THE TWO-COMPONENT SIGNAL TRANSDUCTION SYSTEMS OF
PSEUDOMONAS AERUGINOSA
by
Jessica Richard
A professional paper submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Microbiology
MONTANA STATE UNIVERSITY
Bozeman, Montana
May 2008
©COPYRIGHT
by
Jessica Richard
2008
All Rights Reserved
ii
APPROVAL
of a professional paper submitted by
Jessica Richard
This professional paper has been read by each member of the
professional paper committee and has been found to be satisfactory regarding
content, English usage, format, citation, bibliographic style, and consistency, and
is ready for submission to the Division of Graduate Education.
Dr. Michael Franklin
Approved for the Department Microbiology
Dr. Tim Ford
Approved for the Division of Graduate Education
Dr. Carl A. Fox
iii
STATEMENT OF PERMISSION TO USE
In presenting this professional paper in partial fulfillment of the
requirements for a master’s degree at Montana State University, I agree that the
Library shall make it available to borrowers under rules of the Library.
If I have indicated my intention to copyright this thesis by including a
copyright notice page, copying is allowable only for scholarly purposes,
consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for
permission for extended quotation from or reproduction of this thesis in whole or
in parts may be granted only by the copyright holder.
Jessica Richard
May, 2008
iv
ACKNOWLEDGEMENTS
First, I would like to thank Dr. Michael Franklin for his guidance and
support and the opportunity to receive my degree at Montana State University.
I would like to thank all the members of the Franklin lab and my committee for
their assistance and advice over the years.
I would also like to thank my family and friends for always supporting my
decisions and showing great patience in difficult times.
v
TABLE OF CONTENTS
1. INTRODUCTION AND REVIEW OF TWO-COMPONENT SYSTEMS............. 1
Sensor Histidine Kinases ................................................................................. 3
Response Regulators ...................................................................................... 8
2. TWO-COMPONENT SYSTEMS IN PSEUDOMONAS AERUGINOSA PA01 14
Introduction.................................................................................................... 14
Computational Methods................................................................................. 14
Results .......................................................................................................... 16
Sensor Histidine Kinase........................................................................... 16
Response Regulator ................................................................................ 23
Conclusions ................................................................................................... 33
Summary and Perspectives........................................................................... 34
REFERENCES ................................................................................................... 37
vi
LIST OF TABLES
Table
Page
2.1 Histidine kinases ..................................................................................... 22
2.2 Response regulators ............................................................................... 31
vii
LIST OF FIGURES
Figure
Page
1.1 Phosphotransfer reactions.............................................................................. 3
1.2 Domain organization in orthodox histidine kinases and hybrid kinases .......... 6
1.3 Cross regulation and branched pathways ...................................................... 7
1.4 Response regulator domain organization ..................................................... 10
2.1 The conserved histidine residue of sensor kinases in Pseudomonas
aeruginosa PA01 .......................................................................................... 16
2.2 Phylogenetic tree of the 55 histidine kinases................................................ 18
2.3 Neighbor-Joining tree of the clade I sequences............................................ 19
2.4 Transmembrane domain occurrence............................................................ 20
2.5 Neighbor-Joining tree of the 67 response regulators.................................... 25
2.6 Neighbor-Joining tree of the subtree I sequences ........................................ 27
2.7 Conserved aspartate residue of the response regulators ............................. 28
2.8 Sequence logos with the conserved aspartate residue ................................ 29
2.9 3D model of the P. aeruginosa PA2657 response regulator......................... 30
viii
ABSTRACT
Two-component signal transduction systems are important components
for bacteria, because they allow the bacteria to sense environmental conditions
and rapidly adapt to changes in the environment. Two-component systems
generally contain a sensor histidine kinase, which detects an environmental
signal and responds by autophosphorylation at a histidine residue using ATP as
the phosphate donor. The phosphate group is then transferred to an aspartate
residue in the receiver domain of the second component, the response regulator,
which in its activated form responds by stimulating or repressing gene expression
or motility, as needed for the physiological responses of the cell. The structural
versatility of two-component systems reflects the wide range of signals to which
bacteria respond. Despite this versatility, histidine kinases and response
regulators show a conserved mechanism of signal transfer.
Pseudomonas aeruginosa is a versatile organism that can use a variety of
nutrient sources and is found in many different environments. It is a human
pathogen in nosocomial infections as well as in pulmonary fluid of patients with
cystic fibrosis. P. aeruginosa encodes genes for over 60 two-component
regulatory systems.
In this review, I discuss the structure and function of two-component
systems in bacteria, and conduct a phylogenetic analysis of the P. aerugionsa
two-component systems. Finally, I develop a model of the calcium-responsive
two-component system of P. aeruginosa, PA2656/PA2657, which is closely
related to the magnesium responsive PmrAB and phosphate responsive PhoPQ
two-component systems. The results provide insight on how P. aeruginosa is
able to detect and respond to changing environmental conditions.
1
CHAPTER 1
INTRODUCTION AND REVIEW OF TWO-COMPONENT SYSTEMS
Bacteria are often exposed to changes in environmental conditions.
Therefore, they need mechanisms to respond and adapt to those changes
immediately, ensuring survival. Approximately 25 years ago a mechanism to help
ensure survival by adaptation was identified and termed two-component system
for signal transduction [72]. Signalling transduction systems are widespread, and
they have been identified in bacteria, archaea, fungi, molds and plants [62].
Signalling systems in prokaryotes and eukaryotes are often distinguished
by their mechanism of phosphorylation. Eukaryotic pathways include protein
kinases with autophosphorylation activity at specific serine, threonine or tyrosine
residues to regulate a response. Those pathways often display more complex
steps such as three step transfer of the phosphate group, termed phosphorelay.
Although Ser/Thr/Tyr systems are more abundant in eukaryotes, they have also
been found in prokaryotes [30, 53, 87].
Over the past decades signal transduction systems have been identified in
prokaryotes as well, and are called two-component signal transduction systems.
They have been shown to be a primary mechanism enabling bacterial cells to
sense changes in their surrounding environment and to respond to those
changes by regulating gene expression, and two-component systems have also
been found in eukaryotes [8, 44, 57, 62, 72-74, 83].
2
As the name implies, a typical two-component system consists of two
conserved proteins: a membrane bound sensor histidine kinase and a response
regulator located in the cytoplasm. After sensing an environmental signal, the
sensor is autophosphorylated at a histidine residue, followed by the transfer of
the phosphate group to an aspartate residue at the response regulator [1, 54].
The response regulator then plays regulatory roles, such as inducing gene
expression or changing cell motility (chemotaxis). The number of two-component
systems is not the same for all bacteria. Bacteria with larger numbers of twocomponent systems are often able to adapt to a wider range of environmental
signals than organisms with fewer two-component systems [83, 88]. Another
thing to consider when looking at two-component systems is the possible “crosstalk” between two different pathways which in some cases may be advantageous
for the cell. When cross-talk has beneficial effects it is referred to as “crossregulation” [81].
With improving bioinformatics tools, more two-component systems are being
discovered and more functions may be assigned to these systems.
Sensor Histidine Kinases
The histidine kinase, generally found in the bacterial inner membrane, is
the part of the system connecting the cellular environment with the adaptive
response. Histidine kinases possess two conserved domains (i) a site of
phosphorylation and (ii) an ATP-binding domain [26, 56]. In a two-component
3
system, the phosphorylation domain catalyzes the covalent bond of a phosphate
group from MgATP to its histidine residue. The phosphate group is then
transferred to an aspartic acid residue in the response regulator (Figure 1.1).
Phosphotransfer reactions:
i) HK-His + ATP Æ HK-His-P + ADP
ii) HK-His-P + RR-Asp Æ RR-Asp-P + HK-His
iii) RR-Asp-P + H2O Æ Asp + Pi
Figure 1.1: Phosphotransfer reactions
i) The phosphate group is transferred from ATP to the histidine residue of the histidine kinase. ii)
The response regulator then catalyzes the transfer of the phosphate group from the histidine
residue to its own aspartate residue. iii) The last step is a hydrolysis reaction transferring the
phosphate from the aspartate to water.
The phosphotransfer is different from the previously known Ser/Thr/Tyr
kinases where the phosphate group is transferred to the protein substrate instead
of a response regulator. Histidine kinases often have two membrane-spanning
domains. However, some histidine kinases have one, three or more membranespanning domains. In contrast, histidine kinases associated with chemotaxis are
found entirely in the cytoplasm. Analysis of transmembrane domains in histidine
kinases of Pseudomonas aeruginosa revealed that 49% of the analyzed kinases
contain two membrane-spanning domains, and 24% are soluble kinases having
no transmembrane domains. The remaining histidine kinases have between 3
and 12 transmembrane domains (Figure 2.4). The sensor domain of the histidine
kinases usually resides in the periplasmic space while the transmitter domain is
in the cytoplasm. The sensors react to a variety of stimuli including metabolites,
ions, and even photons [8].
4
Due to different sensing functions, the sequences and structures of
sensing domains are very diverse, whereas the transmitter domains show five
conserved motifs, including the H, N, G1, G2 and F boxes (Figure 1.2) [73].
Their names reflect the characteristic amino acids found in the motifs. The H box
is the domain with the most diverged sequences and is positioned at the Nterminus of the histidine residue where autophosphorylation occurs. Blocks N,
G1, G2 and F are located at the C-terminal part of the sensor and function as the
nucleotide binding pocket. G1 and G2 describe glycine rich sequences that are
separated by a spacer containing the F block. Those boxes define the kinase
core, which is the location of ATP binding and trans-phosphorylation. [57, 62,73].
Two different types of histidine kinases are known to exist. These include
the “orthodox” or “archetypal” histidine kinases and the hybrid kinases (Figure
1.2). Hybrid kinases are more abundant in eukaryotes whereas orthodox kinases
are usually found in prokaryotes [72]. An example of an orthodox histidine kinase
is the E.coli osmosensor protein EnvZ. EnvZ is a periplasmic membrane sensor
containing two transmembrane domains with an N-terminal periplasmic sensor
domain and a C-terminal kinase core located in the cytoplasm [29, 88]. This
structure is found in many histidine kinases, but proteins with more
transmembrane domains are also known [72]. A different type of orthodox
histidine kinases are soluble kinases which are not membrane bound. An
example is the NtrB protein, responsible for the carbon and nitrogen utilization
[47, 55]. Soluble kinases react to intracellular signals and/or to the interaction
5
with other proteins in the cytoplasm [62, 72]. Orthodox kinases also contain two
distinct functional motifs, the HisKA and a HATPase_c domain. The HisKA
domain contains the phosphoacceptor site as well as the dimerization domain
which links the sensor domain to the to the N-terminal domain containing the five
conserved boxes. HATPase_c domains are usually found in ATP binding
proteins such as histidine kinases or the DNA gyrase B.
Hybrid kinases consist of several phosphodonor and phosphoacceptor
sites and use a phosphorelay system which is more complex than the
phosphotransfer in orthodox kinases. The phosphorelay system shows several
phosphotransfer steps between histidine and aspartate residues. In hybrid
kinases the histidine and aspartate residues are found in the same domain [62,
72, 83]. An example of a hybrid kinase is the TodS protein found in
Pseudomonas putida consisting of two identical kinase cores, with conserved
histidine domains [43]. Hybrid kinases also contain a receiver domain usually
found in response regulators, but in this case the receiver domain does not
interact with an effector domain.
6
a) Orthodox histidine kinase
P
H
Sensor domain
N G1 F G2
ATP binding
domain
b) Hybrid kinase
Sensor domain
P
P
H
D
N G1 F G2
ATP binding
domain
Figure 1.2: Domain organization in orthodox histidine kinases and hybrid kinases a) A orthodox
kinase contains the periplasmic sensor domain with in most cases two transmembrane domains
(orange). This domain is linked to the cytoplasmic H box containing the conserved histidine
residue (yellow) followed by the N box as well as the G1 and G2 boxes separated by the F box
which build the ATP binding domain. The domain containing the H box also acts as the
dimerization domain. b) Hybrid kinases contain the same conserved domains as orthodox
kinases as well as a response regulator receiver domain (dark blue) including the conserved
aspartate residue (brown) as the site of phosphorylation.
Another feature that distinguishes histidine kinases is the position of the H
box. In unorthodox kinases as well as orthodox histidine kinases like EnvZ the H
box is linked to the N, G1, G2 and F boxes whereas in the case of CheA, a
soluble histidine kinase, the H box is located away from the other four boxes [8,
62, 72]. CheA is a histidine kinase important for chemotaxis where it
phosphorylates the CheY and CheB response regulators [33].
Yet another function of histidine kinases is the dephosphorylation. In this
case they are bifunctional and the histidine kinase also acts as a phosphatase
thereby regulating the level of phosphorylation of the response regulator. When
the sensor then recognizes a signal it changes to a kinase activity instead of
7
functioning as a phosphatase. Those bifunctional kinases are usually found in
pathways that need to be shut down quickly [62, 72].
Although two-component systems are specific to certain signals and the
phosphate group is transferred to its cognate response regulator, communication
between different two-component systems may occur. This communication is
called cross-talk. Cross-talk (in the case of beneficial aspects also called crossregulation) has to be distinguished from the necessary interaction of pathways
such as pathways that are branched. Those include a sensor kinase which
phosphorylates two or more response regulators. Another branched pathway
would be the case when several sensor kinases phosphorylate the same
response regulator (Figure 1.3) [8, 44].
a
c
b
H
P
D
D
H
H
P
D
H
P
P
D
D
H
P
Figure 1.3: Cross regulation and branched pathways
(a) Cross talk, the communication between two pathways. (b) Branched pathways showing the
phosphotransfer from one histidine kinase two several response regulators or (c) the transfer of
the phosphate group from several histidine kinases to the same response regulator.
8
An example for a pathway where an interaction is necessary is the
chemotaxis controlled by the CheA, CheB and CheY proteins in E.coli. CheA, the
histidine kinase, transfers the phosphate group to the CheB and CheY response
regulators. The phosphorylation of both response regulators is necessary for the
whole pathway to function [44, 52].
Although histidine kinases have been intensively studied over the past
years, new features continue to be discovered. It is likely that some functions
are still unknown and need to be identified. For many two-component systems
the exact signals resulting in the phosphorylation of histidine kinases are not yet
defined. Another characteristic of histidine kinases for further research are the
possible cross-regulation and cross-talk between several independent twocomponent systems.
Response Regulators
The response regulators carry out intracellular responses which in most
cases are the regulation of gene expression. However, response regulators also
control other cellular responses such as chemotaxis and motility toward or away
from a stimulus. Most common response regulators consist of a regulatory
domain, also called receiver domain, at the N-terminus and a C-terminal effector
domain [40, 73].
Receiver domains of the response regulators interact with the
phosphorylated form of histidine kinases. After interacting with the histidine
9
kinase, the receiver domain catalyzes the phosphate group transfer from the
histidine residue of the sensor to a conserved aspartate residue in the receiver
domain [1, 54]. Hybrid histidine kinases also contain a receiver domain. In these
cases the receiver domains generally do not interact with an effector domain.
Effector domains of response regulators carry out the final response. The
sequences of effector domains are very diverse within each subfamily of
response regulators. This reflects the variety of possible output responses for
response regulators and their different regulation mechanisms. Most effector
domains function as DNA-binding sites to activate or repress the expression of
certain genes [72, 83].
Two functions of response regulators are autodephosphorylation and the
regulation of its effector domain. Phosphotransfer from a histidine residue at the
sensor kinase to an aspartate residue at the response regulator results in its
activation and a response. Lukat et al. [45] showed that response regulators
catalyze their own phosphorylation resulting in their activation. Small-molecule
phosphodonors, for example acetyl-phosphate and carbamyl-phosphate, can
also be used as phosphodonors. Once the response regulator is activated, it
carries out an intracellular response and often acts as a transcriptional regulator.
Many response regulators bind to their cognate promoter sequence and either
up- or down-regulate gene expression as a result of an environmental stimulus.
Response regulators also undergo specific conformational changes to facilitate
an output response [10, 41]. Those conformational changes reflect the variety of
10
regulatory mechanisms which are specialized according to the requirements of
each system [72, 83].
In addition to catalyzing their own phosphorylation, response regulators
also act as phosphatases thereby limiting the time span of their activity. This
phosphatase activity is not the same for all response regulators, and half-lives of
activity may range from seconds to several hours. Thus, the lifespan of the
response regulators may vary according to their role in the physiological
responses of the cell [62, 83].
The majority of response regulators are classified into three subfamilies,
OmpR, NtrC and NarL, based on their sequence identity of their effector domains
(Figure 1.4).
a) OmpR
D
Trans_reg_C
b) NtrC
D
54
σ -interaction
domain
c) NarL
D
HTH_luxR
d) CheY
D
Figure 1.4: Response regulator domain organization. All classes of response regulators contain a
N-terminal receiver domain (blue) including the domain with the conserved aspartate residue
(brown). a) Response regulators in the OmpR class have the receiver domain linked to an
effector consisting of a trans_reg_C domain (yellow). b) The effector domain of NtrC-like
response regulators contains a domaine where the σ54_holoenzyme form of the RNA Polymerase
interacts (grey). c) NarL-like response regulators have their receiver domain linked to an effector
domain consisting of a helix_turn_helix-luxR site (red). d) The response regulators in the CheY
group are lacking the effector domain.
11
The OmpR family of response regulators has the most known members. OmpR
response regulators act as both activators or repressors and generally regulate
gene expression of proteins involved in osmoregulation. In E.coli OmpR is the
cognate response regulator to the EnvZ histidine kinase important for sensing the
changes in osmolarity in the cells environment [37, 68]. Once activated, OmpR
regulates the expression of OmpC and OmpF [64]. The N-terminal domain of
OmpR interacts with the EnvZ histidine kinase whereas the C-terminal domain
interacts with the OmpC and OmpF promoter regions [51]. OmpR acts as a
repressor as well as an activator. Under low osmolarity OmpR is in a nonphosphorylated form and activates OmpC expression while repressing OmpF
expression. In contrast, OmpR activates OmpF expression while repressing
OmpC under high osmolarity [29, 77, 86].
Another family is the NtrC-like response regulators. NtrC is a nitrogen
regulatory protein enhancing transcription to activate the σ54-holoenzyme form of
the RNA polymerase [66, 72]. When nitrogen is present, NtrC has an inactive
state in which it is not able to activate transcription of the glnA gene encoding a
glutamine synthetase and therefore acts as a repressor. Conversely, under
nitrogen limiting conditions NtrC is phosphorylated by NtrB resulting in its activity
enhancing transcription of the glnA promoter [35, 38, 40]
Response regulators in the subfamily represented by NarL are activators
and repressors of gene expression like the regulators grouped in the OmpR
subfamily. NarL is part of the nitrate and nitrite metabolism. The operons
12
regulated by NarL are also regulated by the Fnr transcription factor [18, 60, 72.]
and encode respiratory-related genes for nitrate and nitrite regulation under
anaerobic conditions. Fnr is a transcription factor that becomes activated under
anaerobic conditions and induces synthesis for respiratory enzymes needed for
the electron transport [9, 18].
Besides those three subfamilies other response regulators have also been
identified. They are distinguished from the three subfamilies by their C-terminal
domains which may have other enzymatic activities. For example, CheB is a
response regulator that also has methyltransferase activity, catalyzing the
demethylation of receptors during chemotaxis [25, 72]. While interacting with
CheR, a methyltransferase, CheB regulates the amount of receptor methylation,
thus influencing the receptor activities and adaptation. CheB demethylates
methylglutamate residues and also deaminates glutamine residues in the
receptors [3]. When methyl-accepting chemotaxis proteins are methylated a
methylester is formed at the C5-position of glutamate [78].
Other response regulators may lack the effector domain [62, 72]. For
example, CheY is a response regulator that does not regulate gene expression.
Instead, CheY interacts with the CheA histidine kinase to receive a phosphate
group. Once CheY is phosphorylated it interacts with the FliM protein, one of the
three proteins on the cytoplasmic site of the flagellar motor switch complex. By
interacting with FliM, CheB enhances clockwise rotation of the flagellar motor
instead of a counterclockwise rotation [13], resulting in tumbling motility rather
13
than smooth swimming. The activity of CheY is limited by the dephosphorylation
by the CheZ phosphatase [11, 27]
Response regulators are the part of a two-component system actually
carrying out the final response to an environmental stimulus. Phosphorylation
results in conformational changes allowing protein-protein interactions [10, 41].
Although response regulators differ in their mechanisms of action, the regulation
of phosphotransfer is the common part of these systems.
14
CHAPTER 2
TWO-COMPONENT SYSTEMS IN PSEUDOMONAS AERUGINOSA PA01
Introduction
Pseudomonas aeruginosa is a versatile organism. It is an opportunistic
pathogen and a major agent of nosocomial infections [59]. The bacteria are
harmful to a large number of hosts, including humans where they can cause
infections of the pulmonary tract i.e. cystic fibrosis, the urinary tract, burns or
wounds. Due to its metabolic diversity P. aeruginosa is found in many
environments, including soil, marshes, plants and animal tissues [31].
Approximately 10% of the genetic capacity of P. aeruginosa is composed of
regulatory proteins, with the two-component signal transduction systems
accounting for a large percentage of these regulators [75]. These regulators play
a major role in the versatility of this organism, and enable it to adapt to new and
changing environments. They also play an important role in virulence [63, 75].
Computational Methods
The Pseudomonas aeruginosa Community Annotation Project web site
(www.pseudomonas.com) [75, 85] was searched for all known and putative twocomponent systems. This search was done either by searching specifically for
histidine kinases and response regulators or by a Blast search using PA2656 and
PA2657 as query sequences using the default setting of the pseudomonas
15
database. The sequences were then used for sequence alignment analyses
using the PROMALS webserver (http://prodata.swmed.edu/promals/promals.php)
[58]. The resulting alignments were converted into the MEGA 4 program [76] and
Neighbor-joining trees were constructed. All sequences used for the alignments
were then used to search for motifs. Motif searches were performed using the
MEME/MAST program (http://meme.sdsc.edu/meme/intro.html) [6] and the
resulting motifs were converted into graphical logos using WebLogo
(http://weblogo.berkeley.edu/) [17].
The analysis for the transmembrane domains was performed using the TMHHM
(http://www.cbs.dtu.dk/services/TMHMM), Tmpred
(http://www.ch.embnet.org/software/TMPRED_form.html) and Sosui
(http://bp.nuap.nagoya-u.ac.jp/sosui/) servers [34, 36, 42].
Histidine kinases as well as response regulators were grouped according
to their Pfam and NCBI annotations. For the homology modeling of the PA2657
response regulator the template was found using the HHpred webserver
(http://toolkit.tuebingen.mpg.de/hhpred) [69, 70]. The modeling was then
performed using the DeepView software [32] and the model was displayed and
color coded using the PyMol program (www.pymol.org). Model Evaluation was
done using the Verify3D (http://nihserver.mbi.ucla.edu/Verify_3D/) [12], ProQ
(http://www.sbc.su.se/~bjornw/ProQ/ProQ.cgi) [80], ProSa
(https://prosa.services.came.sbg.ac.at/prosa.php) [67, 84] and MolProbity
(http://molprobity.biochem.duke.edu/) webserver [19].
16
Results
The search revealed 149 proteins involved in two-component regulation
including sensor/response regulator hybrids and single sensors and regulators.
For the analysis below, 55 histidine kinases and 67 response regulators were
included.
Sensor Histidine Kinase
The 55 histidine kinases retrieved from both types of searches were used
for the analysis (Table 2.1). The lengths of the 55 sequences range from 216
(PA1979) to 1212 (PA3946) amino acids. All 55 sequences were analyzed for
functional motifs and gene regions to classify them into orthodox and unorthodox
sensors. Sensors containing a HisKA and a HATPase_c domain were classified
a
b
*
*
Figure 2.1: The conserved histidine residue of sensor kinases in Pseudomonas aeruginosa. a)
Alignment of the conserved site of the nine subtree sequences with the predicted site of
phosphorylation (*). b) Graphical representation of the conserved site around the histidine residue
generated using the 55 original sensor sequences.
17
as orthodox sensors. Fourty out of the 55 sequences analyzed were found to be
orthodox sensors. Eleven sequences were found to be unorthodox sensors.
Members of the group of unorthodox sensors are hybrid kinases containing the
input and phosphate transmitter domain followed by a response regulator
receiver domain.
Two sequences were found to be CheA-like sensors (PA1458 and
PA0178) and the remaining two sequences of PA0471 and PA3900 are
unclassified sensors. Analysis of the multiple sequence alignment for the
histidine kinases was followed by constructing a phylogenetic tree using the
bootstrap test for Neighbor-Joining phylogenetic trees. Although some
sequences are very diverse with low sequence identities, a similar site of
phosphorylation is conserved throughout all sequences (Figure 2.1b). Unlike the
response regulators, not all orthodox sensors can be found as one cluster. The
unorthodox sensors are spread throughout the whole tree with just a few
grouping together. Only the two CheA-like sensors and the two unclassified
sensors group together with bootstrap values of 87 for PA1458 and PA0178, the
CheA-like histidine kinases, and a value of 99 for the two unclassified sensors
(PA0471 and PA3900) as can be seen in Figure 2.2.
P
A
51
PA
65
/1
44
-6
94
12
/1
-4
22
PA4
293
PprA
/1-92
PA
2
419
7/1
PA
-75
13
8
36
/163
3
PA2571/1
-470
PA4398/1-698
71
82/1-3
PA28
95
/1-5
484
PA5
358
02
/1trB
1-4
S/
4N
1
74
512
Fle
8
/1PA
9
2
4
10
04
21
PA
A3
-1
P
/1
46
39
A
P
PA
48
56
PA5512/1588
18
PA
Re
34
tS
PA
62
/139
/1
94
74
-9
2
La
19
dS
PA4
/
1
725
-79
Cbr
5
A/1983
PA281
0 CopS
/1-443
85
4
-8
43
/1
D
1/
p
02
Kd
41
0
36
A
6
44
P
1
/11
0
PA
8
-48
24
8/1
3
PA
4
1
PA
63
/1-4
8
8 6
PA4
-472
PA2524/1
PA4982/1-998
PA2687 PfeS/1-446
PA0930/1
-445
PA
320
6/1
PA04
-44
64 C
5
reC/1
-474
PA
51
99
En
Pm
vZ
PA
rB
/1
11
/180
-4
4
39
Ph
7
7
oQ
/144
8
PA
47
77
22
-6
/1
X
ar
N
78
38
PA
97
-7
/1
00
6
21
06
/1PA
79
19
769
PA
1
/
spE
53
4W
/1-7
370
458
PA
PA1
-639
PA0178/1
PA0471/1-323
PA3900/1-3
17
0.1
PA403
6/1-76
6
PA1
396
PA
/1-5
40
32
PA
71
19
/111
92
5
9
/1
-5
64
81
-8
/1
76
19
A
P
-530
PA4546 PilS/1
460
57/1PA07
43
/1-4
hoR
P
1
536
PA
1
26
45
43
-4
-4
/1/1
0/1
078
6
3
8
3
5
PA
43
47
26
1PA
/
PA
91
31
A
P
PA1798/1-428
PA115
8/1-45
2
Figure 2.2: Phylogenetic tree of the 55 histidine kinases using the Mega 4 Neighbor-Joining
method. The blue lines outline the subtree including the PA2656 histidine kinase. Gene numbers
are according to those of PseudoCAP. The numbers behind the gene name display the number
of amino acid of the sequence. Arrows indicate the different classes of histidine kinases:
Unorthodox
, CheA
and unclassified
. The unmarked kinases are all orthodox
histidine kinases.
19
100
56
PA2687 PfeS/1-446
PA0930/1-445
57
PA3206/1-445
PA0464 CreC/1-474
PA5199 EnvZ/1-439
PA3191/1-473
96
PA4777 PmrB/1-477
PA2656/1-445
93
PA1180 PhoQ/1-448
97
0.2
Figure 2.3: Neighbor-Joining trees constructed using the alignment with the histidine kinase
sequences found in the subtree grouping with PA2656.
The phylogenetic tree also shows a clearly distinguished subtree
containing the PA2656 two-component sensor. All sensors in this subtree are
orthodox sensor kinases (Figure 2.3). Nine sequences from the subtree were
then aligned and a Neighbor-Joining tree was constructed . The alignment of
those nine sequences shows several conserved sites including the putative site
of phosphorylation (Figure 2.1a) [24, 50]. Five out of the nine subtree sequences
have an assigned function (Figure 2.3). The PfeS sensor detects enterobactin
and stimulates its cognate regulator PfeR which then activates the ferric
enterobactin receptor (PfeA) expression, and is therefore important in iron uptake
[22, 23]. EnvZ is a osmosensor phosphorylating the OmpR response regulator to
control the expression of porin genes [16] depending on the level of OmpR
20
phosphorylation. PmrB and PhoQ are two related histidine kinases. Both respond
to limiting Mg2+ conditions and regulate resistance to polymyxin B and cationic
Transmembrane Domain Occurance
60
O
v
e
ra
ll
P
e
rc
e
n
ta
g
e
50
40
30
20
10
0
1
2
3
4
5
6
7
12
Number of TM domains per Histidine kinase
0
Figure 2.4: Overall percentage of the occurrence of transmembrane domains in the 55 used
histidine kinases. Transmembrane domains were determined using the TMHMM, Tmpred and
SOSUI servers.
antimicrobial peptides [48, 49], via modification of the LPS lipid A with arabinose
groups. The other characterized histidine kinase found in this subtree is the CreC
sensor also known as PhoM. PhoM transfers its phosphate group to PhoB, a
transcriptional activator, under phosphate limiting conditions [2]. PA2656 an
uncharacterized histidine kinase is most closely related to PhoQ and PmrB
(bootstrap value of 97), which suggests that PA2656 may also respond to
essential divalent cations or anions. In fact, microarray work in the Franklin lab
21
has demonstrated that PA2656 is highly upregulated at high concentrations of
Ca2+ (unpublished data).
The analysis of predicted transmembrane domains shows that 49% (27
out of the 55) of the sensor kinases have two transmembrane domains (Figure
2.4). 25 of the histidine kinases with two transmembrane domains are classified
as orthodox kinases, the other two are unorthodox kinases (PA3044, PA4982).
Two histidine kinases, one orthodox (CbrA) and one unorthodox (PA3271), have
twelve transmembrane domains, and for thirteen histidine kinases no
transmembrane domains were predicted. One unclassified and three orthodox
kinases are predicted to have one transmembrane domain, and the remaining
nine histidine kinases contain between three and seven transmembrane
domains.
22
Table 2.1: Histidine kinases
PA
Locus
0178
0464
0471
0600
0757
0930
1098
1158
1180
1336
1396
1438
1458
1636
1798
1976
1979
1992
2480
2524
2571
2656
2687
2810
2882
3044
3078
3191
3206
3271
3462
3704
3878
3900
3946
3974
4036
4102
4197
4293
4380
4398
4494
4546
4725
4777
4856
4886
4982
5124
NP_248868
NP_249155
NP_249162
NP_249291
NP_249448
NP_249621
NP_249789
NP_249849
NP_249871
NP_250027
NP_250087
NP_250129
NP_250149
NP_250327
NP_250489
NP_250666
NP_250669
NP_250682
NP_251170
NP_251214
NP_251261
NP_251346
NP_251377
NP_251500
NP_251572
NP_251734
NP_251768
NP_251881
NP_251896
NP_251961
NP_252152
NP_252393
NP_252567
NP_252589
NP_252635
NP_252663
NP_252725
NP_252791
NP_252886
NP_252983
NP_253070
NP_253088
NP_253184
NP_253236
NP_253413
NP_253465
NP_253543
NP_253573
NP_253669
NP_253811
Gi
Gene
name
15595376
15595661
15595668
15595797
15595954
15596127
15596295
15596355
15596377
15596533
15596593
15596635
15596655
15596833
15596995
15597172
15597175
15597188
15597676
15597720
15597767
15597852
15597883
15598006
15598078
15598240
15598274
15598387
15598402
15598467
15598658
15598899
15599073
15599095
15599141
15599169
15599231
15599297
15599392
15599489
15599576
15599594
15599690
15599742
15599919
15599971
15600049
15600079
15600175
15600317
CreC
FleS
PhoQ
KdpD
PfeS
CopS
WspE
NarX
LadS
PprA
PilS
CbrA
PmrB
RetS
NtrB
TM
domains
0
2
1
2
2
1
0
1
2
2
5
2
0
3
2
0
0
0
2
2
0
2
1
2
0
2
2
2
2
12
6
0
2
0
3
6
4
2
0
0
2
2
5
6
12
2
7
2
2
0
Sequence
length
639
474
323
797
460
445
402
452
448
633
540
481
753
885
428
881
216
564
440
472
470
445
446
443
371
741
431
473
445
1159
919
769
622
317
1212
795
766
434
758
922
426
698
422
530
983
477
942
463
998
358
Class
CheA
orthodox
unclassified
orthodox
orthodox
orthodox
orthodox
orthodox
orthodox
orthodox
unorthodox
orthodox
CheA
orthodox
orthodox
unorthodox
orthodox
unorthodox
orthodox
orthodox
orthodox
orthodox
orthodox
orthodox
orthodox
unorthodox
orthodox
orthodox
orthodox
unorthodox
unorthodox
unorthodox
orthodox
unclassified
unorthodox
unorthodox
orthodox
orthodox
orthodox
orthodox
orthodox
orthodox
orthodox
orthodox
orthodox
orthodox
unorthodox
orthodox
unorthodox
orthodox
23
Table 2.1 continued
PA
Locus
5165
5199
5361
5484
5512
NP_253852
NP_253886
NP_254048
NP_254171
NP_254199
Gi
15600358
15600392
15600554
15600677
15600705
Gene
name
EnvZ
PhoR
TM
domains
2
2
2
2
2
Sequence
length
612
439
443
595
588
Class
orthodox
orthodox
orthodox
orthodox
orthodox
Response Regulator
Compared to the histidine kinases the lengths of the response regulators
do not range as widely. However, the response regulators of P. aeruginosa
range from 121 (PA0179) to 571(PA3346) amino acids. When comparing the
sequence lengths of the four classes of response regulators a certain cluster can
be seen. The four CheY-like response regulators have the smallest number of
amino acids (121 - 135), which can be explained due to their lack of an effector
domain. NarL-like response regulators have a length between 207 and 225
amino acids followed by the OmpR group of regulators with a length between
221 and 247 amino acids and one sequences being 305 amino acids (PA2686).
The group of NtrC-like response regulators ranges from 425 to 490 amino acids
and the unclassified regulators have the widest range of sequence length with
the smallest one having 210 amino acids and the longest one with 571 amino
acids.
Analysis was performed to classify the response regulators according to
their functional domains (Table 2.2). The regulators in the OmpR group have a
“trans_reg_C “ region in their effector domain. Trans_reg_c domains are DNA
24
binding domains that regulate transcription [15, 73]. Response regulators
containing a DNA-binding, helix-turn-helix (HTH) domain and a luxR domain are
classified as NarL-like response regulators [15, 73]. NtrC-like response
regulators have a σ54 interaction domain and the response regulators classified
as CheY-like lack an effector domain.
Classification of the 67 response regulators according to their functional
motifs shows that the largest group is the OmpR family, which includes 24 out of
the 67 response regulators from P. aeruginosa. Of the remaining 43 response
regulators 13 are classified as NarL-like response regulators, ten are members of
the NtrC-family and four belong to the CheY-family. Twelve of the response
regulators are unclassified because they do not have the conserved effector
domain at their C-terminal. Two response regulators are methyltransferases
(PA0173 and PA1459) and the PA0414 and PA3703 response regulators are
methylesterases according to sequence analyses using the Pfam database.
Comparing NCBI annotation results with the phylogenetic tree, it can be
seen that all OmpR-like response regulators form a cluster and build a
distinguishable subtree (clade I on Figure 2.5). Three response regulators from
this clade fall into other families including PA0408 and PA0409 which are CheYlike response regulators, and PA4781 which is unclassified. With the exception of
PA4196 and PA4296, all other NarL-like response regulators are also contained
37
36 8
59/149
PA14
/1-3
173
1
PA0
-57
6/1
334
1
2
PA
93
1
/1- /1-3 0
ilH
1
3
5
5
p
8
-2
33 1-13 09
47
/1
/
4
/1PA
pF
ilG
pE
A0
P
ws 08 p
kd
03
4
0
37
16
PA
PA
PA0179/1
-121
PA14
56 ch
eY/1124
PA19
7
8
/1-22
PA3
1
604
/1 - 2
17
PA
371
PA
4/1
38
PA
-21
79
3
40
80
na
PA
/1rL
2
21
37
/1
4
-2
6/
119
21
3
PA
6
6/1-36
PA439
PA5364/1-300
PA3702 wspR/1-347
25
0
25
-21
214
-2
/1/1
1/1
A
0
0
c
8
ga
06
19
PA 2586
PA
PA
07
09
/1-2
A1/1-2
034
48 roc
PA0
PA39
PA3045/1-207
PA
53
60
P A ph
49 o B
83 /1
/1- -22
PA
24 9
11
PA
4
5
7/1
179
9/1
-23
PA3
-23
192
5 6
gltR
/1-2
PA520
0 ompR42
/1-247
PA4101/1-246
PA07
56/1223
PA2657/1-22
3
PA3204/1-225
-239
PA0929/1 -305
R/1
e
f
p
68 6
PA2
/1
rB
pp
5
27
PA
24
79
79
ph /1PA
oP 2 2
PA0
403
/1 6
463
creB 2/1-23 -225
/1-2
8
29
PA2881/1-303
PA2572
/1-447
PA54
83 a
lgB/1
PA4
-449
493
/1-1
PA
PA
86
10
472
99
6c
PA
brB
PA fle
R/
27
/15
1
478
12
98
-47
5
/1
3
nt
-3
rC
94
/1
-4
76
96
42
PA
11
lR/1-445
PA4547 pi
PA5261 algR/1-248
2
46
/17
66
- 44
1 /1
51
PA PA551 35/1-425
PA13
6/1-214
PA419
P A 3077 /1-223
/1-227
PA4381 -229
7 /1
3
4
PA1
24
/1-2 226
523 R/1- 29
2
p
2
PA
21
co /109 irlR /1-2
8
2
A
A
5
r
P
8
48
pm
PA 776
4
PA
PA
I
II
III
3
34
/1pB
90
ch
1-4
14 leQ/
f
04
PA 097
1
PA
PA4843/1-542
PA3947 rocR/1-392
0.1
Figure 2.5: Neighbor-Joining tree of the 67 response regulator sequences. I, II and III are the
three different subtrees found within the whole tree, distinguished by the blue lines. The gene
numbers are according to those of PseudoCAP. Numbers behind the gene identification number
show the length of the sequence in number of amino acids. The yellow colored clade contains the
PA2657 response regulator sequence. Orange brackets indicate the group of NarL-like response
regulators.
26
in a distinct cluster. For the other groups of response regulators no distinct
clusters can be identified as they have low overall sequence identity.
These proteins are scattered throughout the tree. An alignment was then
conducted using the 27 sequences that grouped into the OmpR-like subtree, that
contained PA2657. The two CheY-like (PA0408 and PA0409) regulators, the one
unclassified regulator (PA4781), as well as PhoB are branched as clear outliers
in this subtree.
In this subtree the PA2657 forms a cluster with four other response
regulators (Figure 2.6). This subtree is similar to that of the histidine kinases.
Two of those have been identified and are of known function (PhoP and PmrA).
Both of these regulators are stimulated by Mg2+ - limiting conditions, and are
important for polymyxin B and cationic antimicrobial resistance [48]. Their
cognate histidine kinases (PhoQ and PmrB) were found to be closest related to
the PA2656 histidine kinase (Figure 2.2). Another response regulator found in the
subtree is PhoB which gets phosphorylated by the PhoM histidine kinase and is
also found in the PA2656 containing subtree (Figures 2.2 and 2.3). PhoB is a
transcriptional activator under phosphate limiting conditions [2]. Apart from the
PhoP and PmrA response regulators others also have an assigned function. The
PfeR response regulator plays a key role in iron uptake and gets phosphorylated
by its cognate histidine kinase PfeS [22]. Once activated PfeR binds to the pfeA
promoter enhancing expression of the ferric enterobactin-receptor PfeA [22, 23].
KdpE regulates the kdpABC operon under osmotic stress as well as K+ limiting
27
PA4885 irlR/1-229
59
PA2809 copR/1-226
69
PA2523/1-224
16
PA1437/1-229
56
PA4381/1-227
PA3077/1-223
54
10
PA2686 pfeR/1-305
PA0929/1-239
99
PA1179 phoP/1-225
9
PA2657/1-223
48
26
PA0756/1-223
PA2479/1-226
33
81
PA4776 pmrA/1-221
PA1637 kdpE/1-230
23
PA0463 creB/1-229
14
PA4032/1-238
46
72
32
PA1799/1-235
PA1157/1-236
PA3204/1-225
PA4101/1-246
15 19
PA3192 gltR/1-242
PA5200 ompR/1-247
12
30
PA4983/1-244
PA5360 phoB/1-229
PA4781/1-393
PA0408 pilG/1-135
PA0409 pilH/1-121
83
0.1
Figure 2.6: Neighbor-Joining tree of the response regulator sequences found in subtree I (Figure
9). Underlined PA numbers indicate the unclassified (_ _ _) and the two CheY-like (___) response
regulators. The other 24 response regulators which are not underlined are members of the OmpR
family of response regulators.
28
a
*
b
*
Figure 2.7: Conserved domain with the site of phosphorylation, Asp51, in the response regulator
(*). a) Alignment of the 27 subtree sequences. b) Sequence logo generated using all 67 response
regulator sequences.
conditions. It is activated by the phosphotransfer from KdpD and then binds to
the kdpABC promoter which encodes genes for the K+-transporter [4, 61]. CopR
is a response regulator important for the Cu2+ resistance and is phosphorylated
by the CopS histidine kinase [50]. It acts as a transcriptional activator binding to
the promoter of the cop operon and it also activates the czsRS operon resulting
in an enhanced Zn2+ resistance [14, 50]. The GltR response regulator is believed
to play a key role in the expression of the glucose transport system [65]. Another
characterized response regulator in clade I of the subtree is the OmpR response
regulator which as previously described either expresses or represses ompF and
ompC expression depending on the osmolarity in the cells environment [16, 29,
29
77, 86]. PilG and PilH are the two CheY-like response regulators found in the
subtree and are involved in twitching motility [20, 21].
The alignment of the OmpR-like subtree sequences also shows some
highly conserved sites, including the conserved aspartate residue at position 51
as the proposed site of phosphorylation (Figure 2.7), with its surrounding amino
acids [24, 50].
a
*
b
c
Figure 2.8: Sequence logos with the conserved aspartate residue (*) as the site of
phosphorylation in Pseudomonas aeruginosa. a) OmpR-like response regulators, b) NarL-like
response regulators and c) NtrC-like response regulators.
Comparing the amino acids flanking Asp51 in the OmpR-like response regulators
with those around the aspartate residue in NtrC- and NarL-like response
30
regulators a certain diversity can be seen due to second or third order
substitutions (Figure 2.8), although some amino acids are highly conserved in all
three classes (DxDxPGxG).
In addition to the analyses above, structural modeling was performed on PA2657
using the solved structure of the Mycobacterium tuberculosis RegX3 response
regulator as a template. Figure 2.9a shows the resulting three-dimensional
structural model with two distinct domains, the C-terminal effector domain and
the N-terminal receiver domain. The PA2657 receiver domain has an alternating
a
b
C
Asp51
C
N
N
Figure 2.9: 3D model of the P. aeruginosa PA2657 response regulator. a) Structure of the
complete response regulator with the N-terminal receiver domain and the C-terminal effector
domain. b) N-terminal receiver domain with the conserved aspartate residue (blue).
α-helix β-sheet folding (β1-α1-β2-α2-β3-α3-β4-α4) consisting of 104 amino acids
with the four parallel β-sheets in the center surrounded by the α-helices.
The above identified aspartate residue (Asp51) as the site of phosphorylation is
located at the C-terminus of β3 (Figure 2.9b).
31
The C-terminal effector domains folds in five α-helices and five β-sheets.
Starting with a helix (α5) linked to the α4-helix at the N-terminal receiver domain
followed by two β-sheets divided from another sheet by a short helix (α6). β7 is
then linked to the remaining three helices and two sheets (α5-β5-β6-α6-β7-α7α8-α9-β8-β9). The model shows that in this part of the protein the α–helices are
in the centre with the β-sheets building a roof around them. Folds similar to the
one described here have also been reported for the NarL, NtrC and PhoB
response regulators [5, 71, 79].
Table 2.2: Response Regulators
PA
Locus
0034
0173
0179
0408
0409
0414
0463
0601
0756
0929
1097
1099
1157
1179
1335
1437
1456
1459
1637
1799
1978
NP_248724
NP_248863
NP_248869
NP_249099
NP_249100
NP_249105
NP_249154
NP_249292
NP_249477
NP_249620
NP_249788
NP_249790
NP_249848
NP_249870
NP_250026
NP_250128
NP_250147
NP_250150
NP_250328
NP_250490
NP_250668
GI
15595377
15585371
15595377
15595605
15595606
15595611
15595660
15595798
15595953
15596126
15596294
15596296
15596354
15596376
15596532
15596634
15596653
15596656
15596834
15596996
15597174
Gene
PilG
PilH
ChpB
CreB
FleQ
FleR
PhoP
CheY
KdpE
Sequence
length
207
349
121
135
121
343
229
210
223
239
490
473
236
225
425
229
124
368
230
235
221
Class
NarL
methylesterase
CheY
CheY
CheY
methylesterase
OmpR
NtrC
OmpR
OmpR
NtrC
NtrC
OmpR
OmpR
NtrC
OmpR
CheY
methyltransferase
OmpR
OmpR
NarL
Table 2.2 Response Regulators continued
PA
Locus
1980
2376
2479
2523
2572
2586
2657
2686
2798
2809
2881
3045
3077
3192
3204
3346
3604
3702
3703
3714
3879
3948
3974
4032
4080
4101
4196
4296
4381
4396
4493
4547
4726
4776
4781
4843
4885
4983
5125
5166
5200
5261
5360
5364
5483
5511
NP_250670
NP_251066
NP_251169
NP_251213
NP_251262
NP_251276
NP_251347
NP_251376
NP_251488
NP_251499
NP_251571
NP_251735
NP_251767
NP_251882
NP_251894
NP_252036
NP_252294
NP_252391
NP_252392
NP_252403
NP_252568
NP_252637
NP_252663
NP_252721
NP_252769
NP_252790
NP_252885
NP_252986
NP_253071
NP_253086
NP_253183
NP_253237
NP_253414
NP_253464
NP_253469
NP_253530
NP_253572
NP_253670
NP_253812
NP_253853
NP_253887
NP_253948
NP_254047
NP_254051
NP_254170
NP_254198
GI
15597176
15597572
15597675
15597719
15597768
15597782
15597853
15597882
15597994
15598005
15598077
15598241
15598273
15598388
15598400
15598542
15598800
15598897
15598898
15598909
15599074
15599143
15599169
15599227
15599275
15599296
15599391
15599492
15599577
15599592
15599689
15599743
15599920
15599970
15599975
15600036
15600078
15600176
15600318
15600359
15600393
15600454
15600553
15600557
15600676
15600704
32
Gene
GacA
PfeR
CopR
GltR
WspR
WspF
NarL
RocA1
LadS
pprB
pilR
cbrB
pmrA
irlR
ntrC
ompR
algR
phoB
algB
Sequence
length
225
213
226
224
447
214
223
305
394
226
303
207
223
242
225
571
217
347
335
213
219
209
392
238
214
246
214
275
227
366
186
445
478
221
393
542
229
244
476
462
247
248
229
300
449
447
Class
NarL
NarL
OmpR
OmpR
unclassified
NarL
OmpR
OmpR
unclassified
OmpR
unclassified
NarL
OmpR
OmpR
OmpR
unclassified
NarL
unclassified
methylesterase
NarL
NarL
NarL
unclassified
OmpR
NarL
OmpR
NarL
NarL
OmpR
unclassified
unclassified
NtrC
NtrC
OmpR
unclassified
unclassified
OmpR
OmpR
NtrC
NtrC
OmpR
unclassified
OmpR
unclassified
NtrC
NtrC
33
Conclusions
When comparing the analyses for the histidine kinases and the response
regulators it can be seen that PA2656 histidine kinase and the PA2657 response
regulator sequences are evolutionarily closely related to the PhoPQ and the
PmrAB two-component systems. This may indicate a similar mode of activation
by the same environmental stimulus, and all three appear to be stimulated by
divalent cations.
The high number of two-component systems in Pseudomonas compared
to other species might explain the occurrence of P. aeruginosa in different
environments, since the two-component systems enable the bacteria to adapt to
a wide range of environmental conditions. This is also reflected in the variety of
signals the systems react to. The high diversity in histidine kinase sensor
domains provides insight on how P. aeruginosa is able to detect and respond to
changing environmental conditions by altering gene expression through response
regulator outputs. This diversity is also reflected in the wide variety of sequence
lengths, ranging from 216 to 1212 amino acids.
The results also show that almost 50% of all analyzed histidine kinases
have two membrane spanning domains followed by 25% of all histidine kinases
which have no transmembrane domains. The soluble kinases reacting to
intracellular signals or they interact with other cytoplasmic proteins. The analysis
of the response regulator sequences shows that the receiver domains including
the site of phosphorylation are highly conserved, whereas the effector domains
34
are the part of response regulators distinguishing them into the four classes
(OmpR, NtrC, NarL and CheY). The individual classes of effector domains show
conservation, but the effector domains differ in sequence for each of the four
classes of response regulators. The divergence of response regulators is much
less pronounced than that of the histidine kinases. The similarity of response
regulators can also be seen in the relatively smaller range of sequence lengths,
between 121 and 571 amino acids, and each class of response regulators falls
within a narrow range of sequence lengths. CheY like response regulators are
the ones with the shortest sequences (121 amino acids) which can be explained
by the lack of the receiver domain.
Overall these results give further insight to the two-component system
organization of Pseudomonas aeruginosa. The findings suggest that the
response regulator domains might have evolved by amino acid substitutions
which results in a higher specificity for DNA binding and therefore a better
adaptation to changing environments.
Summary and Perspectives
Two-component regulatory systems in bacteria have been identified as
complex systems for signal transduction. The variety of stimuli the systems react
to is reflected in the diversity of histidine kinases and the different outputs of the
response regulators. In both parts of the system conserved domains have been
described.
35
Over the past years two-component systems have become a focus in the
development of antimicrobial therapy to fight pathogenic bacteria. Possible
targets can be found throughout the pathway. The histidine kinase activity has
been studied most intensely as a possible target for antimicrobials. For example
the autophosphorylation activity, but also the phosphotransfer reaction to the
response regulator as well as the ligand binding may be useful targets for
antimicrobials. Other targets for antimicrobial therapy are the signal itself, the
interaction of sensor and response regulator, or the dephosphorylation of the
response regulator [7].
Despite advances in understanding the nature of two-component systems,
there are still some unanswered questions. The stimuli for many systems are not
yet identified. Although the phosphotransfer is conserved within all groups of twocomponent systems their functions and regulations are very diverse. Some
functions of two-component systems are difficult to determine because of the
short halflives of the phosphorylated state, which may be as little as a few
seconds [33, 46, 82]. With more two-component systems being identified in
more species the variety of these systems increases and new types are
identified.
The results of the performed study on two-component systems in
Pseudomonas aeruginosa can be compared with previous studies [15, 63] and
confirm the overall picture of two-component systems in regard to classification,
domain organization as well and secondary- and tertiary-structures.
36
Further analyses of the phylogenetic relationships, structural comparisons
at the levels of sequence identity and similarity, including functional motifs, as
well as three-dimensional folding of histidine kinases and response regulators
might give further insight on two-component systems with yet still unknown
functions. In addition to bioinformatic analyses, direct physiological tests via
microarray or RT-PCR can help to further interpret interactions and expression
levels of two-component systems under certain conditions.
37
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