Alkaliphilic Bacteria with Impact on Industrial Applications, Concepts of Early Life Forms, and Bioenergetics of ATP Synthesis

After proton-coupling of alkaliphile ATP synthases was clearly established, a major focus was set next on elements and hypotheses that rationalize the thermodynamic challenge of an apparently insufficient bulk PMF to account for the observed synthesis. Much of the effort and hypotheses have focused on rapid movement of protons from the proton-pumping complexes to the proton-coupled ATP synthase. Studies in support of a more localized movement near the membrane surface included observations by Heberle of proton transfer across bacteriorhodopsin that then extended along the membrane (Heberle, 2000). Proposals of near-membrane barriers to proton equilibration with the bulk phase include compartmentalization and aspects of proton-electrostatics (Mulkidjanian et al., 2006; Liu et al., 2007; Ojemyr et al., 2010; Sandén et al., 2010; Lee, 2012; Wilkens et al., 2013; Rieger et al., 2014). These proposals all have a major focus on the near-membrane environment. However, a comparison of the sequences of c-subunits from Gram-negative alkaliphiles with those from Gram-positive alkaliphiles, as shown in Figure 8, suggests that there may be additional cellular factors besides Fo adaptations that may have roles in maintaining pumped protons in the periplasm or, perhaps, in some instance, enhancing the ability of a particular strain to maintain a lower periplasmic pH. The alignment in Figure 8 includes examples of both Gram-positive and Gram-negative aerobic alkaliphiles as well as neutralophiles from both groups. It is striking that none of the Gram-negative alkaliphiles requires any of the C-terminal PxxExxP motif adaptations within the c-ring that are crucial in Gram-positive alkaliphiles.

Moreover, only Alkalimonas amylolytica N10, an aerobic Gram-negative, NaCl-dependent alkaliphile isolated from Lake Chahanor in China (Ma et al., 2004) has the suite of four N-terminal alanines, but without the V21 that plays an important role in alkaliphilic Bacillus strains. It will be of interest to examine whether the c-ring of A. amylolytica N10 has a higher c-subunit stoichiometry than those of the other Gram-negative alkaliphiles shown, all of which have only a single N-terminal helix alanine. It is also notable that Serpentinomonas raichei A1 grows in an unusually high pH environment, even for alkaliphiles (pH 11.6), without features that are critical to alkaliphilic Bacillus strains (Suzuki et al., 2014).

The Gram-negative alkaliphiles are likely to be protected by the outer membrane that they possess, and perhaps the outer membrane of mitochondria should be considered for playing at least some role in delaying pumped proton equilibration with the bulk phase or cytoplasm. As shown in Figure 9, there are additional strategies that may contribute to delays in proton equilibration with the bulk phase. It has been suggested that particular phospholipids might play a role, and specifically the high cardiolipin level of B. pseudofirmus OF4 might delay proton equilibration (Haines and Dencher, 2002), but at least in B. pseudofirmus OF4, mutants lacking detectable cardiolipin were not impaired in non-fermentative growth at high pH (Liu et al., 2014).

Other delays in proton equilibration might involve the entire periplasmic space, which was shown in Gram-positive bacteria by Matias and Beveridge (2005) is surrounded in bacteria by cell wall layers and additional layers of various secondary cell wall polymers that are often highly charged (Takami, 2010). In B. pseudofirmus OF4, mutational loss of the S-layer gene slp results in a significant deficit in non-fermentative growth at the upper pH range and the S-layer itself is only one of several SLH-motif polymers that contribute to charges on the outside surfaces. Additional contributions are made by charged carbohydrate polymers and a poly-glutamic acid polymer (Gilmour et al., 2000; Janto et al., 2011). In alkaliphilic B. halodurans C-125, the polymers are different but also appear to be candidates for delaying proton equilibration. The polymers in the B. halodurans C-125 surface include teichuronopeptides, teichuronic acids, and poly-glutamate and poly-glucuronate that are proposed to impact very significantly on the actual pH that this alkaliphile can maintain in the periplasm (Aono, 1987; Tsujii, 2002).

Further elements, that are constituents of the membranes themselves, should also be considered in connection with retention of protons at near the membrane surface. Carotenoids have been found to lend the yellow color to many alkaliphile membranes and it is possible that carotenoids might impart properties to the cytoplasmic membrane that enhance closeness of proton pumps and synthases, in addition to their likely role in scavenging reactive oxygen species (Aono and Horikoshi, 1991; Steiger et al., 2015).

Finally, even stronger candidates for promoting micro-domains that might bring the proton pumps into greater proximity with the ATP synthase are membrane flotillins and their associated proteins (Lopez and Kolter, 2010; Bach and Bramkamp, 2013, 2015; Bramkamp and Lopez, 2015). B. halodurans C-125 has an alkali-inducible flotillin-T (Zhang et al., 2005) and B. pseudofirmus OF4 has a flotillin-A candidate as well as several NfeD protein candidates that are associated with flotillins. It will be of interest to see whether mutational loss of any of these diminishes the alkaliphilic capacity of these alkaliphiles.

Alkaliphilic bacteria contribute substantially to numerous technologies, some traditional and others at the cutting edge of new applications of alkaliphile products or their cellular capacities for decontamination of specific commercial settings. Alkaliphiles also are of ecological interest as they are part of serpentinizing sites. The finding that alkaliphilic aerobes that grow non-fermentatively, use energy derived from a proton-pumping respiratory chains and complete oxidative phosphorylation using proton-coupled ATP synthases has challenged a tenet of the formal Mitchellian chemiosmotic hypothesis. Rather than focusing only on narrow solutions to the thermodynamic problem that this re-consideration raises, it is worth considering the broader physiology of alkaliphiles, which may have evolved numerous adaptations that together contribute to their extremophilic capacities.

All five authors participated in planning aspects of the manuscript, provided critical advice about the organization and content during the drafting, which was done by TK, and reviewed the submitted version. LP produced Figures 6, 7, and 9, with input from TM and TK, SS contributed the images for Figure 1 and critical advice and expertise on the serpentinization sections, and DH produced Table 1 and Figures 5 and 8, and drafted Figures 2 and 3 as well as the figure legends with TK.

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.

This work was supported by NIGMS grant RO1 GM28454 to TK. TM would like to express our gratitude to Werner Kühlbrandt for support of TM’s laboratory for the past 9 years. The work in TM’s laboratory was supported by the Collaborative Research Center (SFB) 807 of the German research Foundation (DFG), the Cluster of Excellence “Macromolecular Complexes” (CEF-MC) at the Goethe University Frankfurt (Project EXC 115). NSF-EAR Grant No 1424646 supported the work of SS.