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REVIEW
Prokaryotic 2-component systems and the OmpR/PhoB
superfamily
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Minh-Phuong Nguyen, Joo-Mi Yoon, Man-Ho Cho, and Sang-Won Lee
Abstract: In bacteria, 2-component regulatory systems (TCSs) are the critical information-processing pathways
that link stimuli to specific adaptive responses. Signals perceived by membrane sensors, which are generally
histidine kinases, are transmitted by response regulators (RRs) to allow cells to cope rapidly and effectively with
environmental challenges. Over the past few decades, genes encoding components of TCSs and their responsive
proteins have been identified, crystal structures have been described, and signaling mechanisms have been
elucidated. Here, we review recent findings and interesting breakthroughs in bacterial TCS research. Furthermore, we discuss structural features, mechanisms of activation and regulation, and cross-regulation of RRs, with
a focus on the largest RR family, OmpR/PhoB, to provide a comprehensive overview of these critically important
signaling molecules.
Key words: 2-component system, response regulator, bacterial signal transduction, OmpR/PhoB family.
Résumé : Chez les bactéries, les systèmes régulateurs à 2 composants (SDC) forment les voies cruciales de
traitement de l’information qui lient les stimuli aux réponses adaptatives. Les signaux perçus par les capteurs
membranaires, généralement des histidine kinases, sont transmis par des régulateurs de réponse (RR) permettant
aux cellules de s’adapter rapidement et convenablement aux variations environnementales. Au cours des
dernières décennies, on a identifié les gènes codant les composants des SDC et leurs protéines de réponse, détaillé
des structures cristallines et élucidé les mécanismes de signalisation. Dans le présent ouvrage, nous passons en
revue les récentes trouvailles et percées intéressantes dans le domaine de la recherche sur les SDC. De plus, nous
abordons les aspects structuraux, les mécanismes d’activation et de régulation, et la régulation croisée des RR
(particulièrement la plus importante famille de RR, OmpR/PhoB), afin de brosser un portrait exhaustif de ces
molécules de signalisation au rôle si primordial. [Traduit par la Rédaction]
Mots-clés : système à 2 composants, régulateurs de réponse, transduction de signal chez les bactéries, famille
OmpR/PhoB.
Introduction
Living organisms need to rapidly detect environmental stimuli and respond to survive and proliferate. These
detection and response mechanisms are controlled by
signal transduction pathways. In eukaryotes, signal transduction is often mediated by complex and branched pathways composed of multiple proteins (Schaller et al.
2011), but prokaryotes have simpler systems, including
1-component systems (OCSs) and 2-component systems
(TCSs). OCSs consist of a single protein containing both
input (sensory) and output (regulator) domains but lacking a phosphotransfer domain that is generally found in
typical TCSs. OCSs are less complex, exhibit a greater
diversity of input and output domains, and have a wider
distribution among prokaryotes than TCSs do. Therefore, OCSs have been suggested to be the evolutionary
precursors of TCSs (Ulrich et al. 2005).
The critical signaling pathways in bacteria involve a
TCS (Nixon et al. 1986). TCSs always contain histidine
kinases (HKs), which typically are membrane-bound,
and soluble cytoplasmic response regulators (RRs). Separation of the membrane-bound sensor and the soluble
cytosolic regulator, which are linked via a phosphotransmission pathway, represents a tremendous evolutionary breakthrough in prokaryotic signal transduction.
The separation has allowed bacteria to dramatically expand and diversify their signaling capabilities (Capra and
Laub 2012). It has been suggested that cognate HKs and
Received 28 May 2015. Revision received 17 August 2015. Accepted 19 August 2015.
M.-P. Nguyen* and J.-M. Yoon.* Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea.
M.-H. Cho. Department of Genetic Engineering and Graduate School of Biotechnology, Kyung Hee University, Yongin 446-701, Korea.
S.-W. Lee. Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea; Department of Genetic Engineering and Graduate
School of Biotechnology, Kyung Hee University, Yongin 446-701, Korea.
Corresponding author: Sang-Won Lee (e-mail: [email protected]).
*Minh-Phuong Nguyen and Joo-Mi Yoon are equally contributing co-first authors of this review.
Can. J. Microbiol. 61: 1–12 (2015) dx.doi.org/10.1139/cjm-2015-0345
Published at www.nrcresearchpress.com/cjm on 21 August 2015.
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RRs in TCSs have co-evolved based on phylogenic analysis of their DNA sequences (Grebe and Stock 1999), and
the genes encoding the HK and RR of a specific TCS are
generally adjacent to each other in genomes (Ulrich et al.
2005; Ulrich and Zhulin 2010). The HK transfers its phosphoryl group to a cognate RR following autophosphorylation in response to signal perception by the HK.
Phosphorylation of the RR results in a conformational
change that affects the activity of the output domain
(J.B. Stock et al. 1989; Parkinson and Kofoid 1992). Activated RRs often function as transcription factors, regulating gene expression in response to signals (Stock et al.
2000; Qin et al. 2001). Signal transduction mediated by
TCSs has recently been recognized to be more complex
than originally thought owing to the discovery of diverse
phosphotransmission pathways and cross or joint regulation by TCSs (Mitrophanov and Groisman 2008; Buelow
and Raivio 2010). Furthermore, auxiliary factors, such as
sRNAs (Vogel and Papenfort 2006; De la Cruz and Calva
2010) and other proteins (Mitrophanov and Groisman
2008; Alix and Blanc-Potard 2009; Buelow and Raivio
2010), have been reported to be able to regulate TCS expression.
The critical signaling pathway in
prokaryotes — TCS
Domain structure of HKs and RRs in TCSs
The first component of TCSs is HK, having a sensory
domain and transmitter domain (Fig. 1A) (West and Stock
2001). The sensory domain mostly receives external environmental stimuli, which affect the balance between
autophosphorylation and dephosphorylation of the HK
(Hsing et al. 1998; Gao and Stock 2013). The transmitter
domain contains 2 subdomains: a dimerization domain
with a conserved His residue and an ATPase domain that
catalyzes phosphorylation of the conserved His. RRs, the
second component of TCSs, commonly have a phosphoryl group acceptor (receiver, REC) domain followed
by a diverse output (effector) domain (Fig. 1A). Since the
first description of a REC domain or “response regulator”
with one or more effector domains in a study of chemotaxis proteins (Koshland 1977), more than 60 different
effector domains have been characterized (Galperin
2010). Effector domains in RRs are characterized by their
functional characteristics such as DNA-, RNA-, ligand-, or
protein-binding abilities or enzymatic activity (Galperin
2010).
Diverse phosphotransmission schemes
In addition to the typical phosphotransmission scheme
described above, another scheme, often called a phosphorelay, is available (Fig. 1B). Phosphorelay involves
multiple phosphotransfer steps and often requires more
than 2 proteins (Perraud et al. 1999). Most phosphorelay
processes involve hybrid kinases that have both His- and
Asp-containing domains (Cock and Whitworth 2007) and
a His-containing phosphotransfer (Hpt) protein. In this
Can. J. Microbiol. Vol. 61, 2015
model, the phosphoryl group is transferred from the His
to the Asp of the HK, then to the His of the Hpt, and then
to the Asp of the RR (His-Asp-His-Asp phosphorelay)
(Appleby et al. 1996; Stock et al. 2000). For instance, the
sporulation phosphorelay of Bacillus subtilis (Jiang et al.
2000) begins with autophosphorylation of 1 of 4 HKs:
KinA, KinB, KinC, or KinD. Then the phosphoryl group is
transferred to the response regulator Spo0F, which
serves as a phosphodonor for Spo0B. Finally, the phosphoryl group is transferred from Spo0B to Spo0A, which
regulates a number of genes involved in sporulation. The
phosphorelay signal transduction scheme provides flexibility in signaling strategies and offers a greater number
of potential sites for regulation (West and Stock 2001).
The CheA/CheB/CheY chemosensory TCS is considered
another type of phosphotransmission scheme (Fig. 1C)
(Simon et al. 1989). Unlike most HKs and RRs, HK CheA
has no sensory domain but has an Hpt domain with a His
residue for autophosphorylation. The RR CheY has only a
receiver domain without any effector (A.M. Stock et al.
1989). The RR CheB is a methylesterase and has a multidomain structure (Djordjevic et al. 1998). Signals detected by a methyl-accepting chemotaxis protein (MCP)
induce a conformational change in the protein, which
results in phosphorylation of the His residue in the CheA
Hpt domain. CheW, an adaptor protein, helps regulate
CheA autophosphorylation. The phosphoryl group is
then transferred to CheB and CheY (Hess et al. 1988; Lee
et al. 2001). CheY-P interacts with the flagellar switch
protein FliM to control flagellar rotation. A special feature of this chemosensory system is its ability to adapt to
the presence or absence of a signal.
Classification of TCSs in prokaryotes
HKs are difficult to classify because family members
do not necessarily share the same input domain. Fabret
et al. (1999) categorized B. subtilis HKs into 6 groups based
on conserved sequences surrounding the phosphorylated
His. Although these classes were related to their RRs on
the basis of the homology of B. subtilis RR effectors to
those of E. coli, there was considerable diversity in the
sensory domain within each class of HKs (Fabret et al.
1999). Grebe and Stock (1999) took HKs from multiple
genomes and assigned them into 11 families of related
sequences using a phylogenetic approach. Individual
families of HKs were associated with particular families
of RRs; this classification is now widely used.
RRs are easier to group because they share a conserved
REC domain and only vary in their effector domains. RRs
are divided into DNA-binding domain proteins, RNAbinding domain proteins, protein-binding domain proteins, or proteins with enzymatic activity on the basis of
the effector domain (Galperin 2006, 2010; Gao et al.
2007). The majority of RRs belong to the DNA-binding
group of RRs, also known as transcription factors. They
are further divided into 4 main families: the OmpR/PhoB
family has a winged helix–turn–helix (wHTH) motif; the
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Fig. 1. Phosphotransmission scheme of 2-component systems (TCS) in bacteria. Typical phosphotransmission as typified by
EnvZ-OmpR TCS (A), phosphorelay in Bacillus subtilis sporulation (B), and phosphotransmission in the chemotaxis system
CheABY (C). Sensory, receiving stimuli signals; Transmitter, including dimerization (Dim) and ATPase domains of histidine
kinase (HK); Receiver, regulatory domain of response regulator (RR); Effector, output domain of RR; Hpt, His-containing phosphotransfer protein; MCP, methyl-accepting chemotaxis protein; CheR, methyltransferase; CheB, methylesterase; CheW,
adaptor connecting MCP and CheA; H, histidine residue located in the dimerization domain of HK; P, phosphoryl group;
DD D T K, 5 conserved residues of the receiver domain of the RR; DD, the 2 Asp residues that interact with Mg2+; D (third one),
phosphorylated Asp residue; T, threonine that interacts with the oxygen atom of the phosphoryl group; K, lysine involved in
phosphoryl transfer.
NarL/FixJ and the NtrC/DctD families are characterized
by a classic helix–turn–helix (HTH) motif (Baikalov et al.
1996; Pelton et al. 1999), and the LytR/AgrA family has a
novel structure, an elongated ␤-␤-␤ sandwich, which is
able to bind to a target DNA (Sidote et al. 2008). Information about these prokaryotic RR families can be found in
P2CS (Prokaryotic 2-Component Systems; http://www.p2cs.
org), a TCS-centered database, which categorizes RRs on
the basis of phylogenetic analysis (Barakat et al. 2009,
2011). The Microbial Signal Transduction database (MiST;
http://mistdb.com) contains various domain profiles of
RRs in bacterial and archaeal genomes (Ulrich and
Zhulin 2010). Another useful RR database is found on
NCBI, which provides information of RRs encoded in
bacterial and archaeal genomes (http://www.ncbi.nlm.
nih.gov/Complete_Genomes/RRcensus.html). However,
there are many cases of RRs that cannot easily be assigned to a family using the approaches described above.
For instance, a large group of RRs lacking an effector
domain (REC domain only) are grouped into the CheY
family, because those RR genes are often found in operons with chemosensory HKs (CheA homologues). However, other RRs with only a REC domain are unclassified,
because they are not encoded in the vicinity of CheA
homologues. In addition, some RRs have not yet been
defined, such as multiple REC-domain RRs, multiple
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Fig. 2. Three-dimensional structures of the REC domain (A) and effector domain (B) of PhoB. (A) The active state of the REC
domain dimer (PDB: 1ZES, doi:10.2210/pdb1zes/pdb). Cofactors Mg2+ and BeF3– (beryllofluoride mimics the phosphorylation of
the aspartate residue in the response regulator) are present in the crystal structure. The alpha helices and beta strands are indicated by ␣ and ␤, respectively. (B) The DNA-bound structure of the effector domain of PhoB (PDB: 2Z33, doi:10.2210/pdb2z33/
pdb). The recognition helix ␣3 and recognition wing interact with the major and the minor grooves of DNA, respectively. An
extensive loop between ␣2 and ␣3 is involved in the interaction with RNA polymerase. The numbers following ␣ and ␤ indicate the order of the secondary structures.
effector-domain RRs, and very rare effector-domain RRs
(Whitworth 2012). This diversity of soluble cytoplasmic
regulators may have evolved to facilitate responses to a
huge number of environmental stimuli with a restricted
numbers of regulatory proteins.
OmpR/PhoB family proteins
Structural features of OmpR/PhoB family proteins
Most RRs are OmpR/PhoB family members, thus
these RRs have been studied extensively. OmpR/PhoB
members contain REC and effector domains like other
RRs but are characterized by a unique wHTH domain
(Martinez-Hackert and Stock 1997b) followed by an additional hairpin in the C-terminal effector domain and a
short linker between the REC and effector domains
(Mattison et al. 2002; Walthers et al. 2003). Crystal structures of 5 full-length OmpR/PhoB proteins have been
reported: DrrB and DrrD from Thermotoga maritima
(Buckler et al. 2002; Robinson et al. 2003) and RegX3,
MtrA, and PrrA from Mycobacterium tuberculosis (Nowak
et al. 2006; Friedland et al. 2007; King-Scott et al. 2007).
These crystal structures revealed the arrangement of interdomain interfaces between the REC and the effector
domain and provided insight into the binding mechanism between the target DNA and effector domain.
The receiver (REC) domain
The REC domain is an N-terminal module responsible
for acceptance of a phosphoryl group from HKs following perception of a signal. The REC domain is conserved
in most RRs and has a (␤␣)5 fold with a central ␤-sheet
formed by 5 parallel ␤-strands surrounded by 3 and 2
␣-helices on both sides (Fig. 2A). The conserved Asp for
phosphorylation lies at the beginning of the loop between ␤3 and ␣3 (L␤␣3). Two other Asp residues involved
in coordinating a Mg2+ ion reside in the L␤␣1 loop. Addi-
tional catalytic residues are Thr and Ser, which interact
with one of the oxygen atoms of the phosphoryl group in
the L␤␣4 loop. A conserved Lys residue plays a key role in
phosphotransfer in the L␤␣5 loop (Gao and Stock 2009;
Bourret 2010).
The REC domain has 3 activities (West and Stock 2001).
Firstly, the domain contributes to transmission of a phosphoryl group to an Asp residue by interaction with phosphorylated HKs. This was demonstrated by in vitro
phosphorylation of CheB, CheY, and NtrC in Escherichia
coli by low-molecular-weight phosphorylated compounds,
such as acetyl phosphate, carbamoyl phosphate, and phosphoramidate, in the absence of the cognate HK protein
(Feng et al. 1992; Lukat et al. 1992). Secondly, the domain
catalyzes autodephosphorylation, with H2O serving as
the nucleophile, which limits the lifetime of the active
state (West and Stock 2001). Lastly, the REC domain modulates the activity of associated effector domains in a
phosphorylation-dependent manner, which leads to the
binding of the wHTH effector domain to target DNA
(Toro-Roman et al. 2005a; Gao et al. 2008).
The wHTH effector domain
The most distinguishable feature of the OmpR/PhoB
family is the C-terminal wHTH effector domain, which is
responsible for binding to a target gene. The wHTH motif
(␣1-␤1-␣2-T-␣3-␤2-W-␤3) with a turn (T) between ␣2 and
␣3 and a wing (W) formed by the loop connecting ␤2 and
␤3 (Lai et al. 1991; Brennan 1993) has been reported in
many DNA-binding proteins, such as BirA (Wilson et al.
1992), CAP (Schultz et al. 1991; Parkinson et al. 1996),
histone H5 (Ramakrishnan et al. 1993), HNF-3␥ (Clark
et al. 1993), MuA (Clubb et al. 1994), and heat shock transcription factor (Harrison et al. 1994). However, the
wHTH of the OmpR/PhoB family is unique because it
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contains an additional 4-stranded antiparallel ␤-sheet before the classic wHTH motif (␤1-␤2-␤3-␤4-␣1-␤5-␣2-␣3␤6-␤7) (Fig. 2B) (Martinez-Hackert and Stock 1997b;
Yamane et al. 2008). The amino acid sequence of the
4-stranded antiparallel ␤-sheet is not well conserved,
which might indicate that the 4-stranded antiparallel
␤-sheet is involved in specific interactions with target
genes. The most conserved residues form a portion of
recognition helix ␣3 and recognition wing W. These residues are therefore presumably necessary for correct
binding to the DNA of the response protein, and these
interactions are likely conserved for all members of the
family (Martinez-Hackert and Stock 1997a). Residues critical for DNA-binding have been reported for OmpR (Rhee
et al. 2008), PhoB (Blanco et al. 2002), PhoP (Chen et al.
2004), KdpE (Narayanan et al. 2012), and HP1043 (Hong
et al. 2007). Amino acids in helices ␣1 and ␣2 as well as
residues in the hydrophobic core formed by 7 ␤-strands
and 3 ␣-helices are highly conserved. Members in this
family likely have the same secondary structural elements.
DNA-binding features
The effector domain normally binds to a cis-acting element in the promoter region of regulons and controls
gene expression. The cis-acting elements are 6 bp sequences that are located in the opposite phase to the –35
region of the promoter as reported for VirR, PhoB, and
OmpR (Aoyama and Oka 1990). Most binding sites of
OmpR/PhoB members are direct repeats of DNA, such as
the tor box, which is the target of TorR (Simon et al. 1995),
or the kdpFABC promoter, which is the target of KdpE
(Narayanan et al. 2012). The ability of RRs to access DNA
is controlled by several factors. The first factor is
dimerization of the effector domain. Investigation of
PhoB protein binding to the pho box showed that the
PhoB DNA-binding domain specifically recognizes DNA
on its own and can act as a constitutive activator even
when the regulatory domain of PhoB is removed. Consequently, dimerization appears to influence the binding
of RRs to DNA (Ritzefeld et al. 2013). The second factor is
the binding motif of the RRs. The binding motif TGTCA
and the A/T-rich minor groove of DNA are essential for
the overall recognition process (Ritzefeld et al. 2013).
These factors are likely to be important in other wHTH
OmpR/PhoB proteins as well. A recent report suggested
that DNA topology is a fundamental factor that controls
access of the RRs to target promoters. In Salmonella
enterica, relaxation of DNA supercoiling results in an increase in OmpR binding to DNA, and OmpR binding to
relaxed DNA appears to generate a locally supercoiled
state, which may assist promoter activation by relocating supercoiling stress-induced destabilization of DNA
strands (Cameron and Dorman 2012). Additionally, DNA
relaxation accompanies acid stress in S. typhimurium and
enhances OmpR binding to DNA (Quinn et al. 2014).
5
The linker between the REC and effector domain
An additional characteristic of OmpR/PhoB family
members is a short linker between the REC and DNAbinding effector domain. Linkers of DrRRA (Liu et al.
2012), MtrA (Friedland et al. 2007), and PrrA (Nowak et al.
2006) are only 5, 9, and 12 amino acid residues long,
respectively, while other RR families, such as the CheB
and NarL families, have linkers that are approximately
30 amino acids in length (West et al. 1995; Baikalov et al.
1996). Differences in linker lengths may play some role in
communication between the regulatory (REC) and effector domains (Mattison et al. 2002; Walthers et al. 2003),
but no specific function for this region has been established.
Activation and regulation of OmpR/PhoB family
RRs
Phosphorylation-dependent activation
The common activation mechanism of RRs is
phosphorylation-dependent and relies on the autophosphorylation of HKs triggered by perception of stimuli.
Both cis and trans modes of autophosphorylation have
been proposed. In trans mode, a phosphoryl group is
transferred to the His of the opposite HK subunit (e.g.,
E. coli NtrB and EnvZ HK). In cis mode, the phosphoryl
group is transferred to the His of the same subunit (e.g.,
HK853, PhoR and ArcB HKs) (Ninfa et al. 1993; Cai and
Inouye 2003; Casino et al. 2009; Pena-Sandoval and
Georgellis 2010).
Conformational changes and oligomerizations of the
RRs are induced by phosphorylation during signal transduction of TCSs. Conformational changes of RRs have
been reported for several OmpR/PhoB RRs (Birck et al.
1999; Kern et al. 1999; Lewis et al. 1999; Halkides et al.
2000; Bachhawat et al. 2005). The effector domain of the
RR binds to target DNA to trigger the downstream response, following PiC (Walthers et al. 2003). It was defined as Y–T coupling model of RR CheY (Cho et al. 2000),
which had been the main model of RR activation for the
past few decades. In the inactive state, the hydroxyl
group of the Ser–Thr side chain at the C-terminal end of
␤4 points away from the active site, and the Tyr–Phe
residue found in the middle of ␤5 extends outwards from
the surface of the regulatory domain. However, in the
active state, the Ser–Thr side chain points toward the
active site and interacts with the phosphorylated Asp,
and the Tyr–Phe residue is buried in the hydrophobic
pocket of the REC domain (Bachhawat et al. 2005). These
coordinated movements induce a conformational change
in the exposed ␣4-␤5-␣5 surface of the REC domain. However, a contradictory result has been reported, that NtrC,
which has an HTH motif in the REC domain, is not correlated with the Y–T coupling model or the activation process
(Villali et al. 2014), and a number of hydrogen bonds might
participate in the active–inactive states transition that occurs through multiple pathways (Pontiggia et al. 2015;
Vanatta et al. 2015). Moreover, some results have suggested
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6
that the phosphorylation of RRs stabilizes a preexisting
conformational change (Volkman et al. 2001; Bourret 2010).
It is not clear that the phosphorylation acts to induce or
stabilize the conformational changes in RR.
Similarly, oligomerization of RRs is common and
necessary in the OmpR/PhoB family to enhance DNA
binding and transcriptional regulation. Dimerization
of OmpR after phosphorylation is the driving force for
phosphorylation-mediated regulation of OmpR–DNA
binding (Barbieri et al. 2013). However, a previous report noted that a heterodimer (one phosphorylated
and one unphosphorylated monomer) can enhance
autophosphorylation of RR PhoB and might participate in gene regulation (Creager-Allen et al. 2013).
Therefore, gene regulation of the OmpR/PhoB family
might not require dimerization with phosphorylated
monomers. The most general form of oligomerization in
the OmpR/PhoB family is a 4-5-5 dimer at the ␣4-␤5-␣5
surface, as seen for the receiver domain of E. coli PhoB.
The REC domain of PhoB forms a 2-fold symmetric dimer
using ␣4-␤5-␣5 faces, and the DNA-binding domains then
bind to DNA with tandem symmetry (Fig. 2A) (Bachhawat
et al. 2005). However, polymerization of RRs via the REC
domain can occur even prior to phosphorylation; for example, the N terminus of PhoB, (PhoBN) is a dimer in the
inactive state, with strengthening of this interaction in
the activated state (Bachhawat et al. 2005). Not all activated RRs are dimers, for example, dimers of ArcA REC
domains form an octamer upon activation (Jeon et al.
2001).
Phosphorylation-independent activation and atypical RRs
It was previously believed that phosphorylation was
mandatory for RR activation and DNA binding. However,
cases of phosphorylation-independent RR activation
have been reported. Some OmpR/PhoB RRs form dimers
through the 4-5-5 interface in the absence of beryllium
fluoride, an analogue of phosphate (Bachhawat et al.
2005; Toro-Roman et al. 2005b), which suggests that the
active form of the dimer exists without phosphorylation.
Mycobacterium tuberculosis PhoP binds to its own promoter
in the unphosphorylated state (Gupta et al. 2006; Sinha
et al. 2008). These reports suggest that phosphorylation
of the REC domain does not directly induce DNA binding of the output domain; rather, phosphorylation likely
facilitates or stabilizes dimerization of the receiver
domains, bringing the DNA-binding domains into close
proximity (Menon and Wang 2011). Phosphorylationindependent RR activation is not unique to OmpR/PhoB
RRs. DesR, a member of the NarL/LuxR family, acts as a
molecular thermometer with DesK in B. subtilis. DesR
shows phosphorylation-independent activation by DesK
(Trajtenberg et al. 2014). HK DesK binds to RR DesR to
regulate the functional state through an HK-dependent
allosteric activation mechanism.
Atypical RR (ARR) is an abnormal RR lacking conserved residues important for phosphorylation and ac-
Can. J. Microbiol. Vol. 61, 2015
tivation. The existence of ARRs is further evidence of
phosphorylation-independent activation. ChxR from
Chlamydia is structurally poised to bind to direct repeats
of DNA (Barta et al. 2014). However, ChxR lacks several
conserved residues involved in phosphorylation, including the phosphor-accepting Asp, and is able to activate
transcription of target genes in the absence of phosphorylation (Koo et al. 2006; Hickey et al. 2011). Unlike
ChxR, HP1043, an orphan RR in Helicobacter pylori, binds
to inverted repeat DNA sequence motifs but not direct
repeats (Delany et al. 2002), and its regulatory activity
remains unclear (Hong et al. 2007). In HP1043, several
conserved amino acids are substituted with other residues, e.g., the Asp12 is a Glu, and the Asp that is phosphorylated is replaced by a Lys (Delany et al. 2002). The
OmpR/PhoB ARR GlnR was recently reported in actinomycetes. This is an orphan RR that globally coordinates
the expression of genes related to nitrogen metabolism
(Lin et al. 2014). GlnR Asp50 in the N-terminal receiver
domain is unphosphorylated but is necessary for interaction with Arg52 and Thr9 to maintain the proper conformation for homodimerization. GlnR is naturally
active in its dimer status; how GlnR triggers a response is
unknown. NblR contains a putative phosphorylatable
Asp (Asp57) but lacks other conserved residues required
to chelate to Mg2+, which is necessary for Asp phosphorylation (Ruiz et al. 2008). Espinosa et al. (2012) found
that Tyr104 is a key residue in the NblR dimerization
interface and that the signaling mechanism is similar to
that of other RRs in the OmpR/PhoB family (Espinosa
et al. 2012). Some ARRs are activated by binding to end
products or late biosynthetic intermediates of secondary
metabolites, such as antibiotics (Wang et al. 2009; Liu
et al. 2013). In JadR1 of Streptomyces venezuelae, 2 important Asp residues in the N-terminal receiver domain are
replaced by Glu49 and Ser50, respectively. JadR1 is activated by binding to the end product of antibiotic jadomycin B (Liu et al. 2013). The discovery of ARRs and
evidence of phosphorylation-independent activation
raises questions about the true role of phosphorylation
and the real mechanism of RR activation.
Regulation of TCS-mediated gene expression
RRs can cooperate with other regulatory factors, such
as small RNAs (sRNA) (Vogel and Papenfort 2006), to regulate genes. Most sRNAs modulate gene expression at
the post-transcription level, which means they directly
interact with the target RNA to inhibit translation. For
example, outer membrane protein (OMP) expression in
enterobacteria is controlled by a variety of other sRNAs
in concert with OmpR (De la Cruz and Calva 2010). Furthermore, the sRNAs MicC, MicA, and MicF act specifically on a single omp mRNA, whereas others (OmrA,
OmrB, RybB, and RseX) control multiple omp mRNA targets (Vogel and Papenfort 2006; De la Cruz and Calva
2010).
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Nguyen et al.
Auxiliary proteins can also control OmpR/PhoB
regulon expression by binding to either the HKs or RRs
of TCSs (Mitrophanov and Groisman 2008; Alix and
Blanc-Potard 2009; Buelow and Raivio 2010). Auxiliary
proteins that target RRs can modify the phosphorylation
state, inhibit DNA binding, or act as anti-adaptors. For
example, the small protein PmrD in Salmonella was reported to bind phosphorylated PmrA of the PmrB–PmrA
system. The binding of PmrD to the phosphorylated RR
of the TCS prevented its intrinsic dephosphorylation
that was promoted by PmrB, which in turn activated the
system (Kato and Groisman 2004). Auxiliary proteins
that interact with HKs can turn on or off the signal transduction of a TCS. An example is the E. coli periplasmic
protein CpxP, which interacts with the periplasmic domain of the sensor CpxA and turns off the CpxA–CpxR
TCS (Fleischer et al. 2007; Buelow and Raivio 2010; Kwon
et al. 2012; Debnath et al. 2013). These additional mechanisms for fine regulation can also be viewed as ways to
accommodate changes in the circumstances of a singlecelled organism.
Crosstalk between the OmpR/PhoB family and
other signaling pathways
Crosstalk is defined as communication between 2 distinct signaling pathways (Laub and Goulian 2007) and
has been proposed to be a result of gene duplication and
divergence of TCSs (Capra and Laub 2012; Capra et al.
2012). An example is the crosstalk observed between
PhoR–PhoB and VanR–VanS TCSs. PhoR kinase can phosphorylate VanR only in the absence of VanS, and crosstalk between VanS to PhoB is enhanced not just by the
absence of PhoR but also by the absence of VanR (Fisher
et al. 1995; Novak et al. 1999). In E. coli, crosstalk between
CpxA and OmpR or EnvZ and CpxR of the CpxA–CpxR
and EnvZ–OmpR systems can occur in the absence of the
cognate signaling partners (Siryaporn and Goulian 2008).
Unlike eukaryotes, in which crosstalk is very common,
there is very limited crosstalk in bacteria because it can
have disastrous consequences for cell fitness. However,
some cases of crosstalk are permitted or used as a means
of either integrating multiple signals or diversifying the
response from a single input; this is referred to as “crossregulation” to distinguish it from unwanted crosstalk.
Escherichia coli PhoR and PhoM–CreC (Wanner 1993),
ArcB–ArcA, and EnvZ–OmpR (Matsubara et al. 2000), and
B. subtilis PhoR–PhoP and YycG–YycF (Howell et al. 2003)
interactions are examples of cross-regulation interactions.
Crosstalk appears to be disadvantageous in prokaryotes because it results in decreased responses and
the need for increased levels of active RRs to maintain
signal transduction (Batchelor and Goulian 2003). Therefore, avoiding crosstalk appears to be a major selective
pressure driving the diversification of specific residues
following gene duplication events (Zarrinpar et al. 2003;
7
Podgornaia and Laub 2013). Bacteria possess 3 key
mechanisms for ensuring the specificity of 2-component
pathways at the level of phosphotransfer: molecular recognition, phosphatase activity, and substrate competition (Podgornaia and Laub 2013). Systematic analyses of
phosphotransfer have shown that HKs possess the intrinsic ability to discriminate their cognate RRs from other
noncognate substrates (Skerker et al. 2005). Analyses of
amino acid co-evolution in cognate signaling proteins
have identified key specificity-determining residues in
HKs and RRs (Skerker et al. 2008; Casino et al. 2009;
Weigt et al. 2009; Bell et al. 2010; Capra et al. 2010). Moreover, some HKs are bifunctional; they can dephosphorylate their cognate RRs in the absence of an input
signal (Dutta and Inouye 1996; Gao and Stock 2009) and
phosphorylate RRs in response to an input signal. In addition to the phosphatase activity of bifunctional HKs,
there is evidence that RR competition also help limit
crosstalk between some signaling pathways. A recent
study that used a mathematical modeling approach reported that the pressure to avoid crosstalk has influenced the evolution of new TCS pairs, driving rapid
sequence divergence in protein interaction interfaces
immediately postduplication (Rowland and Deeds 2014).
Perspectives
TCS proteins are critical signal transduction components in bacteria. Rapid developments in sequencing
technology have resulted in the sequencing of over 5000
bacteria genomes and 164 277 proteins, including 74 029
of HKs, and more than 500 000 of RRs have been predicted to be TCS proteins (P2CS: http://www.p2cs.org).
This massive amount of information and the wealth of
TCS studies over the past few decades have resulted in
elucidation of signaling mechanisms mediated by TCS
proteins. However, there are still several issues that remain to be resolved in this field. Many sensors for TCSs
have been reported, but only a few stimuli (or ligands)
that are specifically recognized by HKs have been identified. Furthermore, the mechanisms by which these signals trigger autophosphorylation of HKs to start signal
transduction have yet to be clarified. In addition, the
discovery of phosphorylation-independent activation
of RRs has raised questions about the role of phosphorylation in TCS signaling mediated by RRs and the
factors that control binding affinity to DNA. Knowledge
about TCS-mediated signaling is also still fragmented.
Atypical phosphotransmission, the discovery of a hybrid
form of TCS proteins and ARRs, and cross-regulation between TCSs suggest that the pathways linking a particular HK to a particular RR may not be as simple as
previously thought. The development of diverse pathways is most likely due to demands from continuous,
simultaneous, and frequent environmental changes.
Although it is not yet clear if complex and integrative
regulation is beneficial, interference or cooperation in
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regulatory systems can occur in bacteria cells. Recently,
our group generated single knock-out mutant strains of
all RRs in the rice pathogenic bacterium, Xanthomonas
oryzae pv. oryzae. In simple expression profiling experiments in the knock-out strains, we observed that a lack
of a RR altered the expression of other RRs. Previous
publications and our observation suggest that TCSs have
complicated connections not only at the expression level
but also at the signaling circuit level. To answer the questions posed above and to enhance our understanding of
bacterial biology, integrative analyses of TCSs are required.
Acknowledgements
This research was supported by a grant from the Basic
Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry
of Education (NRF-2013R1A1A2009726).
References
Alix, E., and Blanc-Potard, A.B. 2009. Hydrophobic peptides:
novel regulators within bacterial membrane. Mol. Microbiol.
72(1): 5–11. doi:10.1111/j.1365-2958.2009.06626.x. PMID:19210615.
Aoyama, T., and Oka, A. 1990. A common mechanism of transcriptional activation by the three positive regulators, VirG,
PhoB, and OmpR. FEBS Lett. 263(1): 1–4. doi:10.1016/00145793(90)80691-B. PMID:2185030.
Appleby, J.L., Parkinson, J.S., and Bourret, R.B. 1996. Signal
transduction via the multi-step phosphorelay: not necessarily a road less traveled. Cell, 86(6): 845–848. doi:10.1016/S00928674(00)80158-0. PMID:8808618.
Bachhawat, P., Swapna, G.V., Montelione, G.T., and Stock, A.M.
2005. Mechanism of activation for transcription factor PhoB
suggested by different modes of dimerization in the inactive
and active states. Structure, 13(9): 1353–1363. doi:10.1016/j.str.
2005.06.006. PMID:16154092.
Baikalov, I., Schroder, I., Kaczor-Grzeskowiak, M.,
Grzeskowiak, K., Gunsalus, R.P., and Dickerson, R.E. 1996.
Structure of the Escherichia coli response regulator NarL. Biochemistry, 35(34): 11053–11061. doi:10.1021/bi960919o. PMID:
8780507.
Barakat, M., Ortet, P., Jourlin-Castelli, C., Ansaldi, M., Mejean, V.,
and Whitworth, D.E. 2009. P2CS: a two-component system
resource for prokaryotic signal transduction research. BMC
Genomics, 10: 315. doi:10.1186/1471-2164-10-315. PMID:19604365.
Barakat, M., Ortet, P., and Whitworth, D.E. 2011. P2CS: a database of prokaryotic two-component systems. Nucleic Acids
Res. 39(Database issue): D771–D776. doi:10.1093/nar/gkq1023.
PMID:21051349.
Barbieri, C.M., Wu, T., and Stock, A.M. 2013. Comprehensive
analysis of OmpR phosphorylation, dimerization, and DNA
binding supports a canonical model for activation. J. Mol.
Biol. 425(10): 1612–1626. doi:10.1016/j.jmb.2013.02.003. PMID:
23399542.
Barta, M.L., Hickey, J.M., Anbanandam, A., Dyer, K.,
Hammel, M., and Hefty, P.S. 2014. Atypical response regulator ChxR from Chlamydia trachomatis is structurally poised for
DNA binding. PLoS One, 9(3): e91760. doi:10.1371/journal.pone.
0091760. PMID:24646934.
Batchelor, E., and Goulian, M. 2003. Robustness and the cycle
of phosphorylation and dephosphorylation in a twocomponent regulatory system. Proc. Natl. Acad. Sci. U.S.A.
100(2): 691–696. doi:10.1073/pnas.0234782100. PMID:12522261.
Bell, C.H., Porter, S.L., Strawson, A., Stuart, D.I., and
Can. J. Microbiol. Vol. 61, 2015
Armitage, J.P. 2010. Using structural information to change
the phosphotransfer specificity of a two-component chemotaxis signalling complex. PLoS Biol. 8(2): e1000306. doi:10.
1371/journal.pbio.1000306. PMID:20161720.
Birck, C., Mourey, L., Gouet, P., Fabry, B., Schumacher, J.,
Rousseau, P., et al. 1999. Conformational changes induced by
phosphorylation of the FixJ receiver domain. Structure, 7(12):
1505–1515. doi:10.1016/S0969-2126(00)88341-0. PMID:10647181.
Blanco, A.G., Sola, M., Gomis-Ruth, F.X., and Coll, M. 2002. Tandem DNA recognition by PhoB, a two-component signal
transduction transcriptional activator. Structure, 10(5): 701–
713. doi:10.1016/S0969-2126(02)00761-X. PMID:12015152.
Bourret, R.B. 2010. Receiver domain structure and function in
response regulator proteins. Curr. Opin. Microbiol. 13(2):
142–149. doi:10.1016/j.mib.2010.01.015. PMID:20211578.
Brennan, R.G. 1993. The winged-helix DNA-binding motif: another helix-turn-helix takeoff. Cell, 74(5): 773–776. doi:10.1016/
0092-8674(93)90456-Z. PMID:8374950.
Buckler, D.R., Zhou, Y., and Stock, A.M. 2002. Evidence of intradomain and interdomain flexibility in an OmpR/PhoB homolog from Thermotoga maritima. Structure, 10(2): 153–164.
doi:10.1016/S0969-2126(01)00706-7. PMID:11839301.
Buelow, D.R., and Raivio, T.L. 2010. Three (and more) component regulatory systems — auxiliary regulators of bacterial
histidine kinases. Mol. Microbiol. 75(3): 547–566. doi:10.1111/
j.1365-2958.2009.06982.x. PMID:19943903.
Cai, S.J., and Inouye, M. 2003. Spontaneous subunit exchange
and biochemical evidence for trans-autophosphorylation in
a dimer of Escherichia coli histidine kinase (EnvZ). J. Mol. Biol.
329(3): 495–503. doi:10.1016/S0022-2836(03)00446-7. PMID:
12767831.
Cameron, A.D., and Dorman, C.J. 2012. A fundamental regulatory mechanism operating through OmpR and DNA topology
controls expression of Salmonella pathogenicity islands SPI-1
and SPI-2. PLoS Genet. 8(3): e1002615. doi:10.1371/journal.pgen.
1002615. PMID:22457642.
Capra, E.J., and Laub, M.T. 2012. Evolution of two-component
signal transduction systems. Annu. Rev. Microbiol. 66: 325–
347. doi:10.1146/annurev-micro-092611-150039. PMID:22746333.
Capra, E.J., Perchuk, B.S., Lubin, E.A., Ashenberg, O.,
Skerker, J.M., and Laub, M.T. 2010. Systematic dissection and
trajectory-scanning mutagenesis of the molecular interface
that ensures specificity of two-component signaling pathways. PLoS Genet. 6(11): e1001220. doi:10.1371/journal.pgen.
1001220. PMID:21124821.
Capra, E.J., Perchuk, B.S., Skerker, J.M., and Laub, M.T. 2012.
Adaptive mutations that prevent crosstalk enable the expansion of paralogous signaling protein families. Cell, 150(1):
222–232. doi:10.1016/j.cell.2012.05.033. PMID:22770222.
Casino, P., Rubio, V., and Marina, A. 2009. Structural insight
into partner specificity and phosphoryl transfer in twocomponent signal transduction. Cell, 139(2): 325–336. doi:10.
1016/j.cell.2009.08.032. PMID:19800110.
Chen, Y., Abdel-Fattah, W.R., and Hulett, F.M. 2004. Residues
required for Bacillus subtilis PhoP DNA binding or RNA polymerase interaction: alanine scanning of PhoP effector domain transactivation loop and alpha helix 3. J. Bacteriol.
186(5): 1493–1502. doi:10.1128/JB.186.5.1493-1502.2004. PMID:
14973033.
Cho, H.S., Lee, S.-Y., Yan, D., Pan, X., Parkinson, J.S., Kustu, S.,
et al. 2000. NMR structure of activated CheY. J. Mol. Biol.
297(3): 543–551. doi:10.1006/jmbi.2000.3595. PMID:10731410.
Clark, K.L., Halay, E.D., Lai, E., and Burley, S.K. 1993. Co-crystal
structure of the HNF-3/fork head DNA-recognition motif resembles histone H5. Nature, 364(6436): 412–420. doi:10.1038/
364412a0. PMID:8332212.
Clubb, R.T., Omichinski, J.G., Savilahti, H., Mizuuchi, K.,
Gronenborn, A.M., and Clore, G.M. 1994. A novel class of
Published by NRC Research Press
Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by 2.81.42.47 on 09/21/15
For personal use only.
Nguyen et al.
winged helix–turn–helix protein: the DNA-binding domain
of Mu transposase. Structure, 2(11): 1041–1048. doi:10.1016/
S0969-2126(94)00107-3. PMID:7881904.
Cock, P.J., and Whitworth, D.E. 2007. Evolution of prokaryotic
two-component system signaling pathways: gene fusions
and fissions. Mol. Biol. Evol. 24(11): 2355–2357. doi:10.1093/
molbev/msm170. PMID:17709334.
Creager-Allen, R.L., Silversmith, R.E., and Bourret, R.B. 2013. A
link between dimerization and autophosphorylation of the
response regulator PhoB. J. Biol. Chem. 288(30): 21755–21769.
doi:10.1074/jbc.M113.471763. PMID:23760278.
De la Cruz, M.A., and Calva, E. 2010. The complexities of porin
genetic regulation. J. Mol. Microbiol. Biotechnol. 18(1): 24–36.
doi:10.1159/000274309. PMID:20068355.
Debnath, I., Norton, J.P., Barber, A.E., Ott, E.M., Dhakal, B.K.,
Kulesus, R.R., and Mulvey, M.A. 2013. The Cpx stress response
system potentiates the fitness and virulence of uropathogenic Escherichia coli. Infect. Immun. 81(5): 1450–1459. doi:10.
1128/IAI.01213-12. PMID:23429541.
Delany, I., Spohn, G., Rappuoli, R., and Scarlato, V. 2002.
Growth phase-dependent regulation of target gene promoters for binding of the essential orphan response regulator
HP1043 of Helicobacter pylori. J. Bacteriol. 184(17): 4800–4810.
doi:10.1128/JB.184.17.4800-4810.2002. PMID:12169605.
Djordjevic, S., Goudreau, P.N., Xu, Q., Stock, A.M., and
West, A.H. 1998. Structural basis for methylesterase CheB
regulation by a phosphorylation-activated domain. Proc.
Natl. Acad. Sci. U.S.A. 95(4): 1381–1386. doi:10.1073/pnas.95.4.
1381. PMID:9465023.
Dutta, R., and Inouye, M. 1996. Reverse phosphotransfer from
OmpR to EnvZ in a kinase/phosphatase mutant of EnvZ
(EnvZN347D), a bifunctional signal transducer of Escherichia
coli. J. Biol. Chem. 271(3): 1424–1429. doi:10.1074/jbc.271.3.
1424. PMID:8576133.
Espinosa, J., Lopez-Redondo, M.L., Miguel-Romero, L., Neira, J.L.,
Marina, A., and Contreras, A. 2012. Insights into the mechanism of activation of the phosphorylation-independent response regulator NblR. Role of residues Cys69 and Cys96.
Biochim. Biophys. Acta, 1819(5): 382–390. doi:10.1016/j.
bbagrm.2012.01.007. PMID:22306661.
Fabret, C., Feher, V.A., and Hoch, J.A. 1999. Two-component
signal transduction in Bacillus subtilis: how one organism sees
its world. J. Bacteriol. 181(7): 1975–1983. PMID:10094672.
Feng, J., Atkinson, M.R., McCleary, W., Stock, J.B., Wanner, B.L.,
and Ninfa, A.J. 1992. Role of phosphorylated metabolic intermediates in the regulation of glutamine synthetase synthesis
in Escherichia coli. J. Bacteriol. 174(19): 6061–6070. PMID:1356964.
Fisher, S.L., Jiang, W., Wanner, B.L., and Walsh, C.T. 1995. Crosstalk between the histidine protein kinase VanS and the
response regulator PhoB: characterization and identification of a VanS domain that inhibits activation of PhoB.
J. Biol. Chem. 270(39): 23143–23149. doi:10.1074/jbc.270.39.
23143. PMID:7559459.
Fleischer, R., Heermann, R., Jung, K., and Hunke, S. 2007. Purification, reconstitution, and characterization of the CpxRAP
envelope stress system of Escherichia coli. J. Biol. Chem. 282(12):
8583–8593. doi:10.1074/jbc.M605785200. PMID:17259177.
Friedland, N., Mack, T.R., Yu, M., Hung, L.W., Terwilliger, T.C.,
Waldo, G.S., and Stock, A.M. 2007. Domain orientation in the
inactive response regulator Mycobacterium tuberculosis MtrA
provides a barrier to activation. Biochemistry, 46(23): 6733–
6743. doi:10.1021/bi602546q. PMID:17511470.
Galperin, M.Y. 2006. Structural classification of bacterial response regulators: diversity of output domains and domain
combinations. J. Bacteriol. 188(12): 4169–4182. doi:10.1128/JB.
01887-05. PMID:16740923.
Galperin, M.Y. 2010. Diversity of structure and function of re-
9
sponse regulator output domains. Curr. Opin. Microbiol.
13(2): 150–159. doi:10.1016/j.mib.2010.01.005. PMID:20226724.
Gao, R., and Stock, A.M. 2009. Biological insights from structures
of two-component proteins. Annu. Rev. Microbiol. 63: 133–154.
doi:10.1146/annurev.micro.091208.073214. PMID:19575571.
Gao, R., and Stock, A.M. 2013. Probing kinase and phosphatase
activities of two-component systems in vivo with concentrationdependent phosphorylation profiling. Proc. Natl. Acad. Sci. U.S.A.
110(2): 672–677. doi:10.1073/pnas.1214587110. PMID:23267085.
Gao, R., Mack, T.R., and Stock, A.M. 2007. Bacterial response
regulators: versatile regulatory strategies from common domains. Trends Biochem. Sci. 32(5): 225–234. doi:10.1016/j.tibs.
2007.03.002. PMID:17433693.
Gao, R., Tao, Y., and Stock, A.M. 2008. System-level mapping of
Escherichia coli response regulator dimerization with FRET hybrids. Mol. Microbiol. 69(6): 1358–1372. doi:10.1111/j.1365-2958.
2008.06355.x. PMID:18631241.
Grebe, T.W., and Stock, J.B. 1999. The histidine protein kinase
superfamily. Adv. Microb. Physiol. 41: 139–227. doi:10.1016/
S0065-2911(08)60167-8. PMID:10500846.
Gupta, S., Sinha, A., and Sarkar, D. 2006. Transcriptional autoregulation by Mycobacterium tuberculosis PhoP involves recognition of novel direct repeat sequences in the regulatory
region of the promoter. FEBS Lett. 580(22): 5328–5338. doi:
10.1016/j.febslet.2006.09.004. PMID:16979633.
Halkides, C.J., McEvoy, M.M., Casper, E., Matsumura, P., Volz, K.,
and Dahlquist, F.W. 2000. The 1.9 Å resolution crystal structure of phosphono-CheY, an analogue of the active form of
the response regulator, CheY. Biochemistry, 39(18): 5280–
5286. doi:10.1021/bi9925524. PMID:10819997.
Harrison, C.J., Bohm, A.A., and Nelson, H.C. 1994. Crystal structure of the DNA binding domain of the heat shock transcription factor. Science, 263(5144): 224–227. doi:10.1126/science.
8284672. PMID:8284672.
Hess, J.F., Oosawa, K., Kaplan, N., and Simon, M.I. 1988. Phosphorylation of three proteins in the signaling pathway of
bacterial chemotaxis. Cell, 53(1): 79–87. doi:10.1016/00928674(88)90489-8. PMID:3280143.
Hickey, J.M., Lovell, S., Battaile, K.P., Hu, L., Middaugh, C.R., and
Hefty, P.S. 2011. The atypical response regulator protein
ChxR has structural characteristics and dimer interface interactions that are unique within the OmpR/PhoB subfamily.
J. Biol. Chem. 286(37): 32606–32616. doi:10.1074/jbc.M111.
220574. PMID:21775428.
Hong, E., Lee, H.M., Ko, H., Kim, D.U., Jeon, B.Y., Jung, J., et al.
2007. Structure of an atypical orphan response regulator protein supports a new phosphorylation-independent regulatory mechanism. J. Biol. Chem. 282(28): 20667–20675. doi:10.
1074/jbc.M609104200. PMID:17491010.
Howell, A., Dubrac, S., Andersen, K.K., Noone, D., Fert, J.,
Msadek, T., and Devine, K. 2003. Genes controlled by the
essential YycG/YycF two-component system of Bacillus subtilis
revealed through a novel hybrid regulator approach. Mol.
Microbiol. 49(6): 1639–1655. doi:10.1046/j.1365-2958.2003.
03661.x. PMID:12950927.
Hsing, W., Russo, F.D., Bernd, K.K., and Silhavy, T.J. 1998. Mutations that alter the kinase and phosphatase activities of the
two-component sensor EnvZ. J. Bacteriol. 180(17): 4538–4546.
PMID:9721293.
Jeon, Y., Lee, Y.S., Han, J.S., Kim, J.B., and Hwang, D.S. 2001. Multimerization of phosphorylated and non-phosphorylated ArcA is
necessary for the response regulator function of the Arc twocomponent signal transduction system. J. Biol. Chem. 276(44):
40873–40879. doi:10.1074/jbc.M104855200. PMID:11527965.
Jiang, M., Shao, W.L., Perego, M., and Hoch, J.A. 2000. Multiple
histidine kinases regulate entry into stationary phase and
sporulation in Bacillus subtilis. Mol. Microbiol. 38(3): 535–542.
doi:10.1046/j.1365-2958.2000.02148.x. PMID:11069677.
Published by NRC Research Press
Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by 2.81.42.47 on 09/21/15
For personal use only.
10
Kato, A., and Groisman, E.A. 2004. Connecting two-component
regulatory systems by a protein that protects a response
regulator from dephosphorylation by its cognate sensor.
Genes Dev. 18(18): 2302–2313. doi:10.1101/gad.1230804. PMID:
15371344.
Kern, D., Volkman, B.F., Luginbuhl, P., Nohaile, M.J., Kustu, S.,
and Wemmer, D.E. 1999. Structure of a transiently phosphorylated switch in bacterial signal transduction. Nature,
402(6764): 894–898. doi:10.1038/47273. PMID:10622255.
King-Scott, J., Nowak, E., Mylonas, E., Panjikar, S., Roessle, M.,
Svergun, D.I., and Tucker, P.A. 2007. The structure of a fulllength response regulator from Mycobacterium tuberculosis
in a stabilized three-dimensional domain-swapped, activated state. J. Biol. Chem. 282(52): 37717–37729. doi:10.1074/
jbc.M705081200. PMID:17942407.
Koo, I.C., Walthers, D., Hefty, P.S., Kenney, L.J., and
Stephens, R.S. 2006. ChxR is a transcriptional activator in
Chlamydia. Proc. Natl. Acad. Sci. U.S.A. 103(3): 750–755. doi:10.
1073/pnas.0509690103. PMID:16407127.
Koshland, D.E. , Jr. 1977. A response regulator model in a simple
sensory system. Science, 196(4294): 1055–1063. doi:10.1126/
science.870969. PMID:870969.
Kwon, E., Kim, D.Y., Ngo, T.D., Gross, C.A., Gross, J.D., and
Kim, K.K. 2012. The crystal structure of the periplasmic domain of Vibrio parahaemolyticus CpxA. Protein Sci. 21(9): 1334–
1343. doi:10.1002/pro.2120. PMID:22760860.
Lai, E., Prezioso, V.R., Tao, W.F., Chen, W.S., and Darnell, J.E.
1991. Hepatocyte nuclear factor 3 alpha belongs to a gene
family in mammals that is homologous to the Drosophila homeotic gene fork head. Genes Dev. 5(3): 416–427. doi:10.1101/
gad.5.3.416. PMID:1672118.
Laub, M.T., and Goulian, M. 2007. Specificity in two-component
signal transduction pathways. Annu. Rev. Genet. 41: 121–145.
doi:10.1146/annurev.genet.41.042007.170548. PMID:18076326.
Lee, S.Y., Cho, H.S., Pelton, J.G., Yan, D., Henderson, R.K.,
King, D.S., et al. 2001. Crystal structure of an activated response regulator bound to its target. Nat. Struct. Biol. 8(1):
52–56. doi:10.1038/83053. PMID:11135671.
Lewis, R.J., Brannigan, J.A., Muchova, K., Barak, I., and
Wilkinson, A.J. 1999. Phosphorylated aspartate in the structure of a response regulator protein. J. Mol. Biol. 294(1): 9–15.
doi:10.1006/jmbi.1999.3261. PMID:10556024.
Lin, W., Wang, Y., Han, X.B., Zhang, Z.L., Wang, C.Y., Wang, J.,
et al. 2014. Atypical OmpR/PhoB subfamily response regulator GlnR of actinomycetes functions as a homodimer,
stabilized by the unphosphorylated conserved Asp-focused
charge interactions. J. Biol. Chem. 289(22): 15413–15425. doi:
10.1074/jbc.M113.543504. PMID:24733389.
Liu, G., Chater, K.F., Chandra, G., Niu, G., and Tan, H. 2013.
Molecular regulation of antibiotic biosynthesis in Streptomyces.
Microbiol. Mol. Biol. Rev. 77(1): 112–143. doi:10.1128/MMBR.
00054-12. PMID:23471619.
Liu, Y., Gao, Z.Q., She, Z., Qu, K., Wang, W.J., Shtykova, E.V.,
et al. 2012. The structural basis of the response regulator
DrRRA from Deinococcus radiodurans. Biochem. Biophys.
Res. Commun. 417(4): 1206–1212. doi:10.1016/j.bbrc.2011.12.
110. PMID:22227191.
Lukat, G.S., McCleary, W.R., Stock, A.M., and Stock, J.B. 1992.
Phosphorylation of bacterial response regulator proteins by
low molecular weight phospho-donors. Proc. Natl. Acad.
Sci. U.S.A. 89(2): 718–722. doi:10.1073/pnas.89.2.718. PMID:
1731345.
Martinez-Hackert, E., and Stock, A.M. 1997a. The DNA-binding
domain of OmpR: crystal structures of a winged helix transcription factor. Structure, 5(1): 109–124. doi:10.1016/S09692126(97)00170-6. PMID:9016718.
Martinez-Hackert, E., and Stock, A.M. 1997b. Structural relationships in the OmpR family of winged-helix transcription fac-
Can. J. Microbiol. Vol. 61, 2015
tors. J. Mol. Biol. 269(3): 301–312. doi:10.1006/jmbi.1997.1065.
PMID:9199401.
Matsubara, M., Kitaoka, S., Takeda, S., and Mizuno, T. 2000.
Tuning of the porin expression under anaerobic growth conditions by His-to-Asp cross-phosphorelay through both the
EnvZ-osmosensor and ArcB-anaerosensor in Escherichia coli.
Genes Cells, 5(7): 555–569. doi:10.1046/j.1365-2443.2000.
00347.x. PMID:10947842.
Mattison, K., Oropeza, R., and Kenney, L.J. 2002. The linker region plays an important role in the interdomain communication of the response regulator OmpR. J. Biol. Chem. 277(36):
32714–32721. doi:10.1074/jbc.M204122200. PMID:12077136.
Menon, S., and Wang, S. 2011. Structure of the response regulator PhoP from Mycobacterium tuberculosis reveals a dimer
through the receiver domain. Biochemistry, 50(26): 5948–
5957. doi:10.1021/bi2005575. PMID:21634789.
Mitrophanov, A.Y., and Groisman, E.A. 2008. Signal integration
in bacterial two-component regulatory systems. Genes Dev.
22(19): 2601–2611. doi:10.1101/gad.1700308. PMID:18832064.
Narayanan, A., Paul, L.N., Tomar, S., Patil, D.N., Kumar, P., and
Yernool, D.A. 2012. Structure–function studies of DNA binding domain of response regulator KdpE reveals equal affinity
interactions at DNA half-sites. PLoS One, 7(1): e30102. doi:10.
1371/journal.pone.0030102. PMID:22291906.
Ninfa, E.G., Atkinson, M.R., Kamberov, E.S., and Ninfa, A.J. 1993.
Mechanism of autophosphorylation of Escherichia coli nitrogen
regulator II (NRII or NtrB): trans-phosphorylation between subunits. J. Bacteriol. 175(21): 7024–7032. PMID:8226644.
Nixon, B.T., Ronson, C.W., and Ausubel, F.M. 1986. Twocomponent regulatory systems responsive to environmental
stimuli share strongly conserved domains with the nitrogen
assimilation regulatory genes ntrB and ntrC. Proc. Natl. Acad.
Sci. U.S.A. 83(20): 7850–7854. doi:10.1073/pnas.83.20.7850.
PMID:3020561.
Novak, R., Henriques, B., Charpentier, E., Normark, S., and
Tuomanen, E. 1999. Emergence of vancomycin tolerance in
Streptococcus pneumoniae. Nature, 399(6736): 590–593. doi:10.
1038/21202. PMID:10376600.
Nowak, E., Panjikar, S., Konarev, P., Svergun, D.I., and
Tucker, P.A. 2006. The structural basis of signal transduction
for the response regulator PrrA from Mycobacterium tuberculosis.
J. Biol. Chem. 281(14): 9659–9666. doi:10.1074/jbc.M512004200.
PMID:16434396.
Parkinson, G., Wilson, C., Gunasekera, A., Ebright, Y.W.,
Ebright, R.E., and Berman, H.M. 1996. Structure of the CAP–
DNA complex at 2.5 Å resolution: a complete picture of the
protein–DNA interface. J. Mol. Biol. 260(3): 395–408. doi:10.
1006/jmbi.1996.0409. PMID:8757802.
Parkinson, J.S., and Kofoid, E.C. 1992. Communication modules
in bacterial signaling proteins. Annu. Rev. Genet. 26(1): 71–
112. doi:10.1146/annurev.ge.26.120192.000443. PMID:1482126.
Pelton, J.G., Kustu, S., and Wemmer, D.E. 1999. Solution structure of the DNA-binding domain of NtrC with three alanine
substitutions. J. Mol. Biol. 292(5): 1095–1110. doi:10.1006/jmbi.
1999.3140. PMID:10512705.
Pena-Sandoval, G.R., and Georgellis, D. 2010. The ArcB sensor
kinase of Escherichia coli autophosphorylates by an intramolecular reaction. J. Bacteriol. 192(6): 1735–1739. doi:10.1128/JB.
01401-09. PMID:20097862.
Perraud, A.L., Weiss, V., and Gross, R. 1999. Signalling pathways in two-component phosphorelay systems. Trends Microbiol. 7(3): 115–120. doi:10.1016/S0966-842X(99)01458-4. PMID:
10203840.
Podgornaia, A.I., and Laub, M.T. 2013. Determinants of specificity
in two-component signal transduction. Curr. Opin. Microbiol.
16(2): 156–162. doi:10.1016/j.mib.2013.01.004. PMID:23352354.
Pontiggia, F., Pachov, D.V., Clarkson, M.W., Villali, J.,
Hagan, M.F., Pande, V.S., and Kern, D. 2015. Free energy landPublished by NRC Research Press
Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by 2.81.42.47 on 09/21/15
For personal use only.
Nguyen et al.
scape of activation in a signalling protein at atomic resolution. Nat. Commun. 6: 7284. doi:10.1038/ncomms8284. PMID:
26073309.
Qin, L., Yoshida, T., and Inouye, M. 2001. The critical role of DNA
in the equilibrium between OmpR and phosphorylated
OmpR mediated by EnvZ in Escherichia coli. Proc. Natl. Acad.
Sci. U.S.A. 98(3): 908–913. doi:10.1073/pnas.98.3.908. PMID:
11158569.
Quinn, H.J., Cameron, A.D., and Dorman, C.J. 2014. Bacterial
regulon evolution: distinct responses and roles for the identical OmpR proteins of Salmonella typhimurium and Escherichia
coli in the acid stress response. PLoS Genet. 10(3): e1004215.
doi:10.1371/journal.pgen.1004215. PMID:24603618.
Ramakrishnan, V., Finch, J.T., Graziano, V., Lee, P.L., and
Sweet, R.M. 1993. Crystal structure of globular domain of
histone H5 and its implications for nucleosome binding.
Nature, 362(6417): 219–223. doi:10.1038/362219a0. PMID:
8384699.
Rhee, J.E., Sheng, W., Morgan, L.K., Nolet, R., Liao, X., and
Kenney, L.J. 2008. Amino acids important for DNA recognition by the response regulator OmpR. J. Biol. Chem. 283(13):
8664–8677. doi:10.1074/jbc.M705550200. PMID:18195018.
Ritzefeld, M., Walhorn, V., Anselmetti, D., and Sewald, N. 2013.
Analysis of DNA interactions using single-molecule force
spectroscopy. Amino Acids, 44(6): 1457–1475. doi:10.1007/
s00726-013-1474-4. PMID:23468137.
Robinson, V.L., Wu, T., and Stock, A.M. 2003. Structural analysis
of the domain interface in DrrB, a response regulator of the
OmpR/PhoB subfamily. J. Bacteriol. 185(14): 4186–4194. doi:
10.1128/JB.185.14.4186-4194.2003. PMID:12837793.
Rowland, M.A., and Deeds, E.J. 2014. Crosstalk and the evolution
of specificity in two-component signaling. Proc. Natl. Acad.
Sci. U.S.A. 111(15): 5550–5555. doi:10.1073/pnas.1317178111.
PMID:24706803.
Ruiz, D., Salinas, P., Lopez-Redondo, M.L., Cayuela, M.L., Marina, A.,
and Contreras, A. 2008. Phosphorylation-independent activation of the atypical response regulator NblR. Microbiology,
154(10): 3002–3015. doi:10.1099/mic.0.2008/020677-0. PMID:
18832306.
Schaller, G.E., Shiu, S.H., and Armitage, J.P. 2011. Twocomponent systems and their co-option for eukaryotic signal
transduction. Curr. Biol. 21(9): R320–R330. doi:10.1016/j.cub.
2011.02.045. PMID:21549954.
Schultz, S.C., Shields, G.C., and Steitz, T.A. 1991. Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees.
Science, 253(5023): 1001–1007. doi:10.1126/science.1653449.
PMID:1653449.
Sidote, D.J., Barbieri, C.M., Wu, T., and Stock, A.M. 2008. Structure of the Staphylococcus aureus AgrA LytTR domain bound to
DNA reveals a beta fold with an unusual mode of binding.
Structure, 16(5): 727–735. doi:10.1016/j.str.2008.02.011. PMID:
18462677.
Simon, G., Jourlin, C., Ansaldi, M., Pascal, M.C., Chippaux, M.,
and Mejean, V. 1995. Binding of the TorR regulator to cisacting direct repeats activates tor operon expression.
Mol. Microbiol. 17(5): 971–980. doi:10.1111/j.1365-2958.1995.
mmi_17050971.x. PMID:8596446.
Simon, M.I., Borkovich, K.A., Bourret, R.B., and Hess, J.F. 1989.
Protein phosphorylation in the bacterial chemotaxis system.
Biochimie, 71(9–10): 1013–1019. doi:10.1016/0300-9084(89)
90105-3. PMID:2512992.
Sinha, A., Gupta, S., Bhutani, S., Pathak, A., and Sarkar, D. 2008.
PhoP–PhoP interaction at adjacent PhoP binding sites is influenced by protein phosphorylation. J. Bacteriol. 190(4):
1317–1328. doi:10.1128/JB.01074-07. PMID:18065544.
Siryaporn, A., and Goulian, M. 2008. Cross-talk suppression
between the CpxA–CpxR and EnvZ–OmpR two-component
11
systems in E. coli. Mol. Microbiol. 70(2): 494–506. doi:10.1111/
j.1365-2958.2008.06426.x. PMID:18761686.
Skerker, J.M., Prasol, M.S., Perchuk, B.S., Biondi, E.G., and
Laub, M.T. 2005. Two-component signal transduction pathways regulating growth and cell cycle progression in a bacterium: a system-level analysis. PLoS Biol. 3(10): e334. doi:10.
1371/journal.pbio.0030334. PMID:16176121.
Skerker, J.M., Perchuk, B.S., Siryaporn, A., Lubin, E.A.,
Ashenberg, O., Goulian, M., and Laub, M.T. 2008. Rewiring
the specificity of two-component signal transduction systems. Cell, 133(6): 1043–1054. doi:10.1016/j.cell.2008.04.040.
PMID:18555780.
Stock, A.M., Mottonen, J.M., Stock, J.B., and Schutt, C.E. 1989.
Three-dimensional structure of CheY, the response regulator
of bacterial chemotaxis. Nature, 337(6209): 745–749. doi:10.
1038/337745a0. PMID:2645526.
Stock, A.M., Robinson, V.L., and Goudreau, P.N. 2000. Twocomponent signal transduction. Annu. Rev. Biochem. 69(1):
183–215. doi:10.1146/annurev.biochem.69.1.183. PMID:10966457.
Stock, J.B., Ninfa, A.J., and Stock, A.M. 1989. Protein phosphorylation and regulation of adaptive responses in bacteria.
Microbiol. Rev. 53(4): 450–490. PMID:2556636.
Toro-Roman, A., Mack, T.R., and Stock, A.M. 2005a. Structural
analysis and solution studies of the activated regulatory domain of the response regulator ArcA: a symmetric dimer
mediated by the ␣4-␤5-␣5 face. J. Mol. Biol. 349(1): 11–26. doi:
10.1016/j.jmb.2005.03.059. PMID:15876365.
Toro-Roman, A., Wu, T., and Stock, A.M. 2005b. A common
dimerization interface in bacterial response regulators
KdpE and TorR. Protein Sci. 14(12): 3077–3088. doi:10.1110/ps.
051722805. PMID:16322582.
Trajtenberg, F., Albanesi, D., Ruetalo, N., Botti, H.,
Mechaly, A.E., Nieves, M., et al. 2014. Allosteric activation of
bacterial response regulators: the role of the cognate histidine kinase beyond phosphorylation. mBio, 5(6): e02105. doi:
10.1128/mBio.02105-14. PMID:25406381.
Ulrich, L.E., and Zhulin, I.B. 2010. The MiST2 database: a comprehensive genomics resource on microbial signal transduction. Nucleic Acids Res. 38(Database issue): D401–D407. doi:
10.1093/nar/gkp940. PMID:19900966.
Ulrich, L.E., Koonin, E.V., and Zhulin, I.B. 2005. One-component
systems dominate signal transduction in prokaryotes.
Trends Microbiol. 13(2): 52–56. doi:10.1016/j.tim.2004.12.006.
PMID:15680762.
Vanatta, D.K., Shukla, D., Lawrenz, M., and Pande, V.S. 2015. A
network of molecular switches controls the activation of the
two-component response regulator NtrC. Nat. Commun. 6:
7283. doi:10.1038/ncomms8283. PMID:26073186.
Villali, J., Pontiggia, F., Clarkson, M.W., Hagan, M.F., and
Kern, D. 2014. Evidence against the “Y–T coupling” mechanism of activation in the response regulator NtrC. J. Mol.
Biol. 426(7): 1554–1567. doi:10.1016/j.jmb.2013.12.027. PMID:
24406745.
Vogel, J., and Papenfort, K. 2006. Small non-coding RNAs and
the bacterial outer membrane. Curr. Opin. Microbiol. 9(6):
605–611. doi:10.1016/j.mib.2006.10.006. PMID:17055775.
Volkman, B.F., Lipson, D., Wemmer, D.E., and Kern, D. 2001.
Two-state allosteric behavior in a single-domain signaling
protein. Science, 291(5512): 2429–2433. doi:10.1126/science.
291.5512.2429. PMID:11264542.
Walthers, D., Tran, V.K., and Kenney, L.J. 2003. Interdomain
linkers of homologous response regulators determine their
mechanism of action. J. Bacteriol. 185(1): 317–324. doi:10.1128/
JB.185.1.317-324.2003. PMID:12486069.
Wang, L., Tian, X., Wang, J., Yang, H., Fan, K., Xu, G., et al. 2009.
Autoregulation of antibiotic biosynthesis by binding of
the end product to an atypical response regulator. Proc.
Published by NRC Research Press
Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by 2.81.42.47 on 09/21/15
For personal use only.
12
Natl. Acad. Sci. U.S.A. 106(21): 8617–8622. doi:10.1073/pnas.
0900592106. PMID:19423672.
Wanner, B.L. 1993. Gene regulation by phosphate in enteric bacteria. J. Cell. Biochem. 51(1): 47–54. doi:10.1002/jcb.240510110.
PMID:8432742.
Weigt, M., White, R.A., Szurmant, H., Hoch, J.A., and Hwa, T. 2009.
Identification of direct residue contacts in protein–protein
interaction by message passing. Proc. Natl. Acad. Sci. U.S.A.
106(1): 67–72. doi:10.1073/pnas.0805923106. PMID:19116270.
West, A.H., and Stock, A.M. 2001. Histidine kinases and response regulator proteins in two-component signaling systems. Trends Biochem. Sci. 26(6): 369–376. doi:10.1016/
S0968-0004(01)01852-7. PMID:11406410.
West, A.H., Martinez-Hackert, E., and Stock, A.M. 1995. Crystal
structure of the catalytic domain of the chemotaxis receptor
methylesterase, CheB. J. Mol. Biol. 250(2): 276–290. doi:10.
1006/jmbi.1995.0376. PMID:7608974.
Can. J. Microbiol. Vol. 61, 2015
Whitworth, D.E. 2012. Classification and organization of twocomponent systems. In Two-component systems in bacteria.
Horizon Scientific Press, Norfolk, UK. pp. 1–20.
Wilson, K.P., Shewchuk, L.M., Brennan, R.G., Otsuka, A.J., and
Matthews, B.W. 1992. Escherichia coli biotin holoenzyme synthetase/bio repressor crystal structure delineates the biotinand DNA-binding domains. Proc. Natl. Acad. Sci. U.S.A. 89(19):
9257–9261. doi:10.1073/pnas.89.19.9257. PMID:1409631.
Yamane, T., Okamura, H., Ikeguchi, M., Nishimura, Y., and
Kidera, A. 2008. Water-mediated interactions between DNA
and PhoB DNA-binding/transactivation domain: NMR-restrained
molecular dynamics in explicit water environment. Proteins,
71(4): 1970–1983. doi:10.1002/prot.21874. PMID:18186481.
Zarrinpar, A., Park, S.H., and Lim, W.A. 2003. Optimization
of specificity in a cellular protein interaction network by
negative selection. Nature, 426(6967): 676–680. doi:10.1038/
nature02178. PMID:14668868.
Published by NRC Research Press