8 Characterization and Quantification of Compatible Solutes in (Hyper)thermophilic Microorganisms

Santos, Helena; Lamosa, Pedro; Borges, Nuno

doi:10.1016/s0580-9517(08)70011-4

Cited by 22 publications

(23 citation statements)

References 34 publications

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“…Aliquots were removed for determination of total protein. The remaining cell extract was treated twice with boiling 80% ethanol as described previously (30). Freeze-dried extracts were dissolved in 2 H 2 O and analyzed by nuclear magnetic resonance (30).…”

Section: Methodsmentioning

confidence: 99%

Thermococcus kodakar ensis Mutants Deficient in Di- myo -Inositol Phosphate Use Aspartate To Cope with Heat Stress

et al. 2010

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Many of the marine microorganisms which are adapted to grow at temperatures above 80°C accumulate di-myo-inositol phosphate (DIP) in response to heat stress. This led to the hypothesis that the solute plays a role in thermoprotection, but there is a lack of definitive experimental evidence. Mutant strains of Thermococcus kodakarensis (formerly Thermococcus kodakaraensis), manipulated in their ability to synthesize DIP, were constructed and used to investigate the involvement of DIP in thermoadaptation of this archaeon. The solute pool of the parental strain comprised DIP, aspartate, and ␣-glutamate. Under heat stress the level of DIP increased 20-fold compared to optimal conditions, whereas the pool of aspartate increased 4.3-fold in response to osmotic stress. Deleting the gene encoding the key enzyme in DIP synthesis, CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase, abolished DIP synthesis. Conversely, overexpression of the same gene resulted in a mutant with restored ability to synthesize DIP. Despite the absence of DIP in the deletion mutant, this strain exhibited growth parameters similar to those of the parental strain, both at optimal (85°C) and supraoptimal (93.7°C) temperatures for growth. Analysis of the respective solute pools showed that DIP was replaced by aspartate. We conclude that DIP is part of the strategy used by T. kodakarensis to cope with heat stress, and aspartate can be used as an alternative solute of similar efficacy. This is the first study using mutants to demonstrate the involvement of compatible solutes in the thermoadaptation of (hyper)thermophilic organisms.Hyperthermophilic bacteria and archaea isolated from saline environments accumulate unusual organic solutes in response to osmotic as well as heat stress. Mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, mannosyldi-myo-inositol phosphate (DIP), diglycerol phosphate, and glycero-phospho-myo-inositol are examples of compatible solutes highly restricted to thermophiles and hyperthermophiles (27,31). Our team has, over several years, examined the compatible solute composition in a large number of hyperthermophiles and their accumulation under stressful conditions. The data reveal a trend toward specialization of roles in thermoadaptation and osmoadaptation. Indeed, mannosylglycerate and diglycerol phosphate typically accumulate in response to increased NaCl concentration in the growth medium, whereas the levels of DIP and derivatives consistently increase at supraoptimal growth temperatures (11,16,17,27,31).

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Section: Methodsmentioning

confidence: 99%

Thermococcus kodakar ensis Mutants Deficient in Di- myo -Inositol Phosphate Use Aspartate To Cope with Heat Stress

et al. 2010

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“…C ellular responses to abiotic stresses include the accumulation and release of organic solutes called osmolytes (1)(2)(3). Relevant stressors include noxious chemicals, such as urea, as well as osmotic pressures and temperatures that are sub-or supraoptimal for cell survival and population growth.…”

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confidence: 99%

“…Relevant stressors include noxious chemicals, such as urea, as well as osmotic pressures and temperatures that are sub-or supraoptimal for cell survival and population growth. Osmolyte accumulation clearly promotes bacterial growth in high-osmolality medium, and some osmolytes also confer thermal and/or urea stress tolerance on prokaryotic and eukaryotic cells (1)(2)(3)(4)(5). Naturally occurring osmolytes include amino acids, sugars, and related compounds that are highly water soluble and uncharged or zwitterionic.…”

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Analysis of Strains Lacking Known Osmolyte Accumulation Mechanisms Reveals Contributions of Osmolytes and Transporters to Protection against Abiotic Stress

Murdock

Burke

Coumoundouros

et al. 2014

Appl Environ Microbiol

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Osmolyte accumulation and release can protect cells from abiotic stresses. In Escherichia coli, known mechanisms mediate osmotic stress-induced accumulation of K ؉ glutamate, trehalose, or zwitterions like glycine betaine. Previous observations suggested that additional osmolyte accumulation mechanisms (OAMs) exist and their impacts may be abiotic stress specific. Derivatives of the uropathogenic strain CFT073 and the laboratory strain MG1655 lacking known OAMs were created. CFT073 grew without osmoprotectants in minimal medium with up to 0.9 M NaCl. CFT073 and its OAM-deficient derivative grew equally well in high-and low-osmolality urine pools. Urine-grown bacteria did not accumulate large amounts of known or novel osmolytes. Thus, CFT073 showed unusual osmotolerance and did not require osmolyte accumulation to grow in urine. Yeast extract and brain heart infusion stimulated growth of the OAM-deficient MG1655 derivative at high salinity. Neither known nor putative osmoprotectants did so. Glutamate and glutamine accumulated after growth with either organic mixture, and no novel osmolytes were detected. MG1655 derivatives retaining individual OAMs were created. Their abilities to mediate osmoprotection were compared at 15°C, 37°C without or with urea, and 42°C. Stress protection was not OAM specific, and variations in osmoprotectant effectiveness were similar under all conditions. Glycine betaine and dimethylsulfoniopropionate (DMSP) were the most effective. Trimethylamine-N-oxide (TMAO) was a weak osmoprotectant and a particularly effective urea protectant. The effectiveness of glycine betaine, TMAO, and proline as osmoprotectants correlated with their preferential exclusion from protein surfaces, not with their propensity to prevent protein denaturation. Thus, their effectiveness as stress protectants correlated with their ability to rehydrate the cytoplasm.

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“…Aliquots were removed for determination of total protein content. The remaining cell extract was treated twice with boiling 80% ethanol as described previously (31). Freeze-dried extracts were dissolved in 2 H 2 O and analyzed by NMR (31).…”

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confidence: 99%

“…The ethanolic cell extract, obtained as described above, was loaded onto a QAE-Sephadex A-25 column previously equilibrated with 5 mM sodium bicarbonate (pH 9.8), and elution was performed with a linear gradient of the same buffer (5 mM to 1 M). Fractions were analyzed by 31 P NMR to monitor the presence of MDIP. Samples containing this compound were desalted using a column of activated Dowex 50W-X8.…”

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A Unique β-1,2-Mannosyltransferase of Thermotoga maritima That Uses Di- myo -Inositol Phosphate as the Mannosyl Acceptor

et al. 2009

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In addition to di-myo-inositol-1,3-phosphate (DIP), a compatible solute widespread in hyperthermophiles, the organic solute pool of Thermotoga maritima comprises 2-(O-␤-D-mannosyl)-di-myo-inositol-1,3-phosphate (MDIP) and 2-(O-␤-D-mannosyl-1,2-O-␤-D-mannosyl)-di-myo-inositol-1,3-phosphate (MMDIP), two newly identified ␤-1,2-mannosides. In cells grown under heat stress, MDIP was the major solute, accounting for 43% of the total pool; MMDIP and DIP accumulated to similar levels, each corresponding to 11.5% of the total pool. The synthesis of MDIP involved the transfer of the mannosyl group from GDP-mannose to DIP in a single-step reaction catalyzed by MDIP synthase. This enzyme used MDIP as an acceptor of a second mannose residue, yielding the di-mannosylated compound. Minor amounts of the tri-mannosylated form were also detected. With a genomic approach, putative genes for MDIP synthase were identified in the genome of T. maritima, and the assignment was confirmed by functional expression in Escherichia coli. Genes with significant sequence identity were found only in the genomes of Thermotoga spp., Aquifex aeolicus, and Archaeoglobus profundus. MDIP synthase of T. maritima had maximal activity at 95°C and apparent K m values of 16 mM and 0.7 mM for DIP and GDP-mannose, respectively. The stereochemistry of MDIP was characterized by isotopic labeling and nuclear magnetic resonance (NMR): DIP selectively labeled with carbon 13 at position C1 of the L-inositol moiety was synthesized and used as a substrate for MDIP synthase. This ␤-1,2-mannosyltransferase is unrelated to known glycosyltransferases, and within the domain Bacteria, it is restricted to members of the two deepest lineages, i.e., the Thermotogales and the Aquificales. To our knowledge, this is the first ␤-1,2-mannosyltransferase characterized thus far.Thermotoga maritima was first isolated from hot marine sediments on Vulcano Island, Italy, being able to grow between 55°C and 90°C (14). This strictly anaerobic bacterium ferments a variety of simple and complex carbohydrates to acetate, hydrogen, and CO 2 (10). In line with these metabolic traits, a substantial percentage of the genes annotated in the genome of this hyperthermophile are allocated to the metabolism of mono-and polysaccharides (8, 23). Therefore, T. maritima has been pointed out as a source of glycoside hydrolases with potential industrial relevance, namely, in processes of conversion of biomass into biofuels (3, 34).Like many other hyperthermophiles isolated from marine environments, Thermotoga maritima is slightly halophilic (optimum NaCl concentration of 2.7%, wt/vol) and has developed biochemical strategies to counterbalance the external osmotic pressure. The accumulation of low-molecular-mass organic compounds in the cytoplasm is the most common osmoadaptation mechanism, which enables a rapid response to fluctuations in the salinity of the external medium. Interestingly, the organic solutes encountered in organisms adapted to thrive in hot environments are clearly different from those ...

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8 Characterization and Quantification of Compatible Solutes in (Hyper)thermophilic Microorganisms

Cited by 22 publications

References 34 publications

Thermococcus kodakar ensis Mutants Deficient in Di- myo -Inositol Phosphate Use Aspartate To Cope with Heat Stress

Thermococcus kodakar ensis Mutants Deficient in Di- myo -Inositol Phosphate Use Aspartate To Cope with Heat Stress

Analysis of Strains Lacking Known Osmolyte Accumulation Mechanisms Reveals Contributions of Osmolytes and Transporters to Protection against Abiotic Stress

A Unique β-1,2-Mannosyltransferase of Thermotoga maritima That Uses Di- myo -Inositol Phosphate as the Mannosyl Acceptor

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