Evidence that the cell wall of Bacillus subtilis is protonated during respiration

Abstract

Several independent experiments suggest that cell walls of Bacillus subtilis are protonated during growth. When cells were grown in the presence of fluorescein-labeled dextran to saturate the cell walls, centrifuged, and suspended in PBS, fluorescence-activated cell sorter analyses revealed the bacteria were only poorly fluorescent. In contrast, when the bacteria were purged with N(2) to dissipate protonmotive force (pmf) fluorescence became intense. Upon reconstitution of the pmf with phenazine methosulfate, glucose, and oxygen, fluorescence declined. Another approach used pH-dependent chemical modification of cell walls. The walls of respiring B. subtilis cells were amenable to carboxylate modification by [(14)C]ethanolamine and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The carbodiimide activation of carboxylate groups occurs only in acidic conditions. Upon dissipation of pmf the walls were refractory to chemical modification. Ammonium groups can be condensed with FITC in alkaline medium, but the condensation is very slow in acidic buffers. It was found that the derivatization of the walls with FITC could occur in the absence of pmf. The use of pH-dependent fluorophores and pH-dependent chemical modification reactions suggest that cell walls of respiring B. subtilis cells have a relatively low pH environment. This study shows a bacterium has a protonated compartment. Acidification of cell walls during growth may be one means of regulating autolytic enzymes.

Figure 1

Histograms of B. subtilis 168 cells using FACS. B. subtilis cells were labeled with FITC-dextran and purged with N₂ gas, and the pmf was reconstituted with phenazine methosulfate, glucose, and oxygen. Black line, unlabeled cells; green fill, cells labeled with FITC-dextran; orange line, cells labeled with FITC-dextran and purged with N₂ gas; blue line, cells labeled with FITC-dextran, purged with N₂ gas with the pmf reconstituted.

Figure 2

Coupling of FITC to B. subtilis 168 in the presence and absence of an energy poison. Cells in PBS-glucose buffer at pH 8.0 (A). Cells in acetate-glucose buffer at pH 5.0 (B). Energy poison was 100 μM DCCD. Black line, untreated control cells; green line, cells treated with FITC; orange line, cells subjected to DCCD and treated with FITC.

Figure 3

Fluorescence micrographs of B. subtilis 168 cells derivatized with FITC. Cells PBS (pH 8) treated with FITC (50 μg/3 ml of cells) (A). Cells in PBS purged with N₂ gas and then treated with FITC (50 μg/3 ml of cells) (B).

Figure 4

Modification of B. subtilis 168 by ethanolamine and EDAC. Cells in PBS-glucose buffer (pH 7.3) were mixed with EDAC and [¹⁴C]ethanolamine at room temperature in the presence and absence of 40 mM sodium azide. At intervals, samples were removed, rapidly filtered, washed, and counted by liquid scintillation. The results shown represent the average of three separate trials. The curves were generated by graphpad software.