Relationships between the osmoadaptation strategy, amino acid composition of bulk protein, and properties of certain enzymes of haloalkaliphilic bacteria

Haloalkaliphilic microorganisms isolated from soda lakes were compared in terms of the amino acid composition of bulk protein and the reaction of a number of key enzymes to salts and pH of the medium. In the extremely haloalkaliphilic bacterium Natroniella acetigena (selt-in osmoadaptation strategy), acidic amino acids (glutamic and aspartic) made up 30.91 mol % of the total of bulk protein amino acids. In the moderate haloalkaliphiles Tindallia magadiensis, Halomonas campisalis, and Halomonas sp. AIR-2 (compatible-solutes osmoadaptation strategy), the proportion of acidic amino acids (24.36, 23.15, and 23.58 mol %, respectively) was lower than in N. acetigena but higher than in the freshwater Acetobacterium paludosum (20.77 mol %). The excess of acidic amino acids over basic amino acids (lysine and arginine) increased with the degree of halophily. The enzymes of haloalkaliphiles proved to be tolerant to salts and high pH values, although the degree of tolerance varied. The activity of N. acetigena CO dehydrogenase was maximum in the presence of 0.7 M NaCl, but it was virtually independent of the NaHCO₃ concentration. The hydrogenase and CO dehydrogenase of T. magadiensis exhibited maximum activity in the absence of NaCl; the Co dehydrogenase was most active at 0.25 M NaHCO₃, and hydrogenase activity was only weakly dependent on NaHCO₃ in the concentration range of 0–1.2 M. The nitrate reductases of H. campisalis and Halomonas sp. AIR-2 were active in broad ranges of NaCl and KCl concentrations; the activity maxima were recorded at moderate concentrations of these salts. The pH optima of most of the studied enzymes of haloalkaliphiles were in the alkaline zone. Thus, it was shown that the amino acid composition of bulk protein is determined by the osmoadaptation strategy employed by the bacterium. A correlation was found between the salt tolerance of enzymes and the proportion of acidic amino acids in the bulk protein. The ability of enzymes to function at high pH values is one of the mechanisms of adaptation of microorganisms to high pH values.     

Osmoadaptation in representatives of haloalkaliphilic bacteria from soda lakes

The adaptation of microorganisms to life in brines allows two strategies: the accumulation of organic osmoregulators in the cell (as in many moderate halophiles, halomonads in particular) or the accumulation of inorganic ions at extremely high intracellular concentrations (as, for example, in haloanaerobes). To reveal the regularities of osmoregulation in haloalkaliphiles developing in soda lakes, Halomonas campisalis Z-7398-2 and Halomonas sp. AIR-2 were chosen as representatives of halomonads, and Natroniella acetigena, as a representative of haloanaerobes. It was established that, in alkaliphilic halomonads, the intracellular concentrations of inorganic ions are insufficient for counterbalancing the environmental osmotic pressure and balance is attained due to the accumulation of organic osmoregulators, such as ectoine and betaine. On the contrary, the alkaliphilic haloanaerobe N. acetigena employs K+, Na+, and Cl- ions for osmoregulation. High intracellular salt concentrations increasing with the content of Na+ in the medium were revealed in this organism. At a concentration of 1.91 M Na+ in the medium, N. acetigena accumulated 0.83 M K+, 0.91 M Na+, and 0.29 M Cl- in cells, and, with an increase in the Na+ content in the medium to 2.59 M, it accumulated 0.94 M K+, 1.98 M Na+, and 0.89 M Cl-, which counterbalanced the external osmotic pressure and provided for cell turgor. Thus, it was shown that alkaliphilic microorganisms use osmoregulation strategies similar to those of halophiles and these mechanisms are independent of the mechanism of pH homeostasis.     

Response of Halomonas campisalis to saline stress: changes in growth kinetics, compatible solute production and membrane phospholipid fatty acid composition

The haloalkaliphile Halomonas campisalis, isolated near Soap Lake, Washington, was grown under both aerobic and denitrifying conditions from 0 to 260 g L(-1) NaCl, with optimal growth occurring at 20 and 30 g L(-1) NaCl, respectively. Halomonas campisalis was observed to produce high concentrations of compatible solutes, most notably ectoine (up to 500 mM within the cytoplasm), but hydroxyectoine and glycine betaine were also detected. The types and amounts of compatible solutes produced depended on salinity and specific growth rate, as well as on the terminal electron acceptor available (O(2) or NO(3) (-)). A decrease in ectoine production was observed with NO(3) (-) as compared with O(2) as the terminal electron acceptor. In addition, changes in the phospholipid fatty acid composition were measured with changing salinity. An increase in trans fatty acids was observed in the absence of salinity, and may be a response to membrane instability. Cyclic fatty acids were also observed to increase, both in the absence of salinity, and at very high salinities, indicating cell stress at these conditions.     

Halomonas campisalis, an obligatorily alkaliphilic, nitrous oxide-reducing denitrifier with a Mo-cofactor-lacking nitrate reductase

We isolated eight strains of denitrifying bacteria that reduce nitrate and nitrous oxide at pH 10 from Lake Magadi (Kenya). Despite certain differences between the strains, they are similar in terms of G+C content (66.1-68.1 mol %) and DNA-DNA homology (75-92%) and represent different morphotypes of the same species. Based on the results of partial 16S rRNA sequencing, strain Z-7398-2 was most closely related to the Halomonas campisalis isolate from Alkali Lake (USA). The DNA-DNA homology level between the tested strain and the type strain of H. campisalis 4A was 88%. These two strains were also similar phenotypically. However, the culture isolated by us was characterized by peculiar properties, such as obligate alkaliphily, which manifested itself in the culture dependence on carbonates and lack of growth at pH values below 7, a nitrous oxide-reducing capacity, and an unusual nitrate reductase that lacked molybdenum and a Mo cofactor.     

Cloning, expression, purification and activation by Na ion of halophilic alkaline phosphatase from moderate halophile Halomonas sp. 593

We have succeeded in the cloning of alkaline phosphatase gene, haalp, from moderate halophile Halomonas sp. 593. A deduced amino acid sequence showed a high ratio of acidic to basic amino acids, characteristic of halophilic proteins. The gene product was efficiently expressed in Escherichia coli BL21 Star (DE3) pLysS, but in an inactive form. The purified recombinant HaALP was separated into four fractions by gel filtration. When they were dialyzed against 50 mM Tris-HCl (pH 8.0)/2 mM MgCl? buffer containing 3 M NaCl, one of these four fractions was activated to almost full activity. This fraction contained a folding intermediate that was converted to the native structure by the salt. Among the additional salts tested, i.e., KCl, KBr, LiCl, MgCl?, (NH?)?SO?, and Na?SO?, only Na?SO? was effective, suggesting the importance of Na ion.     

rHsp90 gene expression in response to several environmental stresses in rice (Oryza sativa L.).

In this study, the gene for a rice (Oryza sativa L.) 90 kDa heat shock protein (rHsp90, GenBank accession no. AB037681) was identified by screening rice root cDNAs that were up-regulated under carbonate (NaHCO(3)) stress using the method of differential display, and cloned. The open-reading-frame of rHsp90-cDNA was predicted to encode a protein containing 810 amino acids, which showed high similarity to proteins in Hordeum vulgare (accession no. X67960) and Catharathus roseus (accession no. L14594). Further studies showed that rHsp90 mRNA accumulated following exposure to several abiotic stresses, including salts (NaCl, NaHCO(3) and Na(2)CO(3)), desiccation (using polyethylene glycol), high pH (8.0 and 11.0) and high temperature (42 and 50 degrees C). Yeast (Saccharomyces cerevisiae) over-expressing rHsp90 exhibited greater tolerance to NaCl, Na(2)CO(3) and NaHCO(3) and tobacco seedlings over-expressing rHsp90 could tolerate salt concentrations as high as 200 mM NaCl, whereas untransformed control seedlings couldn't. These results suggest that rHsp90 plays an important role in multiple environmental stresses.     

Halomonas campaniensis sp. nov., a haloalkaliphilic bacterium isolated from a mineral pool of Campania Region, Italy

A Gram-negative, aerobic, motile and rod-shaped haloalkaliphilic bacterial strain 5AGT (DSM 15293 and ATCC BAA-966) was isolated from water with algal mat of a mineral pool in Malvizza site (Campania-Italy) and was subjected to a polyphasic study. The isolate grew at temperature of 10.0-43.0 degrees C with an optimum at 37.0 degrees C. Strain 5AGT grew optimally in the presence of 10% NaCl and grew also in the absence of salt. The isolate grew in the pH range 7.0-10.0 with an optimum at pH 9.0. It accumulated glycine-betaine, ectoine, and glutamate, as osmoprotectants. Strain 5AGT was also characterized chemotaxonomically by having ubiquinone-8 (Q8) as the predominant isoprenoid quinone, phosphoethanolamine (PEA), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG), as major polar lipids and aiC16:0 and C18:1cis as the major fatty acids. The DNA G+C content was 63.7mol%. Phylogenetic analyses based on 16S rRNA gene sequence showed that the isolate belonged to the genus Halomonas. The DNA-DNA hybridization of the type strain 5AGT with the most related Halomonas campisalis showed a re-association value of 35.0%.On the basis of phenotypic properties and phylogeny, strain 5AGT should be placed in the genus Halomonas as a member of a novel species for which we propose the name Halomonas campaniensis sp. nov.     

Halomonas mongoliensis sp. nov. and Halomonas kenyensis sp. nov., new haloalkaliphilic denitrifiers capable of reducing N2O, isolated from soda lakes

In the course of the search for N2O-utilizing microorganisms, two novel strains of haloalkaliphilic denitrifying bacteria, Z-7009 and AIR-2, were isolated from soda lakes of Mongolia and Kenya. These microorganisms are true alkaliphiles and grow in the pH ranges of 8.0-10.5 and 7.5-10.6, respectively. They are facultative anaerobes with an oxidative type of metabolism, able to utilize a wide range of organic substrates and reduce nitrate, nitrous oxide, and, to a lesser extent, nitrite to gaseous nitrogen. They can oxidize sulfide in the presence of acetate as the carbon source and nitrous oxide (strain Z-7009) or nitrate (strain AIR-2) as the electron acceptor. The strains require Na+ ions. They grow at medium mineralization levels of 0.16-2.2 M Na+ (Z-7009) and 0.04-2.2 M Na+ (AIR-2). The G+C contents of the DNA of strains Z-7009 and AIR-2 are 67.9 and 65.5 mol %, respectively. According to the results of 16S rRNA gene sequencing and DNA-DNA hybridization, as well as on the basis of physiological properties, the strains were classified as new species of the genus Halomonas: Halomonas mongoliensis, with the type strain Z-7009T (=DSM 17332, =VKM B2353), and Halomonas kenyensis, with the type strain AIR-2T (=DSM 17331, =VKM B2354).     

Amino acid composition of bulk protein and salt relationships of selected enzymes of Salinibacter ruber,an extremely halophilic bacterium

The extremely halophilic bacterium Salinibacter ruber was previously shown to have a high intracellular potassium content, comparable to that of halophilic Archaea of the family Halobacteriaceae. The amino acid composition of its bulk protein showed a high content of acidic amino acids, a low abundance of basic amino acids, a low content of hydrophobic amino acids, and a high abundance of serine. We tested the level of four cytoplasmic enzymatic activities at different KCl and NaCl concentrations. Nicotinamide adenine dinucleotide (NAD)-dependent isocitrate dehydrogenase functioned optimally at 0.5-2 M KCl, with rates of 60% of the optimum value at 3.3 M. NaCl provided less activation: 70% of the optimum rates in KCl were found at 0.2-1.2 M NaCl, and above 3 M NaCl, activity was low. We also detected nicotinamide adenine dinucleotide phosphate (NADP)-dependent isocitrate activity, which remained approximately constant between 0-3.2 M NaCl and increased with increasing KCl concentration. NAD-dependent malate dehydrogenase functioned best in the absence of salt, but rates as high as 25% of the optimal values were measured in 3-3.5 M KCl or NaCl. NAD-dependent glutamate dehydrogenase, assayed by the reductive amination of 2-oxoglutarate, showed low activity in the absence of salt. NaCl was stimulatory with optimum activity at 3-3.5 M. However, no activity was found above 2.5 M KCl. Although the four activities examined all function at high salt concentrations, the behavior of individual enzymes toward salt varied considerably. The results presented show that Salinibacter enzymes are adapted to function in the presence of high salt concentrations.     

Is the alkaline tide a signal to activate metabolic or ionoregulatory enzymes in the dogfish shark (Squalus acanthias)?

Experimental metabolic alkalosis is known to stimulate whole-animal urea production and active ion secretion by the rectal gland in the dogfish shark. Furthermore, recent evidence indicates that a marked alkaline tide (systemic metabolic alkalosis) follows feeding in this species and that the activities of the enzymes of the ornithine-urea cycle (OUC) for urea synthesis in skeletal muscle and liver and of energy metabolism and ion transport in the rectal gland are increased at this time. We therefore evaluated whether alkalosis and/or NaCl/volume loading (which also occurs with feeding) could serve as a signal for activation of these enzymes independent of nutrient loading. Fasted dogfish were infused for 20 h with either 500 mmol L(-1) NaHCO3 (alkalosis + volume expansion) or 500 mmol L(-1) NaCl (volume expansion alone), both isosmotic to dogfish plasma, at a rate of 3 mL kg(-1) h(-1). NaHCO3 infusion progressively raised arterial pH to 8.28 (control = 7.85) and plasma [HCO3-] to 20.8 mmol L(-1) (control = 4.5 mmol L(-1)) at 20 h, with unchanged arterial P(CO2), whereas NaCl/volume loading had no effect on blood acid-base status. Rectal gland Na+,K+-ATPase activity was increased 50% by NaCl loading and more than 100% by NaHCO3 loading, indicating stimulatory effects of both volume expansion and alkalosis. Rectal gland lactate dehydrogenase activity was elevated 25% by both treatments, indicating volume expansion effects only, whereas neither treatment increased the activities of the aerobic enzymes citrate synthase, NADP-isocitrate dehydrogenase, or the ketone body-utilizing enzyme beta-hydroxybutyrate dehydrogenase in the rectal gland or liver. The activity of ornithine-citrulline transcarbamoylase in skeletal muscle was doubled by NaHCO3 infusion, but neither treatment altered the activities of other OUC-related enzymes (glutamine synthetase, carbamoylphosphate synthetase III). We conclude that both the alkaline tide and salt loading/volume expansion act as signals to activate some but not all of the elevated metabolic pathways and ionoregulatory mechanisms needed during processing of a meal.     

<Emphasis Type="Italic">Halomonas mongoliensis</Emphasis> sp. nov. and <Emphasis Type="Italic">Halomonas kenyensis</Emphasis> sp. nov., new haloalkaliphilic denitrifiers capable of N<Subscript>2</Subscript>O reduction,isolated from soda lakes

In the course of the search for N₂O-utilizing microorganisms, two novel strains of haloalkaliphilic denitrifying bacteria, Z-7009 and AIR-2, were isolated from soda lakes of Mongolia and Kenya. These microorganisms are true alkaliphiles and grow in the pH ranges of 8.0–10.5 and 7.5–10.6, respectively. They are facultative anaerobes with an oxidative type of metabolism, able to utilize a wide range of organic substrates and reduce nitrate, nitrous oxide, and, to a lesser extent, nitrite to gaseous nitrogen. They can oxidize sulfide in the presence of acetate as the carbon source and nitrous oxide (strain Z-7009) or nitrate (strain AIR-2) as the electron acceptor. The strains require Na⁺ ions. They grow at 0.16–2.2 M Na⁺ (Z-7009) and 0.04–2.2 M Na⁺ (AIR-2) in the medium. The G+C contents of the DNA of strains Z-7009 and AIR-2 are 67.9 and 65.5 mol %, respectively. According to the results of 16S rRNA gene sequencing and DNA-DNA hybridization, as well as on the basis of physiological properties, the strains were classified as new species of the genus Halomonas: Halomonas mongoliensis, with the type strain Z-7009^T (=DSM 17332, =VKM B2353), and Halomonas kenyensis, with the type strain AIR-2^T (=DSM 17331, =VKM B2354).     

Regulation of carbon monoxide dehydrogenase and hydrogenase in Rhodospirillum rubrum: effects of CO and oxygen on synthesis and activity.

Exposure of the photosynthetic bacterium Rhodospirillum rubrum to carbon monoxide led to increased carbon monoxide dehydrogenase and hydrogenase activities due to de novo protein synthesis of both enzymes. Two-dimensional gels of [35S]methionine-pulse-labeled cells showed that induction of CO dehydrogenase synthesis was rapidly initiated (less than 5 min upon exposure to CO) and was inhibited by oxygen. Both CO dehydrogenase and the CO-induced hydrogenase were inactivated by oxygen in vivo and in vitro. In contrast to CO dehydrogenase, the CO-induced hydrogenase was 95% inactivated by heating at 70 degrees C for 5 min. Unlike other hydrogenases, this CO-induced hydrogenase was inhibited only 60% by a 100% CO gas phase.     

长穗薄冰草（Thinopyrum ponticum）对盐碱胁迫的生理响应

实验采用不同浓度（以干土重0.3%、0.5%、0.7%和0.9%计）Na2SO4、NaCl、NaHCO3、Na2CO3,Na2SO4：NaCl=1：1、NaHCO3：Na2CO3=1：1和Na2SO4：NaCl：NaHCO3：Na2CO3=1：1：1：1对长穗薄冰草进行胁迫,研究长穗薄冰草对盐碱胁迫的生理响应.结果表明：根系活力、SOD活性随盐碱浓度的增加逐渐下降,脯氨酸含量呈上升趋势,叶绿素含量除Na2SO4处理组外,呈先上升后下降的趋势.其中碱性盐（NaHCO3、Na2CO3、NaHCO3：Na2CO3=1：1、Na2SO4：NaCl：NaHCO3：Na2CO3=1：1：1：1）对长穗薄冰草的胁变效应较中性盐（Na2SO4、NaCl、Na2SO4：NaCl=1：1）大,以Na2SO4处理组对长穗薄冰草的影响最小,0.7% Na2CO3是长穗薄冰草正常生长的临界浓度.     

Development of a minimally defined medium for the acetogen Clostridium thermoaceticum

A minimally defined medium was developed for the cultivation of the acetogen Clostridium thermoaceticum. The medium contained glucose as the carbon and energy source, ammonium sulfate as the nitrogen source, nicotinic acid as the sole essential vitamin, reductant, a phosphate-bicarbonate buffer, mineral salts and chelator, and a CO2 gas phase. Adaptation of C. thermoaceticum from undefined medium containing yeast extract and tryptone to the minimally defined medium required sequential passage on defined medium supplemented with amino acids and vitamins. Growth and cell yields were reduced on the minimal medium, but the activities of carbon monoxide dehydrogenase, hydrogenase, and formate dehydrogenase were comparable between undefined and minimal media.     

Oxidation of Carbon Monoxide in Cell Extracts of Pseudomonas carboxydovorans

Extracts of aerobically, CO-autotrophically grown cells of Pseudomonas carboxydovorans were shown to catalyze the oxidation of CO to CO(2) in the presence of methylene blue, pyocyanine, thionine, phenazine methosulfate, or toluylene blue under strictly anaerobic conditions. Viologen dyes and NAD(P)(+) were ineffective as electron acceptors. The same extracts catalyzed the oxidation of formate and of hydrogen gas; the spectrum of electron acceptors was identical for the three substrates, CO, formate, and H(2). The CO- and the formate-oxidizing activities were found to be soluble enzymes, whereas hydrogenase was membrane bound exclusively. The rates of oxidation of CO, formate, and H(2) were measured spectrophotometrically following the reduction of methylene blue. The rate of carbon monoxide oxidation followed simple Michaelis-Menten kinetics; the apparent K(m) for CO was 45 muM. The reaction rate was maximal at pH 7.0, and the temperature dependence followed the Arrhenius equation with an activation energy (DeltaH(0)) of 35.9 kJ/mol (8.6 kcal/mol). Neither free formate nor hydrogen gas is an intermediate of the CO oxidation reaction. This conclusion is based on the differential sensitivity of the activities of formate dehydrogenase, hydrogenase, and CO dehydrogenase to heat, hypophosphite, chlorate, cyanide, azide, and fluoride as well as on the failure to trap free formate or hydrogen gas in coupled optical assays. These results support the following equation for CO oxidation in P. carboxydovorans: CO + H(2)O --> CO(2) + 2 H(+) + 2e(-) The CO-oxidizing activity of P. carboxydovorans differed from that of Clostridium pasteurianum by not reducing viologen dyes and by a pH optimum curve that did not show an inflection point.     

盐碱混合生态条件的人工模拟及其对羊草胁迫作用因素分析

将中性盐NaCl和Na2SO4,碱性盐NaHCO3和Ｎa2CO3按不同比例混合,模拟出３０种盐度和pH各不相同的盐碱生态条件,并对羊草苗进行盐碱混合胁迫处理,测定其日相对生长率（ＲＧＲ）等７项胁变指标,用数学方法分析盐度,缓冲量等各种胁迫因素与诸项胁变指标间的相互关系,结果表明：30种处理均匀覆盖了总盐度50-350mmol/L,pH7.14-10.81范围内的各种盐碱条件,用盐度,缓冲量,pH和[Cl^-]即可代表盐碱混合胁迫的所有胁迫作用因素,诸胁变指标与这4因素间均具有高度线性相关性,4因素对胁变的贡献明显不同,其中缓冲量和盐度是决定性的主导因素,pH和[Cl^-]的作用明显次之,有时甚至可以忽略,不同胁变指标与各因素的关系也有不同。分析结果表明,对于盐碱混合胁迫来说,以盐度加缓冲量代表总胁强较为合理。     

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.     

Properties of purified carbon monoxide dehydrogenase from Clostridium thermoaceticum, a nickel, iron-sulfur protein

Carbon monoxide dehydrogenase from Clostridium thermoaceticum has been purified to homogeneity using a strict anaerobic procedure. The enzyme has a molecular weight of about 440,000 and it consists of three each of two different subunits giving the composition alpha 3 beta 3. The molecular weight of the alpha-subunit is 78,000 and that of the beta-subunit is 71,000. Pore limit gel electrophoresis gave a molecular weight of 161,000 indicating that the enzyme dissociates to form a dimer with an alpha beta structure. The dimer apparently contains per mol 2 nickel, 1 zinc, 11 iron, and 14 acid-labile sulfur. The anaerobic enzyme has an iron-sulfur type spectrum, which is changed in the presence of the substrate, CO. In the presence of oxygen, which destroys the activity or CO2, the spectrum is that of a typical iron-sulfur protein. Under acidic conditions a low molecular weight nickel factor separates from the enzyme. Viologens, methylene blue, ferredoxin, flavodoxin, and rubredoxin serve as electron acceptors. Of these rubredoxin is by far the most efficient. The enzyme has a pH optimum around 8.4. At this pH and 50 degrees C under 100% CO atmosphere, the apparent Km for methyl viologen is 3.03 mM and Vmax is 750 mumols of CO oxidized min-1 mg-1. Cyanide and methyl iodide inhibit the enzyme. CO reverses the cyanide inhibition but promotes the reaction with methyl iodide. The pure enzyme has no hydrogenase or formate dehydrogenase activity.