Abstract
P>Natranaerobius thermophilus is an unusual extremophile because it is halophilic, alkaliphilic and thermophilic, growing optimally at 3.5 M Na+, pH55 degrees C 9.5 and 53 degrees C. Mechanisms enabling this tripartite lifestyle are essential for understanding how microorganisms grow under inhospitable conditions, but remain unknown, particularly in extremophiles growing under multiple extremes. We report on the response of N. thermophilus to external pH at high salt and elevated temperature and identify mechanisms responsible for this adaptation. N. thermophilus exhibited cytoplasm acidification, maintaining an unanticipated transmembrane pH gradient of 1 unit over the entire extracellular pH range for growth. N. thermophilus uses two distinct mechanisms for cytoplasm acidification. At extracellular pH values at and below the optimum, N. thermophilus utilizes at least eight electrogenic Na+(K+)/H+ antiporters for cytoplasm acidification. Characterization of these antiporters in antiporter-deficient Escherichia coli KNabc showed overlapping pH profiles (pH 7.8-10.0) and Na+ concentrations for activity (K-0.5 values 1.0-4.4 mM), properties that correlate with intracellular conditions of N. thermophilus. As the extracellular pH increases beyond the optimum, electrogenic antiport activity ceases, and cytoplasm acidification is achieved by energy-independent physiochemical effects (cytoplasmic buffering) potentially mediated by an acidic proteome. The combination of these strategies allows N. thermophilus to grow over a range of extracellular pH and Na+ concentrations and protect biomolecules under multiple extreme conditions.