Environmental Microbiology (2016) 00(00), 00–00
13397
Organization and mode of action of two component
system signaling circuits from the various kingdoms
of life
Adrian F. Alvarez, Carlos Barba-Ostria,
Hortencia Silva-Jime nez and Dimitris Georgellis*
Departamento de Gene  tica Molecular, Instituto de
Fisiologıa Celular, Universidad Nacional Auto  noma de
Me xico, 04510 Me  xico City, Me
 xico.
Summary
Two-component system (TCS) signaling circuits reg-
ulate numerous cellular processes in response to
environmental cues in both prokaryotic and eukaryo-
tic cells. These signaling circuits are all based on
phosphoryl-group transfers between histidine and
aspartate containing modules of sensor kinase and
response regulator proteins. Curiously, the architec-
ture and organization of prokaryotic and eukaryotic
two-component systems reveal notable variations,
raising the question of whether the input-response
specificity that governs the majority of prokaryotic
TCSs also governs the eukaryotic ones. In this
review, we contrast the TCS architecture and signal-
ing circuits of prokaryotes and eukaryotes, and
discuss their possible consequences on signaling
specificity.
Introduction
The ability of organisms to survive in ever-changing habi-
tats requires the coordination of multiple environmentally
regulated cellular responses. Such regulation is often
achieved by a group of signal transduction proteins that
constitute the large family of two-component systems
(TCS) (Nixon et al., 1986; Ronson et al., 1987). Typically, a
TCS comprises a sensor histidine kinase (HK) and its cog-
nate response regulator (RR) that are involved in sensing
and signaling environmental cues, resulting in adaptive
gene expressions. The diversity of environmental signals
perceived by TCSs is extensive and therefore many differ-
Received 21 April, 2016; revised 17 May, 2016; accepted 23 May,
2016. *For correspondence. E-mail: dimitris@ifc.unam.mx; Tel.
1(52) 55 5622 5738; Fax: (152) 55 5622 5611.
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doi:10.1111/1462-2920.
ent molecular mechanisms of signal recognition by HKs
have evolved (Cheung and Hendrickson, 2010; Mascher
et al., 2006; Krell et al., 2010).These signaling circuits are
widely distributed across bacteria, and are also present in
many archaeal genomes (Ashby, 2006; Galperin, 2005;
Ulrich and Zhulin, 2010; Wuichet et al., 2010). Although
they have not been found in animals or protists (Koretke
et al., 2000; Wolanin et al., 2002) they are present in sev-
eral other eukaryotes, including fungi, slime molds, social
amoebas, and plants (reviewed by Catlett et al., 2003;
Santos and Shiozaki, 2001; Schaller et al., 2011; Thoma-
son and Kay, 2000). While retaining functional
characteristics, the TCSs of organisms from diverse king-
doms of life show significant differences in architecture and
organization. Consequently, the principles that govern the
signal–response specificity of the vast majority of bacterial
TCSs, partially based in their linear architecture, may not
fully apply to eukaryotic TCSs. Here, we describe the
architectures and signaling circuits of the bacterial TCSs,
and contrast them to the ones of plants and filamentous
fungi. Finally we discuss the possible consequences of
such differences on signaling specificity.
Bacterial TCS signaling: architecture, signaling
circuitry and specificity
The canonical TCS
The prototypical two-component system comprises a HK
protein and a RR protein that contain transmitter domains
(TD) and receiver domains (RD) (Fig. 1A) (Kofoid and Par-
kinson, 1988; Ronson et al., 1987). Transmitter domains
have a conserved kinase core with an invariant histidine
residue, whereas receiver domains have an invariant
aspartate residue. A typical HK is anchored to the cyto-
plasmic membrane and consists of a signal sensing (or
input) domain and a transmitter domain, whereas a typical
RR consists of a receiver domain and an output or effector
domain. Signal reception by the HK is believed to propa-
gate conformational changes in the protein that stimulate
an ATP-dependent autophosphorylation at the conserved
His residue in its transmitter domain. It is of interest to
mention that HKs act as homodimers (Dutta et al., 1999;
  nez and D. Georgellis
2 A. F. Alvarez, C. Barba-Ostria, H. Silva-Jime

Levit et al., 1996; Tanaka et al., 1998; West and Stock, coccus aureus, respectively) subunit reaction (Casino
2001), and their autophosphorylation can occur by either et al., 2009; Ninfa et al., 1993; Stock et al., 2000; Swanson
an inter- (e.g. EnvZ and NtrB of E. coli) or intra- (e.g. et al., 1993; Yang and Inouye, 1991). The phosphorylated
HK853 and PhoR of Thermotoga maritime and Staphylo- HK then donates the phosphoryl-group (P) to the
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 Two-component system signaling 3

Fig. 1. Schematic representation of two-component signaling circuitries.

A. In a prototypical two-component system, signal perception by the histidine kinase (HK) promotes its autophosphorylation at the conserved
His (H) residue in the transmitter domain. The phospho-kinase, then, donates the phosphoryl-group to the conserved Asp (D) residue in the
receiver domain of the cognate response regulator (RR), thereby rendering it functional and resulting in a specific output response. In the
absence of signal, the RR undergoes dephosphorylation either by spontaneous hydrolysis of the phospho-aspartyl bond or by the
phosphatase activity of the cognate HK or another protein (phosphatase X).
B. Hybrid histidine sensor kinases (HHKs) consist of a canonical transmitter domain (TD), a receiver domain (RD), and a phosphotransfer
domain (HPt) that can be present in the same protein (tripartite kinases) or exist in two or three different proteins. In such HHK proteins, the
P is transferred through a multi-step phosphorelay involving three subsequent His to Asp steps (TD ! RD ! HPt ! RD of the RR).
C. Representation of the TD1 ! RD ! TD2 ! RD phosphorelay operating in the TodS-like HHKs. Although autophosphorylation of TD2
(represented by a dotted arrow) occurs in vitro, TD2 autophosphorylation in response to a specific signal has not yet been demonstrated.
D. Representation of the reverse phosphorelay (RD of the RR ! HPt ! RD ! Pi), responsible for signal silencing, carried out by HHKs in the
absence of stimulus.

conserved Asp residue in the receiver domain of the cog-
nate RR, thereby rendering it functional, in general as a
transcriptional regulator. Upon cessation of signaling, both
the cognate RR and the HK undergo dephosphorylation
(Fig. 1A), which results in silencing of the system
(reviewed by Stock et al., 2000). Generally, the dephos-
phorylation of the RR-P is controlled by the inherent lability
of the mixed anhydride phospho-aspartyl bond and/or a
phosphatase-like activity embodied in the cognate HK or
another protein (reviewed by Stock et al., 2000).
Hybrid sensor kinases, differential module
arrangements, and phosphorelay
The hybrid histidine sensor kinases (HHKs), present in
prokaryotes and predominant in eukaryotes, provide a
common variant of two component signaling. These HHKs
participate in a more elaborate signal transmission path-
way, which involves the sequential transfer of the P in a
His ! Asp ! His !Asp phosphorelay (Fig. 1B) (Burbulys
et al., 1991; Georgellis et al., 1997; Takeda et al., 2001;
Uhl and Miller, 1996b; Williams and Whitworth, 2010).
Although the participating His and Asp residues, present in
transmitter (TD), receiver (RD) and phosphotranfer (HPt)
domains, are identifiable by homology, the architecture of
these systems displays an extensive diversity in the
arrangement of domains (Fig. 1B). That is, the three TD,
RD, and HPt domains can be present in the same protein
or exist as different proteins. For instance, in the ArcB/A
system of Escherichia coli, the three domains are present
in the same protein, the tripartite kinase ArcB. It is of inter-
est to mention that each of the three TD, RD, and HPt
containing polypeptides of ArcB has evolved sufficient
autonomy to fold into functional tertiary structures, with or
without covalent linkage of the contiguous domains,
emphasizing the intrinsic modular nature of this class of
HHKs (Georgellis et al., 1997; 1998; Kwon et al., 2000).
An alternative arrangement, present in a few bacterial
TCS, is a bipartite sensor kinase with the TD and RD
domains on one protein and the HPt domain on a separate
protein. The RcsC-YojN-RcsB and CckA-ChpT-CtrA signal-
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ing pathways of E. coli and Caulobacter crescentus,
respectively, are examples of this organization (Biondi
et al., 2006; Takeda et al., 2001) (Fig. 1B). Finally, each of
the three TD, RD, and HPt domains can exist on a different
protein as exemplified by the Spo pathway of B. subtilis
(Burbulys et al., 1991) (Fig. 1B). In such systems, the P
is transferred through three subsequent His to Asp steps
(TD ! RD ! HPt ! RD of the RR) constituting a multi-
step phosphorelay (Fig. 1B) (Burbulys et al., 1991;
Georgellis et al., 1997; Takeda et al., 2001; Uhl and Miller,
1996b). Yet, a different phosphorelay variant is repre-
sented by the TodS/TodT TCS of Pseudomonas putida
and TmoS/TmoT of Pseudomonas mendocina (Busch
et al., 2009; Silva-Jime nez et al., 2012), where a second
TD substitutes the HPt domain in the HHK (Fig. 1C).
Nevertheless, transphosphorylation of the RR occurs
via a His ! Asp ! His ! Asp phosphorelay, although
it has been demonstrated that the second TD is able to
autophosphorylate in vitro. Whether in vivo autophos-
phorylation of the second TD in response to a specific
signal is physiologically relevant remains to be demon-
strated (Busch et al., 2009).
Also, as is the case of the canonical HKs, HHKs act as
homodimers and autophosphorylate by either an inter-
(Cotter and Jones, 2003) or intra-molecular reaction
(Pen ~a-Sandoval and Georgellis, 2010). Furthermore, in
the absence of stimulus, HHKs act as specific phospha-
tases that dephosphorylate their cognate RRs through a
reverse phosphorelay (RD of the RR ! HPt ! RD ! Pi)
(Fig. 1D), resulting to the silencing of the system (Georgel-
~a-Sandoval et al., 2005; Uhl and Miller,
lis et al., 1998; Pen
1996a). Although the advantages of phosphorelay path-
ways over the canonical ones are not clear, the availability
of different check points that could allow various inputs to
be integrated in a multilevel control mechanism has been
suggested to contribute to the fine tuning and robustness
of the response (Hoch and Silhavy, 1995; Kim and Cho,
2006; Malpica et al., 2006; Silversmith, 2010). In fact, ArcB
may provide such an example, as it has been shown that
the fermentative metabolite D-lactate plays a physiological
role as an allosteric effector that is able to accelerate, but
  nez and D. Georgellis
4 A. F. Alvarez, C. Barba-Ostria, H. Silva-Jime
not to activate the kinase activity of ArcB (Georgellis et al.,
1999; Rodriguez et al., 2004).

Acetyl phosphate can phosphorylate RR in a sensor-

independent manner
Many RRs have been found to catalyze their own phos-
phorylation, in vitro and in vivo, at the expense of certain
high-energy phosphate compounds, including acetyl phos-
phate and carbamoyl phosphate (Lukat et al., 1992; Wolfe,
2005). These compounds have been suggested to main-
tain the basal level of many RRs in the phosphorylated
state, and also to signal global metabolic conditions
(McCleary et al., 1993), a hypothesis supported by the
finding that the cellular levels of acetyl-P can vary from
<0.04 mM to 1.2 mM (McCleary and Stock, 1994).
However, it is doubtful whether such acetyl phosphate-
dependent RR-autophosphorylation significantly modu-
lates the RR-P levels in the presence of the cognate HK,
as the in vivo evidence comes exclusively from studies
using mutants that lack the HK protein. In this context, it is
relevant to mention that the steady-state level of the RR-P
Fig. 2. Phosphorylation of UvrY occurs by two independent routes.
depends on the balance between the kinase and the phos- Acetate promotes the phosphorylation of the RR UvrY by two
phatase activities of the HK. Consequently, in the absence simultaneous mechanisms. Escherichia coli cells growing on
glucose produce acetyl-CoA, which in part is incorporated into the
of stimuli, where the phosphatase activity of the sensor
Tricarboxylic Acid Cycle (TCA) but also converted to acetate. The
protein is expected to increase as its kinase activity dimin- produced acetate is secreted and accumulated in the medium. At
ishes, the phosphatase activity of most HKs may play a late exponential phase of growth the concentration of acetate peaks
and acts as a direct stimulus for BarA, which autophosphorylates
key role in suppressing a possible acetyl-P dependent RR
and transphosphorylates UvrY. At the same time acetate is
phosphorylation. Therefore the relative contribution by assimilated and converted to acetyl-P, by the Pta–AckA pathway.
acetyl-P might be significantly exaggerated in the absence Acetyl-P, in turn, is used by UvrY to autophosphorylate, resulting in
two distinct but physiologically relevant ways to generate UvrY-P
of the HK (Liu et al., 2009). Nevertheless, from an evolu-
(AckA, acetate kinase; Pta, phosphotransacetylase; PoxB, pyruvate
tionary point of view, acetyl-P could provide the means to oxidase).
primitive microorganisms, where the HKs were not fully
cognate phosphorylations have only been observed in
evolved, to respond to metabolic fluctuations by direct
phosphorylation of RR proteins (Wolfe, 2010). Acetyl-P cells where the cognate HK has been deleted (Fig. 3B) or
could play a physiological role in the E. coli BarA/UvrY when the noncognate HK has been overexpressed. This
TCS because acetate, which acts as an activating signal can be partially explained by the fact that over-expression
for BarA, also promotes acetyl-P synthesis via the Pta- of the noncognate HK or deletion of the cognate HK, which
AckA pathway, resulting in UvrY phosphorylation by both shows a kinetic preference for binding and phosphorylating
routes (Fig. 2) (Camacho et al., 2015; Chavez et al., its partner RR (Laub and Goulian, 2007; Skerker et al.,
2010). 2005; 2008), promote the interaction of noncognate pairs
of proteins. Also, the absence of the cognate HK results in
the elimination of the HK-dependent phosphatase activity,
Crosstalk
which acts as a noise suppressor by eliminating nonspe-
HKs and RRs constitute the two largest paralogous gene cific and undesired phosphorylation of the cognate RRs
families in bacteria (Galperin, 2005) and share significant (Fig. 3A), minimizing possible cross phosphorylation and
sequence and structural similarity. Moreover, multiple preventing crosstalk (Guckes et al., 2013; Siryaporn and
TCSs are usually present in a cell at the same time with Goulian, 2008). Therefore, crosstalk is unlikely to be physi-
similar cellular localization patterns, raising the possibility ologically relevant to the wild-type organism.
of cross interactions and crosstalk; that is, phosphorylation
between noncognate pairs of proteins (Fig. 3). Indeed,
Cross-regulation
several studies have provided evidence that crosstalk
occurs both in vitro and in vivo (Siryaporn and Goulian, In contrast to cross talk, cross regulation refers to the con-
2008; Verhamme et al., 2002). Again, these in vivo non- trol of a RR or HK of a given TCS by a different regulatory
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Fig. 3. The phosphatase activity embedded in sensor kinases
annuls nonphysiological crosstalk outcomes.
A. Schematic representation of the TCS1 and TCS2 in a wild type
bacterial cell, in the presence of the specific stimulus for TCS2. In
this example, HHK2 is able to transphosphorylate its cognate
response regulator RR2, but also RR1, although much less
efficiently. However, HHK1 in the absence of its stimulus acts as a
phosphatase that dephosphorylates specifically its cognate
response regulator RR1-P and suppresses its regulatory activity.
B. On the other hand, in a Dhhk1 background, the
transphosphorylation of RR1 by HK2 result in accumulation of RR1-P
resulting in a TCS1-specific output response generated by crosstalk.
system, which may or may not be a two-component regu-
latory system, under physiological conditions (Eguchi and
Utsumi, 2005; Eguchi et al., 2012; Goulian, 2010; Wanner,
1992). Frequently, cross regulation can involve auxiliary
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Two-component system signaling 5
proteins, referred to as connector proteins, able to modu-
late the activity of a HK or RR, and whose expression is
controlled by a different TCS (Fig. 4A). For instance, the
Salmonella PhoQ/P and PmrB/A TCS illustrates such regu-
lation (Kato and Groisman, 2004). The phosphorylated
form of the RR PhoP activates the expression of the small
protein PmrD, which in turn binds to the phosphorylated
form of the RR PmrA, preventing both its intrinsic dephos-
phorylation and the one promoted by PmrB (Kox et al.,
2000; Kato and Groisman, 2004). PmrA-activated loci
mediate resistance to the antibiotic polymyxin B. There-
fore, the PmrD connector protein enables S. enterica to be
resistant to polymyxin B not only in response to the Fe31
signal that is sensed by the PmrB protein (Wo €sten et al.,
2000), but also in low-Mg21 environments that activate the
PhoQ/PhoP system (Garcıa Ve scovi et al., 1996). Other
examples of pathways linked by connectors include the
CpxA/CpxR-EnvZ/OmpR and the PhoQ/P-EvgA/S TCSs
that are connected by the MzrA and SafA proteins (Gerken
et al., 2009; Eguchi et al., 2010), respectively. However,
cross-regulation can also be observed between TCSs with-
out the participation of connector proteins. Such a case is
exemplified by the Pseudomonas aeruginosa RetS and
GacS/A TCS (Fig. 4B). In this case, RetS, an orphan HHK,
directly interacts with the GacS HHK. RetS binding to
GacS competes with GacS homodimerization, thereby
inhibits GacS autophosphorylation and stimulates its
dephosphorylation (Fig. 4B). This results in decreased lev-
els of GacA-P and elimination of GacA promoted
transcriptional control. Noteworthy, the RetS–GacS func-
tion does not require the predicted RetS phosphorelay
residues, thereby providing a mechanism for integrating
signals without cross-phosphorylation of a RR by a non-
cognate HK (Goodman et al., 2009). The presence of
auxiliary proteins between TCS could represent an advant-
age for the cells, as it constitutes an important mechanism
to integrate responses in a controlled way, forming a net-
work rather than isolated linear pathways.
Nonlinear bacterial signaling circuits
In addition to linear signaling pathways, some of which
involve connector proteins, there are several examples in
which the phosphorylation pathway is branched, involving
many HKs and one or few RRs, or one HK and many RRs
[reviewed by (Laub and Goulian, 2007)]. These nonlinear
signaling circuits differ from cross talk, which is of little or
no physiological relevance (harmful to the cell), in that they
are finely controlled to produce a desired response under
certain growth conditions and in response to a given stimu-
lus. Therefore, the physical and functional interactions
between the participant HK and RR proteins, despite the
apparent degree of promiscuity, are physiologically rele-
vant. For instance, the cyanobacterium Synechococcus
  nez and D. Georgellis
6 A. F. Alvarez, C. Barba-Ostria, H. Silva-Jime
Fig. 4. Cross regulation between TCSs can occur under
physiological conditions.
A. Cross regulation could involve a connector protein (orange oval),
whose expression is controlled by a TCS (TCS1). This connector
protein modulates the activity/stability of the HK/HK-P or the RR/
RR-P of another TCS (TCS2), thereby affecting the extent of its
response (output 2).
B. The GacS/A TCS of Pseudomonas aeruginosa. The activity of
the GacS/A TCS is controlled by RetS, an orphan HHK, which
directly interacts with the GacS HHK in the absence of its specific
stimulus. RetS binding to GacS impedes GacS homodimerization
and inhibits GacS autophosphorylation but promotes its
dephosphorylating activity. This results in decreased levels of
GacA-P, and thereby silencing of the system.
elongatus NblS sensor kinase and RpaB and SrrA RRs
represent a good example of a “one-to-many” architecture,
in which environmental cues determine the preference of
two expression programs regulated by a branched TCS
pez-Redondo et al., 2010). NblS-dependent phospho-
(Lo
rylation of RpaB occurs under low-light conditions,
promoting a determined expression program that includes
srrA repression, thereby preventing interference of SrrA
with the RpaB-P regulation of expression program
(Fig. 5A). On the other hand, under the initial period of
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high-light conditions, RpaB-P dephosphorylation occurs,
resulting in derepression of srrA (Fig. 5A). Subsequent
acclimatization to high-light conditions results in the activa-
tion of NblS as a kinase, which, because of its strong
preference to SrrA, leads to SrrA phosphorylation. SrrA-P,
in turn, activates the light-specific programs enabling the
photosynthesis machinery to adapt to the high-light condi-
tions (Lo pez-Redondo et al., 2010). Many-to-one
architectures are also found in bacteria, and allow the inte-
gration of several input signals to generate one output
response. This is exemplified by the quorum-sensing sys-
tem of Vibrio harveyi, in which the three different HKs,
LuxN, LuxQ, and CqsS autophosphorylate in response to
different auto-inducers, and transphosphorylate the RR
LuxO, which in turn controls the level of the master regula-
tor LuxR (Fig. 5B) (Henke and Bassler, 2004). Other
examples include the sporulation phosphorelay of B. subti-
lis, where the four kinases KinA-D phosphorylate the
regulator Spo0F (Jiang et al., 2000) and the DosS-DosT/
DosR system from Mycobacterium tuberculosis (Kumar
et al., 2007).
These architectures may be more common than
expected in bacteria, since a comparative genomics analy-
sis of 165 000 regulatory proteins from 850 prokaryotic
genomes suggests that organisms having high numbers of
TCS proteins also present more interconnected and
branched two-component networks (Seshasayee and Lus-
combe, 2011).
Architecture and mode of action of TCS in different
kingdoms of life
Distribution of signaling proteins in prokaryotic, archaeal
and eukaryotic organisms
Analysis of several bacterial genomes, reveals that sensor
HK and RR proteins appear to be evenly distributed and in
most cases maintain an almost one-to-one ratio (Williams
and Whitworth, 2010). However, cyanobacterial genomes
contain a high amount of orphan RRs, resulting in a 1:2
HK/RR ratio. Likewise, plants encode a larger number of
RR than HK. For example, the Arabidopsis thaliana
genome encodes 16 sensor kinases (9 HHK and 7 HKs), 6
histidine-phosphotranfer proteins and 32 RRs (Hwang
et al., 2002). However, 2 HHKs, 6 HK, one HPt, and 9 RRs
lack the conserved His or Asp residues that provide the
sites of phosphorylation, resulting in 1 HK, 7 HHK, 5 HPt,
and 23 RR functional proteins. In contrast, fungal genomes
encode between 1 and 21 HHKs (Bahn, 2008; Catlett
et al., 2003; Pott et al., 2000; Virginia et al., 2000), a single
HPt protein and two or three RRs. For example, the Asper-
gillus nidulans genome encodes 15 hybrid HKs, one
intermediary HPt, and four RRs, one of which lacks the
conserved Asp residue and another that lacks the effector
or output domain (Vargas-Pe rez et al., 2007). Finally, the
 Two-component system signaling 7

Fig. 5. Schematic representation of branched TCS circuitries. Branched TCSs allow either the coordination of differential expression programs
in response to a stimulus or the integration of multiple stimuli in a sole specific response.
A. “One to many”. In the cyanobacterium Synechococcus elongatus NblS/RpaB-SrrA TCS, NblS-dependent phosphorylation of RpaB occurs
under low-light conditions, promoting a determined expression program that includes srrA repression. Under the initial period of high-light
conditions, RpaB-P undergoes dephosphorylation, resulting in derepression of srrA. After acclimatization to high-light conditions, NblS
transphosphorylates, preferencially, SrrA, because of its strong preference to SrrA as compared to RpaB, leading to the activating of another
expression program.
B. “Many to one”. In the Vibrio harveyi quorum-sensing system, three different HHKs, LuxN, LuxQ, and CqsS, autophosphorylate in response
to different autoinducers, and transphosphorylate the RR LuxO through the Hpt protein LuxU. LuxO-P, in turn, controls the level of the quorum-
sensing master regulator LuxR.

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  nez and D. Georgellis
8 A. F. Alvarez, C. Barba-Ostria, H. Silva-Jime
less studied archaeal TCS signaling components that are
present only in organisms from phyla Euryarchaeota and
Thaumarchaeota, but not Nanoarchaeota, Korarchaeota
and Crenarchaeota (MiST2 database) (Ulrich and Zhulin,
2010), appear to embrace all above examples. For
instance, Natrinema pellirubrum contains 21 HKs and 21
RRs, whereas the Methanocella paludicola and Methano-
spirillum hungatei genomes encode for 53 HKs and 9 RRs
and 43 HKs and 84 RRs, respectively (Becker et al., 2014;
Gunsalus et al., 2016; Sakai et al., 2011).
Interestingly, in diverse fungi and plants, but also in arch-
aea, RRs can be found with a canonical receiver domain
but lacking an obvious effector domain. Such RR proteins
are believed to act as response modulators because they
compete with the canonical RRs for binding and receiving
the P from the HK proteins. Moreover, in both types of
eukaryotic organisms, it is common to find RR-like proteins
that have diverged from typical RRs by substituting the
essential phospho-accepting Asp residue with a Glu resi-
due. Such an Asp to Glu substitution has been shown, in
some cases, to mimic the phosphorylated, and thus active
form of the RR (Arribas-Bosacoma et al., 2007; Gao et al.,
2006; Klose et al., 1993; Siam and Marczynski, 2003). At
last, hybrid response regulators (HRR), which contain a N-
terminal RD followed by a transmitter module instead of an
output domain, are commonly found in archaea, constitut-
ing 30% of the RRs (MiST2 database) (Ulrich and Zhulin,
2010), whereas they are rare in bacteria (3% of the RRs)
and absent in eukaryotic organisms (Wuichet et al., 2010).
Furthermore, in contrast to bacterial and archaeal TCSs,
where HHKs comprise less than 20 and 2% of sensor
kinases, respectively, more than 90% of plant and 100% of
fungal sensor kinases are HHKs (Wuichet et al., 2010).
Interestingly, the eukaryotic HHKs comprise a TD and a
RD in one protein whereas the HPt domains are on a sep-
arate protein, enabling them to easily shuttle between
cellular compartments irrespective of their phosphorylation
state (Lu et al., 2003; Punwani et al., 2010). Another inter-
esting difference is that almost all bacterial and plant HKs
and HHKs are membrane anchored proteins, whereas
almost all fungal HHKs are soluble proteins.
Evolutionary considerations
As mentioned above, TCS proteins can be found in all
kingdoms of life, but with the strongest prevalence in pro-
karyotes. It has been predicted that one-component
systems, having the input domain and the output domain
fused in the same protein, preceded the bacterial TCS
(Ulrich et al., 2005). Subsequently, these apparently simple
signaling systems, although abundant and with a vast vari-
ety of input and output domains, evolved to give rise to the
more complex TCSs (Krell et al., 2009; Ulrich et al., 2005;
Wuichet et al., 2010).
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Two plausible models for the evolution of novel TCSs
have been put forward: the recruitment model and the co-
evolution model. The first proposes that novel TCSs evolve
through gene duplication of one component and recruit-
ment of a component from a heterologous system as the
cognate partner, thereby giving rise to a novel TCS. The
coevolution model suggests that novel TCS arise by dupli-
cation of all the components of a signaling system followed
by differentiation through complementary adaptation of the
components to yield new specificity (Koretke et al., 2000).
There are several “pros and cons” for both these models
that are the subject of several excellent works (Capra and
Laub, 2012; Chen et al., 2004; Koretke et al., 2000; Wui-
chet et al., 2010; Zhang and Shi, 2005). Furthermore, the
rise of TCS in archaea and eukaryotic organisms is likely
the result of multiple and independent horizontal gene
transfer events from bacteria (Capra and Laub, 2012; Kim
and Forst, 2001; Koretke et al., 2000; Wuichet et al.,
2010). Phylogenetic analyses have shown that archaeal
and eukaryotic HKs and RRs cluster with distinct groups of
bacterial signaling components (Kim and Forst, 2001;
Koretke et al., 2000). Therefore, it appears that the hori-
zontal gene transfer events occurred after the separation
of the three kingdoms of life from a common primitive
ancestor.
Plant two component proteins play redundant roles in
signal transduction
Sequence analysis of plant genomes revealed a large
number of TCS proteins, similar to those found in bacte-
ria. However, detailed studies indicate that the majority
of these proteins obey mechanisms of action that signifi-
cantly differ from the ones followed by their bacterial
counterpart proteins. For example, A. thaliana contains
16 sensor kinases, 9 HHK and 7 HKs (Hwang et al.,
2002) (Fig. 6). These proteins are divided in two main
groups: ethylene receptors (ER) and nonethylene recep-
tors. The first group includes the HHKs ETR1, ETR2 and
EIN4, and also the orthodox HKs ERS1 and ERS2, all of
which contain an ethylene binding trans-membrane
domain. However, only two ERs (ETR1 and ERS1)
contain the well-preserved features that permit auto-
phosphorylation and subsequent phosphotransfer
activity, typical of bacterial TCS signaling. The others
lack either the conserved His residue, the site of auto-
phosphorylation, or the nucleotide binding pocket. All
these ERs have been reported to control the activity of
the CTR1–EIN2 pathway. That is, in the absence of eth-
ylene, the ERs activate CTR1, a Ser/Thr kinase, which
phosphorylates the C-terminus of EIN2, thereby prevent-
ing its localization in the nucleus. In the presence of
ethylene, the ERs undergo conformational changes that
result in CTR1 inactivation and consequent EIN2
 Two-component system signaling 9

Fig. 6. TCS homologous proteins found in A. thaliana. A. thaliana possess 16 sensor kinases, 6 histidine-phosphotranfer proteins and 32
response regulators. H and D represent the conserved histidine and aspartate residues, that is, the phosphorylation and transphosphorylation
sites, in the transmitter and receiver domains. A glutamate residue (E) substitutes the conserved aspartate residue (D) of the receiver domain
in the pseudo-RRs. The presence of transmembrane domains in HK/HHK is represented by short perpendicular lines. The CCT motif is a
nuclear localization signal, common in plants. The GARP motif is a multifunctional domain responsible for both nuclear localization and DNA
binding. The first group of histidine kinases includes the ethylene receptors, from which only two (ETR1 and ERS1) contain the well-preserved
features that permit autophosphorylation and subsequent phosphotransfer activity. The second group comprises the phytochrome receptors
Phy1–Phy5 that lack the essential motifs for histidine kinase activity. The third group includes the cytokinin receptors, known as the AHK
family, which in combination with the AHP1–AHP5 HPt proteins and the 11 type-B RRs are involved in typical TCS His to Asp P transfers.

dephosphorylation. Consequently, the C-terminal portion
of the dephosphorylated EIN2 is proteolytically cleaved,
and the truncated protein migrates to the nucleus where
it activates a family of transcriptional factors, resulting in
the ethylene-promoted response [reviewed by (Shakeel
et al., 2013)]. An alternative ETR1 and/or ERS1 His-Asp
phosphorelay mediated ethylene response cannot be
fully ruled out, because phosphorylation should promote
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V
conformational changes in ERs that could affect the
interaction between ERs and other proteins (i.e. CTR1)
(Bisson and Groth, 2010; Hall et al., 2012), and also
because ctr1 mutants conserve a residual ethylene
response (Hall and Bleecker, 2003; Larsen and Cancel,
2003). Nevertheless, despite their sequence similarity to
prokaryotic sensor HKs, ERs cannot be considered con-
stituents of two-component signaling pathways.
  nez and D. Georgellis
10 A. F. Alvarez, C. Barba-Ostria, H. Silva-Jime
The nonethylene receptor group includes 5 photorecep-
tors, 5 cytokinin receptors, and one putative osmosensor.
Again, all phytochrome receptors involved in diverse bio-
logical responses to red light lack the essential motifs for
histidine kinase activity. Therefore, their participation in
His-Asp phosphotransfer seems to be improbable. Instead,
the mode of action of these receptors involve their light-
dependent migration from the cytosol to the nucleus,
where they directly interact with transcriptional factors (Ni
et al., 1998). In addition, it has been proposed that they
may be functional as Ser-Thr kinases (Han et al., 2010;
Kim et al., 2004; Yeh and Lagarias, 1998). Thus, although
plant TCS proteins have a bacterial origin, only half of
them preserved the typical His to Asp phosphotransfer,
whereas the others were integrated within the typical
eukaryotic signaling pathways (ESP). Whether these TCS
were adapted and integrated to the existing ESP or
whether they coevolved with the ESP remains to be
elucidated.
Consequently, only the 6 remaining HHK of A. thaliana,
known as the AHK family, are involved in TCS-typical His
to Asp phosphotransfer. This family of true HHK includes
AHK1, a putative osmo-receptor (Urao, 1999; Wohlbach
et al., 2008), AHK2, AHK3, AHK4 and CKI1, all cytokinin
receptors [although the role of CKI1 in cytokinin signaling
is only observed when over-expressed (Deng et al.,
2010)], and CKI2 (AHK5), which is a cytoplasmic HHK with
a, thus far, unknown role in signaling. AHK1 is referred to
as a plant osmosensor, similar to the S. cerevisiae HHK
SLN1, because it has been shown to functionally comple-
ment a sln1–sho1 yeast mutant (Urao, 1999). However, it
was recently demonstrated that the growth of ahk1
mutants is not more sensitive to salt stress, suggesting
that AHK1 may not be the main plant osmosensor (Kumar
et al., 2013). Finally, AHK2, AHK3, and AHK4 have redun-
dant functions in the perception of and response to the
plant hormone cytokinin (Inoue et al., 2001; Suzuki et al.,
2001), reviewed by (Kakimoto, 2003). These three HHKs,
when active, autophosphorylate and trigger a phosphore-
lay involving 5 HPt proteins (AHP1-5) (Hutchison et al.,
2006; Hwang et al., 2002) and 11 typical RRs (ARR1,
ARR2, ARR10–ARR14, and ARR18–ARR21), referred to
as type-B RRs. The phosphorylated type-B RRs activate
the expression of target genes (Argyros et al., 2008;
Mason et al., 2005), including 10 type-A RRs, all of which
contain a functional receiver domain but not an output/
effector domain (Hwang and Sheen, 2001; Mason et al.,
2005). These type-A RRs function as modulators in the
cytokinin response, acting as negative feedback regula-
tors, because they can receive the P from the
phosphorylated AHP1-5, thereby diverting P to futile flow
(Kiba et al., 2003; To et al., 2004). Despite the apparent
redundancy in this signaling cascade (at least three HHKs
perceive the same signal and phosphorylate 11 RR
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V
through 5 HPts to produce a specific response) more
recent studies suggest that each HHK may have prefer-
ence for a specific HPt protein(s) (Peka rova
 et al., 2011).
The same could be also true for the HPt to RR phosphoryl-
ation step. However, the above-mentioned redundancy
does not sacrifice specificity, because one specific signal
triggers a specific output, but with increased robustness of
the response.
Obligatory branched pathways in fungi
The architecture of fungal TCSs differs significantly from
those of bacteria and plants (Fig. 7). This is mainly
because in the thus far sequenced fungal genomes,
although there can be found two canonical RRs and up to
two pseudo-RRs, the number of sensor kinases can vary
from one, as is the case of S. cerevisiase (Ota and Var-
shavsky, 1993), to 21, as is the case of Cochliobolus
heterostrophus (Catlett et al., 2003). All these sensor
kinases are bipartite, comprising a transmitter and receiver
domain, and all except one (chHHK5), are cytosolic pro-
teins (Fig. 7). Intriguingly, in all the above fungal genomes,
regardless of the number of HHKs, there is only a single
HPt intermediary protein, which has been shown to shuttle
between cytoplasm and nucleus (Lu et al., 2003) and to be
essential for cell viability (Banno et al., 2007; Lee et al.,
2011; Posas et al., 1996; Vargas-Pe rez et al., 2007). Thus,
in S. cerevisiase there is a “one to two” branched pathway,
whereas in filamentous fungi it appears to be a highly con-
vergent pathway with as many as 21 input-accepting
HHKs that transmit signals, through a single HPt, to two
RR proteins. As is the case in plants, the two pseudo-RRs
could play a role in response modulation by either interact-
ing with the HPt protein, thereby preventing interaction with
and thus phosphorylation of the true RRs, or by acting as
negative feedback regulators in a similar manner as the
plant type-A RRs.
The thus far best-studied and simplest fungal TCS cas-
cade is that of S. cerevisiase, where one HHK (Sln1)
phosphorylates a unique HPt (Ypd1), which, in turn, allows
the phosphorylation of two RRs (Ssk1 and Skn7). Under
high osmolarity conditions, the kinase activity of Sln1 is
suppressed, leading to the accumulation of nonphosphory-
lated Ssk1 and Skn7. Skn7 remains inactive as a RR,
whereas Ssk1 in its nonphosphorylated state interacts with
and activates Ssk2 and Ssk22, two mitogen-activated pro-
tein kinase (MAPK) kinase kinases that further stimulate
the Hog1 MAPKK–MAPK cascade, which ultimately con-
trols the concentration of glycerol within the cell. Under
normal osmotic conditions Sln1 is activated leading to the
phosphorylation of Ssk1 and Skn7. Ssk1 phosphorylation
results in the release of Ssk2 and Ssk22 interaction,
thereby silencing the Hog pathway (Maeda et al., 1994;
Posas et al., 1996). Phosphorylated Skn7 acts as a
 Two-component system signaling 11

Fig. 7. Distribution of TCS proteins in filamentous fungi. Filamentous fungi possess as many as 21 bipartite HHK (for clarity only seven are
indicated), one unique HPt protein and two canonical RRs. All HHKs, except one, are soluble proteins. The HPt protein has been shown to be
essential and able to shuttle between cytoplasm and nucleus. One of the two RRs acts by interacting with the MAPKKKs Ssk2 and Ssk22,
which stimulate the Hog1 MAPKK–MAPK cascade involved in osmotic stress response. The other one is believed to be localized in the
nucleus and act as a transcriptional factor. Signaling specificity could be provided by: (i) scaffold proteins that bind multiple components of a
given pathway, thereby accelerating signaling reactions and maintaining fidelity; (ii) given HHKs could be integrated in eukaryotic signaling
pathways and transmit signals through mechanisms distinct to His–Asp phosphorelay; or (iii) some HHKs, in their phosphorylated state, could
migrate to the nucleus and interact with proteins such as transcriptional factors or histone modifying proteins, regulating gene expression in a
RR-independent manner.

transcriptional regulator, which directly affects the tran-
scription of Sln1–Skn7-responsive genes such as the
mannosyltransferase gene Och1 (Brown et al., 1994; Li
et al., 2002). Furthermore, under oxidative stress condi-
tions Skn7 interacts with the transcription factor Yap1, in
an Sln1- and aspartyl phosphorylation-independent man-
ner, allowing the activation of the oxidative stress response
(He and Fassler, 2005; Morgan et al., 1997). It is of interest
to mention that although the Sln1 ! Ypd1 ! Ssk1/Skn7
cascade maintains the defining features of TCS signaling,
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V
it also represents an example of the adaptation and inte-
gration of a TCS signaling protein, i.e. Ssk1, in a typical
eukaryotic regulation pathway, that is the Hog1 MAPKK
cascade.
TCS protein organization in filamentous fungi presents
another interesting architectural variation. Although they
encode two true RR proteins (Ssk1 and Skn7 orthologs)
and only one HPt protein (Ypd1 ortholog), as is the case of
S. cerevisiae, they encode many more HHKs (in the case
of Cochliobolus heterostrophus there are as many as 21
  nez and D. Georgellis
12 A. F. Alvarez, C. Barba-Ostria, H. Silva-Jime
HHKs) (Catlett et al., 2003). It is likely that filamentous
fungi, because of their higher complexity and greater range
of environmental niches in contrast to yeast, have
expanded their sensing capabilities by increasing the num-
ber of HHKs. Thus, the simplest operating scenario that
can be envisaged is as follows: (i) activation of HHKx by its
specific stimulus, let us call it X, should lead to its auto-
phosphorylation resulting in HHKx-P; (ii) the P from
HHKx-P should then be transferred to the unique HPt pro-
tein, which, in turn, (iii) should transfer it to both RR
proteins. Finally, (iv) phosphorylated RRs should produce
an output response. However, if this operating scenario
were to be true, activation of HHKy by its specific stimulus,
which is different from signal X, should produce the very
same output response. This raises the question of whether
as many as 21 signals sensed by the 21 HHKs, which is
the case of Cochliobolus heterostrophus, are integrated
into a single output response, or whether sensing of a sig-
nal by a specific HHK triggers a specific output response.
In this respect it is important to mention that the thus far
studied fungal RRs and HHKs not only present pheno-
types related to osmolarity and oxidative stress resistance
(Hagiwara et al., 2009; Motoyama et al., 2005; Noguchi
et al., 2007; Schumacher et al., 1997; Vargas-Pe rez et al.,
2007; Yoshimi et al., 2005), similar to those of S. cerevi-
siae, but have also been shown to be involved in other
physiological processes such as sexual or asexual repro-
duction, pigmentation, virulence, fungicide resistance,
normal sporulation, and conidiospore viability (Barba-
Ostria et al., 2011; Boyce et al., 2011; Hagiwara et al.,
2013; Kanamaru, 2011; Oide et al., 2010; Vargas-Pe rez
et al., 2007; Zhang et al., 2010). Thus, some signal/output
specificity should be present in these systems. This could
be achieved with the help of scaffold or adaptor proteins.
Signaling adaptors can bind multiple components of a
given pathway, thereby accelerating signaling reactions
and maintaining fidelity (Fig. 7). This is because they
increase the local concentration of the bound signaling
components (Bashor et al., 2008; Matsunaga-Udagawa
et al., 2010) and insulate them from other signaling pro-
teins (Good et al., 2011; Zeke et al., 2009). Another
possibility is that not all HHKs transmit signals through the
two RRs but rather some HHKs were adapted and inte-
grated in other signaling pathways of the host, as is the
case of the plant ERs (Fig. 7). However, this remains a
pure speculation because no experimental evidence from
fungal models supporting this possibility has been provided
thus far. Finally, the soluble nature of almost all fungal
HHKs, permitting their migration from the cytosol to the
nucleus, where they could directly interact with transcrip-
tional factors, as is the case of plant phytochrome
receptors, presents another possibility (Fig. 7). Notably, it
was recently demonstrated that two HHKs of Candida guil-
liermondii, Nik1p and Chk1p, migrate from the cytosol to
C 2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 00, 00–00
V
the nucleus in response to hyperosmotic stress (Foureau
et al., 2014). Also, the phytochrome FphA of A. nidulans
exerts its light-dependent regulatory action on target genes
by migrating to the nucleus and interacting with various
proteins (Hedtke et al., 2015). Moreover, in contrast to the
plant phytochromes that also act in the nucleus but do not
require the conserved histidine residue, the histidine
kinase activity of FphA is essential for its regulatory func-
tion. Thus, HHK migration to the nucleus and interaction
with proteins such as transcriptional factors or histone
modifying proteins could not only extend the scope of fun-
gal TCS response as a whole, but also improve the signal/
response specificity of individual HHKs.
Conclusion
Two-component signaling systems, based on P transfer
between His and Asp containing protein modules, serve as
a signal–response coupling mechanism that enables
organisms to sense, respond, and adapt to a broad range
of environmental changes. Typically, prokaryotic TCSs
consist of cognate pairs of HK/HHK and RR proteins and
employ multiple mechanisms to avoid undesirable cross
talk interactions, to produce highly specific signal–
response signaling cascades. However, a few controlled
interactions between TCSs that coordinately regulate cel-
lular processes, establishing regulatory networks rather
than linear signaling pathways, do also exist.
It has been suggested that these signaling pathways ori-
ginated in bacteria and spread to eukaryotes through
horizontal gene transfer events. Curiously, despite their
common origin, TCSs from different kingdoms of life exhibit
significant differences in their mode of action, regulation,
and integration in their respective organism. This is par-
tially reflected by the distinctive TCS architectures found in
bacteria (predominantly linear signaling pathways), fungi
(convergent pathways), and plants (divergent circuits).
Moreover, around half of the plant HK/HHK and RR pro-
teins lack the conserved features that permit His
phosphorylation and/or Asp trans-phosphorylation, exclud-
ing their participation in a typical TCS transduction
cascade. Many of these proteins appear to have been
adapted and integrated within already existing eukaryotic
signaling cascades, or to control gene expression by
directly interacting with nuclear regulatory proteins.
Finally, the particular architecture of fungal TCS net-
works poses some interesting questions. For instance, do
fungi integrate the signals sensed by as many as 21 a pri-
ori functional HHKs, through one HPt protein and two RRs,
to one common response? Are scaffold proteins or tempo-
ral/spatial differential expression/localization of the
signaling components possible mechanisms that could
allow some degree of signal–response specificity?
Addressing these questions could provide important

insights into the operation of these TCS signal transduction
systems that present an interesting variation on the pro-
karyotic theme.
Acknowledgements
This work was partially supported by the Consejo Nacional de
Ciencia y Tecnologıa, CONACyT [178033], and DGAPA,
UNAM [IN206412 and IA203216].
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