deficient and AmtB-deficient strains
Results
Immunoprecipitation of TnrA with GlnK or
GS in B subtilis
In contrast to what is seen in wild-type cells, in amtB
or glnK knockout mutants TnrA was only detectable
in the soluble fraction of cell-free extracts, but was never membrane-bound and no proteolytic degrada-tion occurred after nitrate depledegrada-tion [12,15] To explain the mechanism of TnrA protection from proteolysis,
we investigated which proteins TnrA is bound to in these mutants, considering GlnK and GS, in particu-lar, as potential partner proteins of TnrA To this end, immunoprecipitation assays were performed with cell-free extracts from both mutant and wild-type nitrate-grown cells, and from cells shifted to nitrogen-depleted medium Cell-free extracts were incubated with TnrA-specific, GlnK-specific or GS-specific antibodies cou-pled to Protein A Sepharose, in the presence of non-ionic detergent These antigen–antibody complexes were collected, and after rigorous washing in nonionic 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
Trang 3was 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-purified with TnrA in the wild-type or AmtB-deficient mutant under either condition (Fig 1A) These obser-vations were confirmed by reversing the immunopre-cipitation experiments, using antibodies against GlnK
or GS TnrA was copurified with immunoprecipitated GlnK from extracts of both wild-type and AmtB-defi-cient cells (Fig 1B), and was recovered by GS immu-noprecipitation only in the GlnK-deficient mutant (Fig 1C) Taken together, these data demonstrate, that TnrA binds constitutively to GlnK in AmtB-deficient mutants, and to GS in GlnK-deficient mutants This constitutive binding in the mutant strains probably protects TnrA from proteolytic degradation
Surface plasmon resonance analysis (SPR) of the GlnK–TnrA interaction
As a next step, the interaction of TnrA with GlnK was investigated in vitro by BIAcore SPR detection For this analysis, a Strep-tag II-tagged variant of GlnK (GlnK-ST) and a His-tagged recombinant TnrA were overproduced in Escherichia coli BL21 and purified to apparent electrophoretic homogeneity [12] His6-tagged TnrA was immobilized on flow cell (FC) 2 of a chelat-ing nitrilotriacetic acid sensor chip, and GlnK-ST was used as an analyte His6-N-acetyl-l-glutamate kinase (NAGK) from Synechococcus elongatus [16] was bound
to the reference cell (FC 1) as a control for nonspecific 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 (His6-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 Mg2+) 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
2 mm 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
grown under nitrogen-limited conditions in SMM supplemented
with 20 m M NaNO 3 (I) At late exponential growth phase, cells
were washed and shifted to combined nitrogen-free medium,
incu-bated at 37 C with shaking for 10 min, and then harvested (II).
The crude cell extracts were used for immunoprecipitation as
described in Experimental procedures The washed
immunoprecipi-tates were analyzed by immunoblotting with antibodies against
TnrA, GlnK, or GS, as indicated on the right.
Trang 4was incubated with various effector molecules, and the
mixture was used as an analyte in SPR analysis ATP
and 2-oxoglutarate are known to be the primary
effec-tors involved in PII signaling, and they strongly affect
interactions of many GlnK proteins with their
recep-tors [17] The divalent cations Mg2+ or Mn2+ were
previously shown to negatively affect the binding of
ATP to GlnK [12] Therefore, we investigated the
binding of TnrA to GlnK in the presence of different
mixtures of Mg2+or Mn2+with the effector molecules
ATP and 2-oxoglutarate As shown in Fig 2B, MgCl2
or MnCl2alone did not affect TnrA binding to GlnK
However, Mg2+ and Mn2+ gradually relieved the
inhibitory effect of ATP on the GlnK–TnrA
inter-action, so that, in the presence of 1 mm Mg2+ or
Mn2+, ATP at 2 mm was not fully inhibitory, and
2 mm Mg2+or Mn2+ 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 Mg2+ or Mn2+ To resolve the inhibitory effect of ATP on the GlnK–TnrA interaction in the absence of divalent cations more clearly, ATP was titrated to the binding assays in the absence or pres-ence of 2-oxolguatarate As shown in Fig 3A, 0.2 mm ATP was sufficient to inhibit 50% of the GlnK–TnrA interaction The inhibitory effect of ATP was not further enhanced by 2-oxoglutarate, in agreement with earlier studies showing that B subtilits GlnK does not respond to 2-oxoglutarate [12] Other nucleotides, such as ADP, AMP, and GTP, at concentrations of
Fig 3 Influence of various effector molecules on the interaction of GlnK with the His6-TnrA surface GlnK was preincubated with effec-tor molecules at the concentrations indicated, and injected onto the His 6 -TnrA surface GlnK incubated in pure HBS buffer served as a control (set as 100% binding) (A) Effect of increasing ATP concen-trations (as indicated), with or without 1 m M 2-oxoglutarate (2-OG) present (B) Effects of various nucleotides (ATP, ADP, AMP, and GTP) and 2-OG at different concentrations on GlnK binding to TnrA.
Fig 2 BIAcore analysis of GlnK–TnrA complex formation and ATP
effect on dissociation of the GlnK–TnrA complex The analyte GlnK
was injected in a volume of 30 lL at a flow rate of 15 lLÆmin)1.
The graph shows the response difference between FC 2 (His 6
-TnrA) and FC 1 (His6-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 His6-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 m M ATP and 1 m M
2-oxo-glutarate (2-OG) present, as indicated GlnK in pure HBS buffer
served as a control (set as 100% binding).
Trang 51–3 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
presence of divalent cations and that the complex is
negatively affected most efficiently by ATP
Isothermal titration calorimetry (ITC)
The affinity of binding of GlnK to nucleotides, which
affected the GlnK–TnrA interaction as revealed by
SPR analysis, was quantified by ITC Previously,
bind-ing of different combinations of ATP and
2-oxogluta-rate to GlnK was measured by this method [12] Under
optimal binding conditions, strong binding of ATP was
observed (Fig 4), which could be perfectly fitted with a
three sequential binding sites model Data analysis
resolved two high-affinity binding sites (dissociation
constant for the first two sites: Kd1= 12 ± 4 lm and
Kd2= 77 ± 15 lm) and one low-affinity site (site 3)
(Kd3= 4 ± 0.05 mm) No binding of other nucleotides (ADP, AMP, and GTP) was detectable (Fig S1), con-firming their weak effect on the GlnK–TnrA interac-tion (Fig 3) These data support the idea that, in
B subtilis, GlnK senses the intracellular ATP level at site 3, as the binding affinity of this site is in the milli-molar range, which is considered to be physiologically relevant, and that this signal is then transmitted to TnrA
BIAcore analysis of the GS–TnrA interaction 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 His6-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 His6-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 incubated with effector molecules for 5 min in ice, and the mixture was used as an analyte in SPR analysis AMP and glutamine are known to be the most effec-tive inhibitors of GS [9] Figure 5B shows the effect of the feedback inhibitors AMP and glutamine on GS binding to a TnrA-coated sensor chip The presence of either AMP or glutamine led to an approximately two-fold signal increase in comparison with non-feedback-inhibited GS ATP, at a physiological concentration, negatively affected GS binding to the TnrA sensor surface At a concentration of 2.5 mm, it decreased complex formation by approximately 60%, and at a concentration of 5 mm, by 80% (Fig 5C) ATP at
10 mm was required to completely abolish complex formation; however, once the complex was formed,
Fig 4 ITC of ATP binding to GlnK The raw data were fitted with a
three-site binding model for a PII trimer The upper panel shows
the raw data in the form of the heat effect during the titration of
25 l M GlnK solution (trimer concentration) with ATP (titration from
2.1 to 73.5 l M ) The lower panels show the binding isotherm and
the best-fit curve according to the three sequential binding sites
model.
Trang 6this concentration could not efficiently dissociate the
complex (see above)
The C-terminus of TnrA is required for interaction
with both GlnK and GS, as well as for
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] Interacdimeriza-tions 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) Truncated forms of TnrA with the C-terminus lacking six or 20 amino acids still bound to GlnK; however, removal of 35 amino acids completely abolished binding
of GlnK (Fig 6A,C) This result implies that a region in TnrA located between 20 and 35 amino acids from the C-terminus is required for GlnK interaction, whereas the ultimate C-terminal amino acids of TnrA are appar-ently needed for GS binding
In addition, the in vitro proteolysis of truncated TnrA variants was investigated, as described previously for full-length TnrA [15] B subtilis 168 (wild-type) cells were grown in SMM medium supplemented with sodium nitrate until the late exponential growth phase, and the cells were then washed and resuspended in SMM without nitrate, and finally incubated for a further 20 min Samples were taken before and after the shift, and soluble cell-free extract was prepared by ultracentrifugation The soluble extract (containing
20 lg of total protein) was supplemented with 50 ng of TnrA, and the mixture was incubated at 37C for
60 min; TnrA incubated in buffer served as a control The fate of TnrA in the samples was then analyzed by immunoblotting with TnrA-specific antibodies (Fig 7) During incubation in the soluble cytoplasmic extract, TnrA6, TnrA20 and wild-type TnrA were almost com-pletely degraded, but not TnrA35 This indicates that a
Fig 5 BIAcore analysis of GS–TnrA complex formation (A) GS–
TnrA interaction First, non-feedback-inhibited GS was injected onto
the His6-TnrA surface After 180 s of washing with HBS buffer,
25 lL of 10 m 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.
Trang 7region 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 TnrA is eliminated from the cells by proteolysis [9,10,15] The findings in the present study strongly imply that, in vivo, TnrA is stable only in a complex with a partner protein In nitrate-grown wild-type cells, TnrA is active and bound to the GlnK–AmtB complex [12] (Fig 1A,B) After shifting of the cells to nitrate-free medium, this complex dissociates and TnrA becomes degraded, whereas no degradation occurs in AmtB-defi-cient or GlnK-defiAmtB-defi-cient cells [12] Our data show that,
in these mutant strains, TnrA interacts constitutively with either soluble GlnK or GS, respectively, and in consequence is protected from proteolysis (Fig 1) The reason for this protection, according to the present study, is that binding of GS or GlnK to the C-terminus
Fig 7 In vitro proteolysis of truncated TnrA proteins Soluble cell-free extracts were prepared from (I) cells that had been shifted into nitrogen-free medium and incubated for 20 min, and (II) nonshifted cells Purified TnrA protein variants, full-length or different C-termi-nal truncations (each 50 ng of protein), were incubated with these extracts for 30 min (as described in [15]) TnrA incubated in assay buffer served as a control (C) Subsequently, proteolytic removal of TnrA was analyzed by western blotting.
Fig 6 The interaction of truncated TnrA proteins with GlnK and
GS (A) BIAcore analysis of GlnK and GS binding to wild-type TnrA
(TnrAwt), TnrA6, TnrA20, and TnrA35 The analyte (40 n M GlnK or
GS oligomers) was injected in a volume of 30 lL onto the TnrA
sur-face at a flow rate of 15 lLÆmin)1 His 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 n M 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 m M destiobiotin (II), and the eluates were
analyzed by western blot with TnrA-specific, GlnK-specific and
GS-specific antibodies, as indicated on the left.
Trang 8of 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
able to activate gene expression The assumption that
GlnK binding does not impair TnrA activity is
consis-tent with the observation that, in nitrate-grown
wild-type cells, TnrA is bound to the AmtB–GlnK complex
despite being transcriptionally active
Nitrate depletion leads to dissociation of the AmtB–
GlnK–TnrA complex and subsequent TnrA
degrada-tion, whereas in AmtB mutants TnrA remains bound
to GlnK and is therefore protected from proteolysis
This suggests a role of AmtB in dissociation of the
GlnK–TnrA complex A possible regulatory role of
AmtB proteins has been suggested previously [20];
however, the mechanism leading to AmtB-dependent
GlnK–TnrA dissociation has remained elusive so far
In B subtilis wild-type cells growing on a poor
nitrogen source (nitrate), GS is active and does not
bind TnrA (Fig 1A,C), and the latter is sequestered by
the AmtB–GlnK complex However, in the GlnK
mutant, TnrA is constitutively bound to GS; a shift to
a nitrate-deprived medium does not lead to
dissocia-tion of the complex, and TnrA remains protected from
proteolysis The constitutive binding of TnrA to GS
seems to contradict previous reports that only
feed-back-inhibited GS is able to bind TnrA [9] However,
the sensitive SPR analysis has demonstrated that
non-feedback-inhibited GS is, in fact, able to bind TnrA,
although with reduced affinity as compared with
feed-back-inhibited GS (Fig 5) The reduced affinity could
account for the fact that this interaction is not detected
by examining it indirectly through TnrA–DNA binding
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 [11] B subtilis cells were grown in Spizizen minimal med-ium (SMM) [22] containing glucose [0.5% (w⁄ v)] as a car-bon source Sodium nitrate (20 mm) served as a nitrogen source l-Tryptophan was added to a final concentration of
50 mg L)1
Protein preparation
TnrA from B subtilis 168 cells and NAGK from S elonga-tus, carrying His6-tags on their N-terminal, were overpro-duced in E coli BL21 with the pET15b expression vector
nitrilotriacetic acid columns to apparent electrophoretic homogeneity, as described previously [12,16] GlnK-ST and GS-ST were overproduced in E coli BL21 with the pDG148 expression vector and purified with a Strep-Tactin column (IBA, Go¨ttingen, Germany), as described in detail in Doc S1
Immunoblot analysis
For immunoblot analysis, the samples were separated on 15% SDS⁄ PAGE gels After electrophoresis, the proteins were transferred to a nitrocellulose membrane by semi-dry electroblotting Antibodies were visualized with secondary antibodies (anti-rabbit IgG–POD) (Sigma-Aldrich, Tauf-kirchen, Germany) and the LumiLight detection system (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
24C in 0.5 mL of NaCl ⁄ Pi (4.3 mm Na2HPO4, 1.8 mm
were harvested by short centrifugation (11 500 g, 30 s,
Trang 94C), and incubated with 0.5 mL of antiserum for 1 h at
24C with gentle shaking After being washed times with
5 mL of 0.2 m Na3BO3(pH 9.0), the Sepharose beads were
dimethyl pimelimidate dihydrochloride was added to a final
concentration of 20 mm The incubation was continued for
30 min at 24C with gentle shaking The Protein A
Sepha-rose beads were washed twice with 5 mL of 0.2 m
ethanol-amine (pH 8.0), resuspended in 5 mL of this, and incubated
beads were washed twice with 5 mL of NaCl⁄ Pi, twice with
5 mL of 100 mm glycine (pH 3.0), and twice with NaCl⁄ Pi,
and resuspended in 0.5 mL of NaCl⁄ Pi
Immunoprecipitation
The immunoprecipitation experiments were performed as
described in [23] Cultures of B subtilis were grown in SMM
with 20 mm NaNO3to a D600 nmof 0.8, harvested by
centri-fugation (8500 g, 10 min, 4C), resuspended in buffer I
FastPrep-24 (M.P Biomedical, Irvine, CA, USA) After
cen-trifugation (15 000 g, 10 min, 4C) 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 24C, 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 4C, Sepharose beads
were harvested by centrifugation (16 000 g, 30 s, 4C), 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
His6-TnrA on the nitrilotriacetic acid biosensor surface,
Ni2+was first bound to the nitrilotriacetic acid surfaces of
both flow chambers through injection of 10 lL of a 5 mm
NiSO4solution Then, His6-TnrA was injected into FC 2 in a
volume of 50 lL at a concentration of 2 nmol⁄ mL in HBS
buffer (10 mm Hepes, 200 mm NaCl, 0.005% Nonidet P-40,
pH 7.5) His6-NAGK from S elongatus was injected into
FC 1 in a volume of 50 lL at a concentration of 2 nmol⁄ mL
in HBS buffer This resulted in increases in resonance units
of 500 in FC 2 and 800 in FC 1 Experiments were per-formed at 25C in HBS buffer at a flow rate of 15 lLÆmin)1, with GlnK-ST or GS-ST as analyte at the concentrations indicated To analyze the effect of small molecules on GlnK
or GS binding to the His6-TnrA surface, the analyte was preincubated for 5 min on ice with the various effector mole-cules as indicated, and was then injected into the sensor chip For novel reloading of the nitrilotriacetic acid sensor chip with fresh His6-TnrA, 50 lL of 0.5 m EDTA was injected to completely remove His6-TnrA and Ni2+ Subsequently, the chip was loaded again with Ni2+ and His6-TnrA or His6 -NAGK as described above This procedure was performed when the performance of analyte binding to the His6-TnrA surface started to decrease
ITC
ITC experiments were performed on a VP-ITC microcalo-rimeter (MicroCal, LCC, New York, USA) in 10 mm
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-is168 Briefly, the tnrA gene coding for the protein with dele-tion of six amino acids from C-terminus was obtained with primers TnrAN (5¢-GCT CGA GGA TCC GAT GAC
ACG GGA TCC GTA CCG TTA GTG AGC ATT AAG-3¢) The PCR products were purified, digested with BamHI, and ligated into the BamHI-digested pET-15b vector (Nov-agen) This vector provides N-terminally His6-tagged protein overexpression in E coli BL21 cells To obtain TnrA pro-teins lacking 20 and 35 amino acids from the C-terminus, TnrA20 (5¢-TCC AGC GGA TCC TTC CGC ACT TAC GGA TC-3¢) and TnrA35 (5¢-TTC TTT GGA TCC CAT ATC CTT TTA AAT CTC TGC-3¢) oligonucleotides were used, respectively, instead of TnrA6 The sequences of all cloned genes were confirmed by DNA sequencing
Pulldown
For these assays, the purified His6-tagged TnrA proteins (wild-type and truncated versions), GlnK-ST and GS-ST
Trang 10were used Initially, the proteins (10 nm each protein) were
diluted in 300 lL of buffer B (100 mm Tris⁄ HCl, pH 8.0,
at 20C for 30 min Afterwards, the protein mixture was
loaded onto Ni2+–nitrilotriacetic acid Sepharose (Qiagen,
Hilden, Germany) or Strep-Tactin Sepharose (IBA,
Go¨ttin-gen, Germany) equilibrated with 10 column volumes
times with five volumes of the same buffer Proteins were
eluted with buffer E (buffer B supplemented with 250 mm
imidazole from Ni2+–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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