Christine Maria Ziegler
email: christine.ziegler@biophys.mpg.de

Erhard Bremer

Reinhard Krämer
email: r.kraemer@uni-koeln.de

The BCCT-family of carriers: from physiology to crystal structure

Keywords: osmoregulation, compatible solutes, crystal structures, ligand binding, sensing of water stress Osmoregulation -compatible solutes -sodium-and proton-coupled transporters -antiporters -sensing of water stress -ligand

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Introduction

One of the key parameters affecting the survival and growth of microorganisms are changes in the osmotic conditions of their habitat [START_REF] Kempf | Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments[END_REF][START_REF] Wood | Osmosensing and osmoregulatory compatible solute accumulation by bacteria[END_REF][START_REF] Poolman | How do membrane proteins sense water stress?[END_REF][START_REF] Altendorf | Chapter 5.4.5, Osmotic Stress. In: EcoSal-Escherichia coli and Salmonella: cellular and molecular biology[END_REF][START_REF] Hagemann | Molecular biology of cyanobacterial salt acclimation[END_REF]. Exposure of the cell to either hypo-or hyper-osmotic surroundings triggers rapid water fluxes across the semi-permeable cytoplasmic membrane (Fig. 1) whose direction is determined by the differential in osmotic potential between the cell's interior and the environment [START_REF] Wood | Osmosensing by bacteria: signals and membrane-based sensors[END_REF][START_REF] Bremer | Coping with osmotic challenges: osmoregulation through accumulation and release of compatible solutes[END_REF][START_REF] Altendorf | Chapter 5.4.5, Osmotic Stress. In: EcoSal-Escherichia coli and Salmonella: cellular and molecular biology[END_REF]. Both the osmotically instigated water fluxes and the resulting changes in turgor, an intracellular hydrostatic pressure, impinge on many physiological processes [START_REF] Record | Responses of E. coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water[END_REF][START_REF] Wood | Osmosensing by bacteria: signals and membrane-based sensors[END_REF]. AqpZ-type aquaporins that are present in a substantial number of microorganisms mediate accelerated water fluxes across the cytoplasmic membrane in response to osmotic gradients [START_REF] Tanghe | Why do microorganisms have aquaporins?[END_REF]. However, these types of water channels are not essential for water fluxes across the cell membrane in bacteria. Most importantly, no single microorganism can actively transport water into or out of the cell. But microorganisms can actively influence the direction of water fluxes across the cell membrane in response to hyper-or hypo-osmotic conditions. They do so by indirectly determining the scale of the osmotically driven water fluxes across the cytoplasmic membrane by actively adjusting the osmotic potential of their interior via active control of their ion and organic solute pools (Fig. 1).

Environmental osmotic up-and downshifts and compensatory microbial reactions

Exposure of a microbial cell to a hypo-osmotic surrounding triggers rapid water influx (Fig. 1). In extreme cases, water entry can drive up turgor to such magnitudes that the stressbearing peptidoglycan sacculus can no longer cope with it and the cell is threaten to bursts [START_REF] Booth | Mechanosensitive channels in bacteria: signs of closure?[END_REF]. To avoid such a catastrophic event, mechanosensitive channels are transiently opened as safety valves through which both solutes and solvents can pass rapidly [START_REF] Perozo | Gating prokaryotic mechanosensitive channels[END_REF][START_REF] Martinac | Ion channels in microbes[END_REF][START_REF] Anishkin | State-stabilizing interactions in bacterial mechanosensitive channel gating and adaptation[END_REF]. Hence, the cell can reduce the osmotic potential of its interior within seconds and thereby curbs water influx and reduces turgor; as a consequence, cell lysis is prevented [START_REF] Booth | Mechanosensitive channels in bacteria: signs of closure?[END_REF].

Exposure of a microbial cell to hyper-osmotic conditions elicits water efflux (Fig. 1), thereby causing dehydration of the cytoplasm, a reduction or even collapse of turgor, and eventually growth arrest [START_REF] Record | Responses of E. coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water[END_REF]. The slowing of cell growth and finally growth arrest are strongly correlated with the diminished amounts of free cytoplasmic water [START_REF] Cayley | Roles of cytoplasmic osmolytes, water, and crowding in the response of Escherichia coli to osmotic stress: biophysical basis of osmoprotection by glycine betaine[END_REF]. The cell can increase the content of free cytoplasmic water by raising the osmotic potential of its interior via accumulation of either ions or organic osmolytes [START_REF] Kempf | Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments[END_REF][START_REF] Wood | Osmosensing by bacteria: signals and membrane-based sensors[END_REF][START_REF] Bremer | Coping with osmotic challenges: osmoregulation through accumulation and release of compatible solutes[END_REF][START_REF] Wood | Osmosensing and osmoregulatory compatible solute accumulation by bacteria[END_REF][START_REF] Oren | Microbial life at high salt concentrations: phylogenetic and metabolic diversity[END_REF][START_REF] Altendorf | Chapter 5.4.5, Osmotic Stress. In: EcoSal-Escherichia coli and Salmonella: cellular and molecular biology[END_REF][START_REF] Hagemann | Molecular biology of cyanobacterial salt acclimation[END_REF].

Properties of compatible solutes

Many microorganisms accumulate a selected class of organic osmolytes, the compatible solutes, to fend off the detrimental effects of high osmolarity on water content and cell physiology [START_REF] Kempf | Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments[END_REF][START_REF] Bremer | Coping with osmotic challenges: osmoregulation through accumulation and release of compatible solutes[END_REF][START_REF] Wood | Osmosensing and osmoregulatory compatible solute accumulation by bacteria[END_REF][START_REF] Empadinhas | Osmoadaptation mechanisms in prokaryotes: distribution of compatible solutes[END_REF]. Compatible solutes are operationally defined as organic osmolytes that can be accumulated to exceedingly high intracellular levels without impairing vital functions of the cell [START_REF] Kempf | Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments[END_REF][START_REF] Burg | Intracellular organic osmolytes: function and regulation[END_REF][START_REF] Empadinhas | Osmoadaptation mechanisms in prokaryotes: distribution of compatible solutes[END_REF]. These solutes have biophysical and biochemical properties that distinguish them from other types of organic compounds, and they have been specifically selected during the course of evolution as cytoprotectants in all three kingdoms of life [START_REF] Yancey | Living with water stress: the evolution of osmolyte systems[END_REF][START_REF] Yancey | Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses[END_REF]. A hallmark of compatible solutes is their preferential exclusion from the immediate hydration shell of proteins [START_REF] Arakawa | The stabilization of proteins by osmolytes[END_REF] due to unfavourable interactions between the osmolytes and the protein backbone [START_REF] Bolen | Structure and energetics of the hydrogen-bonded backbone in protein folding[END_REF]. This uneven distribution of compatible solutes in the cell water generates a thermodynamic driving force that promotes the proper conformation of proteins and the formation of protein assemblies, thereby maintaining their biological functions [START_REF] Bolen | The osmophobic effect: natural selection of a thermodynamic force in protein folding[END_REF][START_REF] Ignatova | Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant[END_REF][START_REF] Street | A molecular mechanism for osmolyteinduced protein stability[END_REF]Rosgen, 2007;[START_REF] Capp | Interactions of the osmolyte glycine betaine with molecular surfaces in water: thermodynamics, structural interpretation, and prediction of m-values[END_REF]Rosgen, 2009;[START_REF] Street | Osmolyte-induced conformational changes in the Hsp90 molecular chaperone[END_REF]. It should be noted however, that compatible solutes that are ubiquitously used by microorganisms (e.g. proline and glycine betaine) interact differently with protein surfaces [START_REF] Auton | Structural thermodynamics of protein preferential solvation: osmolyte solvation of proteins, aminoacids, and peptides[END_REF].

Microbial transport systems for compatible solutes

Most microorganisms that adopt the "salt-out" strategy both synthesize and acquire various types of compatible solutes. Osmotically down-shocked or decaying microbial cells (e.g. after viral attack), excretion products of animals and plants (urine and root exudates, respectively) and of algae are sources of these compounds [START_REF] Welsh | Ecological significance of compatible solute accumulation by microorganisms: from single cells to global climate[END_REF]. Compatible solutes are found in very low concentrations (typically in the µM or nM range) in the environment. To take advantage of these scarce resources as osmostress protectants, microorganisms use osmotically controlled high-affinity uptake systems [START_REF] Kempf | Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments[END_REF][START_REF] Wood | Osmosensing by bacteria: signals and membrane-based sensors[END_REF][START_REF] Bremer | Coping with osmotic challenges: osmoregulation through accumulation and release of compatible solutes[END_REF][START_REF] Wood | Osmosensing and osmoregulatory compatible solute accumulation by bacteria[END_REF][START_REF] Poolman | Bacterial osmosensing: roles of membrane structure and electrostatics in lipid-protein and protein-protein interactions[END_REF][START_REF] Wood | Osmosensing by bacteria[END_REF] [START_REF] Hagemann | Molecular biology of cyanobacterial salt acclimation[END_REF]. The intracellular level to which compatible solutes are amassed is fine-tuned to the degree of osmotic stress perceived by the microbial cell. Microorganisms can accumulate compatible solutes via transport systems against steep concentration gradients, and intracellular concentrations above one molar can be attained [START_REF] Peter | Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine[END_REF][START_REF] Krämer | Osmosensing and osmosignaling in Corynebacterium glutamicum[END_REF]. Uptake of compatible solutes by microorganisms is energetically favourable over de novo synthesis [START_REF] Oren | Bioenergetic aspects of halophilism[END_REF]. Furthermore, the uptake of compatible solutes often represses the expression of genes encoding enzymes required for compatible solute synthesis.

Transport systems for compatible solutes not only serve to capture these compounds from scarce environmental sources but also serve for the re-import of these compounds leaked from microbial producer cells in high osmolarity surroundings [START_REF] Lamark | Efflux of choline and glycine betaine from osmoregulating cells of Escherichia coli[END_REF][START_REF] Mikkat | Molecular analysis of the ggtBCD gene cluster of Synechocystis sp. strain PCC6803 encoding subunits of an ABC transporter for osmoprotective compounds[END_REF][START_REF] Grammann | New type of osmoregulated solute transporter identified in halophilic members of the bacteria domain: TRAP transporter TeaABC mediates uptake of ectoine and hydroxyectoine in Halomonas elongata DSM 2581(T)[END_REF].

Frequently, the transcription of the structural genes encoding compatible solute transporters are up-regulated in response to increases in the osmolarity of the environment.

Typically, gene expression is rapidly induced after the cells experience an osmotic up-shift, promoter activity being adjusted to the degree of the prevalent osmotic stress [START_REF] Spiegelhalter | Osmoregulation of the opuE proline transport gene from Bacillus subtilis -contributions of the σ A -and σ B -dependent stress-responsive promoters[END_REF][START_REF] Cheung | A microarray-based antibiotic screen identifies a regulatoty role for supercoiling in the osmotic stress response of Escherichia coli[END_REF][START_REF] Nagarajavel | Repression by binding of H-NA within the transcription unit[END_REF][START_REF] Romeo | Osmotic regulation of transcription in Lacococcus lactis: ionic-strenght-dependent binding of the BusR repressor to the busA promoter[END_REF][START_REF] Rosenthal | Poising of the Escherichia coli RNA polymerase and its release from the sigma 38 C-terminal tail for osmY transcription[END_REF][START_REF] Altendorf | Chapter 5.4.5, Osmotic Stress. In: EcoSal-Escherichia coli and Salmonella: cellular and molecular biology[END_REF][START_REF] Krämer | Bacterial stimulus perception and signal transduction: response to osmotic stress[END_REF]. Furthermore, the activity of the compatible solute transporter itself is often subjected to osmotic control on the level of activity [START_REF] Wood | Osmosensing by bacteria: signals and membrane-based sensors[END_REF][START_REF] Van Der Heide | Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane[END_REF][START_REF] Van Der Heide | On the osmotic signal and osmosensing mechanism of an ABC transport system for glycine betaine[END_REF][START_REF] Poolman | Bacterial osmosensing: roles of membrane structure and electrostatics in lipid-protein and protein-protein interactions[END_REF][START_REF] Wood | Osmosensing by bacteria[END_REF][START_REF] Krämer | Osmosensing and osmosignaling in Corynebacterium glutamicum[END_REF][START_REF] Mahmood | Engineering of ion sensing by the cystathionine beta-synthase module of the ABC transporter OpuA[END_REF][START_REF] Romantsov | Cardiolipin and the osmotic stress responses of bacteria[END_REF]. Thus, microbial uptake systems for compatible solutes are not only effective transporters but they can also sense and respond to osmotic stress themselves. This permits a practically instantaneous physiological response by allowing the osmotically stressed cell to accumulate types of solutes that restrict water loss and that are at the same time congruous with its other physiological needs. By sensitive adjustment of both the amount of the transport protein(s) via gene expression and de novo protein synthesis and the activity of the transporter itself, the bacterial cell can exert maximum control over its cellular compatible solutes pool.

The preferential exclusion of compatible solutes from protein surfaces is a hallmark of the protective function of these types of solutes [START_REF] Arakawa | The stabilization of proteins by osmolytes[END_REF][START_REF] Bolen | The osmophobic effect: natural selection of a thermodynamic force in protein folding[END_REF][START_REF] Ignatova | Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant[END_REF][START_REF] Street | A molecular mechanism for osmolyteinduced protein stability[END_REF]Rosgen, 2007;[START_REF] Bolen | Structure and energetics of the hydrogen-bonded backbone in protein folding[END_REF][START_REF] Street | Osmolyte-induced conformational changes in the Hsp90 molecular chaperone[END_REF]. However, in transport systems for compatible solutes, highaffinity interactions between the substrates and proteins have to take place in order to archive effective import. Crystallographic studies with soluble ligand-binding proteins from six ABCand two TRAP-transport systems and the secondary transporters BetP and CaiT have recently provided insight into how such a task can be accomplished (Schiefner et al., 2004a;Schiefner et al., 2004b;[START_REF] Horn | Molecular determinants for substrate specificity of the ligand-binding protein OpuAC from Bacillus subtilis for the compatible solutes glycine betaine and proline betaine[END_REF][START_REF] Oswald | Crystal structures of the choline/acetylcholine substratebinding protein ChoX from Sinorhizobium meliloti in the liganded and unligandedclosed states[END_REF][START_REF] Smits | The compatible-solute-binding protein OpuAC from Bacillus subtilis: ligand binding, sitedirected mutagenesis, and crystallographic studies[END_REF][START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF][START_REF] Tang | Crystal structure of the carnitine transporter and insights into the antiport mechanism[END_REF][START_REF] Schulze | Structural basis of Na + -independent and cooperative substrate/product antiport in CaiT[END_REF][START_REF] Wolters | Ligand binding and crystal structures of the substrate-binding domain of the ABC transporter OpuA[END_REF]. In all these systems, aromatic ligand binding boxes of various architectures recognize the bulky head-groups of glycine betaine, choline, L-carnitine, γ-butyrobetaine and DMSA (Fig. 2) via cation-pi interactions.

Hydrogen bonds and salt-bridges then fix and orient the various "tails" of the different substrates (Fig. 2) within the ligand-binding site. Cation-pi interactions also play important roles for the high-affinity binding of ectoine and hydroxyectoine by soluble ligand binding proteins from ABC-or TRAP-transporters, but due to the chemical features of these compatible solutes (Figure 2), the architecture of the ligand-binding sites is different form those that are used to bind compatible solutes such as glycine betaine, proline betaine and DMSA [START_REF] Hanekop | Crystal structure of the ligand-binding protein EhuB from Sinorhizobium meliloti reveals substrate recognition of the compatible solutes ectoine and hydroxyectoine[END_REF][START_REF] Kuhlmann | Osmoregulated transport of compatible solutes in the halophilic bacterium Halomonas elongata: 1.55 A high-resolution structure of the periplasmic ectoinebinding protein from TRAP-transporter TeaABC[END_REF][START_REF] Lecher | The crystal structure of UehA in complex with Ectoine-A comparison with Other TRAP-T binding proteins[END_REF].

The types of functionally characterized microbial transporters for compatible solutes include members of the multi-component ABC (e.g. ProU, OpuA, and Ehu) and TRAP transporter families (Tea and Ueh) and single-component transport systems such as ProP (H + /solute symporter; MSF-family), OpuE (Na + /solute symporter; SSSF-family) and members of the BCCT family (e.g. BetT, BetP, OpuD). The prototypes of these osmotically controlled uptakes systems have been discovered through diligent genetic and physiological studies with just a few bacterial species [START_REF] Kempf | Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments[END_REF][START_REF] Bremer | Coping with osmotic challenges: osmoregulation through accumulation and release of compatible solutes[END_REF][START_REF] Wood | Osmosensing and osmoregulatory compatible solute accumulation by bacteria[END_REF][START_REF] Bremer | Adaptation to changing osmolarity. In: Bacillus subtilis and its closest relatives: from genes to cells[END_REF][START_REF] Altendorf | Chapter 5.4.5, Osmotic Stress. In: EcoSal-Escherichia coli and Salmonella: cellular and molecular biology[END_REF][START_REF] Krämer | Osmosensing and osmosignaling in Corynebacterium glutamicum[END_REF]. However, even a cursory inspection of the more than 1 000 microbial genome sequences available today shows that compatible solute uptake systems related to those mentioned above are found widely in the domain of the Bacteria and of the Archaea.

Three transporters have been studied in detail by biochemical, biophysical and genetic approaches with the aim of unravelling the mechanism(s) and interplay of their osmoregulatory and transporter function. These are the ABC transporter OpuA from Lactococcus lactis, the H + /solute symporter ProP from Escherichia coli and the BCCT carrier BetP from Corynebacterium glutamicum (van der Heide and [START_REF] Van Der Heide | Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane[END_REF]van der Heide et al., 2001;[START_REF] Poolman | Bacterial osmosensing: roles of membrane structure and electrostatics in lipid-protein and protein-protein interactions[END_REF]Culham et al., 2008a;Culham et al., 2008b;[START_REF] Krämer | Osmosensing and osmosignaling in Corynebacterium glutamicum[END_REF][START_REF] Mahmood | Engineering of ion sensing by the cystathionine beta-synthase module of the ABC transporter OpuA[END_REF].

The BCCT carrier BetP from C. glutamicum occupies a special position within this group of osmoregulatory compatible solute transport systems because a well-resolved crystal structure of the BetP protein with bound glycine betaine and sodium ligands has been reported [START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF]. Most recently, three X-ray structures of another BCCT-carrier, the L-carnitine/γ-butyrobetaine antiporter CaiT from E. coli and Proteus mirabilis was solved in complex with its substrate L-carnitine and γ-butyrobetaine [START_REF] Tang | Crystal structure of the carnitine transporter and insights into the antiport mechanism[END_REF][START_REF] Schulze | Nature (in press) * The BetT protein from Haemophilus influenzae was suggested to function in the uptake of [Fan et al[END_REF]. The CaiT proteins from these two organisms are not involved in osmostress responses and instead mediate L-carnitine:γ-butyrobetaine antiport under unaerobic growth conditions [START_REF] Jung | CaiT of Escherichia coli, a new transporter catalyzing L-carnitine/gammabutyrobetaine exchange[END_REF].

The ligand-bound crystal structures of the BCCT carriers BetP [START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF] and CaiT [START_REF] Tang | Crystal structure of the carnitine transporter and insights into the antiport mechanism[END_REF][START_REF] Schulze | Structural basis of Na + -independent and cooperative substrate/product antiport in CaiT[END_REF] reveal common characteristics with respect to their overall fold, their topological organization with the cytoplasmic membrane and the transport function of these proteins. However, distinct features became apparent from these crystal structures that build the foundation for the osmotic control of the transport activity of BetP. Using the structural data of both the BetP and CaiT transporters and a wealth of biochemical data available for BetP [START_REF] Ott | Regulatory properties and interaction of the C-and N-terminal domains of BetP, an osmoregulated betaine transporter from Corynebacterium glutamicum[END_REF][START_REF] Krämer | Osmosensing and osmosignaling in Corynebacterium glutamicum[END_REF][START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF] and CaiT [START_REF] Jung | CaiT of Escherichia coli, a new transporter catalyzing L-carnitine/gammabutyrobetaine exchange[END_REF][START_REF] Schulze | Structural basis of Na + -independent and cooperative substrate/product antiport in CaiT[END_REF] as a point of reference, we will evaluate features for osmotic activity control of BetP to assess the physiological roles of various microbial BCCT carriers.

The BCCT carriers: discovery and general features

The first member (BetT) of the subsequently christened BCCT transporter family was described almost 20 years ago by Arne R. Strom and co-workers in connection with the genetic analysis of the osmoregulatory choline-to-glycine betaine synthesis pathway of E. coli [START_REF] Lamark | DNA sequence and analysis of the bet genes encoding the osmoregulatory cholineglycine betaine pathway of Escherichia coli[END_REF]. BetT was shown to mediate the high-affinity uptake of choline, the biosynthetic precursor for the compatible solute glycine betaine. A L-carnitine transporter protein (CaiT) related to the choline transporter BetT was subsequently discovered when the genes involved in L-carnitine metabolism in E. coli were studied [START_REF] Eichler | Molecular characterization of the cai operon necessary for carnitine metabolism in Escherichia coli[END_REF]. The simultaneous discoveries of the OpuD transporter from Bacillus subtilis [START_REF] Kappes | Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD[END_REF] and BetP from C. glutamicum [START_REF] Peter | Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine[END_REF], both involved in osmotically controlled glycine betaine uptake, led then [START_REF] Kappes | Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD[END_REF] to propose that the afore-mentioned choline, L-carnitine and glycine betaine transporters form a new transporter sub-family with substrate specificity for various types of trimethylammonium compounds (Fig. 2).

Using the substrates of the BetT, CaiT, OpuD and BetP transporters as a point of reference, Milton Saier's laboratory subsequently grouped these transporters into the BCCT family (betaine-choline-carnitine-transporter) [START_REF] Saier | A functional-phylogenetic classification system for transmembrane solute transporters[END_REF] and placed them into sub-group 2.A.15 of the International Union of Biochemistry and Molecular Biology-approved transporter classification (TC) system [http://www.tcdb.org] [START_REF] Saier | The transporter classification database: recent advances[END_REF]. BCCT BetT) or sodium-motive-force-driven (e.g. BetP) symport, or more rarely, by substrate:product antiport (e.g. CaiT) (Table 1).

It was originally thought that a unifying feature of BCCT carriers is the transport of substrates with quaternary ammonium groups [R-N + (CH) 3 ) 3 ] such as choline, L-carnitine and glycine betaine [START_REF] Kappes | Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD[END_REF][START_REF] Saier | A functional-phylogenetic classification system for transmembrane solute transporters[END_REF] (Fig. 2). In recent years, several new substrates for BCCT carriers have been discovered. Of these, proline betaine, acetylcholine, dimethylsulfoniopropionate (DMSP) and dimethylsulfonioacetate (DMSA) all posses fully methylated nitrogen-or sulfonium headgroups (Fig. 2) and, thus, conform in a wider sense to the originally suggested chemical features of substrates for BCCT carriers. However, the chemical structures of the compatible solutes ectoine and hydroxyectoine (Fig. 2), substrates for BCCT carriers detected in various microbial species (Table 1), do not match this general scheme. In addition, some BCCT-type transporters also mediate proline uptake, but usually with rather low affinity. So far, no functionally characterized BCCT carrier has been described to catalyze the uptake of the full range of currently known substrates (Table 1; Fig. 2). However, for some BCCT carriers competition experiments have been carried out that suggests a rather restricted substate specificity for a given BCCT carrier. For instance, in the halotolerant alakaliphilic cyanobacterium Aphanothece halophytica, the BetT-mediated glycine betaine transport (Table 1) was not inhibited by glycine betaine aldehyde, dimethylglycine, sarchosine, choline or proline [START_REF] Laloknam | Halotolerant cyanobacterium Aphanothece halophytica contains a betaine transporter active at alkaline pH and high salinity[END_REF].

Physiological functions of BCCT carriers and transcriptional regulation of their structural genes

The explosion in the number of available genome sequences of Bacteria and Archaea revealed a widespread distribution of BCCT carriers in the microbial world. Under the entry "IPR000060 BCCT transporter", the InterPro database [http://www.ebi.ac.uk/interpro/] lists currently about 2 200 protein sequences. Most of these protein sequences originate from members of the Bacteria and only 22 are from members of the Archaea. The InterPro database also records a limited number of BCCT carriers from Eukarya, but nothing is known about their function.

Most BCCT carriers have a common hydropathy profile with 12 predicted transmembrane (TM)-spanning segments and exhibit N-and C-terminal extensions of various lengths (Table 1) that are predicted to protrude into the cytoplasm. An important functional role of these N-and C-terminal domains for the control of transport activity and osmotic regulation was first discovered for the glycine betaine transporter BetP (Peter et al., 1998a).

Subsequent studies with the choline transporters BetT of E. coli [START_REF] Tondervik | Membrane topology and mutational analysis of the osmotically activated BetT choline transporter of Escherichia coli[END_REF] and Pseudomonas syringae [START_REF] Chen | Pseudomonas syringae BetT is a low-affinity choline transporter that is responsible for superior osmoprotection by choline over glycine betaine[END_REF], revealed the contribution of the Cterminal domain for the proper functioning of these BCCT carriers as well. The most conserved part of BCCT carriers comprises TM8 and the connecting loop to TM9 and these segments contain the tryptophane motif that is the signature sequence of BCCT carriers [START_REF] Kappes | Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD[END_REF][START_REF] Saier | A functional-phylogenetic classification system for transmembrane solute transporters[END_REF].

In the Pfam database [http://pfam.sanger.ac.uk/] some physiologically uncharacterized BCCT carrier related proteins are listed that possess additional domains (Usp, CoaE, TrkA_C) or membrane-spanning segments fused to the canonical 12 TM core structure of BCCT carriers. Twenty-two microbial BCCT carriers have so far been functionally characterized at least to some extent (Table 1). Using the C. glutamicum BetP glycine betaine transporter protein as the search template, these 22 proteins exhibit a range from 48% down to 25% identical amino acid residues (Table 1). However, it should be noted that functionally uncharacterized BCCT carriers can readily be found in database searches that are much more closely related to the BetP protein from C. glutamicum then those listed in Table 1.

Only two of the functionally characterized transporters of the BCCT-family are physiologically not connected with microbial osmotic adjustment processes: the CaiT proteins from E. coli and Proteus mirabilis (Table 1). The CaiT proteins are unusual members of the BCCT-family, since they do not require a sodium-or proton-gradient to fuel transport and instead function according to the substrate:product antiport principle. L-carnitine (Fig. 2) is taken up in E. coli via CaiT under anaerobic growth conditions when no other electron acceptor or glucose is present and it is then metabolized to γ-butyrobetaine (Fig. 2) that is subsequently excreted via the CaiT antiporter [START_REF] Jung | CaiT of Escherichia coli, a new transporter catalyzing L-carnitine/gammabutyrobetaine exchange[END_REF]. The genetic regulation of the caiTABCDE gene cluster reflects this physiological role. It is controlled by the globally acting regulators CRP and FNR (both positively acting) and the nucleoid-associated protein H-NS (negatively acting) of E. coli and by the carnitine-responsive activator protein CaiF [START_REF] Buchet | Regulation of the carnitine pathway in Escherichia coli: investigation of the cai-fix divergent promoter region[END_REF]. A BCCT carrier (DddT) was recently shown to mediate uptake of sulfur-containing compatible solute dimethylsulfoniopropionate (DMSP) (Fig. 2) in a relative of Halomonas venusta [START_REF] Todd | Molecular dissection of bacterial acrylate catabolism -unexpected links with dimethylsulfoniopropionate catabolism and dimethyl sulfide production[END_REF]. DMSP is widespread in marine environments and can be used by microorganisms as an effective osmoprotectant or as a versatile sulfur source (Pichereaux et al., 1998;Dickschat et al., 20110;Vila-Costa et al., 2010). DddT-mediated uptake of DMSP in the studies Halomonas strain is connected with catabolism since expression of the dddT gene is induced by DMSP and two of its metabolites [START_REF] Todd | Molecular dissection of bacterial acrylate catabolism -unexpected links with dimethylsulfoniopropionate catabolism and dimethyl sulfide production[END_REF].

The majority (19 out of 22) of the currently functionally characterized BCCT carriers (Table 1) are involved in microbial adjustment to high osmolarity surroundings via uptake of compatible solutes or their biosynthetic precursors. In line with these physiological functions, expression of their structural genes is typically induced in cells grown at high osmolarity.

With the exception of the proton-coupled choline transporter BetT from E. coli and from Pseudomonas syringae and CutT from Staphylococcus xylosus [START_REF] Lamark | DNA sequence and analysis of the bet genes encoding the osmoregulatory cholineglycine betaine pathway of Escherichia coli[END_REF][START_REF] Rosenstein | The choline-converting pathway in Staphylococcus xylosus C2A: genetic and physiological characterization[END_REF][START_REF] Tondervik | Membrane topology and mutational analysis of the osmotically activated BetT choline transporter of Escherichia coli[END_REF][START_REF] Chen | Pseudomonas syringae BetT is a low-affinity choline transporter that is responsible for superior osmoprotection by choline over glycine betaine[END_REF], substrate uptake via BCCT carriers is driven by sodium symport (Table 1). As an example, BetPmediated import of one molecule of glycine betaine is accompanied by the uptake of two sodium ions [START_REF] Farwick | Glycine betaine uptake after hyperosmotic shift in Corynebacterium glutamicum[END_REF][START_REF] Peter | Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine[END_REF]. Since glycine betaine concentration above 1 mol/l can be attained by C. glutamicum, massive amounts of Na + ions will be pumped into the cell under osmotic stress conditions. It is apparent that the functioning of Na + -driven BCCT carrires for osmoprotectants (Table 1) must be physiologically coupled to the functioning of Na + extrusion systems in order to keep the intracellular concentration of this cytotoxic cation low [START_REF] Padan | Alkaline pH homeostasis in bacteria: new insights[END_REF].

Functional data, such as K M and V max values, sodium-or proton-coupling and a description of their activity regulation are available only for a few of the BCCT carriers systems listed in Table 1. We refer the readers for a detailed account of these topics to the original publication (Table 1). A convenient way to assess the substrate specificity of a given BCCT carriers, or the genetic section for BCCT carriers-encoding genes form different microorganisms by functional complementation [START_REF] Kappes | Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD[END_REF][START_REF] Peter | Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine[END_REF]Ly et al., 2004), is the use of an E. coli K-12 mutant strain (MKH13), which lacks the genes for choline uptake via BetT and glycine betaine synthesis (betBA) and that is simultaneously defective in the ProU and ProP compatible solute transport systems [START_REF] Haardt | The osmoprotectant proline betaine is a major substrate for the binding-protein-dependent transport system ProU of Escherichia coli K-12[END_REF].

It is generally not firmly understood at the molecular level how bacteria sense osmotic stress and how this environmental cue ultimately results in altered gene transcription [START_REF] Bremer | Coping with osmotic challenges: osmoregulation through accumulation and release of compatible solutes[END_REF][START_REF] Cheung | A microarray-based antibiotic screen identifies a regulatoty role for supercoiling in the osmotic stress response of Escherichia coli[END_REF][START_REF] Nagarajavel | Repression by binding of H-NA within the transcription unit[END_REF][START_REF] Romeo | Osmotic regulation of transcription in Lacococcus lactis: ionic-strenght-dependent binding of the BusR repressor to the busA promoter[END_REF][START_REF] Rosenthal | Poising of the Escherichia coli RNA polymerase and its release from the sigma 38 C-terminal tail for osmY transcription[END_REF][START_REF] Altendorf | Chapter 5.4.5, Osmotic Stress. In: EcoSal-Escherichia coli and Salmonella: cellular and molecular biology[END_REF][START_REF] Krämer | Bacterial stimulus perception and signal transduction: response to osmotic stress[END_REF]. Information on osmotically controlled gene expression is available for only a few of the functionally characterized BCCT carriers (Table 1). For instance, osmotic induction of betP transcription is governed by the two-component MtrAB regulatory system of C. glutamicum (Moker et al., 2004;Moker et al., 2007). Expression of the B. subtilis opuD gene is partially dependent on the alternative Virgibacillus pantothenticus (Kuhlmann et al.; unpublished data). The substrates of osmotically induced BCCT carriers typically do not trigger gene expression of their corresponding structural genes, with the exception of the choline transporters BetT from E. coli [START_REF] Lamark | The complex bet promoters of Escherichia coli: regulation by oxygen (ArcA), choline (BetI), and osmotic stress[END_REF] and CudT from Staphylococcus xylosus [START_REF] Rosenstein | The choline-converting pathway in Staphylococcus xylosus C2A: genetic and physiological characterization[END_REF] where unrelated choline-responsive repressor proteins (BetI and CudC) have been identified.

Osmoregulation and simultaneous substrate induction of betT and cudT transcription is of physiological relevance because the function of choline as an osmoprotectant is entirely dependent on its conversion into glycine betaine.

It is increasingly recognized that compatible solutes not only function as microbial osmoprotectants, but serve other stress protective functions as well [START_REF] Yancey | Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses[END_REF][START_REF] Empadinhas | Osmoadaptation mechanisms in prokaryotes: distribution of compatible solutes[END_REF]. In particular, a compatible-solute-mediated temperature stress protection both at the upper and lower boundaries of growth has been observed in various microbial species. BCCT carriers participate in these processes in several microorganisms. Examples are the BetT-mediated uptake of choline in E. coli [START_REF] Caldas | Thermoprotection by glycine betaine and choline[END_REF] and the OpuD-mediated uptake of glycine betaine in B. subtilis as heat stress protectants [START_REF] Holtmann | Thermoprotection of Bacillus subtilis by exogenously provided glycine betaine and structurally related compatible solutes: involvement of Opu transporters[END_REF], the chill stress protection by glycine betaine via BetL-mediated import in Listeria monocytogenes [START_REF] Angelidis | Role of the glycine betaine and carnitine transporters in adaptation of Listeria monocytogenes to chill stress in defined medium[END_REF][START_REF] Sheehan | Heterologous expression of BetL, a betaine uptake system, enhances the stress tolerance of Lactobacillus salivarius UCC118[END_REF] and OpuD in B. subtilis (Hoffmann and Bremer; unpublished data) and the EctTmediated uptake of ectoine/hydroxyectoine in V. pantothenticus (Kuhlmann et al.; unpublished data).

The BetP transporter from C. glutamicum and other BCCT carriers: structure, function and regulation

Function of BetP from C. glutamicum in response to osmotic stress

In comparison to other bacteria such as E. coli [START_REF] Altendorf | Chapter 5.4.5, Osmotic Stress. In: EcoSal-Escherichia coli and Salmonella: cellular and molecular biology[END_REF] or B. subtilis [START_REF] Bremer | Adaptation to changing osmolarity. In: Bacillus subtilis and its closest relatives: from genes to cells[END_REF], which employ a combination of primary transporters (the ABC transporters) and secondary transporters, uptake of compatible solutes in C. glutamicum is mediated entirely by osmoregulated secondary transporters (Peter et al., 1998b). Three of them, BetP, EctP, and LcoP, are members of the BCCT family. These transporters vary in substrate specificity, affinity, and transcriptional regulation, but all respond to hyperosmotic stress by regulating the level of transport activity [START_REF] Krämer | Osmosensing and osmosignaling in Corynebacterium glutamicum[END_REF]. BetP is by far the best described, both with respect to transport and regulation. BetP is specific for glycine betaine and couples its uptake to the electrochemical Na + potential (smf) by co-transport with two Na + ions, leading to extremely high accumulation ratios up to 10 6 and internal concentrations above 1 mol/l [START_REF] Farwick | Glycine betaine uptake after hyperosmotic shift in Corynebacterium glutamicum[END_REF][START_REF] Peter | Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine[END_REF].

The activity of BetP is close to zero in the absence of hyperosmotic stress. Upon osmotic challenge, BetP starts taking up betaine in less than a second (Peter et al., 1998a).

When transport activity is plotted against the extent of osmotic stress, a steep sigmoidal increase in activity is observed until an optimum is reached; in intact C. glutamicum cells this occurs around 1.3 osmol/kg (Fig. 3). Consequently, besides its catalytic activity (Na + -coupled glycine betaine uptake), BetP acts both as an osmosensor and an osmoregulator by responding to osmotic stress with instant regulation of its transport activity. This fact was documented both by heterologous expression of the betP gene in E. coli [START_REF] Peter | Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine[END_REF], and by reconstitution of the purified BetP protein in proteoliposomes [START_REF] Rübenhagen | Osmosensor and osmoregulator properties of the betaine carrier BetP from Corynebacterium glutamicum in proteoliposomes[END_REF]. In these two artificial membrane surroundings, the BetP transporter from C. glutamicum retained all its functions, indicating that no other proteinaceous activating factors are involved in controlling BetP activity. The differences in the activation pattern, observed in Fig. 3, can be explained by a significantly different share of negatively charged phospholipids in the membranes of the two different organisms [START_REF] Schiller | Influence of membrane composition on osmosensing by the betaine carrier BetP from Corynebacterium glutamicum[END_REF]. Activation of BetP is likely modulated through transitions between at least two different functional and conformational states: inactive or active. Hyperosmotic stress will shift the balance towards the active state, while hypo-osmotic stress will favour the inactive conformation of BetP. Thus, the function of BetP can be described by a kinetic scheme (Fig. 4) comprising two functional cycles: (i) The catalytic cycle of glycine betaine transport corresponds to that of a secondary transporter. It includes a conformational change between outward and inward facing carrier molecules [START_REF] Jardetzky | Simple allosteric model for membrane pumps[END_REF] and a cycle between substrate binding and release. The turnover in this cycle depends on both glycine betaine availability and the energetic driving force. (ii) The balance between the two-state regulatory cycles of BetP depends on certain stimuli; some of them are related to osmotic stress. An assumption inherent in the regulatory cycle of BetP as depicted in Fig. 4 is that the occluded states of the transporter has been chosen as the points where the two cycles connect, but the mechanistically correct connection point is not known so far. During both the catalytic and the regulatory cycle, BetP passes through several functional states and, consequently, must adopt different conformations. BCCT carriers involved in osmotic stress response catalyze Na + -or H + -symport, during which the smf or pmf drives the conformational change from an outward-facing to an inwardfacing state according to the alternating access mechanism of secondary transporters [START_REF] Jardetzky | Simple allosteric model for membrane pumps[END_REF] (Fig. 4). Structural data are required in order to understand the mechanisms of ion-coupled osmolyte symport by BCCT carriers. Crystal structures of two members of this transporter family are available to date: the structure of BetP from C. glutamicum, which was solved by X-ray crystallography to 3.35 Å resolution in complex with its substrate glycine betaine [START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF] [PDB entry 2WIT], a 3.15Å structure of CaiT from E. coli in complex with four L-carnitine molecules [START_REF] Tang | Crystal structure of the carnitine transporter and insights into the antiport mechanism[END_REF] [PDB entry 3HFX], a 3.5 Å structure of CaiT from E. coli in complex with two γ-butyrobetaine molecules [PDB code: 2WSX], and an apo structure of CaiT [PDB entry 2WSW] from P. mirabilis at 2.3Å [START_REF] Schulze | Structural basis of Na + -independent and cooperative substrate/product antiport in CaiT[END_REF]. Although CaiT shares the least sequence identity to BetP of all functionally characterized BCCT carriers listed in Table 1, the crystal structures of BetP and CaiT superimpose quite well with an r.m.s.d. deviation of ~2.5 Å. Both the purified BetP and CaiT proteins form trimers [START_REF] Jung | CaiT of Escherichia coli, a new transporter catalyzing L-carnitine/gammabutyrobetaine exchange[END_REF][START_REF] Ziegler | Projection structure and oligomeric state of the osmoregulated sodium/glycine betaine symporter BetP of Corynebacterium glutamicum[END_REF][START_REF] Vinothkumar | Oligomeric structure of the carnitine transporter CaiT from Escherichia coli[END_REF] and both proteins also crystallize as trimers (Fig. 5). Each subunit consisting of twelve TM αhelices and contain a curved amphipathic α-helix (helix 7) that runs parallel to the plane of the cytoplasmic membrane and a long bend TM helix (TM2), which both provide a hydrophobic scaffold surrounding the centre of the transporter (Fig. 6). Helix 7 is located at the trimer centre of both the BetP and CaiT transporters, close to the periplasmic membrane surface, where it mediates contacts between all three subunits within the trimer at the periplasmic side.

A feature that distinguishes BetP from CaiT is a α-helical C-terminal domain protruding into the cytoplasm (Fig. 5B). In BetP, the C-terminal domain on the cytoplasmic side contributes to trimer contacts and as a result, a slightly different subunit arrangement within the trimers of the BetP and CaiT crystal structures are observed (Fig. 5B). In comparison to BetP, each subunit within the trimer assembly is tilted somewhat resulting in a larger cavity in the trimer centre of CaiT (Fig. 5B, black arrow). TM3, TM4, TM8 and TM9 form an iris-shaped 4-helix bundle close to the centre of both the BetP and CaiT subunit, with aromatic side chains in TM4 and TM8 contributing to the substrate-binding site (Fig. 7B). The 3.15 Å crystal structure of CaiT from E. coli reveals four L-carnitine molecules (Fig. 7A) most likely because the protein was crystallized with an excess of L-carnitine. The primary binding site at the center of the protein and the secondary substrate-binding site at the bottom of the intracellular vestibule have been confirmed by site-directed mutagenesis studies F o r P e e r R e v i e w [START_REF] Tang | Crystal structure of the carnitine transporter and insights into the antiport mechanism[END_REF], while the two others L-carnitine molecules might by associated with the crystallized CaiT protein for structural reasons. The ligand-binding site in one of the E. coli CaiT structure contains a bound γ-butyrobetaine (Schulze et al., 20210), the substrate that is exported from the E. coli cell after metabolism of L-carnitine under anerobic growth conditions [START_REF] Eichler | Molecular characterization of the cai operon necessary for carnitine metabolism in Escherichia coli[END_REF][START_REF] Jung | CaiT of Escherichia coli, a new transporter catalyzing L-carnitine/gammabutyrobetaine exchange[END_REF]. As observed in the CaiT structure in complex with L-carnitine, the bulky headgroup of γ-butyrobetaine is coordinated by cation-pi interactions and van-der-Waals contacts. In contrast, the central binding site of the P. mirabilis CaiT structure does not contain any bound substrate. A second γ-butyrobetaine molecule is bound in the extracellular cavity of the E. coli CaiT protein (Fig. 7A). Instead of γ-butyrobetaine, two ordered water molecules occupy the corresponding position in the P. mirabilis CaiT protein, most likely because access of the substrate is blocked by crystal contacts. The functional role of this putative second ligand-binding site is unclear. The different CaiT crystal structures revealed that two of the L-carnitine binding sites are identical to those containing γ-butyrobetaine, while the other two L-carnitine sites most likely contain non-specifically bound substrate [START_REF] Tang | Crystal structure of the carnitine transporter and insights into the antiport mechanism[END_REF][START_REF] Schulze | Structural basis of Na + -independent and cooperative substrate/product antiport in CaiT[END_REF].

The central binding site (Fig. 7B) of L-carnitine and γ-butyrobetaine in CaiT correspond to the glycine betaine-binding site present in BetP [START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF]. However, the bound L-carnitine and γ-butyrobetaine molecules in the CaiT structures are accessible from the cytoplasm, while in the BetP structure, the access to the bound glycine betaine molecule is occluded from both sides of the membrane (Fig. 7C). In TM4 and TM8 of BCCT carriers, highly conserved aromatic residues, including those that are part of the BCCT signature sequence [START_REF] Kappes | Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD[END_REF][START_REF] Saier | A functional-phylogenetic classification system for transmembrane solute transporters[END_REF], stabilize the 4 TM helix bundle in the CaiT and BetP subunits. The amino acid sequence of TM3 in the BetP and CaiT proteins is not conserved; however, both crystal structures revealed that this trans-membrane region has a locally unwound segment approximately halfway across the membrane (Fig. 7A). In BetP, TM3 harbours one of the Na + ion binding sites. This sodium ion contributes to the coordination of the carboxyl group of glycine betaine (Fig. 2) and, thereby, stabilizes the ligand within in its binding site [START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF], while the second sodium ion stabilizes the unwound segment of TM3. Unlike BetP, CaiT is a Na + -independent transporter and the functional role of the locally unwound segment in TM3 is therefore not immediately obvious. unwound helix in TM3. The coordination of the carboxyl group of the γ-butyrobetaine ligand is maintained in CaiT by the sulfur of a Met residue that is highly conserved in CaiT-related proteins but it is absent in BetP-related proteins.

The crystal structures of BetP [START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF] and of CaiT (Tang et al., 20210: Schulze et al., 2010) share a strikingly similar architecture with that of other Na + -coupled symporters like LeuT [START_REF] Yamashita | Crystal structure of a bacterial homologue of Na + /Cl --dependent neurotransmitter transporters[END_REF][START_REF] Singh | Antidepressant binding site in a bacterial homologue of neurotransmitter transporters[END_REF][START_REF] Zhou | LeuT-desipramine structure reveals how antidepressants block neurotransmitter uptake[END_REF][START_REF] Singh | A competitive inhibitor traps LeuT in an open-to-out conformation[END_REF], vSglT [START_REF] Faham | The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na + /sugar symport[END_REF] and Mhp1 [START_REF] Weyand | Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter[END_REF], with H + -coupled symporters like ApcT [START_REF] Shaffer | Structure and mechanism of a Na + -independent amino acid transporter[END_REF] and antiporters like AdiC (Gao et al., 2010), despite the fact that they are not related at the amino acid sequence level. Each of these transporters possesses a core domain consisting of 10 TM helices. Hydropathy profile alignments [START_REF] Lolkema | The major amino acid transporter superfamily has a similar core structure as Na + -galactose and Na + -leucine transporters[END_REF] confirm the structural similarity between transporters of the neurotransmitter:sodium:symporter family (LeuT), the sodium:solute:symporter family (vSglT), and the aminoacid:polyamine:organocation family (ApcT) [START_REF] Gouaux | The molecular logic of sodium-coupled neurotransmitter transporters[END_REF][START_REF] Krishnamurthy | Unlocking the molecular secrets of sodium-coupled transporters[END_REF][START_REF] Shaffer | Structure and mechanism of a Na + -independent amino acid transporter[END_REF]. These ten C-terminal TMs (corresponding to TM3-12 in BetP) contain an inverted repeat of two 5 TM helixes each. Repeat 1 (TM3-TM7 in BetP) is a structural homolog of repeat 2 and they are related by pseudo two-fold symmetry axis running parallel to the membrane (Fig. 6B). Substrate and co-substrate binding sites are all located in the interface between the two intertwined repeats. A superposition of the core structures of this group of transporters reveals an r.m.s.d. of 3.8-4.5 Å, indicating not only a high degree of structural conservation with respect to the overall fold but also to the position of the substrate and co-substrate binding sites [START_REF] Caplan | Molecular mechanism of ion-ion and ion-substrate coupling in the Na + -dependent leucine transporter LeuT[END_REF][START_REF] Noskov | Control of ion selectivity in LeuT: two Na + binding sites with two different mechanisms[END_REF][START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF]. The common core structure, the similar location of the substrates in these transporters, and the fact that the crystal structures reflect distinct conformations, were recently used to propose a common molecular mechanism for Na + -and H + -coupled symport and antiport [START_REF] Abramson | Structure and function of Na + -symporters with inverted repeats[END_REF][START_REF] Gouaux | The molecular logic of sodium-coupled neurotransmitter transporters[END_REF][START_REF] Krishnamurthy | Unlocking the molecular secrets of sodium-coupled transporters[END_REF][START_REF] Shaffer | Structure and mechanism of a Na + -independent amino acid transporter[END_REF].

The Na + -coupled glycine betaine symporter BetP and the L-carnitine/γ-butyrobetaine antiporter CaiT represent different subgroups within the BCCT carriers (Table 1). However, the striking similarities of their crystal structures provide now a solid basis for formulating unifying concepts that describe functional mechanisms operating within the BCCT family. In our comparisons of a large number of BCCT carriers, we noted a high amino acid sequence conservation of those residues that are involved in coupling and substrate binding within the BCCT family. The degree of sequence identity in TM3, TM4 and TM8 is very high within the BCCT family. However, there are two distinct pattern of conservation: (a) residues involved in ion-coupling or counter-substrate coupling and (b) residues involved in binding of the fully methylated head group of various compatible solutes (Fig. 2 and7B). Therefore, the structures of BetP and CaiT probably can be used to explain transport of other methylated ammonium or sulfur compounds such as choline and DMSP and permits structure-guided speculations on the transport mechanism of non-quaternary ammonium substrates of BCCT carriers such as ectoine and hydroxyectoine (Fig. 2). Thus, the BetP and CaiT structures allow us to search for key residues that are likely to be involved in functionally relevant conformational changes in BCCT carriers.

Substrate binding: what determines substrate specificity and coupling mode in the BCCT family?

In general, it is not possible to predict with confidence the substrate profile of a given BCCT carrier solely by inspecting and aligning amino acid sequences. However, the BetP and CaiT structures reveal a striking pattern in substrate binding that allows one, at least to some extent, to predict the substrate profile of other BCCT carriers based on sequence alignments. Glycine betaine transporters of the BCCT family show a substantial degree of sequence relatedness (between 48-40%) in the 10 TM transporter core (excluding the N-terminal and C-terminal extensions). In contrast, the amino acid sequence relatedness of the 10 TM core falls below 40% for ectoine and choline transporters and even lower (below 30 %) for L-carnitine/γbutyrobetaine antiporters.

The BetP crystal structure revealed the architecture of the binding site for its ligand glycine betaine and the functional importance of key-residues within this binding site were confirmed by site-directed mutagenesis studies [START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF]. The architecture of the ligand-binding site present in BetP is nearly identical with the primary ligand-binding site for L-carnitine and γ-butyrobetaine in CaiT (Tang et al., 20210;[START_REF] Schulze | Structural basis of Na + -independent and cooperative substrate/product antiport in CaiT[END_REF]. Hence, the crystal structures of BetP and CaiT revealed for the first time the molecular determinants for substrate binding of trimethylammonium compounds by members of the BCCT carriers. In particular, the indole groups of three tryptophane residues, one positioned in TM4 and two positioned in TM8, are arranged in form of an aromatic cage in which the fully methylated head groups of glycine betaine, L-carnitine and γ-butyrobetaine (Fig. 2) are stabilized via cation-pi interactions. The carboxyl groups of these ligands protrude out from this aromatic cage and are coordinated via hydrogen bonding interactions within the ligand-binding site (Fig. 7B). The coordination of the glycine betaine, L-carnitine and γ-butyrobetaine substrates within the BetP and CaiT transporters show striking similarities to that found in the ligand- binding proteins (ProX) of the glycine betaine ABC-type uptake system from E. coli (Schiefner et al., 2004a) and the hyperthermophilic archaeon Archeoglobus fulgidus (Schiefner et al., 2004b). The ProX proteins are soluble and function in the context of ABCtransporters and are thus not evolutionarily closely related to the integral membrane proteins BetP and CaiT. Hence, nature has used common design principles to achieve the selective and high-affinity binding of this group of compatible solutes by proteins, compounds which are typically preferentially excluded from the hydration shell of the protein backbone [START_REF] Arakawa | The stabilization of proteins by osmolytes[END_REF][START_REF] Bolen | The osmophobic effect: natural selection of a thermodynamic force in protein folding[END_REF][START_REF] Street | A molecular mechanism for osmolyteinduced protein stability[END_REF]Rosgen, 2007;[START_REF] Bolen | Structure and energetics of the hydrogen-bonded backbone in protein folding[END_REF][START_REF] Street | Osmolyte-induced conformational changes in the Hsp90 molecular chaperone[END_REF]. The crystal structures of ligand-binding proteins from ABC-and TRAP-transport systems revealed that the binding of compatible solutes in substrate-selective binding sites present in transporter proteins seems to require a certain number of aromatic residues for substrate coordination via cation-pi and van der Waals interactions in order to provide a surface that minimally repels osmolytes (Schiefner et al., 2004a;[START_REF] Horn | Molecular determinants for substrate specificity of the ligand-binding protein OpuAC from Bacillus subtilis for the compatible solutes glycine betaine and proline betaine[END_REF][START_REF] Hanekop | Crystal structure of the ligand-binding protein EhuB from Sinorhizobium meliloti reveals substrate recognition of the compatible solutes ectoine and hydroxyectoine[END_REF][START_REF] Kuhlmann | Osmoregulated transport of compatible solutes in the halophilic bacterium Halomonas elongata: 1.55 A high-resolution structure of the periplasmic ectoinebinding protein from TRAP-transporter TeaABC[END_REF][START_REF] Smits | The compatible-solute-binding protein OpuAC from Bacillus subtilis: ligand binding, sitedirected mutagenesis, and crystallographic studies[END_REF][START_REF] Capp | Interactions of the osmolyte glycine betaine with molecular surfaces in water: thermodynamics, structural interpretation, and prediction of m-values[END_REF]Lercher et al., 2009;[START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF]Tang et al., 20210;Schulze et al., 20210;[START_REF] Wolters | Ligand binding and crystal structures of the substrate-binding domain of the ABC transporter OpuA[END_REF]. Several crystal structures of binding proteins from ABC-or TRAP-type transport systems in complex with compatible solutes used by BCCT carriers (Table 1) have been reported (Schiefner et al., 2004a;Schiefner et al., 2004b;[START_REF] Horn | Molecular determinants for substrate specificity of the ligand-binding protein OpuAC from Bacillus subtilis for the compatible solutes glycine betaine and proline betaine[END_REF][START_REF] Hanekop | Crystal structure of the ligand-binding protein EhuB from Sinorhizobium meliloti reveals substrate recognition of the compatible solutes ectoine and hydroxyectoine[END_REF][START_REF] Kuhlmann | Osmoregulated transport of compatible solutes in the halophilic bacterium Halomonas elongata: 1.55 A high-resolution structure of the periplasmic ectoinebinding protein from TRAP-transporter TeaABC[END_REF][START_REF] Oswald | Crystal structures of the choline/acetylcholine substratebinding protein ChoX from Sinorhizobium meliloti in the liganded and unligandedclosed states[END_REF][START_REF] Smits | The compatible-solute-binding protein OpuAC from Bacillus subtilis: ligand binding, sitedirected mutagenesis, and crystallographic studies[END_REF][START_REF] Lecher | The crystal structure of UehA in complex with Ectoine-A comparison with Other TRAP-T binding proteins[END_REF][START_REF] Wolters | Ligand binding and crystal structures of the substrate-binding domain of the ABC transporter OpuA[END_REF]. Hence, the coordination of the substrates in these soluble proteins might give valuable clues to substrate binding site(s) present in BCCT carriers for which no crystal structure is yet available (e.g. transporters for ectoines and DMSP).

Most of the BCCT carriers show a high degree of conservation of the Trp residues in TM4 and TM8 that coordinate the trimethlyammonium ligands in BetP and CaiT [START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF][START_REF] Tang | Crystal structure of the carnitine transporter and insights into the antiport mechanism[END_REF]Schulze et al. 20210) but these residues can not be identified in ectoine/hydroxyectoine transporters of the BCCT-family. From these patterns of conservation, we conclude that the coordination of ectoine or hydroxyectoine, which lack a quaternary ammonium group (Fig. 2), is different from that of glycine betaine, γbutyrobetaine or L-carnitine, while coordination of choline is likely to be similar. This latter suggestion is supported by the molecular determinants governing choline binding by the periplasmic ligand binding ChoX protein from Sinorhizobium meliloti that also archives the binding of the methylated head group of choline through cation-pi interactions in an aromatic ligand-binding cage [START_REF] Oswald | Crystal structures of the choline/acetylcholine substratebinding protein ChoX from Sinorhizobium meliloti in the liganded and unligandedclosed states[END_REF]. Like glycine betaine, ectoine and hydroxyectoine carry a negatively charged carboxylate (Fig. 2). Hence, it is tempting to speculate that a sodium ion might interact directly with the carboxylate of ectoine and hydroxyectoine in a similar way to that found in BetP [START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF]. The coordinating Na + ion in the BetP crystal structure is located halfway across TM3, close to an unwound, glycine-rich segment that separates TM3 into two segments (Fig. 7B). This flexible segment in BetP contains a GMGIG motif that is conserved in glycine betaine-specific transporters of the BCCT family. TM3 in the Na + -independent substrate:product antiporter CaiT shows now sequence homology in this region, while ectoine-specific BCCT carriers show sequence modifications at the corresponding position.

Substrate-coordination by residues in TM3 might also be crucial for proton-coupled BetT-like proteins of the BCCT family. The chemical nature of glycine betaine and choline differs with respect to their carboxyl and alcohol groups (Fig. 2). While salt bridges to the substrate are maintained in Na + -coupled transporters by the coupling ion, this might not be the case for choline, which carries a positive charge (Fig. 2). However, the choline transporter BetT from E. coli comprises a charged residue in the flexible segment in TM3. The presence of a side chain capable of reversible protonation during the transport cycle might be a pre-requisite for the H + -coupled transport, since such an arrangement was also observed in the H + -coupled amino acid transporter ApcT from Methanocaldococcus jannaschii [START_REF] Shaffer | Structure and mechanism of a Na + -independent amino acid transporter[END_REF].

There might be substrates for BCCT carriers that are different from those compiled in Fig. 2. Some putative BCCT carriers harbour conserved, but significant different motifs of the glycine betaine, choline, ectoine or carnitine transporters of the BCCT-family. This functionally uncharacterized group of BCCT carriers is predominantly found in members of the Firmicutes, and they also form the major group of BCCT carriers present in Synechococcus, a subclass of cyanobacteria. Depending on the degree of their salt stress resistance these microorganisms synthesize sucrose, trehalose, glucosylglycerol and glycine betaine under salt stress [START_REF] Hagemann | Molecular biology of cyanobacterial salt acclimation[END_REF]. Some marine γ-Proteobacteria possess BCCT carriers with a very low degree of sequence homology to BetP (approximately 20%) and, most strikingly, they lack TM1. Accordingly, these transporters have a predicted transmembrane topology that is different from that of BetP. These considerations suggests that it might be rewarding to study these so-far uncharacterized BCCT carriers with respect to their potential substrates, their transport activity profiles and topological organization within the cytoplasmic membrane The oligomeric state of BCCT carriers and their terminal domains BetP and CaiT are both trimeric in detergent solution [START_REF] Ziegler | Projection structure and oligomeric state of the osmoregulated sodium/glycine betaine symporter BetP of Corynebacterium glutamicum[END_REF][START_REF] Vinothkumar | Oligomeric structure of the carnitine transporter CaiT from Escherichia coli[END_REF], as well as in the membrane when analyzed by 2D electron crystallography, and in the 3D crystal [START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF][START_REF] Tang | Crystal structure of the carnitine transporter and insights into the antiport mechanism[END_REF][START_REF] Schulze | Structural basis of Na + -independent and cooperative substrate/product antiport in CaiT[END_REF]Tsai et al., submitted). The observed trimer formation is a consequence of the unique structural elements characterizing the fold of the BCCT carriers, which are the amphiphatic helix 7 and the long bend TM2. They provide a hydrophobic interface, which force a triangular subunit arrangement for stability reasons. In fact, BetP and CaiT have proven to be very stable both in detergent solution and when reconstituted back into the membrane (Tsai et al. 2007). It is thus tempting to speculate that trimer-formation is a general feature of the BCCT carriers with functional impact for transport and/or regulation. We have analyzed the differences in trimer contacts in the osmotically regulated BetP and the osmotically non-regulated CaiT proteins to validate this assumption. The three subunits within the CaiT and BetP trimer form a central conical cavity lined by aromatic side chains from TM2, TM3 and h7 (Fig. 5A). The hydrophobic cavity in the trimer centre is most likely filled with lipid as observed for the crystallized BetP protein [START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF]. Residues contributing to the formation of this cavity are highly conserved in members of the BCCT carriers. On the periplasmic side, the amphipathic helix 7 is the key player in maintaining trimer contacts (Fig. 6). Helix h7 in BetP and CaiT is rich in Leu and Asp residues and shows characteristics of a non-specific lipid interaction site that stabilizes the trimer assembly. This helix represents a structurally distinct feature of the BCCT carriers since it is not present in any other transporter sharing the same overall inverted structural repeat (e.g. LeuT). In BetP and CaiT, helix h7 separates the two structural repeats from each other and, therefore, cannot be attributed to either of the repeat parts of the transpiorter proteins. Contacts involve mainly hydrophobic residues in helix h7, TM2, TM3 and loop7 of adjacent subunits. Residues participating in trimer contact via helix h7 are not entirely conserved within the BCCT carriers. CaiT shares only 17% sequence identity in this region to the BetP protein. However, the two structurally important polar interactions observed in BetP are also found in CaiT and are predicted to occur in eight other BCCT carriers listed in Table 1. In CaiT and nine other BCCT carriers, additional subunitsubunit contacts are likely to be promoted by ionic interactions, resulting in a quite rigid trimer assembly; in contrast, in BetP they are formed by more flexible hydrophobic interactions. However, only BetP Cg and OpuD Vc (Table 1) seem to have exclusively hydrophobic interactions in their trimer contact regions as found in BetP. The majority of BCCT carriers show (i) a conserved length of h 7 and (ii) the potential for both hydrophobic sterically. The C-terminal helix of BetP is not found in CaiT (Fig. 5). Th additional trimer contact formed by the C-terminal helix of BetP within the BetP trimeric structure is of particular interest because biochemical studies have provided compelling evidence for the involvement of this domain in osmosensing and regulation [START_REF] Ott | Regulatory properties and interaction of the C-and N-terminal domains of BetP, an osmoregulated betaine transporter from Corynebacterium glutamicum[END_REF]. The C-terminal domain of BetP contains several positively charged residues clustered at the N-terminal part of the C-terminal domain (cluster 1: R554, R558, R565, R567, and R568) and near the end of the C-terminal domain (cluster 2: R576, K581, R582-584, R586, and K587). It lacks Gly and/or Pro residues, except close to its very end, and thus forms a straight α-helix (Fig. 5B and 6B). Biochemical data revealed the functional importance of the conformation and position of the first charge cluster within the C-terminal domain of BetP [START_REF] Ott | Regulatory properties and interaction of the C-and N-terminal domains of BetP, an osmoregulated betaine transporter from Corynebacterium glutamicum[END_REF][START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF]. In the context of trimer contacts in BetP, arginines in the first cluster form salt bridges to a glutamate separating both clusters in the C-terminal domain of the adjacent subunit. In the 22 functionally characterized microbial BCCT carriers (Table 1), the Cterminal domains do not have a conserved sequence, but three different motifs can be observed: (i) BetP-type C-terminal domains feature two clusters with opposite charges positioned several residues apart from each other, but lack flexible stretches of Gly and/or Pro residues. (ii) CaiT-type C-terminal domains have, at most, very short charged clusters.

Consequently, the BetP-type C-terminal domains present in BetP Cg , BetT Ah , CudT, BetH Ht , OpuD Bs , EctM, ButA and EctP (Table 1) are likely to contribute to trimer contacts, while CaiT-type C-terminal domains present in BetM and DddT (Table 1), do not do so. (iii) There is a third type of C-terminal domain in members of the BCCT carriers. It consists of charged clusters but is also rich in Gly and/or Pro residues, suggesting that these C-terminal domains are not entirely α-helical. This type of C-terminal domain is often very long (> 70 aa) and is 1). For several members of the BCCT carriers, namely BetL, BetP Vp , OpuD Vc , and LcoP (Table 1), the assignment to the three types of C-terminal domains is not a trivial task, since they all comprise charged amino acid clusters together with flexible stretches of glycine and/or proline residues. A BetP-like helix h7 and a BetP-like C-terminal domain are found together only in the glycine betaine transporter ButA from Tetragenococcus halophila (Table 1) [START_REF] Baliarda | Isolation and characterization of ButA, a secondary glycine betaine transport system operating in Tetragenococcus halophila[END_REF].

Interactions of C-terminal domains with the cytoplasmic side of adjacent subunits are one of the main differences between the BetP (Ressl et al., 2008) and the CaiT structures [START_REF] Tang | Crystal structure of the carnitine transporter and insights into the antiport mechanism[END_REF][START_REF] Schulze | Structural basis of Na + -independent and cooperative substrate/product antiport in CaiT[END_REF]. The C-terminal interactions are not symmetrical in the BetP trimer resulting in a break in non-crystallographic threefold symmetry. A break in symmetry was also observed for BetP embedded in the membrane in 2D crystals [START_REF] Ziegler | Projection structure and oligomeric state of the osmoregulated sodium/glycine betaine symporter BetP of Corynebacterium glutamicum[END_REF], while the CaiT trimer exhibits a perfect threefold symmetry. Unfortunately, electron crystallographic data of CaiT are not available yet at the required resolution, but with respect to trimer formation it is very likely that CaiT is also a symmetric trimer in the membrane. In contrast, each subunit in the BetP trimer adopts a distinct conformation (Tsai et al., submitted). The different conformations of each subunit in membrane-embedded BetP, the structural flexibility of the C-terminal domains and of the BetP trimer suggest a role of trimerization far beyond protein stabilization in BetP. Together with the fact the C-terminal domains acts as osmoregulator, the observed C-terminal interactions point towards a functional role in transport activity regulation in BetP [START_REF] Krämer | Osmosensing and osmosignaling in Corynebacterium glutamicum[END_REF]. A possible scenario would be that the subunits communicate their conformational state via the trimer interface to synchronize their transport activity. Thereby, the BetP glycine betaine transporter would take advantage of the trimeric state in a very elegant fashion.

Sensing and signal transduction by BetP at the biochemical level

A considerable number of the BCCT carriers listed in Table 1 respond to osmotic stress but this issue has only been studies in detail for a few of threes transporters. In view of the multiplicity of potential physical stimuli directly or indirectly related to osmotic stress [START_REF] Wood | Osmosensing by bacteria: signals and membrane-based sensors[END_REF][START_REF] Bremer | Coping with osmotic challenges: osmoregulation through accumulation and release of compatible solutes[END_REF][START_REF] Poolman | Bacterial osmosensing: roles of membrane structure and electrostatics in lipid-protein and protein-protein interactions[END_REF][START_REF] Wood | Osmosensing by bacteria[END_REF][START_REF] Krämer | Osmosensing and osmosignaling in Corynebacterium glutamicum[END_REF][START_REF] Altendorf | Chapter 5.4.5, Osmotic Stress. In: EcoSal-Escherichia coli and Salmonella: cellular and molecular biology[END_REF], the analysis of the sensory input into a transporter protein is an experimentally difficult task. For BetP, the situation was simplified by the fact that the reconstituted protein in proteoliposomes was fully capable of responding to osmotic stress [START_REF] Rübenhagen | Osmosensor and osmoregulator properties of the betaine carrier BetP from Corynebacterium glutamicum in proteoliposomes[END_REF]. Consequently, individual physical stimuli could be tested separately and their relevance for carrier activation could be carefully evaluated.

The physical stimuli found to be relevant for the osmotic control of BetP activity can been divided into two classes: (i) An increase in K + (and Rb + or Cs + ) triggers BetP activity but only when applied at the cytoplasmic side [START_REF] Rübenhagen | The osmoreactive betaine carrier BetP from Corynebacterium glutamicum is a sensor for cytoplasmic K +[END_REF]. The observed specificity with respect to the activating agent practically excludes the unspecific effect of a change in the degree of BetP hydration as a stimulus. An unspecific influence of osmolytes and macromolecules has been proposed as a stimulus for osmotic control of the MFS-type transporter ProP from E. coli [START_REF] Culham | Osmosensor ProP of Escherichia coli responds to the concentration, chemistry, and molecular size of osmolytes in the proteoliposome lumen[END_REF]. (ii) BetP activity is also affected by stimuli originating from the membrane. A modulating effect of local anaesthetics inserted into the membrane affects BetP-mediated glycine betaine transport [START_REF] Steger | LcoP, an osmoregulated betaine/ectoine uptake system from Corynebacterium glutamicum[END_REF]. This is not an unspecific effect, since these types of compounds did not affect the BCCT carrier EctP from C. glutamicum. It was also observed that the surface charge density of the surrounding membrane strongly modulates BetP activity; the concentration of K + necessary for BetP activation increased in parallel to the content of negatively charged lipids [START_REF] Schiller | Influence of membrane composition on osmosensing by the betaine carrier BetP from Corynebacterium glutamicum[END_REF]. In addition to osmotic stress, BetP was also activated by chill stress (Özcan et al., 2005). Since this effect was independent of changes in K + content the relevant stimulus should originate from changes in the physical state of the membrane. Finally, membranes with changed lipid composition but unchanged surface charge density exhibit altered regulation of the BetP transporter (Özcan et al., 2007). Taken together, signal input leading to stimulation of BetP transporter activity seems to be of two types: a rise in the intracellular K + concentration that follows an osmotic up-shift and a stimulus originating from the membrane into which the BetP transporter is embedded.

Mutational studies of BetP have provided substantial information on the contribution of selected domains with respect to its transport function and regulation of its activity by high osmolarity. A stepwise truncation of the C-terminal domain of BetP showed that the Cterminal region containing the second charge cluster could be deleted without significant impact on transporter function. Further truncation of the C-terminal domain by 25 amino acid residues or more, including the first charge cluster, led to BetP transporter derivatives with decreased but constitutive activity (Peter et al., 1998a;[START_REF] Schiller | The C-terminal domain of the betaine carrier BetP of Corynebacterium glutamicum is directly involved in sensing K + as an osmotic stimulus[END_REF]. Full removal of the C-terminal domain rendered BetP inactive. Interestingly, activation of BetP activity at low temperature, probably by a stimulus derived from the surrounding membrane, was not influenced by a truncation of the C-terminal domain (Özcan et al., 2005). The functional significance of the C-terminal domain of BetP was further studied by amino acid scanning mutagenesis (amino acid replacements with Ala or Pro residues) [START_REF] Ott | Regulatory properties and interaction of the C-and N-terminal domains of BetP, an osmoregulated betaine transporter from Corynebacterium glutamicum[END_REF]. These experiments led to two important conclusions: (i) a particular region in the middle of the Cterminal domain of BetP was found to be critical for sensory function with respect to its conformational properties rather than its charge distribution, and (ii) interaction of the Cterminal domain with particular loops at the cytoplasmic face of BetP and with the N-terminal domain was detected [START_REF] Ott | Regulatory properties and interaction of the C-and N-terminal domains of BetP, an osmoregulated betaine transporter from Corynebacterium glutamicum[END_REF]. Consequently, both terminal domains, and in particular the C-terminus, are critically involved in the activation of BetP by osmotic stress.

BetT-like choline transporter proteins have C-terminal domains that are substantially longer then that present in BetP (Table 1). In both E. coli and P. syringae, BetT proteins with truncated C-terminal domains gradually lose their capacity for osmotic activation, indicating that these domains are involved in osmosensing and/or osmoregulation of transporter activity [START_REF] Tondervik | Membrane topology and mutational analysis of the osmotically activated BetT choline transporter of Escherichia coli[END_REF][START_REF] Chen | Pseudomonas syringae BetT is a low-affinity choline transporter that is responsible for superior osmoprotection by choline over glycine betaine[END_REF].

Sensing and signal transduction by BetP: correlation of structure with function

Biochemical data elucidated three key players for sensing and signal transduction by BetP: (i) the C-terminal domain, (ii) cytoplasmic loops, and (iii) the N-terminal domain (Fig. 8). While the N-terminal domain is not resolved in the X-ray structure of BetP [START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF], biochemical data concerning the functional impact of cytoplasmic loops, especially of loop 2 and loop 8, and of the C-terminal domain on osmotic activation and transporter function can be correlated to the BetP crystal structure. Loop 2 preceding TM3 contains a cluster of negatively charged residues . Mutations at Asp-131 and Glu-132 somehow prevent expression of the betP structural gene and a mutation at Glu-135 reduces transport activity [START_REF] Ott | Regulatory properties and interaction of the C-and N-terminal domains of BetP, an osmoregulated betaine transporter from Corynebacterium glutamicum[END_REF]. The BetP structure reveals an interaction of Asp-131 via a salt bridge with an Arg residue located in the C-terminal helix of the adjacent subunit, and Glu-132 with an Arg residue in loop 8, which connects TM8 and TM9 and is part of the signature sequence of the BCCT family [START_REF] Kappes | Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD[END_REF][START_REF] Saier | A functional-phylogenetic classification system for transmembrane solute transporters[END_REF]. Within this network, changes in orientation of the C-terminal helix in one subunit of BetP would affect the orientation and conformation of loop 2 and loop 8 in an adjacent subunit. It seems likely that such interactions would have consequences for structural features of TM3 and TM8, both of which are involved in substrate binding and Na + coupling. Thus, the structure of BetP suggests a "locking" interaction of the C-terminal domain with these loops, which might translate into an inhibition of transport activity and, consequently, seems to be partly responsible for regulating the transport cycle of BetP. Based on biochemical data, activation of BetP by a rise in the internal K + concentration occurs due to K + binding to the BetP protein at the cytoplasmic site, a fact that has so far not been confirmed by structural data. Presumably, K + binding influences the interaction network of the C-terminal domain with the intracellular loops 2 and 8. Furthermore, it is reasonable to assume a direct interaction of positively charged residues in the C-terminal domain with negatively charged lipids of the membrane. Lipids occupying the clefts between the individual BetP subunits or in the hydrophobic cavity in the centre of the trimer (Fig. 5) might affect the orientation of the C-terminal helices of BetP, which, in turn, might render a regulatory K + -binding site more accessible. One can further assume that the flexible trimeric state of the transporter may be a crucial point for the activity regulation of BetP. This would involve in all likelihood the amphipathic helix h7 on the periplasmic side of BetP, a structure that is buried in the membrane interface (Fig. 6). A regulatory mechanism that senses differences in membrane composition or membrane state at the periplasmic side via helix h7 would be in agreement with a physical stimulus for BetP activity originating from within the membrane. Stimulus perception by helix h7 could be structurally translated into the flexible trimer by rearrangement of the BetP subunits.

The question of which transporters might form additional trimer contacts by their Cterminal domains is of interest within the context of the regulatory properties of BCCT carrier. Furthermore, it is of interest whether the C-terminal interaction with loop 2 and 8, as observed biochemically [START_REF] Ott | Regulatory properties and interaction of the C-and N-terminal domains of BetP, an osmoregulated betaine transporter from Corynebacterium glutamicum[END_REF] and structurally [START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF] in BetP, is also found in other members of the BCCT carriers. Loop 8 is nearly 100% conserved in the BCCT carriers but this is not the case for loop 2. However, six BCCT carriers (Table 1) have a negatively charged cluster at the positions of Asp-131 and Glu-132 and charged cluster in the C-terminal domain at the same position as in BetP. Two additional transporters from C. glutamicum, LcoP and EctP, which share with BetP the described profile of charged residues in loop2 and in their C-terminal domain, are indeed both osmoregulated transporters. Lacking glycines and prolines in their C-terminal domains, it can be assumed that these domains are α-helical and these transporters might very well be regulated in a similar way as BetP.

Subunit interactions within the trimer seem to be an essential feature of regulation of transport activity in members of the BCCT carriers. This might explain some differences in regulatory behaviour of osmoregulated BCCT carriers such as BetP compared to osmoregulated transporters of other transporter families, e.g. ProP from E. coli (Culham et al., 2008a;Culham et al., 2008b) and OpuA from L. lactis (van der Heide and [START_REF] Van Der Heide | Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane[END_REF][START_REF] Van Der Heide | On the osmotic signal and osmosensing mechanism of an ABC transport system for glycine betaine[END_REF][START_REF] Mahmood | Engineering of ion sensing by the cystathionine beta-synthase module of the ABC transporter OpuA[END_REF]. In fact, this idea has already been suggested in a previous review [START_REF] Poolman | Bacterial osmosensing: roles of membrane structure and electrostatics in lipid-protein and protein-protein interactions[END_REF]. However, the concepts derived for the osmotic activity control of transporters involved in compatible solute acquisition must be viewed with caution, since the transporter families, the structure of the OpuA, ProP and BetP transporters and their subunit composition are rather different.

Moreover, the putative primary trigger that controls transport activity in response to a high osmolarity challenge seems to be rather different as well: a change in intracellular water availability in the case of ProP [START_REF] Culham | Osmosensor ProP of Escherichia coli responds to the concentration, chemistry, and molecular size of osmolytes in the proteoliposome lumen[END_REF], a change in the intracellular ionic strength for OpuA (van der Heide and [START_REF] Van Der Heide | Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane[END_REF][START_REF] Van Der Heide | On the osmotic signal and osmosensing mechanism of an ABC transport system for glycine betaine[END_REF][START_REF] Mahmood | Engineering of ion sensing by the cystathionine beta-synthase module of the ABC transporter OpuA[END_REF], and altered cytoplasmic K + concentration in the case of BetP [START_REF] Rübenhagen | The osmoreactive betaine carrier BetP from Corynebacterium glutamicum is a sensor for cytoplasmic K +[END_REF]. Nevertheless, some common features can be deduced from the biochemical analysis the ProP, OpuA and BetP transport systems. Turgor was not found to be responsible for activation of any of these transporters; a switching behaviour of terminal domains as a mechanistic element in regulation has been suggested for all three; and an impact of membrane conditions and composition on carrier activity was found to be relevant for ProP, OpuA and BetP (van der Heide et al., 2001;[START_REF] Poolman | Bacterial osmosensing: roles of membrane structure and electrostatics in lipid-protein and protein-protein interactions[END_REF][START_REF] Schiller | Influence of membrane composition on osmosensing by the betaine carrier BetP from Corynebacterium glutamicum[END_REF][START_REF] Mahmood | Engineering of ion sensing by the cystathionine beta-synthase module of the ABC transporter OpuA[END_REF][START_REF] Romantsov | Cardiolipin and the osmotic stress responses of bacteria[END_REF]. It is not possible to generalise, however, on the particular nature of the transport activity-controlling stimulus, the mechanistic details of stimulus sensing, and the concept of how the transport cycle should be connected to the regulatory cycle of the various transporters under study.

The BCCT carriers: conclusions and perspectives

When initially recognized in 1996, the BCCT family of transporters comprised just 4 functionally characterized members [START_REF] Kappes | Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD[END_REF]. Since then, the number of functionally studied BCCT carriers has modestly risen to 22 (Table 1). The explosion in the number of available genome sequences of microorganisms revealed a widespread occurrence of BCCT carriers (currently about 2 200 representatives) that are predominantly present in members of the Bacteria. Substrates for BCCT carriers different form those originally found [START_REF] Kappes | Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD[END_REF] have also been detected in the meantime (Fig. 2). Most notably are transporters for ectoines and for DMSP.

BCCT carriers for ectoine and hydroxyectoine (Table 1), widely synthesized as osmostress-and thermostress-protectants by members of the Bacteria [START_REF] Garcia-Estepa | The ectD gene, which is involved in the synthesis of the compatible solute hydroxyectoine, is essential for thermoprotection of the halophilic bacterium Chromohalobacter salexigens[END_REF][START_REF] Bursy | Osmotically induced synthesis of the compatible solute hydroxyectoine is mediated by an evolutionarily conserved ectoine hydroxylase[END_REF][START_REF] Bursy | Synthesis and uptake of the compatible solutes ectoine and 5hydroxyectoine by Streptomyces coelicolor A3(2) in response to salt and heat stress[END_REF]Kuhlmann et al., unpublished results), are of interest from a crystallographic point of view because the chemical structures of these compatible solutes differ considerably from those of other substrates for BCCT carriers (Fig. 2). Crystal structures of soluble ectoine and hydroxyectoine binding proteins operating in the framework of either ABC-or TRAP-type transporters have recently been determined [START_REF] Hanekop | Crystal structure of the ligand-binding protein EhuB from Sinorhizobium meliloti reveals substrate recognition of the compatible solutes ectoine and hydroxyectoine[END_REF][START_REF] Kuhlmann | Osmoregulated transport of compatible solutes in the halophilic bacterium Halomonas elongata: 1.55 A high-resolution structure of the periplasmic ectoinebinding protein from TRAP-transporter TeaABC[END_REF][START_REF] Lecher | The crystal structure of UehA in complex with Ectoine-A comparison with Other TRAP-T binding proteins[END_REF]). It will be interesting to see if, similar to the situation found for the BetP transporter and the periplasmic binding protein ProX [START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF]Schiefner et al., 2004a), the ligand-binding site present in ectoine/hydroxyectoine-specific BCCT carriers will correspond to that of the aforementioned soluble ectoine/hydroxyectoine binding proteins.

The second type of BCCT carrier that is of considerable physiological interest is that for the compatible solute dimethylsulfoniopropionate (DMSP) (Fig. 2) which is synthesized in huge amounts (about 1 billion tons per year) primarily by marine plankton. It can be used by microorganisms as a sulphur source and can be catabolized to the volatile dimethyl sulfide, a climate-active trace gas [START_REF] Todd | Molecular dissection of bacterial acrylate catabolism -unexpected links with dimethylsulfoniopropionate catabolism and dimethyl sulfide production[END_REF][START_REF] Dickschat | Pathways and substrate specificity of DMSP catabolism in marine bacteria of the Roseobacter clade[END_REF]Vila-Kosta et al., 2010).

Since DMSP is also an effective microbial osmoprotectant [START_REF] Pichereau | Differential effects of dimethylsulfoniopropionate, dimethlysulfonioacetate, and other Smethylated compounds on the growth of Sinorhizobium meliloti at low and high osmolarities[END_REF] it would not be surprising if BCCT carriers for the uptake if DMSP would be discovered in addition to those involved in DMSP uptake (e.g. DddT) for catabolism [START_REF] Todd | Molecular dissection of bacterial acrylate catabolism -unexpected links with dimethylsulfoniopropionate catabolism and dimethyl sulfide production[END_REF].

The crystal structures of Na + -coupled BetP transporter [START_REF] Ressl | Molecular basis of transport and regulation in the Na + /betaine symporter BetP[END_REF] and the substrate:product antiporter CaiT [START_REF] Tang | Crystal structure of the carnitine transporter and insights into the antiport mechanism[END_REF][START_REF] Schulze | Structural basis of Na + -independent and cooperative substrate/product antiport in CaiT[END_REF] provided the opportunity to correlate structural features of functionally distinct members of the BCCT family with a wealth of biochemical and physiological data [START_REF] Jung | CaiT of Escherichia coli, a new transporter catalyzing L-carnitine/gammabutyrobetaine exchange[END_REF][START_REF] Vinothkumar | Oligomeric structure of the carnitine transporter CaiT from Escherichia coli[END_REF][START_REF] Schiller | Influence of membrane composition on osmosensing by the betaine carrier BetP from Corynebacterium glutamicum[END_REF]Peter et al., 1998a;[START_REF] Rübenhagen | The osmoreactive betaine carrier BetP from Corynebacterium glutamicum is a sensor for cytoplasmic K +[END_REF][START_REF] Ott | Regulatory properties and interaction of the C-and N-terminal domains of BetP, an osmoregulated betaine transporter from Corynebacterium glutamicum[END_REF][START_REF] Krämer | Osmosensing and osmosignaling in Corynebacterium glutamicum[END_REF]. With respect to BetP, the combination of biochemical and crystallographic analysis provided unprecedented insight into the functioning and activity control of a secondary transporter responsive to osmotic stress. The regulatory response of BetP to increased osmolarity was found to require a partly α-helical C-terminal domain of the transporter and, furthermore, evidence has been provided that the trimeric state may be an requirement for functional regulation. Based on the biochemical characteristics and crystal structures of BetP and CaiT, we suggest that many BCCT carriers should form trimeric complexes within the membrane. Our analysis of BetP and CaiT indicates that there might be a relationship between the flexibility of the BetP-type trimer and the osmotic regulation of transport activity and the rigidity of the CaiT-type trimer that is not able to regulate transport activity in response to osmotic challenges. However, we note that structural determinants of trimerization apparently show significant variation with the BCCT carriers. A combination of X-ray crystallography and biochemical analysis will be required to further unravel substrate From a crystallographic and mechanistic point of view, a high-resolution crystal structure of an H + -coupled BetT-type choline transporter (Table 1) would be highly desirable.

With the advent of such a structure, a reference protein for each of the three types of transporters grouped together into the BCCT-family [START_REF] Saier | A functional-phylogenetic classification system for transmembrane solute transporters[END_REF][START_REF] Saier | The transporter classification database: recent advances[END_REF] Top-down view on the trimer of the BetP BCCT-carrier and substrates for BetP (glycine betaine; blue) and the L-carnitine:γ-butyrobetaine (red and green, respectively) antiporter CaiT, a member of the BCCT-family.

  systems that are energized either by proton-motive-force-driven (e.g.

  (F. Spiegelhalter and E. Bremer; unpublished data), the master regulator of the general stress regulon. SigB also controls the osmotic induction of ectT from

  structures of BetP and CaiT: structural models for members of the BCCT family

  However, two residues present in TM3 of CaiT are involved in interactions with the Lcarnitine molecule trapped in both the primary and secondary binding site. Interestingly, an Arg residue in TM7 in CaiT occupies the position of the second Na + -binding site present in BetP. Similar to Na + , the positive charge of the Arg amino acid side chain stabilizes the

  via helix h7. Both parameters favour a triangular interface, while a dimer or a tetramer formation is not possible. Therefore we can safely assume that the majority of BCCT carriers form trimers in the plane of the cytoplasmic membrane. It seems that BetP and CaiT represent two different structural solutions within the BCCT carriers to foster trimerization. It is an intriguing question whether the flexible trimer interactions observed in BetP in comparison to those found in CaiT can be related to BetPs osmoregulatory abilities. Subunit interactions within the BetP trimer not only involve helix h7 but also involve the C-terminal helix of each BetP subunit within the trimer assembly; this C-terminal extension interacts with loop 2 and the C-termini of the other two BetP subunits. Only when the subunits are arranged in a trimer, individual C-terminal domains of BetP would not hinder each other

  BetT Ec , BetT Ps , BetP Hi , BetS and BetU transporters (Table

  mechanism and activity regulation of additional members of the BCCTcarriers in response to environmental stimuli.

Fig. 1 .

 1 Fig. 1. Osmotically driven water fluxes across the cytoplasmic membrane of a microbial cell exposed either to hyper-or hypo-osmotic surroundings.

Fig. 2 .

 2 Fig. 2. Known substrates for BCCT carriers. The positively charged nitrogen or sulfur headgroups and the delocalized positive charge present in ectoine and hydroxyectoine are highlighted.

Fig. 3 .

 3 Fig. 3. Activation profile of the BetP transporter embedded in different membranes. The rate of glycine betaine uptake was measured as a function of the osmolarity of the surrounding medium, either in the native membrane of C. glutamicum (red line), after heterologous expression in E. coli (green line), or after isolation, purification and reconstitution into proteoliposomes (blue line). The maximum activity measured was set to 100%, the absolute

Fig. 4 .

 4 Fig. 4. Schematic representation of the catalytic and the activation (regulatory) cycle of the BetP transporter. In the activation cycle, BetP (C = carrier) occurs either in the active (C*, blue and yellow colour) or the inactive state (C, red colour). In the catalytic cycle, BetP exposes its glycine betaine substrate-binding site either to the periplasmic space (C* e , carrier protein symbol open to the top) or to the cytoplasmic space (C* i , carrier protein symbol open to the bottom). Alternatively, the substrate-binding site is occluded (yellow colour) from the surroundings. Substrate (S) denotes one molecule of glycine betaine (B; black triangel) together with 2 Na + ions (green circles). In the course of one round of the catalytic cycle of the BetP carrier, one molecule of glycine betaine and two Na + ions are translocated across the plasma membrane.

Fig. 5 .

 5 Fig. 5. Crystal structure of BetP from C. glutamicum and CaiT from E. coli in complex with their ligands. (A) Periplasmic view on the BetP trimer (resolved to a resolution of 3.35Å) in spiral presentation and on the CaiT trimer (resolved to a resolution of 3.5Å). Individual subunits are coloured in blue, red and green (B) Side view on the BetP and CaiT trimers. The C-terminal domain of subunit 1 from the BetP trimer assembly is shown to protrude 35 Å into the cytoplasm.

Fig. 6 .

 6 Fig. 6. Toplogical organization of BetP within the plain of the cytoplasmic membrane. (A) Top view on a BetP subunit with a colouring of the TM helices assigned according to BetP's topology (B) showing the organization of the two structural repeats.

Fig. 7 .

 7 Fig. 7. Structural features and ligand binding sites of BetP and CaiT. (A) Side view on the C. glutamicum BetP structure in complex with one molecule of glycine betaine; the CaiT structure from E. coli in complex with four L-carnitine molecules and the CaiT structure from P. mirabilis in complex with two γ-butyrobetaine molecules. (B) Structural features of the central ligand-binding site in BetP and CaiT. The aromatic ligand-binding boxes coordinating the substrate glycine betaine, L-carnitine or γ-butyrobetaine are highlighted. (C) Crosssection through the BetP and CaiT structures revealing an occluded state for BetP and an inward-facing state for CaiT, respectively.

Fig. 8 .

 8 Fig. 8. Osmoregulatory interactions within the BetP trimer. Spatial interaction of the Cterminal domain of one subunit (monomer 1) of BetP with charged residues in loop 2 and loop 8 of the adjacent BetP subunit (monomer 3), which is suggested to be of significance for activity regulatory. (A) Under low external osmolarity and/or absence of K + ions (inactive conditions) the BetP carrier is locked in a resting conformation with the bound two Na + ions (green circles) and glycine betaine (B; black triangle) occluded from the cytoplasmic side. (B) Under hyperosmotic conditions and increasing concentrations of K + ions (grey spheres) in the cytoplasm and stimuli perceived via the membrane, the BetP carrier initiates a orientation/conformation change of its C-terminal domain which alters the interaction network and allows a conformational change of the TM helices of the inverted repeats. As a consequence, glycine betaine can be released into the cytoplasm. The role of the N-terminal domain of BetP (monomer 3) in this interaction switching is currently not firmly understood. It is suggested that the negative charge clusters in BetP (red spheres) interact with the positive charge clusters (blue spheres) present the C-terminal domain of BetP (picture kindly provided by Susanne Ressl).

F

Table 1 .

 1 Functionally characterized members of the BCCT family 1316

					Predicted		Sequence	N-	C-
	Transporter [ [ [ [citation]	Organism (Accession number)	Substrate	driving force/	Total number	identity to BetP	terminal domain	terminal 40 domain
					direction	aa	[%]	[aa]	[aa]
	BetP [1]	Corynebacterium	GB	smf /	595	100	58
			glutamicum		symport
			CAA63771.1
	1315
	BetP [1]	Corynebacterium	GB	smf /	595	100	58
			glutamicum		symport
			CAA63771.1
	OpuD [2]	Bacillus subtilis	GB	smf /	512	43	4
			AAC44368.1		symport
	BetL [3]	Listeria monocytogenes	GB	smf /	507	41	3
			AAD30266.1		symport
	BetH [4]	Halobacillus trueperi	GB	smf /	505	41	3
			AAT3760.1		symport
	BetM [5]	Marinococcus halophilus	GB	smf /	493	38	3
			AAQ67247.1		symport
	ButA [6]	Tetragenococcus	GB	smf /	608	34	51
			halophila		symport
			AAP06751.1
	OpuD [7] BetT [8]	Vibrio cholerae AAF944381.1 AAC23352.1 Haemophilus influenza o r GB ?*	smf / symport ?	539 669	33 32	34 7
	BetT BetP [7] [9] BetU [10] Escherichia coli Aphanothece halophytica BAF03065.1 P GB Vibrio parahaemolyticus BAC60168. GB e AAQ10261.1 GB e smf / symport smf / symport smf / symport AAL37253.1 symport r BetS [11] Sinorhizobium meliloti GB/PB smf /	564 523 667 706	32 32 31 31	36 41 17 52
	DddT [12] Psychrobacter sp. ACY02893.1 LcoP [13] Corynebacterium glutamicum CAF20998.1 EctT [14] Virgibaccilus pantothenticus AAL16076.1 EctM [5] Marinococcus halophilus AAQ67247.1 glutamicum EctP [15] Corynebacterium	GB/ DMSP E/GB E/HE E/HE E/P/GB	smf / symport R smf / symport e 550 630 smf / 501 v symport i smf / 493 e 28 37 36 33 symport symport w smf / 615 29	25 43 6 10 21
			CAA04760.1
	BetT	[16] Pseudomonas syringae	Ch/ACh	pmf /	664	30	14
			ZP_03399554		symport
	BetT	[17] Escherichia coli	Ch	pmf /	677	30	14
			CAQ30790.1		symport
	CudT [18] Staphylococcus xylosus	Ch	pmf /	540	30	7
			AAD23898		symport
	CaiT	[19] Escherichia coli	CT/γ-BB Substrate:	504	25	10
			CAQ30560.1		product
					antiport
	PmCaiT [20] Proteus mirabilis	CT/γ-BB Substrate:	514	25	15
			YP_002152355.1		product
					antiport
	1317
	1318

Acknowledgements

Work in the author's laboratories on bacterial osmoregulation and the biochemical and structural analysis of BCCT carriers was funded by grants from the Deutsche Forschungsgemeinschaft (DFG), the Max-Planck Society, the Fonds der Chemischen Industrie and the LOEWE program of the State of Hessen (SynMicro LOEWE Centre; Marburg). We are grateful to Vickie Koogle for her kind help in editing of the manuscript.

We apologize to all our colleagues whose important work on microbial osmoadaptation and compatible solute transporters could not be appropriately cited due to space limitations.