Formation of Proton Motive Force Under Low-Aeration Alkaline Conditions in Alkaliphilic Bacteria

Abstract

In Mitchell's chemiosmotic theory, a proton (H⁺) motive force across the membrane (Δp), generated by the respiratory chain, drives F₁F_o-ATPase for ATP production in various organisms. The bulk-base chemiosmotic theory cannot account for ATP production in alkaliphilic bacteria. However, alkaliphiles thrive in environments with a H⁺ concentrations that are one-thousandth (ca. pH 10) the concentration required by neutralophiles. This situation is similar to the production of electricity by hydroelectric turbines under conditions of very limited water. Alkaliphiles manage their metabolism via various strategies involving the cell wall structure, solute transport systems and molecular mechanisms on the outer surface membrane. Our experimental results indicate that efficient ATP production in alkaliphilic Bacillus spp. is attributable to a high membrane electrical potential (ΔΨ) generated for an attractive force for H⁺ on the outer surface membrane. In addition, the enhanced F₁F_o-ATPase driving force per H⁺ is derived from the high ΔΨ. However, it is difficult to explain the reasons for high ΔΨ formation based on the respiratory rate. The Donnan effect (which is observed when charged particles that are unable to pass through a semipermeable membrane create an uneven electrical charge) likely contributes to the formation of the high ΔΨ because the intracellular negative ion capacities of alkaliphiles are much higher than those of neutralophiles. There are several variations in the adaptation to alkaline environments by bacteria. However, it could be difficult to utilize high ΔΨ in the low aeration condition due to the low activity of respiration. To explain the efficient ATP production occurring in H⁺-less and air-limited environments in alkaliphilic bacteria, we propose a cytochrome c-associated "H⁺ capacitor mechanism" as an alkaline adaptation strategy. As an outer surface protein, cytochrome c-550 from Bacillus clarkii possesses an extra Asn-rich segment between the region anchored to the membrane and the main body of the cytochrome c. This structure may contribute to the formation of the proton-binding network to transfer H⁺ at the outer surface membrane in obligate alkaliphiles. The H⁺ capacitor mechanism is further enhanced under low-aeration conditions in both alkaliphilic Bacillus spp. and the Gram-negative alkaliphile Pseudomonas alcaliphila.

Keywords: Bacillus; Donnan effect; Pseudomonas; alkaliphilic; bioenergetic mechanism; cytochrome c; membrane electrical potential; proton condenser.

FIGURE 1

Cytochrome content of cell extracts of Gram-positive obligately alkaliphilic Bacillus clarkii K24-1U (grown at pH 10) under low-aeration and high-aeration conditions. HA, HB, and HC are the cytochrome a, b, and c levels, respectively, under high-aeration conditions. LA, LB, and LC are the cytochrome a, b, and c levels, respectively, under low-aeration conditions. This difference in cytochrome c content may indicate that cytochrome c has an important function under low-aeration conditions at pH 10. Low-aeration conditions were produced by using 15 L of a medium in a 20-L stainless-steel fermenter (Takasugi Seisakusho, Tokyo, Japan) with an agitation speed of 106 rpm and an air flow rate of 20 Lmin^-1, while high-aeration conditions were produced by using 15 L of a medium in a 30-L stainless-steel fermenter (Marubishi, Tokyo, Japan) with an agitation speed of 250 rpm and an air flow rate of 20 Lmin^-1. This figure was made according to the data of Hijikata (2004).

FIGURE 2

Hypothetical model of the function of membrane-bound cytochrome c-550 in the respiratory system of B. clarkii K24-1U. Cytochrome c-550 contains the specific structure Gly₂₂-Asn₃₄ (Asn-rich) at the N-terminal region of its sequence, which may facilitate H⁺ transfer at the interface of the outer surface membrane. The tetrameric structure is predicted to be important for enhancement of the H-bound network. The production of cytochrome c-550 was enhanced under low-aeration conditions. This enhanced cytochrome c-550 on the outer surface of the membrane led to the accumulation of electrons, H⁺ and the H⁺-condenser construct. This structure facilitates the growth of the microorganism, especially under high-pH and low-aeration conditions. This figure was produced as an original hypothetical model for this review.

FIGURE 3

Growth characteristics of the facultative alkaliphile Pseudomonas alcaliphila AL15-1^T (wild type: blue symbols) and the cytochrome c-522 deletion mutant derived from the wild-type strain (mutant: red symbols) under high-aeration (A) and low-aeration (B) conditions at pH 10 (squares) and pH 7 (circles). The reproducibility of the results was confirmed by performing three independent experiments. This figure was reproduced from Matsuno and Yumoto (2015).

FIGURE 4

Hypothetical model of the function of cytochrome c-552 in the respiratory system of the Gram-negative facultative alkaliphilic P. alcaliphila AL15-1^T. This bacterium possesses cytochrome c-552 (monoheme), cytochrome c-554 (monoheme), and cytochrome 551 (diheme), all of which tend to retain the reduced state. Cytochrome c-552 is a major soluble cytochrome c constituent in the periplasmic space. The features of cytochrome c-552 cause in high electron retention and attract H⁺, and the development of electron- and H⁺ -retention conditions occurs in the periplasmic space of microorganisms concomitant with an abundance of this cytochrome c under low-aeration conditions at pH 10. Thus, Gram-negative P. alcaliphila AL15-1^T expresses an electron and H⁺ condenser in the periplasmic space. This figure was reproduced based on a hypothetical model presented in Matsuno and Yumoto (2015).