Computational Investigation of BMAA and Its Carbamate Adducts as Potential GluR2 Modulators.

Beta-N-methylamino-l-alanine (BMAA) is a potential neurotoxic nonprotein amino acid, which can reach the human body through the food chain. When BMAA interacts with bicarbonate in the human body, carbamate adducts are produced, which share a high structural similarity with the neurotransmitter glutamate. It is believed that BMAA and its l-carbamate adducts bind in the glutamate binding site of ionotropic glutamate receptor 2 (GluR2). Chronic exposure to BMAA and its adducts could cause neurological illness such as neurodegenerative diseases. However, the mechanism of BMAA action and its carbamate adducts bound to GluR2 has not yet been elucidated. Here, we investigate the binding modes and the affinity of BMAA and its carbamate adducts to GluR2 in comparison to the natural agonist, glutamate, to understand whether these can act as GluR2 modulators. Initially, we perform molecular dynamics simulations of BMAA and its carbamate adducts bound to GluR2 to examine the stability of the ligands in the S1/S2 ligand-binding core of the receptor. In addition, we utilize alchemical free energy calculations to compute the difference in the free energy of binding of the beta-carbamate adduct of BMAA to GluR2 compared to that of glutamate. Our findings indicate that carbamate adducts of BMAA and glutamate remain stable in the binding site of the GluR2 compared to BMAA. Additionally, alchemical free energy results reveal that glutamate and the beta-carbamate adduct of BMAA have comparable binding affinity to the GluR2. These results provide a rationale that BMAA carbamate adducts may be, in fact, the modulators of GluR2 and not BMAA itself.

How does the neurotoxin ß-N-methylamino-L-alanine exist in biological matrices and cause toxicity?

The neurotoxin ß-N-methylamino-L-alanine (BMAA) has been deemed as a risk factor for some neurodegenerative diseases such as amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC). This possible link has been proved in some primate models and cell cultures with the appearance that BMAA exposure can cause excitotoxicity, formation of protein aggregates, and/or oxidative stress. The neurotoxin BMAA extensively exists in the environment and can be transferred through the food web to human beings. In this review, the occurrence, toxicological mechanisms, and characteristics of BMAA were comprehensively summarized, and proteins and peptides were speculated as its possible binding substances in biological matrices. It is difficult to compare the published data from previous studies due to the inconsistent analytical methods and components of BMAA. The binding characteristics of BMAA should be focused on to improve our understanding of its health risk to human health in the future.

Bacteria at Work - Experimental and Theoretical Studies Reveal the Catalytic Mechanism of Ectoine Synthase.

Ectoine synthase (EctC) catalyses the ultimate step of ectoine biosynthesis, a kosmotropic compound produced as compatible solute by many bacteria and some archaea or eukaryotes. EctC is an Fe2+-dependent homodimeric cytoplasmic protein. Using Mössbauer spectroscopy, molecular dynamics simulations and QM/MM calculations, we determined the most likely coordination number and geometry of the Fe2+ ion and proposed a mechanism of the EctC-catalysed reaction. Most notably, we show that apart from the three amino acids binding to the iron ion (Glu57, Tyr84 and His92), one water molecule and one hydroxide ion are required as additional ligands for the reaction to occur. They fill the first coordination sphere of the Fe2+-cofactor and act as critical proton donors and acceptors during the cyclization reaction.

Recent advances in production and applications of ectoine, a compatible solute of industrial relevance.

Extremophilic bacteria growing in saline ecosystems are potential producers of biotechnologically important products including compatible solutes. Ectoine/hydroxyectoine are two such solutes that protect cells and associated macromolecules from osmotic, heat, cold and UV stress without interfering with cellular functions. Since ectoine is a high value product, overviewing strategies for improving yields become relevant. Screening of natural isolates, use of inexpensive substrates and response surface methodology approaches have been used to improve bioprocess parameters. In addition, genome mining exercises can aid in identifying hitherto unreported microorganisms with a potential to produce ectoine that can be exploited in the future. Application wise, ectoine has various biotechnological (protein protectant, membrane modulator, DNA protectant, cryoprotective agent, wastewater treatment) and biomedical (dermatoprotectant and in overcoming respiratory and hypersensitivity diseases) uses. The review summarizes current updates on the potential of microorganisms in the production of this industrially relevant metabolite and its varied applications.

Strategies for the biological production of ectoine by using different chassis strains.

As an amino acid derivative and a typical compatible solute, ectoine can assist microorganisms in resisting high osmotic pressure. Own to its long-term moisturizing effects, ectoine shows extensive applications in cosmetics, medicine and other fields. With the rapid development of synthetic biology and fermentation engineering, many biological strategies have been developed to improve the ectoine production and simplify the production process. Currently, the microbial fermentation has been widely used for large scaling ectoine production. Accordingly, this review will introduce the metabolic pathway for ectoine synthesis and also comprehensively evaluate both wild-type and genetically modified strains for ectoine production. Furthermore, process parameters affecting the ectoine production efficiency and adoption of low cost substrates will be evaluated. Lastly, future prospects on the improvement of ectoine production will be proposed.

A UPLC-MS/MS method for quantification of ß-N-methylamino-L-alanine (BMAA) in Cycas sphaerica roxb. and its use in validating efficacy of a traditional BMAA removal method.

The presence of neurotoxin ß-N-Methylamino-L-alanine (BMAA) in the seeds of Cycas sphaerica is reported for first time. We developed a UPLC-MS/MS method for BMAA quantification by derivatizing with dansyl chloride. The method successfully differentiated L-BMAA from its structural isomer 2,4-diaminobutyric acid (DAB). The extracting mixture 0.1M TCA: ACN 4:1 v/v had a recovery level of >95%. The method is a high throughput sensitive chromatographic technique with 16.42 ng g-1 Limit of Quantification. BMAA was present in the endosperm of C. sphaerica, and was not detected in the leaves and pith. Washing of seeds in running cold water for 48 h reduced BMAA content by 86%. The local communities also treat the seeds under running cold water, but only for 24 h. The results of the study thus validated the traditional BMAA removal process through cold water treatment, but recommend for increase in the treatment period to 48 h or more.

Diamine Oxidase as a Therapeutic Enzyme: Study of Germination from Vegetal Sources and Investigation of the Presence of ß-N-Oxalyl-L-α,ß-diaminopropionic Acid (ß-ODAP) Using LC-MS/MS.

Vegetal diamine oxidase (vDAO), an enzyme proposed to relieve symptoms of histaminosis, shows better reactivity with histamine and aliphatic diamines, as well as higher enzymatic activity than DAO of animal origin. The objective of this study was to evaluate the enzyme activity of vDAO from germinating grains from Lathyrus sativus (grass pea) and Pisum sativum (pea), and to verify the presence of a neurotoxin, ß-N-Oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), in the crude extract obtained from their seedlings. A targeted liquid chromatography-multiple-reaction monitoring mass spectrometry method was developed and used to quantify ß-ODAP in the analysed extracts. An optimized sample preparation procedure, involving protein precipitation with acetonitrile followed by mixed-anion exchange solid-phase extraction, allowed for high sensitivity and good peak shape for ß-ODAP detection. The Lathyrus sativus extract exhibited the highest vDAO enzyme activity of the extracts, followed by the extract from pea cultivar Amarillo from the Crop Development Centre (CDC). The results have also shown that even though ß-ODAP was present in the crude extract from L. sativus, its content was far below the toxicity threshold (300 mg of ß-ODAP/kg body/day). CDC Amarillo showed 5000-fold less ß-ODAP than the undialysed L. sativus extract. It was concluded that both species can be considered as convenient sources of vDAO for potential therapeutic use.

Microbial conversion of carbon dioxide and hydrogen into the fine chemicals hydroxyectoine and ectoine.

This study explores a novel conversion of CO2 into the chemicals hydroxyectoine and ectoine, which are compounds with high retail values in the pharmaceutical industry. Firstly, 11 species of microbes able to use CO2 and H2 and that have the genes for ectoines synthesis (ectABCD) were identified through literature search and genomic mining. Laboratory tests were then conducted to ascertain the capacity of these microbes to produce ectoines from CO2. Results showed that the most promising bacteria for CO2 to ectoines bioconversion areHydrogenovibrio marinus, Rhodococcus opacus, and Hydrogenibacillus schlegelii.Upon salinity and H2/CO2/O2 ratio optimization,H. marinus accumulated 85 mg of ectoine g biomass-1. Interestingly, R.opacusand H. schlegelii mainly produced hydroxyectoine (53 and 62 mg g biomass-1), which has a higher commercial value. Overall, these results constitute the first proof of a novel valorization platform of CO2 and lay the foundation for a new economic niche aimed at CO2 recircularization into pharmaceuticals.

Degradation of cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) using ozone process: influencing factors and mechanism.

ß-N-methylamino-L-alanine (BMAA), which has been considered as an environmental factor that caused amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) or Alzheimer's disease, could be produced by a variety of genera cyanobacteria. BMAA is widely present in water sources contaminated by cyanobacteria and may threaten human health through drinking water. Although oxidants commonly used in drinking water plants such as chlorine, ozone, hydrogen peroxide, and hydroxyl radicals have been shown to effectively degrade BMAA, there are limited studies on the mechanism of BMAA degradation by different oxidants, especially ozone. This work systematically explored the effectiveness of BMAA ozonation degradation, investigated the effect of the operating parameters on the effectiveness of degradation, and speculated on the pathways of BMAA decomposition. The results showed that BMAA could be quickly eliminated by ozone, and the removal rates of BMAA were nearly 100% in pure water, but the removal rates were reduced in actual water. BMAA was primarily degraded by direct oxidation of ozone molecules in acidic and near-neutral conditions, and indirect oxidation of •OH accounted for the main part under strong alkaline conditions. The pH value had a significant effect on the decomposition of BMAA, and the degradation rate of BMAA was fastest at near-neutral pH value. The degradation rates of TOC were significantly lower than that of BMAA, indicating that by-products were generated during the degradation process. Three by-products ([M-H]+ = 105, 90, and 88) were identified by UPLC-MS/MS, and the degradation pathways of BMAA were proposed. The production of by-products was attributed to the fracture of the C-N bonds. This work is helpful for the in-depth understanding on the mechanism and demonstration of the feasibility of the oxidation of BMAA by the ozone process. HIGHLIGHTS: • The reaction of ozonation BMAA was easy to occur. • The degradation rate was fast under near-neutral conditions. • Direct oxidation under neural conditions was the main pathway for ozone degradation of BMAA. • Three products were detected, and the reaction path was inferred.

Optimization of ectoine production from Nesterenkonia xinjiangensis and one-step ectoine purification.

In the current study, the optimization of ectoine production byNesterenkonia xinjiangensisand purification of ectoine from the bacterial cell extract were performed for the first time. Various carbon sources (glucose, sucrose, maltose, lactose, mannitol, and xylose) and nitrogen sources (ammonium nitrate, ammonium phosphate, ammonium chloride, ammonium oxalate, ammonium sulphate, and ammonium acetate), were used to optimize ectoine production. Subsequently, the effects of salt, pH and, concentrations of carbon and nitrogen source on ectoine production were optimized by response surface methodology (RSM). Ultimately, high pure (over 99%) and yield (98%) of ectoine from bacterial cells extracted was obtained by a single-step process using cation exchange chromatography. This study provides information that higher ectoine production can be achieved from this bacterial isolate by optimizing the factors influencing ectoine production and thus can be used as a new and alternative ectoine producer.

Extraction of Non-Protein Amino Acids from Cyanobacteria for Liquid Chromatography-Tandem Mass Spectrometry Analysis.

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of NPAAs can provide crucial information about cellular uptake and/or function, metabolic pathways, and potential toxicity. ß-methylamino-L-alanine (BMAA) is a neurotoxic NPAA produced by various algae species and is associated with an increased risk for neurodegenerative diseases, which has led to significant research interest. There are numerous ways to extract AAs for analysis, with liquid chromatography-tandem mass spectrometry being the most common, requiring protein precipitation followed by acid hydrolysis of the protein pellet. Studies on the presence of BMAA in algal species provide contradictory results, with the use of unvalidated sample preparation/extraction and analysis a primary cause. Like most NPAAs, protein precipitation in 10% aqueous TCA and hydrolysis with fuming HCl is the most appropriate form of extraction for BMAA and its isomers aminoethylglycine (AEG) and 2,4-diaminobutyric acid (2,4-DAB). The present protocol describes the steps in a validated NPAA extraction method commonly used in research and teaching laboratories.

The Changes in Cyanobacterial Concentration of ß-Methylamino-L-Alanine during a Bloom Event.

ß-N-methylamino L-alanine (BMAA) is a neurotoxin linked to high incidences of neurodegenerative disease. The toxin, along with two of its common isomers, 2,4-diaminobuytric acid (2,4-DAB) and N-(2-aminoethyl)glycine (AEG), is produced by multiple genera of cyanobacteria worldwide. Whilst there are many reports of locations and species of cyanobacteria associated with the production of BMAA during a bloom, there is a lack of information tracking changes in concentration across a single bloom event. This study aimed to measure the concentrations of BMAA and its isomers through the progression and end of a cyanobacteria bloom event using liquid chromatography-triple quadrupole-mass spectrometry. BMAA was detected in all samples analysed, with a decreasing trend observed as the bloom progressed. BMAA's isomers were also detected in all samples, however, they did not follow the same decreasing pattern. This study highlights the potential for current sampling protocols that measure a single time point as representative of a bloom's overall toxin content to underestimate BMAA concentration during a bloom event.

Influence of operational conditions on the performance of biogas bioconversion into ectoines in pilot bubble column bioreactors.

The application of biogas as a low-priced substrate for the production of ectoines constitutes an opportunity to decrease their production costs and to enhance the viability of anaerobic digestion. The influence of operational conditions on CH4-biogas biodegradation and on ectoines production yields was assessed in continuous pilot bubble column bioreactors. The rise in biomass concentration from 1 to 3 g L-1 resulted in a decrease in the specific ectoine content from 42 ± 8 to 30 ± 4 mgectoine gVSS-1. The concentration of Cu2+ and Mg2+ did not impact process performance, while the use of ammonium as N source resulted in low CH4 biodegradation and ectoine yields (13 ± 7 mgectoine gVSS-1). The increase in CH4 content from 4.5 to 9 %v·v-1 enhanced CH4 removal efficiency. Process operation at NaCl concentrations of 3 %w·w-1 instead of 6 %w·w-1 decreased the ectoine yield to 17 mgectoine gVSS-1. Finally, Methylomicrobiumburyatense was identified as the dominant species.

Genomic insights into the biosynthesis and physiology of the cyanobacterial neurotoxin 3-N-methyl-2,3-diaminopropanoic acid (BMAA).

Cyanobacteria are an ancient clade of photosynthetic prokaryotes, present in many habitats throughout the world, including water resources. They can present health hazards to humans and animals due to the production of a wide range of toxins (cyanotoxins), including the diaminoacid neurotoxin, 3-N-methyl-2,3-diaminopropanoic acid (ß-N-methylaminoalanine, BMAA). Knowledge of the biosynthetic pathway for BMAA, and its role in cyanobacteria, is lacking. Present evidence suggests that BMAA is derived by 3-N methylation of 2,3-diaminopropanoic acid (2,3-DAP) and, although the latter has never been reported in cyanobacteria, there are multiple pathways to its biosynthesis known in other bacteria and in plants. Here, we used bioinformatics analyses to investigate hypotheses concerning 2,3-DAP and BMAA biosynthesis in cyanobacteria. We assessed the potential presence or absence of each enzyme in candidate biosynthetic routes known in Albizia julibrissin, Lathyrus sativus seedlings, Streptomyces, Clostridium, Staphylococcus aureus, Pantoea agglomerans, and Paenibacillus larvae, in 130 cyanobacterial genomes using sequence alignment, profile hidden Markov models, substrate specificity/active site identification and the reconstruction of gene phylogenies. Most enzymes involved in pathways leading to 2,3-DAP in other species were not found in the cyanobacteria analysed. Nevertheless, two species appear to have the genes sbnA and sbnB, responsible for forming the 2,3-DAP constituent in staphyloferrin B, a siderophore from Staphylococcus aureus. It is currently undetermined whether these species are also capable of biosynthesising BMAA. It is possible that, in some cyanobacteria, the formation of 2,3-DAP and/or BMAA is associated with environmental iron-scavenging. The pam gene cluster, responsible for the biosynthesis of the BMAA-containing peptide, paenilamicin, so far appears to be restricted to Paenibacillus larvae. It was not detected in any of the cyanobacterial genomes analysed, nor was it found in 93 other Paenibacillus genomes or in the genomes of two BMAA-producing diatom species. We hypothesise that the presence, in some cyanobacterial species, of the enzymes 2,3-diaminopropionate ammonia-lyase (DAPAL) and reactive intermediate deaminase A (RidA) may explain the failure to detect 2,3-DAP in analytical studies. Overall, the taxonomic distribution of 2,3-DAP and BMAA in cyanobacteria is unclear; there may be multiple and additional routes, and roles, for the biosynthesis of 2,3-DAP and BMAA in these organisms.

[Advances in the microbial production of the compatible solute ectoine: a review].

Ectoine is an amino acid derivative and an important natural product in halophilic microorganisms. It plays an important role in protecting cells and stabilizing biological macromolecules, and can be widely used in biomedical fields such as drug preparation adjuvants, organ transplantation and preservation, skin wound repair and cosmetics. Due to the medical value and commercial market demand of ectoine, this article summarized the recent advances in the microbial production of ectoine, including the mutation and breeding of hyper-producing strains, construction of genetically and metabolically engineered strains, optimization of fermentation processes, and extraction and purification processes. The application of multi-omics technologies and computational biology to develop an ectoine producing cell factory was prospected, with the aim to provide a reference for ectoine overproduction.

Enhancing ectoine production by recombinant Escherichia coli through step-wise fermentation optimization strategy based on kinetic analysis.

In this study, the recombinant ectoine-producing Escherichia coli ET01 was constructed by introducing the ectABC operon from Halomonas venusta ZH. To further improve ectoine production, the regulation of the fermentation process was systematically investigated. First, the effects of the initial glucose concentrations and glucose feeding mode on ectoine production were analyzed. Using a combination of pH-feedback feeding and glucose-controlled feeding, the ectoine titer reached 25.5 g/L, representing an 8.8-fold increase over standard batch culture. Then, the effects of dissolved oxygen (DO) levels (50, 40, 30, or 20%) on ectoine production were studied, and a DO control strategy was developed based on the fermentation kinetics. When the final optimized two-stage fermentation strategy was used, the ectoine titer reached 47.8 g/L, which was the highest level of ectoine produced by E. coli fermentation. The fermentation regulation strategy developed in this study might be useful for scaling up the commercial production of ectoine.

On the Differential Roles of Mg2+, Zn2+, and Cu2+ in the Equilibrium of ß-N-Methyl-Amino-L-Alanine (BMAA) and its Carbamates.

ß-N-methyl-amino-L-alanine (BMAA) in the presence of bicarbonate (HCO3-) undergoes structural modifications generating two carbamate species, α-carbamate and ß-carbamate forms of BMAA. The chemical structure of BMAA and BMAA-carbamate adducts strongly suggest they may interact with divalent metal ions. The ability of BMAA to cross the blood-brain barrier and possibly interact with divalent metal ions may augment the neurotoxicity of these molecules. To understand the effects of divalent metal ions (Mg2+, Zn2+, and Cu2+) on the overall dynamic equilibrium between BMAA and its carbamate adducts, a systematic study using nuclear magnetic resonance (NMR) is presented. The chemical equilibria between BMAA, its carbamate adducts, and each of the divalent ions were studied using two-dimensional chemical exchange spectroscopy (EXSY). The NMR results demonstrate that BMAA preferentially interacts with Zn2+ and Cu2+, causing an overall reduction in the production of carbamate species by altering the dynamic equilibria. The NMR-based spectral changes due to the BMAA interaction with Cu2+ is more drastic than with the Zn2+, under the same stoichiometric ratios of BMAA and the individual divalent ions. However, the presence of Mg2+ does not significantly alter the dynamic equilibria between BMAA and its carbamate adducts. The NMR-based results are further validated using circular dichroism (CD) spectroscopy, observing the n ➔ π interaction in the complex formation of BMAA and the divalent metal ions, with additional verification of the interaction with Cu2+ using UV-Vis spectroscopy. Our results demonstrate that BMAA differentially interacts with divalent metal ions (Mg2+ < Zn2+ < Cu2+), and thus alters the rate of formation of carbamate products. The equilibria between BMAA, the bicarbonate ions, and the divalent metal ions may alter the total population of a specific form of BMAA-ion complex at physiological conditions and, therefore, add a level of complexity of the mechanisms by which BMAA acts as a neurotoxin.

A Single Laboratory Validation for the Analysis of Underivatized ß-N-Methylamino-L-Alanine (BMAA).

ß-N-Methylamino-L-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria that can accumulate in ecosystems and food webs. Human exposure to cyanobacterial and algal blooms may be a risk factor for neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. Analytical chemists have struggled to find reliable methods for BMAA analysis in complex sample matrices. Analysis of BMAA is complicated by at least 3 naturally occurring isomers: N-(2-aminoethyl)glycine (AEG), 2,4-diaminobutyric acid (DAB), and ß-aminomethyl-L-alanine (BAMA). More than 350 publications have reported detection and quantification of BMAA and its isomers, but varying results have led to controversy in the literature. The objective of this study was to perform a single laboratory validation (SLV) of a frequently published method for BMAA analysis using a ZIC-HILIC column. We investigated the selectivity, linearity, accuracy, precision, and sensitivity of the method and our data show that this HILIC method fails many of the criteria for a validated method. The method fails the criterion for selectivity as the chromatography does not separate BMAA from its isomer BAMA. Sensitivity of the method greatly decreased over the experimental period and it demonstrated a higher limit of detection (LOD) (7.5 pg on column) and a higher lower limit of quantification (LLOQ) (30 pg on column) than other published validated methods. The method demonstrated poor precision of repeated injections of standards of BMAA with % relative standard deviation (%RSD) values that ranged from 37 to 107% while HorRat values for BMAA had a fail rate of 80% and BAMA had a fail rate of 73%. No HorRat values between 0.5 and 2 were found for repeated injections of standards of AEG and DAB. Recovery of 13C3,15N2-BMAA in a cyanobacterial matrix was < 10% in experiments and we were also unable to accurately detect other protein amino acids including methionine, cysteine, or alanine, indicating matrix effects. The results of this study demonstrate that the ZIC-HILIC column is not fit for purpose for the analysis of BMAA in cyanobacterial matrices and further provides explanations for the high level of negative results reported by researchers using this method.

The ups and downs of ectoine: structural enzymology of a major microbial stress protectant and versatile nutrient.

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.

Halotolerance mechanisms of the methanotroph Methylomicrobium alcaliphilum.

Methylomicrobium alcaliphilum is an alkaliphilic and halotolerant methanotroph. The physiological responses of M. alcaliphilum to high NaCl concentrations, were studied using RNA sequencing and metabolic modeling. This study revealed that M. alcaliphilum possesses an unusual respiratory chain, in which complex I is replaced by a Na+ extruding NQR complex (highly upregulated under high salinity conditions) and a Na+ driven adenosine triphosphate (ATP) synthase coexists with a conventional H+ driven ATP synthase. A thermodynamic and metabolic model showing the interplay between these different components is presented. Ectoine is the main osmoprotector used by the cells. Ectoine synthesis is activated by the transcription of an ect operon that contains five genes, including the ectoine hydroxylase coding ectD gene. Enzymatic tests revealed that the product of ectD does not have catalytic activity. A new Genome Scale Metabolic Model for M. alcaliphilum revealed a higher flux in the oxidative branch of the pentose phosphate pathway leading to NADPH production and contributing to resistance to oxidative stress.