TY - JOUR A1 - Martin-Sanchez, Pedro Maria A1 - Gorbushina, Anna A1 - Kunte, Hans-Jörg A1 - Toepel, Jörg T1 - A novel qPCR protocol for the specific detection and quantification of the fuel-deteriorating fungus Hormoconis resinae N2 - A wide variety of fungi and bacteria are known to contaminate fuels and fuel systems. These microbial contaminants have been linked to fuel system fouling and corrosion. The fungus Hormoconis resinae, a common jet fuel contaminant, is used in this study as a model for developing innovative risk assessment methods. A novel qPCR protocol to detect and quantify H. resinae in, and together with, total fungal contamination of fuel systems is reported. Two primer sets, targeting the markers RPB2 and ITS, were selected for their remarkable specificity and sensitivity. These primers were successfully applied on fungal cultures and diesel samples demonstrating the validity and reliability of the established qPCR protocol. This novel tool allows clarification of the current role of H. resinae in fuel contamination cases, as well as providing a technique to detect fungal outbreaks in fuel systems. This tool can be expanded to other well-known fuel-deteriorating microorganisms. KW - Microbial contamination KW - Real-time quantitative PCR KW - Microbiologically influenced corrosion; KW - Diesel biodeterioration KW - Fouling KW - Indicator PY - 2016 U6 - https://doi.org/10.1080/08927014.2016.1177515 SN - 0892-7014 VL - 32 IS - 6 SP - 635 EP - 644 PB - Taylor & Francis Group CY - Abingdon AN - OPUS4-37337 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 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 U6 - 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 - 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 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 - CONF A1 - Kunte, Hans-Jörg A1 - Davies, N. W. A1 - Galinski, E. A. A1 - Witt, K. J. A1 - Grammann, K. A1 - Volke, A. A1 - Sauer, T. T1 - How to shift and exploit the unfavourable equilibrium of ADPC formation T2 - VAAM Jahrestagung CY - Bremen, Germany DA - 2013-03-10 PY - 2013 AN - OPUS4-28375 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Weinhart, M. A1 - Becherer, T. A1 - Schnurbusch, N. A1 - Schwibbert, Karin A1 - Kunte, Hans-Jörg A1 - Haag, R. T1 - Linear and hyperbranched polyglycerol derivatives as excellent bioinert glass coating materials N2 - The non-specific adsorption of proteins to surfaces in contact with biofluids constitutes a major problem in the biomedical and biotechnological field, due to the initiation of biofilm formation and the resulting improper function of devices. Therefore, non-fouling surfaces modified with poly(ethylene glycol) (PEG) are usually applied. In this study, we report the synthesis of triethoxysilane modified glycerol based polymers of linear and branched architecture for the preparation of covalently attached monolayers on glass. Evaluation of the biocompatibility of these surfaces was performed in comparison to bare non-coated glass, hydrophobic hexadecane modified glass, and mPEG modified glass as the controls. Protein adsorption of BSA and fibrinogen (1 mg · mL-1 in PBS) after 4 and 24 h immersion was reduced by more than 96 and 90%, respectively, compared to the adsorption on bare glass substrates. In addition, mouse NIH-3T3 fibroblast cells showed only marginal adhesion on the polyglycerol and mPEG coated slides after 3 and 7 d incubation in cell suspension, which demonstrates the long-term stability of the applied glass coatings. The non-adhesive properties of these coatings were further reflected in bacterial adhesion tests of Escherichia coli K12 and three clinically relevant Gram-positive and negative strains (Staphylococcus aureus, Pseudomonas aeruginosa, and Aeromonas hydrophila), since linear polyglycerol (LPG(OH)), linear poly(methyl glycerol) (LPG(OMe)), and hyperbranched polyglycerol (HPG) reduced the adhesion for all tested strains by more than 99% compared to bare glass. Therefore, polyglycerol derivatives present an excellent non-fouling surface coating as an alternative to PEG with feasibility for surface modification of various substrates. KW - Protein adsorption KW - Non-fouling-surfaces KW - Linear polyglycerol KW - Hyperbranched polyglycerol KW - Bacterial adhesion PY - 2011 U6 - https://doi.org/10.1002/adem.201180012 SN - 1438-1656 VL - 13 IS - 12 SP - B501 EP - B510 PB - Wiley-VCH Verl. CY - Weinheim AN - OPUS4-25221 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Marinelli, F. A1 - Kuhlmann, S.I. A1 - Grell, E. A1 - Kunte, Hans-Jörg A1 - Ziegler, C. M. A1 - Faraldo-Gómez, J.D. T1 - Evidence for an allosteric mechanism of substrate release from membrane-transporter accessory binding proteins N2 - Numerous membrane importers rely on accessory water-soluble proteins to capture their substrates. These substrate-binding proteins (SBP) have a strong affinity for their ligands; yet, substrate release onto the low-affinity membrane transporter must occur for uptake to proceed. It is generally accepted that release is facilitated by the association of SBP and transporter, upon which the SBP adopts a conformation similar to the unliganded state, whose affinity is sufficiently reduced. Despite the appeal of this mechanism, however, direct supporting evidence is lacking. Here, we use experimental and theoretical methods to demonstrate that an allosteric mechanism of enhanced substrate release is indeed plausible. First, we report the atomic-resolution structure of apo TeaA, the SBP of the Na+-coupled ectoine TRAP transporter TeaBC from Halomonas elongata DSM2581T, and compare it with the substrate-bound structure previously reported. Conformational free-energy landscape calculations based upon molecular dynamics simulations are then used to dissect the mechanism that couples ectoine binding to structural change in TeaA. These insights allow us to design a triple mutation that biases TeaA toward apo-like conformations without directly perturbing the binding cleft, thus mimicking the influence of the membrane transporter. Calorimetric measurements demonstrate that the ectoine affinity of the conformationally biased triple mutant is 100-fold weaker than that of the wild type. By contrast, a control mutant predicted to be conformationally unbiased displays wild-type affinity. This work thus demonstrates that substrate release from SBPs onto their membrane transporters can be facilitated by the latter through a mechanism of allosteric modulation of the former. KW - Binding thermodynamics KW - Periplasmic binding protein KW - Secondary transporter KW - ABC transporter KW - Replica-exchange metadynamics PY - 2011 U6 - https://doi.org/10.1073/pnas.1112534108 SN - 0027-8424 SN - 1091-6490 VL - 108 IS - 49 SP - E1285 EP - E1292 PB - National Academy of Sciences CY - Washington, DC AN - OPUS4-25049 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kunte, Hans-Jörg T1 - Osmoregulation in halophilic bacteria: Synthesis and transport of osmoprotectants in halomonas elongata T2 - Max-Planck-Institut für Biochemie CY - Planegg, Germany DA - 2006-04-26 PY - 2006 AN - OPUS4-14156 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kunte, Hans-Jörg T1 - Mechanismen der Anpassung an Wasserstress: Synthese und Tansport von Zellschutzstoffen in halophilen Bacteria T2 - Humboldt Universität CY - Berlin, Germany DA - 2006-12-19 PY - 2006 AN - OPUS4-14157 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Kunte, Hans-Jörg T1 - Osmoregulation in Bacteria: Compatible Solute Accumulation and Osmosensing N2 - Bacteria and Archaea have developed two basic mechanisms to cope with osmotic stress, the salt-in-cytoplasm mechanism, and the organic-osmolyte mechanism. Organic osmolytes or so-called compatible solutes can be accumulated in molar concentration in the cytoplasm and allow for the adaptation of bacterial cells to varying salt concentrations. The biosynthetic pathways of compatible solutes and different compatible solute transport systems are described. A model for osmoregulatory compatible solute accumulation is introduced. KW - Archaea KW - Compatible solutes KW - Halophilic KW - Halotolerant KW - Osmoregulated transporter PY - 2006 U6 - https://doi.org/10.1071/EN06016 SN - 1448-2517 SN - 1449-8979 VL - 3 IS - 2 SP - 94 EP - 99 PB - CSIRO Publishing CY - Collingwood, Vic. AN - OPUS4-14258 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - 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 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 U6 - 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 -