JBA-06947; No of Pages 9

Biotechnology Advances xxx (2015) xxx–xxx

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Biotechnology Advances

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Mechanisms of acid tolerance in bacteria and prospects in biotechnology

and bioremediation
Yuping Liu a,b, Hongzhi Tang a,b,⁎, Zhanglin Lin c, Ping Xu a,b,⁎
a
State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China
b
School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China
c
Department of Chemical Engineering, Tsinghua University, One Tsinghua Garden Road, Beijing 100084, People's Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Acidogenic and aciduric bacteria have developed several survival systems in various acidic environments to
Received 24 July 2014 prevent cell damage due to acid stress such as that on the human gastric surface and in the fermentation medium
Received in revised form 2 June 2015 used for industrial production of acidic products. Common mechanisms for acid resistance in bacteria are proton
Accepted 2 June 2015
pumping by F1–F0–ATPase, the glutamate decarboxylase system, formation of a protective cloud of ammonia,
Available online xxxx
high cytoplasmic urease activity, repair or protection of macromolecules, and biofilm formation. The field of
Keywords:
synthetic biology has rapidly advanced and generated an ever-increasing assortment of genetic devices and
Acid resistance biological modules for applications in biofuel and novel biomaterial productions. Better understanding of aspects
F1–F0–ATPase such as overproduction of general shock proteins, molecular mechanisms, and responses to cell density adopted
Glutamate decarboxylase by microorganisms for survival in low pH conditions will prove useful in synthetic biology for potential industrial
Macromolecule and environmental applications.
Synthetic biology © 2015 Elsevier Inc. All rights reserved.

1. Introduction modifications. Strains such as Escherichia coli, Salmonella enterica,

Bacteria play important roles in the industrial production of
various products, in human health, and in environmental gover-
nance. In order to survive sudden, potentially lethal challenges,
microorganisms must possess effective mechanisms against various
environmental stresses such as antibiotics, acids, organic solvents,
and heat (Winfield and Groisman, 2003). Among these, an acidic
condition is the most common condition encountered by several
bacteria; therefore, biologists have focused a lot of attention on
acid resistance in microorganisms. Acid mine drainage causes
serious environmental problems (Akcil and Koldas, 2006), strongly
affecting routine human life. Several environmental pollutants such
as mine drainage basins contaminated with polycyclic aromatic
hydrocarbons (PAHs) are acidic in nature, owing to the utilization
of HCl or H2SO4. Moreover, industrial production of products such
as lactic acid (Michelson et al., 2006; Zhang et al., 2014) and amino
acids (Shih and Van, 2001) have limiting acidic conditions such as a
low pH environment, which negatively influence the growth of
microorganisms. It is therefore mandatory to add chemicals such
as calcium carbonate to neutralize the protons of acids to increase
the pH in the medium. It is important to understand the acid-
resistance mechanisms (ARMs; the survival systems used in
acidic conditions) in microorganisms to determine their adaptive
⁎ Corresponding authors.
E-mail addresses: tanghongzhi@sjtu.edu.cn (H. Tang), pingxu@sjtu.edu.cn (P. Xu).
and Shigella flexneri are highly resistant to low pH and can survive
in acidic conditions of the mammalian stomach as well, which can
decrease rapidly to an approximate pH of 2.0 (Kanjee and Houry,
2013; Spector and Kenyon, 2012; Waterman and Small, 2003).
These microorganisms can remain viable for several hours at a pH
as low as 2.5 in a stationary phase, mainly owing to their ARMs
(Waterman and Small, 2003). The two general ARMs adopted by
microbes for maintaining their pH homeostasis during growth are
as follows: i) use of H+ antiport systems such as H+-ATPase activity,
acid end-product efflux, and decreased proton permeability to main-
tain a low intracellular concentration of protons; and ii) synthesis of
alkali products to neutralize acid generated during extracellular
metabolism. Several bacteria have evolved diverse resistance or tol-
erance mechanisms against the normally lethal pH values of ≤ 2.5,
mainly through acid-tolerance responses (ATRs), which provide
the ability to sense, respond and adapt to an acidified environment,
and ARMs (Spector and Kenyon, 2012). In addition, Pseudomonas
aeruginosa (Williams and Camara, 2009) and Streptococcus mutans
(Li et al., 2001) form dense biofilms to protect cells against extracellular
acid shock.
In this article, we review some model acid-resistant bacteria and
their related ARMs (Fig. 1). Breakthroughs in genetic engineering and
genomic research related to acid-resistance systems of microorganisms
can supply genetic devices and biological modules for applications in
industrial biotechnology and bioremediation. In addition, promising
applications of ARMs in synthetic biology are discussed.

http://dx.doi.org/10.1016/j.biotechadv.2015.06.001
0734-9750/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Liu, Y., et al., Mechanisms of acid tolerance in bacteria and prospects in biotechnology and bioremediation, Biotechnol
Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.06.001
2 Y. Liu et al. / Biotechnology Advances xxx (2015) xxx–xxx

Fig. 1. Common acid-resistance mechanisms in microorganisms (partly adapted, with permission, from Matsui and Cvitkovitch, 2010). A: Gad system; gadA/B encode glutamate
decarboxylase GadA/B, which convert Glu to GABA, and GadC (encoded by gadC), which acts as the Glu/GABA antiporter (Capitani et al., 2003). B: Biofilm and cell density; comCDE
(Li et al., 2001) and luxS (Wen and Burne, 2004) are involved in a quorum sensing system that is essential for biofilm formation, and the las system is necessary for the exogenous Pseu-
domonas quinolone signaling molecule to stimulate biofilm formation (Williams and Camara, 2009). C: F1–F0–ATPase; as a proton pump, this complex pumps H+ out to increase intracel-
lular pH. D: Protection of macromolecules; RecA (Van der Veen and Abee, 2011) and UvrA (Croteau et al., 2008), as well as AP endonuclease (Hahn et al., 1999), are involved in DNA repair;
DnaK (Tomoyasu et al., 1998) is a protein repair chaperone, and IrrE (Earl et al., 2002) is a global regulatory protein that can stimulate recA transcription. E: Alkali production; urea is trans-
formed into NH3 by urease to neutralize protons (Maroncle et al., 2006); The Adi and Agd systems can also produce NH3 through several reactions (Griswold et al., 2004). Glu: glutamate;
Adc: arginine decarboxylase; Agd: agmatine deiminase; Ptc: putative putrescine transcarbamylase; Adi: arginine deiminase; Otc: ornithine transcarbamylase; Ck: carbamate kinase.

2. Common mechanisms of acid resistance
Microorganisms developed various effective mechanisms to
survive the acidic environment, among which the most common
mechanisms are the Gad system, biofilm formation and cell density,
the F1–F0–ATPase proton pump, protection or repair of macromolecules
and alkali production. We discuss these acid resistance mechanisms in
detail below.
2.1. The glutaminase and Gad system
The Gad-dependent acid-resistance system is present in a variety
of bacteria such as E. coli (Kanjee and Houry, 2013), S. flexneriis
(Waterman and Small, 2003), Listeria monocytogenes (Cotter et al.,
2005), and Lactobacillus reuteri (Su et al., 2011). The Gad system plays
important roles in the ARMs of these strains (Capitani et al., 2003; Su
et al., 2011; Teixeira et al., 2014). In addition, the acid resistance system
in E. coli relies on L-glutamine (Gln), which is converted to L-glutamate
(Glu) by the acid-activated glutaminase YbaS, with concomitant release
of dissolved ammonia. YbaS and the amino acid antiporter GadC are suf-
ficient for E. coli survival in extremely acidic environments (Lu et al.,
2013). In general, the Gad system requires one or two Gad enzymes
(pyridoxal phosphate-dependent Gad: GadA and GadB) as structural
constituents and one Glu/γ-aminobutyrate (GABA) antiporter (GadC)
(Fig. 1), which is comprised of 12 transmembrane segments (Ma et al.,
2012). The Gad system is assumed to catalyze the conversion of
protonated Glu to GABA, whereas the antiporter exports GABA in ex-
change for a new extracellular Glu molecule (Fig. 2). At intracellular
pH below 4.25, uncharged glutamate is the main ion species and
glutamine deamidation consumes one intracellular proton (Feehily
and Karatzas, 2012; Teixeira et al., 2014). This process consumes
protons in cells, which eventually increases the intracellular pH,
protecting the cell from the damage caused by acid shock.
If the extracellular proton concentration abruptly increases to a
lethal level, the intracellular pH becomes lower. Then, GadC is activated
and selectively exchanges Glu− or Glu0 with GABA+ by switching
between its outward and inward-open conformations, resulting in an
effective proton extrusion of N0.9 H+ per turnover to counter proton in-
vasion into acid-challenged bacteria (Ma et al., 2012; Tsai et al., 2013).
Then, Glu is protonized and transformed into GABA intracellularly by
GadA or GadB (Capitani et al., 2003).
The by-product of this progress, GABA, is exported via the antiporter
GadC along with another Glu molecule that is pumped into the cell
(De Biase and Pennacchietti, 2012). In this reaction, one proton is con-
sumed to eventually yield a nearly neutral intracellular pH. In E. coli,
gadA exists in the acid fitness island (AFI), which comprises 14 genes
contributing to acid resistance (De Biase and Pennacchietti, 2012).
Among these genes, gadX and gadW encode two regulators of acid resis-
tance. GadX is an AraC-like regulator that activates the expression of
gadA and gadBC at any pH, whereas GadW inhibits the reaction by
repressing gadX. When GadX is absent, GadW functions as the gadA
and gadBC activator (Ma et al., 2002). The putative regulatory protein
YhiF, lipoprotein Slp, and the periplasmic chaperone HdeA, all encoded
by genes in the AFI, protect E. coli from organic acid metabolites
produced during fermentation once the external pH is reduced to 2.5.
Two other genes, yhiD and hdeD, encode putative membrane proteins

Please cite this article as: Liu, Y., et al., Mechanisms of acid tolerance in bacteria and prospects in biotechnology and bioremediation, Biotechnol
Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.06.001
 Y. Liu et al. / Biotechnology Advances xxx (2015) xxx–xxx 3

330-kDa hexamer GadB has been determined at acidic and neutral

pHs, and determination of the structural change due to acidic
pH provides a complete description of its intracellular activity
(Capitani et al., 2003).
The Gad system plays important roles in several microorganisms and
can ensure survival of the bacteria in extremely acidic environments.

2.2. Biofilm and cell density

As biofilm formation offers the cells an important strategy for surviv-

Fig. 2. The principle of the Gad system (partly adapted, with permission, from De Biase and
Pennacchietti, 2012). When cells are under low pH, the intracellular Glu (0) is catalyzed to
GABA (+) by GadA/GadB, with the consumption of one proton. Then, GABA (+) is
transported out in exchange for a molecule of Glu (0) by GadC, acting as a transporter
(Teixeira et al., 2014; Tsai et al., 2013). At intracellular pH below 4.25, uncharged
glutamate is the main ion species and glutamine deamidation consumes one intracellular
proton (Feehily and Karatzas, 2012; Teixeira et al., 2014).
only at high cell densities (Mates et al., 2007). The gadA gene is
usually cotranscribed with gadX, while gadB is constantly cotranscribed
with gadC (Tramonti et al., 2002) (Fig. 3). The crystal structure of the
al in various harmful environments (Hall-Stoodley and Stoodley, 2009),
several bacteria in the environment grow as a biofilm or communities
that are surface-attached (Fig. 1). Among all the ARMs in bacteria, the
formation of biofilms is the most interesting as it involves cell-to-cell
communication (Li et al., 2001). Biofilms of some organisms show
strong resistance to low pH, while those of planktonic cells are extreme-
ly acid-sensitive (McNeill and Hamilton, 2003), which explains why
certain proteins with unknown functions present in these organisms
are absent in planktonic cells. In addition, starvation can induce resis-
tance of S. mutans to acid shock, implying that oral bacteria may be
more resistant to acid stress between meals as they can form a biofilm
during this period (Zhu et al., 2001).
Adhesion to a surface is essential for the development of acid
resistance in biofilm cells, and the presence of fluoride can inhibit the
induction of ATR in S. mutans (Welin-Neilands and Svensater, 2007).
At the same time, biofilm formation was shown to be inhibited by
salicylic acid, which acts as a competitive analog to the LasR regulator
in P. aeruginosa (Williams and Camara, 2009). As a biofilm can protect
cells against extracellular acid shock, living cells are usually observed
in the innermost part of the biofilm. Although biofilm formation gener-
ally enhances the survival ability of microorganisms, it ceases to be
effective in the presence of internal proton shock due to production
of acidic metabolites during cell growth or other metabolic activities

Fig. 3. The Gad system in different microorganisms. The genes encoding glutamate decarboxylase are gadA (located in the acid fitness island) and gadB. A: Usually, gadB is located upstream of
the antiporter-encoding gene gadC, whereas in some other organisms, the locus is located downstream. B: The acid fitness island comprises gadA, the regulator encoding genes gadX and gadW,
the putative regulatory protein, lipoprotein and periplasmic chaperone encoding genes yhiF, slp and hdeA, separately, as well as the putative membrane protein-encoding genes yhiD and hdeD.

Please cite this article as: Liu, Y., et al., Mechanisms of acid tolerance in bacteria and prospects in biotechnology and bioremediation, Biotechnol
Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.06.001
4 Y. Liu et al. / Biotechnology Advances xxx (2015) xxx–xxx
(Takahashi and Yamada, 1999). In S. mutans BM71, formation of biofilm
has been proven to modulate the acid adaption of the log-phase ATR
because pre-adapted cells from a dense biofilm show significantly
higher resistance to lethal pHs as compared with cells at a lower cell den-
sity; furthermore, the log-phase ATR could be induced by a neutralized
culture filtrate collected from a low-pH culture, indicating that the culture
filtrate of S. mutans possesses some extracellular induction components
related to acid adaptation. Moreover, mutant defective experiments
showed that the genes comC, comD, and comE (encoding a precursor to
the competence stimulating peptide, a histidine kinase, and a response
regulator, respectively), in the quorum-sensing system of S. mutans,
play important roles in the biofilm-dependent log-phase ATR (Fig. 1)
(Li et al., 2001). LuxS is also involved in biofilm formation of S. mutans
(Wen and Burne, 2004). In addition, biofilm formation of P. aeruginosa
is controlled by the quorum-sensing mechanism involved in complicated
cell-to-cell communications (Williams and Camara, 2009).
2.3. F1–F0–ATPase proton pump
The multisubunit enzyme F1–F0–ATPase encompasses a hydrophilic
enzyme (F1) composed of α, β, γ, δ, and ε subunits, as well as a hydro-
phobic membrane channel (F0) composed of a, b, and c subunits. The
F1 protein catalyzes intracellular ATP hydrolysis or synthesis, whereas
the F0 protein plays an important role in proton translocation (Fig. 1).
These mechanisms are utilized by various bacteria to regulate their
cytoplasmic pH and are operated efficiently by hydrolyzing ATP to
pump out H+ from cells, thereby maintaining the pH homeostasis
and protecting cells from damage induced by an acidic environment.
In several microorganisms, transcription of the F1–F0 operon is
induced by exposure to acidic pH, including Streptococcus pneumoniae
(Martin-Galiano et al., 2001) and Lactobacillus acidophilus (Kullen and
Klaenhammer, 1999). Moreover, the F1–F0–ATPase was found to be
transcriptionally upregulated (Kuhnert et al., 2004) in S. mutans
confronted with an acidic environment, suggesting its critical role in
acid resistance.
2.4. Protection or repair of macromolecules
The stability of membrane protein oxidases is correlated with
acid resistance of bacterial strains (Trcek et al., 2006). This strongly
implies that macromolecular stability is an important selective trait for
acidophiles. Researchers (Francois and Kappock, 2007; Mullins et al.,
2012) showed that there are similar acid stability enhancements in
cytoplasmic proteins that increase acid resistance without energy
input (e.g., proton pumping). Thus, in the optimal acid-resistance
system, the presence of a variety of macromolecules such as DNA and
proteins is essential (Fig. 1). In acid stress conditions, macromolecules
such as those in Dps and RecA systems may get damaged or lose their
function; therefore, it is essential to protect or repair these macromole-
cules after exposure to such challenging conditions. The association be-
tween the repair of DNA and acid adaptation exists in several organisms.
The DNA-binding protein from starved cells (Dps) was described as
a DNA-binding protein with regulatory and protective roles in E. coli.
The Dps system could provide multifaceted protection such as DNA
binding, iron sequestration, iron and hydrogen peroxide detoxification
through its ferroxidase activity, and resistance to an acidic environment
(Calhoun and Kwon, 2010).
RecA plays a pivotal role in biological processes that require
homologous DNA repair and recombination as well as an SOS response
(Adikesavan et al., 2011). RecA-mutant strains of Helicobacter pylori
proved to be sensitive to DNA-damaging agents and showed reduced
homologous gene conversion related to outer membrane protein ex-
pression, resulting in a reduced survival capacity in acidic environments
(Amundsen et al., 2008; Thompson and Blaser, 1995). This suggests the
existence of a RecA-dependent acid-tolerance system. Mutagenesis and
biochemical analysis of Bacillus caldontenax UvrA showed that UvrA and
Please cite this article as: Liu, Y., et al., Mechanisms of acid tolerance in bacteria and prospects in biotechnology and bioremediation, Biotechnol
Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.06.001
UvrB played important roles in DNA cooperative damage recognition
(Croteau et al., 2008). A recent study showed that the LuxS-mediated
quorum sensing played an important part in the regulation of
physiological functions in several bacteria, and LuxS-deficient
mutants of S. mutans were more susceptible to acid shock; in
addition, the expression of RecA, SmnA (AP endonuclease), and
Nth (endonuclease) was downregulated in mutant strains (Wen and
Burne, 2004). AP endonuclease, which is independent of RecA, was
found to be essential for S. mutans in acid adaptation, and its function
was similar to that of DNA repair enzymes in other organisms (Hahn
et al., 1999). The ultraviolet (UV) excinuclease gene (uvrA), associated
with low-pH adaptation, was shown to be involved in the repair of
acid-induced DNA damage in S. mutans, because the S. mutans uvrA
mutant was more sensitive than the wild-type strain growing at a pH
of 5.0 (Hanna et al., 2001). Proteins like DnaK from E. coli (Zou et al.,
1998) and IrrE from Deinococcus radiodurans (Earl et al., 2002) ensure
proper functioning of the DNA repair mechanisms, instead of directly
protecting the bacteria against acid-induced damage.
As macromolecules play an important role in acid-resistant
microorganisms, several repair or regulatory proteins involved in
the production or repair of antacid-related macromolecules may be
beneficial to protect organisms against acid-induced damages.
2.5. Alkali production
Some microorganisms produce alkaline compounds to neutralize
acid against an acidic environment. There are two major substrates for
alkali generation, urea and arginine. Urea is rapidly hydrolyzed to
ammonia and CO2 by ureases, while arginine is catabolized to ornithine
with the release of ammonia and CO2. Urease and arginine deiminase
(Adi) or arginine dihydrolase system (Ads) plays important roles to
obtain alkali, commonly in the form of ammonia (Fig. 1). When exposed
to an acidic environment, cells produce ammonia, which combines with
the intracellular protons to neutralize the internal pH (Burne and
Marquis, 2000; Griswold et al., 2004).
The urease system consisting of the ureIABCEFGD operon is more
widespread and can protect some oral bacteria such as L. reuteri against
acid-induced damages (Cotter and Hill, 2003; Wilson et al., 2014).
The nickel-containing enzyme urease catalyzes the reaction of urea
hydrolysis, yielding ammonia and carbon dioxide (Fig. 1), in which the
ammonia molecule plays a proton-consuming role in the cell (Maroncle
et al., 2006; Scott et al., 1998; Sissons et al., 1990). Specifically,
Streptococcus salivarius generates a large quantity of urease, with
100-fold higher activity at pH 5.5 than at neutral pH (Sissons et al.,
1990). In addition, the amount of urease produced can be determined
by the regulation of the growth pH (Cotter and Hill, 2003). In H. pylori,
the metabolism range of pH was 3.5–8.6. Owing to the pH-regulated
urease system, the metabolism pH can extend to a larger range up to
pH 2.5 (Rektorschek et al., 1998). The gene ureI in H. pylori and
S. salivarius encodes a membrane-bound, H+-gated urea transporter
that can facilitate uptake of urea in acidic environment. H. pylori urease
exists not only in the cytoplasm but also in the outer surface of the cell
(Scott et al., 2002), facilitating better efficiency of the mechanism on
exposure to acid stress.
The Adi or Ads pathway is involved in cellular protection against acid
stress, and provides ATP for microbial growth (Burne and Marquis,
2000; Lu, 2006). The system functions by the generation of ammonia
and carbon dioxide through the breakdown of arginine, which could
function at a pH below the minimal pH required for growth of
L. reuteri (Rollan et al., 2003). The genes encoding for the Ads were
cloned and identified from S. sanguis, and it was shown that this highly
acid-tolerant pathway is mainly comprised of three enzymes, encoded
by the genes arcA, arcB and arcC (Burne et al., 1989). During the
first step, arginine is converted to citrulline with the production of
by-product NH3 catalyzed by Ads (encoded by arcA); then, citruline is
transformed into ornithine and carbamyl phosphate by ornithine
 Y. Liu et al. / Biotechnology Advances xxx (2015) xxx–xxx
transcarbamylase (encoded by arcB); and, finally, carbamyl phosphate
is catabolized to generate ammonia, carbon dioxide, and ATP by
carbamate kinase ArcC (Lu, 2006) (Fig. 1). However, the system is
not always active; it is stimulated through energy reduction of the
cells in the stationary phase or by adaptation of exponentially growing
cells of L. reuteri under acidic conditions (Rollan et al., 2003). The gene clus-
ter from Streptococcus gordonii DL1 has six open reading frames. Compared
with the wild-type and ArcR-deficient strains of S. gordonii DL1, ArcR acts
as a transcriptional regulator necessary for the expression of the gene clus-
ter (Dong et al., 2002). Regulation is contributed by multiple factors, in-
cluding the transcriptional regulator ArgR, the alternative stress sigma
factor σB, and the central virulence regulator PrfA (Gruening et al., 2006;
Ryan et al., 2009). In addition, the arcABC operon contributes greatly to
the growth and survival of Streptococcus suis under acid stress (Gruening
et al., 2006). The system could be induced by low pH or arginine and is sup-
pressed by excess oxygen pressure (Ryan et al., 2009). Furthermore, two
pathways provide acid tolerance in the same genomic region in Lactobacil-
lus brevis, including the agmatine deiminase (Agd) (Fig. 1) and tyrosine
catabolic pathways (Lucas et al., 2007).
Another promising research area is malolactic fermentation by the
strain S. mutans and Oenococcus oeni. During the process, an acid
commonly found in fruits, can contribute to the alkalinization of the
cytoplasm relative to the environment through the production of CO2
and possible diffusion of CO2 itself out of the cytoplasm (Sheng and
Marquis, 2007). In addition, the cell membrane plays a crucial role in
survival of bacteria and archaea in extreme environments. Transport
of solutes across the membrane is mainly catalyzed by primary ATP
driven transport systems or by proton or sodium motive force driven
secondary transport systems (Konings et al., 2002; Salema et al., 1996).
In summary, alkali production mechanisms are efficient for the
survival of bacteria under different acidic conditions.
3. Bacteria possessing specialized acid survival strategies
In order to survive in a highly acidic environment, several bacterial
strains such as S. mutans (Griswold et al., 2004), Streptococcus pyogenes
(Cusumano and Caparon, 2015), S. enterica (Spector and Kenyon, 2012),
L. monocytogenes (Davis et al., 1996), acetic acid bacteria (Nakano and
Fukaya, 2008), H. pylori (Stingl et al., 2002), and E. coli (Kanjee and
Houry, 2013) have developed various ARMs.
S. mutans was first discovered in 1924 (Clarke, 1924); it is an
acidogenic bacterium that is highly efficient in producing acid from
fermenting carbohydrates within a wide pH range, and is also the
major causative agent in dental plaque (Len et al., 2004). S. mutans
uses adaptive mechanisms to withstand the fluctuating pH conditions
in fermentation media. In acidic environments, the intracellular cyto-
plasm may become acidified, leading to structural damage of proteins
or DNA molecules involved in the maintenance of basic functions
(Matsui and Cvitkovitch, 2010). It has been reported that increased syn-
thesis of protein-repair chaperone DnaK (Jayaraman et al., 1997), has
many effects including increased expression of the eukaryotic signal
recognition particle gene Ffh (Gutierrez et al., 1999), generation of am-
monia by the Agd system (Griswold et al., 2004) and Adi pathway
(Matsui and Cvitkovitch, 2010), increased amino acid metabolism
(Santiago et al., 2012), alterations in metabolic pathways (Lemos and
Burne, 2008), induction of H+-ATPase (Kuhnert et al., 2004), upregula-
tion of DNA damage regulatory-repair protein RecA, and upregulation of
alkaline phosphatase (AP) endonuclease activity (Hahn et al., 1999), all
of which may be related to the repair of acid-induced cellular damage in
S. mutans. Moreover, S. mutans can also endure highly acidic environ-
ments by forming a biofilm, which is an important microbial survival
strategy. Biofilm cells have lower acid susceptibility as compared
with planktonic cells (Welin-Neilands and Svensater, 2007); studies
conducted in a chemostat showed that the ATRs of biofilm cells resulted
in higher resistance to a lethal pH of 3.5 as compared to those of
planktonic cells (McNeill and Hamilton, 2003). Proteomic analysis of
Please cite this article as: Liu, Y., et al., Mechanisms of acid tolerance in bacteria and prospects in biotechnology and bioremediation, Biotechnol
Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.06.001
5
planktonic and biofilm cells of S. mutans showed that 57 proteins
were 1.3-fold upregulated in the biofilm system, which may contribute
to its acid resistance under low pH conditions (Svensater et al., 2001).
In addition, S. mutans can prolong survival with the induction of both
fructans and glucans. These compounds may be used as carbon and en-
ergy sources or they may somehow play a protective role, preventing
the cells from being damaged during survival (Busuioc et al., 2009).
In S. pyogenes, the major defense mechanism against acid stress is
the arginine deiminase (ADI) pathway, which catabolizes arginine to
generate two ammonia molecules and one molecule of ATP from acid
stress. During the protection provided by arginine utilization, the
protection provided by citrulline catabolism was pH independent,
requiring the generation of ATP via the ADI pathway and a functional
F1–F0–ATP synthase (Cusumano and Caparon, 2015).
S. enterica is a facultative intracellular pathogen with an outstanding
ability to cope with extreme environments both in its natural habitats
and within an infected host. It encounters the acidic pH of the stomach
during host infection, indicating strong endurance in the face of high
acid concentrations. The ARMs of S. enterica are highly influenced by
the stage of growth and the presence of certain amino acids such as
arginine and lysine. The systems involved are mainly ATRs and ARMs
(Spector and Kenyon, 2012). The arginine–agmatine antiporter and
decarboxylase in extreme acid resistance were also found in E. coli
(Iyer et al., 2003; Lin et al., 1996). By exposing exponential-phase cells
to a sublethal pH, it was shown that the cells can withstand a pH of
approximately 3.0 (adjusted by HCl), which is lethal for S. enterica
(Tiwari et al., 2004). Cells with log-phase ATRs have enhanced acid
tolerance as compared with non-adapted cells. In addition, the amino
acid-dependent ARMs may play vital roles in S. enterica. The genes
cadA, cadB, and adiA, encoding lysine decarboxylase, lysine–cadeverine
antiporter, and arginine decarboxylase, respectively, have been associ-
ated with ARMs. Pre-adapting cells with arginine or lysine at pH 4.5,
followed by challenging with a pH of 2.5 (adjusted by HCl) increased
the survival rates of the cells as compared with those of non-adapted
cells (Alvarez-Ordonez et al., 2010). Moreover, RpoS, the primary
sigma factor, is responsible for the expression of stress resistance related
genes and has accordingly been described as the master regulator of the
general stress response in Salmonella and E. coli (Klauck et al., 2007;
Loewen and Hengge-Aronis, 1994; Wilson et al., 2002). The gene ydcI,
being part of the rpoS regulon involved in stress resistance, is necessary
for acid resistance in S. enterica. In addition, the gene ydcI (encoding a
DNA-binding protein) is conserved in various gram-negative bacteria;
when induced in S. enterica, it can form biofilms. The ydcI-mutant strain
displayed increased sensitivity to both organic and inorganic acid
attacks (Jennings et al., 2011). In S. enterica, cells will induce their fatty
acids composition modifications and tolerance when it entered the
stationary phase, which will help them to overcome nonoptimal condi-
tions such as in acid (Dubois-Brissonnet et al., 2011). The mechanism
was also found in E. coli, and membrane cyclopropane fatty acid content
is a major factor in acid resistance (Chang and Cronan, 1999).
L. monocytogenes, a food-borne pathogen, shows several fatal
outbreaks when confronted with harsh survival conditions, especially
acidic stress (Cotter and Hill, 2003). Owing to its pathogenic lifestyle,
this organism must develop a variety of mechanisms to rapidly adjust
to the adverse environments. The glutamate decarboxylase (Gad)
system (Karatzas et al., 2012), operon encoding F0–F1–ATPase (Cotter
et al., 2000), arginine deiminase (Adi) system (Ryan et al., 2009), DNA
repair system, activator of the SOS response (RecA) (Van der Veen and
Abee, 2011), and the gene encoding for the nucleotide excision repair
pathway (uvrA) (Kim et al., 2006) have been reported to be important
for pH homeostasis in L. monocytogenes.
Acetic acid bacteria are of great significance in the industrial produc-
tion of vinegar owing to their ability to ferment ethanol and their signif-
icant resistance to acetic acid. Acetic acid bacteria such as Acetobacter
have complex mechanisms to cope with severe conditions of acetic
acid, including the citric acid cycle-related aarABC (Fukaya et al., 1990)
6 Y. Liu et al. / Biotechnology Advances xxx (2015) xxx–xxx

and aconitase (acn) genes (Nakano et al., 2004) and the aatA gene
encoding a putative ABC transporter (Nakano et al., 2006). The aarA
gene encodes citrate synthase (Francois et al., 2006), aarB encodes
a protein upregulating the TCA cycle (later named to sixA (Mullins
et al., 2008)), and aarC encodes an enzyme catalyzing the reaction of
succinyl-CoA and acetate to form succinate and acetyl-CoA (Mullins
et al., 2008). In addition, the typical stress proteins GroES and GroEL
(Okamoto-Kainuma et al., 2002) and a proton motive force-dependent
efflux system (Matsushita et al., 2005) also contribute to the acetic
acid resistance of Acetobacter. The ethanol oxidation-related enzymes
play important roles in acetic acid resistance, as pyrroloquinoline
quinone-dependent alcohol dehydrogenase enables some acetic acid
bacteria to grow in a medium containing high concentrations of acetic
acid (Trcek et al., 2006).
H. pylori, a urease-producing pathogenic bacterium, can survive for
several hours at pH 1 (Stingl et al., 2002). The cytoplasmic ammonia
generated by enzyme-catalyzed urea decomposition produces alkali to
protect the cells from an inorganic acidic environment (Ha et al.,
2001). In addition, an acid resistance-related two-component regulator
system has been reported in the genome sequence of probiotic bacteria
L. acidophilus NCFM, which protects the bacteria from acid stress
(Azcarate-Peril et al., 2005). Moreover, complicated acid-resistance sys-
tems in E. coli have efficient Adi and Gad systems (Kanjee and Houry,
2013). An acid-activated glutaminase YbaS, which catalyzes the
reaction, that converts Gln to Glu and releases ammonia to neutralize
H+, also contributes greatly to acid resistance in E. coli (Lu et al., 2013).
In conclusion, some bacteria probably employ more than one ARM
for their survival in acidic environments; these mechanisms efficiently
work together to ensure proper metabolism and growth of the microbe
in hostile conditions. Considering the challenging living conditions in
nature, bacteria have developed several types of biological systems to
adapt to acid stress. Knowledge about these mechanisms will definitely
contribute further insights into detection of other stress-tolerant
microorganisms and their survival mechanisms.
4. Promising applications in synthetic biology
Synthetic biology has enormous potential to generate biological
modules of unprecedented applications by combining the basic ele-
ments of biology (Khalil and Collins, 2010). Of the basic elements in
microorganisms that may contribute greatly to synthetic biology,
acid-resistant components or ARMs can find great utilization in
industrial bioprocesses and environmental pollution treatments. Lactic
acid fermentation during industrial production creates an acidic (lactic
acid fermentation) condition in the fermentation medium, which
indicates that lactic acid bacteria possess mechanisms to withstand
the severe habitat during fermentation. Acid-resistant systems in lactic
acid bacteria include pH homeostatic and macromolecular repair and
protection mechanisms (Cotter and Hill, 2003), as well as the Gad sys-
tem (Kim et al., 2007) and a glutamine-dependent acid resistance
mechanism in L. reuteri (Teixeira et al., 2014). In addition, industrial pro-
duction of acetic acid and butyric acid also demand the fermentative
bacteria to be highly resistant to acidic environments (Nakano and
Fukaya, 2008; Wu and Yang, 2003), necessitating the development
of natural and artificial genetic devices that can aid cells to deal with
acid stress. Apart from the industrial applications, research on acid

Table 1
Common acid-resistance genes in microorganisms.

Acid resistance-related Microorganism Function Reference

gene

Glutamate decarboxylase system

gadA/BC Escherichia coli In the Gad system, GadA/B converts glutamate to GABA, De Biase and Pennacchietti (2012), Su et al. (2011),
Lactobacillus reuteri with the consumption of one proton; GadC transports Waterman and Small (2003), Cotter et al. (2005)
Shigella flexneriis out GABA in exchange of one molecule of glutamate.
Listeria monocytogenes

Biofilm and cell density

las system Pseudomonas aeruginosa Essential for exogenous Pseudomonas quinolone signal Williams and Camara (2009)
molecule to stimulate biofilm formation
comC/D/E operon Streptococcus mutans Encodes a quorum-sensing system essential for cell Li et al. (2001)
biofilm formation
luxS Streptococcus mutans LuxS-mediated quorum sensing-regulated biofilm Wen and Burne (2004)
formation and stress tolerance

F1–F0–ATPase proton pump

F1–F0–ATPase Streptococcus mutans Pumps out protons from cells to maintain a relatively Kuhnert et al. (2004), Kullen and Klaenhammer (1999)
Lactobacillus acidophilus neutral intracellular pH

Protection or repair of macromolecules

recA Listeria monocytogenes Important factor in DNA repair and the activator of the Van der Veen and Abee (2011), Amundsen et al. (2008),
Helicobacter pylori SOS response Hahn et al. (1999)
Streptococcus mutans
uvrA Bacillus caldontenax Repair of DNA damage during the process of Croteau et al. (2008), Hanna et al. (2001), Kim et al. (2006)
Streptococcus mutans nucleotide-excision repair
Listeria monocytogenes
irrE Deinococcus radiodurans As a novel regulatory protein, IrrE stimulates recA Earl et al. (2002)
transcription
dnaK Escherichia coli A highly sensitive stress sensor that controls the Tomoyasu et al. (1998), Jayaraman et al. (1997)
Streptococcus mutans expression of heat shock genes directly in response to
protein misfolding

Alkali production
arcABC Lactobacillus reuteri In the Adi system, enzymes encoded by arcABC convert Rollan et al. (2003), Burne et al. (1989), Gruening et al. (2006)
Streptococcus sanguis arginine to CO2 and ammonia.
Streptococcus suis
ureABIEFGH Helicobacter pylori Urease gene cluster. Urease-catalyzed ureolysis, yielding Scott et al. (2002), Wilson et al. (2014) Sissons et al. (1990)
Lactobacillus reuteri ammonia to neutralize protons
Streptococcus salivarius

Please cite this article as: Liu, Y., et al., Mechanisms of acid tolerance in bacteria and prospects in biotechnology and bioremediation, Biotechnol
Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.06.001
 Y. Liu et al. / Biotechnology Advances xxx (2015) xxx–xxx
components may also benefit environmental pollutant treatment, such
as that of polycyclic aromatic hydrocarbons PAHs polluted areas. PAHs
are common contaminants present in extremely acidic environments
such as mine drainage basins and petroleum-polluted areas.
Biodegradation of PAHs can be efficiently conducted by acidophilic
bacteria (Stapleton et al., 1998), which have complicated ARMs. Several
common acid-resistance related genes and mechanisms exist in a mi-
croorganism (Table 1), and introduction of artificially manufactured
acid-resistant components into environmental organisms such as
Pseudomonas putida B6-2 (Tang et al., 2011) and P. aeruginosa DQ8
(Zhang et al., 2011) or into industrial bacteria such as the lactic acid pro-
ducing bacteria Bacillus coagulans (Xu and Xu, 2014; Zhang et al., 2014)
and Lactobacillus delbrueckii (Michelson et al., 2006) and the acetic acid
bacterium Acetobacter aceti (Ory et al., 2004) would definitely be condu-
cive to future studies on improving the acid-contaminated environment
as well as in industrial production. Therefore, stress-tolerant microor-
ganisms have great potential for applications in synthetic biology.
5. Summary and discussion
Bacteria have developed several distinct strategies to survive in
acidic environments. The ARMs adopted by bacteria include pumping
out of protons, generation of alkali, protection of macromolecules, and
formation of biofilms. In addition, these mechanisms are basically
regulated by diverse environmental factors, of which sensing of mild
acidification is the most efficient as it allows the organisms to adapt to
lethal stress (Cotter and Hill, 2003). There are some examples for suc-
cessful engineering of improved acid resistance in biotechnologically
important bacteria. Heterogeneous expression of dnaK from E. coli in
L. lactis NZ9000 improved its stress tolerance (towards lactic acid, salt,
ethanol and heat) and lactic acid fermentation efficiency (Abdullah Al
et al., 2010). Owing to the expression of laboratory-evolved mutants
of irrE from D. radiodurans, Chen et al. enhanced multiple stress
tolerances of E. coli, including tolerance of ethanol, butanol, acetate,
and inorganic acid (Chen et al., 2011). Through engineering the global
regulator cAMP receptor protein, it was shown that it had successfully
improved the tolerance of E. coli towards various stresses (Chong
et al., 2013). By heterogeneously expressing acid tolerance-related
genes and mechanisms, we can surely enhance acid tolerance of some
microorganisms, thus benefiting industrial production and environ-
mental management. Synthetic biology presents a systematic approach
allowing programmability and robustness for use in any biological
engineering endeavor. Modifying the primary DNA sequence to match
the codon methods is used in particular microbial chassis. It is therefore
of great importance to completely understand the acid-tolerance
mechanisms of microorganisms, because use of this information in
combination with synthetic biology may contribute greatly to the
industrial processes involving acid-resistant microbes.
Acknowledgments
This work was supported, in part, by the grants from the National
Basic Research Program of China (2013CB733901), and from the
Chinese National Science Foundation for Excellent Young Scholars
(31422004). We also acknowledge the “Shanghai Rising-Star
Program” (13QA1401700) and the “Chen Xing” project from Shanghai
Jiaotong University.
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Please cite this article as: Liu, Y., et al., Mechanisms of acid tolerance in bacteria and prospects in biotechnology and bioremediation, Biotechnol
Adv (2015), http://dx.doi.org/10.1016/j.biotechadv.2015.06.001
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