Measurement of cytoplasmic pH of the alkaliphile Bacillus lentus C-125 with a fluorescent pH probe

Abstract

A method was established to measure the cytoplasmic pH of the facultative alkaliphilic strain, Bacillus lentus C-125. The bacterium was loaded with a pH-sensitive f luorescent probe, 2',7'-bis-(2=carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF), and cytoplasmic pH was determined from the intensity of fluorescence of the intracellular BCECF. The activity of the organism to maintain neutral cytoplasmic pH was assessed by measuring the cytoplasmic pH of the cells exposed to various pH conditions. The cytoplasmic pH maintenance activity of G I 2 5 increased with increasing culture pH, indicating that the activity was regulated in response to the culture pH.

Contribution of the Cell Wall Component Teichuronopeptide to pH Homeostasis and Alkaliphily in the Alkaliphile Bacillus lentus C-125

“…The BCECF-loaded cells were incubated in buffers [0.1 M N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid-NaOH (pH 6.0 to 8.6) or 0.1 M glycine-NaOH (pH 8.0 to 12.0)] containing 0.1 M NaCl-0.1 M KCl-0.1% glucose. The intracellular BCECF was excited at 450 or 510 nm, and emission was measured at 535 nm (9). Immediately after measurement of fluorescence, the pH of the suspension was measured by means of a glass electrode, and then the suspension was centrifuged at 6,000 ϫ g for 5 min.…”

Identification of Antibacterial Property of Different Medicinal Plant Extracts

“…Natural products are typically secondary metabolites, produced by plants and microorganisms in response to external stimuli such as nutritional changes. They are widely used in the pharmaceutical industry for their remarkable structural diversity and range of pharmacological activities [3]. Plants have been used for centuries to treat infectious diseases and are considered as an important source of new antimicrobial agents [4].…”

Decrease in Cytoplasmic pH-Homeostastatic Activity of the AlkaliphileBacillus LentusC-125 by a Cell Wall Defect

“…Two parameters, the ‰uorescence intensity of BCECF at 510 nm (F510) and the ratio of the intensities at 510 and 450 nm (Ratio510 W 450 ), were used as measures of cytoplasmic pH. 13,18) Ionophore , and C-125-90 carrying a plasmid containing tupA (a broken line with a dot), that were reported previously, 5,14) are shown as references. 219 pH Homeostasis of Cell Wall-defective Mutants of Alkaliphile gramicidin (Sigma Chem.)…”

“…was used to relate the parameters to cytoplasmic pH for each batch of the cell suspension. 14) Cytoplasmic pH maintenance of the cell wall component-defective mutants in alkaline environments…”

“…12,13) BCECF was used to measure cytoplasmic pH of C-125 previously. 14) Estimation of cytoplasmic pH by ‰uorescence intensity of intracellular BCECF All the strains used are derivatives of C-125. Strain C-125-11 is a mutant lacking TUA.…”