Characterization of the ectoine biosynthesis genes of haloalkalotolerant obligate methanotroph “Methylomicrobium alcaliphilum 20Z”

The genes involved in biosynthesis of the major compatible solute ectoine (1,4,5,6-tetrahydro-2-methylpyrimidine carboxylic acid) in halotolerant obligate methanotroph “Methylomicrobium alcaliphilum 20Z” were studied. The complete nucleotide sequences of the structural genes encoding l-aspartokinase (Ask), l-2,4-diaminobutyric acid transaminase (EctB), l-2,4-diaminobutyric acid acetyltransferase (EctA), and l-ectoine synthase (EctC) were defined and shown to be transcribed as a single operon ectABCask. Phylogenetic analysis revealed high sequence identities (34–63%) of the Ect proteins to those from halophilic heterotrophs with the highest amino acid identities being to Vibrio cholerae enzymes. The chromosomal DNA fragment from “M. alcaliphilum 20Z” containing ectABC genes and putative promoter region was expressed in Escherichia coli. Recombinant cells could grow in the presence of 4% NaCl and synthesize ectoine. The data obtained suggested that despite the ectoine biosynthesis pathway being evolutionary well conserved with respect to the genes and enzymes involved, some differences in their organization and regulation could occur in various halophilic bacteria.Dedicated to the 70th birthday of Professor Gerhard Gottschalk who inspired our studies on methylotrophic haloalkaliphiles.     

Construction and characterization of an NaCl-sensitive mutant of Halomonas elongata impaired in ectoine biosynthesis

Using transposon mutagenesis we generated a salt-sensitive mutant of the halophilic eubacterium Halomonas elongata impaired in the biosynthesis of the compatible solute ectoine. HPLC determinations of the cytoplasmic solute content showed the accumulation of a biosynthetic precursor of ectoine, l-2,4-diaminobutyric acid. Ectoine and hydroxyectoine were not detectable. This mutant failed to grow in minimal medium with NaCl concentrations exceeding 4%. However, when supplemented with organic osmolytes, the ability to grow in high-salinity medium (15% and higher) was regained. We cloned and sequenced the regions flanking the transposon insertion in the H. elongata chromosome. Sequence comparisons with known proteins revealed significant similarity of the mutated gene to the l-2,4-diaminobutyric acid acetyltransferase from the ectoine biosynthetic pathway in Marinococcus halophilus. Analysis of a PCR product demonstrated that the ectoine biosynthetic genes (ectABC) follow the same order as in M. halophilus.     

Membrane fluidity of halophilic ectoine-secreting bacteria related to osmotic and thermal treatment

In response to sudden decrease in osmotic pressure, halophilic microorganisms secrete their accumulated osmolytes. This specific stress response, combined with physiochemical responses to the altered environment, influence the membrane properties and integrity of cells, with consequent effects on growth and yields in bioprocesses, such as bacterial milking. The aim of this study was to investigate changes in membrane fluidity and integrity induced by environmental stress in ectoine-secreting organisms. The halophilic ectoine-producing strains Alkalibacillus haloalkaliphilus and Chromohalobacter salexigens were treated hypo- and hyper-osmotically at several temperatures. The steady-state anisotropy of fluorescently labeled cells was measured, and membrane integrity assessed by flow cytometry and ectoine distribution. Strong osmotic downshocks slightly increased the fluidity of the bacterial membranes. As the temperature increased, the increasing membrane fluidity encouraged more ectoine release under the same osmotic shock conditions. On the other hand, combined shock treatments increased the number of disintegrated cells. From the ectoine release and membrane integrity measurements under coupled thermal and osmotic shock conditions, we could optimize the secretion conditions for both bacteria.     

Continuous Synthesis and Excretion of the Compatible Solute Ectoine by a Transgenic, Nonhalophilic Bacterium

The compatible solute 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) acts in microorganisms as an osmotic counterweight against halostress and has attracted commercial attention as a protecting agent. Its production and application are restricted by the drawbacks of the discontinuous harvesting procedure involving salt shocks, which reduces volumetric yield, increases reactor corrosion, and complicates downstream processing. In order to synthesize ectoine continuously in less-aggressive media, we introduced the ectoine genes ectABC of the halophilic bacterium Chromohalobacter salexigens into an Escherichia coli strain using the expression vector pASK-IBA7. Under the control of a tet promoter, the transgenic E. coli synthesized 6 g liter⁻¹ ectoine with a space-time yield of 40 mg liter⁻¹ h⁻¹, with the vast majority of the ectoine being excreted.     

Inhibitory effect of ectoine on melanogenesis in B16-F0 and A2058 melanoma cell lines

Skin injuries, congenital lesions, melasma, Addison's disease and many pigment abnormalities prompt us to search for an effective whitening agent. Ideal whitening agent is a natural compound that can inhibit melanogenesis and has no cytotoxic effects. In a previous study, we have developed an optimum method for the production and characterization of ectoine from a halophilic bacterium isolated from a salt environment in Taiwan was identified as Marinococcus sp. In the present study, we screened the whitening properties of the biosynthesized ectoine using mouse and human melanoma cell lines, B16-F0 and A2058. Here, we examined the cell viabilities of melanoma cells after ectoine treatment at various concentrations up to 500 μM. Also, we addressed the melanin synthesis of melanoma cells after treatment with ectoine. The inhibitory effects of ectoine on tyrosinase activity were assessed in both mushroom tyrosinase and cellular tyrosinase. Furthermore, we investigated the type of inhibition of mushroom tyrosinase using Lineweaver–Burk enzyme kinetic. The melanogenesis-related gene expression (tyrosinase, TRP1, TRP2 and MITF) and their protein secretion were determined by the assays of quantitative real-time PCR and western blots, respectively. Our results demonstrated that ectoine is a safe and effective whitening agent, inhibited melanin synthesis, reduced both mushroom tyrosinase and cellular tyrosinase, and had various inhibitory effects on the expressions of melanogenesis-related genes and secretion of proteins in mouse and human melanoma cell lines. Thus, we suggest that ectoine can serve as a useful and safe new agent in cosmetic and clinical applications.     

Genome analysis provides insights into crude oil degradation and biosurfactant production by extremely halotolerant Halomonas desertis G11 isolated from Chott El-Djerid salt-lake in Tunisian desert

Here, we report the genomic features and the bioremediation potential of Halomonas desertis G11, a new halophilic species which uses crude oil as a carbon and energy source and displays intrinsic resistance to salt stress conditions (optimum growth at 10% NaCl). G11 genome (3.96 Mb) had a mean GC content of 57.82%, 3622 coding sequences, 480 subsystems and 64 RNA genes. Annotation predicted 38 genes involved in osmotic stress including the biosynthesis of osmoprotectants glycine-betaine, ectoine and osmoregulated periplasmic glucans. Genome analysis revealed also the versatility of the strain for emulsifying crude oil and metabolizing hydrocarbons. The ability of G11 to degrade crude oil components and to secrete a glycolipid biosurfactant with satisfying properties was experimentally confirmed and validated. Our results help to explain the exceptional capacity of G11 to survive at extreme desertic conditions, and highlight the metabolic features of this organism that has biotechnological and ecological potentialities.     

Synthesis of osmoprotectants by halophilic and alkaliphilic methanotrophs

The¹H-NMR analysis of methanol extracts of halophilic and halotolerant alkaliphilic methanotrophs isolated from the soda lakes of Southern Transbaikal and Tuva showed that bacterial cells grown at an optimum salinity accumulated mainly sucrose and 5-oxo-1-proline, whereas cells adapted to 0.5–1.0 M NaCl additionally synthesized ectoine. A more detailed study showed that nitrogen deficiency in the growth medium ofMethylobacter alcaliphilus 20Z decreased the synthesis of nitrogen-contaihing osmoprotectants, ectoine and 5-oxo-1-proline.M. alcaliphilus 20Z cells exhibited activities of UDP-glucose pyrophosphorylase and sucrose-phosphate synthase involved in sucrose synthesis. Glutamine synthetase in vitro did not require NH ₄ ⁺ ions, which implies that this enzyme is involved in 5-oxo-1-proline synthesis. Cells grown at high salinity exhibited elevated levels of aspartate kinase, aspartate-semialdehyde dehydrogenase, and ectoine synthase. This suggests that ectoine is synthesized via aspartate and aspartate-semialdehyde, i.e., via the route earlier established for extremely halophilic bacteria.     

Metabolic engineering of Escherichia coli for efficient osmotic stress-free production of compatible solute hydroxyectoine

Compatible solutes are key for the ability of halophilic bacteria to resist high osmotic stress. They have received wide attention from researchers for their excellent osmotic protection properties. Hydroxyectoine is a particularly important compatible solute, but its production by microbes faces several challenges, including low titer/yield, the presence of the byproduct ectoine, and the requirement of high salinity. Here, we aimed to metabolically engineer Escherichia coli to efficiently produce hydroxyectoine in the absence of osmotic stress without accumulating the byproduct ectoine. First, combinatorial optimization of the expression strength of key genes in the ectoine synthesis module and hydroxyectoine synthesis module was conducted. After optimization of the expression of these genes, 12.12 g/L hydroxyectoine and 0.24 g/L ectoine were obtained at 36 h in shake-flask fermentation with the addition of the co-substrate α-ketoglutarate. Further optimization of the addition of α-ketoglutarate achieved the sole production of hydroxyectoine (i.e., no ectoine accumulation), indicating that the supply of α-ketoglutarate is critically important for sole hydroxyectoine production. Finally, quorum sensing-based auto-regulation of intracellular α-ketoglutarate pool was implemented as an alternative to α-ketoglutarate addition by coupling the expression of sucA with the esaI/esaR circuit, which led to 14.93 g/L hydroxyectoine with a unit cell yield of 1.678 g/g and no ectoine accumulation in the absence of osmotic stress. This is the highest reported titer of sole hydroxyectoine production under salinity-free fermentation to date.     

高盐盐湖可分离嗜盐耐盐菌的种群多样性及四氢嘧啶产量评价

为了解柴达木盆地茶卡盐湖、柯柯盐湖和小柴旦盐湖等三大硫酸镁亚型高盐盐湖可分离嗜盐耐盐菌的种群多样性,采用RM中、高盐培养基筛选分离可培养的嗜盐菌和耐盐菌,扩增16S rRNA基因序列进行种属鉴定和环境因子典范对应分析(CCA),选取优势菌属构建系统发育树,并采用高效液相色谱法(HPLC)检测次级代谢产物四氢嘧啶(Ect...     

Biosynthesis and fate of compatible solutes in extremely halophilic phototrophic eubacteria

Abstract Most halophilic and halotolerant eubacteria are able to accumulate compatible solutes from their environment during salt stress. They are usually able to synthesize sugars and amino acids. These compatible solutes, however, are of less importance in extremely halophilic eubacteria, where usually glycinebetaine or ectoine is required. Extremely halophilic phototrophic sulfur bacteria of the genus Ectothiorhodospira are able to synthesize the three compatible solutes glycinebetaine, trehalose and ectoine. While glycinebetaine is the major compatible solute under all conditions the percentage of trehalose and ectoine varies depending on the availability of nitrogen sources. Using acetate plus bicarbonate as simultaneous substrates the three compatible solutes were analysed by ¹³C-NMR spectroscopy. The label found indicated the following biosynthesis pathways: glycine, derived from glyoxylate out of the Kornberg cycle, undergoes a three-fold methylation with S-adenosylmethionine as methyl donor. The latter is derived from the tetrahydrofolate pathway. Several enzymes of this pathway have been found and are under investigation. The labelling of trehalose indicates that the Calvin cycle is blocked in the presence of acetate. The two glucose moieties of trehalose are linked by trehalose-6-phosphate synthase. The enzyme was characterized. Ectoine is synthesized from aspartate via aspartophosphate, aspartate semialdehyde and α, β-diaminobutyrate. Dilution stress leads to rapid excretion of betaine and ectoine, followed by immediate uptake to balance overshoot excretion. Trehalose is not excreted under dilution stress but is degraded by trehalase and subsequently metabolized.     

Role of Nγ-Acetyldiaminobutyrate as an Enzyme Stabilizer and an Intermediate in the Biosynthesis of Hydroxyectoine

Strain CHR63 is a salt-sensitive mutant of the moderately halophilic wild-type strain Halomonas elongata DSM 3043 that is affected in the ectoine synthase gene (ectC). This strain accumulates large amounts of Nγ-acetyldiaminobutyrate (NADA), the precursor of ectoine (D. Cánovas, C. Vargas, F. Iglesias-Guerra, L. N. Csonka, D. Rhodes, A. Ventosa, and J. J. Nieto, J. Biol. Chem. 272:25794–25801, 1997). Hydroxyectoine, ectoine, and glucosylglycerate were also identified by nuclear magnetic resonance (NMR) as cytoplasmic organic solutes in this mutant. Accumulation of NADA, hydroxyectoine, and ectoine was osmoregulated, whereas the levels of glucosylglycerate decreased at higher salinities. The effect of the growth stage on the accumulation of solutes was also investigated. NADA was purified from strain CHR63 and was shown to protect the thermolabile enzyme rabbit muscle lactate dehydrogenase against thermal inactivation. The stabilizing effect of NADA was greater than the stabilizing effect of ectoine or potassium diaminobutyrate. A ¹H NMR analysis of the solutes accumulated by the wild-type strain and mutants CHR62 (ectA::Tn1732) and CHR63 (ectC::Tn1732) indicated that H. elongata can synthesize hydroxyectoine by two different pathways—directly from ectoine or via an alternative pathway that converts NADA into hydroxyectoine without the involvement of ectoine.     

Involvement of EupR,a response regulator of the NarL/FixJ family,in the control of the uptake of the compatible solutes ectoines by the halophilic bacterium <Emphasis Type="Italic">Chromohalobacter salexigens</Emphasis>

Background  

Osmosensing and associated signal transduction pathways have not yet been described in obligately halophilic bacteria. Chromohalobacter salexigens is a halophilic bacterium with a broad range of salt tolerance. In response to osmotic stress, it synthesizes and accumulates large amounts of the compatible solutes ectoine and hydroxyectoine. In a previous work, we showed that ectoines can be also accumulated upon transport from the external medium, and that they can be used as carbon sources at optimal, but not at low salinity. This was related to an insufficient ectoine(s) transport under these conditions.     

Biosynthesis of ectoine by the methanotrophic bacterial consortium isolated from Bogdanka coalmine (Poland)

Ectoine belongs to the family of compatible solutes, which are known to contribute mainly to the adaptation of the cell to osmotic stress by mediation of a constant turgor. In addition, the cell’s essential functions are maintained under difficult conditions like high salinity, heat, or aridity stress. Biosynthesis of ectoine has been found in halophilic and halotolerant microorganisms. We showed that the methanotrophic bacterial consortium (MBC) isolated from coalbed rocks from coalmine Bogdanka (Poland) and resistant to extreme environmental conditions (low content of moisture) was able to synthesize ectoine. MBC was cultured in mineral nitrate mineral salts medium supplied with NaCl at atmospheric air enriched with 10% of methane. The levels of methanotrophic activity were determined by the gas chromatography technique (943.05 ± 30.73 ? 94.14 ± 0.85 μM CH₄ gDW^?1 day^?1) and the biomass concentration of MBC was evaluated based on OD₆₀₀, as well as biosynthesis of ectoine in relation to the salinity (0–5% NaCl) of the medium. The levels of ectoine tested by NIR measurements ranged from 1.33 ± 0.10 mg gDW^?1 to 0.42 ± 0.08 mg gDW^?1 depending on the salinity of the solution. In addition, we identified MBC based on the pmoA gene.