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

A previous study suggested that cytochrome c has an important role in the adaptation to alkaline environments based on its abundance (Lewis et al., 1980; Guffanti et al., 1986; Yumoto et al., 1991, 1997). In addition, cytochrome c is likely located at the outer surface membrane and associated with H+ transfer in the vicinity of the membrane. Therefore, we attempted to elucidate the precise structure of membrane-bound cytochrome c in alkaliphilic Bacillus spp. An experiment was performed to isolate intact protein from the obligate alkaliphile B. clarkii K24-1U. Bacillus spp. generally exhibit strong protease activity, making it difficult to isolate intact cytochrome c from their cells. This strong protease activity often causes isolated cytochrome c to exhibit an anchor-less, soluble protein. First, an attempt was made to isolate obligate alkaliphilic Bacillus spp. that exhibit low protease activity. Screening was performed using soil samples from approximately 10 sites in the Hokkaido region of Japan. One strain, K24-1U, isolated from Yuubari in Hokkaido Japan (43°04′ N, 141°58′ E), was found to be an obligate alkaliphile that exhibited very weak protease activity. The cytochrome c-550 gene sequence was determined and cloned, and the protein was purified and characterized (Ogami et al., 2009). Purified cytochrome c-550 was attached to a diacylglycerol-cysteine moiety. According to the analyzed gene sequence of cytochrome c-550, a signal peptide was present at the 5′ end of the gene. During the processing of mature cytochrome c-550, expressed cytochrome c-550 was translocated to the extracellular side of the membrane by the signal peptide. After the signal peptide was dissociated, the terminal cystatin was modified by attaching diacylglycerol and acetyl moieties. Cytochrome c-550 binds to fatty acids with carbon lengths of C15, C16, and C17 via glycerol-Cys18.

Although the length of the internal carbon chain is always C15, the external chain length varies from C15-C17. The diacylglycerol moiety exhibits flexibility in its fatty acid molecular species. Therefore, if the expressed amount of cytochrome c-550 is dependent on the culture conditions, the fatty acid composition of the membrane may be minimally influenced.

The amino acid sequence of cytochrome c-550 was deduced from the gene sequence and aligned with the amino acid sequences of cytochromes c from obligate and facultative alkaliphilic and neutralophilic Bacillus spp. and related taxa (Ogami et al., 2009). The results indicated that cytochrome c-550 contains only two basic amino acids, including histidine, in the heme c axial ligand. This scarcity of basic amino acids is more pronounced in obligate alkaliphilic Bacillus spp. than in facultative alkaliphiles and neutralophiles. Cytochrome c-550 contains the distinct amino acid sequence Gly22-Asn34, which is absent in facultative alkaliphilic and neutralophilic Bacillus spp. Thus, this sequence exists specifically for adaptation to alkaline environments. The amino acid sequence Gly22-Asn34 contains the H+-transferable amino acids Asp and Glu at ratios of 3/13 and 1/13, respectively. The most prominent constituent of the Gly22-Asn34 sequence is Asn. Asn is present at a ratio of 7/17 in the Asn21-Asn37 region and may play an important role in the H+ transfer network in the vicinity of the outer surface membrane.

Although Asn is theoretically H+-transferable, there have been a few examples of the contribution of H+ transfer processes due to weak hydrogen binding of the involved residues. Doukov et al. (2007) reported a very interesting hypothetical model of a hydrogen-bond network involving the H+-transferable characteristics of Asn in the methyltetrahydrofolate (MTHF) corrinoid-iron-sulfur protein methyltransferase. The enzyme catalyzes the transfer of the methyl group of MTHF to cob(I)amide. This transfer reaction requires electrophilic activation of the methyl group of MTHF, which includes proton transfer to the N5 group of the pterin ring of MTHF. However, the resolved crystal structure of the methyltransferase revealed no obvious H+ donor within hydrogen-bonding distance of the N5 position of MTHF. Combining kinetic and structural evidence, it was predicted that the extended hydrogen-bond network contributes to H+ transfer to the N5 group of the pterin ring of MTHF. This extended hydrogen-bonding network contains an Asn, a conserved Asp and a water molecule. The evidence from this study suggests that even amino acid residues that exhibit weak hydrogen bonding, such as Asn can contribute to a cumulative hydrogen-bond network such that the overall effect on this transitional state is greater than expected based on the individual components alone. If this knowledge is applied to the Asn21-Asn37 region in cytochrome c-550 in B. clarkii K24-1U, then the region contributes to a cumulative hydrogen-bond network.

Based on the structures of other reported membrane-bound cytochromes c (David et al., 2000), it can be assumed that the region from Asn21 to Asn37 is located outside the α-helical domain from the N-terminus to the surrounding heme c. Thus, the region is located in the vicinity of the outer surface membrane. In addition, we attempted to elucidate the structure of cytochrome c-550 in B. clarkii K24-1U at the original membrane. Cytochrome c-550 with a membrane-anchoring diacylglycerol-cysteine moiety exhibits a tetrameric structure in the presence of the detergent Triton X-100. In addition, a mutant protein containing an N-terminal Cys18 to Met mutation in the mature protein also exists as a tetramer in the absence of Triton X-100. This finding indicates that cytochrome c-550 may exist as a tetramer on the outer surface membrane. This structure may be important not only for the regulation of the redox reaction involving four redox centers, as in the case of cytochrome c3 in Desulfovibrio gigas (Messias et al., 2006) but also for the formation of the hydrogen-bond network.

The above-described H+/O ratio in B. clarkii DSM 8720T (H+/O ratio = 3.6) cannot be explained by the conventional understanding of the function of respiratory chain because the theoretical H+/O ratio in Bacillus spp. should be 6 (complex III, 4 plus complex IV, 2 = 6) (Sone et al., 1999). The H+/O ratio in B. clarkii DSM 8720T, which is lower than the theoretical value, may be attributable to the high amount of energy required to translocate H+ across the membrane under a larger ΔΨ value than those of neutralophiles. Due to the low translocation of H+ to the outer surface of the membrane, mechanisms for retaining H+ and/or regulating H+ behavior on the outer surface membrane are necessary. It is expected that the membrane-bound cytochrome c concomitant with physicochemical factors assumes these functions on the outer surface of the membrane.

Electron transfer-coupled H+ transfer was studied by Murgida and Hildebrandt (2001) via deuterium substitution. Horse heart cytochrome c was bound as a self-assembled monolayer (SAM) on a Ag electrode produced using different chain lengths (C2-C16) of ω-carboxyl alkanethiols. Cytochrome c redox reactions were performed by changing the electrical potential of the Ag electrode and monitoring changes in the Raman spectra. When the distance between cytochrome c and the electrode was short (C2; distance between cytochrome c and the electrode: 6.3 Å), the electron transfer rate between cytochrome c and the Ag electrode was slower in D2O (33 s-1) than that in H2O (132 s-1). However, there was no difference in the electron transfer rate (0.073-0.074 s-1) between cytochrome c and the Ag electrode when the distance between cytochrome c and the electrode was large (C16; distance between cytochrome c and the electrode: 24 Å), regardless of whether the aqueous solvent was D2O or H2O. These results indicate that the D+ exchange rate with amino acids in cytochrome c was not the rate-limiting step when the electron transfer rate between cytochrome c and the Ag electrode was low. H+-exchange-coupled electron transfer between cytochrome c and the Ag electrode may be the rate-limiting step if the rate of electron transfer between cytochrome c and the Ag electrode is high. The above-described rate-limiting H+/D+ transfer-coupled electron transfer was observed during self-assembly but not in solution. Therefore, differentiated H+/D+ transfer-coupled electron transfer can be observed only in the accumulated nanolayers of cytochromes affected by changes in the electric field (Coulomb’s force) of the Ag electrode when an electron is taken in and out concomitant with electron transfer (associated H+/D+ transfer).

The fluctuating electric field may be equivariant to the local electric field strength at biological interfaces (Murgida and Hildebrandt, 2004). This finding suggests that the H+ transfer rate between cytochrome c and the H+ transfer network on the outer surface membrane is affected not only by electron transfer to cytochrome c but also by the local electric field strength on the outer surface membrane. Electron transfer and local electric field strength on the outer surface membrane may fluctuate depending on the electron transfer events in the respiratory chain.

The sulfate-reducing bacterium D. gigas, which reduces sulfuric acid to hydrogen sulfide, possesses large quantities of cytochrome c3, with four heme molecules in one protein (Coutinho and Xavier, 1994). The redox potential of cytochrome c changes depending on the pH, affecting the electric field and reducing the sequence of these hemes. Cytochrome c3 has been reported to facilitate the transfer of H+ to the electron transfer complex or F1Fo-ATPase by cooperative H+/e- linkage (redox-Bohr effect) (Louro et al., 1997; Messias et al., 2006). The midpoint redox potentials of heme I, heme II, heme III, and heme IV of cytochrome c3 are -306, -327, -308, and -297 mV, respectively, in solution but -332, -384, -381, and -457 mV, respectively, on the SAM on the electrode. The differences in midpoint redox potentials between the solution and the SAM follow the sequence heme IV (160 mV) > heme III (73 mV) > heme II (57 mV) > heme I (16 mV), which is consistent with the sequence for the distance between hemes and the electrode (shorter distances with larger differences in redox potentials) (Rivas et al., 2005). This finding indicates that the midpoint redox potential is affected by structural changes in the protein concomitant with changes in the electric field (Coulomb’s force). Thus, the redox potential of heme c is affected by localized structural changes in the vicinity of heme c. Changes in the intensity of the redox potential may be accounted for by the reduced distances between the hemes, and the electrode may be affected by the enhanced electric field.

To summarize the above-described phenomena regarding the behavior of redox reactions concomitant with H+ exchange in the aqueous phase in cytochrome c (or heme c), H+ transfer via the H+ exchange network is affected not only by the redox change in heme c but also by the change in the intensity of the electric field on the SAM of cytochrome c. In addition, the same cumulative configuration of the structure of the assembled cytochrome c was indispensable for these events.

In this section, we consider respiratory regulatory mechanisms based on the obtained experimental data for B. clarkii DSM 8720T and B. clarkii K24-1U because both strains belong to the same species. As described above, the cytochromes c from alkaliphilic Bacillus spp. exhibited lower redox potentials (+47-+95 mV) than those from neutralophilic bacteria (+170-+230 mV) (Hicks and Krulwich, 1995; Yumoto, 2002; Goto et al., 2005) due to electron transport across the membrane between the redox center located in the outer membrane to the intramembrane side and/or transport of the heavier H+ from the intracellular to the extracellular side in the presence of a large ΔΨ. Although the midpoint redox potential of cytochrome c-550 in B. clarkii K24-1U was +83 mV, based on redox titration, the potential was even lower when determined by cyclic voltammetric measurements (Ogami et al., 2009), probably because the redox potential was affected by the electric field of the electrode during cyclic voltammetric measurement. In the abovementioned example of cytochrome c in the SAM, the midpoint redox potential and H+ transfer behavior of cytochrome c-550 were also affected by ΔΨ. For example, the ΔΨ of B. clarkii DSM 8720T was lower under low-aeration conditions than under high-aeration conditions (Goto et al., 2016). Thus, electron transport via the respiratory chain concomitant with H+ transport across the membrane is relatively easy under low-aeration conditions, whereas the Δp per H+ is lower under low-aeration conditions than under high-aeration conditions. Under high-aeration conditions, the cytochrome c content of B. clarkii K24-1U (0.23 nmolmg protein-1) in the membrane is lower than that (1.38 nmolmg protein-1) under low-aeration conditions (Figure 1). This finding suggested that the membrane-bound cytochrome c in B.

clarkii plays an important role under low-aeration conditions at a pH of approximately 10. Growth characteristics were studied under both conditions for alkaliphilic B. clarkii K24-1U (Table 1). The μmax and OD650,max of B. clarkii under low-aeration conditions were 0.26 h-1 and 0.96 (18 h), respectively, whereas the μmax and OD650,max under high-aeration conditions were 0.21 h-1 and 1.27 (12 h), respectively. These results indicated that although the growth rate and intensity of B. clarkii were greatly influenced by aeration conditions, the bacterium retained a high growth rate under low-aeration conditions. In addition, despite a culture duration of 21 h, the growth intensity remained high. This finding is quite remarkable considering the low H+ concentration at the outer surface membrane and the low number of terminal electron acceptors (low O2 concentration) in the conducting respiratory system. Therefore, it can be assumed that an increased level of membrane-bound cytochrome c affects H+ transfer at the outer surface of the membrane as follows: H+ condensation at the outer surface membrane via both (i) the electrical force (in reduced form) (ii) the hydrogen-bond network produced by the chemical characteristics of the protein (via Asn-rich structures). Thus, we hypothesize that the H+-condensation mechanism produced by cytochrome c in certain alkaliphilic Bacillus spp. plays very significant role under condition of limited aeration (Figure 2).

Gram-negative alkaliphiles have been studied far less investigated than Gram-positive alkaliphiles, probably because most of the sources for alkaliphile isolation have been terrestrial samples, such as soil. The facultative alkaliphile P. alcaliphila AL15-21T was isolated from seawater obtained from the coast of Rumoi, Hokkaido, Japan (43°56′N 141°38′E) (Yumoto et al., 2001), and the cytochromes c of this bacterium was studied because Gram-negative bacteria possess soluble cytochromes c (Matsuno et al., 2007, 2009; Matsuno and Yumoto, 2015). This property is due to the presence of periplasmic space in Gram-negative bacteria on the outer side of the membrane. Pseudomonas spp. belonging to the same node as P. alcaliphila (such as P. mendocina and P. toyotomiensis) in a phylogenetic tree based on the 16S rRNA gene sequence are able to grow at pH 10 (Hirota et al., 2011). This relationship between alkaline adaptation and phylogenetic position based on 16S rRNA gene sequences is similar to that observed for alkaliphilic Bacillus spp.

The soluble cytochrome c content in cells grown at pH 7-10 under low- or high-aeration conditions was estimated (Matsuno et al., 2007). The highest amount of cytochrome c was observed in cells grown at pH 10 under low-aeration conditions. The cytochrome c content in cells grown at pH 10 under low-aeration conditions (0.47 ± 0.05 nmolmg protein-1) was 3.6 times higher than that in cells grown at pH 7 under high-aeration conditions, which was the lowest cytochrome c content among the tested samples (0.13 ± 0.05 nmolmg protein-1) (Matsuno et al., 2009). The increased cytochrome c content at high pH under low-aeration conditions was similar to that observed for facultative alkaliphilic Bacillus spp. such as B. clarkia K24-1U (Figure 1; Hijikata, 2004).

The soluble fraction of P. alcaliphila AL15-21T contains three types of cytochrome c: cytochrome c-552, cytochrome c-554, and cytochrome c-551. Cytochrome c-522 is the major soluble cytochrome c component, constituting 64% of the total cytochrome c content in P. alcaliphila AL15-21T (Matsuno and Yumoto, 2015). One particular characteristic of cytochrome c-552 is that the resting state of this protein is similar to its fully reduced state (Matsuno et al., 2009). Thus, cytochrome c-552 possesses electron-retention characteristics. The molecular mass of this protein is 7.5 kDa, as determined by SDS-PAGE, which is somewhat smaller than the reported masses of cytochrome c proteins isolated from neutralophilic P. aeruginosa (9-15 kDa). A phylogenetic analysis performed using the amino acid sequence of cytochrome c-552 classified it as a small cytochrome c5 belonging to group 4 of class I cytochrome c proteins (Matsuno et al., 2007; Matsuno and Yumoto, 2015). Class I cytochromes c consist of six groups, and group four contains monoheme cytochromes c form Gram-negative bacteria such as Pseudomonas spp. and Shewanella spp. (Sone and Toh, 1994; Bertini et al., 2006; Matsuno and Yumoto, 2015). The midpoint redox potential of cytochrome c-552 determined by redox titration (+228 mV) was almost the same as that determined by cyclic voltammetry (+224 mV) (Matsuno et al., 2009). Cytochrome c-552 reacts with the terminal oxidase in the respiratory system (Matsuno et al., 2009).

The pH dependence of the cytochrome c-552 reduction rate was determined by estimating the reduction rate under anaerobic conditions (Matsuno et al., 2009). Cytochrome c-552 was fully reduced after 40 h at pH 8.5 but was fully reduced after 4 h at pH 10 in the presence of the electron mediator TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine). The reduction rate exhibited first-order reaction constants of 0.07 and 0.56 h-1 at pH 8.5 and pH 10, respectively. The oxidation rates of cytochrome c-552 and horse heart cytochrome c were estimated at pH 6-10 under ambient conditions. Cytochrome c-552 was oxidized very slowly at pH 8-10, with the slowest rate observed at pH 8, but was oxidized rapidly from pH 6-7. The oxidation rates of horse heart cytochrome c were consistently high at pH 6-10. The results demonstrated that cytochrome c-552 possessed distinctive electron retention characteristics. If electron-transfer-coupled H+ transfer (redox-Bohr effect) is possible, then cytochrome c-552 retains H+ in the periplasmic space.

To understand the physiological function of P. alcaliphila AL15-21T cytochrome c-552, an antibiotic-marker-less cytochrome c-552-deficient mutant was constructed to exclude the effects of antibiotics (Matsuno et al., 2011). The growth features of the wild-type P. alcaliphila AL15-21T and cytochrome c-552 deletion mutant strains were compared at pH 10 and 7 under low- and high-aeration conditions by estimating the maximum specific growth rate (μmax [h-1]) and maximum cell turbidity (OD660,max) (Figure 3 and Table 2). The most significant differences in the growth parameters were observed at pH 10 under low-aeration conditions between the wild-type (μmax [h-1] = 0.85, OD660,max = 0.27) and the cytochrome c-522 deletion mutant (μmax [h-1] = 0.69, OD660,max = 0.20). The μmax (h-1) of the deletion mutant was 1.5 times higher, whereas the OD660,max was 26% lower than that of the wild-type strain (Table 2).

The oxygen consumption rates of cell suspensions of the wild-type strain and the cytochrome c-522 deletion mutant of P. alcaliphila AL15-21T were assessed under different conditions (i.e., pH 7 or pH 10; high or low aeration) (Matsuno et al., 2011). The oxygen consumption rates in the cytochrome c-522 deletion mutant cells under low- and high-aeration conditions increased by 12 and 17%, respectively, compared to those in the wild type. This finding indicates that cytochrome c-552 hinders the direct electron transfer in the respiratory chain. A possible role of this protein might be the formation of an electron bypass to construct an electron reservoir in the periplasmic space. This hypothesis is consistent with the finding that cytochrome c-552 has strong electron retention ability at a high pH values (Figure 4).

Bacteria utilize several strategies to adapt to high pH, avoid OH- and manage scarce H+ resources via secondary cell wall components, the Na+-based transportation system and flagellar rotation. However, for the respiratory system, the management of energy production under one-thousandth of the ambient concentrations of H+ is difficult. In the case of alkaliphilic Bacillus spp., a large ΔΨ is indispensable for adaptation at high pH. This high value not only compensates for the deficient ΔpH but also attracts H+ moieties that are translocated by the respiratory chain and affect the redox potential of the cytochrome c bound to the outer surface membrane. However, although we have not determined the ΔΨ of alkaliphilic Pseudomonas spp., ΔΨ is not believed to greatly influence the cytochrome c in the periplasmic space. This difference may be attributable to differences in the proteins present in the periplasmic space in Gram-negative Pseudomonas spp. Thus, the effect of ΔΨ is assumed to encompass the outer surface membrane, although this parameter hardly affects the entire periplasmic space. The main region involved in alkaline adaptation in alkaliphilic Bacillus spp. is the outer surface membrane, comprising the ΔΨ (physical parameter) and proteins (associated with the acidic nature and retention and transportation of H+ and electrons) located at the outer surface membrane. In contrast, the main region involved in adaptation in alkaliphilic Pseudomonas spp. is the periplasmic space and the corresponding localized proteins (associated with the acidic nature and retention and transportation of H+ and electrons).

Cytochrome c is a well-known electron carrier in the respiratory chain. However, as described above, despite several reports regarding the H+-transferring characteristics of cytochrome c, it is difficult to conclude that these characteristics are well understood. Studies of cytochrome c in alkaliphilic bacteria have shown that not only the H+-transferring characteristics but also the H+-retention features of cytochrome c are important for energy production, especially under low-aeration conditions. We propose that cytochrome c participates in the “H+ capacitor mechanism” as an energy production strategy under low-aeration and alkaline conditions. Surprisingly, although the cell surface structure is completely different between Gram-positive and Gram-negative bacteria, the H+ and electron retention characteristics of cytochrome c are important. However, there are several differences (the charge in redox potential depends on ΔΨ, the value of the redox potential and electron retention ability) among cytochrome c proteins. These changes may be attributable to strategic differences in the reaction site, which is an interfacial surface (outer surface membrane) or space (periplasmic space). As described above, cytochrome c has multiple functions (electron carrier or reservoir, H+ carrier or reservoir and acidic nature). However, pronounced expression of cytochrome c is not considered indispensable for adaptation under alkaline conditions because some alkaliphilic Bacillus spp. do not express large amounts of cytochrome c. According to the experimental results described above, alkaliphiles that express large amounts of cytochrome c likely exhibit superior growth features at a high pH at low-oxygen concentrations.

KoY, NI, HiM, KaY, and IY designed the research. TM, TG, SO, and HaM performed the research. KaY and IY analyzed the data and wrote the paper.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.