{'fraction_non_alphanumeric': 0.036775778711262586, 'fraction_numerical': 0.019418245224696838, 'mean_word_length': 4.531784305129329, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 6, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
pH Stress Alters Cytoplasmic Membrane Fluidity and atpB Gene Expression in Streptococcus mutans

Streptococcus mutans (S. mutans), which plays a major role in the etiology of human dental caries, is able to tolerate exposure to acid shock in addition to its acidogenicity. We investigated the effects of pH stress on membrane fluidity, activities and expression levels of F-ATPase, and proton permeability in S. mutans. Using 1,6-diphenyl-1,3,5-hexatriene, we observed membrane ordering at pH 4.8 and pH 8.8. The ordering effects were larger at pH 4.8 in cytoplasmic membranes isolated from S. mutans (CMSM). Increasing pH resulted in a decrease in the activities and expression levels of F-ATPase. The proton permeability was decreased at both acidic and alkaline pHs, and the lowest permeability was observed at pH 4.8. The lower permeability at pH 8.8 than pH 6.8 is likely to be caused by the decreased proton influx due to the decreased CMSM fluidity. In addition, it seems to be evident that extremely low permeability at pH 4.8 was caused by the decreased proton influx due to the decreased CMSM fluidity as well as the increased proton efflux due to the increased activity and expression level of F-ATPase. It is likely that CMSM fluidity and F-ATPase activity are two major key factors that determine proton permeability in S. mutans. We suggest that CMSM fluidity plays an important role in the determination of proton permeability, which sheds light on the possibility of using nonspecific membrane fluidizers, e.g., ethanol, for anti-caries purposes.


Introduction
Today, dental caries remains one of the most common diseases afflicting humans throughout the world. Although 200-300 bacterial species have been found associated with dental biofilm, only the presence of Streptococcus mutans (S. mutans) has been consistently linked with the formation of human dental caries [17]. This gram-positive bacterium is able to demineralize dental enamel by generation of a localized acidic environment in plaque by rapid glycolysis of dietary sugars to lactic acid. In addition to its acidogenicity, S. mutans is able to tolerate exposure to continual and rapid cycles of acid shock [10]. It has been known that acid tolerance of S. mutans is primarily based on the presence of the membrane-bound, proton-pumping F-ATPase that can maintain the intracellular pH [5,7,11,16,25]. However, very few results have been reported for the expression of S. mutans F-ATPase in response to external pH.
The integrity of the bacterial cytoplasmic membrane is essential in maintaining the viability of cells and their metabolic functions particularly under stress. Bacteria actively adjust membrane fluidity through changes in lipid composition in response to alterations in temperature, pressure, ion concentrations, nutrient availability, xenobiotics and pH [19]. Unsaturated fatty acids (UFAs) have crucial roles in maintenance of membrane structure and function in many groups of aerobic bacteria. UFAs are key molecules in the regulation of cellular membrane fluidity. Quivey et al. [23] showed that S. mutans grown at pH 5.0 had increased levels of mono-unsaturated fatty acids and longer chain fatty acids than cells grown at pH 7.0. Fozo and Quivey [8] reported that as the growth pH is lowered there is an incremental change from short-chained, saturated fatty acids (C14:0 and C16:0) to long-chained, mono-unsaturated fatty acids (C18:1 and C20:1). However, no attempt has been made yet to determine membrane fluidity alterations in response to environmental acidification in S. mutans.
Proton permeability is an important factor permitting cells to maintain the proton-motive force (pmf) for vital energy transducing processes. pmf is essential for S. mutans activity.
In addition to its high-affinity phosphotransferase sugar transport system, S. mutans has the second, low-affinity sugar uptake system, which is activated by pmf [12]. This is why S. mutans can survive under acidic conditions which are unfavorable to other bacteria. It has been shown that the proton permeability correlates with the physical state of the membrane; in particular, the permeability increases with increasing membrane fluidity [26].
The aims of this research are threefold: (1) to study the effects of pH stress on the fluidity of the cytoplasmic membranes isolated from S. mutans (CMSM); (2) to check the expression levels of atpB gene of S. mutans under pH stress; and (3) to examine the relationship between proton permeability, CMSM fluidity, and F-ATPase. We observed CMSM ordering at both acidic and alkaline pHs, however, the ordering effects were larger at acidic pH. Our results suggest that CMSM fluidity plays a pivotal role in the viability of S. mutans under pH stress. Cytoplasmic contamination was assessed by assaying the presence of glucokinase by the method of Porter and Chassy [22]. Total protein contents of membranes were determined by the method of Lowry et al. [18].

Fluorescence Measurements
Aliquots of CMSM were suspended in phosphate buffer (pH 4.8, 6.8 or 8.8) at a protein concentration of 50 μg/ml. The anisotropy of DPH is calculated by where the intensity of the components of the fluorescence that were parallel (IVV) and perpendicular (IVH) to the direction of the vertically polarized excitation light was determined by measuring the emitted light through polarizers oriented vertically and horizontally. The grating correction factor G is given by the ratio of the fluorescence intensities of the vertical (IHV) to horizontal (IHH) components when the exciting light is polarized in the horizontal direction.

F-ATPase assay
CMSM were added to Tris-maleate buffer (pH 7.0) with 10 mM MgSO4, and the mixtures were warmed to 37°C. The ATPase reaction was initiated by addition of ATP (pH 7.0) and incubated at 37°C for 15 min. The reaction was stopped by adding 10% trichloroacetic acid. As the negative control, we did the same procedure, but added TCA at time zero in order to inactivate the enzyme first. The released phosphate was determined using the method of Bencini et al. [6].

Real-time PCR
The resulting cDNA and negative controls were amplified by ABI PRISM 7700 real-time PCR system with SYBR Green Each reaction tube contained 20 μl of reaction mixture, including 1X SYBR Green PCR Master Mix, cDNA, template, and 2 μl of 10 pM forward and reverse primers. The cycle profile was as follows: 1 cycle at 50°C for 5 min and initial denaturation at 95°C for 10 min, followed by 40 cycle amplification consisting of denaturation at 95°C for 15 s, annealing at 54°C for 30 s, and extension at 72°C for 30 s.
The relative expression was calculated by normalizing the mRNA of the atpB and atpD genes with that of the 16S rRNA gene, which served as the reference gene.

Proton flux assay
The proton permeability was assessed using the procedures described previously [4,21].

Statistical analysis
The data are presented as mean ± SEM. Student's t-test was performed to determine the significant difference. Values were considered statistically significant when P value was <0.05. The statistical analyses were performed using IBM SPSS Statistics 23 (SPSS Inc., Chicago, IL, USA).

Results and Discussion
Effects of pH stress on the fluidity of CMSM First of all, we compared the growth rates of S. mutans cultured under pH stress. The measured growth rates, calculated as c = Δlog2(OD420 ×1,000)/Δt, are depicted in Fig. 1A.
It is seen that pH stress did not allow a full recovery of the growth rate measured at optimum pH of 6.8. Compared to pH 6.8 (c=0.0352 min -1 ), the growth rates were reduced to 0.0119 and 0.  Membrane ordering at acidic pH is consistent with the results obtained previously using Oenococcus oeni [19] and Bacillus subtilis [20]. However, Bacillus subtilis showed membrane fluidization by alkaline pH [20], which does not coincide with our result. We assume that membrane effects of pH are different depending on the organisms.
Among the nine combinations of culture and buffer pHs, the CMSM with culture and buffer pH 6.8 were the most fluid. On the other hand, CMSM were the most ordered at culture pH 8.8 and buffer pH 4.8. When the CMSM of culture pH 4.8 were exposed to pH 8.8 buffer, the extent of CMSM ordering was much smaller. This may be due to the acid tolerance of S. mutans. Buffer pH resulted in a comparable fluidity change to culture pH. In view of the fact that CMSM are composed of not only lipids but also proteins, it is highly probable that the pH changes result in a significant change in protein conformations as well. Effects of pH stress on the F-ATPase activities and the expression levels of the atpB gene in S. mutans Although recent studies have shown that a large proportion of the S. mutans genome is required for acid tolerance [2,3,24], the F-ATPase is still considered as one of the key components of the acid tolerance response. The F-ATPase activity is critical for establishing and maintaining pmf across CMSM. The F-ATPase activity might be influenced by CMSM ordering under pH stress. The order of F-ATPase activity was: pH 4.8 > pH 6.8 > pH 8.8 ( Fig. 2A).

A B
Our results agree with those of others who showed increased levels of F-ATPase activity in acidic environment [5,7,11,16,25]. The lowest F-ATPase activity was obtained with CMSM from S. mutans cultured at pH 8.8. The F-ATPase operon promoter [13,15] and atp genes [2,9] of S. mutans were transcriptionally upregulated at acidic pH. We checked the relative quantities of mRNA expression of the atpB gene which encodes the catalytic β-subunit of F-ATPase (Fig. 2B). The mRNA expression levels were: pH 4.8 > pH 6.8 > pH 8.8. The S. mutans F-ATPase was transcriptionally upregulated at pH 4.8 and downregulated at pH 8.8. Similar results were obtained for the expression of atpD (regulatory α-subunit gene). Increasing pH resulted in a decrease in the activities and the expression levels of F-ATPase.
Relationship between fluidity, proton permeability and F-ATPase activity Petrackova et al. [20] reported decreased proton permeability at both acidic and alkaline pH in Bacillus subtilis.
Hence, it is highly probable that the above rigidification is related to CMSM's permeability to protons and/or other ions. We performed an acidic pH-jump assay in intact cells adapted to pH 4.8, 6.8 and 8.8. Fig. 3A shows the results of the proton flux assay. It can be seen that, following the initial pH pulse, the pH of the cell suspension starts to gradually increase as a consequence of cellular proton uptake.
The rate of the pH increase was found to be the highest for S. mutans cultivated at pH 6.8 and the lowest at pH 4.8.
We examined the relationship between cytoplasmic membrane fluidity, F-ATPase activity and proton permeability in S. mutans. The upper model of Fig. 3B shows the proton flux in S. mutans when we consider only F-ATPase activity.
Without considering CMSM fluidity, equal amounts of protons will pass through CMSM at all three pHs. Under these circumstances, the order of proton permeability will be: pH 8.8 > pH 6.8 > pH 4.8. Of course, this was not the case. Under our experimental conditions, S. mutans grown at pH 6.8 showed the highest proton permeability (Fig. 3A). The lower permeability at pH 8.8 than pH 6.8 is likely to be caused by decreased proton influx due to decreased CMSM fluidity. The decreased proton influx seems to offset the effect of the lowest proton efflux. In addition, very low permeability of S. mutans grown at pH 4.8 can be better explained when considering CMSM fluidity together (lower model in proton permeability in S. mutans.
The suggestion that CMSM fluidity plays an important role in determination of proton permeability sheds light on the possibility of the use of nonspecific membrane fluidizers, e.g., ethanol, for anti-caries purposes. Ethanol might influence S. mutans viability, or, at least, be used as an adjunct to fluoride dentifrices and anti-plaque mouthrinses. However, it should be pointed out that we used S. mutans in suspensions. More convincing results can be obtained using S. mutans in biofilms which contain the extracellular polysaccharide matrix [14]. Additionally, only steady-state fluorescence measurements were performed. Time-resolved anisotropy decay will give us information about the rate and range of rotational motions of DPH within CMSM.
To our knowledge, this report is the first to examine CMSM fluidity and to elucidate the relationship between membrane fluidity, the activities and the expression levels of F-ATPase, and proton permeability in S. mutans under pH stress.