TY - JOUR A1 - Vandrich, Jasmina A1 - Pfeiffer, F. A1 - Alfaro Espinoza, Gabriela A1 - Kunte, Hans-Jörg T1 - Contribution of mechanosensitive channels to osmoadaptation and ectoine excretion in Halomonas elongata N2 - For osmoadaptation the halophilic bacterium Halomonas elongata synthesizes as its main compatible solute the aspartate derivative ectoine. H. elongata does not rely entirely on synthesis but can accumulate ectoine by uptake from the surrounding environment with the help of the osmoregulated transporter TeaABC. Disruption of the TeaABC-mediated ectoine uptake creates a strain that is constantly losing ectoine to the medium. However, the efflux mechanism of ectoine in H. elongata is not yet understood. H. elongata possesses four genes encoding mechanosensitive channels all of which belong to the small conductance type (MscS). Analysis by qRT-PCR revealed a reduction in transcription of the mscS genes with increasing salinity. The response of H. elongata to hypo- and hyperosmotic shock never resulted in up-regulation but rather in downregulation of mscS transcription. Deletion of all four mscS genes created a mutant that was unable to cope with hypoosmotic shock. However, the knockout mutant grew significantly faster than the wildtype at high salinity of 2 M NaCl, and most importantly, still exported 80% of the ectoine compared to the wildtype. We thus conclude that a yet unknown system, which is independent of mechanosensitive channels, is the major export route for ectoine in H. elongata. KW - Halomonas elongata KW - Ectoine KW - Osmotic shock KW - Solute excretion KW - Osmoadaptation KW - Mechanosensitive channel KW - MscS PY - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-507229 VL - 24 SP - 421 EP - 432 PB - Springer AN - OPUS4-50722 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Moghaddam, J. A. A1 - Böhringer, N. A1 - Burdziak, A. A1 - Kunte, Hans-Jörg A1 - Galinski, E. A. A1 - Schäberle, T. F. T1 - Different strategies of osmoadaptation in the closely related marine myxobacteria Enhygromyxa salina SWB007 and Plesiocystis pacifica SIR-1 N2 - Only a few myxobacteria are known to date that are classified as marine, owing to their salt dependency. In this study, the salt tolerance mechanism of these bacteria was investigated. To this end, a growth medium was designed in which the mutated Escherichia coli strain BKA13 served as sole food source for the predatory, heterotrophic myxobacteria. This enabled measurement of the osmolytes without any background and revealed that the closely related strains Enhygromyxa salina SWB007 and Plesiocystis pacifica SIR-1 developed different strategies to handle salt stress. Ple. pacifica SIR-1, which was grown between 1 and 4% NaCl, relies solely on the accumulation of amino acids, while Enh. salina SWB007, which was grown between 0.5 and 3% NaCl, employs, besides betaine, hydroxyectoine as the major compatible solute. In accordance with this analysis, only in the latter strain was a locus identified that codes for genes corresponding to the biosynthesis of betaine, ectoine and hydroxyectoine. KW - Osmoadaptation PY - 2016 U6 - https://doi.org/10.1099/mic.0.000250 VL - 162 SP - 651 EP - 661 PB - SOC General Microbiology CY - Berks, UK AN - OPUS4-35782 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Moghaddam, J. A. A1 - Boehringer, N. A1 - Burdziak, A. A1 - Kunte, Hans-Jörg A1 - Galinski, E. A. A1 - Schäberle, T. F. T1 - Different strategies of osmoadaptation in the closely related marine myxobacteria Enhygromyxa salina SWB007 and Plesiocystis pacifica SIR-1 N2 - Only a few myxobacteria are known to date that are classified as marine, owing to their salt dependency. In this study, the salt tolerance mechanism of these bacteria was investigated. To this end, a growth medium was designed in which the mutated Escherichia coli strain BKA13 served as sole food source for the predatory, heterotrophic myxobacteria. This enabled measurement of the osmolytes without any background and revealed that the closely related strains Enhygromyxa salina SWB007 and Plesiocystis pacifica SIR-1 developed different strategies to handle salt stress. Ple. pacifica SIR-1, which was grown between 1 and 4% NaCl, relies solely on the accumulation of amino acids, while Enh. salina SWB007, which was grown between 0.5 and 3% NaCl, employs, besides betaine, hydroxyectoine as the major compatible solute. In accordance with this analysis, only in the latter strain was a locus identified that codes for genes corresponding to the biosynthesis of betaine, ectoine and hydroxyectoine. KW - Osmoregulation KW - Myxobacteria KW - Betaine KW - Ectoine PY - 2016 U6 - https://doi.org/10.1099/mic.0.000250 VL - 162 IS - 4 SP - 651 EP - 661 AN - OPUS4-39115 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - 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 - 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 freeenergy 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 AN - OPUS4-37681 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CHAP A1 - Kunte, Hans-Jörg A1 - Schwarz, T. A1 - Galinski, E. A. ED - Lee, N. M. T1 - The compatible solute ectoine: protection mechanisms, strain development, and industrial production N2 - Bacteria, Archaea, and Eukarya can adapt to saline environments by accumulating compatible solutes in order to maintain an osmotic equilibrium. Compatible solutes are of diverse chemical structure (sugars, polyols, amino acid derivatives) and are beneficial for bacterial cells not only as osmoregulatory solutes but also as protectants of proteins by mitigating detrimental effects of freezing, drying, and high temperatures. The aspartate derivative ectoine is a widespread compatible solute in Bacteria and possesses additional protective properties compared with other compatible solutes and stabilizes even whole cells against stresses such as ultraviolet radiation or cytotoxins. Here, it is our intention to go beyond a simple description of effects, but to depict the molecular interaction of ectoine with biomolecules, such as proteins, membranes, and DNA and explain the underlying principles. The stabilizing properties of ectoine attracted industry, which saw the potential to market ectoine as a novel active component in health care products and cosmetics. In joint efforts of industry and research, a large-scale fermentation procedure has been developed with the halophilic bacterium Halomonas elongata used as a producer strain. The development and application of ectoine-excreting mutants from H. elongata (“leaky” mutants) allow for the annual production of ectoine on a scale of tons. The details of the strain development and fermentation processes will be introduced. KW - Ectoine KW - Biotechnology KW - Compatible solute KW - Preferential exclusion KW - Osmophobic effect PY - 2020 SN - 978-3-11-042773-8 SP - 121 EP - 136 PB - De Gruyter AN - OPUS4-51472 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Huang, Y.-L. A1 - Zheng, A1 - Zhang, A1 - Lu, A1 - Wang, A1 - Kunte, Hans-Jörg A1 - Sand, W. T1 - Forecast on Long Term Chemical Environment Evolution on Surface of Nuclear Waste Container N2 - Surface environment changes of nuclear waste container, mainly including oxygen contents, buffer pore water components and pH under the conditions of “borehole” type and “In-floor” type with bentonite buffer/backfill and concrete buffer were summarized. This summarization provides a basic corrosion environment reference for the corrosion evolution re- search of high-level radioactive waste disposal repository in our country. KW - Corrosion evolution KW - Nuclear waste KW - Disposal repository KW - Corrosion environment PY - 2018 U6 - https://doi.org/10.7643/ issn.1672-9242.2018.10.017 SN - 1672-9242 VL - 15 IS - 10 SP - 103 EP - 108 PB - Zhongguo Bingqi Gongye di-wujiu Yanjiusuo CY - Chongqing AN - OPUS4-46952 LA - zho AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Hobmeier, K. A1 - Oppermann, M. A1 - Stasinski, N. A1 - Kremling, A. A1 - Pflüger-Grau, K. A1 - Kunte, Hans-Jörg A1 - Marin Sanguino, A. T1 - Metabolic engineering of Halomonas elongata: Ectoine secretion is increased by demand and supply driven approaches N2 - The application of naturally-derived biomolecules in everyday products, replacing conventional synthetic manufacturing, is an ever-increasing market. An example of this is the compatible solute ectoine, which is contained in a plethora of treatment formulations for medicinal products and cosmetics. As of today, ectoine is produced in a scale of tons each year by the natural producer Halomonas elongata. In this work, we explore two complementary approaches to obtain genetically improved producer strains for ectoine production. We explore the effect of increased precursor supply (oxaloacetate) on ectoine production, as well as an implementation of increased ectoine demand through the overexpression of a transporter. Both approaches were implemented on an already genetically modified ectoine-excreting strain H. elongata KB2.13 (ΔteaABC ΔdoeA) and both led to new strains with higher ectoine excretion. The supply driven approach led to a 45% increase in ectoine titers in two different strains. This increase was attributed to the removal of phosphoenolpyruvate carboxykinase (PEPCK), which allowed the conversion of 17.9% of the glucose substrate to ectoine. For the demand driven approach, we investigated the potential of the TeaBC transmembrane proteins from the ectoine-specific Tripartite ATP-Independent Periplasmic (TRAP) transporter as export channels to improve ectoine excretion. In the absence of the substrate-binding protein TeaA, an overexpression of both subunits TeaBC facilitated a three-fold increased excretion rate of ectoine. Individually, the large subunit TeaC showed an approximately five times higher extracellular ectoine concentration per dry weight compared to TeaBC shortly after its expression was induced. However, the detrimental effect on growth and ectoine titer at the end of the process hints toward a negative impact of TeaC overexpression on membrane integrity and possibly leads to cell lysis. By using either strategy, the ectoine synthesis and excretion in H. elongata could be boosted drastically. The inherent complementary nature of these approaches point at a coordinated implementation of both as a promising strategy for future projects in Metabolic Engineering. Moreover, a wide variation of intracelllular ectoine levels was observed between the strains, which points at a major disruption of mechanisms responsible for ectoine regulation in strain KB2.13. KW - Osmoadaptation KW - Metabolic engineering PY - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-555644 SN - 1664-302X VL - 13 SP - 1 EP - 13 PB - Frontiers Media CY - Lausanne AN - OPUS4-55564 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Hobmeier, K. A1 - Goëss, M. C. A1 - Sehr, C. A1 - Schwaminger, S. A1 - Berensmeier, S. A1 - Kremling, A. A1 - Kunte, Hans-Jörg A1 - Pflüger-Grau, K. A1 - Marin-Sanguino, A. T1 - Anaplerotic Pathways in Halomonas elongata: The Role of the Sodium Gradient N2 - Salt tolerance in the γ-proteobacterium Halomonas elongata is linked to its ability to produce the compatible solute ectoine. The metabolism of ectoine production is of great interest since it can shed light on the biochemical basis of halotolerance as well as pave the way for the improvement of the biotechnological production of such compatible solute. Ectoine belongs to the biosynthetic family of aspartate-derived amino-acids. Aspartate is formed from oxaloacetate, thereby connecting ectoine production to the anaplerotic reactions that refill carbon into the tricarboxylic acid cycle (TCA cycle). This places a high demand on these reactions and creates the need to regulate them not only in response to growth but also in response to extracellular salt concentration. In this work, we combine modeling and experiments to analyze how these different needs shape the anaplerotic reactions in H. elongata. First, the stoichiometric and thermodynamic factors that condition the flux distributions are analyzed, then the optimal patterns of operation for oxaloacetate production are calculated. Finally, the phenotype of two deletion mutants lacking potentially relevant anaplerotic enzymes: phosphoenolpyruvate carboxylase (Ppc) and oxaloacetate decarboxylase (Oad) are experimentally characterized. The results show that the anaplerotic reactions in H. elongata are indeed subject to evolutionary pressures that differ from those faced by other gram-negative bacteria. Ectoine producing halophiles must meet a higher metabolic demand for oxaloacetate and the reliance of many marine bacteria on the Entner-Doudoroff pathway compromises the anaplerotic efficiency of Ppc, which is usually one of the main enzymes fulfilling this role. The anaplerotic flux in H. elongata is contributed not only by Ppc but also by Oad, an enzyme that has not yet been shown to play this role in vivo. Ppc is necessary for H. elongata to grow normally at low salt concentrations but it is not required to achieve near maximal growth rates as long as there is a steep sodium gradient. On the other hand, the lack of Oad presents serious difficulties to grow at high salt concentrations. This points to a shared role of these two enzymes in guaranteeing the supply of oxaloacetate for biosynthetic reactions. KW - Metabolic flux analysis KW - Halophilic bacteria KW - Halomonas elongata KW - Metabolic modeling PY - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-513124 VL - 11 SP - 561800 AN - OPUS4-51312 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Hobmeier, K. A1 - Cantone, M. A1 - Nguyen, Q. A. A1 - Pflüger-Grau, K. A1 - Kremling, A. A1 - Kunte, Hans-Jörg A1 - Pfeiffer, F. A1 - Marin-Sanguino, A. T1 - Adaptation to varying salinity in Halomonas elongata: Much more than ectoine accumulation N2 - The halophilic γ-proteobacterium Halomonas elongata DSM 2581T thrives at salt concentrations well above 10 % NaCl (1.7 M NaCl). A well-known osmoregulatory mechanism is the accumulation of the compatible solute ectoine within the cell in response to osmotic stress. While ectoine accumulation is central to osmoregulation and promotes resistance to high salinity in halophilic bacteria, ectoine has this effect only to a much lesser extent in non-halophiles. We carried out transcriptome analysis of H. elongata grown on two different carbon sources (acetate or glucose), and low (0.17 M NaCl), medium (1 M), and high salinity (2 M) to identify additional mechanisms for adaptation to high saline environments. To avoid a methodological bias, the transcripts were evaluated by applying two methods, DESeq2 and Transcripts Per Million (TPM). The differentially transcribed genes in response to the available carbon sources and salt stress were then compared to the transcriptome profile of Chromohalobacter salexigens, a closely related moderate halophilic bacterium. Transcriptome profiling supports the notion that glucose is degraded via the cytoplasmic Entner-Doudoroff pathway, whereas the Embden-Meyerhoff-Parnas pathway is employed for gluconeogenesis. The machinery of oxidative phosphorylation in H. elongata and C. salexigens differs greatly from that of non-halophilic organisms, and electron flow can occur from quinone to oxygen along four alternative routes. Two of these pathways via cytochrome bo' and cytochrome bd quinol oxidases seem to be upregulated in salt stressed cells. Among the most highly regulated genes in H. elongata and C. salexigens are those encoding chemotaxis and motility proteins, with genes for chemotaxis and flagellar assembly severely downregulated at low salt concentrations. We also compared transcripts at low and high-salt stress (low growth rate) with transcripts at optimal salt concentration and found that the majority of regulated genes were down-regulated in stressed cells, including many genes involved in carbohydrate metabolism, while ribosome synthesis was up-regulated, which is in contrast to what is known from non-halophiles at slow growth. Finally, comparing the acidity of the cytoplasmic proteomes of non-halophiles, extreme halophiles and moderate halophiles suggests adaptation to an increased cytoplasmic ion concentration of H. elongata. Taken together, these results lead us to propose a model for salt tolerance in H. elongata where ion accumulation plays a greater role in salt tolerance than previously assumed. KW - Ectoine KW - Osmoadaptation PY - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-545608 SN - 1664-302X VL - 13 SP - 1 EP - 19 PB - Frontiers Media CY - Lausanne AN - OPUS4-54560 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -