TY - JOUR A1 - Kuhlmann, S. A1 - Terwisscha van Scheltinga, A. C. A1 - Bienert, Ralf A1 - Kunte, Hans-Jörg A1 - Ziegler, C. M. T1 - 1.55 Å Structure of the Ectoine Binding Protein TeaA of the Osmoregulated TRAP-Transporter TeaABC from Halomonas elongata JF - Biochemistry N2 - TeaABC from the moderate halophilic bacterium Halomonas elongata belongs to the tripartite ATP-independent periplasmic transporters (TRAP-T), a family of secondary transporters functioning in conjunction with periplasmic substrate binding proteins. TeaABC facilitates the uptake of the compatible solutes ectoine and hydroxyectoine that are accumulated in the cytoplasm under hyperosmotic stress to protect the cell from dehydration. TeaABC is the only known TRAP-T activated by osmotic stress. Currently, our knowledge on the osmoregulated compatible solute transporter is limited to ABC transporters or conventional secondary transporters. Therefore, this study presents the first detailed analysis of the molecular mechanisms underlying substrate recognition of the substrate binding protein of an osmoregulated TRAP-T. In the present study we were able to demonstrate by isothermal titration calorimetry measurements that TeaA is a high-affinity ectoine binding protein (Kd = 0.19 μM) that also has a significant but somewhat lower affinity to hydroxyectoine (Kd = 3.8 μM). Furthermore, we present the structure of TeaA in complex with ectoine at a resolution of 1.55 Å and hydroxyectoine at a resolution of 1.80 Å. Analysis of the TeaA binding pocket and comparison of its structure to other compatible solute binding proteins from ABC transporters reveal common principles in compatible solute binding but also significant differences like the solvent-mediated specific binding of ectoine to TeaA. KW - Halophilic Bacteria KW - Halomonas elongata KW - Substrate binding protein KW - TRAP-transporter KW - TeaABC KW - osmoregulation KW - compatible solutes KW - ectoine KW - hydroxyectoine PY - 2008 DO - https://doi.org/10.1021/bi8006719 SN - 0006-2960 SN - 1520-4995 VL - 47 IS - 36 SP - 9475 EP - 9485 PB - ACS Publ. CY - Washington, DC AN - OPUS4-18560 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Kunte, Hans-Jörg ED - Gunde-Cimerman, N. ED - Oren, A. ED - Plemenitas, A. T1 - K+ Transport and its Role for Osmoregulation in a Halophilic Member of the Bacteria Domain: Characterization of the K+ Uptake Systems from Halomonas elongata T2 - Adaptation to life at high salt concentrations in Archaea, Bacteria, and Eukarya KW - Osmoregulation KW - Potassium KW - Glutamate KW - Potassium transport KW - TrkH KW - TrkA KW - Trkl KW - Halomonas elongata KW - Halophilic Bacteria PY - 2005 SN - 1-402-03632-9 N1 - Serientitel: Cellular Origin, Life in Extreme Habitats and Astrobiology – Series title: Cellular Origin, Life in Extreme Habitats and Astrobiology IS - 9 SP - 289 EP - 300 PB - Springer CY - Berlin AN - OPUS4-11668 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Kraegeloh, A. A1 - Amendt, B. A1 - Kunte, Hans-Jörg T1 - Potassium Transport in a Halophilic Member of the Bacteria Domain: Identification and Characterization of the K+ Uptake Systems TrkH and TrkI from Halomonas elongata DSM 2581T JF - Journal of bacteriology N2 - The halophilic bacterium Halomonas elongata accumulates K+, glutamate, and the compatible solute ectoine as osmoprotectants. By functional complementation of Escherichia coli mutants defective in K+ uptake, we cloned three genes that are required for K+ uptake in H. elongata. Two adjacent genes, named trkA (1,374 bp) and trkH (1,449 bp), were identified on an 8.5-kb DNA fragment, while a third gene, called trkI (1,479 bp), located at a different site in the H. elongata chromosome, was found on a second 8.5-kb fragment. The potential protein expressed by trkA is similar to the cytoplasmic NAD+/NADH binding protein TrkA from E. coli, which is required for the activity of the Trk K+ uptake system. The deduced amino acid sequences of trkH and trkI showed significant identity to the transmembrane protein of Trk transporters. K+ transport experiments with trkH and trkI mutants of H. elongata revealed that TrkI exhibits a Km value of 1.12 mM, while the TrkH system has a half-saturation constant of 3.36 mM. Strain KB12, relying on TrkH alone, accumulated K+ with a lower Vmax and required a higher K+ concentration for growth in highly saline medium than the wild type. Strain KB15, expressing only TrkI, showed the same phenotype and the same K+ transport kinetics as the wild type, proving that TrkI is the main K+ transport system in H. elongata. In the absence of both transporters TrkH and TrkI, K+ accumulation was not detectable. K+ transport was also abolished in a trkA deletion mutant, indicating that TrkI and TrkH depend on one type of TrkA protein. Reverse transcriptase PCR experiments and Northern hybridization analyses of the trkAH locus revealed cotranscription of trkAH as well as a monocistronic transcript with only trkA. KW - Potassium transport KW - Osmoregulation KW - Halophilic Bacteria KW - Halomonas elongata KW - TrkH KW - TrkA PY - 2005 DO - https://doi.org/10.1128/JB.187.3.1036-1043.2005 SN - 0021-9193 SN - 1098-5530 VL - 187 IS - 3 SP - 1036 EP - 1043 PB - American Society for Microbiology CY - Washington, DC AN - OPUS4-6294 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -