Extreme Alkaliphiles: Experts at Alkaline Ph Homeostasis and Able to Grow When Cytoplasmic Ph Rises Above the Limit for Growth of Non-Alkaliphiles
International Symposium on Extremophiles and Their Applications 2005
Extreme Alkaliphiles: Experts at Alkaline pH Homeostasis and Able to Grow when Cytoplasmic pH Rises Above the Limit for Growth of Non-Alkaliphiles
Terry Ann Krulwich* 1Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029 USA *E-mail: [email protected]
Abstract Alkaline pH homeostasis at pH ! 10 has been most extensively studied in extremely alkaliphilic Bacillus pseudofirmus OF4. Comparisons between B. pseudofirmus OF4 and neutrophile Bacillus subtilis show that the capacity for alkaline pH homeostasis is more robust in the alkaliphile and that it sets the upper pH limit for alkaliphile growth. Active pH homeostasis mechanisms are essential, including a Na+ cycle in which cytoplasmic accumulation of H+ relative to the outside is mediated by Na+/H+ antiporters. Additional transporters have a role in H+ capture and/or retention, e.g. a specially adapted H+-ATP synthase and an ammonium transporter of alkaliphiles. Several alkaliphile adaptations in support of pH homeostasis disadvantage their growth at pH " 7.5, i.e. alkaliphiles are “hard-wired” for alkaliphily. At pH values at which their capacity for pH homeostasis no longer maintains a cytoplasmic pH < 8, extreme alkaliphiles still exhibit growth. They grow well when the cytoplasmic pH is 8.3-9.6, values that preclude most neutrophile growth. This indicates that there are adaptations of cytoplasmic processes of alkaliphiles.
Keywords; alkaliphile, neutrophile, Na+/H+ antiporter, Mrp, S-layer, ATP synthase
1. Introduction
Alkaline pH homeostasis is the central bioenergetic challenge of extreme alkaliphily, setting the upper limit of pH for growth and the growth rate at the alkaline edge of the pH range for alkaliphile growth [1-3]. Alkaline pH homeostasis by “the pro” (Fig. 1), the professional alkaliphile, has been most extensively studied in the extremely alkaliphilic Bacillus species such as Bacillus halodurans C-125 and Bacillus pseudofirmus OF4 and the best comparative framework among alkali-adaptable, non-alkaliphilic Bacillus species is provided by Bacillus subtilis [4, 5]. Comparative studies of alkaline pH homeostasis that include the effects of mutations in key surface molecules or transporters have not yet been conducted on other alkaliphilic and neutrophilic bacteria of the same species, probably because of the paucity of genetically accessible alkaliphiles aside from B. pseudofirmus OF4. The reason that B. pseudofirmus OF4 was chosen for development as a genetically accessible model alkaliphile was that it was among the most extremely alkaliphilic of the Bacillus species that can also grow at near neutral pH, i.e. are facultative alkaliphiles [6-8]. The extreme, facultative alkaliphily of B. pseudofirmus OF4 makes it possible to do comparative physiological experiments in an external pH range of 7.5 to > 11 [1] and to isolate mutant strains with site-directed changes or targeted gene deletions that are non-alkaliphilic but exhibit robust growth at pH 7.5 [9, 10].
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Alkaline pH Homeostasis: Professional Alkaliphily ( Bacillus Fig. 1 A summary of differences between pseudofirmus OF4) vs. Talented Amateurs ( B. subtilis or E. coli ) extreme alkaliphiles such as B. pseudofirmus OF4 and neutrophiles such as B. subtilis with respect to the capacity The Pro The Adaptable Amateur for pH homeostasis Grows well at pH ! 10.5, Grows sub-optimally up to pH 9 and sub-optimally at pH " 7 and optimally at pH 6 -7.5
Grows well when pH in > 8.2 Growth stops when pH in > 8.2
Hard-wired for alkaliphily Hard-wired for neutrophily
More passive mechanisms Some passive mechanisms than in neutrophiles ?
Active pH homeostasis Active pH homeostasis mechanisms, e.g. Na +-cycle, mechanisms use K + and Na + functioning at high levels and at lower aggregate levels
B. pseudofirmus OF4 grows with a doubling time of 54 minutes at pH 7.5 in malate- containing continuous cultures with rigorous pH control. The growth rate is faster at external pH values from 8.5-10.5, a range in which the doubling time is 38 minutes [1, 2]. At external pH values of 7.5-9.5, the cytoplasmic pH is kept near 7.5 [1], which is in the optimal range of cytoplasmic pH for most bacteria [3, 11]. By contrast, the cytoplasmic pH at pH 10.5 is 8.2. This represents a 2.3 unit net acidification of the cytoplasm relative to the bulk external pH, a remarkable capacity for pH homeostasis. Nonetheless, a cytoplasmic pH of 8.2 is higher than is tolerable for growth of many neutrophiles, let alone compatible with an optimal growth rate as it is in B. pseudofirmus OF4. This suggests that in addition to possessing adaptations that facilitate pH homeostasis, alkaliphiles have adaptations of key cytoplasmic processes that render them more alkali-tolerant than homologous processes in neutrophiles [12, 13]. Structural features of at least one cytoplasmic enzyme from an extreme alkaliphile are consistent with this hypothesis [14]. Another important feature of the pH homeostasis capacity of extreme alkaliphiles that contrasts with that of neutrophiles is the ability of the extremophile to sustain its cytoplasmic pH below 8.5 upon a sudden alkaline shift of pH 8.5- adapted cells to an external pH of 10.5 [2, 15]. No transient alkalinization of the cytoplasm is detected by standard assays as long as the active Na+ cycle that is required for cytoplasmic pH homeostasis is supported. This requires the presence of sufficient added Na+ and the presence of solutes whose entry is coupled to Na+ uptake so that the level of cytoplasmic Na+ is high enough support rapid exchange for external H+, i.e. support the Na+/H+ antiport activity that is the Na+ cycle element that is directly responsible for net cytoplasmic H+ accumulation in respiring, H+-extruding cells (see Fig. 3 below). By contrast to the alkaliphile’s ability to manage an acute alkalinization of the external milieu, neutrophilic Escherichia coli can adjust to a more modest pH shift of pH 7.2 to 8.3 only if the shift is not imposed suddenly. Even then, ultimate homeostasis is achieved after there is first an alkalinization of the cytoplasm to approximately the new external pH [3, 13].
2. Mechanisms of alkaline pH homeostasis
2.1 Involvement of passive properties
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Several passive properties have been suggested for trapping H+ and/or Na+ within the cytoplasm or near the external surface of the cytoplasmic membrane. These include trapping of cation between the cell wall layers and the outside of the cytoplasmic membrane by Secondary Cell Wall Polymers (SCWP) [16]. SCWP make a clear contribution to alkaliphile pH homeostasis as shown by the compromised alkaliphily of mutants in production of teichuronopeptide and teichuronic acids in Bacillus halodurans C-125 [17-19]. Similarly, the S-layer of B. pseudofirmus OF4 makes a contribution to pH homeostasis as assessed by the pH range for growth as well as cytoplasmic pH homeostasis during a sudden alkaline shift, as shown by comparisons of S-layer (SlpA protein) mutants and the wild-type [9]. Interestingly, the S-layer of B. pseudofirmus OF4 is expressed well across the pH range even though it is disadvantageous at pH 7.5. The slpA mutant grows better than wild-type at pH 7.5 rather than worse, as it does at pH 11 [9]. The adverse expression of slpA at pH 7.5 is an example of hard-wiring of the extremophile so that it is prepared to successfully confront sudden alkalinization. A second property that has been modeled as applying to all membranes is a putative interfacial barrier between the region near the phospholipid headgroups of the outer side of the cytoplasmic membrane and the bulk water phase [20, 21]. Such a barrier could well prevent the near-membrane region from attaining the same high pH as the bulk medium. However, an extrapolation of the model to suggest that this barrier renders the near- membrane pH indistinguishable from that of neutrophiles [20] is negated by several lines of evidence including:
! the use of Na+-coupled motility and solute transport by alkaliphiles, which would not be necessary if the near membrane pH is not unusually high in alkaliphiles, thus lowering the protonmotive force in that region; ! special properties of the membrane-embedded ATP synthase [10], some of which will be noted as part of active pH homeostasis mechanisms; ! special properties of alkaliphile membranes, e.g. high cardiolipin and squalene contents in alkaliphilic Bacillus species [22-24]; ! genomic evidence that externally exposed protein loops of alkaliphile membrane proteins that are close to the membrane surface show consistent substitutions of acidic residues for neutral/basic residues that are present in homologues [13](Fig. 2).
Fig. 2 Membrane protein loops that are just outside the cytoplasmic membrane of alkaliphiles show distinct, consistent patterns of amino acid substitution from the comparable regions of homologous membrane proteins from neutrophiles, constituting genomic evidence for a difference in peri-membrane pH of alkaliphiles and neutrophiles. Examples are the FtsH stress protein, the cytochrome c-containing domain of a terminal oxidase, and a segment of flagellar motor stators [25-27]; the regions in red have a much higher acidic/basic amino acid composition in alkaliphiles than in neutrophiles (data are in [13]).
Passive mechanisms ameliorate what would otherwise be a greater differential between alkaliphiles and neutrophiles in the pH elevation and protonmotive force decrease
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near the membrane. The increased acidic amino acid content in many near membrane protein loops may contribute to cation trapping while it also reflects the fact that the pH is unusually high just outside the alkaliphile membrane. No other special passive adaptations have yet been identified in alkaliphiles vs. neutrophiles but they may well exist. In sum, however, passive mechanisms do not render the near membrane pH profile comparable to that of neutrophiles, nor do they eliminate the requirement for special sequence features of the membrane-embedded ATP synthase that carries out H+-coupled ATP synthesis and the dependence of cytoplasmic pH homeostasis on active mechanisms [10, 28].
2.2 Active mechanisms, including an essential Na+-cycle that results in cytoplasmic H+ accumulation
The Na+ cycle. The most intensively studied active mechanism for alkaline pH homeostasis is the Na+-cycle in which a complement of Na+/H+ antiporter catalyze net + + cytoplasmic accumulation of H and the cytoplasmic Na required to sustain antiport activity is provided by Na+ entry routes. The aggregate level of antiport is much higher in extreme alkaliphiles than in neutrophiles [3-5, 13, 29], consistent with the greater burden of alkaliphile pH homeostasis because of the higher external pH values involved and with the sparing contribution of K+/H+ antiport to the alkaline pH homeostasis of neutrophiles whereas pH homeostasis in free-living alkaliphiles is completely dependent upon Na+/H+ antiport [3, 4]. The likely reason for the specificity of antiport-based pH homeostasis for Na+ is the avoidance of depletion of cytoplasmic K+, depletion that would compromise optimal function of cytoplasmic processes [3, 4]. As depicted in Fig. 3, the neutrophile has both Na+- and H+- coupled solute uptake systems whereas those of the alkaliphile are all Na+-coupled [13]. Similarly, B. subtilis has Na+- and H+-coupled motility channels, MotPS and MotAB respectively, whereas the alkaliphile has only MotPS [3, 27]. In addition to these two routes for Na+ uptake, B. halodurans C-125 and B. pseudofirmus OF4 both have a voltage-gated + Na channel [30-32] (called NaChBac and NavBP, respectively, in the two species) that has been shown in B. pseudofirmus OF4 to have a role in several processes that are most active at very high pH, i.e. pH homeostasis, motility and chemotaxis [31].
It is not yet known what specific properties make a particular antiporter system more important than others for pH homeostasis as opposed to Na+-resistance. In alkaliphilic Bacillus species, the Mrp antiporter (also called Sha or Mnh by some investigators) has a dominant role in pH homeostasis whereas in B. subtilis that role is played by the multi- functional TetL antiporter while Mrp is critical for Na+-resistance [33, 34]. The Mrp-type antiporters are particularly complex in requiring multiple gene products for full activity. We have hypothesized that Mrp is a consortium of transporters whose functions are somehow synergistic and interdependent [33]. It is also not yet known what all of the independent antiporters of any particular organism contribute although it appears as that their differences in specificity, affinity, pH optimum, activation properties, etc. create added value for each of them under specific conditions. For example, the NhaD-type antiporter of the soda lake alkaliphile Alkalimonas amylolytica is most active at unusually high pH and [Na+] relative to homologous antiporters from other bacteria, suggesting that these conditions are encountered with some frequency in the cytoplasm of the organism or that this particular antiporter has only an emergency role in A. amylolytica [35].
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Fig. 3 The Na+ cycles and other transporters related to Na+, K+ and H+ homeostasis in extremely alkaliphilic Bacillus species and in neutrophilic Bacillus subtilis. Characterized or predicted (www.membranetransport.org) Na+/H+ antiporters are shaded in gray except for the multi-functional TetL antiporter that catalyzes both tetracycline-metal efflux and monovalent cation (Na+ and K+)-H+ exchange. Features highlighted by blue shading in the alkaliphile are elements that play special roles in alkaliphiles: the Na+ channel and the respiratory chain complexes and ATP synthase that participate in oxidative phosphorylation. Features highlighted by red shading in B. subtilis indicate differences from the alkaliphilic Bacillus sp.: use of TetL as the dominant antiporter involved in pH homeostasis and the use of both Na+- and H+-coupled solute uptake and motility as opposed to solely Na+-coupling as in the alkaliphiles.
Additional active mechanisms. It is likely that other important mechanisms of active pH homeostasis complement the role of the Na+ cycle in alkaliphiles and some of those are beginning to emerge. An ammonium transporter, AmhT, in B. pseudofirmus OF4 was shown to prevent cytoplasmic ammonium for subverting the work of the Na+ cycle when the organism is growing on media with a high amine-nitrogen content [36]. An even more + striking role was observed for H capture by the ATP synthase during oxidative phosphorylation. This role was observed in studies comparing the ability of wild-type B. pseudofirmus OF4 and mutant strains with site-directed changes in the ATP synthase to synthesize ATP to a high phosphorylation potentials when ATP-depleted cells were re- energized concomitant with a pH shift from 8.5 to 10.5 in a buffered malate solution. The phosphorylation potential (!Gp, reflecting the [ATP]/[ADP]) increased relative to the low protonmotive force, !p, over a 3 hour re-synthesis period at pH 10.5, whereas synthesis occurred at pH 7.5 without a change in the !Gp/!p ratio (Fig. 4a). The capacity of the ATP synthase to support this characteristic alkaliphile pattern of oxidative phosphorylation was better in mutant cT33A than in wild-type and better in wild-type than in mutant cP51A. This capacity correlated directly with the acidification observed of the cytoplasm relative to the external medium, i.e. better acification in the cT33A mutant than in wild-type and better in the wild-type than in the cP51A mutant [10]. In addition, the a-subunit of the alkaliphile ATP synthase has an apparent gating feature, mediated by a pair of unusual residues, Lys180 and Gly212 in the putative proton uptake pathway of the ATP synthase [37] (Fig. 4b). This gate prevents proton fluxes to and from the bulk phase at pH " 9.2, a feature that prevents loss of cytoplasmic H+ through the ATP synthase when the cells are confronted by acute alkalinization even though it may disadvantage the alkaliphile at near-neutral pH [10]. In the context of cytoplasmic H+ retention, a generally low basal hydrolytic activity has been noted 54 for alkaliphile ATP synthases [38], and the pKa of the c-subunit carboxylate (of Glu ) is higher than those measured in several other ATP synthases [39].
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a) b)
Fig. 4 The alkaliphile ATP synthase contributes to pH homeostasis and has special features that contribute to cytoplasmic H+ retention. a) The data are taken from Fig. 3 of Wang et al. [10] depicting part of a bioenergetic profile of a panel of mutants in which six alkaliphile-specific sequence features of the membrane-embedded a- and c-subunits of the ATP synthase were changed to the consensus sequence for non-alkaliphilic Bacillus. In this experiment, ATP-depleted cells equilibrated at pH 8.5 were re-energized with malate concomitant with a shift to buffer malate at an external pH of either 7.5 or 10.5. The ability of the wild-type and mutants were compared with respect to the alkaliphile capacity to generate a large phosphorylation potential, !Gp, relative to the protonmotive force, !p, at high pH. Thus the !Gp/!p ratio at pH 7.5 is a bit below the typical range of ~ 3-4 during ATP synthesis whereas, at peak, wild-type B. pseudofirmus OF4 synthesizes ATP with a !Gp/!p ratio ~ 13. The capacity to generate high phosphorylation potentials at high pH depends upon special ATP synthase features. Although, the c-subunit T33A mutation allows an even greater !Gp/!p ratio in this protocol, the growth yield on malate is adversely affected by the mutation at pH 10.5 but not 7.5; growth on glucose, which does not require oxidative phosphorylation, is not. The cP51A mutant exhibits a non-alkaliphilic growth phenotype on malate (but not on glucose). The capacity to acidify the cytoplasm at pH of 10.5 (upper panels, red line) correlates with the ATP synthesis capacity at high pH (upper panels, blue line). b) A model taken from Fig. 1 of Wang et al. [10] that was prepared by Mark Girvin of the a-subunit region containing he Lys180 and Gly212. These residues are implicated in pH-dependent gating of proton fluxes through the putative H+ uptake pathway [37] and preventing H+ loss during sudden alkalinization of the external medium [10].
Metabolic production of acids should also be noted as an adaptive strategy to alkaline pH homeostasis that is found in both neutrophiles and alkaliphiles, although when alkaliphiles grow fermentatively their upper pH limit for growth and capacity for pH homeostasis during an alkaline shift is not quite as robust as observed in alkaliphiles growing non-fermentatively [9, 40].
3. Conclusion
Future insights into alkaliphile pH homeostasis will benefit greatly if more extreme alkaliphiles are made genetically tractable so that studies of mutants of diverse alkaliphiles can clarify the different mechanisms that may dominate in different groups. Additional active + mechanisms apart from the Na -cycle are likely to be found and there are also key questions concerning the Na+-cycle, such as: what is the added value of the complicated Mrp antiporter system in alkaliphile pH homeostasis, does this antiporter function as a complex and does it serve a comparable function in Gram-negative alkaliphiles? Finally, it appears that there are important adaptations of cytoplasmic enzymes and/or processes that account for the
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alkaliphile’s ability to grow when the cytoplasmic pH is high enough to arrest growth of neutrophiles. This is an important area of investigation.
4. References
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