Prevalence of the Alkaliphile ATP Synthase Sequence Motif in Alkaliphilic Bacillus Strains

1. Prevalence of the Alkaliphile ATP Synthase Sequence Motif in Alkaliphilic Bacillus Strains Dr. Mack Ivey, Tanushree Thote Department of Biological Sciences, University of Arkansas ABSTRACT: Bacteria growing at high pH must acidify the cytoplasm, and as a result must synthesize ATP using a very low protonmotive force. The ATP synthases of two species of alkaliphilic bacteria have been reported to contain unusual amino acid sequence motifs in the energy transducing a and c subunits of FO-ATPase. As part of an effort to assess the prevalence and importance of this set of motifs, we have developed a set of degenerate oligonucleotide primers for PCR amplification of the atp operon. Long PCR amplification techniques were employed using these primers, along with template chromosomal DNA from six alkaliphilic Bacillus species. For each of the alkaliphiles, the predicted 5.2 kb product was obtained. The ATP synthase sequence signature has now been identified in four alkaliphilic bacterial strains, representing at least three distinct species, and has been observed in no non-alkaliphilic organism. This lends support to the suggestion that this set of motifs is required for oxidative phosphorylation at high pH. Fig 1 : Electrochemical gradient in transmembrane region INTRODUCTION: Alkaliphilic bacteria present an important challenge to the chemiosmotic hypothesis, which is the central organizing concept in bioenergetics. Alkaliphiles maintain a cytoplasmic pH that is much lower than the pH of the medium, and yet synthesize ATP by oxidative phosphorylation mechanisms Materials and Methods Bacterial strains and growth conditions. The bacterial strains of E. coli utilized were grown aerobically at 37ºC in Luria Broth (LB) medium with kanamycin (50mg/ml) and/or a combination of ampicillin (20mg/ml) and methicillin (80mg/ml) as appropriate to promote selective pressure. DNA manipulations.Ligations, restriction endonuclease digests, and nucleotide fill-ins, and transformations were done by the method of Sambrook, et al., and according to protocols provided by the vendors. Recombinant strains of E. coli were constructed by transforming TSB competent BL21(DE3)[pLysS] cells with plasmid DNA mini-prepped with a Wizard Plus SV kit. Plasmid isolation was performed by the alkaline lysis procedure with a Wizard Plus SV mini-prep kit (Promega). Dideoxy sequencing was performed using T3 and T7 primers (Sequenase). Sequencing reactions were run on an automated DNA sequencer (Li-Cor, Inc., Lincoln, NE) using the Sequitherm DNA polymerase as described by the manufacturer (Epicenter Tech.). All sequence analyses employed the Wisconsin Genetics Computer Group Sequence Analysis Software Package. that seem to be in opposition to the chemiosmotic hypothesis. pH homeostasis involves respiration-linked proton extrusion, a proton-coupled F1FO-type of ATP synthase (see figure 1), and secondary Na+/H+ antiporters. Figure 3. Cloning of the ctaCD genes from B. subtilis. In support of this model, unusual motifs, perhaps associated with the pH gate and/or the direct interaction between FO and the cytochrome oxidase, have been revealed in the deduced amino acid sequences of the alkaliphile FO subunits a and c , including a lysine residue in place of a conserved glycine at a position in the a subunit is involved in H+ translocation. The alkaliphile subunit a lysine has been suggested to be part of the extracellular pH sensing and gating mechanism of the ATP synthase (87). The alkaliphile c subunits are unique in having two proline residues in the C-terminal transmembrane region. These prolines flank the buried, DCCD-binding carboxylate residue that is required for H+-translocation. Discussion Mutational analysis of the E. coli F1FO-ATPase suggests that the domains upon which we have focused in the alkaliphiles are particularly crucial for proton translocation through FO. In each of these regions, the alkaliphile FO differs in significant ways from that of neutrophilic Bacillus species and other prokaryotes. Acknowledgements Dr. Ivey for his guidance and support on the project Dr. Sears for his help with the research.