Báo cáo khoa học: NrpRII mediates contacts between NrpRI and general transcription factors in the archaeon ¨ Methanosarcina mazei Go1 pot

NrpRII mediates contacts between NrpRI and general
transcription factors in the archaeon
Methanosarcina mazei Go
¨
1
Katrin Weidenbach, Claudia Ehlers, Jutta Kock and Ruth A. Schmitz
Institut fu
¨
r Allgemeine Mikrobiologie, Christian-Albrechts Universita
¨
t zu Kiel, Germany
Keywords
Archaea; Methanosarcina mazei;
transcription regulation; NrpR; 2-oxoglutarate
Correspondence
R. A. Schmitz, Institut fu
¨
r Allgemeine
Mikrobiologie, Universita
¨
t Kiel, Am
Botanischen Garten 1-9, 24118 Kiel,
Germany
Fax: +49 (431) 8802194
Tel: +49 (431) 8804334
E-mail:
(Received 24 June 2010, revised 3 August
2010, accepted 13 August 2010)
doi:10.1111/j.1742-4658.2010.07821.x
We report here on the formation of a complex between the two NrpR
homologs present in Methanosarcina mazei Go

¨
1 and their binding proper-
ties to the nifH and glnK
1
promoters. Reciprocal co-chromatography dem-
onstrated that NrpRI forms stable complexes with NrpRII (at an
NrpRI : NrpRII molar ratio of  1 : 3), which are not affected by 2-oxo-
glutarate. Promoter-binding, analyses using DNA-affinity chromatography
and electrophoretic gel mobility shift assays, verified that NrpRII is not
able to bind to either the nifH promoter or the glnK
1
promoter except
when in complex with NrpRI. Specific binding of NrpRI to the nifH and
glnK
1
promoters was shown to be highly sensitive to 2-oxoglutarate,
regardless of whether only NrpRI, or NrpRI in complex with NrpRII,
bound to the promoter. Finally, strong interactions between NrpRII and
the general transcription factors TATA-binding proteins (TBP) 1–3 and the
general transcription factor TFIIB (TFB) were demonstrated, interactions
which are also sensitive to 2-oxoglutarate. On the basis of these findings we
propose the following: under nitrogen sufficiency NrpRII binds from solu-
tion to either the nifH promoter or the glnK
1
promoter by simultaneously
contacting NrpRI and TBP plus TFB, resulting in full repression of tran-
scription; whereas, under nitrogen limitation, increasing 2-oxoglutarate
concentrations significantly decrease the binding of NrpRI to the operator
as well as the binding of NrpRII to TBP and TFB, ultimately allowing
recruitment of RNA polymerase to the promoter.

Structured digital abstract
l
MINT-7990058: NrpRII (uniprotkb:Q8PVJ4) physically interacts (MI:0915) with TBP3 (uni-
protkb:
Q8PUZ4)bypull down (MI:0096)
l
MINT-7989998: NrpRII (uniprotkb:Q8PVJ4) physically interacts (MI:0915) with TBP1 (uni-
protkb:
Q8PY37)bypull down (MI:0096)
l
MINT-7989971, MINT-7989984: NrpRII (uniprotkb:Q8PVJ4) physically interacts (MI:0915)
with NrpRI (uniprotkb:
Q8PXY1)bypull down (MI:0096)
l
MINT-7990028: NrpRII (uniprotkb:Q8PVJ4) physically interacts (MI:0915) with TBP2 (uni-
protkb:
Q8PY36)bypull down (MI:0096)
l
MINT-7990087: NrpRII (uniprotkb:Q8PVJ4) physically interacts (MI:0915) with TFP (uni-
protkb:
Q977U3)bypull down (MI:0096)
l
MINT-7990149: NrpRII (uniprotkb:Q8PVJ4) and NrpRI (uniprotkb:Q8PXY1) bind
(
MI:0407)bymolecular sieving (MI:0071)
Abbreviations
EMSA, electrophoretic gel mobility shift assays; IPTG, isopropyl thio-b-
D-galactoside; MBP, maltose binding protein; TFB, homologues of the
eucaryotic general txn factor FTIIB; TBP, TATA-binding protein.
4398 FEBS Journal 277 (2010) 4398–4411 ª 2010 The Authors Journal compilation ª 2010 FEBS

Introduction
Global regulatory mechanisms allow microorganisms
to survive periods of nutrient starvation or stress
resulting from drastic changes in the environment.
Besides the regulatory mechanism controlling uptake
and metabolism of the carbon sources, the system
responsible for regulating uptake and assimilation of
different nitrogen sources is of significant importance
for surviving under nutrient starvation. Regulation of
nitrogen metabolism and regulation of nitrogen fixa-
tion in diazotrophes in response to environmental
changes – mostly proceeding at the transcriptional and
post-translational levels – is well understood in bacte-
ria (reviewed in [1–6]). In contrast, still little is known
about the global regulation of nitrogen metabolism
and nitrogen fixation in archaea. Generally different
regulatory mechanisms are expected as the archaeal
transcription and translation machineries have many
features more similar to their eukaryotic than their
bacterial counterparts [7–11].
Within the methanogenic archaea, studies on the
regulation of nitrogen metabolism and nitrogen fixa-
tion have been pioneered in Methanococcus maripalu-
dis. Leigh and coworkers demonstrated that the
transcriptional regulation of nitrogen-regulated genes
in M. maripaludis differs significantly from that known
in bacteria [12–16]. They identified a global nitrogen
regulator (NrpR), which binds as a dimer to an opera-
tor sequence and inhibits transcription in the presence
of sufficient nitrogen [12,17–20]. They further showed

that the affinity of NrpR to its operator is modulated
by 2-oxoglutarate, resulting in transcription initiation
under nitrogen limitation. This finding indicates that
increasing cellular concentrations of the metabolite 2-
oxoglutarate provides the intracellular signal for nitro-
gen limitation [16,21,22], which has also been proposed
for a variety of nitrogen sensors and regulators in
methanogenic archaea [16,23–25].
Two homologs of NrpR (NrpRI and NrpRII) have
been identified in Methanosarcina mazei strain Go
¨
1
[26], a methylotrophic methanogen of the order Meth-
anosarcinales [27–29], which is also able to fix molecu-
lar nitrogen [30]. Studies of global gene expression in
M. mazei in response to nitrogen, using DNA micro-
arrays combined with biochemical and genetic
approaches, recently demonstrated that NrpRI repre-
sents the global nitrogen regulator in M. mazei and led
to the identification of the corresponding operator,
which differs from that identified in M. maripaludis
[31,32]. However, the function of the second NrpR
homolog in M. mazei, which lacks a DNA-binding
domain, is not known, and it is thus of specific interest
to elucidate whether NrpRII is crucial for nitrogen
regulation. There are currently two findings that may
indicate a potential modulating function of NrpRII in
nitrogen regulation in M. mazei First, an nrpRI mutant
strain retained approximately 10% nitrogen regulation
compared with the wild-type strain [32] and, second,

both NrpR homologs (i.e. an NrpR domain, with or
without an N-terminal DNA-binding domain) identi-
fied in Methanosarcina acetivorans, a close relative of
M. mazei, had to be expressed in trans in order to
restore regulated repression in an M. maripaludis nrpR
deletion mutant [26]. Thus, the goal of this work was
to elucidate the role of NrpRII in nitrogen regulation
in M. mazei.
Results
The presence of two constitutively expressed NrpR
homologs in M. mazei, and the observed residual nitro-
gen regulation in the absence of the main regulator,
NrpRI [32], strongly indicate that NrpRII plays a modu-
lating role in nitrogen regulation in M. mazei, poten-
tially acting in concert with NrpRI. However, even in
the absence of NrpRI, NrpRII appears to inhibit tran-
scription of nitrogen-regulated promoters to some extent
[32]. Consequently, potential formation of a complex
between NrpRI and NrpRII, as well as between NrpRII
and general transcription factors, were investigated and
the effects of 2-oxoglutarate on binding were analysed.
Complex formation between NrpRI and NrpRII
In order to study potential interactions between NrpRI
and NrpRII, we performed co-chromatography experi-
ments using Ni-nitrilotriacetic acid agarose and amy-
lose resin to detect complexes between NrpRI with an
N-terminally fused maltose binding protein (MBP)
(MBP–NrpRI) and NrpRII fused to an N-terminal
His-tag [(His)
6

–NrpRII]. MBP–NrpRI and (His)
6
–
NrpRII were individually expressed in Escherichia coli,
cell-free extracts were prepared and affinity chromatog-
raphy using Ni-nitrilotriacetic acid agarose was per-
formed as described in the Materials and methods.
The elution fractions were analysed by western blotting
using antibodies directed against the MBP-fusion and
the His-tag to detect MBP–NrpRI and (His)
6
–NrpRII,
respectively, clearly demonstrating that significant
amounts of MBP–NrpRI co-eluted with (His)
6
–NrpRII
from Ni-nitrilotriacetic acid (Fig. 1A). Degradation of
MBP–NrpRI into two major products was frequently
observed, as was the case for purified MBP–NrpRI
K. Weidenbach et al. Role of NrpRII in M. mazei
FEBS Journal 277 (2010) 4398–4411 ª 2010 The Authors Journal compilation ª 2010 FEBS 4399
control proteins, and is apparently based upon protein
instability. In order to verify formation of the com-
plex, complementary co-chromatography was per-
formed using the same cell-free extracts, but amylose
resin for affinity purification. Western blot analysis
confirmed that (His)
6
–NrpRII co-eluted with MBP–
NrpRI (Fig. 1B). Control experiments further clearly

demonstrated that neither nonspecific binding of
MBP–NrpRI to Ni-nitrilotriacetic acid nor nonspecific
binding of (His)
6
–NrpRII to the amylose matrix
occurred (Fig. 1C), confirming complex formation
between NrpRI and NrpRII. Moreover, those com-
plexes appear to form independently of an NrpR-regu-
lated promoter as the cell extracts were free of DNA.
Furthermore, we studied the potential effects of
2-oxoglutarate on the formation of a complex between
NrpRII and NrpRI. The respective E. coli cell-free
extracts containing MBP–NrpRI and (His)
6
–NrpRII
were combined in a 1 : 1 ratio and separated into three
equal aliquots. The aliquots were supplemented with 0,
2or10mm 2-oxoglutarate (the aliquot with no 2-oxo-
glutarate served as the control). After incubation with
Ni-nitrilotriacetic acid agarose and washing the matrix
with the respective buffer supplemented with 2-oxo-
glutarate according to the incubation conditions,
(His)
6
–NrpRII and interacting MBP–NrpRI were
eluted in the presence of the respective 2-oxoglutarate
concentration (0, 2 or 10 mm 2-oxoglutarate). The pro-
tein ratios of the complexes were determined by quan-
titative western blot analysis using known amounts of
purified proteins. Under all conditions MBP–NrpRI

bound to (His)
6
–NrpRII at an NrpRI : NrpRII molar
ratio of approximately 1 : 3 (Fig. 2), strongly indicat-
ing that the stoichiometry of the NrpRI ⁄ NrpRII com-
plexes is not affected by 2-oxoglutarate.
Purified MBP–NrpRI and (His)
6
–NrpRII were fur-
ther analysed by gel-filtration, which demonstrated
that MBP–NrpRI elutes in a single peak corresponding
to a molecular mass of > 700 kDa (Fig. S1A left
panel). As monomeric MBP–NrpRI has a molecular
mass of 70 kDa, the native protein appears to exist in
a higher oligomeric conformation, consisting of at least
10 subunits. Analysing MBP–NrpRI in the presence of
10 mm 2-oxoglutarate further indicated that the pres-
ence of 2-oxoglutarate may negatively effect the stabil-
ity of higher oligomeric NrpRI conformations to some
extent (Fig. S1B). Purified (His)
6
–NrpRII eluted in a
single elution peak corresponding to a molecular mass
of at least 700 kDa, and 2-oxoglutarate had no effect
(Fig. S1 CD). Taking the apparent molecular mass of
the monomer into account, the eluting (His)
6
–NrpRII
appears to be in a complex consisting of more than 23
monomers.

Promoter-binding assays
In order to analyse specific binding of NrpRI, NrpRII
and NrpRI ⁄ NrpRII complexes to nitrogen-regulated
A
1 2 3 4 5 6
86 kDa
34 kDa
MBP–NrpRI
(His)
6
–NrpRII
100 mM 250 mM
B
1 2 3 4 5 6
86 kDa
34 kDa
MBP–NrpRI
(His)
6
–NrpRII
C
c 1 2 3 4 5
c 1 2 3 4 5
MBP–NrpRI
(His)
6
–NrpRII
86 kDa
34 kDa
Fig. 1. Co-chromatography of MBP–NrpRI with (His)

6
–NrpRII. (A)
(His)
6
–NrpRII in 40 mg of cell extract was immobilized to 1 mL of
Ni-nitrilotriacetic acid agarose, and 40 mg of cell extract containing
MBP–NrpRI was added. After washing with buffer A containing
20 m
M imidazole, (His)
6
–NrpRII and potentially interacting proteins
were eluted in the presence of 100 and 250 m
M imidazole, each in
three 0.5-mL fractions. (B) MBP–NrpRI in 40 mg of crude extract
was immobilized to 1 mL of amylose resin, and 40 mg of cell
extract containing (His)
6
–NrpRII was added. After washing with
MBP buffer, MBP–NrpRI and potential interaction partners were
eluted in the presence of 10 m
M maltose (in six, 0.5-mL fractions).
(C) Controls. Upper panel, 40 mg of crude extract containing MBP–
NrpRI was incubated with 1 mL of Ni-nitrilotriacetic acid agarose
for 1 h. Wash and elution steps were performed as described for
(A). Lower panel, 40 mg of crude extract containing (His)
6
-NrpRI
was incubated with 1 mL of amylose resin for 1 h, washed and
eluted as described for (B). The elution fractions were analysed by
western blotting using antibodies directed against the His-tag and

the MBP–fusion. Protein detection and quantification were
performed using the ECL Plus system (GE-Healthcare, Munich,
Germany), a fluoroimager (DianaIII; Raytest, Straubenhardt,
Germany) and the AIDA Image Analyzer, as described in the
Materials and methods. (A) Lanes 1–3, elution fractions 1–3 in the
presence of 100 m
M imidazole; lanes 4–6, elution fractions 4–6 in
the presence of 250 m
M imidazole. (B) Lanes 1–6, elution fractions
1–6 in the presence of 10 m
M maltose. (C) Upper panel: c, 0.5 lg
of purified MBP–NrpRI; lane 1, flow-through; lanes 2 and 3, wash
fractions; lane 4, combined elution fractions (100 m
M imidazole);
lane 5, combined elution fractions (250 m
M imidazole). Lower
panel: c, 0.5 lg of purified (His)
6
–NrpRII; lane 1, flow-through; lanes
2 and 3, wash fractions; lane 4, combined elution fractions 1–3;
lane 5, combined elution fractions 4–6.
Role of NrpRII in M. mazei K. Weidenbach et al.
4400 FEBS Journal 277 (2010) 4398–4411 ª 2010 The Authors Journal compilation ª 2010 FEBS
promoters, promoter-binding assays were performed
by DNA affinity chromatography and electrophoretic
gel mobility shift assays (EMSAs). Biotinylated PCR
products carrying the nifH promoter region containing
the operator (ACC-GGCTTCC-GGT) or mutated ver-
sions, and the BRE and TATA-box that bind general
transcription factors, were linked to streptavidin-

coated magnetic beads, as recently described [32].
First, dialysed E. coli extracts containing MBP–NrpRI
were incubated with the magnetic beads, and proteins
bound nonspecifically were removed from the nifH
promoter by several elution steps at low-salt concen-
trations (Figs 3 and 4, lanes 1–4). Specifically bound
proteins were then eluted in the presence of 1.0 m
NaCl (Figs 3 and 4, lanes 5, 6). Western blot analysis
of the elution fractions verified that MBP–NrpRI
binds specifically to the wild-type operator (ACC-
GGCTTCC-GGT) and elutes exclusively in the pres-
ence of 1.0 m NaCl (Fig. 3A, lane 6), whereas in the
case of the mutated operators M1 (AAA-GGCTTCC-
GGT) and M2 (AAA-GGCTTCC-CCT), the majority
of MBP–NrpRI eluted at 250 and 500 mm NaCl
(Fig. 3A, M1 and M2, lanes 1–4), indicating that bind-
ing of MBP–NrpRI is significantly affected by these
mutations. The elution of NrpRI from the different
operators was quantified and is depicted in Fig. 3B.
To determine whether 2-oxoglutarate affects the bind-
ing affinity of NrpRI to the operator, promoter-bind-
ing assays were performed in the presence of 2-
oxoglutarate. Cell extracts containing MBP–NrpRI
were supplemented with 2 or 10 mm 2-oxoglutarate,
incubated with the magnetic beads coupled to the
wild-type nifH promoter, and washing plus elution
steps were performed with the buffers supplemented
with the respective 2-oxoglutarate concentration.
Quantification of NrpRI in the respective elution frac-
tions by western blot analysis demonstrated that in the

presence of 2 mm 2-oxoglutarate, NrpRI eluted at low
salt concentrations (Fig. 3C middle panel, lanes 1–4;
and Fig. 3D), indicating that binding of NrpRI to the
operator is significantly decreased, whereas no binding
at all was obtained in the presence of 10 mm 2-oxo-
glutarate (Fig. 3C bottom panel, lanes 1–6; and
Fig. 3D).
Next, binding of NrpRI to the nifH promoter in the
presence of NrpRII was analysed. E. coli cell extracts
containing MBP–NrpRI and (His)
6
–NrpRII were com-
bined in a 1 : 1 ratio and promoter-binding assays
were performed as described above. Western blot anal-
ysis demonstrated binding of (His)
6
–NrpRII to the
nifH promoter exclusively in the presence of NrpRI
(Fig. 4B,C), indicating that NrpRII binds to the opera-
tor indirectly by formation of a complex with NrpRI.
Furthermore, NrpRI binding to the promoter in
MBP–NrpRI
(His)
6
–NrpRII
c
1
c
2
c

3
123
c
1
c
2
c
3
1 2 3
NrpRII/NrpRI (mol·mol
–1
)
Without 2-OG
+ 2 m
M
2-OG
+ 10 m
M
2-OG
0
1
2
3
4
A
B
Fig. 2. Co-chromatography of MBP–NrpRI with (His)
6
–NrpRII in the presence of 2-oxoglutarate. Sixty milligrams of cell extract containing
(His)

6
–NrpRII and 60 mg of cell extract containing MBP–NrpRI were combined and separated into three aliquots. After adding 0, 2 or 10 mM
2-oxoglutarate, the cell extracts were incubated with 0.5 mL of Ni-nitrilotriacetic acid agarose for 1h. Then, the matrix was washed with
10 mL of buffer A containing 20 m
M imidazole (control), or 20 mM imidazole + 2 or 10 mM 2-oxoglutarate, and (His)
6
–NrpRII and potentially
interacting proteins were eluted in the presence of 250 m
M imidazole (1.5 mL) supplemented with the corresponding 2-oxoglutarate concen-
trations in three, 0.5-mL fractions. The respective elution fractions 1–3 were combined and analysed by western blotting using antibodies
directed against the His-tag and the MBP-fusion. Protein quantification was performed using the ECL-System (GE Healthcare, Mu
¨
nchen,
Germany), a fluoroimager (DianaIII, Raytest, Straubenhardt, Germany) and the AIDA Image Analyser. (A) The relative amount of MBP–NrpRI
bound to (His)
6
–NrpRII was calculated as described in the Materials and methods. The respective standard deviations of at least two to three
independent experiments are indicated. (B) Exemplary original western blotting data. Lanes c
1
–c
3
, 0.125, 0.25 and 0.5 lg of purified (His)
6
–
NrpRII (lower panel) and 0.125, 0.25 and 0.5 lg of purified MBP–NrpRI (upper panel); elution in the absence of 2-oxoglutarate (lane 1), and
in the presence of 2 m
M (lane 2) or 10 mM (lane 3). 2-oxoglutarate. 2-OG, 2-oxoglutarate.
K. Weidenbach et al. Role of NrpRII in M. mazei
FEBS Journal 277 (2010) 4398–4411 ª 2010 The Authors Journal compilation ª 2010 FEBS 4401
complex with NrpRII was also shown to be highly sen-

sitive to 2-oxoglutarate (Fig. 4D).
Binding of NrpRI to the operator sequence was fur-
ther verified by EMSA, as described in the Materials
and methods, using a radiolabeled PCR product of the
glnK
1
promoter, including the BRE- and TATA-box,
which has been shown to be under direct NrpRI con-
trol [32]. As demonstrated for the nifH promoter, puri-
fied MBP–NrpRI bound to the glnK
1
–promoter,
resulting in a distinct shift of the promoter (Fig. 5A,
lanes 3–5; Fig. 5B, lane 2), whereas purified (His)
6
–
NrpRII did not (Fig. 5B, lane 1). However, combining
MBP–NrpRI (10 lg) with (His)
6
–NrpRII (10 lg)
before the analysis resulted in a single second promi-
nent shift (Fig. 5B, lane 3), which was not detectable
in the presence of exclusively MBP–NrpRI (Fig. 5B,
lane 2), further verifying formation of a complex
between NrpRI and NrpRII. The presence of 10 mm
2-oxoglutarate significantly decreased the binding of
MBP–NrpRI to the operator as well as the binding of
the MBP–NrpRI ⁄ (His)
6
–NrpRII complex (Fig. 5B,

lanes 4 and 5, respectively).
NrpRII binding properties to general archaeal
transcription factors
To allow more detailed insights into the molecular
repression mechanism of nitrogen-regulated promoters,
we studied the binding properties of NrpRII to the
general transcription factors – TATA-binding protein
(TBP) and the archaeal homolog of the eukaryotic
general transcription factor TFIIB (TFB). MBP–
NrpRII, the three M. mazei (His)
6
–TBPs and (His)
6
–
TFB were individually expressed in E. coli and co-
chromatography was performed using amylose resin
(see the Materials and methods). MBP–NrpRII and
potentially interacting (His)
6
-tagged proteins were
analysed using quantitative western blotting. Interest-
ingly, all three TBPs co-eluted with MBP–NrpRII
(Fig. 6A–C), and the molar ratio of the NrpRII : TBP
complexes was approximately 1 : 0.6, as exemplarily
determined for TBP2 (see Fig. 7). Complex formation
between NrpRII and TBPs was further confirmed by
retaining the native TBPs from M. mazei cell extracts
by Ni-nitrilotriacetic acid-immobilized (His)
6
–NrpRII

12 3 4 5 6
123456
WT
WT
M1
M1
M2
M2
250 m
M
500 m
M
1
M
NaCl
250 m
M
500 m
M
1
M
NaCl
250 m
M
500 m
M
1
M
NaCl
100%

MBP–NrpRI eluting
at 1
M
NaCl (%)
0
50
100
0
50
100
0
0
0
0
0 0
100%
74.5 ± 1.4%
8.3 ± 1.7%
35.6 ± 1.5%
25.5 ± 1.2%
MBP–NrpRI (%)

2 m
M
10 m
M
2-OG
cc
2
c

3
A
B
[2-OG]
D
1
C
Fig. 3. DNA affinity chromatography of NrpRI. The nifH promoter (264 bp) or mutant derivatives including the BRE and TATA boxes as well
as the operator motifs [wild-type (WT), ACC-GGCTTCC-GGT; mutant 1 (M1), A
AA-GGCTTCC-GGT; and mutant 2 (M2), AAA-GGCTTCC-CCT]
were coupled to magnetic beads (Dynal, Oslo, Norway). DNA affinity chromatography was performed using crude extracts of Escherichia coli
containing MBP–NrpRI, as described in the Materials and methods. (A) Western blot analysis of the respective elution fractions (20 lL) of
DNA affinity chromatography (WT, M1 and M2) using antibodies directed against the MBP-fusion. The original data are representative of one
single experiment. (B) Quantification of MBP–NrpRI was performed as described in Fig. 1. The amount of MBP–NrpRI bound to the wild-
type operator was set to 100%. The respective standard deviations of three independent experiments are indicated. (C) DNA affinity chro-
matography was performed in the presence of 2-oxoglutarate. Elution fractions were precipitated with trichloroacetic acid and analyzed by
western blotting. Lanes c
1
–c
3
, controls containing 0.125, 0.25 or 0.5 lg of MBP–NrpRI; lanes 1 and 2, elution fractions in the presence of
250 m
M NaCl; lanes 3 and 4, elution fractions in the presence of 500 mM NaCl; lanes 5 and 6, elution fractions in the presence of 1 M NaCl.
The original data represent the results of a single experiment. (D) Quantification of MBP–NrpRI of three independent experiments. The over-
all amounts of MBP–NrpRI eluting in the absence of 2-oxoglutarate [first column (grey) at the respective NaCl concentration], in the presence
of 2 m
M 2-oxoglutarate [second column (dark) at the respective NaCl concentration, dark] or in the presence of 10 mM 2-oxoglutarate [third
column (dark) at the respective NaCl concentration], were set to 100%. 2-oxoglutarate. 2-OG, 2-oxoglutarate.
Role of NrpRII in M. mazei K. Weidenbach et al.
4402 FEBS Journal 277 (2010) 4398–4411 ª 2010 The Authors Journal compilation ª 2010 FEBS

(data not shown). In addition, complex formation
between NrpRII and TFB was demonstrated; however,
the interactions between NrpRII and TFB appeared to
be weaker, given that NrpRII : TFB molar ratios of
1 : 0.3 were obtained (Fig. 7). Control experiments
ruled out the possibility that the three TBPs and TFB
bind nonspecifically to the amylose resin in the absence
of NrpRII. Co-chromatography with MBP–NrpRII
and (His)
6
–GlnK
1
further ruled out that MBP–NrpRII
binds specifically to the (His)
6
-tag of a fusion protein
(data not shown). These findings confirmed that
NrpRII is able to bind all three TBPs efficiently and to
bind TFB less efficiently. Finally, the potential effects
of 2-oxoglutarate on complex formation between
NrpRII and the general transcription factors were
studied. In the exponential phase the respective genes
of the three TBPs were expressed and induced under
nitrogen limitation, as demonstrated by quantitative
RT-PCR analysis (see Table 1). Thus, we exemplarily
analysed the influence of 2-oxoglutarate on the binding
of NrpRII to TBP2 and TFB. As depicted in Fig. 7,
the presence of 10 mm 2-oxoglutarate significantly
decreased the formation of a complex with NrpRII
and either of these transcription factors to approxi-

mately 20.5% (TBP2) and 36.6% (TFB).
Studying potential interactions between MBP–NrpRI
and the three TBPs and TFB demonstrated that NrpRI
also binds these general transcription factors, which
may occur through the conserved NrpR domains of
NrpRI and NrpRII; however, the stoichiometries of
the complexes in the case of NrpRI were significantly
lower and represented < 2% of the respective NrpRII
complexes (data not shown).
Discussion
NrpRII interacts simultaneously with NrpRI and
the general transcription factors
We recently demonstrated that the DNA-binding
homolog, NrpRI, plays a major role in nitrogen regu-
lation, acting as a repressor binding to its operator
under nitrogen sufficiency [31,32]. In order to analyse
the physiological role of the second NrpR homolog,
NrpRII, and elucidate the requirement of NrpRII for
full repression of the nitrogen-regulated genes in
M. mazei, formation of a complex between NrpRI
and NrpRII was investigated. Using two independent
approaches, we obtained conclusive experimental evi-
dence that NrpRII interacts and forms stable com-
plexes with NrpRI in M. mazei. In the first approach,
co-chromatography demonstrated that immobilized
(His)
6
–NrpRII interacts directly with NrpRI, which
was confirmed by reverse co-chromatography (Fig. 1).
In the second approach, because of the missing HTH

domain, NrpRII is not able to bind to nifH or glnK
1
promoters; however, in complex with NrpRI, binding
to nifH and glnK
1
promoters was demonstrated in
two independent promoter-binding assays (Figs 4
and 5).
Recent in silico analysis revealed that euryarchaeal
NrpR homologs exist in three different domain configu-
rations (see Fig. S2): one with two tandem NrpR
domains fused to an N-terminal HTH domain (e.g.
M. maripaludis NrpR); one with a single NrpR domain
and an N-terminal HTH domain (e.g. NrpRI of
M. mazei and M. acetivorans); and one with a single
NrpR domain (e.g. NrpRII of M. mazei and M. acetivo-
rans) [20,21,26]. Furthermore, genetic complementation
c
1
c
2
c
3
A
B
C
250 m
M
500 m
M

1
M
NaCl
D
NrpRI
NrpRII
NrpRI
+
NrpRII
NrpRI
+
NrpRII
2-OG
c
1
c
2
c
3
c
1
c
2
c
3
c
1
c
2
c

3
1234 56
123456
1 2 3 4 5 6
1 2 3 4 5 6
c
1
c
2
c
3
NrpRI
NrpRII
NrpRI
+
NrpRII
NrpRI
+
NrpRII
2 m
M
2-OG
c
1
c
2
c
3
c
1

c
2
c
3
c
1
c
2
c
3
Fig. 4. Promoter-binding assays using NrpRI and NrpRII. DNA-affin-
ity chromatography was performed using the nifH promoter cou-
pled to magnetic beads and different cell extracts. (A) DNA-affinity
chromatography was performed with crude extracts from Escheri-
chia coli containing MBP–NrpRI; elution fractions were analysed by
western blotting using antibodies directed against the MBP-fusion.
(B) DNA-affinity chromatography using E. coli crude extract contain-
ing (His)
6
–NrpRII; the elution fractions were analysed by western
blotting using antibodies directed against the His-tag. E. coli cell
extracts containing MBP–NrpRI and (His)
6
–NrpRII were combined
in a 1 : 1 ratio and analysed by DNA-affinity chromatography in the
absence (C) or presence of 2 m
M 2-oxoglutarate (D); western blot
analysis was performed using antibodies directed against the MBP-
fusion (upper panel) or against the His-tag (lower panel). M, pre-
stained high-molecular-weight marker (MBI Fermentas, St Leon-

Rot, France) Lanes c
1
–c
3
, controls, 0.125, 0.25 or 0.5 lg of MBP–
NrpRI and 0.125, 0.25 or 0.5 l g of (His)
6
–NrpRII, respectively; elu-
tion fractions in the presence of 250 m
M NaCl (lanes 1 and 2),
500 m
M NaCl (lanes 3 and 4), 1 M NaCl (lanes 5 and 6). 2-oxogluta-
rate. 2-OG, 2-oxoglutarate.
K. Weidenbach et al. Role of NrpRII in M. mazei
FEBS Journal 277 (2010) 4398–4411 ª 2010 The Authors Journal compilation ª 2010 FEBS 4403
of an M. maripaludis nrpR mutant strain demonstrated
that in order to restore regulated repression of the nif
promoter, both identified NrpR homologs of M. acetiv-
orans had to be co-expressed [26], strongly indicating
that both NrpR homologs are simultaneously required
to efficiently inhibit RNA polymerase recruitment. Tak-
ing those results and our finding of stable complex for-
mation between NrpRII and NrpRI into account, we
propose that in M. mazei, and presumably also in
M. acetivorans, NrpRII takes over the function of the
second NrpR domain of M. maripaludis NrpR. We fur-
ther hypothesize that preformed NrpRI ⁄ NrpRII com-
plexes bind to nitrogen-regulated promoters, or
alternatively NrpRII binds from solution to NrpRI
already bound to its operator.

Both potential mechanisms of NrpRI ⁄ NrpRII com-
plex binding to the operator strongly indicate that the
general archaeal transcription factors (TBP and TFB)
may be involved in tethering NrpRII or the
NrpRI ⁄ NrpRII heterooligomeric complex to the pro-
moter, which ultimately results in complete inhibition
of RNA polymerase recruitment to the promoter.
Indeed, we obtained strong evidence by various
co-chromatography experiments that NrpRII interacts
with the three TBPs present in M. mazei, which are all
expressed under nitrogen sufficiency, and interacts in
BA
c
1
12345
MBP-NrpRI
c
1
c
2
1 2 3 4 5
2-OG
c
1
12345
MBP–NrpRI
c
1
c
2

1 2 3 4 5
2-OG
NrpRI
NrpRII
N
rpRI+II
N
rpR
NrpRI+II
Fig. 5. EMSAs of the glnK
1
promoter by MBP–NrpRI and (His)
6
–NrpRII. (A)
32
P-labelled DNA fragments (50 ng) of the glnK
1
promoter were
incubated without additions (c
1
) and with purified MBP–NrpRI (2.5, 5, 10, 15 and 20 lg) (lanes 1–5, respectively). (B) Effects of 2-oxogluta-
rate: 2 m
M 2-oxoglutarate was added to the assays as indicated. c
1
and c
2
,
32
P-labelled glnK
1

promoter, with and without 2-oxoglutarate,
respectively; lane 1, 10 lg of (His)
6
–NrpRII; lane 2, 10 lg of MBP–NrpRI; lane 3, 10 lg of MBP–NrpRI + 10 lg of (His)
6
–NrpRII; lane 4,
10 lg of MBP–NrpRI + 2 m
M 2-oxoglutarate; and lane 5, 10 lg of MBP–NrpRI, 10 lg (His)
6
–NrpRII + 2 mM 2-oxoglutarate. The original shift
assay data are representative of at least three independent experiments. 2-oxoglutarate. 2-OG, 2-oxoglutarate.
A
MBP–NrpRII
(His)
6
–TBP1
86 kDa
25 kDa
1 2 3 4 5 6 7
C
(His)
6
–TBP3
MBP–NrpRII
86 kDa
25 kDa
1 2 3 4 5 6 7
B
(His)
6

–TBP2
MBP–NrpRII
86 kDa
25 kDa
1 2 3 4 5 6 7
D
(His)
6
–TFB
MBP–NrpRII
86 kDa
50 kDa
1 2 3 4 5 6 7
MBP–NrpRII
(His)
6
–TBP1
86 kDa
25 kDa
1 2 3 4 5 6 7
(His)
6
–TBP3
MBP–NrpRII
86 kDa
25 kDa
1 2 3 4 5 6 7
B
(His)
6

–TBP2
MBP–NrpRII
86 kDa
25 kDa
1 2 3 4 5 6 7
D
(His)
6
–TFB
MBP–NrpRII
86 kDa
50 kDa
1 2 3 4 5 6 7
(His)
6
–TBP2
MBP–NrpRII
86 kDa
25 kDa
1 2 3 4 5 6 7
(His)
6
–TFB
MBP–NrpRII
86 kDa
50 kDa
1 2 3 4 5 6 7
Fig. 6. Co-elution of MBP–NrpRII and (His)
6
–TBP or (His)

6
–TFB. Fifty milligrams of cell-free extract containing MBP–NrpRII was combined
with 50 mg of cell-free extract containing (His)
6
–TBP1 (A), (His)
6
–TBP2 (B), (His)
6
–TBP3 (C) or (His)
6
–TFB (D) and incubated with 0.5 mL of
amylose resin. After washing with MBP-buffer, MBP–NrpRII and potential interacting partners were eluted in the presence of 10 m
M malt-
ose (seven, 0.5-mL fractions). The elution fractions were analysed by western blotting using antibodies directed against the MBP-fusion
(upper panels) and the His-tag (lower panels). (A–D) Lanes 1–7, elution fractions 1–7 in the presence of 10 m
M maltose.
Role of NrpRII in M. mazei K. Weidenbach et al.
4404 FEBS Journal 277 (2010) 4398–4411 ª 2010 The Authors Journal compilation ª 2010 FEBS
addition also with TFB, albeit less efficiently (Fig. 6).
This is, to our knowledge, the first report of interac-
tions between an archaeal repressor and the general
archaeal transcription factors TBP and TFB besides
the recent documentation of interactions between the
transcriptional activator GvpE and all five TBPs of
Halobacterium salinarium [33].
In analogy, these findings suggest that the second
NrpR domain of M. maripaludis NrpR also contacts
the general transcription factors when bound to its
operator via the DNA-binding domain. In a recent
genetic approach, Leigh and Lie showed that changing

amino acid 390 located in the second NrpR domain of
M. maripaludis NrpR (S390A) resulted in a complete
loss of repression in M. maripaludis, whereas the muta-
tion of the corresponding amino acid in the first NrpR
domain (S168A) retained a normal regulation [22]. If
the second NrpR domain of M. maripaludis NrpR
indeed contacts the general transcription factors, this
may indicate that S390 of M. maripaludis NrpR and
potentially also the homologus amino acid in M. mazei
NrpRII (S92) are essential for the interaction with
TBP and ⁄ or TFB. As Methanopyrus kandleri contains
a sole NrpR consisting of one single NrpR domain
with a N-terminal HTH domain [26], it is of specific
interest to elucidate whether the single NrpR domain
of M. kandleri is also binding to the general transcrip-
tion factors.
TBP2/NrpRII (mol·mol
–1
)
12
–
+
2-OG
12
+
2-OG
c
1
c
2

c
3
1 2 c
1
c
2
c
3
1 2
MBP-
NrpRII
(His)
6
(His)
6
+
2-OG
– +
2-OG
c
1
c
2
c
3
1 2 c
1
c
2
c

3
1 2
MBP-
NrpRII
(His)
6
–TBP2
(His)
6
–TFB
A
+
2-OG
–
+
2-OG
+
2-OG
+
2-OG
12
+
2-OG
TFB/NrpRII (mol·mol
–1
)
12
–
+
2-OG

0
0.2
0.4
0.6
B
12
+
2-OG
12
+
2-OG
12
+
2-OG
0
0.2
0.4
0.6
12
+
2-OG
12
+
2-OG
Fig. 7. Effects of 2-oxoglutarate on complex formation between NrpRII and general transcription factors. Fifty milligrams of crude extract
containing MBP–NrpRII was incubated with 50 mg of crude extract containing (His)
6
–TBP2 or (His)
6
–TFB in the absence ()) or presence of

10 m
M 2-oxoglutarate (+). To each mixture, 0.5 mL of amylose resin was added. After washing with MBP buffer or MBP buffer containing
10 m
M 2-oxoglutarate, MBP–NrpRI and (His)
6
–TBP2 or (His)
6
–TFB were eluted using MBP buffer supplemented with 10 mM maltose in the
absence ()) or presence of 10 m
M 2-oxoglutarate (+). Pools of the elution fractions 1–4 were analysed by western blotting using antibodies
directed against the MBP-fusion (upper panel) and the His-tag (lower panel) (A). Lanes c
1
–c
3
, 0.05, 0.125 or 0.25 lg of MBP–NrpRII and
0.05, 0.125 or 0.25 lg of (His)
6
–TBP2 or (His)
6
–TFB, respectively; lane 1, combined elution fractions 1–4 purified in the absence of 2-oxoglut-
arate; and lane 2, combined elution fractions 1–4 purified in the presence of 10 m
M 2-oxoglutarate. Protein quantification was performed
using the ECL Plus system (see Fig. 1). The relative amount of MBP–NrpRII binding to (His)
6
–TBP2 or to (His)
6
–TFB in the absence or pres-
ence of 10 m
M 2-oxoglutarate was calculated, and the respective standard deviations of at least three independent experiments are indi-
cated (B). 2-oxoglutarate. 2-OG, 2-oxoglutarate.

Table 1. Relative transcript levels of MM1028, MM2184 and MM1772 under nitrogen limitation versus nitrogen sufficiency and at different
growth phases determined by quantitative RT-PCR analysis.
ORF number
(according to
a previous
publication [46]) Protein [46]
Fold regulation
a
N
2
vs. NH
4
þ
exponential
growth phase
Exponential
vs. lag
phase (NH
4
þ
)
Exponential vs.
stationary growth
phase (NH
4
þ
)
MM1028
b
MM1027

TBP2
TBP1
2.2 ± 0.3 1.6 ± 0.4 0.8 ± 0.2
MM2184 TBP3 2.1 ± 0.4 1.6 ± 0.5 0.4 ± 0.1
MM1772 TFB 1.1 ± 0.5 2.8 ± 1.6 1.4 ± 0.1
a
Data and respective standard deviations are representative of at least three independent experiments.
b
MM1028 and MM1027 are orga-
nized in an operon, as indicated.
#
K. Weidenbach et al. Role of NrpRII in M. mazei
FEBS Journal 277 (2010) 4398–4411 ª 2010 The Authors Journal compilation ª 2010 FEBS 4405
2-Oxoglutarate directly affects NrpRI binding to
the operator and NrpRII binding to the general
transcription factors
Increasing amounts of the intracellular nitrogen metab-
olite, 2-oxoglutarate, under nitrogen limitation have
been demonstrated to modulate a variety of nitrogen
sensors and regulators and have been proposed to
provide the intracellular signal for nitrogen limitation
in cyanobacteria and methanogenic archaea, microor-
ganisms with an incomplete oxidative tricarboxylic
acid cycle (TCA) cycle that has excluxively anabolic
functions [16,23–25,34,35]. Consistent with this, the
binding affinity of M. mazei NrpRI to its operator has
been shown to be strongly affected in the presence of
2-oxoglutarate (Figs 3 and 5) potentially resulting in
transcription initiation under nitrogen-limiting condi-
tions, as recently demonstrated for M. maripaludis

NrpR by Leigh and coworkers [21,22]. Moreover, this
negative effect of 2-oxoglutarate on binding to the
operator was also observed for NrpRI bound to the
nifH promoter in complex with NrpRII (Figs 4C,D
and 5B), indicating that the 2-oxoglutarate-binding
site(s) are still accessible in the NrpRI ⁄ NrpRII com-
plex. For M. maripaludis NrpR it was recently shown
that both NrpR domains participate and are required
for the full 2-oxoglutarate response. Evidence was
obtained that the conserved amino acids C148 and
L195 located in the first, and C389 and H435 located
in the second, NrpR domain are essential for the 2-
oxoglutarate response, either by directly binding 2-oxo-
glutarate or responding with a conformational change
that decreases DNA binding [22]. Thus, it is tempting
to speculate that the corresponding amino acids in the
two M. mazei NrpR proteins (NrpRI C166 and I213;
NrpRII C91 and L138; see Fig. S2) are required for
the 2-oxoglutarate response in M. mazei.
Moreover, we obtained strong evidence that 2-oxo-
glutarate simultaneously affects the binding affinity
of NrpRII to the general transcription regulators,
ultimately leading to complete dissociation of the
hetero-oligomeric NrpRI ⁄ NrpRII complex from the
promoter, allowing transcription initiation.
Hypothetical model for translational regulation of
nitrogen-regulated genes in M. mazei
Based on our findings, we propose the following work-
ing model of nitrogen regulation in M. mazei, which is
depicted in Fig. 8. Under nitrogen sufficiency, NrpRII

binds from solution to the nifH promoter by simulta-
neously contacting NrpRI and the general transcrip-
tion factors, resulting in full repression of
transcription. External nitrogen limitation is perceived
by sensing the internal concentration of 2-oxogluta-
rate, which increases owing to reduced consumption
by the ammonium-dependent glutamine synthetase ⁄
glutamine oxoglutarate aminotransferase (GS/GOGAT)
way. Under these conditions, the binding of 2-oxoglut-
arate to the NrpRI ⁄ NrpRII complex decreases the
binding affinity of NrpRI to the operator as well as
decreasing the NrpRII binding affinity to the general
transcription factors, potentially by inducing a confor-
mational change of the protein which ultimately leads
to the removal of the NrpRI ⁄ NrpRII complex. This
finally allows the recruitment of RNA polymerase to the
promoter and transcription initiation.
Based on the recent finding that nitrogen-regulated
promoters have similar BRE- and TATA-boxes, which
differ significantly from the consensus sequence in
M. mazei [36], we propose that in the absence of
NrpRI, NrpRII is able to inhibit the transcription of
N-regulated genes to some extent by binding the
general transcription factors from solution and thus
TATABRE
ACC GGT
TBP
NrpRI
NrpRII
TFB

RNAP
TATABRE
ACC GGT
TBP
NrpRI
NrpRII
TFB
RNAP
N
2
ACC GGT
TATABRE
TBP
TFB
2-OG
2-OG
NrpRII
NrpRI
NH
4
+
2-OG
2-OG
N
2
TATABRE
ACC GGT
NrpRII NrpRI
N
2

N
2
ACC GGT
TATABRE
TBP
TFB
2-OG
2-OG
NrpRII
NrpRI
ACC GGT
TATABRE
TBP
TFB
2-OG
2-OG
NrpRII
NrpRI
NH
4
+
NH
4
+
2-OG
2-OG
2-OG
2-OG
N
2

N
2
TATABRE
ACC GGT
NrpRII NrpRI
TATABRE
ACC GGT
NrpRII NrpRI
TFB
RNAP
TBP
TFB
RNAP
TBP
TFB
RNAP
TBP
Fig. 8. Hypothetical model for NrpR-mediated repression in M. mazei. Under nitrogen sufficiency, complexes of NrpRI and NrpRII bind to
the operator, inhibiting recruitment of the RNA polymerase (RNAP) thus resulting in repression of transcription. Under nitrogen limitation,
increased 2-oxoglutarate (2-OG) levels decrease the binding affinity of NrpRI to the operator as well as the binding affinity of NrpRII to TBP
and TFB, ultimately leading to the removal of the NrpRI ⁄ NrpRII complex, allowing RNAP recruitment and transcription.
Role of NrpRII in M. mazei K. Weidenbach et al.
4406 FEBS Journal 277 (2010) 4398–4411 ª 2010 The Authors Journal compilation ª 2010 FEBS
affecting efficient binding of the general transcription
factors to the BRE- and TATA-boxes, resulting in a
decrease of RNA polymerase recruitment to the
respective nitrogen-regulated promoter. This scenario
may explain the residual nitrogen regulation (approxi-
mately 10%) in a DnrpRI mutant [32]; however, this
has still to be experimentally proven.

Materials and methods
Strains and plasmids
The strains and plasmids used in this study are listed in
Table S1. Plasmid DNA was transformed into E. coli
according to the method of Inoue [37].
Construction of plasmids
pRS236 and pRS524 were constructed as follows. MM1969
encoding NrpRII was amplified from M. mazei genomic
DNA using Taq polymerase and primers (5¢-TTA
CTGGAGGGTT
CATATGC; 5¢-AACAGACTCGAGTTT
CAGAC) that added flanking NdeI and XhoI sites (under-
lined). The 756-bp PCR fragment was cloned into the NdeI
and XhoI sites of the expression vector pET28a (Novagen,
Madison, WI, USA) yielding pRS236 and fusing six histi-
dine codons in front of the nrpRII start codon (N-terminal
His-tag). MM1969 was amplified with the primer set
MM1969for (5¢-C
CTGCAGTTACTGGAGGGTTG) and
MM 1969rev (5¢-GGTTCAAACAGA
CTGCAGTTTCAG),
which added flanking PstI sites (underlined). The 760-bp
PCR fragment was cloned into the PstI site of pMalC2
(New England Biolabs, Ipswich, MA, USA) fusing malE –
which encodes the E. coli maltose-binding protein – in front
of MM1969. The correct insertion and sequence were con-
firmed by DNA sequencing, and the resulting plasmid was
designated pRS524.
Plasmids for heterologus expression of M. mazei TBPs
and the archaeal homolog of the eukaryotic TFB were con-

structed as follows: MM1027 (TBP1) and MM1028 (TBP2)
coding for two out of the three TBPs present in M. mazei,
and MM1772 coding for TFB, were amplified from chro-
mosomal DNA using the primer sets (Mm1027 His.for
5¢-GGGTGGA
CATATGAGCGAATC ⁄ Mm1027 His.rev
5¢-GTTTG
AAGCTTTTATAAAAGACCCATAC; Mm1028
His.for 5¢-GGTTGA
CATATGAGCGAATC ⁄ Mm1028 His.
rev 5¢-G
AAGCTTCTTATAAAAGCCCC; and Mm 1772
His.for 5 ¢-GGTGATAT
CATATGGTAGAAGTCG ⁄ Mm 1772
His.rev 5¢-GAAGA
AAGCTTTAGAGGATAATCTCG) add-
ing flanking NdeI and HindIII sites (underlined). The 550-
bp TBP fragments and the 1040-bp TFB fragment were
cloned into the NdeI and HindIII sites of the expression
vector pET28a. This resulted in fusing six histidine codons
upstream of the respective genes, ultimately resulting in an
N-terminal His-tag. The obtained plasmids were designated
pRS475 (MM1027), pRS231 (MM1028) and pRS476
(MM1772). MM2184 (TBP3) was amplified using primers
(Mm 2184 His.for 5¢-CCAAATACA
GGATCCATGGA-
ATCTAC and Mm 2184 His.rev 5¢-GA
GAATTCATTT-
AATAAAGAAGTCCTAAG) that added flanking BamHI
and EcoRI sites and facilitated cloning the 580-bp fragment

into the BamHI and EcoRI sites of pET28a, fusing six
histidine codons in front of MM2184. The resulting plasmid
was designated pRS469. Correct insertions and sequences
were in general confirmed by DNA sequencing of both
strands.
Cell extracts and protein purification
For heterologus expression and purification of M. mazei
(His)
6
–NrpRII, pRS236 was transformed into E. coli BL21-
CodonPlus
Ò
-RIL (Stratagene, La Jolla, CA, USA). One-
liter cultures were grown aerobically in Luria–Bertani (LB)
medium at 37 °C, and expression of His–NrpRII was
induced with 100 lm isopropyl thio-b-d-galactoside (IPTG)
respectively, when cells reached a turbidity of 0.6 at
600 nm. After a 2 h induction at 37 °C, the cells were
harvested and cell extracts were prepared by disruption in
buffer A (50 mm NaH
2
PO
4
, 300 mm NaCl, pH 8.0), supple-
mented with the protease inhibitor cocktail for bacterial cell
extracts (Sigma, Mu
¨
nchen, Germany), using a French pres-
sure cell at 4135 · 10
6

NÆm
)2
, followed by centrifugation at
20 000 g for 30 min. (His)
6
–NrpRII was purified from the
supernatant by Ni-affinity chromatography using Ni-nitrilo-
triacetic acid agarose (Qiagen, Hilden, Germany), according
to the manufacturer’s instructions. (His)
6
–NrpRII was
eluted from Ni-nitrilotriacetic acid agarose in the presence
of 100 and 250 mm imidazole, dialysed into buffer A and
stored at -70 °C.
One-liter cultures of E. coli BL21-CodonPlus
Ò
-RIL ⁄
pRS454 were grown in LB medium, and synthesis of MBP–
NrpRI was induced from pRS454 [32] for 2 h with 100 lm
IPTG at a turbidity of 0.6 at 600 nm. MBP–NrpRI was
purified by amylose affinity chromatography, as described
by Weidenbach et al. [32]. Samples of each purification
step were analysed by 12.5% SDS ⁄ PAGE, according to
Laemmli [38], and protein concentrations were determined
via the method of Bradford [39] with the Bio-Rad Labora-
tories (Bio-Rad Laboratories GmbH, Mu
¨
nchen, Germany)
protein assay using BSA as the standard.
Complex analysis by gel filtration

For gel filtration, an analytic Superdex 200 column (GE
Healthcare, Mu
¨
nchen, Germany) and buffer A (50 mm
Tris ⁄ HCl, 150 mm NaCl, pH 8.0) were used; proteins
were detected by monitoring the absorbance at 280 nm.
Protein was eluted from the column using a flow rate of
K. Weidenbach et al. Role of NrpRII in M. mazei
FEBS Journal 277 (2010) 4398–4411 ª 2010 The Authors Journal compilation ª 2010 FEBS 4407
0.5 mLÆmin
)1
, and 0.5-mL fractions were collected. Calibra-
tion of the column was performed using the gel-filtration
mass standard (Bio-Rad Laboratories) containing thyro-
globulin (670 kDa), IgG (158 kDa), ovalbumin (44 kDa),
myoglobulin (17 kDa) and vitamin B12 (1.35 kDa). Fifty
nanograms of purified MBP–NrpRI, or 50 lg of purified
(His)
6
–NrpRII, in the absence or presence of 10 mm 2-oxo-
glutarate, were loaded onto the Supderdex 200 column and
eluted with buffer A alone or buffer A supplemented with
10 mm 2-oxoglutarate, respectively. In order to analyse
complex formation between NrpRI and NrpRII, 50 lgof
purified MBP–NrpRI and 50 lg of purified (His)
6
–NrpRII
were incubated in a total volume of 200 lL of buffer A for
10 min at room temperature before applying to the column.
When analysing the effect of 2-oxoglutarate, the effector

molecule was added to a final concentration of 10 mm
followed by 10 min incubation at room temperature before
gel-filtration analysis, and buffer A was supplemented
with 2-oxoglutarate to the respective final concentration
(10 mm). Elution fractions were analysed by western
blotting.
Analyses of complex formation by
co-chromatography
To analyse whether NrpRI interacts with NrpRII, binding
assays using affinity chromatography were performed.
MBP–NrpRI and (His)
6
–NrpRII were individually
expressed in E. coli BL21-CodonPlus
Ò
-RIL, and the respec-
tive cell-free cell extracts were prepared as described above.
(His)
6
–NrpRII of 40 mg of cell-free cell extract was immo-
bilized to 1 mL of Ni-nitrilotriacetic acid agarose for
30 min at 4 °C. Next, 40 mg of cell-free cell extract contain-
ing MBP–NrpRI was applied to the Ni-nitrilotriacetic acid
agarose (Qiagen, Hilden, Germany) and incubated for 1 h
at 4 °C with slow shaking. Then, unbound protein was
washed from the columns two times with 8 mL of buffer A
supplemented with 20 mm imidazole, followed by elution of
(His)
6
–NrpRII and potentially interacting proteins in the

presence of 100 mm (in three, 0.5 mL fractions) and
250 mm imidazole (in three, 0.5 mL fractions). Reverse
chromatography was performed by immobilizing MBP–
NrpRI from 40 mg of cell-free cell extract to 1 mL of amy-
lose resin (New England Biolabs, Ipswich, MA, US) for
30 min at 4 °C. Then, 40 mg of cell-free cell extract con-
taining (His)
6
–NrpRII was added and further incubated for
1 h at 4 °C. The matrix was washed twice with 10 mL of
MBP–buffer (see above) followed by elution of MBP–
NrpRI and potentially interacting proteins in the presence
of 10 mm maltose in 3 mL (in six, 0.5 mL fractions). Aliqu-
ots of the wash and elution fractions of both co-chromatog-
raphy experiments were analysed by western blotting.
In order to study the effects of 2-oxoglutarate on com-
plex formation between NrpRI and NrpRII, 60 mg of cell-
free cell extract containing (His)
6
–NrpRII and 60 mg of
cell-free cell extract containing MBP–NrpRI were combined
and separated into three aliquots. Two aliquots were
supplemented with 2 mm (A) or 10 mm (B) 2-oxoglutarate
(final concentration) and the third aliquot was used as a
control (C). After the addition of 0.5 mL Ni-nitrilotriacetic
acid agarose to each aliquot, the binding assays were incu-
bated for 1 h at 4 °C in a column. After washing the resin
with 10 mL of buffer A containing 20 mm imidazole (C),
20 mm imidazole + 2 mm 2-oxoglutarate (A) or 20 mm
imidazole + 10 mm 2-oxoglutarate (B), (His)

6
–NrpRII and
potentially interacting proteins were eluted from the column
in the presence of 100 mm (1.5 mL) and 250 mm (1.5 mL)
imidazole supplemented with the corresponding concentra-
tions of 2-oxoglutarate in 0.5-mL fractions. The respective
elution fractions 1–3 were combined and analysed by
western blotting.
Potential complex formation between MBP–NrpRII and
the three TBPs and TFB was analysed by affinity chroma-
tography on amylose resin. MBP–NrpRII, the three (His)
6
–
TBPs and (His)
6
–TFB were individually expressed in E. coli
BL21-CodonPlus
Ò
-RIL and the respective cell-free extracts
were prepared as described above. Fifty milligrams of cell-
free extract containing MBP–NrpRII was combined with
50 mg of cell-free extracts containing (His)
6
–TBP (1–3) or
(His)
6
–TFB and incubated with 0.5 mL of amylose resin
for 60 min at 4 °C. After removing unbound protein with
10 mL of MBP buffer, MBP–NrpRII and potentially inter-
acting (His)

6
-tagged proteins were eluted in the presence of
10 mm maltose (7 · 0.5 mL) and analysed by western blot-
ting. No difference in complex formation was obtained
when MBP–NrpRII from 50 mg of cell-free extracts was
first immobilized to amylose resin and extensively washed
before adding 50 mg of cell extract containing the respec-
tive (His)
6
–TBP or (His)
6
–TFB. In order to study the
effects of 2-oxoglutarate on complex formation, the respec-
tive cell extracts were combined and split into two samples,
one of which was supplemented with 10 mm 2-oxoglutarate.
Co-chromatography was performed as described above, but
for the samples of complex formation in the presence of
2-oxoglutarate all buffers used during the purification were
supplemented with 10 mm 2-oxoglutarate.
Western blot analyses
After purification of potential complexes, proteins from the
respective elution fractions of the different co-chromatogra-
phy were separated on 12.5% denaturating polyacrylamide
gels and transferred to nitrocellulose membranes (Bio-
Trace
Ò
NT; Pall GmbH, Dreieich, Germany) [40]. Mem-
branes were exposed to specific antibodies directed against
MBP (New England Biolabs, Ipswich, MA, US) and the
His-tag (Qiagen, Hilden, Germany) in order to detect

MBP–NrpRI or MBP–NrpRII and (His)
6
-tagged versions
of NrpRII, TBPs and TFB, respectively. The membranes
were then washed and incubated with a second antibody
Role of NrpRII in M. mazei K. Weidenbach et al.
4408 FEBS Journal 277 (2010) 4398–4411 ª 2010 The Authors Journal compilation ª 2010 FEBS
that bound to mouse IgG. Protein bands were detected with
secondary antibodies directed against mouse IgG and cou-
pled to horseradish peroxidase (Bio-Rad Laboratories
GmbH, Mu
¨
nchen, Germany) and visualized using the ECL-
plus system (GE Healthcare, Mu
¨
nchen, Germany) with a
fluoroimager (DianaIII; Raytest, Straubenhardt, Germany).
The protein bands of the complexes were quantified from
at least three independent experiments using the DianaIII
software (Raytest, Straubenhardt, Germany) and known
amounts of the respective purified proteins, which were
simultaneously detected and quantified on the same mem-
brane for each experiment, as described recently [41]. Quan-
tification of purified proteins MBP–NrpRI and (His)
6
–
NrpRII was linear within absolute amounts of 0.125–0.5 lg
per lane. All protein quantifications were performed within
this linear range of the detection system. Degradation of
MBP–NrpRI in the elution fraction was frequently

observed, as was the case for purified standard proteins.
This degradation is based upon protein instability, even at
a low temperature. In the case of degradation, the fusion
protein and the major degradation product detected by the
immunoblot were quantified together.
Promoter-binding assays
Promoter binding was analysed by DNA-affinity chroma-
tography performed essentially as previously described
[32,42,43]. In short, the nifH promoter probe and mutant
derivatives were generated by PCR, as described by
Weidenbach et al. [32], using the primer set nifHfor and
nifH rev-bio tagged with biotin via a TEG linker (MWG-
Biotech AG, Ebersberg, Germany). Approximately
100 pmol of purified biotin-labeled PCR product was
coupled to 3 mg of Dynabeads-conjugated streptavidin (In-
vitrogen, Hamburg, Germany), and noncoupled DNA was
removed by magnetic separation according to the manufac-
turer’s protocol. The resulting coupled Dynabeads were
stored at 4 °C for a maximum of 7 days. Directly before
incubation with cell-free cell extracts containing MBP–
NrpRI or (His)
6
–NrpRII, the coupled Dynabeads were
equilibrated with 400 lL of binding buffer [10 mm
Tris ⁄ HCl (pH 7.5), 1 mm EDTA, 1 mm dithiothreitol] for
2 min. Cells of 1-L cultures containing MBP–NrpRI or
(His)
6
–NrpRII (see above) were disrupted in 4 mL of pro-
tein-binding buffer [100 mm NaCl, 20 mm Tris ⁄ HCl, 1 mm

dithiothreitol, 1 mm EDTA, 10% (v ⁄ v) glycerol, 0.05%
Triton X-100 [v ⁄ v]) using a French pressure cell at
4135 · 10
6
NÆm
)2
, followed by centrifugation at 20 000 g.
Cell-free crude extract was dialysed and concentrated over-
night at 4 °C using protein-binding buffer supplemented
with 20% polyethylene glycol 4000 (PEG4000). The dialy-
sed crude extracts (about 400 lL containing up to 40 mg of
cell protein), together with 10 lL of competitor DNA (her-
ring sperm DNA, 10 mgÆmL
)1
), were incubated with the
coupled and equilibrated Dynabeads for 2 h at room
temperature with vigorous shaking. Unbound proteins were
removed by magnetic separation using a magnet particle
concentrator (Dynal, Oslo, Norway) and two washing steps
of the coupled Dynabeads, each time using 300 lL of pro-
tein-binding buffer (see above). Potentially DNA-bound
proteins were subsequently eluted step-by-step using 200 lL
of binding buffer containing increasing amounts of NaCl
(100, 150, 200, 250, 500 and 1000 mm). Eluted fractions
were collected and analysed by western blotting for the
presence of MBP–NrpRI or (His)
6
–NrpRII. When analyz-
ing the effect of 2-oxoglutarate, the effector molecule was
added to a final concentration of 2 or 10 mm to the respec-

tive cell-free cell extracts and buffers that were used.
EMSA
The glnK
1
promoter was amplified from M. mazei
chromosomal DNA using the primer set glnK
1
for (5¢-TTG
AACCCGGGTTGATCGAATTC-3¢) and glnK
1
rev (5 ¢-AC
GAAGATCTTTCCGCTTCCAAC-3¢). After gel purifica-
tion, the 418-bp PCR products obtained were end-labelled
using [
32
P]dATP[cP] and T4 polynucleotide kinase and then
purified using Illustra Micro Spin G-50 Columns (GE
Healthcare, Freiburg, Germany). Radiolabelled probes in
binding buffer [10 mm Tris ⁄ HCl (pH 8.0), 150 mm KCl,
0.2 mm dithiothreitol, 0.1% Triton X-100, 12.5% glycerol
and 0.5 mm EDTA], 15.1 lm BSA and 100 ng poly(dI–dC))
were mixed with various amounts of purified MBP–NrpRI
and ⁄ or (His)
6
–NrpRII in a total volume of 30 lL, incu-
bated for 30 min at 37 °C and run on a native 5% (w ⁄ v)
polyacrylamide gel in TBE buffer [90 mm Tris ⁄ borate (pH
8.0), 2 mm EDTA] at 150 V at room temperature. 2-Oxo-
glutarate was added to the mixtures to a final concentration
of 10 mm. Radioactive band intensities were detected using

a PhosphorImager FLA-5000 (Fuji, Tokyo, Japan) and the
aida software (Raytest).
Quantitative RT-PCR
M. mazei was grown at 37 °C in 50-mL cultures, under
nitrogen sufficiency or nitrogen limitation, as recently
described [30]. Samples for quantitative RT-PCR were
taken from cultures in the lag-, mid-exponential- and
stationary growth phases. After rapid cooling, the cultures
were harvested by centrifugation and total RNA was
Table 2. Primer sets used for quantitative RT-PCR analysis.
ORF Protein Primer set Sequence 5¢fi3¢
MM1028 TBP2 MM1028rt for AAACGTTGCTGACGTGCACAC
MM1028rt rev CGATATTCTCAAGCCCAAGCC
MM2184 TBP3 MM2184rt for TGATGGAGTCTGGGTTGGAAG
MM2184rt rev AGCCAGATTGATTATGGCCGC
MM1772 TFB MM1772rt for ATTCGGACACCCTTGAAAGGG
MM1772rt rev ATAGACAAGTCCGCAGTCCCC
K. Weidenbach et al. Role of NrpRII in M. mazei
FEBS Journal 277 (2010) 4398–4411 ª 2010 The Authors Journal compilation ª 2010 FEBS 4409
extracted using the RNeasy Mini Kit (Qiagen, Hilden, Ger-
many). The RNA species were quantified and monitored
for purity, as recently described [32]. Quantitative RT-PCR
assays were performed with the QuantiTect Probe RT-PCR
Kit (Qiagen, Hilden, Germany) using a 7300 real-time PCR
system (ABI, Foster City, CA, USA). The primers used for
quantitative RT-PCR reactions are summarized in Table 2.
The fold change in transcript abundance for the genes of
interest was determined by comparison with the cycle
threshold (C
t

) of the transcripts of three control genes
(MM1621, MM2181 and MM1215). Transcripts of these
genes remain at constant levels, irrespective of growth
rate, growth phase or nitrogen source [31,44]. The fold
change in the abundance of a transcript was calculated
using the formula: fold change = 2
)DDCt
, as described
previously [45].
Acknowledgement
This research was financially supported by the Deut-
sche Forschungsgemeinschaft (DFG) as part of the
priority program SPP 1112 ‘‘Genome function and gene
regulation in Archaea’’ (SCHM1052 ⁄ 6-3).
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Supporting information
The following supplementary material is available:
Fig. S1. Gel filtration analysis of NrpRI and NrpRII.
Fig. S2. Comparison of Methanosarcina mazei NrpR
homologs with Methanococcus maripaludis NrpR.
Table S1. Strains and plasmids used in this study.
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.
K. Weidenbach et al. Role of NrpRII in M. mazei
FEBS Journal 277 (2010) 4398–4411 ª 2010 The Authors Journal compilation ª 2010 FEBS 4411