The c-rings of ATP synthases consist of individual c-subunits, all of which harbor a conserved motif of repetitive glycine residues (GxGxGxG) important for tight transmembrane α-helix packing. The cring stoichiometry determines the number of ions transferred during enzyme operation and has a direct impact on the ion-to-ATP ratio, a cornerstone parameter of cell bioenergetics. In the extreme alkaliphile Bacillus pseudofirmus OF4, the glycine motif is replaced by AxAxAxA. We performed a structural study on two mutants with alanine-to-glycine changes using atomic force microscopy and X-ray crystallography, and found that mutants form smaller c 12 rings compared with the WT c 13 . The molar growth yields of B. pseudofirmus OF4 cells on malate further revealed that the c 12 mutants have a considerably reduced capacity to grow on limiting malate at high pH. Our results demonstrate that the mutant ATP synthases with either c 12 or c 13 can support ATP synthesis, and also underscore the critical importance of an alanine motif with c 13 ring stoichiometry for optimal growth at pH >10. The data indicate a direct connection between the precisely adapted ATP synthase c-ring stoichiometry and its ionto-ATP ratio on cell physiology, and also demonstrate the bioenergetic challenges and evolutionary adaptation strategies of extremophiles.
Summary Bacillus pseudofirmus OF4 is an extreme but facultative alkaliphile that grows non-fermentatively in a pH range from 7.5 to above 11.4 and can withstand large sudden increases in external pH. It is a model organism for studies of bioenergetics at high pH, at which energy demands are higher than at neutral pH because both cytoplasmic pH homeostasis and ATP synthesis require more energy. The alkaliphile also tolerates a cytoplasmic pH > 9.0 at external pH values at which the pH homeostasis capacity is exceeded, and manages other stresses that are exacerbated at alkaline pH, e.g. sodium, oxidative and cell wall stresses. The genome of B. pseudofirmus OF4 includes two plasmids that are lost from some mutants without viability loss. The plasmids may provide a reservoir of mobile elements that promote adaptive chromosomal rearrangements under particular environmental conditions. The genome also reveals a more acidic pI profile for proteins exposed on the outer surface than found in neutralophiles. A large array of transporters and regulatory genes are predicted to protect the alkaliphile from its overlapping stresses. In addition, unanticipated metabolic versatility was observed, which could ensure requisite energy for alkaliphily under diverse conditions.
Typically, biochemical screens that employ pure macromolecular components focus on single targets or a small number of interacting components. Researches rely on whole cell screens for more complex systems. Bacterial DNA replicases contain multiple subunits that change interactions with each stage of a complex reaction. Thus, the actual number of targets is a multiple of the proteins involved. It is estimated that the overall replication reaction contains up to 100 essential targets, many suitable for discovery of antibacterial inhibitors. We have developed an assay, using purified protein components, where inhibitors of any of the essential targets can be detected through a common readout. Use of purified components allows each protein to be set within the linear range where the readout is proportional to the extent of inhibition of the target. By performing assays against replicases from model Gram-negative and Gram-positive bacteria in parallel, we show that it is possible to distinguish compounds that inhibit only a single bacterial replicase from those that exhibit broad spectrum potential.
The ATP synthase of the alkaliphile Bacillus pseudofirmus OF4 has a tridecameric c-subunit rotor ring. Each c-subunit has an AxAxAxA motif near the center of the inner helix, where neutralophilic bacteria generally have GxGxGxG. Here, we studied the impact of four single and six multiple Ala-to-Gly chromosomal mutations in the A16xAxAxA22 motif on the capacity for non-fermentative growth and, for most of the mutants, on ATP synthesis by ADP + Pi-loaded membrane vesicles at pH 7.5 and 10.5. SDS-PAGE analyses of the holo-ATP synthases were used to probe stability of the mutant c-rotors and mobility properties of the c-rotors as well as the monomeric c-subunits that are released from them by trichloroacetic acid treatment. Mutants containing an Ala16-to-Gly mutation exhibited the most severe functional defects. On SDS-PAGE, most of the mutant c-monomers exhibited increased mobility relative to the WT c-subunit, but among the intact c-rings, only Ala16-to-Gly containing mutants exhibited significantly increased mobility relative to the WT c-ring. The hypothesis that these c-rings have a decreased c-subunit stoichiometry is still untested but the functional impact of an Ala16-to-Gly mutation clearly depended upon additional Ala-to-Gly mutation(s) and their positions. The double A16/20G mutant exhibited a larger functional deficit than both the A16G and A16/18G mutants. Most of the mutant c-rings showed in vitro instability relative to wild-type (WT) c-ring. However, the functional deficits of mutants did not correlate well with the extent of c-ring stability loss, so this property is unlikely to be a major factor in vivo.
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 octylglucosidestimulated ATPase activity and -subunit levels at least as high as wild-type. Purified mutant F 1 F 0 -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. Proton-coupled F 1 F 0 -ATP synthases are centrally important for non-fermentative cells that energize ATP synthesis using the energy of an electrochemical proton gradient, the PMF, 3 across the cytoplasmic or thylakoid membrane (bacteria) and across the mitochondrial or chloroplast thylakoid membrane (eukaryotes) (1-3). ATP synthases are composed of two domains, with bacterial synthases having simpler structures than eukaryotic homologues. The cytoplasmically located, soluble F 1 domain encompasses three catalytic ␣-and -subunit pairs and single ␥-, ␦-, and ⑀-subunits. The membrane-associated F 0 domain is composed of a single a-subunit, two b-subunits, and multiple c-subunits (2, 4 -6). ATP synthases function as rotary nano-machines, in which inward translocation of protons through the F 0 domain leads to rotation of a membrane-embedded ring-like rotor (2, 3, 7-10). The rotor is formed from 10 -15 hairpin-like c-subunits, depending upon the organism (11-16). Essential steps in coupling of ATP synthesis to the PMF include the protonation of successive c-subunits of the rotor and, after full rotation of a protonated c-subunit, de-protonation of that subunit through interactions of c-subunits of the rotor with the a-subunit stator component (4,5,7). No high resolution structural data are yet available for the a-subunit, but extensive biochemical and genetic evidence indicates that this ATP synthase subunit plays roles in providing the proton path from outside the membrane surface to the carboxylates of interacting c-subunits of the rotor (4, 17-24). An essential, conserved arginine in TMH4 (Arg-210 in Escherichia coli) is proposed to prevent proton short-cutting to the cytoplasm without rotation (25) and to cause a shift in the pK a of the essential carboxylate so that the proton that has completed rotation dissociates and enters the proton exit pathway leading to the cytoplasm. That proton exit pathway is also likely to be within the a-subunit (4, 5, 18, 23, 26 -28).Valuable insights into the mechanism of ATP synthase have been obtained from studies of bacterial synthases because of the ease of introducing and analyzing effects of mutations (8, 14, 15, 29 -31). Our own studies have focused on the ATP synthase of alkaliphilic Bacillus species. The model extreme alka...
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