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Erhard Bremer - One of the best experts on this subject based on the ideXlab platform.
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the ups and downs of Ectoine structural enzymology of a major microbial stress protectant and versatile nutrient
Biological Chemistry, 2020Co-Authors: Lucas Hermann, Laura Czech, Sander H J Smits, Christophernils Mais, Gert Bange, Erhard BremerAbstract:Ectoine and its derivative 5-hydroxyEctoine are compatible solutes and chemical chaperones widely synthesized by Bacteria and some Archaea as cytoprotectants during osmotic stress and high- or low-growth temperature extremes. The function-preserving attributes of Ectoines led to numerous biotechnological and biomedical applications and fostered the development of an industrial scale production process. Synthesis of Ectoines requires the expenditure of considerable energetic and biosynthetic resources. Hence, microorganisms have developed ways to exploit Ectoines as nutrients when they are no longer needed as stress protectants. Here, we summarize our current knowledge on the phylogenomic distribution of Ectoine producing and consuming microorganisms. We emphasize the structural enzymology of the pathways underlying Ectoine biosynthesis and consumption, an understanding that has been achieved only recently. The synthesis and degradation pathways critically differ in the isomeric form of the key metabolite N-acetyldiaminobutyric acid (ADABA). γ-ADABA serves as preferred substrate for the Ectoine synthase, while the α-ADABA isomer is produced by the Ectoine hydrolase as an intermediate in catabolism. It can serve as internal inducer for the genetic control of Ectoine catabolic genes via the GabR/MocR-type regulator EnuR. Our review highlights the importance of structural enzymology to inspire the mechanistic understanding of metabolic networks at the biological scale.
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degradation of the microbial stress protectants and chemical chaperones Ectoine and hydroxyEctoine by a bacterial hydrolase deacetylase complex
Journal of Biological Chemistry, 2020Co-Authors: C N Mais, Laura Czech, Erhard Bremer, Lucas Hermann, Alexandra A Richter, Florian Altegoer, Andreas Seubert, Isa Wernersbach, Gert BangeAbstract:When faced with increased osmolarity in the environment, many bacterial cells accumulate the compatible solute Ectoine and its derivative 5-hydroxyEctoine. Both compounds are not only potent osmostress protectants, but also serve as effective chemical chaperones stabilizing protein functionality. Ectoines are energy-rich nitrogen and carbon sources that have an ecological impact that shapes microbial communities. Although the biochemistry of Ectoine and 5-hydroxyEctoine biosynthesis is well understood, our understanding of their catabolism is only rudimentary. Here, we combined biochemical and structural approaches to unravel the core of Ectoine and 5-hydroxyEctoine catabolisms. We show that a conserved enzyme bimodule consisting of the EutD Ectoine/5-hydroxyEctoine hydrolase and the EutE deacetylase degrades both Ectoines. We determined the high-resolution crystal structures of both enzymes, derived from the salt-tolerant bacteria Ruegeria pomeroyi and Halomonas elongata. These structures, either in their apo-forms or in forms capturing substrates or intermediates, provided detailed insights into the catalytic cores of the EutD and EutE enzymes. The combined biochemical and structural results indicate that the EutD homodimer opens the pyrimidine ring of Ectoine through an unusual covalent intermediate, N-α-2 acetyl-L-2,4-diaminobutyrate (α-ADABA). We found that α-ADABA is then deacetylated by the zinc-dependent EutE monomer into diaminobutyric acid (DABA), which is further catabolized to L-aspartate. We observed that the EutD-EutE bimodule synthesizes exclusively the α-, but not the γ-isomers of ADABA or hydroxy-ADABA. Of note, α-ADABA is known to induce the MocR/GabR-type repressor EnuR, which controls the expression of many Ectoine catabolic genes clusters. We conclude that hydroxy-α-ADABA might serve a similar function.
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biosynthesis of the stress protectant and chemical chaperon Ectoine biochemistry of the transaminase ectb
Frontiers in Microbiology, 2019Co-Authors: Alexandra A Richter, Laura Czech, Sander H J Smits, Christophernils Mais, Gert Bange, Kyra Geyer, A Hoeppner, Tobias J Erb, Erhard BremerAbstract:Bacteria frequently adapt to high osmolarity surroundings through the accumulation of compatible solutes. Ectoine is a prominent member of these types of stress protectants and is produced via an evolutionarily conserved biosynthetic pathway beginning with the L-2,4-diaminobutyrate (DAB) transaminase (TA) EctB. Here, we studied EctB from the thermo-tolerant Gram-positive bacterium Paenibacillus lautus (Pl) and show that this tetrameric enzyme is highly tolerant to salt, pH, and temperature. During Ectoine biosynthesis, EctB converts L-glutamate and L-aspartate-beta-semialdehyde into 2-oxoglutarate and DAB, but it also catalyzes the reverse reaction. Our analysis unravels that EctB enzymes are mechanistically identical to the PLP-dependent gamma-aminobutyrate TAs (GABA-TAs) and only differ with respect to substrate binding. Inspection of the genomic context of the ectB gene in P. lautus identifies an unusual arrangement of juxtapositioned genes for Ectoine biosynthesis and import via an Ehu-type binding-protein-dependent ABC transporter. This operon-like structure suggests the operation of a highly coordinated system for Ectoine synthesis and import to maintain physiologically adequate cellular Ectoine pools under osmotic stress conditions in a resource-efficient manner. Taken together, our study provides an in-depth mechanistic and physiological description of EctB, the first enzyme of the Ectoine biosynthetic pathway.
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biosynthesis of the stress protectant and chemical chaperon Ectoine biochemistry of the transaminase ectb
Frontiers in Microbiology, 2019Co-Authors: Alexandra A Richter, Laura Czech, Sander H J Smits, Gert Bange, C N Mais, Kyra Geyer, A Hoeppner, Erhard BremerAbstract:Bacteria frequently adapt to high osmolarity surroundings through the accumulation of compatible solutes. Ectoine is a prominent member of these types of stress protectants and is produced via an evolutionarily conserved biosynthetic pathway beginning with the L-2,4-diaminobutyrate (DAB) transaminase EctB. Here, we studied EctB from the thermo-tolerant Gram-positive bacterium Paenibacillus lautus and show that this tetrameric enzyme is highly tolerant to salt, pH, and temperature. During Ectoine biosynthesis, EctB converts L-glutamate and L-aspartate-beta-semialdehyde into 2-oxoglutarate and L-2,4-diaminobutyrate, but it also catalyzes the reverse reaction. Our analysis unravels that EctB enzymes are mechanistically identical to the PLP-dependent gamma-aminobutyrate transaminases (GABA-TAs) and only differ with respect to substrate binding. Inspection of the genomic context of the ectB gene in P. lautus identifies an unusual arrangement of juxtapositioned genes for Ectoine biosynthesis and import via an Ehu-type binding-protein-dependent ABC transporter. This operon-like structure suggests the operation of a highly coordinated system for Ectoine synthesis and import to maintain physiologically adequate cellular Ectoine pools under osmotic stress conditions in a resource-efficient manner. Taken together, our study provides an in-depth mechanistic and physiological description of EctB, the first enzyme of the of the Ectoine biosynthetic pathway.
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role of the extremolytes Ectoine and hydroxyEctoine as stress protectants and nutrients genetics phylogenomics biochemistry and structural analysis
Genes, 2018Co-Authors: Laura Czech, Nadine Stoveken, Johann Heider, Lucas Hermann, Alexandra A Richter, Astrid Hoppner, Sander H J Smits, Erhard BremerAbstract:Fluctuations in environmental osmolarity are ubiquitous stress factors in many natural habitats of microorganisms, as they inevitably trigger osmotically instigated fluxes of water across the semi-permeable cytoplasmic membrane. Under hyperosmotic conditions, many microorganisms fend off the detrimental effects of water efflux and the ensuing dehydration of the cytoplasm and drop in turgor through the accumulation of a restricted class of organic osmolytes, the compatible solutes. Ectoine and its derivative 5-hydroxyEctoine are prominent members of these compounds and are synthesized widely by members of the Bacteria and a few Archaea and Eukarya in response to high salinity/osmolarity and/or growth temperature extremes. Ectoines have excellent function-preserving properties, attributes that have led to their description as chemical chaperones and fostered the development of an industrial-scale biotechnological production process for their exploitation in biotechnology, skin care, and medicine. We review, here, the current knowledge on the biochemistry of the Ectoine/hydroxyEctoine biosynthetic enzymes and the available crystal structures of some of them, explore the genetics of the underlying biosynthetic genes and their transcriptional regulation, and present an extensive phylogenomic analysis of the Ectoine/hydroxyEctoine biosynthetic genes. In addition, we address the biochemistry, phylogenomics, and genetic regulation for the alternative use of Ectoines as nutrients.
Carmen Vargas - One of the best experts on this subject based on the ideXlab platform.
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New insights into hydroxyEctoine synthesis and its transcriptional regulation in the broad‐salt growing halophilic bacterium Chromohalobacter salexigens
'Wiley', 2021Co-Authors: Montserrat Argandona, Joaquin J Nieto, Francine Piubeli, Mercedes Reina‐bueno, Carmen VargasAbstract:Summary Elucidating the mechanisms controlling the synthesis of hydroxyEctoine is important to design novel genetic engineering strategies for optimizing the production of this biotechnologically relevant compatible solute. The genome of the halophilic bacterium Chromohalobacter salexigens carries two Ectoine hydroxylase genes, namely ectD and ectE, whose encoded proteins share the characteristic consensus motif of Ectoine hydroxylases but showed only a 51.9% identity between them. In this work, we have shown that ectE encodes a secondary functional Ectoine hydroxylase and that the hydroxyEctoine synthesis mediated by this enzyme contributes to C.␣salexigens thermoprotection. The evolutionary pattern of EctD and EctE and related proteins suggests that they may have arisen from duplication of an ancestral gene preceding the directional divergence that gave origin to the orders Oceanospirillales and Alteromonadales. Osmoregulated expression of ectD at exponential phase, as well as the thermoregulated expression of ectD at the stationary phase, seemed to be dependent on the general stress factor RpoS. In contrast, expression of ectE was always RpoS‐dependent regardless of the growth phase and osmotic or heat stress conditions tested. The data presented here suggest that the AraC‐GlxA‐like EctZ transcriptional regulator, whose encoding gene lies upstream of ectD, plays a dual function under exponential growth as both a transcriptional activator of osmoregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main Ectoine hydroxylase EctD. Inactivation of ectZ resulted in a higher amount of the total Ectoines pool at the expenses of a higher accumulation of Ectoine, with maintenance of the hydroxyEctoine levels. In addition to the transcriptional control, our results suggest a strong post‐transcriptional regulation of hydroxyEctoine synthesis. Data on the accumulation of Ectoine and hydroxyEctoine in rpoS and ectZ strains pave the way for using these genetic backgrounds for metabolic engineering for hydroxyEctoine production
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Insights into metabolic osmoadaptation of the Ectoines-producer bacterium Chromohalobacter salexigens through a high-quality genome scale metabolic model
Microbial Cell Factories, 2018Co-Authors: Francine Piubeli, Manuel Salvador, Montserrat Argandona, Jose M Pastor, Vicente Bernal, Manuel Canovas, Joaquin J Nieto, Carmen VargasAbstract:Background The halophilic bacterium Chromohalobacter salexigens is a natural producer of Ectoines, compatible solutes with current and potential biotechnological applications. As production of Ectoines is an osmoregulated process that draws away TCA intermediates, bacterial metabolism needs to be adapted to cope with salinity changes. To explore and use C. salexigens as cell factory for Ectoine(s) production, a comprehensive knowledge at the systems level of its metabolism is essential. For this purpose, the construction of a robust and high-quality genome-based metabolic model of C. salexigens was approached. Results We generated and validated a high quality genome-based C. salexigens metabolic model ( i FP764). This comprised an exhaustive reconstruction process based on experimental information, analysis of genome sequence, manual re-annotation of metabolic genes, and in-depth refinement. The model included three compartments (periplasmic, cytoplasmic and external medium), and two salinity-specific biomass compositions, partially based on experimental results from C. salexigens. Using previous metabolic data as constraints, the metabolic model allowed us to simulate and analyse the metabolic osmoadaptation of C. salexigens under conditions for low and high production of Ectoines. The i FP764 model was able to reproduce the major metabolic features of C. salexigens . Flux Balance Analysis (FBA) and Monte Carlo Random sampling analysis showed salinity-specific essential metabolic genes and different distribution of fluxes and variation in the patterns of correlation of reaction sets belonging to central C and N metabolism, in response to salinity. Some of them were related to bioenergetics or production of reducing equivalents, and probably related to demand for Ectoines. Ectoines metabolic reactions were distributed according to its correlation in four modules. Interestingly, the four modules were independent both at low and high salinity conditions, as they did not correlate to each other, and they were not correlated with other subsystems. Conclusions Our validated model is one of the most complete curated networks of halophilic bacteria. It is a powerful tool to simulate and explore C. salexigens metabolism at low and high salinity conditions, driving to low and high production of Ectoines. In addition, it can be useful to optimize the metabolism of other halophilic bacteria for metabolite production.
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Quantitative RNA-seq Analysis Unveils Osmotic and Thermal Adaptation Mechanisms Relevant for Ectoine Production in Chromohalobacter salexigens
Frontiers Media S.A., 2018Co-Authors: Manuel Salvador, Montserrat Argandona, Laszlo N Csonka, Joaquin J Nieto, Emilia Naranjo, Francine Piubeli, Carmen VargasAbstract:Quantitative RNA sequencing (RNA-seq) and the complementary phenotypic assays were implemented to investigate the transcriptional responses of Chromohalobacter salexigens to osmotic and heat stress. These conditions trigger the synthesis of Ectoine and hydroxyEctoine, two compatible solutes of biotechnological interest. Our findings revealed that both stresses make a significant impact on C. salexigens global physiology. Apart from compatible solute metabolism, the most relevant adaptation mechanisms were related to “oxidative- and protein-folding- stress responses,” “modulation of respiratory chain and related components,” and “ion homeostasis.” A general salt-dependent induction of genes related to the metabolism of Ectoines, as well as repression of Ectoine degradation genes by temperature, was observed. Different oxidative stress response mechanisms, secondary or primary, were induced at low and high salinity, respectively, and repressed by temperature. A higher sensitivity to H2O2 was observed at high salinity, regardless of temperature. Low salinity induced genes involved in “protein-folding-stress response,” suggesting disturbance of protein homeostasis. Transcriptional shift of genes encoding three types of respiratory NADH dehydrogenases, ATP synthase, quinone pool, Na+/H+ antiporters, and sodium-solute symporters, was observed depending on salinity and temperature, suggesting modulation of the components of the respiratory chain and additional systems involved in the generation of H+ and/or Na+ gradients. Remarkably, the Na+ intracellular content remained constant regardless of salinity and temperature. Disturbance of Na+- and H+-gradients with specific ionophores suggested that both gradients influence Ectoine production, but with differences depending on the solute, salinity, and temperature conditions. Flagellum genes were strongly induced by salinity, and further induced by temperature. However, salt-induced cell motility was reduced at high temperature, possibly caused by an alteration of Na+ permeability by temperature, as dependence of motility on Na+-gradient was observed. The transcriptional induction of genes related to the synthesis and transport of siderophores correlated with a higher siderophore production and intracellular iron content only at low salinity. An excess of iron increased hydroxyEctoine accumulation by 20% at high salinity. Conversely, it reduced the intracellular content of Ectoines by 50% at high salinity plus high temperature. These findings support the relevance of iron homeostasis for osmoadaptation, thermoadaptation and accumulation of Ectoines, in C. salexigens
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Presentation_1_Quantitative RNA-seq Analysis Unveils Osmotic and Thermal Adaptation Mechanisms Relevant for Ectoine Production in Chromohalobacter salexigens.PDF
2018Co-Authors: Manuel Salvador, Montserrat Argandona, Laszlo N Csonka, Joaquin J Nieto, Emilia Naranjo, Francine Piubeli, Carmen VargasAbstract:Quantitative RNA sequencing (RNA-seq) and the complementary phenotypic assays were implemented to investigate the transcriptional responses of Chromohalobacter salexigens to osmotic and heat stress. These conditions trigger the synthesis of Ectoine and hydroxyEctoine, two compatible solutes of biotechnological interest. Our findings revealed that both stresses make a significant impact on C. salexigens global physiology. Apart from compatible solute metabolism, the most relevant adaptation mechanisms were related to “oxidative- and protein-folding- stress responses,” “modulation of respiratory chain and related components,” and “ion homeostasis.” A general salt-dependent induction of genes related to the metabolism of Ectoines, as well as repression of Ectoine degradation genes by temperature, was observed. Different oxidative stress response mechanisms, secondary or primary, were induced at low and high salinity, respectively, and repressed by temperature. A higher sensitivity to H2O2 was observed at high salinity, regardless of temperature. Low salinity induced genes involved in “protein-folding-stress response,” suggesting disturbance of protein homeostasis. Transcriptional shift of genes encoding three types of respiratory NADH dehydrogenases, ATP synthase, quinone pool, Na+/H+ antiporters, and sodium-solute symporters, was observed depending on salinity and temperature, suggesting modulation of the components of the respiratory chain and additional systems involved in the generation of H+ and/or Na+ gradients. Remarkably, the Na+ intracellular content remained constant regardless of salinity and temperature. Disturbance of Na+- and H+-gradients with specific ionophores suggested that both gradients influence Ectoine production, but with differences depending on the solute, salinity, and temperature conditions. Flagellum genes were strongly induced by salinity, and further induced by temperature. However, salt-induced cell motility was reduced at high temperature, possibly caused by an alteration of Na+ permeability by temperature, as dependence of motility on Na+-gradient was observed. The transcriptional induction of genes related to the synthesis and transport of siderophores correlated with a higher siderophore production and intracellular iron content only at low salinity. An excess of iron increased hydroxyEctoine accumulation by 20% at high salinity. Conversely, it reduced the intracellular content of Ectoines by 50% at high salinity plus high temperature. These findings support the relevance of iron homeostasis for osmoadaptation, thermoadaptation and accumulation of Ectoines, in C. salexigens.
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contribution of rpos to metabolic efficiency and Ectoines synthesis during the osmo and heat stress response in the halophilic bacterium chromohalobacter salexigens
Environmental Microbiology Reports, 2015Co-Authors: Manuel Salvador, Montserrat Argandona, Jose M Pastor, Vicente Bernal, Manuel Canovas, Laszlo N Csonka, Joaquin J Nieto, Carmen VargasAbstract:Chromohalobacter salexigens is a halophilic γ-proteobacterium that responds to osmotic and heat stresses by accumulating Ectoine and hydroxyEctoine respectively. Evolution has optimized its metabolism to support high production of Ectoines. We analysed the effect of an rpoS mutation in C. salexigens metabolism and Ectoines synthesis. In long-term adapted cells, the rpoS strain was osmosensitive but not thermosensitive and showed unaltered Ectoines content, suggesting that RpoS regulates Ectoine(s)-independent osmoadaptive mechanisms. RpoS is involved in the regulation of C. salexigens metabolic adaptation to stress, as early steps of glucose oxidation through the Entner-Doudoroff pathway were deregulated in the rpoS mutant, leading to improved metabolic efficiency at low salinity. Moreover, a reduced pyruvate (but not acetate) overflow was displayed by the rpoS strain at low salt, probably linked to a slowdown in gluconate production and/or subsequent metabolism. Interestingly, RpoS does not seem to be the main regulator triggering the immediate transcriptional response of Ectoine synthesis to osmotic or thermal upshifts. However, it contributed to the expression of the ect genes in cells previously adapted to low or high salinity.
Joaquin J Nieto - One of the best experts on this subject based on the ideXlab platform.
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New insights into hydroxyEctoine synthesis and its transcriptional regulation in the broad‐salt growing halophilic bacterium Chromohalobacter salexigens
'Wiley', 2021Co-Authors: Montserrat Argandona, Joaquin J Nieto, Francine Piubeli, Mercedes Reina‐bueno, Carmen VargasAbstract:Summary Elucidating the mechanisms controlling the synthesis of hydroxyEctoine is important to design novel genetic engineering strategies for optimizing the production of this biotechnologically relevant compatible solute. The genome of the halophilic bacterium Chromohalobacter salexigens carries two Ectoine hydroxylase genes, namely ectD and ectE, whose encoded proteins share the characteristic consensus motif of Ectoine hydroxylases but showed only a 51.9% identity between them. In this work, we have shown that ectE encodes a secondary functional Ectoine hydroxylase and that the hydroxyEctoine synthesis mediated by this enzyme contributes to C.␣salexigens thermoprotection. The evolutionary pattern of EctD and EctE and related proteins suggests that they may have arisen from duplication of an ancestral gene preceding the directional divergence that gave origin to the orders Oceanospirillales and Alteromonadales. Osmoregulated expression of ectD at exponential phase, as well as the thermoregulated expression of ectD at the stationary phase, seemed to be dependent on the general stress factor RpoS. In contrast, expression of ectE was always RpoS‐dependent regardless of the growth phase and osmotic or heat stress conditions tested. The data presented here suggest that the AraC‐GlxA‐like EctZ transcriptional regulator, whose encoding gene lies upstream of ectD, plays a dual function under exponential growth as both a transcriptional activator of osmoregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main Ectoine hydroxylase EctD. Inactivation of ectZ resulted in a higher amount of the total Ectoines pool at the expenses of a higher accumulation of Ectoine, with maintenance of the hydroxyEctoine levels. In addition to the transcriptional control, our results suggest a strong post‐transcriptional regulation of hydroxyEctoine synthesis. Data on the accumulation of Ectoine and hydroxyEctoine in rpoS and ectZ strains pave the way for using these genetic backgrounds for metabolic engineering for hydroxyEctoine production
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Insights into metabolic osmoadaptation of the Ectoines-producer bacterium Chromohalobacter salexigens through a high-quality genome scale metabolic model
Microbial Cell Factories, 2018Co-Authors: Francine Piubeli, Manuel Salvador, Montserrat Argandona, Jose M Pastor, Vicente Bernal, Manuel Canovas, Joaquin J Nieto, Carmen VargasAbstract:Background The halophilic bacterium Chromohalobacter salexigens is a natural producer of Ectoines, compatible solutes with current and potential biotechnological applications. As production of Ectoines is an osmoregulated process that draws away TCA intermediates, bacterial metabolism needs to be adapted to cope with salinity changes. To explore and use C. salexigens as cell factory for Ectoine(s) production, a comprehensive knowledge at the systems level of its metabolism is essential. For this purpose, the construction of a robust and high-quality genome-based metabolic model of C. salexigens was approached. Results We generated and validated a high quality genome-based C. salexigens metabolic model ( i FP764). This comprised an exhaustive reconstruction process based on experimental information, analysis of genome sequence, manual re-annotation of metabolic genes, and in-depth refinement. The model included three compartments (periplasmic, cytoplasmic and external medium), and two salinity-specific biomass compositions, partially based on experimental results from C. salexigens. Using previous metabolic data as constraints, the metabolic model allowed us to simulate and analyse the metabolic osmoadaptation of C. salexigens under conditions for low and high production of Ectoines. The i FP764 model was able to reproduce the major metabolic features of C. salexigens . Flux Balance Analysis (FBA) and Monte Carlo Random sampling analysis showed salinity-specific essential metabolic genes and different distribution of fluxes and variation in the patterns of correlation of reaction sets belonging to central C and N metabolism, in response to salinity. Some of them were related to bioenergetics or production of reducing equivalents, and probably related to demand for Ectoines. Ectoines metabolic reactions were distributed according to its correlation in four modules. Interestingly, the four modules were independent both at low and high salinity conditions, as they did not correlate to each other, and they were not correlated with other subsystems. Conclusions Our validated model is one of the most complete curated networks of halophilic bacteria. It is a powerful tool to simulate and explore C. salexigens metabolism at low and high salinity conditions, driving to low and high production of Ectoines. In addition, it can be useful to optimize the metabolism of other halophilic bacteria for metabolite production.
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Quantitative RNA-seq Analysis Unveils Osmotic and Thermal Adaptation Mechanisms Relevant for Ectoine Production in Chromohalobacter salexigens
Frontiers Media S.A., 2018Co-Authors: Manuel Salvador, Montserrat Argandona, Laszlo N Csonka, Joaquin J Nieto, Emilia Naranjo, Francine Piubeli, Carmen VargasAbstract:Quantitative RNA sequencing (RNA-seq) and the complementary phenotypic assays were implemented to investigate the transcriptional responses of Chromohalobacter salexigens to osmotic and heat stress. These conditions trigger the synthesis of Ectoine and hydroxyEctoine, two compatible solutes of biotechnological interest. Our findings revealed that both stresses make a significant impact on C. salexigens global physiology. Apart from compatible solute metabolism, the most relevant adaptation mechanisms were related to “oxidative- and protein-folding- stress responses,” “modulation of respiratory chain and related components,” and “ion homeostasis.” A general salt-dependent induction of genes related to the metabolism of Ectoines, as well as repression of Ectoine degradation genes by temperature, was observed. Different oxidative stress response mechanisms, secondary or primary, were induced at low and high salinity, respectively, and repressed by temperature. A higher sensitivity to H2O2 was observed at high salinity, regardless of temperature. Low salinity induced genes involved in “protein-folding-stress response,” suggesting disturbance of protein homeostasis. Transcriptional shift of genes encoding three types of respiratory NADH dehydrogenases, ATP synthase, quinone pool, Na+/H+ antiporters, and sodium-solute symporters, was observed depending on salinity and temperature, suggesting modulation of the components of the respiratory chain and additional systems involved in the generation of H+ and/or Na+ gradients. Remarkably, the Na+ intracellular content remained constant regardless of salinity and temperature. Disturbance of Na+- and H+-gradients with specific ionophores suggested that both gradients influence Ectoine production, but with differences depending on the solute, salinity, and temperature conditions. Flagellum genes were strongly induced by salinity, and further induced by temperature. However, salt-induced cell motility was reduced at high temperature, possibly caused by an alteration of Na+ permeability by temperature, as dependence of motility on Na+-gradient was observed. The transcriptional induction of genes related to the synthesis and transport of siderophores correlated with a higher siderophore production and intracellular iron content only at low salinity. An excess of iron increased hydroxyEctoine accumulation by 20% at high salinity. Conversely, it reduced the intracellular content of Ectoines by 50% at high salinity plus high temperature. These findings support the relevance of iron homeostasis for osmoadaptation, thermoadaptation and accumulation of Ectoines, in C. salexigens
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Presentation_1_Quantitative RNA-seq Analysis Unveils Osmotic and Thermal Adaptation Mechanisms Relevant for Ectoine Production in Chromohalobacter salexigens.PDF
2018Co-Authors: Manuel Salvador, Montserrat Argandona, Laszlo N Csonka, Joaquin J Nieto, Emilia Naranjo, Francine Piubeli, Carmen VargasAbstract:Quantitative RNA sequencing (RNA-seq) and the complementary phenotypic assays were implemented to investigate the transcriptional responses of Chromohalobacter salexigens to osmotic and heat stress. These conditions trigger the synthesis of Ectoine and hydroxyEctoine, two compatible solutes of biotechnological interest. Our findings revealed that both stresses make a significant impact on C. salexigens global physiology. Apart from compatible solute metabolism, the most relevant adaptation mechanisms were related to “oxidative- and protein-folding- stress responses,” “modulation of respiratory chain and related components,” and “ion homeostasis.” A general salt-dependent induction of genes related to the metabolism of Ectoines, as well as repression of Ectoine degradation genes by temperature, was observed. Different oxidative stress response mechanisms, secondary or primary, were induced at low and high salinity, respectively, and repressed by temperature. A higher sensitivity to H2O2 was observed at high salinity, regardless of temperature. Low salinity induced genes involved in “protein-folding-stress response,” suggesting disturbance of protein homeostasis. Transcriptional shift of genes encoding three types of respiratory NADH dehydrogenases, ATP synthase, quinone pool, Na+/H+ antiporters, and sodium-solute symporters, was observed depending on salinity and temperature, suggesting modulation of the components of the respiratory chain and additional systems involved in the generation of H+ and/or Na+ gradients. Remarkably, the Na+ intracellular content remained constant regardless of salinity and temperature. Disturbance of Na+- and H+-gradients with specific ionophores suggested that both gradients influence Ectoine production, but with differences depending on the solute, salinity, and temperature conditions. Flagellum genes were strongly induced by salinity, and further induced by temperature. However, salt-induced cell motility was reduced at high temperature, possibly caused by an alteration of Na+ permeability by temperature, as dependence of motility on Na+-gradient was observed. The transcriptional induction of genes related to the synthesis and transport of siderophores correlated with a higher siderophore production and intracellular iron content only at low salinity. An excess of iron increased hydroxyEctoine accumulation by 20% at high salinity. Conversely, it reduced the intracellular content of Ectoines by 50% at high salinity plus high temperature. These findings support the relevance of iron homeostasis for osmoadaptation, thermoadaptation and accumulation of Ectoines, in C. salexigens.
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contribution of rpos to metabolic efficiency and Ectoines synthesis during the osmo and heat stress response in the halophilic bacterium chromohalobacter salexigens
Environmental Microbiology Reports, 2015Co-Authors: Manuel Salvador, Montserrat Argandona, Jose M Pastor, Vicente Bernal, Manuel Canovas, Laszlo N Csonka, Joaquin J Nieto, Carmen VargasAbstract:Chromohalobacter salexigens is a halophilic γ-proteobacterium that responds to osmotic and heat stresses by accumulating Ectoine and hydroxyEctoine respectively. Evolution has optimized its metabolism to support high production of Ectoines. We analysed the effect of an rpoS mutation in C. salexigens metabolism and Ectoines synthesis. In long-term adapted cells, the rpoS strain was osmosensitive but not thermosensitive and showed unaltered Ectoines content, suggesting that RpoS regulates Ectoine(s)-independent osmoadaptive mechanisms. RpoS is involved in the regulation of C. salexigens metabolic adaptation to stress, as early steps of glucose oxidation through the Entner-Doudoroff pathway were deregulated in the rpoS mutant, leading to improved metabolic efficiency at low salinity. Moreover, a reduced pyruvate (but not acetate) overflow was displayed by the rpoS strain at low salt, probably linked to a slowdown in gluconate production and/or subsequent metabolism. Interestingly, RpoS does not seem to be the main regulator triggering the immediate transcriptional response of Ectoine synthesis to osmotic or thermal upshifts. However, it contributed to the expression of the ect genes in cells previously adapted to low or high salinity.
Montserrat Argandona - One of the best experts on this subject based on the ideXlab platform.
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New insights into hydroxyEctoine synthesis and its transcriptional regulation in the broad‐salt growing halophilic bacterium Chromohalobacter salexigens
'Wiley', 2021Co-Authors: Montserrat Argandona, Joaquin J Nieto, Francine Piubeli, Mercedes Reina‐bueno, Carmen VargasAbstract:Summary Elucidating the mechanisms controlling the synthesis of hydroxyEctoine is important to design novel genetic engineering strategies for optimizing the production of this biotechnologically relevant compatible solute. The genome of the halophilic bacterium Chromohalobacter salexigens carries two Ectoine hydroxylase genes, namely ectD and ectE, whose encoded proteins share the characteristic consensus motif of Ectoine hydroxylases but showed only a 51.9% identity between them. In this work, we have shown that ectE encodes a secondary functional Ectoine hydroxylase and that the hydroxyEctoine synthesis mediated by this enzyme contributes to C.␣salexigens thermoprotection. The evolutionary pattern of EctD and EctE and related proteins suggests that they may have arisen from duplication of an ancestral gene preceding the directional divergence that gave origin to the orders Oceanospirillales and Alteromonadales. Osmoregulated expression of ectD at exponential phase, as well as the thermoregulated expression of ectD at the stationary phase, seemed to be dependent on the general stress factor RpoS. In contrast, expression of ectE was always RpoS‐dependent regardless of the growth phase and osmotic or heat stress conditions tested. The data presented here suggest that the AraC‐GlxA‐like EctZ transcriptional regulator, whose encoding gene lies upstream of ectD, plays a dual function under exponential growth as both a transcriptional activator of osmoregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main Ectoine hydroxylase EctD. Inactivation of ectZ resulted in a higher amount of the total Ectoines pool at the expenses of a higher accumulation of Ectoine, with maintenance of the hydroxyEctoine levels. In addition to the transcriptional control, our results suggest a strong post‐transcriptional regulation of hydroxyEctoine synthesis. Data on the accumulation of Ectoine and hydroxyEctoine in rpoS and ectZ strains pave the way for using these genetic backgrounds for metabolic engineering for hydroxyEctoine production
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Insights into metabolic osmoadaptation of the Ectoines-producer bacterium Chromohalobacter salexigens through a high-quality genome scale metabolic model
Microbial Cell Factories, 2018Co-Authors: Francine Piubeli, Manuel Salvador, Montserrat Argandona, Jose M Pastor, Vicente Bernal, Manuel Canovas, Joaquin J Nieto, Carmen VargasAbstract:Background The halophilic bacterium Chromohalobacter salexigens is a natural producer of Ectoines, compatible solutes with current and potential biotechnological applications. As production of Ectoines is an osmoregulated process that draws away TCA intermediates, bacterial metabolism needs to be adapted to cope with salinity changes. To explore and use C. salexigens as cell factory for Ectoine(s) production, a comprehensive knowledge at the systems level of its metabolism is essential. For this purpose, the construction of a robust and high-quality genome-based metabolic model of C. salexigens was approached. Results We generated and validated a high quality genome-based C. salexigens metabolic model ( i FP764). This comprised an exhaustive reconstruction process based on experimental information, analysis of genome sequence, manual re-annotation of metabolic genes, and in-depth refinement. The model included three compartments (periplasmic, cytoplasmic and external medium), and two salinity-specific biomass compositions, partially based on experimental results from C. salexigens. Using previous metabolic data as constraints, the metabolic model allowed us to simulate and analyse the metabolic osmoadaptation of C. salexigens under conditions for low and high production of Ectoines. The i FP764 model was able to reproduce the major metabolic features of C. salexigens . Flux Balance Analysis (FBA) and Monte Carlo Random sampling analysis showed salinity-specific essential metabolic genes and different distribution of fluxes and variation in the patterns of correlation of reaction sets belonging to central C and N metabolism, in response to salinity. Some of them were related to bioenergetics or production of reducing equivalents, and probably related to demand for Ectoines. Ectoines metabolic reactions were distributed according to its correlation in four modules. Interestingly, the four modules were independent both at low and high salinity conditions, as they did not correlate to each other, and they were not correlated with other subsystems. Conclusions Our validated model is one of the most complete curated networks of halophilic bacteria. It is a powerful tool to simulate and explore C. salexigens metabolism at low and high salinity conditions, driving to low and high production of Ectoines. In addition, it can be useful to optimize the metabolism of other halophilic bacteria for metabolite production.
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Presentation_1_Quantitative RNA-seq Analysis Unveils Osmotic and Thermal Adaptation Mechanisms Relevant for Ectoine Production in Chromohalobacter salexigens.PDF
2018Co-Authors: Manuel Salvador, Montserrat Argandona, Laszlo N Csonka, Joaquin J Nieto, Emilia Naranjo, Francine Piubeli, Carmen VargasAbstract:Quantitative RNA sequencing (RNA-seq) and the complementary phenotypic assays were implemented to investigate the transcriptional responses of Chromohalobacter salexigens to osmotic and heat stress. These conditions trigger the synthesis of Ectoine and hydroxyEctoine, two compatible solutes of biotechnological interest. Our findings revealed that both stresses make a significant impact on C. salexigens global physiology. Apart from compatible solute metabolism, the most relevant adaptation mechanisms were related to “oxidative- and protein-folding- stress responses,” “modulation of respiratory chain and related components,” and “ion homeostasis.” A general salt-dependent induction of genes related to the metabolism of Ectoines, as well as repression of Ectoine degradation genes by temperature, was observed. Different oxidative stress response mechanisms, secondary or primary, were induced at low and high salinity, respectively, and repressed by temperature. A higher sensitivity to H2O2 was observed at high salinity, regardless of temperature. Low salinity induced genes involved in “protein-folding-stress response,” suggesting disturbance of protein homeostasis. Transcriptional shift of genes encoding three types of respiratory NADH dehydrogenases, ATP synthase, quinone pool, Na+/H+ antiporters, and sodium-solute symporters, was observed depending on salinity and temperature, suggesting modulation of the components of the respiratory chain and additional systems involved in the generation of H+ and/or Na+ gradients. Remarkably, the Na+ intracellular content remained constant regardless of salinity and temperature. Disturbance of Na+- and H+-gradients with specific ionophores suggested that both gradients influence Ectoine production, but with differences depending on the solute, salinity, and temperature conditions. Flagellum genes were strongly induced by salinity, and further induced by temperature. However, salt-induced cell motility was reduced at high temperature, possibly caused by an alteration of Na+ permeability by temperature, as dependence of motility on Na+-gradient was observed. The transcriptional induction of genes related to the synthesis and transport of siderophores correlated with a higher siderophore production and intracellular iron content only at low salinity. An excess of iron increased hydroxyEctoine accumulation by 20% at high salinity. Conversely, it reduced the intracellular content of Ectoines by 50% at high salinity plus high temperature. These findings support the relevance of iron homeostasis for osmoadaptation, thermoadaptation and accumulation of Ectoines, in C. salexigens.
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Quantitative RNA-seq Analysis Unveils Osmotic and Thermal Adaptation Mechanisms Relevant for Ectoine Production in Chromohalobacter salexigens
Frontiers Media S.A., 2018Co-Authors: Manuel Salvador, Montserrat Argandona, Laszlo N Csonka, Joaquin J Nieto, Emilia Naranjo, Francine Piubeli, Carmen VargasAbstract:Quantitative RNA sequencing (RNA-seq) and the complementary phenotypic assays were implemented to investigate the transcriptional responses of Chromohalobacter salexigens to osmotic and heat stress. These conditions trigger the synthesis of Ectoine and hydroxyEctoine, two compatible solutes of biotechnological interest. Our findings revealed that both stresses make a significant impact on C. salexigens global physiology. Apart from compatible solute metabolism, the most relevant adaptation mechanisms were related to “oxidative- and protein-folding- stress responses,” “modulation of respiratory chain and related components,” and “ion homeostasis.” A general salt-dependent induction of genes related to the metabolism of Ectoines, as well as repression of Ectoine degradation genes by temperature, was observed. Different oxidative stress response mechanisms, secondary or primary, were induced at low and high salinity, respectively, and repressed by temperature. A higher sensitivity to H2O2 was observed at high salinity, regardless of temperature. Low salinity induced genes involved in “protein-folding-stress response,” suggesting disturbance of protein homeostasis. Transcriptional shift of genes encoding three types of respiratory NADH dehydrogenases, ATP synthase, quinone pool, Na+/H+ antiporters, and sodium-solute symporters, was observed depending on salinity and temperature, suggesting modulation of the components of the respiratory chain and additional systems involved in the generation of H+ and/or Na+ gradients. Remarkably, the Na+ intracellular content remained constant regardless of salinity and temperature. Disturbance of Na+- and H+-gradients with specific ionophores suggested that both gradients influence Ectoine production, but with differences depending on the solute, salinity, and temperature conditions. Flagellum genes were strongly induced by salinity, and further induced by temperature. However, salt-induced cell motility was reduced at high temperature, possibly caused by an alteration of Na+ permeability by temperature, as dependence of motility on Na+-gradient was observed. The transcriptional induction of genes related to the synthesis and transport of siderophores correlated with a higher siderophore production and intracellular iron content only at low salinity. An excess of iron increased hydroxyEctoine accumulation by 20% at high salinity. Conversely, it reduced the intracellular content of Ectoines by 50% at high salinity plus high temperature. These findings support the relevance of iron homeostasis for osmoadaptation, thermoadaptation and accumulation of Ectoines, in C. salexigens
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contribution of rpos to metabolic efficiency and Ectoines synthesis during the osmo and heat stress response in the halophilic bacterium chromohalobacter salexigens
Environmental Microbiology Reports, 2015Co-Authors: Manuel Salvador, Montserrat Argandona, Jose M Pastor, Vicente Bernal, Manuel Canovas, Laszlo N Csonka, Joaquin J Nieto, Carmen VargasAbstract:Chromohalobacter salexigens is a halophilic γ-proteobacterium that responds to osmotic and heat stresses by accumulating Ectoine and hydroxyEctoine respectively. Evolution has optimized its metabolism to support high production of Ectoines. We analysed the effect of an rpoS mutation in C. salexigens metabolism and Ectoines synthesis. In long-term adapted cells, the rpoS strain was osmosensitive but not thermosensitive and showed unaltered Ectoines content, suggesting that RpoS regulates Ectoine(s)-independent osmoadaptive mechanisms. RpoS is involved in the regulation of C. salexigens metabolic adaptation to stress, as early steps of glucose oxidation through the Entner-Doudoroff pathway were deregulated in the rpoS mutant, leading to improved metabolic efficiency at low salinity. Moreover, a reduced pyruvate (but not acetate) overflow was displayed by the rpoS strain at low salt, probably linked to a slowdown in gluconate production and/or subsequent metabolism. Interestingly, RpoS does not seem to be the main regulator triggering the immediate transcriptional response of Ectoine synthesis to osmotic or thermal upshifts. However, it contributed to the expression of the ect genes in cells previously adapted to low or high salinity.
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contribution of mechanosensitive channels to osmoadaptation and Ectoine excretion in halomonas elongata
Extremophiles, 2020Co-Authors: Jasmina Vandrich, Friedhelm Pfeiffer, Gabriela Alfaroespinoza, Hans Joerg KunteAbstract: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 down-regulation 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.
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a blueprint of Ectoine metabolism from the genome of the industrial producer halomonas elongata dsm 2581t
Environmental Microbiology, 2011Co-Authors: Karin Schwibbert, Georg Lentzen, Irina Bagyan, Alberto Marinsanguino, Gabriele Heidrich, Harald Seitz, M Rampp, Stephan C Schuster, Hanspeter Klenk, Friedhelm PfeifferAbstract:The halophilic γ-proteobacterium Halomonas elongata DSM 2581T thrives at high salinity by synthesizing and accumulating the compatible solute Ectoine. Ectoine levels are highly regulated according to external salt levels but the overall picture of its metabolism and control is not well understood. Apart from its critical role in cell adaptation to halophilic environments, Ectoine can be used as a stabilizer for enzymes and as a cell protectant in skin and health care applications and is thus produced annually on a scale of tons in an industrial process using H. elongata as producer strain. This paper presents the complete genome sequence of H. elongata (4 061 296 bp) and includes experiments and analysis identifying and characterizing the entire Ectoine metabolism, including a newly discovered pathway for Ectoine degradation and its cyclic connection to Ectoine synthesis. The degradation of Ectoine (doe) proceeds via hydrolysis of Ectoine (DoeA) to Nα-acetyl-l-2,4-diaminobutyric acid, followed by deacetylation to diaminobutyric acid (DoeB). In H. elongata, diaminobutyric acid can either flow off to aspartate or re-enter the Ectoine synthesis pathway, forming a cycle of Ectoine synthesis and degradation. Genome comparison revealed that the Ectoine degradation pathway exists predominantly in non-halophilic bacteria unable to synthesize Ectoine. Based on the resulting genetic and biochemical data, a metabolic flux model of Ectoine metabolism was derived that can be used to understand the way H. elongata survives under varying salt stresses and that provides a basis for a model-driven improvement of industrial Ectoine production.
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a blueprint of Ectoine metabolism from the genome of the industrial producer halomonas elongata dsm 2581t
Environmental Microbiology, 2011Co-Authors: Karin Schwibbert, Georg Lentzen, Irina Bagyan, Alberto Marinsanguino, Gabriele Heidrich, Harald Seitz, M Rampp, Stephan C Schuster, Hanspeter Klenk, Friedhelm PfeifferAbstract:The halophilic γ-proteobacterium Halomonas elongata DSM 2581T thrives at high salinity by synthesizing and accumulating the compatible solute Ectoine. Ectoine levels are highly regulated according to external salt levels but the overall picture of its metabolism and control is not well understood. Apart from its critical role in cell adaptation to halophilic environments, Ectoine can be used as a stabilizer for enzymes and as a cell protectant in skin and health care applications and is thus produced annually on a scale of tons in an industrial process using H. elongata as producer strain. This paper presents the complete genome sequence of H. elongata (4 061 296 bp) and includes experiments and analysis identifying and characterizing the entire Ectoine metabolism, including a newly discovered pathway for Ectoine degradation and its cyclic connection to Ectoine synthesis. The degradation of Ectoine (doe) proceeds via hydrolysis of Ectoine (DoeA) to Nα-acetyl-l-2,4-diaminobutyric acid, followed by deacetylation to diaminobutyric acid (DoeB). In H. elongata, diaminobutyric acid can either flow off to aspartate or re-enter the Ectoine synthesis pathway, forming a cycle of Ectoine synthesis and degradation. Genome comparison revealed that the Ectoine degradation pathway exists predominantly in non-halophilic bacteria unable to synthesize Ectoine. Based on the resulting genetic and biochemical data, a metabolic flux model of Ectoine metabolism was derived that can be used to understand the way H. elongata survives under varying salt stresses and that provides a basis for a model-driven improvement of industrial Ectoine production.
