Occurrence of 1-glyceryl-1-myo-inosityl phosphate in hyperthermophiles

The accumulation of compatible solutes was studied in the hyperthermophilic bacterium Aquifex pyrophilus as a function of the temperature and the NaCl concentration of the growth medium. Nuclear magnetic resonance analysis of cell extracts revealed the presence of alpha- and beta-glutamate, di-mannosyl-di-myo-inositol phosphate, di-myo-inositol phosphate, and an additional compound here identified as 1-glyceryl-1-myo-inosityl phosphate. All solutes accumulated by A. pyrophilus are negatively charged at physiological pH. The intracellular levels of di-myo-inositol phosphate increased in response to supraoptimal growth temperature, while alpha- and beta-glutamate accumulated in response to osmotic stress, especially at growth temperatures below the optimum. The newly discovered compound, 1-glyceryl-1-myo-inosityl phosphate, appears to play a double role in osmo- and thermoprotection, since its intracellular pool increased primarily in response to a combination of osmotic and heat stresses. This work also uncovered the nature of the unknown compound, previously detected in Archaeoglobus fulgidus (L. O. Martins et al., Appl. Environ. Microbiol. 63:896-902, 1997). The curious structural relationship between diglycerol phosphate (found only in Archaeoglobus species), di-myo-inositol phosphate (a canonical solute of hyperthermophiles), and the newly identified solute is highlighted. This is the first report on the occurrence of 1-glyceryl-1-myo-inosityl phosphate in living systems.     

Contribution of chemical changes in membrane lipids to the osmoadaptation of the halophilic bacterium Chromohalobacter salexigens

The long-term response of the broad-salt growing halophile Chromohalobacter salexigens DSM 3043T to salt stress has been investigated with respect to adaptive changes in membrane lipid composition. This study included the wild-type and three salt-sensitive, ectoine-deficient strains: CHR62 (ectA::Tn1732, unable to grow above 0.75 M NaCl), CHR63 (ectC::Tn1732, unable to grow above 1.5 M NaCl), and CHR64, which was able to grow in minimal medium M63 up to 2.5 M NaCl, but its growth was slower than the wild-type strain at salinities above 1.5 M NaCl. This mutant accumulated ectoine and hydroxyectoine as major compatible solutes, but also the ectoine precursor, N-gamma-acetyldiaminobutyric acid, and was found to be affected in the ectoine synthase gene ectC. The main phospholipids of the wild-type strain were phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin (CL). Major fatty acids were detected as 16:0, 18:1, and 16:1, including significant amounts of cyc-19:0, and cyc-17:0. CL and cyclopropane fatty acids (CFA) levels were elevated when the wild-type strain was grown at high salinity (2.5 M NaCl). Membranes of the most salt-sensitive trains CHR62 and CHR63, but not of the less salt-sensitive strain CHR64, contained lower levels of CL. The proportion of cyc-19:0 in CHR64 was three-fold (at 2.0M NaCl) and 2.5-fold (at 2.5 M NaCl) lower than that of the wild type, suggesting that this mutant has a limited capacity to incorporate CFA into phospholipids at high salt. The addition of 1 mM ectoine to cultures of the wild-type strain increased the ratio PG/CL from 1.8 to 3.3 at 0.75 M NaCl, and from 1 to 6.5 at 2.5 M NaCl, and led to a slight decrease in CFA content. Addition of 1 mM ectoine to the mutants restored the steady-state levels of CL and CFA found in the wild-type strain supplemented with ectoine. These findings suggest that exogenous ectoine might attenuate the osmostress response involving changes in membrane lipids.     

以四氢嘧啶为主要相容性溶质的中度嗜盐菌I15的分离和特性研究

从草地土壤中分离到一株中度嗜盐菌I15,经过16S rDNA(GenBank登录号为DQ010162)序列分析、形态学和生理生化特征分析,该菌株初步鉴定为Virgibacillus marismortui。I15能在0%～25%NaCl的培养基中生长,最适生长NaCl浓度为10%,最适生长温度为30℃,最适pH为7.5～8.0。在高盐条件下,I15细胞内主要的相容性溶质为四氢嘧啶,在15%NaCl培养基中其含量达到1.608mmol/(g\5cdw),占到相容性溶质总摩尔含量的89.6%。渗透冲击试验表明I15细胞内四氢嘧啶在低渗冲击时能够快速分泌到细胞外,在高渗冲击冲剂时能够较快地重新合成。     

Synthesis of the compatible solutes glucosylglycerol and trehalose by salt-stressed cells of Stenotrophomonas strains

In this study, physiological processes were analysed, which are involved in salt acclimation of two Stenotrophomonas species, Stenotrophomonas maltophilia strain DSM 50170 and Stenotrophomonas rhizophila strain DSM 14405. S. maltophilia accumulated trehalose as the only osmolyte, whereas S. rhizophila produced additionally to trehalose glucosylglycerol (GG). The different spectrum and amounts of compatible solutes in these two strains led to differences in terms of their salt tolerance. The human-associated S. maltophilia was able to grow in media containing up to 3% NaCl (w/v). In contrast, S. rhizophila propagated in salinities up to 5% NaCl (w/v). The strain was isolated from the rhizosphere, a microenvironment which is characterised by high and changing salinities. Light microscopic analysis of S. rhizophila cells showed a significant increase in cell length of salt-treated cells in comparison to control cells. Cells of S. rhizophila exposed to more than 2% NaCl excreted GG into the medium during the transition from exponential to stationary growth phase, while the internal trehalose pool remained constant. This feature offers a high potential for the biotechnological production of GG.     

Mannosylglycerate is essential for osmotic adjustment in Thermus thermophilus strains HB27 and RQ-1

We disrupted the mpgS encoding mannosyl-3-phosphoglycerate synthase (MpgS) of Thermus thermophilus strains HB27 and RQ-1, by homologous recombination, to assess the role of the compatible solute mannosylglycerate (MG) in osmoadaptation of the mutants, to examine their ability to grow in NaCl-containing medium and to identify the intracellular organic solutes. Strain HB27 accumulated only MG when grown in defined medium containing 2% NaCl; mutant HB27M9 did not grow in the same medium containing more than 1% NaCl. When trehalose or MG was added, the mutant was able to grow up to 2% of NaCl and accumulated trehalose or MG, respectively, plus amino acids. T. thermophilus RQ-1 grew in medium containing up to 5% NaCl, accumulated trehalose and lower amounts of MG. Mutant RQ-1M1 lost the ability to grow in medium containing more than 3% NaCl and accumulated trehalose and moderate levels of amino acids. Exogenous MG did not improve the ability of the organism to grow above 3% NaCl, but caused a decrease in the levels of amino acids. Our results show that MG serves as a compatible solute primarily during osmoadaptation at low levels of NaCl while trehalose is primarily involved in osmoadaptation during growth at higher NaCl levels.     

Halotolerance of Methanobacterium thermoautotrophicum delta H and Marburg.

Methanobacterium thermoautotrophicum delta H and Marburg were adapted to grow in medium containing up to 0.65 M NaCl. From 0.01 to 0.5 M NaCl, there was a lag before cell growth which increased with increasing external NaCl. The effect of NaCl on methane production was not significant once the cells began to grow. Intracellular solutes were monitored by nuclear magnetic resonance (NMR) spectroscopy as a function of osmotic stress. In the delta H strain, the major intracellular small organic solutes, cyclic-2,3-diphosphoglycerate and glutamate, increased at most twofold between 0.01 and 0.4 M NaCl and decreased when the external NaCl was 0.5 M. M. thermoautotrophicum Marburg similarly showed a decrease in solute (cyclic-2,3-diphosphoglycerate, 1,3,4,6-tetracarboxyhexane, and L-alpha-glutamate) concentrations for cells grown in medium containing > 0.5 M NaCl. At 0.65 M NaCl, a new organic solute, which was visible in only trace amounts at the lower NaCl concentrations, became the dominant solute. Intracellular potassium in the delta H strain, detected by atomic absorption and 39K NMR, was roughly constant between 0.01 and 0.4 M and then decreased as the external NaCl increased further. The high intracellular K+ was balanced by the negative charges of the organic osmolytes. At the higher external salt concentrations, it is suggested that Na+ and possibly Cl- ions are internalized to provide osmotic balance. A striking difference of strain Marburg from strain delta H was that yeast extract facilitated growth in high-NaCl-containing medium. The yeast extract supplied only trace NMR-detectable solutes (e.g., betaine) but had a large effect on endogenous glutamate levels, which were significantly decreased. Exogenous choline and glycine, instead of yeast extract, also aided growth in NaCl-containing media. Both solutes were internalized with the choline converted to betaine; the contribution to osmotic balance of these species was 20 to 25% of the total small-molecule pool. These results indicate that M. thermoautotrophicum shows little changes in its internal solutes over a wide range of external NaCl. Furthermore, they illustrate the considerable differences in physiology in the delta H and Marburg strains of this organism.     

Compatible Solutes in the Thermophilic Bacteria Rhodothermus marinus and "Thermus thermophilus"

(sup13)C nuclear magnetic resonance spectroscopy and (sup1)H nuclear magnetic resonance spectroscopy were used to identify and quantify the organic solutes of several strains of halophilic or halotolerant thermophilic bacteria. Two strains of Rhodothermus marinus and four strains of "Thermus thermophilus" grown in complex medium containing NaCl were examined. 2-O-Mannosylglycerate was a major compatible solute in all strains: the Thermus strains accumulated the (beta)-anomer only, whereas both anomers were found in R. marinus. 2-O-(beta)-mannosylglycerate and 2-O-(alpha)-mannosylglycerate were the major compatible solutes in R. marinus. The former was the predominant solute in cells grown in 2.0 and 4.0% NaCl-containing medium, while the latter was the predominant compatible solute at higher salinities. Glutamate, trehalose, and glucose were also present as minor components. The intracellular K(sup+) concentration, as determined by (sup39)K nuclear magnetic resonance spectroscopy, in R. marinus increased with salinity and was sufficient to balance the negative charges of the mannosylglycerate. In addition to 2-O-(beta)-mannosylglycerate, trehalose was a major compatible solute of "T. thermophilus." 2-O-(beta)-Mannosylglycerate was the main solute in medium containing 1.0 or 2.0% NaCl, while trehalose predominated in cells grown in medium supplemented with 3.0 or 4.0% NaCl. Glycine betaine, in lower concentrations, was also detected in two "T. thermophilus" strains. This is the first report of mannosylglycerate as a compatible solute in bacteria.     

Protein thermal stabilization by charged compatible solutes: Computational studies in rubredoxin from Desulfovibrio gigas

Molecular dynamics simulation studies of rubredoxin from Desulfovibrio gigas (RDG) were used to characterize the molecular mechanism of thermal stabilization by the compatible solute (CS) diglycerol-phospate (DGP). DGP is a negatively charged CS that accumulates under salt stress in Archaeoglobus fulgidus. Experimental results show that a 100 mM DGP solution exerts a strong protection effect in the half-life of RDG at 363 K (Lamosa et al., Appl Environ Microbiol 2000;66:1974-1979). RDG was simulated in four aqueous solutions at 300 and 363 K: pure aqueous media, 100 mM DGP, 100 mM NaCl, and 500 mM DGP. Our work shows that the 100 mM DGP solution is able to maintain the average protein structure when the temperature is increased, preventing the occurrence of large-scale deviation of a mobile loop involved in the first steps of RDG unfolding. The molecular mechanism of thermal denaturation protection by DGP seems to involve the direct interaction between the protein and the CS by hydrogen bond interactions near the mobile loop. Several clusters of DGP molecules are formed and preferentially localized at neutral electrostatic regions of the surface. The increase of DGP concentration to 500 mM did not yield better stabilization of the protein suggesting that the thermal protective role of this charged CS is achieved at low concentrations, as shown experimentally.     

Glycine betaine and potassium ion are the major compatible solutes in the extremely halophilic methanogen Methanohalophilus strain Z7302.

Methanohalophilus strain Z7302 was previously isolated from a hypersaline environment and grows over a range of NaCl concentrations from 1.7 to 4.4 M. We examined the relationships between cell growth rate, cell volume, and intracellular solute concentrations with increasing salinity. This extremely halophilic methanogen synthesized three zwitterionic compounds, beta-glutamine, N epsilon-acetyl-beta-lysine, and glycine betaine, and also accumulated potassium ion as compatible solutes to balance the external and internal osmotic pressures. Potassium and glycine betaine were the predominant compatible solutes when Methanohalophilus strain Z7302 was grown at high external NaCl concentrations and approached intracellular levels of 3 and 4 M, respectively.     

Accumulation and role of compatible solutes in fast-growing Salinivibrio costicola subsp. yaniae

The moderately halophilic bacterium Salinivibrio costicola subsp. yaniae showed an extremely fast growth rate. Optimal growth was observed in artificial seawater containing 1.4 mol/L NaCl and in MM63 media containing 0.6 mol/L NaCl. We analyzed a variety of compatible solutes that had accumulated in this strain grown in the media. The supplementation effect of the compatible solutes glycine betaine, glutamate, and ectoine to the growth of S. costicola subsp. yaniae was examined. Glycine betaine and glutamate had no supplementation effect on the fast growth rate. Growth of salt-sensitive mutants MU1 and MU2, both of which were defective in the ability to synthesize ectoine, was not observed in MM63 medium in the presence of more than 1.0 mol/L NaCl. From these data, we conclude that ectoine was the predominant compatible solute synthesized in this bacterium that effected an extremely fast growth rate.     

Organic solutes in <Emphasis Type="Italic">Rubrobacter xylanophilus</Emphasis>: the first example of di-<Emphasis Type="Italic">myo</Emphasis>-inositol-phosphate in a thermophile

The thermophilic and halotolerant nature of Rubrobacter xylanophilus led us to investigate the accumulation of compatible solutes in this member of the deepest lineage of the Phylum Actinobacteria. Trehalose and mannosylglycerate (MG) were the major compounds accumulated under all conditions examined, including those for optimal growth. The addition of NaCl to a complex medium and a defined medium had a slight or negligible effect on the accumulation of these compatible solutes. Glycine betaine, di-myo-inositol-phosphate (DIP), a new phosphodiester compound, identified as di-N-acetyl-glucosamine phosphate and glutamate were also detected but in low or trace levels. DIP was always present, except at the highest salinity examined (5% NaCl) and at the lowest temperature tested (43°C). Nevertheless, the levels of DIP increased with the growth temperature. This is the first report of MG and DIP in an actinobacterium and includes the identification of the new solute di-N-acetyl-glucosamine phosphate.     

Interrelation between synthesis and uptake of ectoine for the growth of the halotolerant Brevibacterium species JCM 6894 at high osmolarity

The growth of a halotolerant Brevibacterium sp. JCM 6894 was examined in the presence of compatible solutes such as glycine betaine, ectoine (2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine) and ectoine derivatives. The effect of competition between their uptake and synthesis in the cells was subjected to osmotic shift towards the higher salinity. Among each solute examined the supplement of ectoine or hydroxyectoine exhibited a remarkable stimulation on the growth of strain JCM 6894, regardless of the range of osmotic shifts, where the largest was 0-->2 M NaCl, the intermediate was 1-->2 M NaCl, and no shift was 2-->2 M NaCl. The growth rates of this strain were dependent on the amount of ectoine taken up, which was conspicuous for the largest osmotic shift and during the first few hours of incubation after transfer. The cells subjected to 1-->2 M NaCl and 2-->2 M NaCl transfers took up less ectoine and this resulted in lower growth rates than those of cells with the largest osmotic shift (0-->2 M NaCl). The role of other compatible solutes which accumulated is discussed in relation to growth stimulation of strain JCM 6894.     

Biosynthetic pathways of inositol and glycerol phosphodiesters used by the hyperthermophile Archaeoglobus fulgidus in stress adaptation

Archaeoglobus fulgidus accumulates di-myo-inositol phosphate (DIP) and diglycerol phosphate (DGP) in response to heat and osmotic stresses, respectively, and the level of glycero-phospho-myo-inositol (GPI) increases primarily when the two stresses are combined. In this work, the pathways for the biosynthesis of these three compatible solutes were established based on the detection of the relevant enzymatic activities and characterization of the intermediate metabolites by nuclear magnetic resonance analysis. The synthesis of DIP proceeds from glucose-6-phosphate via four steps: (i) glucose-6-phosphate was converted into l-myo-inositol 1-phosphate by l-myo-inositol 1-phosphate synthase; (ii) l-myo-inositol 1-phosphate was activated to CDP-inositol at the expense of CTP; this is the first demonstration of CDP-inositol synthesis in a biological system; (iii) CDP-inositol was coupled with l-myo-inositol 1-phosphate to yield a phosphorylated intermediate, 1,1'-di-myo-inosityl phosphate 3-phosphate (DIPP); (iv) finally, DIPP was dephosphorylated into DIP by the action of a phosphatase. The synthesis of the two other polyol-phosphodiesters, DGP and GPI, proceeds via the condensation of CDP-glycerol with the respective phosphorylated polyol, glycerol 3-phosphate for DGP and l-myo-inositol 1-phosphate for GPI, yielding the respective phosphorylated intermediates, 1X,1'X-diglyceryl phosphate 3-phosphate (DGPP) and 1-(1X-glyceryl) myo-inosityl phosphate 3-phosphate (GPIP), which are subsequently dephosphorylated to form the final products. The results disclosed here represent an important step toward the elucidation of the regulatory mechanisms underlying the differential accumulation of these compounds in response to heat and osmotic stresses.     

A novel limb in the osmoregulatory network of Methanosarcina mazei Gö1: Nε-acetyl-β-lysine can be substituted by glutamate and alanine

N^ε-acetyl-β-lysine is a unique compatible solute found in methanogenic archaea grown at high salinities. Deletion of the genes that encode the lysine-2,3-aminomutase ( ablA ) and the β-lysine acetyltransferase ( ablB ) abolished the production of N^ε-acetyl-β-lysine in Methanosarcina mazei Gö1. The mutant grew well at low and intermediate salinities. Interestingly, growth at high salt (800 mM NaCl) was only slowed down but not impaired demonstrating that in M. mazei Gö1 N^ε-acetyl-β-lysine is not essential for growth at high salinities. Nuclear magnetic resonance (NMR) analysis revealed an increased glutamate pool in the mutant. In addition to α-glutamate, a novel solute, alanine, was produced. The intracellular alanine concentration was as high as 0.36 ± 0.05 μmol (mg protein)⁻¹ representing up to 18% of the total solute pool at 800 mM NaCl. The cellular alanine concentration increased with the salinity of the medium and decreased in the presence of glycine betaine in the medium, indicating that alanine is used as compatible solute by M. mazei Gö1.     

Mannosylglycerate and Di-myo-Inositol Phosphate Have Interchangeable Roles during Adaptation of Pyrococcus furiosus to Heat Stress

Marine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities. Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed for P. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.     

Structural determinants of protein stabilization by solutes. The important of the hairpin loop in rubredoxins

Despite their high sequence homology, rubredoxins from Desulfovibrio gigas and D. desulfuricans are stabilized to very different extents by compatible solutes such as diglycerol phosphate, the major osmolyte in the hyperthermophilic archaeon Archaeoglobus fulgidus[Lamosa P, Burke A, Peist R, Huber R, Liu M Y, Silva G, Rodrigues-Pousada C, LeGall J, Maycock C and Santos H (2000) Appl Environ Microbiol66, 1974-1979]. The principal structural difference between these two proteins is the absence of the hairpin loop in the rubredoxin from D. desulfuricans. Therefore, mutants of D. gigas rubredoxin bearing deletions in the loop region were constructed to investigate the importance of this structural feature on protein intrinsic stability, as well as on its capacity to undergo stabilization by compatible solutes. The three-dimensional structure of the mutant bearing the largest deletion, Delta17/29, was determined by 1H-NMR, demonstrating that, despite the drastic deletion, the main structural features were preserved. The dependence of the NH chemical shifts on temperature and solute concentration (diglycerol phosphate or mannosylglycerate) provide evidence of subtle conformational changes induced by the solute. The kinetic stability (as assessed from the absorption decay at 494 nm) of six mutant rubredoxins was determined at 90 degrees C and the stabilizing effect exerted by both solutes was assessed. The extent of protection conferred by each solute was highly dependent on the specific mutant examined: while the half-life for iron release in the wild-type D. gigas rubredoxin increased threefold in the presence of 0.1 M diglycerol phosphate, mutant Delta23/29 was destabilized. This study provides evidence for solute-induced compaction of the protein structure and occurrence of weak, specific interactions with the protein surface. The relevance of these findings to our understanding of the molecular basis for protein stabilization is discussed.     

Cloning of rel from Listeria monocytogenes as an osmotolerance involvement gene

Transposon insertional mutants of Listeria monocytogenes were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained. The cloned gene from the transposon insertion site of the mutant, named rel, was 2,214 bp in length and had very high homology to relA of Bacillus subtilis, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively designated (p)ppGpp] synthetase during stringent response. The mutant showed a deficiency in (p)ppGpp accumulation. In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift. The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included rel of L. monocytogenes was introduced into the mutant. After exposure to methyl glucoside, the rel mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth. The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl. Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in L. monocytogenes and that its mechanism is different from that for the accumulation of compatible solutes.     

Combined effect of the growth temperature and salinity of the medium on the accumulation of compatible solutes by Rhodothermus marinus and Rhodothermus obamensis

In this study we propose revised structures for the two major compatible solutes of Rhodothermus marinus. We have also examined the accumulation of compatible solutes by the type strains of the slightly halophilic and thermophilic species Rhodothermus marinus and Rhodothermus obamensis at several growth temperatures and salinities. The major solutes of R. marinus were identified as α-mannosylglycerate (α-MG) and α-mannosylglyceramide (α-MGA), whereas R. obamensis accumulated only α-mannosylglycerate. The total osmolyte content was higher during the early exponential phase and decreased abruptly as growth continued into the stationary phase. At low growth temperatures, R. marinus responded to water stress by accumulation of α-mannosylglycerate and its amide, in addition to low levels of trehalose, glutamate, and glucose. At the highest growth temperature, α-mannosylglycerate was the major compatible solute and α-mannosylglyceramide was not detected. When both compounds were present, an increase in the salinity of the growth medium favored the accumulation of α-mannosylglyceramide over α-mannosylglycerate. The absence of α-mannosylglyceramide in R. obamensis at all growth temperatures and salinities constituted the most pronounced difference in the profiles of compatible solute accumulation by the two strains. Trehalose was also a prominent solute in this organism. Both organisms accumulated higher levels of α-mannosylglycerate as the temperature was raised. The importance of the two compounds in the mechanisms of thermoadaptation and osmoadaptation is discussed. Received: February 10, 1998 / Accepted: January 11, 1999     

Gluconeotrehalose is the principal organic solute in the psychrotolerant bacterium <Emphasis Type="Italic">Carnobacterium</Emphasis> strain 17-4

A high proportion of microorganisms that colonise cold environments originate from marine sites; hence, they must combine adaptation to low temperature with osmoregulation. However, little or nothing is known about the nature of compatible solutes used by cold-adapted organisms to balance the osmotic pressure of the external medium. We studied the intracellular accumulation of small organic solutes in the Arctic isolate Carnobacterium strain 17-4 as a function of the growth temperature and the NaCl concentration in the medium. Data on 16S rDNA sequence and DNA–DNA hybridisation tests corroborate the assignment of this isolate as a new species of the bacterial genus Carnobacterium. The growth profiles displayed maximal specific growth rate at 30°C in medium without NaCl, and maximal values of final biomass at growth temperatures between 10 and 20°C. Therefore, Carnobacterium strain 17-4 exhibits halotolerant and psychrotolerant behaviours. The solute pool contained glycine-betaine, the main solute used for osmoregulation, and an unknown compound whose structure was identified as α-glucopyranosyl-(1-3)-β-glucopyranosyl-(1-1)-α-glucopyranose (abbreviated as gluconeotrehalose), using nuclear magnetic resonance and mass spectrometry. This unusual solute consistently accumulated to high levels (0.35 ± 0.05 mg/mg cell protein) regardless of the growth temperature or salinity. The efficiency of gluconeotrehalose in the stabilisation of four model enzymes against heat damage was also assessed, and the effects were highly protein dependent. The lack of variation in the gluconeotrehalose content observed under heat stress, osmotic stress, and starvation provides no clue for the physiological role of this rare solute.     

外源甜菜碱提高恶臭假单胞菌(Pseudomonas putida)DLL-1耐盐性的研究

研究了外源甜菜碱对恶臭假单胞菌(Pseudomonas putida)DLL-1耐盐性的影响并对其渗透保护机制进行了初步的探讨;结果表明培养基中添加甜菜碱可以改善DLL-1细胞在高盐培养基中的生长情况,添加150mg/L的甜菜碱可以使DLL-1在1.2mol/L NaCl的基础盐培养基中生长,添加10mg/L的甜菜碱就足以显著缩短渗透胁迫条件下DLL-1细胞的延滞期和代时,增加生长量;和不添加对照相比,延滞期由24h缩短到6h,代时由60min缩短到35.7min,最大生长量OD610由1.29增长到1.57。在渗透胁迫条件下,细胞从外界快速吸收外源甜菜碱来代替自身相容性溶质的合成。