RESEARCH COMMUNICATIONS CURRENT SCIENCE, VOL. 104, NO. 9, 10 MAY 2013 1216 *For correspondence. (e-mail: krgawai@chem.unipune.ac.in) Alkaliphile-specific motif analysis of Stenotrophomonas species DL18 F 1 F o -ATP synthase c-subunit isolated from Indian alkaline Soda Lake, Lonar Devendra Lingojwar 1,2 , Ravikant Jadhav 2 and Kachru Gawai 1, * 1 Department of Chemistry, University of Pune, Ganeshkhind, Pune 411 007, India 2 ATG LAB, Ganesh Nagar, Pimple Nilakh, Pune 411 027, India The membrane-associated F 1 F o -ATP synthase of bac- teria plays a vital role in the production of energy molecule, i.e. adenosine triphosphate (ATP). However, under alkaline conditions, ATP synthesis in bacteria is not thermodynamically feasible due to external high pH. Various studies reported motifs in ATP synthase c-subunit as alkaliphile-specific features for adapta- tion under alkaline condition. Some conserved residues in alkaliphiles were observed in Stenotrophomonas species DL18 isolated from Indian alkaline Soda Lake, Lonar, which has pH 10.5. The above-mentioned spe- cific amino acid features in the studied alkaliphile may involve proton translocating mechanism for ATP synthesis. The studied motifs of F 1 F o -ATP synthase c- subunit of Stenotrophomonas species DL18 have GXGXGXA in the inner helix and GXXDXXF in the outer helix. The overall interacting residues of the c- subunit structure may be responsible for the ATP syn- thesis in particular pH conditions. Keywords: Alkaliphile, ATP synthase, electrochemical ion gradient, motif analysis. ADENOSINE triphosphate (ATP) acts as energy currency that allows energy to be utilized in a variety of biochemical processes in living cells. ATP molecules are synthesized by ATP synthase complex using the electrochemical ion gradient of H + or Na + across the phospholipid membranes 1 . ATP synthase, also called ATPase complex, comprises two different domains: the water-soluble F 1 and the membrane-embedded F o motor. Therefore, they are known as F 1 F o -ATP synthases of bacteria and perform two critical functions. They catalyse the synthesis of ATP from ADP and inorganic phosphate (P i ) utilizing the elec- trochemical ion gradient. ATP synthase functions as ATPase by generating the electrochemical ion gradient at the expense of ATP under conditions of low driving force by rotating in the opposite direction, i.e. ATP hydrolysis. The membrane potential (Δψ) and a transmembrane ion concentration gradient (ΔpH/ΔpNa) are alike in ion trans- port through F o rotor 1,2 . The cytoplasmic F 1 catalytic subunit consists of α 3 β3γδε subunit complex, whereas membrane-embedded F o consists of ab 2 c 10–15 subunits. F 1 is in contact with F o by a central (γε) and peripheral (b 2 δ) stalk 3 . The c-subunits of the rotor are in direct contact with the a-subunit of F o . Each c-subunit has two α- helices connected by a loop region, which is in contact with the γ- and ε-subunits. Under alkaline condition, the cytoplasmic pH is maintained below 1.5–2.3 pH units than the external environment. Hence, the reversed ΔpH poses a major thermodynamic problem for ATP synthe- sis 3 . This is circumvented by adaptations in the a-subunit and c-subunits. Despite extensive studies of F 1 F o -ATP synthase c-subunit, essential steps of the proton transport mechanism have remained unclear, especially the proton binding as well as release mechanism. In the present study, attempts were made to analyse F 1 F o -ATP synthase c-subunit of Stenotrophomonas species DL18, which was isolated from Indian alkaline Soda Lake, Lonar. Briefly, isolation and 16S rRNA-based identification of facultative alkaliphile was carried out by initial culture studies at pH 9.5, followed by pure culture maintenance and growth studies of orange pigmented bacterium, i.e. Stenotrophomonas species DL18 (GenBank accession number: JN995612) at different pH conditions (pH 7–12). ATP synthase gene was amplified using pfu polymerase and the c-subunit was sequenced by primer walking method. The respective sequence was deposited in the NCBI database (GenBank accession number: JN995615). The blastx program was used to get a total of 105 amino acid sequences of the c-subunit (Protein ID: AET99166). BLAST analysis of ATP synthase F 0 c-subunit of Stenotrophomonas species DL18, which was isolated from Indian alkaline (pH 10.5) Soda Lake, Lonar, sug- gests 98% identity at nucleotide level (283 identical nu- cleotides out of total 289 nucleotides from query coverage) and 100% at amino acid level (90 amino acids identical out of 90 amino acids from 85% of query cover- age) in comparison with Stenotrophomonas maltophilia. Some of the analysed conserved residues in alkaliphiles were observed in Stenotrophomonas species DL18 as Ala 27 , Glu 44 , Arg 48 , Gln 49 , Pro 50 , Glu 51 , Leu 56 , Phe 61 , Ile 62 , Leu 66 , Ile 73 , Phe 83 (Stenotrophomonas species DL18 num- bering system for ATP synthase c-subunit; Figure 1). In addition, loop sequence (RQPE), which interacts with subunits γ and ε of F 1 , connecting the inner (helix-1) and outer (helix-2) α-helices was conserved among the alka- liphiles (Figure 2). In majority of bacteria, glutamate (Glu 54 in Bacillus pseudofirmus OF4) is conserved in the C-terminal helix of c-subunit near the centre of the mem- brane. However, some of the bacterial ATP synthases are found with aspartate (Asp 61 in Escherichia coli). Hence, Figure 1. Some alkaliphilic conserved residues of Stenotrophomonas species DL18 shown in bold.