Molecular aspects of bacterial pH sensing and homeostasis

Abstract

Diverse mechanisms for pH sensing and cytoplasmic pH homeostasis enable most bacteria to tolerate or grow at external pH values that are outside the cytoplasmic pH range they must maintain for growth. The most extreme cases are exemplified by the extremophiles that inhabit environments with a pH of below 3 or above 11. Here, we describe how recent insights into the structure and function of key molecules and their regulators reveal novel strategies of bacterial pH homeostasis. These insights may help us to target certain pathogens more accurately and to harness the capacities of environmental bacteria more efficiently.

Figure 1. Examples of adaptations by neutralophilic bacteria to manage acid or alkali challenge

a Left, Selected E. coli adaptations supporting acid tolerance during passage through the stomach. Right, Strategies proposed for the non-respiratory oral bacterium Streptococcus mutans. b Left, E. coli adaptations supporting alkali-tolerance. Right, Adaptations of non-respiratory Enterococcus hirae (formerly Streptococcus faecalis) supporting alkali-tolerance. See text for description of these adaptations and of additional examples.

Figure 2. Functional organization of the E. coli Na⁺/H⁺ antiporter NhaA

a The overall architecture of NhaA with its 12 trans-membrane segments (TMS) and funnels (black line) is shown. b The residues whose mutation affects the pH response (magenta), the pH response and the translocation (yellow) or the translocation alone (black) are shown on the structure. c The inverted repeat including TMS III, IV, V and TMS X,XI,XII are shown and the discontinuous helices (IV and XI assembly) are colored green. d Schematic cartoon illustrating the conformational changes caused by pH activation and ion transport. The pH sensor on TMS IX (double red lines) is marked.

Figure 3. Periplasmic buffering by H. pylori and its regulation

a Periplasmic buffering by H. pylori. Urea crosses the outer membrane and then the inner membrane via UreI at pH < 6.0. Cytoplasmic urease forms 2NH₃ + H₂CO₃. The latter is converted to CO₂ by cytoplasmic β-carbonic anhydrase. These gases cross the inner membrane and the CO₂ is converted to HCO₃⁻by the membrane bound α-carbonic anhydrase thereby maintaining periplasmic pH at ~6.1, the effective pK_a of the CO₂/HCO₃⁻couple. Exiting NH₃ neutralizes the H⁺ produced by carbonic anhydrase and entering H⁺ and can also exit the outer membrane to alkalize the medium. This allows maintenance of periplasmic pH much higher than medium pH ^,,. b The role of the pH-responsive TCS HP0244 in acid acclimation by H. pylori. Activation of this TCS results in recruitment of the urease proteins to UreI with immediate access of urea to urease and outward transport of CO₂ and NH₃ and NH₄⁺ through UreI increasing the rate of periplasmic buffering and disposal of cytoplasmic NH₄^{+ ,,,}. c A simplified model representing the HP0165-HP0166 TCS regulation of ureAB gene expression by unphosphorylated HP0166 binding to 5′ureB-sRNA at neutral pH and by phosphorylated HP0166 binding to P_ureA at acidic pH. At neutral pH, HP0165 is not activated and the response regulator HP0166 is not phosphorylated. The unphosphorylated HP0166 binds to the 5′ureB-sRNA promoter, leading to transcription of 5′ureB-sRNA and consequent truncation of ureB resulting in a decline of urease activity. This reflects adaptation to non-acidic pH. At acidic pH, HP0165 is activated with phosphorylation of HP0166 and the phosphorylated HP0166 then binds to the P_ureA promoter to positively regulate the transcription of ureAB genes which results in up regulation of ureA and ureB with a consequent increase of urease activity, reflecting acid acclimation. Antisense sRNAs are shown in red, mRNAs in dark blue. Red bent arrows denote promoters, orange and green bars with arrows denote ureAB gene and antisense 5′ureB-sRNA gene, respectively. A bold lightning sign indicates action of RNase III or RNase E. Positive signs ‘+’ denote positive regulation, Question mark ‘?’ denotes not yet experimentally confirmed. Yellow ovals represent HP0166 binding sites .

Figure 4. The hetero-oligomeric Mrp antiporter and other major pH homeostasis strategies of extremely alkaliphilic B. pseudofirmus OF4

a Top: Diagram of the 7-gene mrp operon of alkaliphilic Bacillus sp. indicates oxidoreductase NDH-1 domains in MrpA and MrpD (light gray) and MrpB domains in MrpA and MrpB (dark gray). Bottom: Schematic illustration of the oxidoreductase NDH-1 domains in MrpA, MrpD, and Complex I NuoL, M and N and the MrpB domains in MrpA and MrpB. Red vertical lines mark locations of conserved, functionally important glutamates and lysines^,,,. b Schematic diagram of B. pseudofirmus OF4 showing external and cytoplasmic pH and Δψ, secondary cell wall polymers (SCWP, e.g. S-layer in B. pseudofirmus OF4) and Na⁺ and H⁺ cycles that support pH homeostasis. Active proton uptake occurs via the critical Mrp antiporter, other antiporters and ATP synthase. The energetic driving force is from two proton-pumping respiratory complexes. The dashed lines indicate hypothesized capture of protons by the ATP synthase near the membrane surface, before they fully equilibrate with the outside liquid phase. Na⁺ re-entry, which supports continuous antiport, occurs through Na⁺/solute symporters, the voltage-gated Na_VBP channel and flagellar-associated MotPS channel. c Top: Two motifs of c-subunits in alkaliphilic Bacillus species (names shaded in blue) exemplify adaptations of the ATP synthase required for function at high pH. Bottom: The c-subunit motifs reduce the pronounced hour-glass shape of the overall c- rotor observed for other c-rotors (left) and promote tight proton binding in the ion binding site (right) (see text).