ATP Synthesis and pH Homeostasis; Studies of Two Processes that Necessitate Inward Proton Translocation in Extremely Alkaliphilic Bacillus Species
Terry A. KRULWICH*a, Masahiro ITOb, David B HICKSa, Raymond GILMOURa and Arthur A. GUFFANTIa
a Department of Biochemistry, Mount Sinai School of Medicine, Box 1020, 1 Gustave Levy Place, New York, New York 10029 USA
b Department of Life Science, Toyo University, 1-1-1, Izumino, Itakura-machi, Oura-gun, Gunma 374-01, Japan
Alkaliphilic Bacillus species that are isolated from non-marine, moderate salt and moderate temperature environments offer the opportunity to explore strategies that have developed for solving the energetic challenges of aerobic growth at pH values between 10 and 11 (1). Such bacteria share many structural, metabolic, genomic, and regulatory features and mechanisms with,non-extremophilic species such as Bacillus subtilis. Comparative studies can therefore illuminate the specific features of gene organization, special features of gene products that are homologues of those found in non-extremophiles, and potentially identify novel gene products of importance in alkaliphily. We have focused our studies on the facultative alkaliphile Bacillus firmus OF4 which is routinely grown on malate-containing medium at either pH 7.5 or 10.5. Current work is directed toward clarification of the characteristics and energetics of membrane-associated proteins that must catalyze inward proton movements. One group of such proteins are the Na+/H+ antiporters that enable cells to adapt to a sudden upward shift in pH and to maintain a cytoplasmic pH that is 2-2.3 units below the external pH in the most alkaline range of pH for growth. Another is the proton-translocating ATP synthase that catalyzes robust production of ATP under conditions in which the external proton concentration and the bulk chemiosmotic driving force are low. Three gene loci that are candidates for Na+/H+ antiporter encoding genes with roles in pH homeostasis have been identified. They include an nhaC locus (2) and a homologue of a locus described in alkaliphilic Bacillus lentus C-125 (3) that encompasses seven genes and is multifunctional in B. subtilis. The physiological importance of each of the loci is being explored via targeted gene disruptions. The atp genes that encode the F1F0-ATP synthase have been found to have interesting motifs in areas of putative importance for proton translocation (4). The atp operon is being expressed in atp-minus mutant strains of Escherichia coli and B. subtilis for preliminary evaluation of the role of these "alkaliphile-specific motifs" in catalytic function of the synthase before site-directed changes are selectively made
