Compatible solutes and fungal development.
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ABSTRACT:
SUBMITTER: Dijksterhuis J
PROVIDER: S-EPMC1609922 | biostudies-other | 2006 Oct
REPOSITORIES: biostudies-other
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Project description:Exposure of protein modified surfaces to air may be necessary in several applications. For example, air contact may be inevitable during the implantation of biomedical devices, for analysis of protein modified surfaces, or for sensor applications. Protein coatings are very sensitive to dehydration and can undergo significant and irreversible alterations of their conformations upon exposure to air. With the use of two compatible solutes from extremophilic bacteria, ectoine and hydroxyectoine, the authors were able to preserve the activity of dried protein monolayers for up to >24?h. The protective effect can be explained by the preferred exclusion model; i.e., the solutes trap a thin water layer around the protein, retaining an aqueous environment and preventing unfolding of the protein. Horseradish peroxidase (HRP) immobilized on compact TiO2 was used as a model system. Structural differences between the compatible solute stabilized and unstabilized protein films, and between different solutes, were analyzed by static time-of-flight secondary ion mass spectrometry (ToF-SIMS). The biological activity difference observed in a colorimetric activity assay was correlated to changes in protein conformation by application of principal component analysis to the static ToF-SIMS data. Additionally, rehydration of the denatured HRP was observed in ToF-SIMS with an exposure of denatured protein coatings to ectoine and hydroxyectoine solutions.
| S-EPMC6026024 | biostudies-literature
Project description:BackgroundAsexually developed fungal spores (conidia) are key for the massive proliferation and dispersal of filamentous fungi. Germination of conidia and subsequent formation of a mycelium network give rise to many societal problems related to human and animal fungal diseases, post-harvest food spoilage, loss of harvest caused by plant-pathogenic fungi and moulding of buildings. Conidia are highly stress resistant compared to the vegetative mycelium and therefore even more difficult to tackle.ResultsIn this study, complementary approaches are used to show that accumulation of mannitol and trehalose as the main compatible solutes during spore maturation is a key factor for heat resistance of conidia. Compatible solute concentrations increase during conidia maturation, correlating with increased heat resistance of mature conidia. This maturation only occurs when conidia are attached to the conidiophore. Moreover, conidia of a mutant Aspergillus niger strain, constructed by deleting genes involved in mannitol and trehalose synthesis and consequently containing low concentrations of these compatible solutes, exhibit a sixteen orders of magnitude more sensitive heat shock phenotype compared to wild-type conidia. Cultivation at elevated temperature results in adaptation of conidia with increased heat resistance. Transcriptomic and proteomic analyses revealed two putative heat shock proteins to be upregulated under these conditions. However, conidia of knock-out strains lacking these putative heat shock proteins did not show a reduced heat resistance.ConclusionsHeat stress resistance of fungal conidia is mainly determined by the compatible solute composition established during conidia maturation. To prevent heat resistant fungal spore contaminants, food processing protocols should consider environmental conditions stimulating compatible solute accumulation and potentially use compatible solute biosynthesis as a novel food preservation target.
| S-EPMC10644514 | biostudies-literature
Project description:The effect of salinity and growth temperature on the accumulation of intracellular organic solutes was examined in the hyperthermophilic archaeon Palaeococcus ferrophilus. The genus Palaeococcus represents a deep-branching lineage of the order Thermococcales, which diverged before Thermococcus and Pyrococcus. Palaeococcus ferrophilus accumulated mannosylglycerate, glutamate, and aspartate as major compatible solutes. Unlike members of the genera Pyrococcus and Thermococcus, Palaeococcus ferrophilus did not accumulate di-myo-inositol phosphate, a canonical solute of hyperthermophiles. The level of mannosylglycerate increased in response to both heat and salt stress; glutamate increased at supraoptimal growth temperatures, whereas aspartate increased at supraoptimal salt concentration. Proline, alanine, and trehalose were also found in lesser amounts, but their levels did not respond significantly to any of the stresses imposed. Additionally, the genes involved in the synthesis of mannosylglycerate in Palaeococcus ferrophilus and Thermococcus litoralis were identified. In both organisms the synthesis proceeds via the two-step pathway comprising mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The mpgS and mpgP genes of Palaeococcus ferrophilus were expressed in Escherichia coli and the proteins were characterized. MPGS had maximal activity at 90 degrees C and pH near 7.0, and was strictly dependent on Mg2+. MPGP had optimal activity at 90 degrees C and pH 6.0 and was barely dependent on Mg2+. The half-life values for inactivation of MPGS and MPGP at 83 degrees C were 18 and 25 min, respectively. A comparative discussion of the osmo- and thermoadaptation strategies in these three genera of the Thermococcales is presented.
| S-EPMC1317470 | biostudies-literature
Project description:BACKGROUND:Associated with appropriate crop and soil management, inoculation of legumes with microbial biofertilizers can improve food legume yield and soil fertility and reduce pollution by inorganic fertilizers. Rhizospheric bacteria are subjected to osmotic stress imposed by drought and/or NaCl, two abiotic constraints frequently found in semi-arid lands. Osmostress response in bacteria involves the accumulation of small organic compounds called compatible solutes. Whereas most studies on rhizobial osmoadaptation have focussed on the model species Sinorhizobium meliloti, little is known on the osmoadaptive mechanisms used by native rhizobia, which are good sources of inoculants. In this work, we investigated the synthesis and accumulations of compatible solutes by four rhizobial strains isolated from root nodules of Phaseolus vulgaris in Tunisia, as well as by the reference strain Rhizobium tropici CIAT 899T. RESULTS:The most NaCl-tolerant strain was A. tumefaciens 10c2, followed (in decreasing order) by R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3, R. etli 12a3 and R. gallicum bv. phaseoli 8a3. 13C- and 1H-NMR analyses showed that all Rhizobium strains synthesized trehalose whereas A. tumefaciens 10c2 synthesized mannosucrose. Glutamate synthesis was also observed in R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3 and A. tumefaciens 10c2. When added as a carbon source, mannitol was also accumulated by all strains. Accumulation of trehalose in R. tropici CIAT 899 and of mannosucrose in A. tumefaciens 10c2 was osmoregulated, suggesting their involvement in osmotolerance. The phylogenetic analysis of the otsA gene, encoding the trehalose-6-phosphate synthase, suggested the existence of lateral transfer events. In vivo 13C labeling experiments together with genomic analysis led us to propose the uptake and conversion pathways of different carbon sources into trehalose. Collaterally, the beta-1,2-cyclic glucan from R. tropici CIAT 899 was co-extracted with the cytoplasmic compatible solutes and its chemical structure was determined. CONCLUSIONS:The soil bacteria analyzed in this work accumulated mainly disaccharides in response to NaCl stress. We could not find a direct correlation between the trehalose content of the rhizobial strains and their osmotolerance, suggesting that additional osmoadaptive mechanism should be operating in the most NaCl-tolerant strain R. tropici CIAT 899.
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Project description:Among compatible solutes, glycine betaine has various applications in the fields of nutrition, pharmaceuticals, and cosmetics. Currently, this compound can be extracted from sugar beet plants or obtained by chemical synthesis, resulting in low yields or high carbon footprint, respectively. Hence, in this work we aimed at exploring the production of glycine betaine using the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a photoautotrophic chassis. Synechocystis mutants lacking the native compatible solutes sucrose or/and glucosylglycerol-∆sps, ∆ggpS, and ∆sps∆ggpS-were generated and characterized. Under salt stress conditions, the growth was impaired and accumulation of glycogen decreased by ∼50% whereas the production of compatible solutes and extracellular polymeric substances (capsular and released ones) increased with salinity. These mutants were used as chassis for the implementation of a synthetic device based on the metabolic pathway described for the halophilic cyanobacterium Aphanothece halophytica for the production of the compatible solute glycine betaine. Transcription of ORFs comprising the device was shown to be stable and insulated from Synechocystis' native regulatory network. Production of glycine betaine was achieved in all chassis tested, and was shown to increase with salinity. The introduction of the glycine betaine synthetic device into the ∆ggpS background improved its growth and enabled survival under 5% NaCl, which was not observed in the absence of the device. The maximum glycine betaine production [64.29 µmol/gDW (1.89 µmol/mg protein)] was reached in the ∆ggpS chassis grown under 3% NaCl. Taking into consideration this production under seawater-like salinity, and the identification of main key players involved in the carbon fluxes, this work paves the way for a feasible production of this, or other compatible solutes, using optimized Synechocystis chassis in a pilot-scale.
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Project description:Water activity (aw) is critical for microbial growth, as it is severely restricted at aw < 0.90. Saturating NaCl concentrations (~5.0 M) induce extreme water deprivation (aw ≅ 0.75) and cellular stress responses. Halophilic fungi have cellular adaptations that enable osmotic balance and ionic/oxidative stress prevention to grow at high salinity. Here we studied the morphology, osmolyte synthesis, and oxidative stress defenses of the halophile Aspergillus sydowii EXF-12860 at 1.0 M and 5.13 M NaCl. Colony growth, pigmentation, exudate, and spore production were inhibited at NaCl-saturated media. Additionally, hyphae showed unpolarized growth, lower diameter, and increased septation, multicellularity and branching compared to optimal NaCl concentration. Trehalose, mannitol, arabitol, erythritol, and glycerol were produced in the presence of both 1.0 M and 5.13 M NaCl. Exposing A. sydowii cells to 5.13 M NaCl resulted in oxidative stress evidenced by an increase in antioxidant enzymes and lipid peroxidation biomarkers. Also, genes involved in cellular antioxidant defense systems were upregulated. This is the most comprehensive study that investigates the micromorphology and the adaptative cellular response of different non-enzymatic and enzymatic oxidative stress biomarkers in halophilic filamentous fungi.
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Project description:The main osmoadaptive mechanisms of extremely halophilic archaea include the "salt-in" strategy and the "compatible solutes" strategy. Here we report the osmoadaptive mechanism of an extremely halophilic archaea H. kocurii 2020YC7, isolated from a high salt environment sample. Genomic data revealed that strain 2020YC7 harbors genes trkA, trkH, kch for K+ uptake, kefB for K+ output, treS for trehalose production from polysaccharide, and betaine/carnitine/choline transporter family gene for glycine betaine uptake. Strain 2020YC7 could accumulate 8.17 to 28.67 μmol/mg protein K+ in a defined medium, with its content increasing along with the increasing salinity from 100 to 200 g/L. When exogenous glycine betaine was added, glycine betaine functioned as the primary osmotic solute between 200 and 250 g/L NaCl, which was accumulated up to 15.27 mg/mg protein in 2020YC7 cells. RT-qPCR results completely confirmed these results. Notably, the concentrations of intracellular trehalose decreased from 5.26 to 2.61 mg/mg protein as the NaCl increased from 50 to 250 g/L. In combination with this result, the transcript level of gene treS, which catalyzes the production of trehalose from polysaccharide, was significantly up-regulated at 50-100 g/L NaCl. Therefore, trehalose does not act as an osmotic solute at high NaCl concentrations (more than 100 g/L) but at relatively low NaCl concentrations (50-100 g/L). And we propose that the degradation of cell wall polysaccharide, as a source of trehalose in a low-salt environment, may be one of the reasons for the obligate halophilic characteristics of strain 2020YC7.
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Project description:The intracellular accumulation of small organic solutes was described in the marine bacterium Rhodopirellula baltica, which belongs to the globally distributed phylum Planctomycetes whose members exhibit an intriguing lifestyle and cell morphology. Sucrose, ?-glutamate, trehalose and mannosylglucosylglycerate (MGG) are the main solutes involved in the osmoadaptation of R. baltica. The ratio and total intracellular organic solutes varied significantly in response to an increase in salinity, temperature and nitrogen content. R. baltica displayed an initial response to both osmotic and thermal stresses that includes ?-glutamate accumulation. This trend was followed by a rather unique and complex osmoadaptation mechanism characterized by a dual response to sub-optimal and supra-optimal salinities. A reduction in the salinity to sub-optimal conditions led primarily to the accumulation of trehalose. In contrast, R. baltica responded to salt stress mostly by increasing the intracellular levels of sucrose. The switch between the accumulation of trehalose and sucrose was by far the most significant effect caused by increasing the salt levels of the medium. Additionally, MGG accumulation was found to be salt- as well as nitrogen-dependent. MGG accumulation was regulated by nitrogen levels replacing ?-glutamate as a K(+) counterion in nitrogen-poor environments. This is the first report of the accumulation of compatible solutes in the phylum Planctomycetes and of the MGG accumulation in a mesophilic organism.
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Project description:Responses to NaCl stress were investigated in phototrophically grown Alphaproteobacterium Rhodobacter sphaeroides by transcriptome profiling, mutational analysis, and measurements of compatible solutes and membrane phospholipids. After exposure to salt stress, genes encoding two putative glycine betaine uptake systems, proVWX and betS, were highly upregulated. Mutational analysis revealed that BetS, not ProVWX, was the primary transporter of this compatible solute. Upon the addition of salt, exogenous glycine betaine was taken up rapidly, and maximal intracellular levels were reached within minutes. In contrast, synthesis of another important compatible solute in R. sphaeroides, trehalose, increased slowly following salt stress, reaching maximal levels only after several hours. This accumulation pattern was consistent with the more gradual increase in salt-induced transcription of the trehalose biosynthesis operon otsBA. Several genes encoding putative transcription factors were highly induced by salt stress. Multiple copies of one of these factors, crpO (RSP1275), whose product is a member of the cyclic AMP receptor protein/fumarate and nitrate reduction regulator (CRP/FNR) family, improved NaCl tolerance. When crpO was provided in multicopy, expression of genes for synthesis or transport of compatible solutes was unaltered, but the membrane phospholipid composition became biased toward that found in salt-stressed cells. Collectively, this study characterized transcriptional responses to salt stress, correlated changes in transcription with compatible solute accumulation rates, identified the main glycine betaine transporter and trehalose synthase, characterized salt-induced changes in phospholipid composition, and uncovered a transcription factor associated with changes in phospholipids. These findings set the stage for deciphering the salt stress-responsive regulatory network in R. sphaeroides.
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