Osmoregulated transporters sense intracellular osmotic pressure and respond to hyperosmotic stress by accumulation of osmolytes to restore normal hydration levels. Here we report the determination of the X-ray structure of a member of the family of betaine/choline/carnitine transporters, the Na(+)-coupled symporter BetP from Corynebacterium glutamicum, which is a highly effective osmoregulated uptake system for glycine betaine. Glycine betaine is bound in a tryptophan box occluded from both sides of the membrane with aromatic side chains lining the transport pathway. BetP has the same overall fold as three unrelated Na(+)-coupled symporters. Whereas these are crystallized in either the outward-facing or the inward-facing conformation, the BetP structure reveals a unique intermediate conformation in the Na(+)-coupled transport cycle. The trimeric architecture of BetP and the break in three-fold symmetry by the osmosensing C-terminal helices suggest a regulatory mechanism of Na(+)-coupled osmolyte transport to counteract osmotic stress.

The glycine betaine carrier BetP from Corynebacterium glutamicum responds to changes in external osmolality by regulation of its transport activity, and the C-terminal domain was previously identified to be involved in this process. Here we investigate the structural requirements of the C-terminal domain for osmoregulation as well as interacting domains that are relevant for intramolecular signal transduction in response to osmotic stress. For this purpose, we applied a proline scanning approach and amino acid replacements other than proline in selected positions. To analyze the impact of the surrounding membrane, BetP mutants were studied in both C. glutamicum and Escherichia coli, which strongly differ in their phospholipid composition. A region of approximately 25 amino acid residues within the C-terminal domain with a high propensity for alpha-helical structure was found to be essential in terms of its conformational properties for osmodependent regulation. The size of this region was larger in E. coli membranes than in the highly negatively charged C. glutamicum membranes. As a novel aspect of BetP regulation, interaction of the C-terminal domain with one of the cytoplasmic loops as well as with the N-terminal domain was shown to be involved in osmosensing and/or osmoregulation. These results support a functional model of BetP activation that involves the C-terminal domain shifting from interaction with the membrane to interaction with intramolecular domains in response to osmotic stress.

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We studied the requirement for potassium and for potassium transport activity for the biotechnologically important bacterium Corynebacterium glutamicum, which is used for large-scale production of amino acids. Different from many other bacteria, at alkaline or neutral pH, C. glutamicum is able to grow without the addition of potassium, resulting in very low cytoplasmic potassium concentrations. In contrast, at acidic pH, the ability for growth was found to depend on the presence of K ؉ . For the first time, we provide experimental evidence that a potential potassium channel (CglK) acts as the major potassium uptake system in a bacterium and proved CglK's function directly in its natural membrane environment. A full-length CglK protein and a separate soluble protein harboring the RCK domain can be translated from the cglK gene, and both are essential for full CglK functionality. As a reason for potassium-dependent growth limitation at acidic pH, we identified the impaired capacity for internal pH homeostasis, which depends on the availability and internal accumulation of potassium. Potassium uptake via CglK was found to be relevant for major physiological processes, like the activity of the respiratory chain, and to be crucial for maintenance of the internal pH, as well as for the adjustment of the membrane potential in C. glutamicum.