Acid stress response in enteropathogenic gammaproteobacteria: an aptitude for survival.

Abstract

Enteric bacteria such as Escherichia coli have acquired a wide array of acid stress response systems to counteract the extreme acidity encountered when invading the host's digestive or urinary tracts. These acid stress response systems are both enzyme and chaperone based. The 3 main enzyme-based acid resistance pathways are glutamate-, arginine-, and lysine-decarboxylase pathways. They are under a complex regulatory network allowing the bacteria to fine tune its response to the external environment. HdeA and HdeB are the main chaperones involved in acid stress response. The decarboxylase systems are also found in Vibrio cholera, Vibrio vulnifus, Shigella flexneri, and Salmonella typhimurium, although some differences exist in their functional mechanism and regulation.

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REVIEW / SYNTHE`SE
Acid stress response in enteropathogenic
gammaproteobacteria: an aptitude for survival1
Boyu Zhao and Walid A. Houry
Abstract: Enteric bacteria such as Escherichia coli have acquired a wide array of acid stress response systems to counter-
act the extreme acidity encountered when invading the host’s digestive or urinary tracts. These acid stress response sys-
tems are both enzyme and chaperone based. The 3 main enzyme-based acid resistance pathways are glutamate-, arginine-,
and lysine-decarboxylase pathways. They are under a complex regulatory network allowing the bacteria to fine tune its
response to the external environment. HdeA and HdeB are the main chaperones involved in acid stress response. The de-
carboxylase systems are also found in Vibrio cholera, Vibrio vulnifus, Shigella flexneri, and Salmonella typhimurium,
although some differences exist in their functional mechanism and regulation.
Key words: acid stress, glutamate decarboxylase, arginine decarboxylase, lysine decarboxylase, HdeA/HdeB.
Re ´sume ´ : Les bacte ´ries ente ´riques comme Escherichia coli ont acquis une vaste gamme de syste `mes de re ´ponse au stress
acide afin de contrecarrer l’acidite ´ extre ˆme rencontre ´e lorsqu’elles envahissent les syste `mes digestifs ou urinaires de leurs
ho ˆtes. Ces syste `mes de re ´ponse au stress acide reposent sur des enzymes ou des chaperons. Les trois principales voies de
re ´sistance a ` l’acidite ´ base ´es sur des enzymes sont les voies des glutamate-, arginine- et lysine de ´carboxylases. Elles sont
place ´es sous le contro ˆle d’un re ´seau re ´gulateur complexe qui permet a ` la bacte ´rie de re ´gler finement sa re ´ponse a `
l’environnement externe. HdeA et HdeB sont les deux chaperons principaux implique ´s dans la re ´ponse au stress acide.
Les syste `mes des de ´carboxylases sont aussi trouve ´s chez Vibrio cholera, Vibrio vulnifus, Shigella flexneri et Salmonella
typhimurium, me ˆme s’il existe quelques diffe ´rences entre leur me ´canisme de fonctionnement et leur re ´gulation.
Mots-cle ´s : stress acide, glutamate de ´carboxylase, arginine de ´carboxylase, lysine de ´carboxylase, HdeA/HdeB.
[Traduit par la Re ´daction]
Introduction
Seemingly small and vulnerable, bacteria possess an extra-
ordinarily complicated set of stress response mechanisms
that give them the ability and resilience to survive, or even
thrive, in harsh conditions. Acid resistance in enteric bacte-
ria such as Escherichia coli is one important example of
such an adaptation. These bacteria can colonize the intes-
tines of their host organism, including humans, and cause in-
fection. In the process, they inevitably have to pass through
the gastric acid in the stomach (pH 2.5), which serves as a
natural antibiotic barrier. However, despite being neutro-
philes, they can survive in this hostile acidic condition.
Therefore, understanding the complex regulatory mecha-
nisms and pathways of the bacterial acid stress response is
crucial to developing strategies for controlling bacterial in-
fection. In this review, we will discuss the acid resistance
systems in enteric bacteria, focusing primarily on describing
the mechanism of function of the enzymes and chaperones
involved in the acid resistance pathways. At first, we will
describe the decarboxylase-based and chaperone-based path-
ways in Escherichia coli, as they are the most extensively
studied. Subsequently, we will compare and contrast the
acid stress systems among 5 enteric bacterial species:
Escherichia coli, Vibrio cholera, Vibrio vulnifus, Shigella
flexneri, and Salmonella typhimurium.
Enzyme-based acid stress response systems
in E. coli
Five acid resistance (AR) pathways, AR1–AR5, are
known in E. coli. (Kashiwagi et al. 1992; Foster 2004)
(Fig. 1). The AR1 pathway, though poorly understood, is ac-
tivated when cells are placed in minimal media at pH 2.5
Received 17 September 2009. Revision received 20 November 2009. Accepted 7 December 2009. Published on the NRC Research Press
Web site at bcb.nrc.ca on 18 March 2010.
B. Zhao and W.A. Houry.2Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada.
1This paper is one of a selection of papers published in this special issue entitled ‘‘Canadian Society of Biochemistry, Molecular &
Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases’’ and has undergone the Journal’s usual peer review
process.
2Corresponding author (e-mail: walid.houry@utoronto.ca).
301
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without an external supply of any amino acid after the cells
are grown at pH 5.5 to stationary phase in buffered Luria
broth (Foster 2004). This pathway requires the alternative
signal factor sSand cAMP receptor protein (CRP) to func-
tion. Because CRP is involved, the system is repressed by
glucose (Foster 2004). The AR2–AR5 pathways are all
amino acid decarboxylase-based pathways. In general, they
consist of a decarboxylase enzyme that is induced by low
pH and the presence of a specific amino acid, as well as an
antiporter. The enzymes involved in AR2, AR3, and AR4
are the glutamate, arginine, and lysine decarboxylases, re-
spectively. The AR2 and AR3 pathways enable bacteria to
survive in extreme acidic environments (e.g., pH 2.5)
(Foster 2004). The AR4 and AR5 pathways allow E. coli
cells to survive in moderately acidic environments (e.g.,
pH 4.5). AR5 consists of the inducible ornithine decarboxy-
lase SpeF, which has a pH activity optimum of 6.9, and the
ornithine–putrescine antiporter PotE (Kashiwagi et al. 1992;
Foster 2004). AR5 is not as well studied as AR2–AR4 and
will not be discussed.
AR2: the glutamate decarboxylase system
This system has been extensively studied and constitutes
the major acid response system in E. coli under extreme
acidic conditions. The system consists of paralogous GadA
and GadB decarboxylases and an inner-membrane antiporter
GadC. GadA and GadB are pyridoxal 5’-phosphate (PLP)-
dependent enzymes that convert glutamate to gamma-amino
butyric acid (GABA) and carbon dioxide (CO2) in a reaction
that consumes a cytoplasmic proton (Bearson et al. 1997;
Foster 2004) (Fig. 1). GABA is transported out of the cell
by the inner membrane antiporter GadC in exchange for
more glutamate (Bearson et al. 1997).
Structures of the glutamate decarboxylases
The structures of both GadA and GadB have been solved
by X-ray crystallography (Capitani et al. 2003; Dutyshev et
al. 2005). The two isozymes differ in their primary sequen-
ces at only 5 residues and are therefore very similar in struc-
ture. They form 330 kDa hexamers assembled from
trimerization of GadA(B) dimers. The GadA(B) monomer
can be divided into 3 domains: the N-terminal domain, the
large domain (PLP binding domain), and the C-terminal
small domain (Fig. 2). The N-terminal domain is critical for
the function of GadB because it is responsible for the pref-
erential association of GadB with the inner membrane when
pH is lowered. Deletion of the first 14 residues of GadB di-
minishes its ability to migrate to the inner membrane. By
comparing the structures of GadB at neutral (pH 7.6) and
acidic pHs (pH 4.6), the N-terminal domain is found to
undergo a conformational change from a disordered state
containing little secondary structure at neutral pH to an a-
helix at acidic pH (Capitani et al. 2003) (Fig. 3). This a-he-
lix is oriented perpendicularly to the subunit surface. As a
result, the active (low pH) form of GadB hexamer has a 3-
helical bundle on each of its 2 opposing surfaces (Fig. 3).
The bundles have a hydrophobic core and are charged on
the outside. There are 3 Asp residues and 1 Glu residue in
the first 15 residues of GadB. At least two of them are pro-
tonated upon acidification, resulting in the conformational
changes in GadB N-terminus (Capitani et al. 2003).
More interestingly, halide ions such as Cl–are found, by
X-ray crystallography, to be able to bind to the bottom of
the C-terminus of each of the N-terminal helices in the 2
triple-helix bundles (Gut et al. 2006). The binding fixes the
turn formed by residues 16–19 and, as a result, stabilizes the
triple a-helix bundle required for GadB hexamer interaction
with the inner membrane (Gut et al. 2006). This finding is
significant because it shows an additional function of Cl–
ion on top of its use to protect the membrane potential dur-
ing extreme acid stress (see discussion on the role of Cl–in
acid stress below).
The X-ray structure of GadA hexamer was only solved at
pH 4.6 and the N-terminal helix structure was resolved at
that pH. There is no structure of GadA at a higher pH.
Therefore, although similar biochemical experiments to
GadB are not yet performed with GadA, it is highly likely
that N-terminal domain in GadA also undergoes a conforma-
tional change from a disordered state at neutral pH to an a-
helix at acidic pH.
The active site of GadB (as well as of GadA) is in the
PLP-binding large domain. Lys276, in GadB (GadA), forms
a Schiff base linkage with the C4 atom of the pyridine ring
of PLP. Unlike other PLP-dependent enzymes, GadB uses
Gln163 instead of an aromatic residue for a stacking interac-
tion with the pyridine ring of PLP (Capitani et al. 2003).
When the glutamate substrate interacts with the active site
of GadB, it is held in place by hydrogen binding of its g-
carboxylate group with the protein Phe63 main chain, the
Thr62 side chain, and the carboxylate side chain of Asp86
of the neighboring subunit. This binding explains the maxi-
mum activity of GadB at low pH, since either the Glu sub-
strate or Asp86 of GadB must be protonated for this
interaction to occur. In addition, an Arg422 residue that
binds the a-carboxylate of the substrate in many PLP-
dependent enzymes is kept away from interacting with the
Glu substrate in GadB, so that it does not interfere with the
decarboxylation process (Capitani et al. 2003).
When the pH increases back to neutral, GadB undergoes a
stepwise conformational change to its inactive form. At first,
each of the N-terminal triple helical bundles unfold inde-
pendently. When both are unfolded, an aldimine structure
(imine derived from an aldehyde) forms between the imadi-
zole ring of His465 at the C-terminal end of GadA/B and
Lys276-PLP imine to close the active site (Gut et al. 2006).
This covalent adduct is the 340 nm-absorbing chromophore
that is the signature of the inactive form of GadB (Gut et al.
2006). Hence, at neutral pH (pH 7.6), each active site funnel
is blocked by the C-terminus of the same subunit and by a
b-hairpin from the neighboring subunit.
Regulation of the glutamate decarboxylase system
As the most effective acid stress response pathway under
extreme acid stress conditions, the AR2 system is intricately
regulated. To date, there are over 20 proteins and 3 small
non-coding RNAs that are identified as regulating the Gad
system (Fig. 4A). The proteins and factors include CRP,
Dps, EvgA/S, GadE, GadX, GadW, H-NS, Lon, PhoP/Q,
RNaseE, s70, ss, SspA, TrmE, TopA, TorS/R, and YdeO.
The 3 small non-coding RNAs are DsrA, GadY, and GcvB.
In the E. coli genome, the gadA and gadB genes are lo-
cated 2100 kb apart (Fig. 4A). The gadC gene is located
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downstream of gadB and they form an operon (Dutyshev et
al. 2005). The central transcriptional activator of the gadA
and gadBC genes is GadE (Ma et al. 2003; Foster 2004).
GadE induces the expression of the Gad system by binding
to a 20 bp sequence, termed the gad box, which is located
around 60 bp upstream of the transcription start sites of
gadA and gadBC (Castanie-Cornet and Foster 2001; Ma et
al. 2002).
Apart from the Gad system, GadE also serves as a global
transcriptional activator for many genes. When over-
expressed, GadE is found to induce genes involved in stress
response (e.g., somC, hdeA, and ycgG), in biosynthesis of
glutamate (e.g., gltD and gltH), and also membrane compo-
nents (e.g., rcsA and rfaG) (Hommais et al. 2004).
Other protein factors regulate, directly or indirectly, the
expression of GadE depending on the growth phase of the
cells and on the media (Fig. 4A). Recently, it was found
that there is a 750 bp regulatory region upstream of the tran-
scription start site of the gadE gene (Sayed and Foster
2009). This region contains 3 promoters (P1, P2, and P3)
that allow different regulators to bind and produce 3 gadE
transcripts: T1, T2, and T3. T1 starts at –124, T2 at –324,
and T3 at –566 bases from the gadE start codon. The P1,
P2, and P3 promoters are located about 200 bp upstream of
the start of each transcript. P1 is where GadE acts to auto
induce itself in minimal medium containing glucose (Ma et
al. 2004; Sayed and Foster 2009). This auto activation also
requires ss, the alternative sigma factor responsible for the
Fig. 1. Schematic diagram of the acid resistance (AR) systems in E. coli. The amino acid and decarboxylation products are shown in
chemical notation and the proteins responsible for the reactions are shown under the reaction arrows. All the decarboxylation reactions
consume a proton and release carbon dioxide. AR1 is not shown because the system is not well characterized. The HdeA/B and Hsp31
chaperones are represented by ovals. This figure is adapted from Gajiwala and Burley (2000), Zhao et al. (2003), and Foster (2004).
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transcription of many stress response genes in stationary
phase, and another unidentified factor. P2 and P3 are acti-
vated by GadX (YhiX) and GadW (YhiW) during stationary
phase growth and by the EvgA/S and YdeO pathway during
exponential phase growth in minimal medium at acidic pH
(Ma et al. 2004).
GadX and GadW are both AraC-like regulators; AraC is a
well-studied transcription activator of the arabinose operon
that encodes proteins involved in metabolizing L-arabinose.
Along with ss, GadX and GadW are important for inducing
the Gad system in stationary phase cells grown in either
minimal or rich media (Ma et al. 2002). The transcription
of gadX is induced by ss(Fig. 4A). CRP and H-NS proteins
are two repressors of gadX transcription. CRP represses the
production of ssduring normal cell growth and, thus, indi-
rectly represses gadX transcription (Castanie-Cornet and
Foster 2001). H-NS is a major component of bacterial chro-
matin. It preferentially binds AT-rich DNA sequences, often
found in E. coli promoters, to repress expression of down-
stream genes. H-NS is a repressor of gadA, gadE, and
gadX, but not of gadBC (Giangrossi et al. 2005). Several in
vitro experiments have shown that GadW and GadX can
bind directly to the promoter regions of gadA and gadBC
and induce their expression through different mechanisms
(Ma et al. 2002; Giangrossi et al. 2005; Tramonti et al.
2006). However, it seems that in vivo GadX and GadW ac-
tivate the Gad system indirectly by activating gadE tran-
scription (Fig. 4A), because overexpression of GadX cannot
induce gadA or gadBC in a gadE mutant background (Ma et
al. 2003; Gong et al. 2004; Ma et al. 2004; Sayed et al.
2007). Moreover, overexpression of GadE seems to diminish
the requirement for GadX and GadW in E. coli acid resist-
ance, but not vice versa (Ma et al. 2003). Therefore, the in
vitro results do not exactly agree with the in vivo results. So
Fig. 2. Domain arrangement of the acid stress induced amino acid decarboxylases and chaperones. The domain boundaries for E. coli GadA/B,
AdiA, HdeA, and Hsp31 are based on the solved X-ray structures. Since the structures of E. coli LdcI and ornithine decarboxylase (OrnDC)
are not yet solved, their domain boundaries are defined based on sequence alignment with Lactobacillus 30a OrnDC whose X-ray structure
has been solved (Momany et al. 1995).
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far, the exact mechanism of the GadX/W/E-mediated activa-
tion has not been elucidated. It is probable that subsequent
to binding of GadX/W to the gadA/BC promoters, coopera-
tion with GadE is still required for full activation of the Gad
system (Sayed et al. 2007). Moreover, the mechanism of
GadX/W/E activation maybe different depending on the en-
Fig. 3. X-ray structures of the acid stress induced amino acid decarboxylases, antiporters, and chaperones. The domains of the decarboxy-
lases and chaperones are colour-coded according to Fig. 2. The structures of E. coli GadA (PDB ID:1XEY (Dutyshev et al. 2005)), E. coli
GadB (1PMO, inactive form at pH 7.6; 1PMM, active form at pH 4.6 (Capitani et al. 2003)), E. coli AdiA (2VYC (Andre ´ll et al. 2009)),
E. coli LdcI (EM image (Snider et al. 2006)), and Lactobacillus 30a OrnDC (1ORD (Momany et al. 1995)) are displayed in the same
column for comparison. The X-ray structures of the E. coli AdiC antiporter [3H5M (Gao et al. 2009)] and E. coli ClC Cl–channel (1KPK
(Dutzler et al. 2002)) are also shown. Escherichia coli HdeA (1DJ8 (Gajiwala and Burley 2000)) and Hsp31 (1ONS (Zhao et al. 2003)) are
shown in their physiological dimeric form. (Note: the full-colour versions of Figures 2 and 3 are available from http://bcb.nrc.ca.)
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