DNA-binding characteristics of the regulator SenR in
response to phosphorylation by the sensor histidine
autokinase SenS from Streptomyces reticuli
Gabriele Bogel, Hildgund Schrempf and Darı
´o Ortiz de Orue
´Lucana
FB Biologie ⁄Chemie, Universita
¨t Osnabru
¨ck, Germany
One of the major signal transduction systems govern-
ing bacterial responses and adaptation to environmen-
tal changes is the two-component system (TCS). A
typical TCS consists of an autophosphorylating sensor
histidine kinase (SK) and a cognate response regulator
(RR) [1]. SKs detect stimuli via an extracellular input
domain or intracellular signals via cytoplasmic regions,
or use transmembrane regions and sometimes
additional short extracellular loops for sensing [2]. In
addition to the N-terminal input domain, SKs contain
a C-terminal portion representing the transmitter mod-
ule, with several blocks of amino acid residues being
conserved among these kinase types. Phosphorylation
within a typical SK usually takes place at a conserved
histidine residue; the phosphoryl group of the SK is
subsequently transferred to a conserved aspartic
acid residue within the receiver domain of the RR.
As a result, its C-terminally located output domain
has an altered DNA-binding capacity for the reg-
ulatory region of target gene(s) or operons [3,4]. The
Keywords
DNA binding; phosphorylation;
Streptomyces; two-component system
SenS–SenR
Correspondence
D. Ortiz de Orue
´Lucana, Universita
¨t
Osnabru
¨ck, FB Biologie ⁄Chemie,
Angewandte Genetik der Mikroorganismen,
Barbarastr. 13, 49069 Osnabru
¨ck, Germany
Fax: +49 541 9692804
Tel: +49 541 9693439
E-mail: ortiz@biologie.uni-osnabrueck.de
(Received 13 March 2007, revised 7 June
2007, accepted 7 June 2007)
doi:10.1111/j.1742-4658.2007.05923.x
The two-component system SenS–SenR from Streptomyces reticuli has been
shown to influence the production of the redox regulator FurS, the mycel-
ium-associated enzyme CpeB, which displays heme-dependent catalase and
peroxidase activity as well as heme-independent manganese peroxidase
activity, and the extracellular heme-binding protein HbpS. In addition, it
was suggested to participate in the sensing of redox changes. In this work,
the tagged cytoplasmic domain of SenS (SenS
c
), as well as the full-length
differently tagged SenR, and corresponding mutant proteins carrying speci-
fic amino acid exchanges were purified after heterologous expression in
Escherichia coli.In vitro, SenS
c
is autophosphorylated to SenS
c
Pat
the histidine residue at position 199, transfers the phosphate group to the
aspartic acid residue at position 65 in SenR, and acts as a phosphatase for
SenRP. Bandshift and footprinting assays in combination with competi-
tion and mutational analyses revealed that only unphosphorylated SenR
binds to specific sites upstream of the furS–cpeB operon. Further specific
sites within the regulatory region, common to the oppositely orientated
senS and hbpS genes, were recognized by SenR. Upon its phosphorylation,
the DNA-binding affinity of this area was enhanced. These data, together
with previous in vivo studies using mutants lacking functional senS and
senR, indicate that the two-component SenS–SenR system governs the
transcription of the furS–cpeB operon, senS–senR and the hbpS gene. Com-
parative analyses reveal that only the genomes of a few actinobacteria
encode two-component systems that are closely related to SenS–SenR.
Abbreviations
EMSA, electrophoretic mobility shift assay; LC, liquid chromatography; RR, response regulator; SenRP, phosphorylated SenR; SenS
c
,
cytoplasmic domain of SenS; SenS
c
P, phosphorylated SenS
c
; SK, sensor histidine kinase; TCS, two-component system.
3900 FEBS Journal 274 (2007) 3900–3913 ª2007 The Authors Journal compilation ª2007 FEBS
well-studied receiver domain within the nitrogen regu-
latory protein C )controlling the transcription of
genes involved in nitrogen metabolism )has been
shown to change its topology upon activation by phos-
phorylation [5]. Generally, the signaling pathway
includes a phosphatase that returns the RR to the non-
phosphorylated state. The phosphatase can exist as an
individual protein, or reside on a module, which is
linked either to the RR or to the kinase. A combina-
tion of kinase and phosphatase activity ensures rapid
coordination of the cell response [6].
Streptomycetes are Gram-positive and G + C-rich
bacteria with a complex developmental life cycle. Ger-
mination of spores and subsequent elongation of germ
tubes lead to a network of vegetative hyphae. In
response to nutritional stress and extracellular signa-
ling, aerial hyphae develop, in which spores mature [7].
As soil-dwelling organisms, streptomycetes need to
respond to highly variable conditions. The range of
environmental stimuli to which a bacterium can
respond is expected to correlate with the number of
functional SKs and RRs. These are assumed to have
evolved by selection pressure for different ecophysio-
logic properties of the different strains [8]. The com-
plete genome sequence of Streptomyces coelicolor
A3(2) comprises 84 SK genes and 80 RR genes [9].
The physiologic roles of only a few of them have been
investigated experimentally. For instance, the AbsA1–
AbsA2 system negatively regulates the production of
several antibiotics [10,11], and the VanR–VanS system
activates the expression of vancomycin resistance
[12,13]. Phosphate control of the production of actino-
rhodin and undecylprodigiosin in S. lividans and
S. coelicolor A3(2) is mediated by the two-component
PhoR–PhoP system, which also controls the alkaline
phosphatase gene (phoA) and other phoA-related genes
[14,15]. To date, however, the phosphorylation cascade
between a Streptomyces SK and its cognate RR lead-
ing to altered DNA-binding affinity of the RR has not
been analyzed in detail.
The cellulose degrader S. reticuli has been reported
to contain the neighboring genes senS and senR,
which encode an SK and an RR, respectively. SenS
(42.2 kDa) comprises five predicted membrane-span-
ning portions. SenR (23.2 kDa) has a C-terminal region
with a predicted helix–turn–helix motif, which is char-
acteristic for different DNA-binding proteins [16]. It
was concluded that SenR is the cognate RR for the SK
SenS. Comparative transcriptional and biochemical
studies with a designed S. reticuli senS–senR chromoso-
mal disruption mutant showed that the presence of
SenS–SenR influences the transcription of the furS–
cpeB operon encoding the redox regulator FurS and
the catalase-peroxidase CpeB, and the hbpS gene for
the secreted HbpS, representing a novel type of heme-
binding protein [16]. Physiologic studies showed that
the production of HbpS is positively influenced by
hemin in S. reticuli; this correlated with increased
hemin resistance. Interestingly, the presence of HbpS
leads to enhanced synthesis of the heme-containing
CpeB [17].
In this study, we describe the in vitro phosphoryla-
tion cascade between the purified cytoplasmic domain
of SenS (SenS
c
) and SenR. Using designed mutant pro-
teins, the phosphorylation sites within SenS
c
and SenR
have been investigated. Bandshift and footprinting
analyses have allowed the characterization of the
DNA-binding properties in response to phosphoryla-
tion by the sensorkinase SenS.
Results
Cloning of wild-type and mutant senS
c
and senR
genes and purification of fusion proteins
As shown previously, overexpression of the full-length
senS gene resulted in the synthesis of an insoluble pro-
tein in Escherichia coli [16]. To obtain a truncated
SenS (comprising its predicted cytoplasmic portion; see
Experimental procedures) with an N-terminal Strep-tag
(SenS
c
), the corresponding portion of senS was cloned
into the plasmid pASK-IBA7. Furthermore, using site-
directed mutagenesis, a mutant gene was designed and
cloned into plasmid pASK-IBA7 (see Experimental
procedures), leading to the mutant SenS
c
H199A, which
carried an alanine residue in place of the histidine resi-
due in position 199. After induction with anhydrotetra-
cycline, each of the corresponding E. coli XL1-Blue
transformants produced a SenS
c
fusion type in a sol-
uble form within the cytoplasm. Using streptactin affin-
ity chromatography, the SenS
c
and the SenS
c
H199A
fusion protein, both with a predicted molecular mass of
27.1 kDa, were obtained (96 nmol per 1 L of culture)
in high purity (Fig. 1). After proteolytic treatment with
trypsin, each protein was analyzed by liquid chroma-
tography ⁄mass spectrometry (LC-MS), and was found
to comprise the correct N-terminal and internal
peptides (data not shown).
The full-length senR gene and mutant senR genes (car-
rying designed codon exchanges) were cloned into the
plasmid pET21a. The resulting wild-type protein carry-
ing a C-terminal His-tag (SenR) with a predicted
molecular mass of 24.3 kDa was purified to homogen-
eity from an E. coli BL21(DE3)pLys transformant after
induction with isopropyl thio-b-d-galactoside by Ni
2+
–
nitrilotriacetic acid affinity chromatography (Fig. 1).
G. Bogel et al.Response regulator SenR
FEBS Journal 274 (2007) 3900–3913 ª2007 The Authors Journal compilation ª2007 FEBS 3901
Correspondingly, the mutant SenRD60A and Sen-
RD65A fusion proteins (24.3 kDa), which carried an
alanine instead of the original aspartic acid residue at
position 60 or 65, were purified to homogeneity from
the corresponding E. coli BL21(DE3)pLys transform-
ants by Ni
2+
–nitrilotriacetic acid affinity chromatogra-
phy (Fig. 1). Surprisingly, SenRD60A seemed to be
partially degraded and aggregated. From 1 L of E. coli
culture, about 144 nmol of each SenR type was purified.
SenS
c
acts as a histidine autokinase in vitro
SenS
c
exhibited time-dependent autophosphorylation
during incubation with [
32
P]ATP[cP]. The highest sig-
nal intensity was already achieved after 5 min of incu-
bation (Fig. 2A). The subsequent addition of an excess
of unlabeled ATP resulted in a constant level of phos-
phorylated SenS
c
(SenS
c
P) over a relatively long per-
iod (at least 20 min; Fig. 2B). Sequence alignments
showed that the histidine residue at position 199 within
SenS is predicted to be the phosphorylation site [16].
To corroborate this assumption, the corresponding
H199 codon was replaced by one for alanine using
site-directed mutagenesis (see Experimental proce-
dures). The purified SenS
c
H199A (Fig. 2C, left) failed
to undergo autophosphorylation after incubation with
[
32
P]ATP[cP] (Fig. 2C, right). Chemical stability tests
were applied to characterize the nature of the phos-
pholigand. Thus, after treatment of SenS
c
P with 1 m
HCl, the labeled phosphate group was lost from the
protein, but it was retained in the presence of 1 m
NaOH (Fig. 2D). This is the characteristic feature of a
phosphoamidate, which is stable under alkaline condi-
tions but is sensitive to acidic conditions, under which
rapid aminolysis at pH < 5.5 is induced [18]. Taken
together, the presented data show clearly that SenS is
a histidine autokinase.
SenS
c
phosphorylates and dephosphorylates
SenR
As SenR was predicted to be the cognate RR of the
SK SenS, the transfer of radiolabeled phosphate from
Fig. 1. Expression and purification of SenS
c
and SenR proteins. Sol-
uble protein extracts containing SenS
c
obtained from E. coli XL1-
Blue pASK2 (lane 1) after induction with anhydrotetracycline
(lane 2) were loaded onto a streptactin column. After washing (see
Experimental procedures), SenS
c
was eluted with buffer W contain-
ing 2.5 mMdesthiobiotin (lane 3). SenS
C
H199A was purified in the
same manner (lane 4). To obtain SenR, a cytoplasmic protein
extract (lane 5) containing SenR obtained from E. coli BL21(DE3)-
pLys pETR1 after induction (lane 6) was loaded onto an Ni
2+
–nitrilo-
triacetic acid-containing agarose column. Bound SenR was eluted
with solution A containing 250 mMimidazole (lane 7) as described
under Experimental procedures. SenRD60A (lane 8) and SenRD65A
(lane 9) were purified in the same manner. The molecular masses
of the protein markers (S) are indicated.
Fig. 2. Phosphorylation analysis of SenS
c
. (A) To test its autokinase
activity, the purified SenS
c
protein (74 pmol) was incubated in kin-
ase buffer containing 0.05 lCi of [
32
P]ATP[cP] at 30 C for the indi-
cated period. Each sample was then separated by SDS ⁄PAGE;
subsequently, the gel was dried and exposed on an X-ray-sensitive
film. (B) After 4 min of self-phosphorylation of SenS
c
, an excess of
unlabeled ATP was added to the samples. Each reaction was ter-
minated by adding an equal amount of 4 ·sample buffer. After
electrophoresis, the gel was dried and exposed on an X-ray-sensi-
tive film. (C) SenS
c
(148 pmol) or SenS
c
H199A (148 pmol) was
incubated in the kinase buffer with 0.05 lCi of [
32
P]ATP[cP] for
5 min at 30 C. After the addition of 4 ·sample buffer, the reaction
was stopped, and the mixture was subsequently subjected to
SDS ⁄PAGE. The gel was stained with Coomassie Brilliant Blue
(left), or alternatively dried and exposed on an X-ray-sensitive film
(right). (D) After autophosphorylation of 74 pmol of SenS
c
with
0.05 lCi of [
32
P]ATP[cP] in kinase buffer for 5 min at 30 C, the
reaction was terminated by adding 4 ·sample buffer and subjected
to SDS ⁄PAGE. Each gel was treated with the indicated solutions,
dried, and exposed on an X-ray-sensitive film.
Response regulator SenR G. Bogel et al.
3902 FEBS Journal 274 (2007) 3900–3913 ª2007 The Authors Journal compilation ª2007 FEBS
SenS
c
to SenR was investigated. For this purpose, the
purified SenR was added to the
32
P-autophosphorylat-
ed SenS
c
(see previous section). Very rapid (within 5–
10 s) labeling of SenR was observed, together with a
concomitant reduction of the phospholabel within
SenS
c
(Fig. 3A,B). Autophosphorylation activity of
SenR using [
32
P]ATP[cP] or the phosphodonor acetyl-
phosphate could not be detected (data not shown).
The deduced SenR comprises aspartic acid residues at
position 60 (D60) and position 65 (D65), each of
which is a candidate to participate in the phosphoryla-
tion process [16]. Site-directed mutagenesis showed
that each of the two residues was replaced by an alan-
ine. SenRD60A and SenRD65A were subsequently
purified from corresponding E. coli transformants (see
above). Further transphosphorylation analysis revealed
that the presence of SenS
c
P provoked phospholabe-
ling of wild-type SenR and SenRD60A. In contrast,
the mutant protein SenRD65A was not found to be
phosphorylated by SenS
c
P (Fig. 3C). D65 is therefore
the phosphorylation site within SenR.
As demonstrated by quantitative analysis (using a
PhosphorImager system), during the transphosphoryla-
tion reaction dephosphorylation of phosphorylated
SenR (SenRP) occurred after aproximately 3 min of
incubation (Fig. 3B); during this period, no rephospho-
rylation of SenS
c
was recorded. To investigate this pro-
cess in more detail, phospholabeled SenR (carrying a
His-tag) was separated immediately after phosphoryla-
tion from SenS
c
(carrying a Strep-tag) by Ni
2+
–nitrilo-
triacetic acid affinity chromatography. The addition of
dephosphorylated SenS
c
to a reaction mixture contain-
ing phospholabeled SenR provoked a rapid (within
60 s) loss of the phosphoryl group from SenR
(Fig. 4A,B). In the absence of SenS
c
, autodephospho-
rylation of SenRP occurred only after a longer
(> 120 s) period of incubation (data not shown).
These data show that SenS
c
also acts as a phosphatase
for SenRP.
DNA-binding properties of SenR depend on its
phosphorylated state
Comparative analysis of wild-type S. reticuli and the
senS–senR disruption mutant showed that the presence
of SenS–SenR correlates with a significant reduction of
Fig. 4. Dephosphorylation rate of SenRP. (A) SenR was first phos-
phorylated by SenS
c
P in a transphosphorylation reaction, and sub-
sequently separated from it by Ni
2+
–nitrilotriacetic acid affinity
chromatography. Purified SenRP(82 pmol) was incubated at
30 C alone (top) or with (bottom) 148 pmol of dephosphorylated
SenS for the indicated times. Each reaction was stopped by adding
an equal amount of 4 ·sample buffer, and the products were
analyzed by SDS ⁄PAGE. Gels were dried and exposed on an X-ray-
sensitive film. (B) Dried gels were further analyzed using a Phos-
phorImager. The diagram shows the quantified results representing
the measured radioactivity at the indicated times (j) with SenRP
alone or for the mixture (r) of SenRP and SenS
c
.
Fig. 3. Phosphotransfer from SenS
c
to SenR, SenRD60A or Sen-
RD65A. (A, B) Purified SenS
c
(184 pmol) was incubated with
0.05 lCi of [
32
P]ATP[cP] for self-phosphorylation. After 4 min, equal
amounts of purified SenR were added and incubated for the indica-
ted period at 30 C. The reactions were terminated by adding
4·sample buffer. After SDS ⁄PAGE, the gel was dried and
exposed on an X-ray-sensitive film (A) or quantified by detection of
the radioactivity emitted by SenRP(j) or SenS
c
P(r) using a
PhosphorImager (B). (C) The wild-type SenR or SenR mutant pro-
teins (SenRD60A or SenRD65A), in each case 330 pmol of protein,
were mixed with 260 pmol of SenS
c
P in transphosphorylation
buffer for 1 min at 30 C. Reactions were terminated with 4 ·sam-
ple buffer, subjected to SDS ⁄PAGE, and stained with Coomassie
Brilliant Blue (left), or alternatively the gel was dried and exposed
on an X-ray-sensitive film (right).
G. Bogel et al.Response regulator SenR
FEBS Journal 274 (2007) 3900–3913 ª2007 The Authors Journal compilation ª2007 FEBS 3903
transcripts (furS–cpeB and hbpS) and the correspond-
ing proteins [16]. For further analyses, different DNA
fragments (Fig. 5A) corresponding to the upstream
region (310 bp, named up-furS1) of the furS–cpeB
operon or the upstream region (548 bp, named
up-hbpS1) located between hbpS and senS were ampli-
fied by PCR. Electrophoretic mobility shift assays
(EMSAs) were performed with labeled DNA
(5200 pmol of up-furS1 or 2900 pmol of up-hbpS1) and
increasing quantities (0–16 pmol) of the purified SenR
or SenRP. Interestingly, in contrast to SenRP,
SenR interacted with up-furS1 (Fig. 5B). The addition
of 12 pmol of SenR to the reaction mixture led to an
84% decrease of free up-furS1, whereas the same
amount of SenRP provoked only a 10% reduction
(Fig. 5D). The presence of small quantities (4 and
8 pmol) of SenR led to one type of retarded DNA spe-
cies (Fig. 5B, arrow b); an additional one was formed
if the protein concentration (12 and 16 pmol) was
increased (Fig. 5B, arrow a). These data suggested the
presence of multiple SenR-binding sites. The specificity
of this interaction was verified by competition using
constant amounts of SenR and additional increasing
amounts of unlabeled up-furS1 (Fig. 5B, third box
Fig. 5. Gene organization and EMSAs with isolated SenR proteins. (A) The gene organization of furS–cpeB,hbpS,senS and senR is indica-
ted.The labeled DNA regions are marked in gray. (B, C) The upstream region of the furS–cpeB operon (5200 pmol of up-furS1) (B) or the
intergenic region between hbpS and senS (2900 pmol of up-hbpS1) (C) was incubated without or with increasing amounts (0, 4, 8, 12 or
16 pmol; black triangle) of SenR or SenRP in incubation buffer (see Experimental procedures). For competition experiments, labeled up-
furS1 (5200 pmol) was incubated with constant amounts (16 pmol) of SenR and increasing amounts of unlabeled up-furS1 (0, 5200, 7800,
10 400 or 13 000 pmol; open triangle) (B, third box from left). In the same manner, unlabeled up-hbpS1 (0, 2900, 4350, 5800 or 7250 pmol;
open triangle) was added to the mixture comprising labeled up-hbpS1 (2900 pmol) and constant amounts (16 pmol) of SenR (C, third box
from left). For further corroboration of the binding specificity, SenR (0–16 pmol; black triangle) was incubated with the upstream region of
cpeB (up-cpeB, 5500 pmol) (B, fourth box from left). After incubation at 30 C for 15 min, the mixtures were separated on 5% polyacryla-
mide gels, and then subjected to autoradiography. The retarded DNA fragments are indicated (a, b, c and d). The control DNA in mixtures
without SenR is everywhere marked as lane 0. (D) In addition, gels were dried and analyzed by a PhosphorImager System. The radioactivity
level of the DNA probe alone was set at 100%. The reaction products up-furS1 + SenR (j), up-furS1 + SenRP(h), up-hbpS1 + SenR (r),
and up-hbpS1 + SenRP(e) as well as the quantities of SenR used are indicated.
Response regulator SenR G. Bogel et al.
3904 FEBS Journal 274 (2007) 3900–3913 ª2007 The Authors Journal compilation ª2007 FEBS