Cytoplasmic acidification and the benzoate transcriptome in Bacillus subtilis

PLoS One. 2009 Dec 14;4(12):e8255. doi: 10.1371/journal.pone.0008255.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacillus subtilis / cytology
  • Bacillus subtilis / drug effects*
  • Bacillus subtilis / genetics*
  • Bacillus subtilis / growth & development
  • Benzoates / pharmacology*
  • Cluster Analysis
  • Cytoplasm / drug effects
  • Cytoplasm / metabolism*
  • Down-Regulation / drug effects
  • Gene Expression Profiling*
  • Gene Expression Regulation, Bacterial / drug effects*
  • Green Fluorescent Proteins / metabolism
  • Hydrochloric Acid / pharmacology*
  • Hydrogen-Ion Concentration / drug effects
  • Operon / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Stress, Physiological / drug effects
  • Up-Regulation / drug effects


  • Benzoates
  • Green Fluorescent Proteins
  • Hydrochloric Acid