Stimulus analysis of BetP activation under in vivo conditions.

The secondary active, Na(+) coupled glycine betaine carrier BetP from Corynebacterium glutamicum BetP was shown to harbor two different functions, transport catalysis (betaine uptake) and stimulus sensing, as well as activity regulation in response to hyperosmotic stress. By analysis in a reconstituted system, the rise in the cytoplasmic K(+) concentration was identified as a primary stimulus for BetP activation. We have now studied regulation of BetP in vivo by independent variation of both the cytoplasmic K(+) concentration and the transmembrane osmotic gradient. The rise in internal K(+) was found to be necessary but not sufficient for BetP activation in cells. In addition hyperosmotic stress is required for full transport activity in cells, but not in proteoliposomes. This second stimulus of BetP could be mimicked in cells by the addition of the amphiphile tetracaine which hints to a relationship of this type of stimulus to a change in membrane properties. Determination of the molecular activity of BetP in both cells and proteoliposomes provided experimental evidence that in proteoliposomes BetP exists in a pre-stimulated condition and reaches full activity already in response to the K(+) stimulus.

Molecular basis of transport and regulation in the Na(+)/betaine symporter BetP.

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.

Potassium transport in corynebacterium glutamicum is facilitated by the putative channel protein CglK, which is essential for pH homeostasis and growth at acidic pH.

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.

Regulatory properties and interaction of the C- and N-terminal domains of BetP, an osmoregulated betaine transporter from Corynebacterium glutamicum.

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.

Use of liposomes to study cellular osmosensors.

When cells are exposed to changes in the osmotic pressure of the external medium, they respond with mechanisms of osmoregulation. An increase of the extracellular osmolality leads to the accumulation of internal solutes by biosynthesis or uptake. Particular bacterial transporters act as osmosensors and respond to increased osmotic pressure by catalyzing uptake of compatible solutes. The functions of osmosensing, osmoregulation , and solute transport of these transporters can be analyzed in molecular detail after solubilization, isolation, and reconstitution into phospholipid vesicles. Using this approach, intrinsic functions of osmosensing transporters are studied in a defined hydrophilic (access to both sides of the membrane) and hydrophobic surrounding (phospholipid membrane), and free of putative interacting cofactors and regulatory proteins.

Influence of membrane composition on osmosensing by the betaine carrier BetP from Corynebacterium glutamicum.

The glycine betaine carrier BetP from Corynebacterium glutamicum was recently shown to function as both an osmosensor and osmoregulator in proteoliposomes made from Escherichia coli phospholipids by sensing changes in the internal K+ concentration as a measure of hyperosmotic stress (Rübenhagen, R., Morbach, S., and Krämer, R. (2001) EMBO J. 20, 5412-5420). Furthermore, evidence was provided that a stretch of 25 amino acids of the C-terminal domain of BetP is critically involved in K+ sensing. This K+-sensitive region has been further characterized. Glu572 turned out to be important for osmosensing in E. coli cells and in proteoliposomes made from E. coli phospholipids. BetP mutants E572K, E572P, and E572A/H573A/R574A were unable to detect an increase in the internal K+ concentration in this membrane environment. However, these BetP variants regained their ability to detect osmotic stress in membranes with increased phosphatidylglycerol content, i.e. in intact C. glutamicum cells or in proteoliposomes mimicking the composition of the C. glutamicum membrane. Mutants E572P and Y550P were still insensitive to osmotic stress also in this membrane background. These results led to the following conclusions. (i) The K+ sensor in mutants E572Q, E572D, and E572K is only partially impaired. (ii) Restoration of activity regulation is not possible if the correct conformation or orientation of the C-terminal domain is compromised by a proline residue at position 572 or 550. (iii) Phosphatidylglycerol in the membrane of C. glutamicum seems to stabilize the inactive conformation of BetP C252T and other mutants.