Switching osmolyte strategies: response of Methanococcus thermolithotrophicus to changes in external NaCl

Methanococcus thermolithotrophicus, a thermophilic methanogenic archaeon, produces and accumulates beta-glutamate and L-alpha-glutamate as osmolytes when grown in media with <1 M NaCl. When the organism is adapted to grow in >1 M NaCl, a new zwitterionic solute, N(epsilon)-acetyl-beta-lysine, is synthesized and becomes the dominant osmolyte. Several techniques, including in vivo and in vitro NMR spectroscopy, HPLC analyses of ethanol extracts, and potassium atomic absorption, have been used to monitor the immediate response of M. thermolithotrophicus to osmotic stress. There is a temporal hierarchy in the response of intracellular osmolytes. Changes in intracellular K(+) occur within the first few minutes of altering the external NaCl. Upon hypoosmotic shock, K(+) is released from the cell; relatively small changes occur in the organic osmolyte pool on a longer time scale. Upon hyperosmotic shock, M. thermolithotrophicus immediately internalizes K(+), far more than would be needed stoichiometrically to balance the new salt concentration. This is followed by a decrease to a new K(+) concentration (over 10-15 min), at which point synthesis and accumulation of primarily L-alpha-glutamate occur. Once growth of the M. thermolithotrophicus culture begins, typically 30-100 min after the hyperosmotic shock, the intracellular levels of organic anions decrease and the zwitterion (N(epsilon)-acetyl-beta-lysine) begins to represent a larger fraction of the intracellular pool. The observation that N(epsilon)-acetyl-beta-lysine accumulation occurs in osmoadapted cells but not immediately after osmotic shock is consistent with the hypothesis that lysine 2,3-aminomutase, an enzyme involved in N(epsilon)-acetyl-beta-lysine synthesis, is either not present at high levels or has low activity in cells grown and adapted to lower NaCl. That lysine aminomutase specific activity is 8-fold lower in protein extracts from cells adapted to low NaCl compared to those adapted to 1.4 M NaCl supports this hypothesis.     

Effect of pH on aluminum-driven methanogenesis by Methanococcus thermolithotrophicus

Methanogenesis from various elemental metals as electron sources has been demonstrated before. In this study, we have examined the influence of pH on the methanogenic activity of Methanococcus thermolithotrophicus dependent on cathodic hydrogen produced by elemental aluminum wires. When grown on H₂+CO₂, M. thermolithotrophicus had an optimum pH of 6.2, but when all the H₂ was supplied from A1°, the pH optimum was 5.7, consistent with thermodynamic predictions. The results also indicated that aluminum is quite resistant to anaerobic corrosion when compared to iron, most likely due to adhesion of aluminum oxide or hydroxide layers on the surface of the wires. Correspondence to: R. Boopathy     

13C- and 1H-NMR studies of osmoregulation in Dunaliella

Utilizing ¹³C-labeled algae, and ¹³C- and ¹H-NMR techniques, the following was shown. (a) Dunaliella salina grown at 1.5 M NaCl contains, intracellularly, approx. 1.9 M glycerol, which is osmotically equivalent to 1.25 M NaCl. Other NMR-observed soluble metabolites accounted for the remaining 0.25 M salt-equivalent. (b) The other observed soluble metabolites were dihydroxyacetone, pyruvate, lactate, glucose, alanine and glutamate. (c) Mild heating of the cells released an α-(1 → 4)-glucan into the soluble fraction. (d) A major temporal decrease in glycerol concentration and an increase in α-(1 → 4)-glucan content were observed following a hypoosmotic shock, and the opposite effect following a hyperosmotic shock. Smaller changes in the content of the other soluble metabolites, primarily alanine and glutamate, were also observed. (e) Glycerol was not released into the medium during these osmoregulatory adjustments. Pathways are proposed which can account for the metabolic conversion of α-(1 → 4)-glucan to glycerol following a hypertonic shock, and of glycerol to α-(1 → 4)-glucan following a hypotonic shock.     

Pressure stabilization is not a general property of thermophilic enzymes: the adenylate kinases of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii.

The application of 50-MPa pressure did not increase the thermostabilities of adenylate kinases purified from four related mesophilic and thermophilic marine methanogens. Thus, while it has been reported that some thermophilic enzymes are stabilized by pressure (D. J. Hei and D. S. Clark, Appl. Environ. Microbiol. 60:932-939, 1994), hyperbaric stabilization is not an intrinsic property of all enzymes from deep-sea thermophiles.     

Effect of osmotic stress on protein turnover in Lemna minor fronds

Evidence is presented that although many proteins from the fronds of Lemna minor L. undergo enhanced degradation during osmotic stress, ribulose-1,5-bisphosphate carboxylase (RuBPCase) is not degraded. Instead RuBPCase is converted in a series of steps to a very high-molecular-weight form. The first step involves the induction of an oxidase system which after 24 h of stress converts RuBPCase to an acidic and catalytically inactive form. Subsequently, the oxidised RuBPCase protein is gradually polymerized to a number of very large aggregates (molecular weight of several million).The conversion of RuBPCase to a high-molecular-weight form appears to be correlated with (i) a reduction in the number of-SH residues and (ii) the susceptibility to in-vitro proteolysis. Indeed, the number of-SH groups per RuBPCase molecule decreases from 89 in the native enzyme to 54 and 22 in the oxidised and polymerized forms, respectively. On the other hand, the oxidised enzyme is more susceptible to in-vitro proteolysis than the native form. However, it is the polymerized form of RuBPCase which is particularly susceptible to in-vitro proteolysis.Western-blotting experiments and anti-ubiquitin antibodies were used to detect the presence of ubiquitin conjugates in extracts from osmotically stressed Lemna fronds. The possible involvement of ubiquitin in the formation of the aggregates is discussed.Abbreviations DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - FPLC fast protein liquid chromatography - kDa kilodaltons - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulphonyl fluoride - RuBPCase ribulose bisphosphate carboxylase - SDS sodium dodecyl sulphate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Energetics of the growth of Methanobacterium thermoautotrophicum and Methanococcus thermolithotrophicus on ammonium chloride and dinitrogen

Methanobacterium thermoautotrophicum was grown in continuous culture in a fermenter gassed with H₂ and CO₂ as sole carbon and energy sources, and in a medium which contained either NH₄Cl or gaseous N₂ as nitrogen source. Growth was possible with N₂. Steady states were obtained at various gas flow rates with NH₄Cl and with and the maintenance coefficient varied with the gas input and with the nitrogen source. Growth of Methanococcus thermolithotrophicus in continuous culture in a fermenter gassed with H₂, CO₂ as nitrogen, carbon and energy sources was also examined.Abbreviations molecular growth yield (g dry weight of cells per mol of CH₄ evolved) - growth rate (h^-1) - D dilution rate (h^-1) - rate (h_-1); relation of Neijssel and Tempest and of Stouthamer and Bettenhaussen - energy     

Growth of Methanococcus thermolithotrophicus in batch and continuous culture on H2 and CO2: influence of agitation

Summary A thermophilic methanogenic bacterium, Methanococcus thermolithotrophicus, was grown on H₂ and CO₂ in both batch and continuous culture, in a fermentor equipped with either a straight blade impeller or a Rushton impeller. Production was continued until 470 l CH₄·l^-1 per day was obtained with a biomass of 3.5 g dry wt. l^-1 under batch conditions.     

1H-NMR studies on substance P hexapeptide analogs

¹H-nmr studies of [pGlu⁶]SP_6–11, [gpGlu⁶,mPhe⁷]SP_6–11, and [pGlu⁶,N-CH₃Phe⁷]SP_6–11 in DMSO-d₆ reveal characteristic chemical shifts, ³J_NH-αCH, temperature dependence, as well as deuterium exchange half-times. Marked similarities are revealed for the two first analogs, whereas the N-methylated analog is clearly different. Possible conformations are considered.     

Conformational studies of 13 trinucleoside bisphosphates by 360-MHz 1H-NMR spectroscopy. 1. Ribose protons

The ribose protons of 13 trinucleoside bisphosphates (trimers) were studied, using 360-MHz proton nuclear magnetic resonance spectroscopy. Complete assignments and analyses of the NMR signals of these protons were carried out by the methods of homonuclear decoupling and computer line-shape simulations. It was shown that the trinucleotides preferred the anti, 3' endo, gamma +, beta t and epsilon t/epsilon- conformations for the glycosidic torsions, the ribose rings, the C4'-C5' bonds, the C5'-O5' bonds, and the C3'-O3' bonds, respectively. It was also found that the trimers, especially those which had noticeable population of 'bulged' structures, did not necessarily have a higher population of these preferred local conformations than their component dimers. The overall conformations of the trinucleotides are classified into two categories. The conformations in the first category involve the nearest-neighbor interactions. Each dinucleotide moiety can assume one of the four stable conformations (I, I', II and III) or the open forms of dinucleoside monophosphates. However, due to steric hindrance, there are only four cases in which both dinucleotide moieties can assume one of the four stable conformations at the same time. These four combinations of conformations are I-I, I'-I', I-II and III-I', where the first Roman numeral represents the conformation of the NpN'p-moiety and the second one, that of the -pN'pN' moiety of the trimers. Among them, I-I and I'-I' are helical structures, capable of forming a double helix. The second category contains conformations with bulged structures which have the two dinucleotide moieties in open forms (i.e. no nearest-neighbor interactions) and the bases of the two terminal residues stacking on each other while the middle residue is bulged out. These bulged conformations may serve as structural models for frame-shift mutations.     

Effects of osmotic stress on rabbit corneal endothelium

The effects of osmotic stress on corneal endothelium were investigated by exposing rabbit corneas to anisosmotic conditions, and then perfusing the corneas with isosmotic glutathione bicarbonate Ringer solution for 4 hr at 35 degrees C. During the perfusion, endothelial function was assessed by measuring corneal thickness with a specular microscope. After perfusion, the corneas were prepared for scanning and transmission electron microscopy. Endothelial ultrastructure and function were well maintained after exposure to a wide range of osmolality (0.12-2.7 osmol/kg), but this tolerance of osmotic stress was dependent both on the duration and the temperature of exposure to the anisosmotic conditions. Exposure to an osmolality of 2.7 osmol/kg for 15 min at 23 or 37 degrees C resulted in gross damage to the endothelium when the hyperosmotic agent was sodium chloride. This damage was not the result of increased osmolality per se nor cellular shrinkage because the endothelium tolerated exposure to a sucrose solution of the same osmolality for 15 min at 37 degrees C. The detrimental effect of sodium chloride, however, was mitigated by reducing the temperature of exposure to 0 degrees C or reducing the duration of exposure to 5 min. These results suggest that endothelial cells become more tolerant of high electrolyte concentrations with reducing temperature, and this could be an important factor in the survival of the endothelium in corneal cryopreservation. The results also help to define the limits of osmotic shrinkage and swelling tolerated by endothelial cells. This will be of value in overcoming the detrimental osmotic effects associated with the addition and, in particular, the removal of cryoprotectants.     

13C-NMR, 1H-NMR and gas-chromatography mass-spectrometry studies of the biosynthesis of 13C-enriched L-lysine by Brevibacterium flavum

The basic metabolic pathways of lysine biosynthesis in Brevibacterium flavum, a strain which excretes excessive amounts of L-lysine, have been followed by using two 13C-labeled precursors. 13C- and 1H-NMR spectroscopies in conjunction with gas chromatography mass spectrometry (GC-MS) have revealed the various metabolic pathways leading to L-[13C]lysine. Discrete metabolic pathways give rise to distinct labeling patterns. L-Lysine resulting from [1-13C]glucose fermentation is relatively specifically labeled: L-[3,5-13C]lysine is the main product. Experimental and theoretical approaches based on the 13C-enrichment values of intracellular glutamate, a major intermediate metabolite, allowed us to assess the relative contribution of the major metabolic pathways forming lysine. The labeling pattern of glutamate reflects the isotope distribution in 2-oxoglutarate. When [2-13C]acetate is used as the sole carbon source in the culture, the energy-producing steps of the Krebs cycle are essential. The higher activity of the Krebs cycle, when endogenous carbohydrates are exhausted from the culture, is indicated by the increased 13C enrichment in C-1 of lysine and reveal a high content of isotopomers of four, five and six 13C atoms in the lysine molecule, pointing out that the four-carbon intermediates of the cycle are being derived from the glyoxylate shunt pathway. Such a phenomenon does not occur in glucose fermentation. GC-MS analyses of 13C enrichments and isotopomer distributions in metabolites and end products are in good agreement with the predicted contribution of each metabolic pathway. This new methodological approach of combined NMR and GC-MS has been demonstrated to be applicable to various other metabolic studies.     

Four-dimensional 13C/13C-edited nuclear Overhauser enhancement spectroscopy of a protein in solution: application to interleukin 1 beta

A four-dimensional 13C/13C-edited NOESY experiment is described which dramatically improves the resolution of protein NMR spectra and enables the straightforward assignment of nuclear Overhauser effects involving aliphatic and/or aromatic protons in larger proteins. The experiment is demonstrated for uniformly (greater than 95%) 13C-labeled interleukin 1 beta, a protein of 153 residues and 17.4 kDa, which plays a key role in the immune response. NOEs between aliphatic and/or aromatic protons are first spread out into a third dimension by the 13C chemical shift of the carbon atom attached to the originating proton and subsequently into a fourth dimension by the 13C chemical shift of the carbon atom attached to the destination proton. Thus, each NOE cross peak is labeled by four chemical shifts. By this means, ambiguities in the assignment of NOEs that arise from chemical shift overlap and degeneracy are completely removed. Further, NOEs between protons with the same chemical shifts can readily be detected providing their attached carbon atoms have different 13C chemical shifts. The design of the pulse sequence requires special care to minimize the level of artifacts arising from undesired coherence transfer pathways, and in particular those associated with "diagonal" peaks which correspond to magnetization that has not been transferred from one proton to another.(ABSTRACT TRUNCATED AT 250 WORDS)     

水分胁迫对牛心朴子植株生长及渗透调节物质积累的影响

采用PVC管种植模拟土壤干旱的方法,研究了牛心朴子(Cymanchum komarovii)在水分胁迫下植株生长及渗透调节物质的积累情况。结果表明：牛心朴子植株地上部对土壤水分比较敏感,随水分胁迫程度的加强和胁迫时间的延长,植株生长显著变缓,直至停止生长,而根／冠比值则有所加大;可溶性糖是牛心朴子根系主要的渗透调节物质,随着土壤水分胁迫程度的加重,根系中的可溶性糖呈明显的增加趋势,叶中的可溶性糖则随胁迫的加重而呈下降趋势,说明在干旱胁迫下牛心朴子的同化产物大部分分配于根系之中;Pro在牛心朴子叶、茎、根的渗透调节中也起着重要作用,随土壤水分胁迫的加重,其在根、茎、叶中的积累明显增加;而无机离子在牛心朴子渗透调节过程中的作用很小。     

1H-NMR relaxation studies of glycopeptides: a dynamic structural investigation

The Glycopeptide Man5GlcNAc4Asn (ACCB2) in water solution has been studied by means of 1H NMR relaxation techniques in order to define molecular structure and dynamics. From the analysis of selective and non-selective proton relaxation rates of selected ACCB2 protons, a lack of internal mobility along the polysaccharide chain was observed. The presence of a conformationally well-defined molecular structure for ACCB2 is proposed.     

1H-NMR studies on poly(oxyethylene)-bound oligopeptides

Conformational studies on poly(oxyethylene)-bound homo-, oligo-, guest-host, and sequential peptides synthesized according to the liquid-phase method were carried out by means of ¹H-nmr spectroscopy. The solubilizing effect of the C-terminal polymeric support allowed a thorough investigation of the secondary structure in solution.     

The Drosophila inebriated-encoded neurotransmitter/osmolyte transporter: dual roles in the control of neuronal excitability and the osmotic stress response

Water reabsorption by organs such as the mammalian kidney and insect Malpighian tubule/hindgut requires a region of hypertonicity within the organ. To balance the high extracellular osmolarity, cells within these regions accumulate small organic molecules called osmolytes. These osmolytes can accumulate to a high level without toxic effects on cellular processes. Here we provide evidence consistent with the possibility that the two protein isoforms encoded by the inebriated (ine) gene, which are members of the Na+/Cl--dependent neurotransmitter/osmolyte transporter family, perform osmolyte transport within the Malpighian tubule and hindgut. We show that ine mutants lacking both isoforms are hypersensitive to osmotic stress, which we assayed by maintaining flies on media containing NaCl, KCl, or sorbitol, and that this hypersensitivity is completely rescued by high-level ectopic expression of the ine-RB isoform. We provide evidence that this hypersensitivity represents a role for ine that is distinct from the increased neuronal excitability phenotype of ine mutants. Finally, we show that each ine genotype exhibits a "threshold" [NaCl]: long-term maintenance on NaCl-containing media above, but not below, the threshold causes lethality. Furthermore, this threshold value increases with the amount of ine activity. These data suggest that ine mutations confer osmotic stress sensitivity by preventing osmolyte accumulation within the Malpighian tubule and hindgut.     

1H-NMR studies on the self-association of chloroquine in aqueous solution

The concentration dependences of 1H-NMR chemical shifts and spin-lattice relaxation rates were measured for chloroquine in aqueous solution. The weak self-association constant was evaluated according to a dimerization equilibrium with the formation of self-stacked adducts (Kd = 4.52 +/- 0.68 l mol-1). The motional correlation times were evaluated for the monomer and the dimer by measuring intramolecular dipolar cross-relaxation rates of aromatic vicinal protons (tau cm = 0.06 ns and tau cd = 0.26 ns). The geometry of the stacked dimer was elucidated by measuring intermolecular dipolar cross-relaxation rates and interpreted in terms of partial superposition of quinoline moieties.