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。     

中度嗜盐菌相容性溶质机制的研究进展

生活在高盐环境中的中度嗜盐菌不仅能抗衡外界的高渗透压胁迫,而且还能迅速适应短时间内的渗透冲击。为适应该环境,中度嗜盐菌依赖于一种被称为相容性溶质的物质,以执行渗透保护功能。这类物质属于极性的、易溶的和低分子量的有机化合物,其中包括糖类、氨基酸类、甜菜碱类和四氢嘧啶类等。中度嗜盐菌主要采用相容性溶质机制来适应盐环境。在此,就中度嗜盐菌的盐适应机理、相容性溶质的种类和特点,以及其作用的分子机制进行了阐述和讨论。     

First description of two moderately halophilic and psychrotolerant Mycoplasma species isolated from cephalopods and proposal of Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov

Two moderately halophilic and psychrotolerant new Mycoplasma species were isolated from common cephalopods. Three strains were isolated in pure culture from two individual European flying squid (Todarodes sagittatus), and two individual octopuses (Octopus vulgaris). The strains showed optimal growth at 25 °C and a salinity of 3% (w/v) NaCl. Molecular analyses revealed that the isolates belonged to two new, but phylogenetically related species, divergent from all previously described Mollicutes, representing the first marine isolates of the class, and also the first Mycoplasma strains for which NaCl requirement has been demonstrated. A genome search against all available marine metagenomes and 16S rRNA gene databases indicated that these two species represent a novel non-free-living marine lineage of Mollicutes, specifically associated with marine animals. Morphology and physiology were compatible with other members of this group, and genomic and phenotypic analyses demonstrated that these organisms represent two novel species of the genus Mycoplasma, for which the names Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov. are proposed; the type strains are PE^T (DSM 105487^T, CIP 111404^T) and 5H^T (DSM 105,488^T, CIP 111405^T), respectively.     

中度嗜盐菌DTY1的鉴定及其耐盐机制的初步分析

菌株DTY1分离自山西省五寨县柠条种植区盐碱土壤,可在0~1·2mol/LNaCl的浓度培养基上生长,最适生长温度32℃,最适pH7~10。通过形态观察,生理生化测定与16SrDNA序列分析,将该菌株鉴定为嗜碱芽孢杆菌(Bacillusalcalophilus)。高压液相色谱分析,DTY1菌株在常规LB培养液中能够产生1·40mg/g四氢嘧啶,且在最适盐浓度条件下,盐浓度越高单位干重菌体所产生的四氢嘧啶含量越高。通过PCR介导的方法从DTY1的基因组文库中克隆到四氢嘧啶合成基因ectB。该基因长度为1284bp,编码427个氨基酸的肽链。此肽链与B.haloduransC-125(BAB04638)中二氨基丁酸氨基转移酶同源性达81%。ectB基因可能存在典型的σ70启动子,而且在启动子间有一段明显的23bp的回文序列。     

Community analysis and metabolic pathway of halophilic bacteria for phenol degradation in saline environment

A moderately halophilic bacterial enrichment was able to degrade 120 mg/L of phenol in the presence of 1–2 M of NaCl within 3 d or 2.5–3 M of NaCl within 6 d. The optimal degradation was achieved at 1.5 M of NaCl and 350 mg/L of phenol. PCR-DGGE profile of the enrichment showed that the Acidobacterium sp. and Chloroflexus sp. dominated the community. The phenol-biodegradation pathways consisted of an initial oxidative attack by phenol hydroxylase, and subsequent ring fission by catechol 1,2-dioxygenase and catechol 2,3-dioxygenase. Nuclear magnetic resonance (NMR) spectroscopy profiles showed that ectoine and hydroxyectoine were the main compatible solutes to adjust the bacterial osmotic pressure. This study provides further information on the understanding of phenol-degradation over a wide range of salinity and remediation of phenol as a pollutant in the environment.     

相容性溶质四氢嘧啶及其羟基化衍生物的研究进展

四氢嘧啶类化合物是嗜盐以及耐盐菌胞内合成的一类能够抵御外界高盐胁迫的相容性溶质,概述了四氢嘧啶及其衍生物的理化特征以及在嗜盐微生物中抵御外界高渗透压的作用机理,主要阐述了四氢嘧啶类相容性溶质的生物合成途径、膜运输机理、分泌释放机制、高密度发酵生产等方面在细胞、分子水平上的最新研究进展以及前景展望。并且综述了四氢嘧啶类在精细化工、生物医药及生物制造等行业的应用研究以及发展前景,探讨了未来的研究方向。     

Biomineralization of carbonate and phosphate by moderately halophilic bacteria

We investigated the precipitation of carbonate and phosphate minerals by 19 species of moderately halophilic bacteria using media with variable Mg(2+)/Ca(2+) ratios. The precipitated minerals were calcite, magnesium (Mg) calcite, and struvite (MgNH(4)PO(4) x 6H(2)O) in variable proportions depending on the Mg(2+)/Ca(2+) ratio of the medium. The Mg content of the Mg-calcite decreased with increasing Ca(2+) concentration in the medium. According to the saturation indices, other minerals could also have precipitated. We observed important differences between the morphology of carbonate and phosphate, which may help us to recognize these minerals in natural systems. We studied the growth and pH curves of four bacteria in media specific for carbonate and struvite precipitation. We consider the biomineralization processes that produce carbonate and phosphate minerals, and propose a hypothesis for the lack of struvite in natural environments and ancient rocks.     

The extremely halophilic bacterium Salicola marasensis IC10 accumulates the compatible solute betaine

The extremely halophilic bacterium strain IC10 was isolated from a solar saltern on Isla Cristina (southern Spain). Phylogenetic, genotypic and phenotypic data supported the inclusion of this strain in the species Salicola marasensis. An analysis of intracellular organic osmotic solutes showed glycine betaine to be present, contributing to the overall osmotic balance, and this was the only compatible solute accumulated when S. marasensis IC10 was grown over a wide range of external NaCl concentrations (10–25%, w/v).     

江苏盐城盐场中度嗜盐菌的分离与鉴定

采用高盐的牛肉膏蛋白胨培养基(盐浓度为8%NaCl),研究江苏省盐城市盐场土壤里中度嗜盐菌的分布情况及种群特征。从盐城市的射阳、新滩、灌东三处盐场土壤中共采集和分离得到13株中度嗜盐菌。通过形态观察、生理生化分析、16S rRNA序列分析和系统进化分析等方法进行初步鉴定,结果表明:分离到的中度嗜盐菌分属3个属,Virgibacillus属4株、Halomonas属7株和Marinobacter属2株。研究结果揭示盐城市的盐场存在较为丰富的中度嗜盐菌,具有较高的研究和利用价值。     

Salimicrobium salexigens sp. nov., a moderately halophilic bacterium from salted hides

Two Gram-positive, moderately halophilic bacteria, designated strains 29CMI^T and 53CMI, were isolated from salted hides. Both strains were non-motile, strictly aerobic cocci, growing in the presence of 3–25% (w/v) NaCl (optimal growth at 7.5–12.5% [w/v] NaCl), between pH 5.0 and 10.0 (optimal growth at pH 7.5) and at temperatures between 15 and 40 °C (optimal growth at 37 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed a similarity of 98.7% and were closely related to species of the genus Salimicrobium, within the phylum Firmicutes. Strains 29CMI^T and 53CMI exhibited 16S rRNA gene sequence similarity values of 97.9–97.6% with Salimicrobium album DSM 20748^T, Salimicrobium halophilum DSM 4771^T, Salimicrobium flavidum ISL-25^T and Salimicrobium luteum BY-5^T. The DNA G+C content was 50.7 mol% and 51.5 mol% for strains 29CMI^T and 53CMI, respectively. The DNA–DNA hybridization between both strains was 98%, whereas the values between strain 29CMI^T and the species S. album CCM 3517^T, S. luteum BY-5^T, S. flavidum ISL-25^T and S. halophilum CCM 4074^T were 45%, 28%, 15% and 10%, respectively, showing unequivocally that strains 29CMI^T and 53CMI constitute a new genospecies. The major cellular fatty acids were anteiso-C_15:0, anteiso-C_17:0, iso-C_15:0 and iso-C_14:0. The main respiratory isoprenoid quinone was MK-7, although small amounts of MK-6 were also found. The polar lipids of the type strain consist of diphosphatidylglycerol, phosphatidylglycerol, one unidentified phospholipid and one glycolipid. The peptidoglycan type is A1γ, with meso-diaminopimelic acid as the diagnostic diamino acid. On the basis of the phylogenetic analysis, and phenotypic, genotypic and chemotaxonomic characteristics, we propose strains 29CMI^T and 53CMI as a novel species of the genus Salimicrobium, with the name Salimicrobium salexigens sp. nov. The type strain is 29CMI^T (=CECT 7568^T = JCM 16414^T = LMG 25386^T).     

Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004

An extracellular protease was produced under stress conditions of high temperature and high salinity by a newly isolated moderate halophile, Salinivibrio sp. strain AF-2004 in a basal medium containing peptone, beef extract, glucose and NaCl. A modification of Kunitz method was used for protease assay. The isolate was capable of producing protease in the presence of sodium chloride, sodium sulfate, sodium nitrate, sodium nitrite, potassium chloride, sodium acetate and sodium citrate. The maximum protease was secreted in the presence of 7.5 to 10% (w/v) sodium sulfate or 3% (w/v) sodium acetate (4.6 U ml⁻¹). Various carbon sources including glucose, lactose, casein and peptone were capable of inducing enzyme production. The optimum pH, temperature and aeration for enzyme production were 9.0, 32 °C and 220 rpm, respectively. The enzyme production corresponded with growth and reached a maximum level during the mid-stationary phase. Maximum protease activity was exhibited in the medium containing 1% (w/v) NaCl at 60 °C, with 18% and 41% activity reductions at temperature 50 and 70 °C, respectively. The optimum pH for enzyme activity was 8.5, with 86% and 75% residual activities at pH 10 and 6, respectively. The activity of enzyme was inhibited by EDTA. These results suggest that the protease secreted by Salinivibrio sp. strain AF-2004 is industrially important from the perspectives of its activity at a broad pH ranges (5.0–10.0), its moderate thermoactivity in addition to its high tolerance to a wide range of salt concentration (0–10% NaCl).     

Production,optimization and purification of a novel extracellular protease from the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus karajensis</Emphasis>

The production of a protease was investigated under conditions of high salinity by the moderately halophilic bacterium Halobacillus karajensis strain MA-2 in a basal medium containing peptone, beef extract, maltose and NaCl when the culture reached the stationary growth phase. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed that the maximum secretion occurred at 34°C, pH 8.0–8.5, and in the presence of gelatin. Replacement of NaCl by various concentrations of sodium nitrate in the basal medium also increased the protease production. The secreted protease was purified 24-fold with 68% recovery by a simple approach including a combination of acetone precipitation and Q-Sepharose ion exchange chromatography. The enzyme revealed a monomeric structure with a relative molecular mass of 36 kDa by running on SDS-PAGE. Maximum caseinolytic activity of the enzyme was observed at 50°C, pH 9.0 and 0.5 M NaCl, although at higher salinities (up to 3 M) activity still remained. The maximum enzyme activity was obtained at a broad pH range of 8.0–10.0, with 55 and 50% activity remaining at pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), Pefabloc SC and EDTA; indicating that it probably belongs to the subclass of serine metalloproteases. These findings suggest that the protease secreted by Halobacillus karajensis has a potential for biotechnological applications from its haloalkaline properties point of view.     

Salinivibrio kushneri sp. nov., a moderately halophilic bacterium isolated from salterns

Ten Gram-strain-negative, facultatively anaerobic, moderately halophilic bacterial strains, designated AL184^T, IB560, IB563, IC202, IC317, MA421, ML277, ML318, ML328A and ML331, were isolated from water ponds of five salterns located in Spain. The cells were motile, curved rods and oxidase and catalase positive. All of them grew optimally at 37 °C, at pH 7.2–7.4 and in the presence of 7.5% (w/v) NaCl. Based on phylogenetic analyses of the 16S rRNA, the isolates were most closely related to Salinivibrio sharmensis BAG^T (99.6–98.2% 16S rRNA gene sequence similarity) and Salinivibrio costicola subsp. costicola ATCC 35508^T (99.0–98.1%). According to the MLSA analyses based on four (gyrB, recA, rpoA and rpoD) and eight (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA) concatenated gene sequences, the most closely relatives were S. siamensis JCM 14472^T (96.8–95.4% and 94.9–94.7%, respectively) and S. sharmensis DSM 18182^T (94.0–92.6% and 92.9–92.7%, respectively). In silico DNA–DNA hybridization (GGDC) and average nucleotide identity (ANI) showed values of 23.3–44.8% and 80.2–91.8%, respectively with the related species demonstrating that the ten isolates constituted a single novel species of the genus Salinivibrio. Its pangenome and core genome consist of 6041 and 1230 genes, respectively. The phylogeny based on the concatenated orthologous core genes revealed that the ten strains form a coherent phylogroup well separated from the rest of the species of the genus Salinivibrio. The major cellular fatty acids of strain AL184^T were C_16:0 and C_18:1. The DNA G + C content range was 51.9–52.5 mol% (T_m) and 50.2–50.9 mol% (genome). Based on the phylogenetic-phylogenomic, phenotypic and chemotaxonomic data, the ten isolates represent a novel species of the genus Salinivibrio, for which the name Salinivibrio kushneri sp. nov. is proposed. The type strain is AL184^T (= CECT 9177^T = LMG 29817^T).     

Characterization of exopolysaccharides produced by three moderately halophilic bacteria belonging to the family Alteromonadaceae

Aims:  To study the exopolysaccharides (EPSs) produced by three novel moderately halophilic species belonging to the family Alteromonadaceae to optimize EPS yields, characterize their physical and chemical properties and evaluate possible biotechnological applications for these polymers.
Methods and Results:  EPSs synthesized by Idiomarina fontislapidosi F32^T, Idiomarina ramblicola R22^T and Alteromonas hispanica F23^T were collected and analysed under optimum conditions: MY medium supplemented with 7·5% (w/v) salts; 32°C; and 1% (w/v) glucose. Polymers were synthesized mainly during the early stationary growth phase with yields ranging from 1 to 1·5 g l⁻¹. The Idiomarina species each produced an anionic EPS composed mainly of glucose, mannose and galactose. A. hispanica synthesized an anionic EPS composed mainly of glucose, mannose and xylose. Solutions of all the polymers were low in viscosity and pseudoplastic in their behaviour. They showed emulsifying activity and the capacity to bind some metals.
Conclusions:  The Alteromonadaceae species studied in this work produced EPSs with physical and chemical properties different from those produced by other halophilic and nonhalophilic bacteria, suggesting that the wide diversity of micro-organisms being encountered nowadays in hypersaline environments offers enormous potential resources for biotechnological applications.
Significance and Impact of the Study:  We have optimized the EPS production and analysed new biopolymers produced by some recently described, moderately halophilic bacteria. These biopolymers are chemically and physically different from others already in use in biotechnology and offer hopes for new applications, especially in the case of A. hispanica , which may prove to be a viable source of xylo-oligosaccharides.     

Partial characterization of an extracellular polysaccharide produced by the moderately halophilic bacterium Halomonas xianhensis SUR308

A moderately halophilic bacterium, Halomonas xianhensis SUR308 (Genbank Accession No. KJ933394) was isolated from a multi-pond solar saltern at Surala, Ganjam district, Odisha, India. The isolate produced a significant amount (7.87 g l^?1) of extracellular polysaccharides (EPS) when grown in malt extract–yeast extract medium supplemented with 2.5% NaCl, 0.5% casein hydrolysate and 3% glucose. The EPS was isolated and purified following the conventional method of precipitation and dialysis. Chromatographic analysis (paper, GC and GC-MS) of the hydrolyzed EPS confirmed its heteropolymeric nature and showed that it is composed mainly of glucose (45.74 mol%), galactose (33.67 mol %) and mannose (17.83 mol%). Fourier-transform infrared spectroscopy indicated the presence of methylene and carboxyl groups as characteristic functional groups. In addition, its proton nuclear magnetic resonance spectrum revealed functional groups specific for extracellular polysaccharides. X-ray diffraction analysis revealed the amorphous nature (CI_xrd, 0.56) of the EPS. It was thermostable up to 250°C and displayed pseudoplastic rheology and remarkable stability against pH and salts. These unique properties of the EPS produced by H. xianhensis indicate its potential to act as an agent for detoxification, emulsification and diverse biological activities.     

The effect of compatible solutes on proteins

Summary The ability of the compatible solute, proline, to affect the behavior of proteins has been examined in many different systems by many researches. In the present study of protein solvation, proline has been shown to prevent or diminish, in a concentration-dependent manner, the glutamine synthetase-precipitating ability of polyethylene glycol (PEG). The effects of PEG concentration and molecular weight are reduced by proline, and the interaction is strongly affected by pH.PEG causes precipitation of many proteins, and the ability of proline to reduce the precipitation of two non-enzymatic conjugated proteins, alfalfa mosaic virus and an³H-testosterone/antiserum complex, was also examined. Proline was effective in reducing the PEG-induced precipitation of both proteins. Virus precipitation by PEG and its alleviation by proline are influenced by pH. The increased virus-precipitating effect of PEG in the presence of salt (NaCl) is also alleviated by proline. The precipitation of the radioimmune complex by PEG is diminished by proline and by a mixture of free amino acids.These results indicate the generality of the three-way interaction between proline, protein and PEG. They may be of importance for extraction of proteins from biological systems and in studies of enzyme inactivation or protein denaturation in a cytoplasmic milieu. The results suggest that the protective effects of some amino acids are at least additive and are consistent with the conclusion that the compatible solutes protect protein-containing systems against the unfavorable consequences of dehydration and other stresses, by increasing the tendency of the system to maintain thestatus quo.     

嗜盐细菌中四氢嘧啶和羟基四氢嘧啶的生物合成及其生物学功能

在嗜盐细菌盐适应中,四氢嘧啶(1,4,5,6-四氢-2-甲基-4-嘧啶羧酸)和羟基四氢嘧啶(1,4,5,6-四氢-2-甲基-5-羟基-4-嘧啶羧酸)发挥着十分重要的作用.四氢嘧啶的生物合成以L-天冬氨酸-β-半醛(ASA)为底物,依次由2,4.二氨基丁酸转氨酶(EctB),2,4--氨基丁酸乙酰基转移酶(EctA)和四...     

Halomonas alkaliantarctica sp. nov., isolated from saline lake Cape Russell in Antarctica, an alkalophilic moderately halophilic, exopolysaccharide-producing bacterium

The taxomony of strain CRSS (DSM 15686(T)=ATCC BAA-848(T)) isolated from Cape Russell in Antarctica (Ross Sea, 74 52.35 S 163 53.03 E) was investigated in a polyphasic approach. The morphological, physiological and genetic characteristics were compared with that of related species of the genus Halomonas. The isolate grew optimally at pH 9.0, 10% NaCl at 30 degrees C. The cells were Gram-negative aerobic rods able to produce exopolysaccharide. They accumulated glycine-betaine, as a major osmolyte, with minor components ectoine and glutamate. The strain CRSS biosynthetised alpha-glucosidase. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol as major components. Ubiquinone with nine repetitive unities (Q9) was the only quinone found and the fatty acid composition was dominated by C18:1 (53%). The G+C content of DNA was 55.0mol% and its phylogenetic position was established by 16S rRNA gene sequencing as a member of the genus Halomonas. For physiological, chemotaxonomic and genetic features (DNA-DNA hybridisation) it is proposed to classify the isolate as a new species for which we propose the name Halomonas alkaliantarctica sp. nov.     

Fate of compatible solutes during dilution stress in Ectothiorhodospira halochloris

Abstract Ectothiorhodospira halochloris reacts upon enhancement of the water activity in the environment by excreting its major compatible solute, glycine betaine, thus decreasing the osmotic pressure inside the cell. A suddenly induced dilution stress leads to an overshoot of this reaction, so that more glycine betaine than necessary to compensate the external osmotic change is released. Subsequently the cells take up glycine betaine until they reach osmotic balance with the medium. E. halochloris possesses an active transport system that allows an uptake of glycine betaine against a concentration gradient. Glycine betaine is not metabolized in E. halochloris . Ectoine, a minor compatible solute of E. halochloris , is excreted in a similar manner to that of glycine betaine during dilution stress, whereas no excretion of the third compatible solute, trehalose, was detected.