Cytoplasmic pH Response to Acid Stress in Individual Cells of Escherichia coli and Bacillus subtilis Observed by Fluorescence Ratio Imaging Microscopy

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>The ability of<jats:named-content content-type="genus-species">Escherichia coli</jats:named-content>and<jats:named-content content-type="genus-species">Bacillus subtilis</jats:named-content>to regulate their cytoplasmic pH is well studied in cell suspensions but is poorly understood in individual adherent cells and biofilms. We observed the cytoplasmic pH of individual cells using ratiometric pHluorin. A standard curve equating the fluorescence ratio with pH was obtained by perfusion at a range of external pH 5.0 to 9.0, with uncouplers that collapse the transmembrane pH difference. Adherent cells were acid stressed by switching the perfusion medium from pH 7.5 to pH 5.5. The<jats:named-content content-type="genus-species">E. coli</jats:named-content>cytoplasmic pH fell to a value that varied among individual cells (range of pH 6.2 to 6.8), but a majority of cells recovered (to pH 7.0 to 7.5) within 2 min. In an<jats:named-content content-type="genus-species">E. coli</jats:named-content>biofilm, cells shifted from pH 7.5 to pH 5.5 failed to recover cytoplasmic pH. Following a smaller shift (from pH 7.5 to pH 6.0), most biofilm cells recovered fully, although the pH decreased further than that of isolated adherent cells, and recovery took longer (7 min or longer). Some biofilm cells began to recover pH and then failed, a response not seen in isolated cells.<jats:named-content content-type="genus-species">B. subtilis</jats:named-content>cells were acid shifted from pH 7.5 to pH 6.0. In<jats:named-content content-type="genus-species">B. subtilis</jats:named-content>, unlike the case with<jats:named-content content-type="genus-species">E. coli</jats:named-content>, cytoplasmic pH showed no “overshoot” but fell to a level that was maintained. This level of cytoplasmic pH post-acid shift varied among individual<jats:named-content content-type="genus-species">B. subtilis</jats:named-content>cells (range of pH, 7.0 to 7.7). Overall, the cytoplasmic pHs of individual bacteria show important variation in the acid stress response, including novel responses in biofilms.</jats:p>

pH determines the energetic efficiency of the cyanobacterial CO 2 concentrating mechanism

“…All cells regulate their cytosolic pH due to the inherent pH dependence of biochemical reactions (27). However, cytosolic pH varies substantially between organisms and growth conditions (28,29). Here, we use a mathematical model to examine how the cyanobacterial CCM functions over a range of cytosolic pH values.…”

Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli

“…In addition, other experiments demonstrated that cell homeostasis responses [86][87][88], and the recombinant protein production process occur in a similar time order [99]. Then, we can hypothesize that during those cell responses, cellular microenvironments favor the nucleation and formation of proto-aggregates, which later allow the formation of IBs.…”

“…The cytoplasmic pH is normally maintained within a range of 7.4 to 7.8 when the external pH is in the range of 5.0 to 9.0 in suspension or 5.0 to 8.0 in adherent E. coli cultures [56,[85][86][87][88]. In adherent E. coli cultures the cytoplasmic pH is similar to the external pH of 8.5 to 9.0 [87].…”

Ratiometric Imaging of pH Probes

“…The pH-sensitive polar fluorescein derivative 2 0 ,7 0 -bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF) remains the most widely used pHi indicator. However, experimental limitations imposed by pH-sensitive dyes have more recently led to the development of genetically encoded pH biosensors derived from the jellyfish Aequorea victoria green fluorescent protein (GFP; Martinez et al, 2012;Miesenböck, De Angelis, & Rothman, 1998;Wilks & Slonczewski, 2007). Together, these pH-sensitive dyes and biosensors can be imaged using most standard fluorescence microscopes to measure steadystate and dynamic changes in pH within cells.…”

“…In general, cytoplasmic pH in most organisms from bacteria (Martinez et al, 2012;Wilks & Slonczewski, 2007) to yeasts (Bagar, Altenbach, Read, & Bencina, 2009;Grynkiewicz et al, 1985) to mammalian cells (Fig. 23.1) is slightly basic (pH is $7.0-7.8), with the exception of some acidophilic or basophilic bacteria (Paradiso et al, 1984;Slonczewski, Fujisawa, Dopson, & Krulwich, 2009).…”