The ATP synthase a-subunit of extreme alkaliphiles is a distinct variant: Mutations in the critical alkaliphile-specific residue Lys-180 and other residues that support alkaliphile oxidative phosphorylation
A lysine residue in the putative proton uptake pathway of the ATP synthase a-subunit is found only in alkaliphilic Bacillus species and is proposed to play roles in proton capture, retention and passage to the synthase rotor. Here, Lys-180 was replaced
with alanine (Ala), glycine (Gly), cysteine (Cys), arginine (Arg), or histidine (His) in the chromosome of alkaliphilic Bacillus pseudofirmus OF4. All mutants exhibited octylglucoside-stimulated ATPase activity and β-subunit levels at least as high as wild-type.
Purified mutant F1F0-ATP synthases all contained substantial a-subunit levels. The mutants exhibited diverse patterns of native (no octylglucoside)
ATPase activity and a range of defects in malate growth and in vitro ATP synthesis at pH 10.5. ATP synthesis by the Ala, Gly, and His mutants was also impaired at pH 7.5 in the presence of a
protonophoric uncoupler. Thus Lys-180 plays a role when the protonmotive force is reduced at near neutral, not just at high
pH. The Arg mutant exhibited no ATP synthesis activity in the alkaliphile setting although activity was reported for a K180R
mutant of a thermoalkaliphile synthase (McMillan, D. G., Keis, S., Dimroth, P., and Cook, G. M. (2007) J. Biol. Chem. 282, 17395–17404). The hypothesis that a-subunits from extreme alkaliphiles and the thermoalkaliphile represent distinct
variants was supported by demonstration of the importance of additional alkaliphile-specific a-subunit residues, not found
in the thermoalkaliphile, for malate growth of B. pseudofirmus OF4. Finally, a mutant B. pseudofirmus OF4 synthase with switched positions of Lys-180 (helix 4) and Gly-212 (helix 5) retained significant coupled synthase activity
accompanied by proton leakiness.
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"The added attraction was that they also often had additional capacities, e.g., some with high temperature optima and others with low temperature optima that increased the range of environments in which they were catalytically competent (Kumar and Takagi, 1999; Fujinami and Fujisawa, 2010; Horikoshi, 2011; Sarethy et al., 2011). Examples of alkaliphile enzymes and their uses include alkaline proteases, which are used as detergent additives and for removing hair from hides; starch-degrading amylases with elevated pH optima are also suitable for laundry use and debranching enzymes, together with amylase, play a role in stain removal (Ito et al., 1989; Gessesse et al., 2003; Sarethy et al., 2011); alkaline keratinases can degrade feathers that are often unwanted by-products of other processes (Kojima et al., 2006); and cyclomaltodextrin glucanotransferases (CGTases) from alkaliphilic strains enhance the production of cyclodextrins (CDs), which are used in pharmaceuticals, foodstuffs, and for chemical interactions (Horikoshi, 1999; Qi and Zimmermann, 2005; Fujinami and Fujisawa, 2010). Alkaliphiles also produce useful metabolites, including antibiotics. "
[Show abstract][Hide abstract]ABSTRACT: Alkaliphilic bacteria typically grow well at pH 9, with the most extremophilic strains growing up to pH values as high as pH 12-13. Interest in extreme alkaliphiles arises because they are sources of useful, stable enzymes, and the cells themselves can be used for biotechnological and other applications at high pH. In addition, alkaline hydrothermal vents represent an early evolutionary niche for alkaliphiles and novel extreme alkaliphiles have also recently been found in alkaline serpentinizing sites. A third focus of interest in alkaliphiles is the challenge raised by the use of proton-coupled ATP synthases for oxidative phosphorylation by non-fermentative alkaliphiles. This creates a problem with respect to tenets of the chemiosmotic model that remains the core model for the bioenergetics of oxidative phosphorylation. Each of these facets of alkaliphilic bacteria will be discussed with a focus on extremely alkaliphilic Bacillus strains. These alkaliphilic bacteria have provided a cogent experimental system to probe adaptations that enable their growth and oxidative phosphorylation at high pH. Adaptations are clearly needed to enable secreted or partially exposed enzymes or protein complexes to function at the high external pH. Also, alkaliphiles must maintain a cytoplasmic pH that is significantly lower than the pH of the outside medium. This protects cytoplasmic components from an external pH that is alkaline enough to impair their stability or function. However, the pH gradient across the cytoplasmic membrane, with its orientation of more acidic inside than outside, is in the reverse of the productive orientation for bioenergetic work. The reversed gradient reduces the trans-membrane proton-motive force available to energize ATP synthesis. Multiple strategies are hypothesized to be involved in enabling alkaliphiles to circumvent the challenge of a low bulk proton-motive force energizing proton-coupled ATP synthesis at high pH.
Full-text · Article · Jun 2015 · Frontiers in Bioengineering and Biotechnology
"Proton motive force generation and ATP production is significantly different under these conditions than under neutral pH and low salt conditions . The solutions to these problems that alkaliphilic Bacillus have developed are of continuing interest; for example, some species have modified ATP synthases that allow production at high pH [5,6]. In others, a specific S-layer protein is linked to growth at high pH . "
[Show abstract][Hide abstract]ABSTRACT: Alkaliphilic Bacillus species are intrinsically interesting due to the bioenergetic problems posed by growth at high pH and high salt. Three alkaline cellulases have been cloned, sequenced and expressed from Bacillus cellulosilyticus N-4 (Bcell) making it an excellent target for genomic sequencing and mining of biomass-degrading enzymes.
The genome of Bcell is a single chromosome of 4.7 Mb with no plasmids present and three large phage insertions. The most unusual feature of the genome is the presence of 23 LPXTA membrane anchor proteins; 17 of these are annotated as involved in polysaccharide degradation. These two values are significantly higher than seen in any other Bacillus species. This high number of membrane anchor proteins is seen only in pathogenic Gram-positive organisms such as Listeria monocytogenes or Staphylococcus aureus. Bcell also possesses four sortase D subfamily 4 enzymes that incorporate LPXTA-bearing proteins into the cell wall; three of these are closely related to each other and unique to Bcell. Cell fractionation and enzymatic assay of Bcell cultures show that the majority of polysaccharide degradation is associated with the cell wall LPXTA-enzymes, an unusual feature in Gram-positive aerobes. Genomic analysis and growth studies both strongly argue against Bcell being a truly cellulolytic organism, in spite of its name. Preliminary results suggest that fungal mycelia may be the natural substrate for this organism.
Bacillus cellulosilyticus N-4, in spite of its name, does not possess any of the genes necessary for crystalline cellulose degradation, demonstrating the risk of classifying microorganisms without the benefit of genomic analysis. Bcell is the first Gram-positive aerobic organism shown to use predominantly cell-bound, non-cellulosomal enzymes for polysaccharide degradation. The LPXTA-sortase system utilized by Bcell may have applications both in anchoring cellulases and other biomass-degrading enzymes to Bcell itself and in anchoring proteins other Gram-positive organisms.
"contains at least two crucial amino acids, Lys 180 and Gly 212 , that are not found in the neutrophilic counterpart. Situated at the interface between the a -and the c -ring, Lys 180 and Gly 212 are essential for ATP synthesis at elevated pH, being probably implicated in proton capturing and transferring to the rotor ring of ATP synthase (Fujisawa et al., 2010 ) . Our brief search within the sequence databases has indicated the substitution of Lys 180 and Gly 212 in the haloalkaliphilic Thioalkalivibrio spp., Thioalkalimicrobium spp., Methylomicrobium alcaliphilum, and Thiorhodospira sibirica in an E. coli -like pattern ( Lys 180 /Gly 212 being replaced with Gly/His or Lys). "
[Show abstract][Hide abstract]ABSTRACT: Haloalkaliphiles differ from natronophiles by their requirement for chloride ions in addition to high alkalinity. Natronophilic bacteria grow optimally in soda medium buffered at alkaline pH by a combination of NaHCO3 and Na2CO3. The majority of known haloalkaliphilic and natronophilic prokaryotes are isolated from saline–alkaline ecosystems such as soda lakes and saline–alkaline soils. A great taxonomic and metabolic biodiversity is found in soda systems, enabling the functioning of all the cycles of the essential elements. In spite of the increasing number of haloalkaliphilic and natronophilic isolates, scarce biochemical and functional information on simultaneous adaptation at high salinity and alkalinity is reported. Most of the available data on haloalkaline adaptation can be inferred from the functional characterization of alkaliphilic and halophilic bacterial models as well as from a few haloalkaliphilic and natronophilic genome sequences deposited in databases. At the level of cell envelopes (cell wall and cytoplasmic membrane), the salt and alkaline adaptation strategies are different and relatively conserved between Gram-positive and Gram-negative bacteria. The cell wall of the former group is characterized by the excessive presence of acidic polymers, while cell membranes abound in phospholipids with branched fatty acids. Cell membranes of salt- and alkaline-adapted Gram-negatives contain a large variety of fatty acids as well as significant amounts of nonpolar lipids. Osmotic adaptation mostly depends on the accumulation of organic compatible solutes either by active solute uptake or by combined strategies of importing osmolytes or osmolyte precursors and de novo synthesis of organic compatible solutes. Aerobic and anaerobic haloalkaliphiles are distinguished from each other by very different bioenergetics. Energy conservation in aerobic alkaliphiles and haloalkaliphiles is mainly based on functioning of H+-driven F-type ATP synthase. In spite of the low transmembrane electrochemical proton gradient (equivalent to proton-motive force, pmf) encountered in the alkali-exposed membrane, the energy metabolism remains highly efficient, supporting high growth rate and yield in many aerobic alkaliphiles and haloalkaliphiles. The energetics of haloalkaliphilic anaerobes is less understood, but it seems to involve a greater deal of Na dependency than in their aerobic counterpart. Na+-dependent ATPase activity is reported in a few anaerobic haloalkaliphiles and its role probably deals with active Na+ ejection from the cytoplasm. In haloalkaliphiles and natronophiles, the sodium-motive force (smf) is mainly driving the flagellar movement and sodium/solute symport. Cytoplasmic pH and ion homeostasis in haloalkaliphiles and natronophiles are most probably achieved by a concerted activity of a constellation of alkaline-activated ion transporters, among which Na+/H+ and Mrp-like antiporters have a major contribution.