Coping with acid environments is one of the prerequisites for the soil saprophytic and human pathogenic lifestyle of Bacillus cereus. This minireview highlights novel insights in the responses displayed by vegetative cells and germinating spores of B. cereus upon exposure to low pH as well as organic acids, including acetic acid, lactic acid and sorbic acid. Insights regarding the possible acid-inflicted damage, physiological responses and protective mechanisms have been compiled based on single cell fluorescence microscopy, flow cytometry and transcriptome analyses.
Bacillus cereus is a common human pathogen that can cause two distinct types of food-borne diseases and other types of infection (Kotiranta et al., 2000). Upon ingestion, diarrhoeic strains can produce enterotoxins, such as haemolysin BL, cytotoxin K and non-haemolytic enterotoxin (Schoeni and Wong, 2005), causing abdominal pain and watery diarrhoea (Stenfors Arnesen et al., 2008). The other type of food-borne illness involves intoxication caused by the emetic toxin cereulide produced by some B. cereus strains (Ehling-Schulz et al., 2004). Cereulide is pre-formed in food and because it remains stable upon heat and acid exposures, the toxin is still active after cooking and stomach transit (Kramer and Gilbert, 1989). Upon ingestion of cereulide typical symptoms may occur within 1–6 h that resemble Staphylococcus aureus intoxication (Le Loir et al., 2003), including nausea, vomiting and general malaise. The symptoms are generally mild; however, in rare cases liver failure has been noted resulting in fatalities (Mahler et al., 1997; Dierick et al., 2005). Besides being an important food-borne pathogen, B. cereus is also a notorious food spoilage organism. Food spoilage is caused by growth of unwanted bacteria in food and causes enormous expenses for food industry (Gram et al., 2002). Bacillus cereus mainly causes spoilage of milk and dairy products, because it is able to form endospores. These spores are survival vehicles formed upon nutrient shortage and are metabolically inactive (de Vries, 2006). Spores are extremely resistant to stress conditions, such as radiation, high temperature, freezing, drying and acid conditions (Setlow, 2006).
Spores and vegetative cells of B. cereus can be found in a wide range of environments (Fig. 1), such as soil (von Stetten et al., 1999; Vilain et al., 2006), plant rhizosphere (Berg et al., 2005) and various foods (Choma et al., 2000; Rosenquist et al., 2005). Bacillus cereus can also be isolated from faeces of healthy adults (Ghosh, 1978), suggesting that B. cereus can be part of the microbiota found in the human gastrointestinal tract. The human stomach and small intestine are acidic environments that have to be overcome by spores and/or vegetative cells to become infectious. Outside the human host, B. cereus may also be frequently exposed to acidic conditions including a vast array of foods at low pH, where in specific cases organic acids have been added as preservatives (Keijser et al., 2007). Additionally, the natural reservoir of the soil saprophyte B. cereus may also be acidic upon the exudation of protons and organic acids in the plant rhizosphere (Neumann and Martinoia, 2002). The antimicrobial activity of organic acids is pH-dependent with the maximum effect occurring at low pH values. At these low pH values organic acids are in undissociated states. Because undissociated acid molecules are uncharged and lipophilic, they will penetrate plasma membranes and thus enter cells. Theoretically, the higher-pH environment of the cell's cytoplasm promotes the rapid dissociation of acid molecules into charged protons and anions. These charged molecules cannot subsequently diffuse back across the plasma membrane. Thus, a permeant organic acid stresses the cell by importing protons, depressingcytoplasmic pH, and by concentrating the organic anion within the cytoplasm in proportion to the transmembrane pH difference (Brul and Coote, 1999). These effects may be counteracted by the cell at the expensive ATP when it tries to extrude protons or metabolize undissociated organic acid molecules (Mols et al., 2010b). Apparently, coping with acid conditions is a determining factor in B. cereus' successful colonization of different niches.
Acid stress responses of Gram-negative organisms, such as Escherichia coli and Salmonella Typhimurium (Richardson et al., 2001), and in a select number of Gram-positive bacteria, such as lactic acid bacteria and Listeria monocytogenes (van de Guchte et al., 2002; Cotter and Hill, 2003; Ryan et al., 2009) have been reviewed. These reviews highlight the importance of proton pumps, i.e. F1F0-ATPase, transcriptional regulators, such as RpoS (Gram-negatives) and σB (Gram-positives), proteins involved in protection of macromolecules, such as DnaK and GroESL, and enzymes that produce alkaline compounds, such as the ammonium-forming enzymes urease and arginine deiminase. Until recently, no detailed information was available on the acid stress responses of B. cereus. Fluorescence techniques, physiological studies and transcriptome analyses elucidated acid stress responses of vegetative cells and germinating spores of B. cereus, including novel observations such as the formation of reactive oxygen species (ROS) and the induction of a secondary oxidative stress response (Thomassin et al., 2006; Mols et al., 2009; 2010a,b; den Besten et al., 2010; Biesta-Peters et al., 2010a,b; van Melis et al., 2011a). The aim of this minireview is to provide an overview in the physiological responses, possible acid-inflicted damage and protective mechanisms displayed by B. cereus upon exposure to acid conditions.
Response of vegetative cells to acid stress
The physiological response of vegetative cells of B. cereus upon exposure to acid conditions, including exposure to organic acids, the putative protective mechanisms and the acid-induced secondary oxidative stress are discussed in the following section.
Physiological response to acid conditions
Upon exposure to acid conditions the growth of B. cereus is readily affected (Biesta-Peters et al., 2010a,b; Mols et al., 2010a,b). The growth rate declines and the lag-phase increases when vegetative cells are subjected to a lowered pH with or without additional organic acids (Biesta-Peters et al., 2010a,b). To what extent the growth rate and lag-phase are affected is highly dependent on initial growth rate, strain and acidulant used. During the acid-induced lag-phase B. cereus cells are repairing damage, increasing the internal pH and increasing ATP concentration before growth resumes (E.G. Biesta-Peters, M. Mols, M.W. Reij and T. Abee, unpubl. results). The presence of organic acids next to the lowered pH of the medium increases the growth-diminishing effects of the acid-exposed culture. Exposure to 15 mM lactic acid and 2 mM acetic at pH 5.5 acid stops the growth while without the additional organic acids only the growth rate was decreased (Fig. 2A). When the pH of the environment is acidified further, vegetative cells cannot resume growth and are eventually inactivated (Fig. 2B).
Bacillus cereus displays a so-called acid tolerance response. When vegetative cells were previously exposed to a mildly lowered pH, e.g. pH 6.3, they become resistant to normally lethal pH values, for instance pH 4.6 (Jobin et al., 2002; Thomassin et al., 2006). Additionally, exposure to mildly acidic environments may result in enhanced protection when cells are subsequently exposed to lethal heat or hydrogen peroxide stress, a process often referred to as cross-protection (den Besten et al., 2010). Acid tolerance responses and cross-protection phenomena can have great implications for controlling B. cereus growth and occurrence in food products and food-processing equipment. Therefore, it is necessary to understand the acid stress response of B. cereus and the putative mechanisms deployed to protect against acidic conditions.
General stress response. Mechanisms of acid resistance in other Gram-positive organisms have been reviewed by Cotter and Hill (2003). Most studies were performed on the acid stress response of non-respiring lactic acid bacteria in the presence of oxygen. In contrast, B. cereus actively respires in the presence of oxygen and therefore the results obtained from these lactic acid bacteria should only be extrapolated cautiously to B. cereus. Besides differences between the organisms reviewed by Cotter and Hill (2003) and B. cereus, there are common mechanisms putatively playing a role in acid resistance as indicated by induction of the corresponding genes (Fig. 3 and Table 1). Genes involved not only in acid stress response but also in responses to other stresses, including protein repair chaperones groESL and dnaK and clp genes, were shown to be upregulated upon exposure to acid conditions in B. cereus. Furthermore, several transcriptional regulators are putatively involved in the acid stress response. The expression of sigB, the gene encoding for alternative sigma factor σB, was induced, which is in agreement with previous studies (van Schaik et al., 2004). Heat stress regulators ctsR and hrcA (van de Guchte et al., 2002) were also upregulated upon exposure to low pH in B. cereus, indicating possible common damaging factors and protective mechanisms in different stress conditions. These general stress response mechanisms have been described to be involved in cross-protection of B. cereus exposed to various stress conditions, including low pH, and can be used as biomarkers for bacterial robustness (den Besten et al., 2010).
Table 1. Acid stress responses of B. cereus.
Putative acid resistance mechanism
Expression upon acid shocks
Presence in genome of B. cereus
Repressed upon mild acid shocks and induced upon lethal acid shocks.
Induced at mild pH; however, urease activity did not lead to increased resistance (Mols and Abee, 2008).
Metabolic rearrangements. Metabolic rearrangements, known to be involved in acid resistance in lactic acid bacteria, were also induced by B. cereus upon low pH exposures (Fig. 3 and Table 1). Genes encoding for enzymes catalysing the reaction from pyruvate to acetoin and butanediol, i.e. alsDS, were induced upon exposure to acid shocks. Although such reaction is at the expense of pyruvate, it removes intracellular protons and forms carbon dioxide (CO2). Also in Bacillus subtilis alsSD genes are strongly induced under mild acid stress conditions (Wilks et al., 2009) and in Lactobacillus plantarum activation of the corresponding enzymes contributed to pH homeostasis (Tsau et al., 1992). Genes encoding for alcohol dehydrogenases and lactate dehydrogenases were induced upon exposure to lethal acid shocks. Therefore, the conversion of pyruvate to ethanol or lactate, generating CO2 and consuming protons, may be an ultimate futile response of B. cereus to deal with low intracellular pH (pHi) or restoration of NAD+/NADH balance. Some metabolic rearrangements were found specifically correlated with lactic acid or acetic acid stress, such as metabolic pathways for amino acid metabolism (Mols et al., 2010b), but their functions remain to be established. Metabolomics and mutant analysis will aid in unravelling the role of these metabolic changes in organic acid resistance.
pH homeostasis. Upon exposure to acid conditions, many bacteria activate enzymes contributing to pH homeostasis (Fig. 3 and Table 1). Cells may pump protons out of the cell, prevent protons from leaking in and counteract acidification of the cytoplasm by producing alkaline compounds. Aerobic bacteria, such as B. cereus, use their electron transport machinery to transport protons over the cell membrane generating an excess of protons on the outside of the cell thus generating a proton motive force (PMF). The PMF is subsequently used to generate ATP by inward flux of protons via F1F0-ATPase. In lactic acid bacteria, and presumably also in anaerobically growing B. cereus cells, F1F0-ATPase can also transport protons outside the cell at the expensive of ATP in acid conditions. Bacillus cereus represses the expression of genes encoding for subunits of F1F0-ATPase upon exposure to mild acidic environments (Mols et al., 2010a). Conceivably, B. cereus does not use F1F0-ATPase to pump protons out of the cell in aerobic acid conditions and by repressing F1F0-ATPase genes and lowering the amount of active ATPase, the influx of protons is limited. Notably, upon exposure to lethal levels of acidity these genes are not repressed. Also other proton transporters, such as napA and nhaC, were downregulated upon exposure to mild acid stress. Interestingly, these genes were (highly) induced upon exposure to lethal pHs, indicating a fine balance between proton influx and ATP synthesis on one hand and on the other hand proton pumps regulating pHi at the expense of ATP. In addition, amino acid decarboxylases may contribute to homeostasis of pHi in bacteria (Cotter and Hill, 2003; Foster, 2004). In Gram-positives, especially L. monocytogenes, glutamate decarboxylase (GAD) has been associated with acid resistance (Cotter et al., 2001). Glutamate is converted to gamma-aminobutyric acid (GABA) consuming an intracellular proton by GAD. Subsequently, the product GABA is exchanged with extracellular glutamate by a glutamate/GABA antiporter. Such a transporter has been found to be necessary for optimal GAD-dependant acid resistance in L. monocytogenes. In the genome of the B. cereus type strain ATCC 14579 no GAD system could be identified. Another sequenced B. cereus strain, ATCC 10987, does harbour a glutamate decarboxylase gene (Mols et al., 2007) that was however not found to be differentially expressed upon exposure to low pH values (Mols et al., 2010a), indicating that the role of GAD in the acid resistance of B. cereus ATCC 10987 is limited. An explanation for this phenomenon is the fact that a glutamate/GABA antiporter gene is lacking in the genome of ATCC 10987. Bacteria can also counteract a low internal pH by the production of alkaline compounds, such as ammonia. One of the mechanisms known to produce ammonia and involved in acid resistance in other bacteria is the arginine deiminase pathway (ADI) (Ryan et al., 2009). The ADI pathway converts arginine into ammonia and CO2 via citrulline and carbamoyl-phosphate. Although the role of ADI in acid resistance in other bacteria is evident, in B. cereus the ADI genes are only moderately upregulated upon exposure to acid in aerobic conditions, whereas the ADI pathway was found highly upregulated under mildly acidic anaerobic conditions, suggesting that this system may play a role in acid stress survival in anaerobic conditions (van der Voort and Abee, 2009). Arginase, which also converts arginine to citrulline producing ammonia, was highly induced upon exposure to low pHs, indicating that arginine catabolism may support acid tolerance in B. cereus. Another well-known mechanism of alkali production is the hydrolysis of urea into ammonia and CO2 by the enzyme urease. Urease is known to be involved in the acid resistance of several bacteria and well-studied in Helicobacter pylori and B. subtilis (Mobley et al., 1995; Wray et al., 1997). Urease and concomitant ureolytic activity is shown by strain ATCC 10987, in contrast to type strain, which does not harbour the urease genes. The genes encoding for the urease enzyme were somewhat induced upon exposure to sublethal pH 5.4 in ATCC 10987 (Mols et al., 2010a). However, it was shown that the ureolytic activity in a variety of B. cereus strains, including ATCC 10987, did not provide for acid resistance and that its role was solely in nitrogen metabolism (Mols and Abee, 2008).
Secondary oxidative stress response
The exposure of B. cereus to inorganic acid as well as organic acids, lactic acid and acetic acid in aerobic conditions revealed a major oxidative response (Mols et al., 2010a,b). This secondary oxidative stress response (Mols and Abee, in press) was indicated by the induction of oxidative stress associated genes, including genes encoding for thioredoxins, catalases, superoxide dismutase and the major oxidative stress regulator PerR, upon exposure to acid shocks. Thioredoxins are known to control the reduced state of thiol groups that can be oxidized upon exposure to oxidative stress (Holmgren, 1985). Superoxide dismutase and catalase convert superoxide and hydrogen peroxide into water (Imlay, 2003) and superoxide dismutase has been suggested to be involved in the acid tolerance response of B. cereus (Browne and Dowds, 2002; Jobin et al., 2002). PerR is a hydrogen peroxide sensing transcriptional regulator associated with the expression of genes encoding catalases and peroxidases (Mongkolsuk and Helmann, 2002). The induction of these oxidative stress-associated genes suggests that oxidative compounds are generated upon exposure to low pHs in B. cereus. Indeed, ROS such as hydroxyl (OH·), peroxynitrite (ONOO-) (Mols et al., 2010a) and superoxide (O2-) (M. Mols, M. Ceragioli and T. Abee, unpubl. results) are shown to be generated when B. cereus cells are exposed to bactericidal pHs. These ROS may be generated at specific sites in the aerobic electron transfer chain (ETC). Acid shocks may affect ETC activity since expression of genes encoding alternative electron donor and acceptor mechanisms was found to be induced. In the B. cereus type strain the most prominent induction upon exposure to acid shocks was that of nitrate and nitrite reductase genes (Mols et al., 2010a). Nitrate can act as an alternative electron acceptor and is converted to nitrite in a reaction consuming a proton (Richardson et al., 2001). Subsequently the resultant nitrite can be reduced to ammonium consuming five protons. Whether nitrate and nitrite reductases are induced because they form an alternative ETC, restore NAD+/NADH balance, and/or because the reactions they catalyse consume intracellular protons remains to be elucidated. Also other alternative components of the ETC were associated with mainly lethal levels of organic and inorganic acid shocks (Mols et al., 2010a,b). Cytochrome bd oxidase (cydAB) genes, which may act as an alternative complex IV of the ETC, were highly induced upon exposure to 15 mM acetic acid and at pH 4.5. Cytochrome bd oxidase has been proposed to function in an alternative electron transport chain together with NAD(P)H-dependant dehydrogenases, such as lactate (ldh) and alcohol dehydrogenase (adhA) (Chai et al., 2009). Lactate dehydrogenase (ldh) and cytochrome bd oxidase genes are coordinately expressed together with the lactate permease gene lctP and formate-nitrite transporter gene ywcJ and under control of the negative regulator YdiH (Rex) in B. subtilis (Larsson et al., 2005). Together with the alsSD genes, cydAB, ldh and lctP form a distinct regulon, which is part of the larger anaerobic-responsive Fnr regulon (Reents et al., 2006), indicating a clear association between these upregulated genes and anaerobic conditions. Furthermore, B. cereus is more resistant to acid stress when grown and exposed under oxygen limitation (Mols et al., 2009). Whether activation of these enzymes and pathways contribute to higher acid resistance of B. cereus when grown and exposed without oxygen remains to be elucidated.
Response of spores germinating in acid conditions
Bacillus cereus is able to form endospores that allow the organism to survive adverse conditions. These spores are formed upon nutrient shortage and are metabolically inactive (de Vries, 2006). They can be isolated from many environments and are very resistant to harsh conditions, including low pH and high organic acid concentrations. When spores encounter more favourable conditions, they can germinate into vegetative cells and subsequently grow (Setlow, 2003; Hornstra et al., 2006; Paredes-Sabja et al., 2011). The process of germination is of interest, because it is the transition between inactive spores to metabolically active and possibly virulent vegetative cells. Recently, the effect of low pH with the addition of sorbic acid on the germination of B. cereus has been studied (van Melis et al., 2011a). The germination of B. cereus spores in mildly acidic pH (pH 5.5) is not affected (Fig. 4). The rate of outgrowth into dividing vegetative cells, on the other hand, is decreased. In the presence of sorbic acid such decreased outgrowth rate is also observed. Furthermore, the presence of sorbic acid delays the germination and decreases the germination efficiency. With microscopic observations and flow cytometry, the transition from dormant phase bright spores to phase dark spores was shown to be delayed and the process of germination and outgrowth is stuck at the phase dark spore with no outgrowth visible. The reduced germination efficiency may relate to the hydrophobicity of sorbic acid, causing it to accumulate in the spore's inner membrane. This accumulation may interfere with the signalling cascade that is required for germinant receptor-mediated germination (van Melis et al., 2011b). Transcriptome analysis of spores germinated in the presence of sorbic acid indeed revealed genes involved in membrane biogenesis and cell envelope modifications. Notably, the transcriptome analyses revealed that spores are triggered to germinate and initiate vegetative growth irrespective of whether conditions for outgrowth were acidic or not. Gene expression data showed that genes that are induced in the control and sorbic acid stressed spores largely overlap in the initial stage of germination. This corroborates observations made in B. subtilis, where detailed transcriptome analysis revealed spore germination to occur via a tightly controlled spore outgrowth programme (Keijser et al., 2007). The spore outgrowth programme of B. cereus indeed shows high similarity with that of B. subtilis and is not influenced by the presence of organic acids or a low pH.
The responses of B. cereus vegetative cells to acid environments resemble the responses seen in other Gram-positive organisms. However, there are several crucial differences including the findings that B. cereus does not utilize all possible protective mechanisms, such as transporting protons outwards via F1F0-ATPase and producing ammonium via urease, in aerobic conditions. Furthermore, the rearrangements in energy production and conversion are striking. The induction of alternative ETC components may indicate a protective mechanism; however, the correlation with the generation of a secondary oxidative stress response is more evident. This secondary oxidative stress response originates from acid-induced malfunctioning of the ETC resulting in ROS formation. These ROS are proposed to be part of a common mechanism of cellular death in E. coli exposed to bactericidal antibiotics (Kohanski et al., 2007). Although actively respiring cells and germinating spores induce oxidative stress-associated genes upon exposure to acid environments and ROS are formed, the role of ROS in the cellular death remains to be established. However, not only ROS formation and the induction of oxidative stress responses determine the cell fate, as indicated by the inactivation of anaerobically grown and exposed B. cereus cells. The insights obtained in recent studies and reviewed here contribute to our understanding of Bacillus' physiology in various acid environments and may provide leads to optimize the efficiency of existing and new food preservation strategies.