Adaptive responses of Bacillus cereus ATCC14579 cells upon exposure to acid conditions involve ATPase activity to maintain their internal pH

This study examined the involvement of ATPase activity in the acid tolerance response (ATR) of Bacillus cereus ATCC14579 strain. In the current work, B. cereus cells were grown in anaerobic chemostat culture at external pH (pHe) 7.0 or 5.5 and at a growth rate of 0.2 h−1. Population reduction and internal pH (pHi) after acid shock at pH 4.0 was examined either with or without ATPase inhibitor N,N’-dicyclohexylcarbodiimide (DCCD) and ionophores valinomycin and nigericin. Population reduction after acid shock at pH 4.0 was strongly limited in cells grown at pH 5.5 (acid-adapted cells) compared with cells grown at pH 7.0 (unadapted cells), indicating that B. cereus cells grown at low pHe were able to induce a significant ATR and Exercise-induced increase in ATPase activity. However, DCCD and ionophores had a negative effect on the ability of B. cereus cells to survive and maintain their pHi during acid shock. When acid shock was achieved after DCCD treatment, pHi was markedly dropped in unadapted and acid-adapted cells. The ATPase activity was also significantly inhibited by DCCD and ionophores in acid-adapted cells. Furthermore, transcriptional analysis revealed that atpB (ATP beta chain) transcripts was increased in acid-adapted cells compared to unadapted cells before and after acid shock. Our data demonstrate that B. cereus is able to induce an ATR during growth at low pH. These adaptations depend on the ATPase activity induction and pHi homeostasis. Our data demonstrate that the ATPase enzyme can be implicated in the cytoplasmic pH regulation and in acid tolerance of B. cereus acid-adapted cells.


Introduction
Bacillus cereus is a gram-positive, facultative anaerobe, endospore-forming bacterium that can be isolated from a wide variety of different sites (Kotiranta et al. 2000), and also recognized as one of the major food-borne pathogenic bacteria (McKillip 2000). Bacillus cereus is responsi-ble for two types of food-associated illnesses: emetic (vomiting) and diarrheal syndromes. The former is due to a small-molecular-weight cyclic toxin, cereulide, whereas the diarrheal syndrome results from the production of at least two types of multiple-component enterotoxins, hemolysin BL (HBL), nonhemolytic enterotoxin (NHE) (Stenfors Arnesen et al. 2008).
It has been shown that vegetative B. cereus cells, like many other bacteria are able to induce an acid tolerance response (ATR) (Thomassin et al. 2006;Desriac et al. 2013). Bacillus cereus ATCC14579 and B. cereus TZ415 are more tolerant to acid shocks when cells are cultivated at low pH (Jobin et al. 2002;Thomassin et al. 2006). Recently, it has been shown that B. cereus ATCC14579 cells can employ complex survival strategies involving decarboxylase and deiminase systems which are implicated in intracellular pH (pH i ) homeostasis (Senouci-Rezkallah et al. 2011). In response to low pH, Proton pumps play a major role in pH i homeostasis in Listeria monocytogenes (Cotter et al. 2000). ATPases from different sources have very similar structures (Santana et al. 1994). They consist of two main subcomplexes: F 1 , the extrinsic membrane subcomplex, which contains the catalytic sites for ATP hydrolysis, and F 0 , the integral membrane subcomplex, which forms the proton channel in bacteria (Kanazawa et al. 1981). In bacteria with a respiratory chain, the primary role of the enzyme is to synthesize ATP from the proton gradient of the respiratory chain. On the other hand, its role is to create a proton gradient (used for a variety of transport processes) with the energy provided by ATP hydrolysis and to maintain the intracellular pH via proton extrusion (Kakinuma 1998), this is the case for the oral streptococci Streptococcus mutans and Streptococcus sanguis (Bender et al. 1986), Lactobacillus acidophilus (Kullen and Klaenhammer 1999) and Lactococcus lactis (Koebmann et al. 2000). The proton translocating F 1 F 0 -ATPase enzyme complex plays a significant role in the regulation of intracellular pH in a number of bacteria (Cotter et al. 2000). In Enterococcus faecalis, Lactobacillus brevis and Enterococcus hirae, a high increase in F 1 F 0 -ATPase activity was observed when cells were grown at low pH (Kobayashi et al. 1984(Kobayashi et al. , 1986Arikado et al. 1999). Little is known about the ATPase activity of B. cereus. The enzyme shows similar characteristics as the enzyme isolated from Escherichia coli and B. subtilis (Banfalvi et al. 1981). The ATPase activity was specifically inhibited by N,N'dicyclohexylcarbodiimide (DCCD) and proton translocation by reacting with the conserved Glu (or Asp) residues of the rotor ring of both F-and V-ATPases (Mizutania et al. 2011). Ionophore antibiotics as valinomycin and nigericin act by specifically increasing the ion permeability of the cell membrane (Bakker 1979). Recently, Mols et al. have demonstrated that genes encoding subunits of the F 1 F 0 -ATPase (atpB) were highly downregulated in aerobically grown and exposed B. cereus cells upon exposure to sublethal pH 5.4 (Mols et al. 2010a,b;Mols and Abee 2011).
In our study, the role of ATPase activity in ATR and pH i homeostasis of B. cereus ATCC14579 was determined.
For this goal, anaerobic Chemostat cultures (fermentation) were carried out at constant growth conditions with variation in only one parameter (culture pH). The effect of culture pH, ATPase inhibitor DCCD and ionophores (valinomycin and nigericin) on the ATPase activity, acid survival and thus the internal pH homeostasis of B. cereus cells was established.

Materials and Methods
Bacterial strains and growth conditions (chemostat culture) Bacillus cereus strain ATCC14579 was obtained from the American Type Culture Collection. Growth medium was J-Broth (JB) (Claus and Berkeley 1986). Chemostat cultures were performed in a 2-L bioreactor (Discovery 100 MRU; INCELETECH, Toulouse, France) using a 1-L working volume. All experiments were carried out at 34°C with agitation at 300 rpm. Culture pH was monitored and maintained at pH 5.5 AE 0.04 or 7.0 AE 0.06. During fermentation, the culture pH was continuously sparged with oxygen-free nitrogen gas to ensure anaerobiosis (Senouci-Rezkallah et al. 2011).

ATPase activity measurement
A modification of protocol described by (Fortier et al. 2003) was used for cell permeabilization. Bacillus cereus cells were grown in a chemostat at different pH values (pH 7.0 or 5.5) and a growth rate of 0.2 h À1 (Tourdot-Marechal et al. 1999).
The liberated P i was measured according to (Goffeau and Dufour 1988). ATPase activity was expressed as U mg À1 (lmol L À1 of P i produced per min and per mg of total protein). Protein concentration was determined using a Bio-Rad (France), protein assay.
The effect of DCCD inhibitor and ionophores on ATPase activity of B. cereus cells was investigated by incubation of cells in the presence of DCCD (0.2 mmol L À1 ) at 34°C for 30 min, or by incubation in the presence of 1 lmol L À1 valinomycin and/or nigericin for 2 min at room temperature (SigmaAldrich Inc, St. Louis, MO) before the ATP addition.
Effect of DCCD, valinomycin and/or nigericin on cell survival during acid shock One milliliter aliquot of steady-state B. cereus cells was incubated in the absence and presence of 0.2 or 1 mmol L À1 of DCCD for 30 min at 37°C or of 1 lmol L À1 valinomycin and/or nigericin (prepared in ethanol at 99%) for 2 min at room temperature. The cells were then diluted in 19 mL of JB at pH 4.0 (acid challenge) or at a pH equivalent to growth pH (control condition) and maintained at 34°C with agitation at 100 rpm. Viability loss was determined by viable counts after 40 min. In control experiment, B. cereus cells were preincubated in JB medium in the presence of ethanol before acid shock at pH 4.0. Cells counting was calculated according to the manufacturer's instructions and expressed as colony forming units per mL (CFU mL À1 ) as previously described (Thomassin et al. 2006). The limit of detection was 200 CFU mL À1 .

Internal pH measurements
Internal pH (pH i ) was determined as previously described (Thomassin et al. 2006). Bacillus cereus cells were grown in a chemostat at different pH values (pH 7.0 or 5.5) and at growth rate l = 0.2 h À1 . Steady-state growing cells were preincubated in JB medium at a pH equivalent to growth pH e either with or without of DCCD (0.2 mmol L À1 for 30 min at 34°C) (SigmaAldrich Inc, St. Louis, MO), and submitted to acid shock at pH 4.0 in JB medium. The pH i of B. cereus cells was measured before and after 10 and 40 min of acid shock.

mRNA preparation and quantification
Quantification of mRNA was performed by real-time polymerase chain reaction (RT-PCR) as previously described (Duport et al. 2004). To evaluate the reproducibility of the method, three independent RNA samples were analyzed in parallel for three independent cultures performed at pH 7.0 and 5.5. Samples were quantified using Light-Cycler Software version 3.5 (Roche Diagnostics, Meylan, France), standardized for the 16S rRNA, and quantified using the mathematical model established by Pfaffl (2001). Standard deviation was found to be roughly constant for the atpB gene coded for the ATPase beta chain (BC5306) (ATPase enzyme). The primers used were F: 5 0 -GCAATATGTTCGCCAGCTTC-3 0 , (forward) R: 5 0 -TCGCAGCTTAGCTCTTCG-3 0 (reverse).

Statistical analysis
Acid stress resistance, pH i and ATPase activity measurements were all determined in triplicate at different times on the same chemostat for each pH tested. The mean value and standard deviation were calculated from the data obtained from the three separate experiments. Results were submitted to variance analysis using Systat 9 software (SPSS, Chicago, IL). Analysis of variance was performed for multiple comparisons of means using Tukey's honestly significant difference test at the 5% level.

Effect of growth pH e on level of ATPase
In order to determine the effect of growth pH e on ATPase activity, B. cereus cells were grown at pH e 5.5 or 7.0 and at l = 0.2 h À1 , and the ATPase activity was measured ( Table 1). The ATPase activity was increased as the growth pH e decreased (9.38 AE 0.31 and 5.21 AE 0.12 U mg À1 proteins at pH e 7.0 and 5.5, respectively). We also investigated the effect of DCCD, valinomycin and nigericin on the ATPase activity. In these experimental conditions, DCCD, valinomycin and nigericin were prepared in ethanol at 99%. Hence, we studied the effect of ethanol on the ATPase activity (as control experiment). The results showed that ethanol has no direct effect on ATPase activity of unadapted cells, but it has a visible effect on ATPase activity of acid-adapted cells. For unadapted and acid-adapted cells treated for 30 min with DCCD, ATPase activity was significantly decreased (Table 1). These results show that this activity is completely inhibited by DCCD. So this inhibitor had a clear effect on ATPase activity whatever the growth pH.
The effect of ionophores valinomycin and nigericin on the ATPase activity was also studied. A 1 mL aliquot of steady-state B. cereus cells were incubated for 2 min in the presence of ionophores: 1 lmol L À1 of valinomycin or nigericin, or in the presence of both ionophores for pH i with pH e equilibration (DpH = 0). The ATPase activity was measured as described above. The results showed that the presence of valinomycin decreases slightly the Table 1. Effect of growth pH e and ionophores on ATPase activity of steady-state Bacillus cereus ATCC14579 cells grown at a pH e 5.5 or 7.0 and at a growth rate of 0.2 h À1 .
1 ATPase activity was expressed as lmol L À1 of P i produced per min. ATPase activity in unadapted cells (16%) compared to acid-adapted cells (83%). Although the ATPase activity was inhibited completely by nigericin whatever the growth pH. In the presence of both ionophores, the ATPase activity was inhibited by 75% and 57% in acid-adapted and unadapted cells, respectively. Thus, the presence of valinomycin can decrease the effect of nigericin on ATPase activity of B. cereus by pH i and pH e equilibration.

Effect of DCCD, valinomycin and nigericin on acid survival of acid-adapted cells
In order to investigate whether DCCD had an effect on the acid resistance of B. cereus, steady-state cells grown at pH 7.0 or 5.5 were incubated for 30 min in the absence or presence of DCCD (0.2 or 1 mmol L À1 ). In control condition, the population of cells transferred on JB at pH equivalent to growth pH (7.0 or 5.5) in the presence of 1 mmol L À1 DCCD was stable (Table 2). So, DCCD had no direct effect on B. cereus viability. Indeed, acid shock survival of acid-adapted and unadapted cells is not affected by ethanol added for 30 min before acid shock for control experiment (Fig. 1). So, ethanol had no major effect on the acid survival of B. cereus cells. Pretreatment of B. cereus cells with DCCD followed by acid shock at pH 4.0 for 40 min decreased the population about 1.64-log for acid-adapted cells ( Fig. 1.1B), but had no significant effect on unadapted cells (4-log in absence and presence of DCCD) ( Fig. 1.1A). Thus, DCCD had clear effect on acid survival of acid-adapted cells. These results indicate that ATPase activity is required for acid adaptation of B. cereus cells.
The effect of ionophores (valinomycin/nigericin) on acid survival of B. cereus cells was also studied. Cells grown at pH e 7.0 (unadapted cells) or 5.5 (adapted cells) were incubated for 2 min in the absence and presence of valinomycin or nigericin (1 lmol L À1 each), or in the presence of both ionophores and transferred (1) at a pH equal to the initial value (pH 7.0 and 5.5, respectively) as a control condition, or (2) submitted to acid shock at pH 4.0 for 40 min. The corresponding population decrease was determined. The presence of both ionophores at pH e 7.0 (pH i 7.0, ΔpH 0) show no effect on unadapted cells viability. Acid-adapted cells viability decreased by 1.5 log after 10 and 40 min of incubation at pH 5.5 (pH i 5.5, ΔpH 0) ( Table 2). Thus, the acid resistance of acidadapted cells was strongly affected by ionophores.
After acid shock, the population decrease of unadapted cells was similar under all four conditions, reaching a value of 4-log after 40 min ( Fig. 1.2C). Acid-adapted cells preincubated for 2 min in the presence of ionophores presented an initial one log population reduction at the beginning of the acid shock. The population decrease in cells preincubated in the absence of ionophore in pH 5.5 (pH i 6.2, ΔpH 0.72) peaked after 40 min of acid shock 0.2-log reduction compared to cells preincubated for 2 min in the presence of both ionophores at pH 5.5 (pH i 5.5, ΔpH 0) (3.5-log reduction) ( Fig. 1.2D). After 10 min of acid shock, the population decrease in acid-adapted cells preincubated in the presence of valinomycin is slightly affected compared to the presence of nigericin and both ionophores. Thereafter, the population decrease was similar under all three conditions, reaching a value of 3.5-log after 40 min of acid shock. Thus, the ATPase activity was strongly inhibited by nigericin compared to valinomycin. This suggests that ATPase activity may involved in the acid survival of acid-adapted cells.

Effect of DCCD on pH i maintenance of B. cereus ATCC14579 cells
To examine whether ATPase activity has a role in pH i homeostasis, B. cereus cells were grown in a chemostat at pH e 5.5 or 7.0. Steady-state cells were preincubated in the absence or presence of DCCD (0.2 mmol L À1 ) for 30 min, and subjected to acid shock at pH 4.0 for 40 min. The pH i was measured before and during acid shock, and dpH value was calculated.
In unadapted untreated cells by DCCD, pH i was decreased from 7.1 before acid shock to 5.85 and 6.08 after 10 and 40 min of acid shock, respectively (Table 3). After preincubation of unadapted cells with DCCD (0.2 mmol L À1 ), pH i was decreased to 4.74 and 4.69 after 10 and 40 min of acid shock, respectively. The pH i of unadapted cells was better maintained in the absence of DCCD. Cells from steady-state chemostat cultures grown at a dilution rate of 0.2 h À1 and at pH e 7.0 or 5.5 were transferred on JB at pH identical to the culture pH (7.0 or 5.5) in the presence of 1 mmol L À1 DCCD or 1 lmol L À1 nigericin and valinomycin instead of the acid challenge were included as controls. Log (N/N 0 ) 10 and log (N/N 0 ) 40 values are the means of data for the least three replicate experiments. DCCD, N, N'-dicyclohexylcarbodiimide.
The pH i of acid-adapted cells was slightly decreased after acid shock and the effect of DCCD was more significant. After 40 min of acid shock, pH i was decreased from 6.22 to 5.35 and 4.25 in absence and in the presence of DCCD (0.2 mmol L À1 ), respectively. These results indicate that the presence of DCCD has an effect on pH i of unadapted cells and this effect was more marked in acid-adapted cells. Thus, activity ATPase inhibition affects the acid survival and pH i maintenance of acid-adapted cells more than unadapted cells.
In addition, we calculated a dpH i value defined as pH i regulation for 40 min of acid shock (pH i after 10 or 40 min of acid shockinitial pH i ). After 40 min of acid shock, the dpH i was decreased by 1.97 and 2.41 units in B. cereus cells treated by DCCD (0.2 mmol L À1 ) compared to untreated cells (dpH i was decreased to 0.87 and 1.02 units) in acid-adapted and unadapted cells, respectively (Table 3). So, dpH i was significantly decreased in B. cereus cells treated by DCCD after 40 min of acid shock. Thus, internal pH decrease was affected by growth pH and the presence of DCCD. This suggests that the ATPase activity plays a major role in the ATR of B. cereus by pH i maintenance.

Transcriptional analysis
RT-PCR assays were conducted to determine the effect of growth pH on ATPase gene expressions. atpB (ATP beta chain) transcripts showed a threefold increase in acidadapted cells grown at pH 5.5 compared with unadapted cells grown at pH 7.0 (Fig. 2). After 10 min of acid shock at pH 4.0 atpB (ATP beta chain "BC5306") gene was upregulated in acid-adapted cells (3.5-fold) compared with unadapted cells (twofold). However, we have observed that gene encoding subunits of the F 1 F 0 -ATPase (represented by atpB) were highly downregulated in acidadapted cells (1.6-fold) compared with unadapted cells (onefold) after 40 min of acid shock. Thus the transcription of ATPase gene expression was activated by acid adaptation in B. cereus.

Discussion
Bacillus cereus ATCC14579 is able to survive low pH environments. The induced ATR was previously observed in B. cereus TZ415 strain (Jobin et al. 2002), NCIMB11796 strain (Browne and Dowds 2002) and ATCC14579 strain (Thomassin et al. 2006), and it was established in other bacteria such as L. monocytogenes, E. coli and Salmonella enterica (O'Hara and Glenn 1994; Davis et al. 1996;Tiwari et al. 2004). Bacillus cereus ATCC14579 is able to adapt and to survive the acid stress when it is grown at pH e (5.5) and thus to induce an ATR. These mechanisms of resistance to acid may involve (1) F 1 F 0 -ATPase and/or glutamate or arginine decarboxylases and arginine deiminase, which are involved in intracellular pH (pH i ) homeostasis (Senouci-Rezkallah et al. 2011), (2) metabolic modifications, and (3) protein synthesis to protect and/or repair macromolecules (Cotter and Hill 2003). Bacillus cereus is a significant acid-resistant neutrophilic bacterium that prefers growth near neutral pH but is able to survive transient exposures to pH 4.0 when preadapted to moderately low pH e (pH 5.5). ATR systems such as ATPase activity may contribute to acid resistance in other bacteria (Mandel et al. 1983;Higuchi et al. 1997;Sakamoto et al. 2002). We observed that the acid shock survival (at pH 4.0) of B. cereus cells grown at pH 5.5 (acidadapted cells) was higher compared to cells grown at 7.0 Table 3. Effect of DCCD on internal pH homeostasis and dpH i maintenance of steady-state Bacillus cereus ATCC14579 cells grown at pH e 5.5 or 7.0 and at growth rate of 0.2 h À1 phi with DCCD. Internal pH values were calculated before and after acid shock at pH 4.0 in the absence (control experiment) and presence of 0.2 mmol L À1 DCCD. a (DCCD 1 mol L À1 )pH i before the acid shock incubated in presence of DCCD as negative control. dpH i = pH i a À pH i (b or c) . DCCD, N,N'dicyclohexylcarbodiimide.
1 pH i before the acid shock.  Figure 2. RT-PCR assays conducted on mRNA isolated in steady-state Bacillus cereus un-adapted cells (grown at pH 7.0) (□) and acidadapted cells (grown at pH 5.5) (■) grown in a chemostat at a growth rate of 0.2 h À1 . The expression of atpB gene before and after 0, 10, and 40 min of acid shock at pH 4.0 was measured. Relative gene expression of B. cereus ATCC14579 cells grown at pH 7.0 before acid shock was set at 1.0 (control condition). (unadapted cells). Our results show also that the ATPase activity was increased when cells were grown at low pH 5.5 (acid-adapted cells) compared to cells grown at neutral pH 7.0 (unadapted cells). Similar results were obtained in Oenococcus oeni IOB84 when cells were grown at pH 3.5 compared to pH 5.3 (Fortier et al. 2003). This result demonstrate that ATPase activity was increased with pH e decrease. Therefore, if the F 1 F 0 -ATPase is involved in acid adaptation, treatment with DCCD would be expected to render acid-adapted cells more acid sensitive. We found that such treatment just prior to the acid challenge resulted in the significantly enhanced acid sensitivity of the acid-adapted cells during acid shock. So, B. cereus population reduction was greatly increased after 40 min of acid shock after DCCD treatment. This reduction was more marked in acid-adapted cells than unadapted cells. Comparable results were observed in L. monocytogenes and Salmonella Typhimurium, acid-adapted cells grown in batch culture and treated with DCCD were much more sensitive than treated unadapted cells to exposure to pH 3.5 and 3.3, respectively (Foster and Hall 1991;Cotter et al. 2000). These data show the role of F 1 F 0 -ATPase in B. cereus acid adaptation.
It is interesting to note that DCCD had no significant effect on unadapted cells at low pH, showing that the ATR is not solely dependent on the activity of this complex. Similarly, our data indicate that the ATPase inhibition by DCCD reached 95% and 96% in cells grown at pH e 7.0 and 5.5, respectively. Previous results showed that both the increased and basal ATPase activity were inhibited equally by DCCD in Streptococcus faecalis (Kobayashi et al. 1986). ATPase activity was also significantly inhibited by DCCD in L. brevis (Sakamoto et al. 2002). Likewise, in Lactobacillus sp. the ATPase activity was also inhibited by DCCD (70% at the concentration of 0.5 mmol L À1 ) (Higuchi et al. 1997). So, the ATPase complex plays a major role in the acid resistance mechanisms in B. cereus as previously described in other grampositive bacteria (Cotter and Hill 2003).
We also observed that ATPase activity was completely inhibited in the presence of nigericin whatever the growth pH. Nevertheless, this activity was inhibited by the valinomycin in acid-adapted cells compared to unadapted cells. Valinomycin renders the plasma membrane permeable to potassium ions, though nigericin exchanges potassium for protons, so that the combined actions of these compounds result in an equilibration of both potassium ions and protons across the membrane and thus the ΔpH. Comparable results were observed in Lactobacillus sp. showing that nigericin (2 lmol L À1 ) inhibits completely the ATPase activity, whereas this activity was not inhibited by valinomycin. In the presence of both ionophores, the ATPase activity was inhibited more than 50% in B.
cereus cells whatever the culture pH. However, the results obtained in Lactobacillus sp. show that the activity was completely inhibited (99%) in the presence of valinomycin and nigericin (0.2 lmol L À1 ) (Higuchi et al. 1997). Likewise, it was observed that ATPase activity was inhibited in Staphylococcus aureus by DCCD (20 lmol L À1 ) and by nigericin (0.5 lmol L À1 ) (Mandel et al. 1983). Thereafter, we also demonstrate that the population decrease during acid shock of acid-adapted cells is greatly affected by ionophores nigericin and valinomycin. So, our results suggest that ATPase activity can be implicated in the acid survival of B. cereus.
To better comprehend B. cereus ATR mechanisms at both physiological and genetic levels, it is particularly important to characterize the particular behavior of B. cereus in low pH environments. Acid resistance in Grampositive bacteria involves several strategies, including mechanisms of pH i homeostasis (Cotter and Hill 2003). In B. cereus, we have observed that the pH i decreased with growth pH e while maintaining a pH i compatible with cells physiology (Thomassin et al. 2006;Senouci-Rezkallah et al. 2011). This suggested that pH i maintenance may be involved in acid resistance of B. cereus. This relatively limited decrease in pH i , together with the corresponding increase in DpH, could prevent an even more dramatic decline in pH i at lower pH e . These results suggest that a mechanism of pH i homeostasis is induced at low pH (pH 5.5). Similar results were observed in Mycobacterium smegmatis and Mycobacterium bovis BCG, and the lethal pH i for both strains was less than pH 6.0 (Rao et al. 2001). Browne and Dowds confirmed a similar adaptation of B. cereus NCIMB11796 in nonregulated batch cultures, where cells were found to maintain their pH i at a higher level than the external acid pH e (Browne and Dowds 2002). Comparable results demonstrate that pH i regulation is involved in acid resistance in B. cereus ATCC14579 (Senouci-Rezkallah et al. 2011). We also showed that the pH i values in B. cereus acid-adapted and unadapted cells were greatly decreased by the DCCD treatment. Whereas, the dpH value of acid-adapted cells treated by DCCD is not the one observed in unadapted cells after acid shock. This suggests that other mechanisms of pH i homeostasis were induced in acid-adapted. Conversely, it was shown that the pH i of M. bovis BCG adapted cells grown at pH 5,0 is decreased in the presence of DCCD, but not affected in cells grown at neutral pH (7.0) (Rao et al. 2001). Thus, our data indicate that F 1 F 0 -ATPase activity plays a major role in acid resistance of acid-adapted cells of B. cereus, suggesting that this enzyme may be involved in pH i maintenance. Therefore, ATPase activity may be involved in pH i homeostasis, dpH maintenance and acid resistance in B. cereus acid-adapted cells. Clearly, the increase in proton translocation by ATPase activity could enhance the ability of B. cereus cells to maintain their pH i and their acid resistance. Thomassin et al. (2006) showed that DpH abolition may not allow essential metabolic activities and/or the activity of proteins that are essential for ATR (such as F 1 F 0 -ATPase). The permeability of the cytoplasmic membrane to protons and proton extrusion by F 1 F 0 -ATPase has been established as essential for pH i maintenance in mycobacteria (Rao et al. 2001). Our results show that the ATPase activity in unadapted B. cereus cells appears to increase the ATR. In acid-adapted cells, this system may be induced before and during acid shock. We establish therefore that pH i maintenance at low pH by ATPase activity is important to B. cereus ATCC14579 ATR through the protons extrusion via ATP hydrolysis.
Since B. cereus is a facultative anaerobic bacterium, it could be supposed that this bacterium may use both ATP hydrolysis and synthesis to maintain its pH homeostasis as shown in L. monocytogenes (Desriac et al. 2013). The low pH induces the expression of ATPase operon in B. cereus, since atpB mRNA was upregulated (threefold) in acid-adapted cells compared to unadapted cells. The overexpression of atpB gene was also observed after 10 min of acid shock at pH 4.0. Similar results of pH-dependent increase in ATPase transcription were observed in S. mutans, L. monocytogenes, L. acidophilus and O. oeni (Kullen and Klaenhammer 1999;Cotter et al. 2000;Quivey et al. 2001). In fact, F 1 F 0 -ATPase encoding genes were downregulated in B. cereus ATCC14579 and B. cereus ATCC10987 exposed to nonlethal acid conditions, and were not repressed upon exposure to lethal acid stresses, indicating that B. cereus does not use F 1 F 0 -ATPase to extrude proton in aerobic conditions (Mols et al. 2010a, b). Genes encoding subunits of the F 1 F 0 -ATPase were highly upregulated in anaerobically grown in a chemostat culture and exposed B. cereus cells upon exposure to sublethal pH e . Conversely, B. cereus does not use F 1 F 0 -ATPase to pump protons out of the cell in aerobic acid conditions and by repressing F 1 F 0 -ATPase genes and lowering the amount of active ATPase, the influx of protons is limited (Mols et al. 2010a,b;Mols and Abee 2011). These results suggest that B. cereus ATCC14579 is able to modify atp expression and ATPase activity as response to cytoplasmic pH variations and aerobic or anaerobic condition. Furthermore, downregulation of F 1 F 0 -ATPase genes could be explained by the cells trying to prevent excessive inward flux of proton via this ATPase upon exposure to acid conditions (Mols et al. 2010b). This downregulation has also been demonstrated in S. aureus where the expression of F 1 F 0 -ATPase encoding genes was clearly reduced to about 50% (Bore et al. 2007).
We have also demonstrated that atpB mRNA was downregulated after 40 min of acid shock. Downregula-tion of F 1 F 0 -ATPase is best described either by the population reduction or by the translation of mRNA upon exposure to acid conditions. The ATPase could still play an important role by pumping H+ out of the cells, such as Arikado et al. (1999) suggest that the enzyme regulation happens mostly at the posttranscriptional level. It was also shown that the regulation of the enzyme level of F 1 F 0 -ATPase by the intracellular pH, in S. faecalis, is mainly at the step of enzyme assembly from its subunits. Consequently, F 1 F 0 -ATPase and antiporters gene regulations under lethal and nonlethal conditions in B. cereus cells showed a good equilibrium between ATP synthesis on one hand and proton pumps regulating pH i at the expense of ATP on the other hand (Mols and Abee 2011).
In summary, our study shows that B. cereus is able to survive under acid conditions, because it can develop acid survival strategies involving ATPase activity to face severe acid stress. The ATPase activity may protect it against severe acid stress in two ways. Inducing protons extrusion via ATP hydrolysis would produce a less acidic internal pH and generate a positive DpH that could help repel protons. This system is of great importance in ATRinduced in B. cereus. Complementary work in progress is to construct the ATPase mutant in order to confirm the ATPase activity implication in ATR and pH i maintain of B. cereus ATCC14579.