Bacteria that grow best at high pH adapt to the extreme environment by acidifying the cytoplasmic compartment. This make it possible for essential biochemical reactions inside the cell to proceed, but creates an energetic problem due to the resulting low protonmotive force across the cytoplasmic membrane. ATP synthesis in facultative alkaliphiles is coupled to the movement of protons, but the path that the protons take through the cytoplasmic membrane differs depending on the growth pH. The studies outlined here seek to characterize the two paths of protons using a molecular biological approach. Alkaliphiles grown at pH 10.5 have a respiratory chain that is rich in cytochromes, with an unusually high ratio of heme a to heme b that seems to be important in energy coupling at low protonmotive force. Expression of cytochromes in facultative alkaliphiles varies in response to changing pH, but the signal-response network controlling that expression is unknown. The alkaliphile ATP synthase shows unusual biochemical properties, including a pH-dependent gating mechanism, and unusual molecular features in the deduced amino acid sequences of subunits. Each of these features - the pH-dependent expression of cytochromes, the high ratio of cytochrome a/b, and the extraordinary properties of the ATP synthase - has been incorporated into a localized coupling model to explain the anomalies of alkaliphile oxidative phosphorylation. For that reason, each of these features is addressed in this project. First, site-directed mutagenesis and expression studies are designed to test the importance of the unusual molecular domains of the ATP synthase, both in terms of growth at high pH and in the catalytic mechanism of energy coupling. Second, the prevalence of the molecular domains among the alkaliphiles will be tested by characterizing the amino acid sequence of other alkaliphile ATP synthases. This LEXEN project will include studies of a newly isolated alkaliphilic Bacillus. Also included will be a survey of new extreme alkaliphiles, including non-Bacillus bacterial species, which are to be isolated from alkaline terrestrial and aquatic environments. Third, dissection and manipulation of the regulatory circuit that dictates the ratio of cytochrome bc to cytochrome caa3 in the respiratory chain of a new alkaliphile isolate will be performed. Together, these studies will investigate physiological problems of fundamental biological importance, but from the perspective of an organism thriving in an extreme environment. In addition, alkaliphilic bacteria have proved useful in industrial applications, and efforts aimed at facilitating the genetic manipulation of these organisms will enhance their importance.
