Research Article: Cytoplasmic Acidification and the Benzoate Transcriptome in Bacillus subtilis

Date Published: December 14, 2009

Publisher: Public Library of Science

Author(s): Ryan D. Kitko, Rebecca L. Cleeton, Erin I. Armentrout, Grace E. Lee, Ken Noguchi, Melanie B. Berkmen, Brian D. Jones, Joan L. Slonczewski, Malcolm James Horsburgh.

Abstract: 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.

Partial Text: Bacillus subtilis encounters a wide range of extracellular pH values during growth in various environments, including diverse soils, association with plants, and within the gastrointestinal tract [1], [2]. The bacteria maintain cytoplasmic pH within a narrow range that allows for the stability of nucleic acids and proteins and subsequent growth over several log units of environmental pH [3]–[5]. Bacillus species respond to environmental pH in ways that are important for pathogenesis. For example, food-borne B. cereus encounters acidic environments in the gastrointestinal tract and in food products [6]–[9]. Low external pH magnifies the inhibitory effects of membrane-permeant organic acids such as acetic, benzoic and salicylic acids; but the relationship between pH and the organic compound is often unclear in the literature [9]–[11]. A permeant acid stresses the cell by importing protons, depressing cytoplasmic pH, and by concentrating the organic anion within the cytoplasm in proportion to the transmembrane pH difference (ΔpH) [12]–[16]. Thus, the effects of external pH, cytoplasmic pH, and any exogenous or endogenous permeant acids must be considered together.

We present the first dynamic measurements of B. subtilis cytoplasmic pH on a rapid time scale, under conditions of acid stress and benzoate stress. Bacteria were cultured in a broth medium (LBK) which, based on our long experience, provides a large range of potential metabolic options for reversing pH stress [5]. The resuspension medium for fluorimentry (supplemented M63) differs somewhat in composition from the growth medium, but includes multiple amino acids available as in LBK. Our results for rapid acid treatment and for steady-state growth in LBK with acid stress were generally consistent, in that the cytoplasmic pH levels obtained following extended growth in buffered LBK were similar to those following a rapid external pH shift after resuspension in supplemented M63 (Figs. 2, 3).



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