嗜盐古菌分类学研究进展

嗜盐古菌是一类需要高盐维持生长的古菌。到目前为止,已发现的嗜盐古菌都属于古菌域的广古菌门,主要包括:嗜盐甲烷古菌类群、嗜盐古菌纲的全部成员以及尚不能培养的纳米嗜盐古菌类群。嗜盐古菌是盐环境的土著类群,驱动着盐环境生态系统的生物地球化学循环。作为极端微生物,嗜盐古菌在理论研究和应用领域具有重要的研究价值。本文从嗜盐古菌分类学地位的变迁、分类学方法、分类学研究现状及我国的嗜盐古菌分类学研究等方面综述了嗜盐古菌分类学的最新研究进展。     

极端嗜盐古菌蛋白类抗生素——嗜盐菌素

古菌(Archaea)是一类与细菌及真核生物显著不同的生命的第三种形式[1],大多生活在极端或特殊环境,主要包括产甲烷古菌(Methanogenic Achaea)、极端嗜盐古菌(Extremely Halophilic Archaea)和极端嗜热古菌(Extremely Thermophilic Archaea)等三大类.极端古菌是极端环境微生物的重要成员,也是极端环境微生物资源开发的重要领域.其中,嗜盐古菌可产生一类蛋白类抗生素,称为嗜盐菌素(halocin).     

新疆两盐湖可培养极端嗜盐菌组成及功能多样性研究

【目的】通过分析不同成盐类型盐湖中的极端嗜盐菌群落组成差异,探究可培养极端嗜盐菌的功能特性。【方法】采集新疆硫酸盐型盐湖七角井和碳酸盐型盐湖南湖的土壤样品,通过平板稀释涂布法分离极端嗜盐菌,经过形态学观察、特征分析获取代表菌株,通过耐盐性测定和16S rRNA基因序列测序等对代表菌株进行鉴定,并对极端嗜盐菌的蛋白酶、淀粉酶、纤维素酶和酯酶活性进行筛选,同时检测苯酚降解能力。【结果】本研究共获得1 679株极端嗜盐菌,代表菌株45株,隶属于5门14个属,古菌数量(70.58%)明显多于细菌,最优盐浓度生长范围为18.4%–20.0%。在属水平上,盐湖中优势类群为古菌的Haloterrigena属(32.94%)和Natrialba属(26.03%),以及细菌的Aquisalimonas属(9.85%)和Aliifodinibius属(8.10%)。两盐湖中,盐度较低的南湖物种丰富度高于七角井盐湖,古菌物种组成相似,均以Haloterrigena属为主;细菌群落组成有差异,南湖以Aquisalimonas属为主,而七角井以Aliifodinibius属为主。功能筛选表明,盐湖中80%的嗜盐...     

24株深海链霉菌耐(嗜)盐性及抗MRSA活性的初步研究

对24深海链霉菌的耐(嗜)盐性及抗MRSA活性筛选,结果显示,耐NaCl盐浓度在10%以上的有23株,中等嗜盐菌2株,16株具有抗MRSA活性,占66.7%.表明深海链霉普遍能耐受10%以上的NaCl浓度,而且大多在高盐浓度下代谢抗MRSA活性物质.     

嗜盐菌Haloferax mediterranei R4胞外淀粉酶的研究

HaloferaxmediterraneiR4是从地中海分离得到的一株极端嗜盐古生菌,属于富盐菌属(Haloferax)。该菌在适当条件下可以产胞外淀粉酶,此酶需要高浓度NaCl维持其活性及稳定性,在NaCl浓度为2～5mol·L-1时维持较高活性。在NaCl浓度为3mol·L-1时该酶的最适反应pH为6.0～7.0,最适反应温度为50℃,且最适反应温度与NaCl浓度有一定的相关性。Ca2+对酶反应活性有影响。用KCl代替NaCl,该酶可以保留约50%活性,而用Na2SO4代替NaCl则完全失活。     

安徽定远盐矿可培养嗜盐微生物多样性

【背景】嗜盐微生物多生活于高盐环境,具有独特的生理代谢特征,是一类重要的极端环境微生物资源。【目的】为更好地认识我国陆相盐矿的嗜盐微生物多样性组成,更好地开发利用嗜盐微生物资源积累丰富的微生物菌种。【方法】对安徽定远盐矿盐芯样品进行嗜盐微生物的纯培养分离,并对所分离菌株进行基于16SrRNA基因的测序和序列相似性分析,并对所分离菌株进行物种多样性分析。在此基础上,对代表菌株进行菌落形态和耐盐度及酶活测定。【结果】通过纯培养共分离获得了嗜盐微生物264株,其中嗜盐古菌150株,占56.8%;嗜盐细菌114株,占43.2%。嗜盐古菌物种分别来自于Halorubrum、 Halopenitus、 Haloterrigena、 Natrinema、 Natronoarchaeum和Natronomonas等6个属;嗜盐细菌物种分别来自于Pseudomonas、Aliifodinibius、Halobacillus、Halomonas和Halospina等5个属。通过代表菌株的酶活平板检测,发现产胞外蛋白酶菌株1株,酯酶1株,淀粉酶2株;能液化明胶菌株2株。在物种多样性组成方面,发现嗜盐古菌的物种多样性指数高于嗜盐细菌。【结论】本研究对我国安徽定远陆相盐矿的可培养嗜盐微生物多样性进行探究,积累了丰富的嗜盐微生物菌株资源。     

极端嗜盐古菌蛋白类抗生素——嗜盐菌素

古菌 (Ａｒｃｈａｅａ)是一类与细菌及真核生物显著不同的生命的第三种形式[1] ,大多生活在极端或特殊环境 ,主要包括产甲烷古菌 (ＭｅｔｈａｎｏｇｅｎｉｃＡｃｈａｅａ)、极端嗜盐古菌 (ＥｘｔｒｅｍｅｌｙＨａｌｏｐｈｉｌｉｃＡｒｃｈａｅａ)和极端嗜热古菌 (ＥｘｔｒｅｍｅｌｙＴｈｅｒｍｏｐｈｉｌｉｃＡｒｃｈａｅａ)等三大类。极端古菌是极端环境微生物的重要成员 ,也是极端环境微生物资源开发的重要领域。其中 ,嗜盐古菌可产生一类蛋白类抗生素 ,称为嗜盐菌素 (ｈａｌｏｃｉｎ)。与细菌素相似[2 ] ,嗜盐菌素是由质粒编码、核糖体合…     

嗜盐微生物

高盐环境通常是指那些盐浓度高于海水的环境．在这些环境中能够生存的微生物可划分为三类：一“类是能耐受一定浓度的盐溶液,但在无盐存在条件下生长最好的菌称为耐盐菌．第二类是一定浓度的盐为菌体生长所必需,且在一定浓度的盐溶液中生长最好,称为嗜盐菌．在盐浓度从零至饱和的盐溶液中均能生长,在一定浓度的盐溶液中生长最好的特殊类群称为多能盐生苗。依据嗜盐浓度的不同,嗜盐菌又可分为轻度嗜盐菌（最适盐浓度0．2—0．smol／L）、中度嗜盐菌（最适盐浓度0．5—2．omol／L）和极端嗜盐菌（最适盐浓度＞3mol／U,其中部分极端嗜…     

嗜盐菌HBCC-2的16S rRNA基因测序分析及其培养特性

从连云港台南盐场海盐生产区中分离纯化到一株嗜盐古菌HBCC-2,该菌株经PCR扩增后,测定其16S rRNA基因序列,采用BLAST软件对基因库中基因序列进行同源性比较,选取其相似性序列,采用Clustalx1.8和MEGA3.1软件对其16S rDNA序列进行了系统发育分析研究,结果表明HBCC-2菌株与菌株Halorubrum sp.GSL5.48的相似性达99%,结合其形态观察及生理生化反应特性,初步确定该菌株属于嗜盐红菌属(Halorubrum),菌株HBCC-2的16S rDNA序列已登陆到GenBank,其序列号为EF687739.通过比较不同NaCl浓度、pH和培养温度对该菌株生长的影响情况,研究了该菌株的生长特性,结果表明NaCl浓度为4mol/L、温度为35℃和pH为7.0的培养条件下其生长最佳.     

云南黑井古盐矿可培养极端嗜盐古菌初步研究*

从云南禄丰县黑井古镇古盐矿采集30多个盐土样品,用6种极端嗜盐古菌的培养基进行分离,共挑选出425株嗜盐菌。经过盐浓度耐受等实验筛选并去除可能重复菌株后共有79株极端嗜盐菌,选出15株进行了16S rRNA基因序列测定,结果显示,其中11株为极端嗜盐古菌。对这11株菌进行初步系统发育分析发现,它们广泛分布在极端嗜盐古菌科至少4个不同属中,其中16S rRNA基因和已有效发表种间的序列相似性在97％以上的有6株,分布在Halorubrum,Natronococcus,Natrialba,Halalkalicoccus4个属中;序列相似性低于97％的有5株：菌株YIM-ARC 0032,YIM-ARC 0036,YIM-ARC 0037,YIM-ARC 0050,它们的分类地位有待进一步确定。实验初步显示出了云南黑井盐矿极端嗜盐古菌的多样性和丰富度,值得深入研究。     

Sodium chloride tolerance in strains ofEpidermophyton floccosum andEpidermophyton stockdaleae

The ability of strains of genusEpidermophyton to grow at different concentrations of NaCl (ranged from 0 to 10%) was studied. A reduction in the diameter of colonies was observed as the concentration of NaCl increased. A nearly complete absence of macroconidia were shown even at the lowest concentration assayed. The geophilic speciesE. stockdaleae tolerated higher concentrations of NaCl (> 7%) thanE. floccosum (< 3%).     

Relationships between heat resistance and phospholipid fatty acid composition of Vibrio parahaemolyticus.

Vibrio parahaemolyticus was grown in tryptic soy broth (TSB) containing NaCl levels of 0.5, 3.0, and 7.5% (wt/vol). Cultures incubated at 21, 29, and 37 C were harvested in late exponential phases and thermal death times at 47 C (D47 c; time at 47 C required to reduce the viable population by 90%) were determined in phosphate buffer containing 0.5, 3.0, and 7.5% NaCl. At a given NaCl concentration in the growth medium, D47 c values increased with elevated incubation temperatures and with elevated levels of NaCl in the heating menstrua. Differences in thermal resistance of cells cultured at a particular temperature were greater between those grown in TSB containing 0.5 and 3.0% NaCl than between those grown in TSB containing 3.0 and 7.5% NaCl. D47c values ranged from 0.8 min (grown at 21 C in TSB with 0.5% NaCl) to 6.5 min (grown at 37 C in TSB with 7.5%, heated in 7.5% NaCl buffer). Methyl esters of major phospholipid fatty acids extracted from cells were quantitated. The ratio of saturated to unsaturated fatty acids in cells grown at a given NaCl concentration increased with elevated incubation temperature. At a particular growth temperature, however, saturated to unsaturated fatty acids ratios were lowest for cells grown in TSB containing 3.0% NaCl.     

Study of the effects of temperature, pH, NaCl, and aw on the proteolytic and lipolytic activities of cheese-related lactic acid bacteria by quadratic response surface methodology

The individual and interactive effects of temperature, pH, NaCl, and a_w on the proteolytic and lipolytic activities of Lactobacillus delbrueckii subsp. bulgaricus B397, Lactococcus lactis subsp. lactis T12, and Lb. plantarum 2739 were studied by quadratic response surface methodology. The effects on enzyme activities depended on the interactions among the independent variables, type of activity, substrate and, especially, species. The proteinase activity of strains B397 and T12 was affected differently by pH as individual or interactive terms depending on the type of substrate _sl- or β-casein. The increase of NaCl concentration (2.5–7.5%) under cheese-like conditions had a negative effect on the proteinase activity of strain T12. The effect of NaCl was related to the corresponding decrease in a_w. Aminopeptidases N and A, iminopeptidase and endopeptidase of Lc. lactis subsp. lactis T12 were strongly inhibited by pH 5–6 and NaCl concentration higher than 3.75%. The negative effects of these independent variables was in several cases enhanced by their interaction and/or by the interaction with the lowest temperatures. In contrast, the same peptidases of Lb. plantarum 2739 retained a high activity under the very hostile environmental conditions. Iminopeptidase and especially endopeptidase activities of strain 2739 were stimulated slightly by NaCl at concentrations up to 5%. Lipase/esterase activity of Lb. delbrueckii subsp. bulgaricus B397 was markedly inhibited under cheese-like conditions.     

Energy flux and osmoregulation of Saccharomyces cerevisiae grown in chemostats under NaCl stress.

The energetics and accumulation of solutes in Saccharomyces cerevisiae were investigated for cells grown aerobically in a chemostat under NaCl stress and glucose limitation. Changed energy requirements in relation to external salinity were examined by energy balance determinations performed by substrate and product analyses, with the latter including heat measurements by microcalorimetry. In both 0 and 0.9 M NaCl cultures, the catabolism was entirely respiratory at the lowest dilution rates tested but shifted to a mixed respiratory-fermentative metabolism at higher dilution rates. This shift occurred at a considerably lower dilution rate for salt-grown cells. The intracellular solute concentrations, as calculated on the basis of intracellular soluble space determinations, showed that the internal Na+ concentration increased from about 0.02 molal in basal medium to about 0.18 molal in 0.9 M NaCl medium, while intracellular K+ was maintained around 0.29 molal despite the variation in external salinity. The intracellular glycerol concentration increased from below 0.05 molal at low salinity to about 1.2 molal at 0.9 M NaCl. The concentrations of the internal solutes, however, changed insignificantly with growth rate and energy metabolism. The additional maintenance energy expenditure for growth at 0.9 M NaCl was, depending on the growth rate, 14 to 31% of the total energy requirement for growth at 0 M NaCl. Including the energy conserved in glycerol, the total additional energy demand for growth at 0.9 M NaCl corresponded to 28 to 51% of the energy required for growth at 0 M NaCl.     

Sensory irritation and taste produced by NaCl and citric acid: effects of capsaicin desensitization

Three experiments were conducted to measure the sensory irritationproduced by two prototypical gustatory stimuli: citric acidand NaCl. The stimuli were applied to the tip of the tongueon filter paper disks. The first experiment revealed that solutionsof NaCl and citric acid that produced approximately equal tastesensations also produced similar amounts of irritation; thatthe psychophysical functions for irritation were approximatelytwice as steep as the functions for taste; and that irritationgrew over time for NaCl but not for citric acid. When viewedas a percentage of the taste sensation at 25 s, NaCl irritationaveraged 23% at the lowest concentration and 70% at the highestconcentration; citric acid irritation averaged 44% at the lowestconcentration and 98% at the highest concentration. The secondexperiment investigated whether the irritation produced by thesetwo stimuli was mediated via capsaicin-sensitive (CS) fibers.The experiment included a pre-test, an irritation treatmentwith either capsaicin (a desensitizing agent) or zingerone (anon-desensitizing agent), a 15 min rest period and a post-test.Reductions in irritation and taste occurred following treatmentwith both capsaicin and zingerone. A third experiment demonstratedthat the majority of the effect of zingerone on taste and irritationwas due to a perceptual context effect. After the context effectwas taken into account, capsaicin desensitization remained significantfor both salt taste and salt irritation at the highest concentration.A similar pattern of results for citric acid suggests that bothcitric acid and NaCl produce irritation in part via CS fibers.The results are discussed in terms of the ability of subjectsto discriminate the gustatory and chemesthetic components oforal sensations and the role of salt and acid irritation inflavor perception.     

The Effect of pH and NaCl Levels on the Physicochemical and Emulsifying Properties of a Cruciferin Protein Isolate

The influence of pH (3.0, 5.0, and 7.0) and ionic strength (0, 50, 100 mM NaCl) on the physicochemical and emulsifying properties of a cruciferin-rich protein isolate (CPI) was investigated. Surface charge on the CPI was found to substantially reduced in the presence of NaCl. Surface hydrophobicity was found to be the lowest for CPI at pH 7.0 with 100 mM NaCl, and highest at pH 3.0 without NaCl. Solubility was found to be lowest at pH 5.0 and 7.0 without NaCl (<20 %), however greatly improved for all other pH and NaCl conditions (>80 %). Interfacial tension was found to be lowest at 10–11 mN/m for pH 5.0–0 mM NaCl and pH 7.0–50/100 mM NaCl, whereas under all other conditions interfacial tension was higher (15+ mN/m). Overall, NaCl has no effect on EAI at pH 3.0 where it ranged between 18.8 and 19.4 m²/g. At pH 5.0, EAI decreased from 21.1 to 12.8 m²/g as NaCl levels increased from 0 to 100 mM. At pH 7.0, EAI values were found to decrease from 14.9 to 5.2 m²/g as NaCl levels were raised from 0 to 100 mM. Overall, ESI was reduced with the addition of NaCl from ~15.7 min at 0 mM NaCl to ~11.6 min and ~12.0 min for the 50 and 100 mM NaCl levels, respectively.     

Relative contributions of the fraction of unfrozen water and of salt concentration to the survival of slowly frozen human erythrocytes.

As suspensions of cells freeze, the electrolytes and other solutes in the external solution concentrate progressively, and the cells undergo osmotic dehydration if cooling is slow. The progressive concentration of solute comes about as increasing amounts of pure ice precipitate out of solution and cause the liquid-filled channels in which the cells are sequestered to dwindle in size. The consensus has been that slow freezing injury is related to the composition of the solution in these channels and not to the amount of residual liquid. The purpose of the research reported here was to test this assumption on human erythrocytes. Ordinarily, solute concentration and the amount of liquid in the unfrozen channels are inversely coupled. To vary them independently, one must vary the initial solute concentration. Two solutes were used here: NaCl and the permeating protective additive glycerol. To vary the total initial solute concentration while holding the mass ratio of glycerol to NaCl constant, we had to allow the NaCl tonicity to depart from isotonic. Specifically, human red cells were suspended in solutions with weight ratios of glycerol to NaCl of either 5.42 or 11.26, where the concentrations of NaCl were 0.6, 0.75, 1.0, 2.0, 3.0, or 4.0 times isotonic. Samples were then frozen to various subzero temperatures, which were chosen to produce various molalities of NaCl (0.24-3.30) while holding the fraction of unfrozen water constant, or conversely to produce various unfrozen fractions (0.03-0.5) while holding the molality of salt constant. (Not all combinations of these values were possible). The following general findings emerged: (a) few cells survived the freezing of greater than 90% of the extracellular water regardless of the salt concentration in the residual unfrozen portion. (b) When the fraction of frozen water was less than 75% the majority of the cells survived even when the salt concentration in the unfrozen portion exceeded 2 molal. (c) Salt concentration affected survival significantly only when the frozen fraction lay between 75 and 90%. To find a major effect on survival of the fraction of water that remains unfrozen was unexpected. It may require major modifications in how cryobiologists view solution-effect injury and its prevention.     

Plasma cholesterol concentration and death from coronary heart disease: 10 year results of the Whitehall study.

Ten year mortality from coronary heart disease in 17,718 middle aged men was related to their initial plasma cholesterol concentrations. The relative risk of death from coronary heart disease declined with age, but the absolute excess risk did not. The risk gradient was continuous over the whole range of cholesterol concentrations, the lowest mortality being among men with concentrations below the lowest decile. It seems that, as with blood pressure, the average cholesterol concentration in the blood pressure, the average cholesterol concentration in the population is too high: lowest concentrations are prognostically the best. A quarter of all deaths from coronary heart disease related to cholesterol occurred among men with concentrations above the top decile, but 55% occurred among men with concentrations in the middle three fifths of the distribution; this figure of 55% could be reduced only by a policy aimed at lowering concentrations in the whole population.     

Studies on marine occurring yeasts: Respiration,fermentation and salt tolerance

Summary The effect of various NaCl concentrations on respiration and fermentation rates in cells with or without added glucose as exogenous substrate as well as on respiratory quotients was determined for Debaryomyces hansenii, Saccharomyces cerevisiae, Cryptococcus albidus, and Candida zeylanoides, all yeasts isolated from marine environment. A given strain had about the same respiratory and fermentatory intensity at 0% and 4% NaCl (w/v). A further increase considerably reduced the oxygen uptake or CO₂-evolution. D. hansenii was the most NaCl tolerant yeast tested, giving about 10% activity still at a concentration of 24% NaCl, whether the activities of whole cells or cell homogenates were determined. For S. cerevisiae or Cr. albidus the respiratory activity was reduced to about the same degree at 16% NaCl for whole cells, at 12% NaCl for homogenates of Cr. albidus. A somewhat higher NaCl concentration was evidently tolerated for respiration and fermentation than for growth, very obvious in the case of C. zeylanoides.The minimum values for water activity (a _w) permitting 10% respiration activity were higher when produced by electrolytes (NaCl, KCl, or Na₂SO₄), lower when due to sugars (metabolizable glucose or non-metabolizable lactose) and lowest when due to glycerol. The a _w per se was evidently not solely decisive for the limitation of respiration activity.Attempts were made to assess an effect of high NaCl concentrations on the glucose uptake.The potassium content was higher in cells of the highly halotolerant D. hansenii than in those of the other yeasts and decreased with the increase in external, consequently in internal, Na⁺ concentration. The decrease in K⁺ content can presumably only proceed to a certain extent, below which the ability for growth and respiration was lost.     

Heterogeneity of heparan sulfate chains in a proteoglycan from bovine lung

A heparan sulfate proteoglycan from bovine lung gas-exchange tissue was isolated by extraction of the tissue with 4.0 M guanidine HCl in the presence of multiple protein inhibitors. The proteoglycan was purified by precipitation with cetylpyridinium chloride in 0.5 M KCl followed by CsCl isopycnic centrifugation (po = 1.45) in 4.0 M guanidine/HCl. Further purification was achieved by gel filtration on Sepharose CL-2B and by chromatography in DEAE-Sepharose CL-6B column. The proteoglycan had 14.9% protein and 22.4% uronate. Heparan sulfate chains from the proteoglycan were isolated after beta-elimination. Fractionation of heparan sulfate chains was achieved on Dowex-1 Cl- column, eluting with a stepwise increase in the concentration of NaCl, 1.0 to 2.0 M with 0.2 M increments. Of the total heparan sulfate recovered from the column, about 10% eluted by 1.2 M NaCl, 68% by 1.4 M NaCl, 18% by 1.6 M NaCl and 4% by 1.8 M NaCl. The fractions varied in their total and N-sulfate ester contents and iduronic acid to glucuronic acid ratios. The fraction that eluted from the Dowex-1 Cl- column at 1.6 M NaCl had the highest molecular weight, 37000, and the fraction that eluted at 1.8 M NaCl had the lowest molecular weight, 12000, as determined by gel filtration method, and the greatest sulfate content. The core protein, obtained by digestion of proteoglycan by heparan sulfate lyase, showed mostly a single band in SDS-polyacrylamide gel electrophoresis. The observations indicate a heterogeneity of the composition of heparan sulfate chains in the proteoglycan. This heterogeneity likely contributes to variations in biologic properties of different heparan sulfate proteoglycan preparations.