Chapter 7
Summarizing discussion
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Chapter 7
CSS in the regulation of iron uptake.
Extracytoplasmic function sigma factors (σECF) represent the third fundamental mechanism
of bacterial signal transduction (Staroń et al., 2009). The functional unit of σECF-dependent
signalling is formed by the σECF and its cognate anti-sigma factor, which controls the
post-translational activation of the σECF. Importantly, in Gram-negative bacteria several
σECF/anti-sigma signalling pathways have evolved to respond to extracellular, and thus
environmental, signals. This occurs via the association of the σECF/anti-sigma pair with
a surface-exposed receptor located in the bacterial outer membrane. Together, these
three proteins form a signal transduction cascade known as cell-surface signalling (CSS).
Most CSS systems are involved in the regulation of iron uptake machineries in response
to the presence of specific iron carriers in the environment. One of the first CSS systems
described was the Escherichia coli Fec system (Table 1), which is the sole CSS pathway
of this bacterium and is activated by ferric citrate (Braun, 1997). In recent years it has
become clear that bacteria belonging to the Pseudomonas genus contain a plethora of
these signal transduction systems (Llamas and Bitter, 2010; Llamas et al., 2014). To fulfil
their requirement for iron Pseudomonads rely on a large repertoire of TonB-dependent
receptors, which enable the uptake of specific iron-loaded siderophores or other iron
sources from the environment (Cornelis and Matthijs, 2002; Wandersman and Delepelaire,
2004; Cornelis and Bodilis, 2009). The multiplicity of TonB-dependent receptors in these
bacteria gives them an advantage in the fierce competition for iron with other organisms,
including host organisms of both plant and animal origin, and reflects the diverse
environments that they inhabit. However, the production of these large outer membrane
proteins is an energetically costly process and is only useful when the cognate iron source
is present in the environment. Since the regulation of siderophore receptor expression in
Pseudomonads is often controlled by CSS, it is not surprising that the mechanism behind
this signal transduction cascade has been mainly studied in these bacteria. Although all
CSS systems share common characteristics, they can also present several architectural
Figure 1. Variations in CSS architecture. Three different CSS systems are
drawn. The P. aeruginosa Fox system, which is activated by ferrioxamine,
represents a classical CSS system, in which the sigma (σFoxI) and anti-sigma (FoxR)
factors are produced as two separate proteins and the CSS outer membrane
receptor (FoxA) contains all the typical characteristics. The P. putida Iut system
contains the hybrid protein IutY, in which sigma and anti-sigma factor
domains are fused, and a normal CSS receptor (IutA) that senses and transports
aerobactin. The P. aeruginosa PUMA3 CSS system includes, apart from typical
sigma (σVreI) and anti-sigma (VreR) factors, a novel receptor-like protein (VreA)
that contains the periplasmic signalling protein of CSS receptors but lacks the
β-barrel outer membrane domain. The putative interaction of this protein with
a still unknown outer membrane receptor is shown, as well as the potential
host signal that activates the system. OM, outer membrane; P, periplasm; CM,
cytoplasmic membrane; C, cytoplasm.
Discussion
|
variations (Table 1 and Fig. 1). For example, the P. aeruginosa Fpv system controls the
activity of not one but two σECF (σPvdS and σFpvI) (Lamont et al., 2002; Beare et al., 2003).
The P. aeruginosa PUMA3 system contains a novel receptor protein that is located in the
periplasm instead of the outer membrane and seems to be involved only in signalling and
not in the transport of the signal molecule (Llamas et al., 2009) (Fig. 1). The P. putida Iut
CSS pathway contains a unique sigma/anti-sigma hybrid protein which combines both
functions in a single polypeptide (Bastiaansen et al., 2014) (Fig. 1). In addition, the antisigma component of CSS pathways, although similar in structure, can differ in their mode
of action. Several of these proteins (including P. aeruginosa FpvR) only contain an antisigma function and in this thesis are referred to as ‘mere’ anti-sigma factors. Deletion of
these proteins results in σECF-mediated transcription in absence of the CSS inducing signal
(Missiakas et al., 1997; Mettrick and Lamont, 2009). In contrast, several CSS anti-sigma
factors do not only inhibit their cognate σECF in non-inducing conditions but are also
required for maximal activity in presence of the stimulus (Ochs et al., 1995; Cuív et al.,
2006; Mettrick and Lamont, 2009). These proteins are therefore also called sigma factor
regulators (SFRs) (e.g. E. coli FecR, P. aeruginosa FoxR and FiuR). The pro-sigma activity
of SFRs seems to lie within their cytosolic N-tail (Table 1) (Mettrick and Lamont, 2009;
Bastiaansen et al., 2015).
CSS: beyond the regulation of iron uptake.
Although CSS σECF are predominantly used to regulate iron uptake functions and commonly
referred to as iron-starvation sigma factors (Leoni et al., 2000), the view that they only
control genes involved in iron homeostasis is oversimplified. Several CSS systems also
regulate other genes not involved in iron uptake, including potential virulence factors.
Therefore, CSS systems not only indirectly contribute to pathogenesis by enabling growth
and survival of the bacterium by providing access to the limited supply of iron, but can
also directly regulate virulence factors or other factors required for the interaction with
the environment (Kazmierczak et al., 2005). For example, P. aeruginosa σPvdS is essential for
successful infection of several animals and induces the production of exotoxin A, the PrpL
endoprotease and possibly the AprA alkaline protease (Shigematsu et al., 2001; Wilderman
et al., 2001; Lamont et al., 2002). Furthermore, this sigma factor regulates the synthesis of
its own inducing signal pyoverdine, which is considered a virulence factor itself (Meyer et
al., 1996; Handfield et al., 2000; Xiong et al., 2000; Banin et al., 2005). Another CSS cascade
directly involved in virulence is the PUMA3 system. In fact, the P. aeruginosa σECF VreI of the
PUMA3 pathway seems to be dedicated solely to the regulation of virulence factors (Llamas
et al., 2009). In Chapter 6 we show that expression of the PUMA3 proteins does not occur
in iron-depleted conditions, but in response to Pi starvation through the PhoB regulator, a
condition which is known to activate a virulence phenotype in P. aeruginosa (Lamarche et
al., 2008). Amongst others, σVreI controls expression of the alternative Hxc type II secretion
system and a two-partner Secretion (TPS) system (Llamas et al., 2009; Faure et al., 2014).
Although the inducing signal for σVreI is currently unknown, constitutive induction of this
system increases the mortality in a Danio rerio zebrafish embryo infection model (Llamas
et al., 2009), which underlines its importance in the regulation of virulence in the host.
A function for PUMA3 in iron/nutrient transport has not yet been demonstrated and it
is possible that, in contrast to the other CSS pathways described in this thesis, it is only
used for signalling in response to the environment. Since PUMA3-regulated genes are
transcribed in vivo during infection (Frisk et al., 2004; Chugani and Greenberg, 2007;
149
7
FoxR
σFoxI
FecR
MucA
σAlgT/AlgU
FiuR
σFecI
σFiuI
IutY*
VreR
σIutY*
FpvR
σFpvI/
σPvdS
σVreI
FiuR
FoxR
σFoxI
σFiuI
Antisigma
factor
-
FecA
FiuA
FoxA
IutA
VreA
FpvA
FiuA
FoxA
Receptor
-
Fec
Fiu
Fox
Iut
PUMA3
Pvd
Fiu
Fox
System
name
Iron uptake
Iron uptake
Iron uptake
Iron uptake
Iron uptake and
virulence
Virulence
Iron uptake
Iron uptake
Main
function(s)
Low Fe
Low Fe
Low Fe
Low Fe
Low Pi
Low Fe
Low Fe
Low Fe
Expression
condition
Ferric citrate
Ferrichrome
Ferrioxamine B
Aerobactin
unknown
Pyoverdine
Ferrichrome
Ferrioxamine B
Inducing
signal
unknown
unknown
unknown
Prc
unknown
unknown
unknown
unknown
Site-1
cleavage
unknown
unknown
unknown
unknown
unknown
unknown
unknown
unknown
Trimming
Alginate
Cell envelope stress AlgW
Prc**
production
E. coli
σE
RseA
Stress response Cell envelope stress DegS
Prc**
B. subtilis
σW
RsiW
Stress response Cell envelope stress PrsW
Prc***
* The sigma and anti-sigma factor of the P. putida Iut system are encoded by the hybrid gene iutY.
**
Prc degrades truncated RseA/MucA proteins, but does not seem to be required for signalling through the wild-type protein.
*** The B. subtilis protease responsible for trimming of RsiW has not been identified, but in an E. coli reconstitution system Prc is involved.
Other systems
P. aeruginosa
E. coli
P. putida
P. aeruginosa
CSS systems
σECF
RseP
(MucP)
RseP
RasP
RseP
RseP
RseP
RseP
RseP
RseP
RseP
RseP
Site-2
cleavage
Anti-sigma
unknown
Anti-sigma
Pro-sigma
unknown
unknown
-
Anti-sigma
Anti-sigma
Pro-sigma
Pro-sigma
Activity
N-tail
No
No
Yes
Yes
No
No
No
Yes
Yes
Yes
Initial
cleavage
Chapter 7
Bacterium
|
Table 1. Overview of CSS and stress response systems discussed in this chapter
150
Discussion
|
Llamas et al., 2009), it is likely that this unusual CSS system is induced by a host signal. The
PUMA3 system is not the first example of a CSS system involved in the positive regulation
of virulence upon interaction with the host. The plant pathogen Ralstonia solanacearum,
the causal agent of bacterial wilt, uses a CSS pathway to regulate the expression of the
hrp type III protein secretion system in response to a plant signal (Marenda et al., 1998;
Aldon et al., 2000; Brito et al., 2002). Interestingly, induction of this system requires
the presence of the plant cell wall and is not triggered by diffusible signals (Marenda et
al., 1998; Aldon et al., 2000). Besides virulence, CSS pathways may also be involved in
bacterial competition. P. aeruginosa contains a CSS system that regulates the production
of bacteriocins (named pyocins in this bacterium) (Llamas et al., 2008). Bacteriocins are
proteins produced and secreted by bacteria that kill bacteria of the same or closely related
species through a range of mechanisms (i.e. degradation of nucleic acids, inhibition of
peptidoglycan synthesis, formation of pores in the cytoplasmic membrane) (Parret and De
Mot, 2002). Since bacteria are immune to their own cytotoxins, secreting these proteins
into the environment provides a growth advantage. Interestingly, overexpression of the
CSS σECF PA4896 in P. aeruginosa resulted in upregulation of pyocin gene expression, which
correlated with increased killing activity of the producer strain (Llamas et al., 2008). This
σECF and the associated anti-sigma and CSS receptor proteins contain all the hallmarks
of classical CSS systems. Although the inducing signal has not been identified yet, it is
tempting to speculate that P. aeruginosa uses this CSS system to sense some molecule
produced by its competitors and kill the competing bacteria in response. Pyocins have
been shown to enter the target cell via siderophore receptors (Baysse et al., 1999; Denayer
et al., 2007; Elfarash et al., 2012; Elfarash et al., 2014). Therefore, the inducing signal for
this CSS system might be the siderophore specific for the pyocin-sensitive receptor on
the target cell, which would ensure that the σECF PA4896 is only activated (and thus the
pyocins are only produced) when the target cell expresses the receptor and is susceptible
for killing. These examples show that although most Pseudomonas CSS pathways regulate
the production of iron uptake machineries, their functions can be quite diverse. The
characterization of more CSS systems in the future will no doubt uncover other interesting
roles for this widespread signal transduction pathway.
Proteolytic regulation of CSS activity.
The key regulator of CSS is the transmembrane anti-sigma factor, which binds and
inactivates the σECF in non-inducing conditions but also interacts with the outer membrane
receptor. In order to initiate transcription of target genes, the σECF needs to be released
from its cognate inhibitor. For a long time conformational changes in the anti-sigma
factor in response to the inducing signal were thought to be responsible for this process.
However, the work presented in this thesis has demonstrated that in fact a complex
proteolytic cascade controls CSS activity, which alters the initial general assumption. We
have demonstrated that the main mechanism for CSS σECF activation is through regulated
intramembrane proteolysis (RIP) of its cognate inhibitor. RIP is a conserved feature
from bacteria to humans and involves the cleavage of the transmembrane segment of
cytoplasmic membrane proteins to release a soluble effector molecule (Brown et al., 2000).
Typically, RIP pathways require that the extracytoplasmic (e.g. periplasmic) domain of the
target protein is shortened to ~30 amino acids by a site-1 cleavage to allow processing by
a site-2 protease in the membrane (Brown et al., 2000). Following CSS activation by the
inducing signal, the anti-sigma factor is subjected to a number of proteolytic reactions that
151
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Chapter 7
Figure 2. Model of the complex proteolytic cascade
controlling CSS activation. Left panel: in the absence
of an inducing signal (e.g. an iron-siderophore) the
interaction with the cytosolic N-tail of the anti-sigma
factor keeps the cognate σECF in an inactive state.
Prior to signal recognition, most anti-sigma factors
(although not all) undergo self-cleavage (initial cleavage)
in a GT site located within the periplasmic domain
of the protein (1). Under these conditions, the Prc
protease seems to keep levels of the anti-sigma factor
low (2). Right panel: upon binding of the inducing
signal, the CCS receptor undergoes a conformational
change that allows the interaction of its periplasmic
signalling domain with the C-domain of the anti-sigma
factor. As a result, the anti-sigma factor becomes
susceptible to site-1 cleavage by an as yet unidentified
protease (3). Since site-1 cleavage might occur near
the C-terminus of the protein and the extracytoplasmic
domain of CSS anti-sigma factors is quite large, it is
possible that trimming by unknown protease(s) is
required to reduce this region to ~30-50 amino acids
(4). Subsequently, RseP cleavage takes place in the
transmembrane region of the CSS anti-sigma factor
(5), which releases the N-tail of the CSS anti-sigma
factor complexed with the σECF into the cytosol. The Ntail of cytosolic anti-sigma factors is degraded by ClpXP
proteases (6) to allow σECF interaction with the RNAPc.
The N-tail of SFRs, which contains pro-sigma activity,
is most likely not degraded and remains bound to the
σECF-RNAPc complex.
Discussion
|
153
allow the liberation of the σECF from the anti-sigma factor (Fig. 2) (described in Chapter 2,
3, 5 and 6). The site-2 RseP protease is essential in this process. Importantly, proteolytic
control does not only occur in response to the inducing CSS signal. In fact, we show that
prior to signal recognition most CSS anti-sigma factors (although not all) are subjected to
a proteolytic event that was named initial cleavage (described in Chapter 3 and 4). Despite
the particularities of each system (Table 1 and Fig. 1), processing of the anti-sigma factor
is a common theme in CSS regulation. However, we have found a number of important
variations among different CSS systems, which will be discussed in more detail in the
sections below.
Initial cleavage of CSS anti-sigma factors.
A very interesting finding we described in this thesis is the autoproteolytic processing
of CSS anti-sigma factors. This event occurs prior to signal perception and is therefore
referred to as initial cleavage (not to be confused with the site-1 cleavage) (Fig. 2). In
Chapter 3 we have determined that the initial cleavage takes place between conserved
Gly and Thr residues located in the periplasmic domains of CSS anti-sigma factors, and
in Chapter 4 we propose that this process occurs through an NO acyl rearrangement
in which the nucleophilic character of the OH-group of the threonine residue attacks
the scissile peptide bond of the preceding glycine. This results in the production of two
distinct polypeptides (e.g. the N- and C-domain) that interact with each other and function
together to control the activity of the σECF. However, we also show that blocking the initial
cleavage event has little effect on the functionality of CSS anti-sigma factors and only the
anti-sigma activity of the mutant proteins is partially affected. Moreover, we have identified
several functional CSS anti-sigma factors that do not contain the conserved GT site and are
therefore not initially processed. This raises the question why initial cleavage occurs? The
initial cleavage process is not linked to the pro-sigma or anti-sigma activity of the cytosolic
N-tail, since both types of anti-sigma factors can be subjected to initial cleavage (Table 1).
Perhaps the separation of the CSS anti-sigma factor in two domains allows a faster response
to the presence of the inducing signal. Instead of requiring a site-1 proteolytic cleavage
to remove the C-terminal residues of the anti-sigma factor, transduction in response to
the CSS signal might disturb the interaction between the anti-sigma N- and C-domains
thereby allowing cleavage by the site-2 RseP protease in the transmembrane segment
of the N-domain and σECF activation. The only difference observed upon blocking initial
cleavage of the FoxR anti-sigma factor was a slight increase of σFoxI activity in absence of
ferrioxamine, corresponding to a slight increase in production of the pro-sigma FoxR N-tail
in this condition. These results indicate that the initial cleavage could be required to tightly
regulate σFoxI-mediated transcription.
Site-1 cleavage of CSS anti-sigma factors: role of Prc and other proteases.
The RIP mechanism responsible for the removal of the CSS anti-sigma factor and activation
of the CSS σECF has been only very recently discovered and many of its features are still
unknown. However, the RIP-mediated activation of the periplasmic stress response sigma
factors E. coli σE and P. aeruginosa σAlgT is well known (Qiu et al., 2007; Ades, 2008) (Table
1) (see Fig. 2 of the Introduction). These RpoE-like sigma factors are also controlled by
transmembrane anti-sigma factors (RseA and MucA, respectively), but are not linked to
a surface-exposed receptor. Activation of σE and σAlgT occurs via RIP of RseA and MucA,
respectively, which includes the cleavage of the transmembrane domain of these anti-
7
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Chapter 7
sigma factors by the site-2 protease RseP. This results in the release of the anti-sigma
N-tail complexed with the σECF into the cytosol, which is further degraded by cytoplasmic
proteases (Qiu et al., 2007; Ades, 2008). The site-1 cleavage of RseA is executed by the
DegS protease (Alba et al., 2002) and that of the MucA by the DegS homologue AlgW
(Cezairliyan and Sauer, 2009) (see Fig. 2 of the Introduction). This step represents the ratelimiting step of the σE-mediated stress response, since DegS keeps itself inactive through
an interaction with its own PDZ domain (Walsh et al., 2003). This protease acts as a sensor
for periplasmic stress and is activated by binding of the C-termini of unfolded outer
membrane proteins (Walsh et al., 2003). DegS cleavage removes the last ~70 amino acids
of the periplasmic domain of RseA, thereby producing a truncate that can be processed by
the site-2 protease RseP in the transmembrane segment (Ades, 2008). The first evidence
that a site-1 cleavage also occurs in CSS anti-sigma factors is the accumulation of a slightly
larger fragment than the RseP product in a ∆rseP deletion mutant, which we observed
for P. aeruginosa FoxR (Chapter 3), FiuR (unpublished data), VreR (Chapter 6) and P.
putida IutY (Chapter 2). This indicates that another protease processes these proteins to
produce the RseP substrate. Furthermore, truncation of the P. aeruginosa FoxR and FiuR
(Llamas et al., 2006), P. putida IutY (Chapters 2 and 5) and E. coli FecR (Welz and Braun,
1998) proteins results in constitutive activity of their cognate σECF, likely by bypassing
the requirement for site-1 cleavage. In this thesis, we have identified one protease, Prc,
that most likely mediates the site-1 cleavage of the unique CSS IutY sigma/anti-sigma
hybrid protein (described in Chapter 2). Prc (also known as Tsp) was first reported to
cleave the last 11 C-terminal amino acids of the E. coli penicillin-binding protein 3 (Hara
et al., 1991). This protease was described as an endoprotease that selects its substrates
based on the nature of the residues located at the C-terminal end of the target protein,
where it prefers small hydrophobic amino acids (Silber et al., 1992; Keiler et al., 1995;
Keiler and Sauer, 1996). Prc seems to have the ability to cleave at multiple and diverse
sites in the target protein, thereby trimming the protein from the C-terminus (Keiler et al.,
1995; Beebe et al., 2000). Deletion of Prc in P. putida prevents the RseP-mediated site-2
cleavage of the IutY sigma/anti-sigma hybrid protein. Interestingly, no additional fragment
was observed on Western-blot in the ∆prc mutant, suggesting that Prc is the first protease
acting on IutY. Moreover, overexpression of Prc is by itself enough to generate the σIutY
domain and to increase σIutY-mediated transcription, which also suggests that this protease
mediates the site-1 cleavage. The role of Prc in the processing of IutY has been further
examined in Chapter 5. By creating truncated versions of the 374 amino acids long IutY
protein, we were able to establish that IutY truncates shorter than 236 amino acids were
completely independent of Prc. In contrast, IutY truncates of 260 amino acids in length or
longer required Prc for full activation. These results indicate that Prc trims IutY from the
C-terminus to create a protein truncate containing ~50 periplasmic residues that can be
cleaved by RseP. Interestingly, single point mutations in the C-terminal anti-sigma domain
of IutY can already induce constitutive activity of the cytosolic σIutY domain (described
in Chapter 5). Although activity of most IutY point mutants is Prc-dependent, we have
identified two point mutations, F251S and V253D, that resulted in IutY proteins (mostly)
independent of Prc for activation. The IutY-F251S and -V253D mutant proteins are still
processed by RseP, and, since RseP cannot process IutY proteins that contain more than 50
periplasmic residues, this also suggests that for these IutY variants another protease may
be responsible for producing the RseP substrate. Importantly, the fact that introducing
point mutations in the periplasmic domain of IutY result in constitutive activity of the
Discussion
|
cytosolic σIutY domain indicates that all the necessary components of the signalling pathway
are already present and active under non-inducing conditions. This shows that, unlike the
site-1 protease DegS that becomes active upon sensing unfolded proteins in the periplasm,
there is no signal (e.g. presence of aerobactin) required to trigger the protease function of
Prc. Then, how does Prc-mediated proteolysis of IutY start under inducing conditions? It
has been proposed that Prc prefers to cleave substrates that are not stably folded (Keiler
et al., 1995; Keiler and Sauer, 1996). Therefore, it is tempting to hypothesize that binding
of aerobactin to the IutA CSS receptor results in the partial unfolding of the periplasmic
domain of IutY, thereby exposing the Prc cleavage sites, whereas the protein in noninducing conditions is stably folded and unavailable for Prc. A similar mechanism applies
to the control of the B. subtilis anti-sigma factor RsiV, which in this case is the sensor of
the inducing signal (lysozyme) and becomes partially unfolded and susceptible for site-1
cleavage upon binding of the signal (Hastie et al., 2014). Alternatively, Prc cleavage could
be controlled by an inhibitory factor that would protect IutY from degradation in absence
of the inducing signal. This hypothesis is supported by the fact that overexpression of IutY
results in partial cleavage and activation of the σIutY domain, which suggests that a negatively
regulating element was titrated. A similar protecting mechanism has been reported for the
E. coli RseA anti-sigma factor, which in non-inducing conditions is protected from DegS
site-1 cleavage by the RseB protein (Kim et al., 2010; Chaba et al., 2011) (see Fig. 2 of the
Introduction). RseB is however not involved in IutY protection since overexpression of
this protein in P. putida had no effect on activity of the Iut CSS cascade (unpublished data).
Although our data suggests that Prc is the site-1 protease for the IutY sigma/anti-sigma
hybrid protein, this is not the case for all other CSS anti-sigma factors we have analysed.
Inactivation of Prc only decreases but does not abolish activity of P. aeruginosa and P.
putida σFoxI and σFiuI, showing that this protease is not essential for their function (described
in Chapter 2). In addition, Prc did not seem to be involved in the control of the Fpv CSS
pathway (Draper et al., 2011). The presence of Prc is not required for the site-2 RsePmediated cleavage of the P. aeruginosa FoxR and FiuR anti-sigma factors (shown in Chapter
3), which further implies that Prc is not the site-1 protease of the RIP cascade that controls
these CSS pathways. Interestingly, although deletion of Prc does not affect RseP-mediated
proteolysis, Prc does seem to have a role in the degradation of the P. aeruginosa FoxR and
FiuR proteins since the amount of these proteins in a ∆prc mutant is significantly higher
than in the wild-type strain in both the absence and the presence of the inducing signal
(Chapter 3). Moreover, expression of a proteolytically inactivated version of Prc in P. putida
did completely abolish Fox and Fiu CSS activity, which further shows that Prc does play a
role in the regulation of CSS in Pseudomonas (Chapter 5). Since sigma and anti-sigma factors
are believed to function in a 1:1 ratio, possibly the significant decline in activity of the Fox
and Fiu CSS systems observed in the ∆prc mutant is related to an overrepresentation of
the FoxR or FiuR anti-sigma factors, respectively. Although an interesting finding, it is not
clear why degradation of CSS anti-sigma factors in non-inducing conditions is required.
The genes encoding the CSS sigma and anti-sigma factor are usually located in an operon
and therefore co-transcribed under the same circumstances, which already regulates the
stoichiometry of the signalling pair. Although degradation of the anti-sigma factor in noninducing conditions seems like a rather energy consuming process, it could be useful in
controlling the activity of these proteins. In fact, degradation of regulatory proteins has
been shown to be used by bacteria to rapidly increase the amount of these proteins by
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Chapter 7
simply blocking its degradation instead of producing them de novo (Jenal and HenggeAronis, 2003). Similarly to FoxR and FiuR, the IutY sigma/anti-sigma hybrid protein is
also not stable in conditions in which is not functional (e.g. in absence of aerobactin, in
Δprc, ΔrseP or ΔiutA mutants) (described in Chapter 5). Therefore, it seems that CSS antisigma factors are subject to proteolytic regulation prior to signal perception, and further
research will identify the biological reason for this.
Prc is described to be involved in the activation of the stress-responsive σW in the Grampositive bacterium Bacillus subtilis, but does not execute the first cleavage of the RsiW
anti-sigma factor (Schobel et al., 2004; Ellermeier and Losick, 2006; Heinrich and Wiegert,
2006). Site-1 cleavage by the PrsW protease removes the last ~40 residues of RsiW, but
creates a product that still contains ~60 amino acids of the extracytoplasmic domain
and cannot be processed by the site-2 protease RasP (a RseP homologue) (Heinrich
and Wiegert, 2009). In order to allow RasP cleavage, RsiW is trimmed down to create a
smaller product. Although the B. subtilis protease that mediates this process has not yet
been identified, in a reconstituted E. coli system Prc is responsible for this (Heinrich et al.,
2009). Moreover, in P. aeruginosa Prc is known to degrade truncated versions of the antisigma factor MucA, although this protease is not necessary for signalling through the wildtype MucA (Reiling et al., 2005; Qiu et al., 2007). Therefore, although Prc does seem to be
involved in the regulation of σECF activity, it is not the general site-1 protease of the RIP
pathways that target stress-responsive anti-sigma factors. This also seems to be the case
for CSS anti-sigma factors. At this moment the protease(s) that is responsible for the site1 cleavage of FoxR, FiuR or other Pseudomonas CSS anti-sigma factors remains unknown.
Data indicated that neither DegS nor DegP are required for Fox and Fiu activity in P. putida
(Chapter 2) or Fpv activity in P. aeruginosa (Draper et al., 2011).
Site-2 cleavage of CSS anti-sigma factors: RseP-mediated proteolysis and fate of the
RseP product (N-tail).
Prior to our studies, a role for the RseP protease in the activation of the E. coli Fec CSS system
was already proposed, but not analysed (Braun et al., 2006). Later, Draper and co-workers
showed that this protease was in fact required for the activation of three P. aeruginosa
CSS systems: the Fpv, Fox and Fiu systems (Draper et al., 2011). The work presented in
this thesis has demonstrated that RseP is also required for activation of CSS in P. putida
(described in Chapter 2), showing a general role for this protease in the regulation of CSS
activity. Furthermore, we have shown that RseP cleaves within the transmembrane domain
of CSS anti-sigma factors (Chapters 2 and 3), in agreement with the already described
ability of this protease to cleave a broad range of transmembrane sequences (Akiyama et
al., 2004). Importantly, RseP cleavage generates the cytosolic N-tail domain of the antisigma factor and liberates the σECF factor. RseP-mediated proteolysis has been shown to
be also essential for the activation of the periplasmic stress response sigma factors E. coli
σE and P. aeruginosa σAlgT by cleaving their cognate anti-sigma factor proteins, RseA and
MucA respectively (Qiu et al., 2007; Ades, 2008) (Table 1) (Fig. 2 of the Introduction).
RseP cleavage of RseA/MucA releases the anti-sigma factor N-tail bound to the σECF into the
cytosol (Qiu et al., 2007; Ades, 2008). However, the interaction between the N-tail of RseA
and σE has been shown to be exceptionally tight and to shield the binding determinants of
σE for the RNAP core enzyme (Campbell et al., 2003). Therefore, the RseA N-tail needs to
be degraded in order for the σE protein to become active. This is facilitated by exposure of
Discussion
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a proteolytic tag on RseA following RseP cleavage that delivers the N-tail to the cytosolic
ClpXP protease complex (Flynn et al., 2003; Flynn et al., 2004; Chaba et al., 2007). This
protease complex is also required for activation of P. aeruginosa σAlgT (Qiu et al., 2008).
Interestingly, this cytosolic proteolytic step is not required for all CSS anti-sigma factors.
The pro-sigma activity of sigma factor regulators (SFRs) resides within their cytoplasmic
N-tail, which is necessary to promote σECF activity and is therefore not degraded upon
production. In Chapter 3 we show that, similar to RseA and MucA, RseP produces the N-tail
of the CSS SFR FoxR in response to the inducing signal. However, this N-tail is stable and
does not seem to be further degraded. In contrast, the N-tail of the ‘mere’ anti-sigma factor
VreR is not detectable (Chapter 6), which suggest that it is degraded upon production.
Since RseP cleavage of CSS anti-sigma factors is a common theme, this raises the question
how the cell distinguishes between N-tails that need to be further degraded and N-tails
that need to remain attached to the σECF to promote its activity. The cytosolic domain of P.
aeruginosa FpvR also seems to be eliminated by the ClpXP protease complex (Llamas et
al., 2014), which suggests that, similar to E. coli RseA, classical anti-sigma factors are also
tagged for degradation following cleavage in the transmembrane segment. Possibly, SFRs
do not contain this proteolytic tag, which might explain the different fates of the N-tails of
these two types of anti-sigma factor proteins. Determining the exact processing sites of
RseP will probably shed light on this matter. Interestingly, although all anti-sigma factors
seem to display a common N-terminal fold responsible for the binding to their cognate σECF
(Campbell et al., 2007), in principle these interactions should differ between ‘mere’ antisigma factors and SFRs. Whereas the N-tail of ‘mere’ anti-sigma factors would permanently
shield the RNAPc binding determinants of their cognate σECF (Campbell et al., 2003), the
N-tail of SFRs would enhance the interaction of the σECF with the RNAPc. In addition, since
gene transcription can also occur at the membrane (Görke et al., 2005), activation of σECF
could involve more than just relocation into the cytosol. Variability has been reported for
the regions the σECF uses to contact its cognate antagonist (Li et al., 2002; Campbell et
al., 2003; Campbell et al., 2007; Edgar et al., 2014; Shukla et al., 2014), which perhaps
relates to the difference in function of both anti-sigma factor classes. How SFRs enhance
σECF activity and why this is necessary is still unclear. The cytosolic domain of the E. coli SFR
FecR has been shown to increase the affinity of its cognate σFecI for the RNAPc and has even
been co-purified in complex with this sigma factor (Mahren and Braun, 2003). Perhaps
σECF regulated by classical anti-sigma factors have a higher intrinsic affinity for the RNAPc
and do therefore not require an extra protein fragment to establish a proper interaction
with the enzyme, but this has not been experimentally proven yet. Another possibility
is that σECF controlled by SFRs are more unstable and binding of the SFR N-tail prevents
its degradation in the cytosol. This hypothesis is supported by experiments showing
that the interaction with FecR protects σFecI from trypsin digestion (Mahren and Braun,
2003). In stark contrast, the ‘mere’ CSS anti-sigma factor FpvR of P. aeruginosa promotes
degradation of σPvdS, which is more stable in absence of its binding partner (Spencer et al.,
2008). In Chapter 6 we show that VreR, also a ‘mere’ anti-sigma factor, has a similar effect
on its cognate σVreI. To determine whether the intrinsic stability of CSS σECF is related to
either the pro- or anti-sigma activity of their regulators, more of these proteins need to be
examined. In addition, solving the structures of more σECF in complex with their cognate
transmembrane partner will provide more insights into these matters.
In Chapter 5 we show that truncated versions of the P. putida sigma/anti-sigma hybrid
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Chapter 7
protein IutY that are longer than 236 amino acids require processing by Prc in order to
allow RseP cleavage. This suggests that RseP is unable to directly process IutY protein
variants containing more than 50 periplasmic residues. Similarly, RseP-dependent
processing of the stress responsive anti-sigma factor RseA requires DegS-mediated site-1
cleavage, which generates a protein truncate containing ~30 periplasmic residues (Alba
et al., 2002; Kanehara et al., 2002). Interestingly, RseP processing of full-length RseA
independent of DegS occurred as a result of deletions or mutations in the periplasmic PDZ
domains of this protease (Kanehara et al., 2003; Bohn et al., 2004). Moreover, simultaneous
deletion of the degS and rseB genes also resulted in RseP-dependent cleavage of fulllength RseA (Grigorova et al., 2004). Therefore, it seems that RseP substrate accessibility
needs to be extensively regulated in order to prevent (full-length) RseA cleavage and σE
activation in non-inducing conditions. This regulation involves the site-1 DegS protease,
the RseB protein, the PDZ domains of RseP, and the anti-sigma factor itself (Kanehara et
al., 2003; Grigorova et al., 2004; Grigorova et al., 2006; Koide et al., 2008). Glutamine-rich
regions in RseA have been shown to interact with RseP and to inhibit RseP cleavage. This
interaction needs to be removed by DegS to allow the RseP site-2 processing of RseA.
Although CSS anti-sigma factors do not contain any glutamine-rich motifs, it is not unlikely
that such an interaction with RseP also occurs. The region of possible interaction remains
to be determined, but our results indicate that for IutY it could be located between the
periplasmic residues 236-374, which is the region that the site-1 Prc protease removes
allowing RseP cleavage.
Signal transduction by the CSS receptor.
The property that sets CSS aside from other signal transfer systems (i.e. one- and twocomponent systems) is the involvement of a surface-exposed receptor. CSS receptors
allow the recognition of extracellular signals that do not necessarily need to enter the
bacterial cell to target a response. These receptors usually are bifunctional proteins
involved not only in signal transduction but also in the transport of the signal molecule
(e.g. iron-siderophore) (Fig. 2) (Llamas et al., 2014). These two functions reside in
different domains of the receptor protein. The signal transfer function lies within the
short N-terminal periplasmic domain referred to as signalling domain (SD), which is the
domain that interacts with the anti-sigma factor and determines the specificity of the
signal transduction pathway. The transport function resides in its large C-terminal domain
that forms a pore in the outer membrane. Transport requires energy in the form of proton
motive force and a complex of three cytoplasmic membrane proteins, TonB-ExbB-ExbD, to
provide this energy to the outer membrane (Koebnik, 2005). Binding of the signal to the
CSS receptor is known to induce vast structural changes to activate the transport function
(Noinaj et al., 2010). However, little is known about how the signal transfer function of
the receptor is activated. In the current model signal perception promotes the interaction
between the SD of the receptor and the anti-sigma factor in the periplasm. This model is
mainly based on experiments with the E. coli Fec system, which showed that introduction
of mutations that compromise the interaction between the CSS receptor FecA and the antisigma factor FecR reduce σFecI activity in response to the inducing signal ferric citrate (Enz
et al., 2003b; Breidenstein et al., 2006). Among the mutations described were those in the
FecR leucine heptad motif, which is a conserved motif in the periplasmic regions of antisigma factors (Enz et al., 2003a). However, mutating this Leu rich domain of the P. putida
IutY protein did not abolish CSS activity (described in Chapter 5). In fact, this mutation in
Discussion
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the anti-sigma domain resulted in constitutive activity of the σIutY domain. This indicates
that the IutA-IutY interaction can be abolished without inhibiting the sigma factor activity,
and suggests that CSS receptors could function in a different way as initially thought. The
SD of CSS receptors display a common fold, in which two α-helices are flanked by two
β-sheets (Garcia-Herrero and Vogel, 2005; Wirth et al., 2007). The region that connects
the SD to the outer membrane β-barrel is long and flexible and has been proposed to
enable movement of the SD in the periplasm upon signal perception, thereby promoting
the interaction with the transmembrane anti-sigma factor. Although the orientation of
the SD in respect to the β-barrel in the outer membrane indeed changes in response to
the inducing signal, experiments showed that the SD does not extend further into the
periplasm (Wirth et al., 2007; Mokdad et al., 2012). Importantly, signal perception does
not induce alterations in the overall structure of the SD (Mokdad et al., 2012), which
suggests that an interaction between SD and the anti-sigma factor might already occur in
absence of the inducing signal. This is supported by biochemical interaction studies with
the E. coli FecA and FecR proteins performed in absence of the inducer (Enz et al., 2000).
Moreover, overexpression of the FecA SD inhibited activity of σFecI but did not affect ferric
citrate transport. Interestingly, expression of the SD did not affect the constitutive activity
caused by FecR truncates, indicating that the separate SD binds to the periplasmic domain
of FecR (Kim et al., 1997). Likewise, overexpression of the P. putida IutA SD completely
inhibits σIutY activity in inducing conditions (unpublished results). These data suggest that
the mere binding of the SD does not seem to be sufficient to trigger activity of the CSS
pathway and indicate that the binding of the SD can protect the anti-sigma factor from
downstream proteolytic degradation. This introduces an additional level of complexity in
the CSS signal transduction cascade and shows that the SD of the receptor deserves more
attention in future research.
Concluding remarks.
The results described in this thesis have provided insight into the mechanism of CellSurface Signalling (CSS), which involves extensive regulation by a complex cascade of
proteolytic cleavages. Our experiments have uncovered a number of new aspects and
new players in the control of CSS σECF activity and have shown that there are several other
partners that are still to be identified. Future experiments will have to show which enzyme
executes site-1 cleavage of CSS anti-sigma factors, or whether each pathway requires
their own specific protease. Hopefully, this will reveal how site-1 cleavage is induced
in response to the environmental signal, either by activation of the protease itself or by
increased susceptibility of the anti-sigma factor by a conformational change or release of
a periplasmic inhibitor. In conclusion, the work presented here has considerably advanced
our understanding of how bacteria sense and respond to the environment, and has opened
a broad field of research that will help future studies.
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