Cytoplasmic acidification and the benzoate transcriptome in Bacillus subtilis

Abstract

Background: Bacillus subtilis encounters a wide range of environmental pH. The bacteria maintain cytoplasmic pH within a narrow range. Response to acid stress is a poorly understood function of external pH and of permeant acids that conduct protons into the cytoplasm.

Methods and principal findings: Cytoplasmic acidification and the benzoate transcriptome were observed in Bacillus subtilis. Cytoplasmic pH was measured with 4-s time resolution using GFPmut3b fluorimetry. Rapid external acidification (pH 7.5 to 6.0) acidified the B. subtilis cytoplasm, followed by partial recovery. Benzoate addition up to 60 mM at external pH 7 depressed cytoplasmic pH but left a transmembrane Delta pH permitting growth; this robust adaptation to benzoate exceeds that seen in E. coli. Cytoplasmic pH was depressed by 0.3 units during growth with 30 mM benzoate. The transcriptome of benzoate-adapted cells was determined by comparing 4,095 gene expression indices following growth at pH 7, +/- 30 mM benzoate. 164 ORFs showed > or = 2-fold up-regulation by benzoate (30 mM benzoate/0 mM), and 102 ORFs showed > or = 2-fold down-regulation. 42% of benzoate-dependent genes are regulated up or down, respectively, at pH 6 versus pH 7; they are candidates for cytoplasmic pH response. Acid-stress genes up-regulated by benzoate included drug resistance genes (yhbI, yhcA, yuxJ, ywoGH); an oligopeptide transporter (opp); glycine catabolism (gcvPA-PB); acetate degradation (acsA); dehydrogenases (ald, fdhD, serA, yrhEFG, yjgCD); the TCA cycle (citZ, icd, mdh, sucD); and oxidative stress (OYE-family yqjM, ohrB). Base-stress genes down-regulated by benzoate included malate metabolism (maeN), sporulation control (spo0M, spo0E), and the SigW alkali shock regulon. Cytoplasmic pH could mediate alkali-shock induction of SigW.

Conclusions: B. subtilis maintains partial pH homeostasis during growth, and withstands high concentrations of permeant acid stress, higher than for gram-negative neutralophile E. coli. The benzoate adaptation transcriptome substantially overlaps that of external acid, contributing to a cytoplasmic pH transcriptome.

Figure 1. Fluorescence signal as a function of cytoplasmic pH.

B. subtilis MMB1311 was cultured in buffered LBK to OD₆₀₀ 0.2 and resuspended at OD₆₀₀ 0.4 in supplemented M63 medium buffered as described in the Materials and Methods, with inclusion of 10 µM nigericin. Fluorescence intensity was summed over the excitation range of 480–510 nm, with emission at 545 nm. The mean intensity for three independently grown cultures is shown, with error bars representing SEM.

Figure 2. Effect of external acid shift on cytoplasmic pH.

MMB1311 was cultured in buffered LBK, then resuspended at OD₆₀₀ 0.4 in M63 supplemented with casein hydrolysate (5 mM MES, 5 mM MOPS, pH 7.5). At 2 min, 15.5 mM HCl was added to shift the external pH from pH 7.5 to pH 6.0. (15.5 mM KCl was used as a control for volume and osmotic effects). At 11–12 min, 10 µM nigericin was added to fully collapse ΔpH for the individual replicate standard curve calculations. To obtain a second standard curve point, 12.5 mM KOH was added to raise the pH for the HCl curves and 15.5 mM HCl was added to lower the pH for the KCl curves. Each condition is represented by two out of three independent cultures. Gaps in the data sets represent the time it took to set up for the next phase of the experiment. Fluorescence intensity was converted to pH units using the internal standard curves as described in Materials and Methods.

Figure 3. Effect of various concentrations of sodium benzoate on cytoplasmic pH.

MMB1311 was cultured in buffered LBK, then resuspended at OD₆₀₀ 0.4 in buffered M63 medium with casein hydrolysate. (A) Grown at pH 7.0 (50 mM MOPS); resuspended at pH 7.0 (5 mM MOPS). (B) Grown at pH 7.0 (50 mM MOPS) with or without 30 mM sodium benzoate; resuspended at pH 6.9 (5 mM MOPS) with or without sodium benzoate. (C) Grown at pH 7.0 (50 mM MOPS); resuspended at pH 7.0 (5 mM MOPS). (D) Grown at pH 6.0 (50 mM MES); resuspended at pH 6.0 (5 mM MES, 5 mM MOPS). For all panels, 10 µM nigericin was then added to fully collapse ΔpH for the individual replicate standard curve calculations. To obtain a second standard curve point, KOH (5.5 mM for A, B, and C, and 12.5 mM for D) was added to raise the pH. Each condition is represented by two out of three independent cultures.

Figure 4. Real-time PCR expression ratios of selected genes as a function of benzoate concentration.

B. subtilis strain AG174 overnight cultures in unbuffered LBK were diluted 500-fold into LBK buffered with 50 mM MOPS with 0, 10, or 30 mM sodium benzoate. RNA was isolated and mRNA expression for individual genes was quantified by real-time PCR using an ABI Prism 7500 DNA analyzer (Applied Biosystems) with SYBR Green one-step protocol. All expression levels are presented relative to the expression at 0 mM benzoate. Error bars represent the SEM (n = 3).

Figure 5. Cluster analysis of gene expression from the benzoate and acid transcriptomes.

Hierarchical clustering of probe sets found to be significantly regulated in both the benzoate and previous low pH transcriptomes. Bars in yellow and blue indicate genes that are up- or down-regulated, respectively. Color intensities are proportional to the variation of expression ranging from −2.5 to 2.5 as indicated by the color bar. The group of genes labeled A (vertical yellow bar) indicates those genes up-regulated more by benzoate than by acid and the group labeled B (orange bar) indicates those genes that are up-regulated more by acid than benzoate. The cluster labeled C (blue bar) includes genes that were down-regulated by both benzoate and external acid. A full list of genes included in this analysis can be found in Table S1C.