Culturable bacteriorhodopsin-producing haloarchaea of Tuz Lake (Turkey)

Abstract

Lake Tuz, the largest hypersaline lake in Turkey, has a great variety of microbial communities that adapted to its extreme environment and produce many industrially important compounds such as photosensitive bacteriorhodopsins. So far, the information about the bacteriorhodopsin-producing haloarchaea species of the lake is still limited. In the present study, archaeal bacteriorhodopsin producers were isolated from three locations of the lake. Their bacteriorhodopsin-producing capability was validated by the purification and SDS-PAGE analysis of the delipidated bacteriorhodopsin molecule. The active isolates were identified by the sequencing of partial 16S rDNA gene regions. According to the results, 11 bacteriorhodopsin-producing isolates grouped in Halobacterium salinarum (4), Halobacterium sp. (3), Haloarcula salaria (2), Haloarcula sp. (1) and Halorubrum sp. (1). Our research demonstrated that Lake Tuz is an important natural source of bacteriorhodopsin-producing haloarchaea and the isolates can be valuable for the related technological applications.     

Characterization of extremely halophilic Archaea isolated from the Ayvalik Saltern,Turkey

Seven extremely halophilic strains were isolated from the Ayvalik Saltern in the north-eastern part of Turkey. Chemical analyses of the brine and salt samples were performed to measure their salt content, hardness and pH. Isolated strains were tested for their antibiotic sensitivities; cell and colony morphologies; hydrolysis of casein, starch, gelatin, Tween 20 and Tween 80; and oxidase and catalase activity. All strains were found to belong to the domain Archaea. Characterization of polar lipids by thin layer chromatography indicated that all isolates contained phytanyl diether derivatives of phosphatidylglycerol (PG), the methyl ester of phosphatidyl glycerophosphate (PGP-Me), and phosphatidylglycerosulphate (PGS). Four isolates had triglycosyl diether (TGD-2) as glycolipid, and the other three contained a sulphated diglycosyl diether instead. All isolates were examined for the presence of plasmids by agarose gel electrophoresis. Four strains were found to harbour plasmids ranging in size from 13.8 to 15.3 kbp. Correlation between the protein profiles in SDS–PAGE and the phenotypic properties of the strains was poor. The data presented here provide the first published account of the microbiota of the Ayvalik saltern, which provides a large part of the salt produced in Turkey.     

Prokaryotic diversity in Tuz Lake, a hypersaline environment in Inland Turkey

Tuz Lake is an inland thalassohaline water body located in central Anatolia that contributes to 60% of the total salt production in Turkey per year. The microbiota inhabiting this lake has been studied by FISH, denaturing gradient gel electrophoresis of PCR-amplified fragments of 16S rRNA genes, and 16S rRNA gene clone library analysis. Total cell counts per milliliter (1.38 × 10⁷) were in the range of the values normally found for hypersaline environments. The proportion of Bacteria to Archaea in the community detectable by FISH was one to three. 16S rRNA gene clone libraries indicated that the archaeal assemblage was dominated by members of the Square Haloarchaea of the Walsby group, although some other groups were also found. Bacteria were dominated by members of the Bacteroidetes , including Salinibacter ruber -related phylotypes. Because members of Bacteroidetes are widely present in different hypersaline environments, a phylogenetic analysis of 16S rRNA gene sequences from Bacteroidetes retrieved from these environments was carried out in order to ascertain whether they formed a unique cluster. Sequences retrieved from Tuz Lake and a group of sequences from other hypersaline environments clustered together in a branch that could be considered as the 'halophilic branch' within the Bacteroidetes phylum.     

A note on the brine shrimp Artemia in Tuz Lake (Turkey)

Population density, fecundity andreproduction period of Artemia sp. in TuzLake, Turkey, beside environmental factors, such as oxygen,temperature, salinity, pH and electrical conductivity werestudied during 1993 and 1994.Artemia sp. occurred at two stations at the coast.Overwintering cysts hatched in early May, when the population was composed of 90% metanauplii (93%at Y4 and 99% at Y5) and few adult females. Peak abundanceoccurred in late May, at 106to 114 ind.m^–3.     

Enzymatic activity of an extremely halophilic phosphatase from the Archaea Halobacterium salinarum in reversed micelles

Alkaline p-nitrophenylphosphate phosphatase (pNPPase) from the halophilic archaeon Halobacterium salinarum (previously halobium) was solubilized in reversed micelles of cetyltrimethylammonium bromide (CTAB) in cyclohexane with 1-butanol as cosurfactant. The hydrolysis reaction appears to follow Michaelis–Menten kinetics. The dependency of the maximum reaction rate (V_max) on the water content θ (% v/v) (or ω₀ value: molar ratio of water to surfactant concentrations) showed a bell-shaped curve for 0.3 M CTAB, but not for 0.2 M CTAB. The enzyme activity increased with the surfactant concentration at a constant ω₀ value (10.27). When the surfactant concentration was increased at a constant θ, the enzyme activity decreased. The enzyme was more stable in reversed micelles than in aqueous media.     

Comparisons of the polar lipid and pigment profiles of two solar salterns located in Newark, California, U.S.A., and Eilat, Israel

The whole community pigments and lipids have been examined during a 5-year period in two commercial solar salterns located in the United States and in Israel. There were significant differences in the complexity of the lipid and pigment patterns within the California saltern system, and these differences were not consistent over the sampling period despite examination of ponds with the same salinity. The solar saltern system in Eilat, Israel, showed greater consistency during this sampling period and compared directly with previous studies. The complexity of the saltern in Newark, California, could be explained on the basis of the prevailing weather conditions (cooler and more rainfall) and the nutrient-enriched source water. The Eilat saltern, however, has an oligotrophic water source and has a considerably warmer and drier climate. This difference resulted in more diverse and more complex pigment and lipid patterns and presumably microbial populations in the Newark, California, plant than in the saltern in Eilat, Israel. Received: December 10, 1999 / Accepted: April 6, 2000     

艾丁湖可培养嗜盐菌多样性及功能酶、抗菌活性筛选

【目的】探究艾丁湖可培养嗜盐菌的多样性、功能酶活性以及抗菌活性,进一步了解其次级代谢产物情况,为新型生物活性物质的发掘提供依据。【方法】选用以20种糖及糖的衍生物作为唯一碳源的寡营养培养基从艾丁湖5个样点中分离得到298株嗜盐菌,根据形态特征去重复后,选取62株菌运用16S r RNA基因系统发育分析的方法研究样品中嗜盐菌的多样性;从不同类群选取22株代表菌株,采用点接法进行3种功能酶的筛选,运用平板对峙法检测代表菌株对12种病原菌的抗菌活性。【结果】从5%、10%和15%3个盐浓度中分别分得221、54和23株嗜盐菌,获得的嗜盐菌分布在9个科18个属;其中放线菌分布于4个属,细菌分布于14个属;Nocardiopsis和Pontibacillus属为艾丁湖可培养嗜盐菌的优势类群,分别占17.7%和16.1%;有15株嗜盐菌相似性低于98.5%,可能为潜在新种。所选取的22株代表菌株中,分别有68.2%、22.7%和72.7%的实验菌株具有蛋白酶、淀粉酶和酯酶活性;45.5%的代表菌株对12种病原菌表现出了抗至少1种病原菌的活性,其中一株Nocardiopsis属放线菌能抗9种病原菌,表现出了广谱的抗菌活性。【结论】新疆艾丁湖土样中嗜盐菌的多样性较丰富,而且具有较好的生物活性,为后续进一步研究其次级代谢产物提供了依据。     

嗜盐古菌R1中与真核同源基因rad25启动子的序列分析和启动活性研究

在盐生盐杆菌(Halobacterium halobium)R1中分析了真核同源基因rad25的转录,分析了rad25同源基因启动子片段的序列特征,用β_半乳糖苷酶基因(bgaH)为报告基因,利用启动子探针检测技术验证了rad25同源基因启动子片段在嗜盐古生菌WFD11中的启动功能;缺失分析进一步确认了rad25同源基因启动子具有嗜盐古菌启动子典型特征。从启动子和转录水平上证明盐生盐杆菌R1中真核同源基因rad25存在生物学功能,推测其可能在核苷酸切除修复(NER)起作用。     

新疆两盐湖可培养嗜盐古菌多样性研究

从新疆地区艾比盐湖和艾丁盐湖卤水及泥土样品中分离到86株嗜盐古菌。16S rRNA基因序列分析结果表明,分离自艾比湖的嗜盐古菌分别属于Haloarcula、Halobacterium、Halorubrum、Haloterrigena、Natrinema和Natronorubrum6个属的11个分类单元,而分离自艾丁湖的嗜盐古菌分别属于Haloarcula、Halobiforma、Halorubrum、Haloterrigena、Natrialba、Natrinema6个属的8个分类单元,这一结果表明艾比湖可培养嗜盐古菌生物多样性稍高于艾丁湖。基于16S rRNA基因序列的系统发育分析表明代表菌株ABH15应为Natronorubrum属的中性嗜盐古菌新种,代表菌株ABH07、ABH12、ABH17、ABH19、ABH51和AD30可能是Halobacterium、Halorubrum、Haloterrigena、Haloarcula的新成员。     

极端嗜盐古菌遗传转化系统的研究

对极端嗜盐古菌遗传转化系统的研究进展进行了综述,内容包括抗性标记基因的选择,基因克隆和表达载体系统的发展以及受体系统的改造。     

Characterization of halophiles isolated from solar salterns in Baja California, Mexico

Solar salterns are extreme hypersaline environments that are five to ten times saltier than seawater (150–300 g L⁻¹ salt concentration) and typically contain high numbers of halophiles adapted to tolerate such extreme hypersalinity. Thirty-five halophile cultures of both Bacteria and Archaea were isolated from the Exportadora de Sal saltworks in Guerrero Negro, Baja California, Mexico. 16S rRNA sequence analysis showed that these cultured isolates included members belonging to the Halorubrum, Haloarcula, Halomonas, Halovibrio, Salicola, and Salinibacter genera and what may represent a new archaeal genus. For the first time, metabolic substrate usage of halophile isolates was evaluated using the non-colorimetric BIOLOG Phenotype MicroArray™ plates. Unique carbon substrate usage profiles were observed, even for closely related Halorubrum species, with bacterial isolates using more substrates than archaeal cultures. Characterization of these isolates also included morphology and pigmentation analyses, as well as salinity tolerance over a range of 50–300 g L⁻¹ salt concentration. Salinity optima varied between 50 and 250 g L⁻¹ and doubling times varied between 1 and 12 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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。     

Production,characterization and bioactivities of biosurfactants from newly isolated strictly halophilic bacteria

Eight distinct halophilic bacteria isolated from extreme saline soil samples of Khewra Salt Mines, Pakistan, were investigated for biosurfactant production. Isolates were identified by physiological, phenotypic and genetic characterization. Using 16S rDNA sequence analysis, the strains MB590, MB591, MB593, MB594, MB595 and MB596 were identified as Halomonas elongata, MB588 as Halobacillus karajiensis, and MB589 as Alkalibacillus almallahensis. Preliminary screening of biosurfactant production in halophilic bacteria was done by multiple screening assays. All biosurfactants showed significant emulsification properties and remarkably low surface tension values (up to 16.5 dynes/cm). Structural characterization of partially purified biosurfactants using Gas chromatography–mass spectrometry (GC–MS) and Fourier-transform infrared spectroscopy (FTIR) techniques indicated different fatty acids, glycolipid derivatives and a novel antimicrobial peptide furanomycin. These biosurfactants exhibited strong bioactivities against bacterial/fungal pathogens i.e. Klebsiella pneumoniae (86.5 %), Staphylococcus aureus (97.75 %), Bacillus subtilis (97 %), Enterococcus faecalis (97.6 %), E. coli (54.5 %), Aspergillus niger (87.75 %), Aspergillus fumigatus (93.1 %), Aspergillus flavus (80.4 %), and Fusarium solani (89.05 %). Additionally, biosurfactants also showed 85 % free radical scavenging activity indicating their antioxidant potential. The present study revealed the potential of halophilic bacterial biosurfactants as effective antimicrobial agents against various pathogens, and their possible applications in the biomedical field.     

Optimizing the fermentation conditions and enhanced production of keratinase from Bacillus cereus isolated from halophilic environment

Keratinase degrading Bacillus cereus was isolated from the halophilic environment in Tamilnadu, India and keratinase production was optimized using wheat bran substrate. Of the screened bacterial isolates, four were found to have the ability to produce keratinolytic enzyme. The process parameters were optimized using one-variable-at-a-time approach and response surface methodology. Supplementation of 1% lactose supported more keratinase production (120?U/g). Among the selected nitrogen sources, addition of casein significantly enhanced maximum keratinase production (132.5?U/g). Among the ions, manganese chloride significantly enhanced keratinsase production (102.6?U/g), however addition of zinc sulphate and copper sulphate decreased keratinase production. The maximum keratinase production was obtained in the wheat bran medium containing 1% lactose, 0.5% manganese with 80% moisture (292?U/g). Statistics based contour plots were generated to explore the variations in the response surface and to find the relationship between the keratinase yield and the bioprocess conditions.     

Haloferax sp. D1227, a halophilic Archaeon capable of growth on aromatic compounds

A pink-pigmented halophilic Archaeon, Strain D1227, was isolated from soil contaminated with oil brine and shown to be a member of the genus Haloferax, based on: (1) its hybridization with a 16S rRNA probe universal for the Archaea; (2) its resistance to a broad spectrum of antibiotics that affect Bacteria; (3) its requirement for at least 0.86 M NaCl and 25 mM Mg²⁺ for growth; (4) its possession of C₅₀-carotenoids characteristic of the halophilic Arachaea; (5) the thin layer chromatographic pattern of its polar lipids, which was identical to that of other species of Haloferax; and (6) its pleomorphic cell morphology. However, in contrast to the known species of Archaea, Haloferax strain D1227 was able to use aromatic substrates (e.g., benzoate, cinnamate, and phenylpropanoate) as sole carbon and energy sources for growth. Physiologically similar organisms, such as Haloferax volcanii, Haloferax mediterrani, Haloarcula vallismortis, and Haloarcula hispanica, could not grow on these aromatic substrates. When grown on ¹⁴C-benzoate, strain D1227 mineralized 70% of the substrate and assimilated 19% of the ¹⁴C-label into cell biomass. In addition to growth on aromatic substrates, D1227 was also capable of growth on a variety of carbohydrates and organic acids. Optimum growth of strain D1227 occurred at 45°C in media containing 1.7–2.6 M NaCl and 100 mM Mg²⁺. Under optimum growth conditions, the cell shape varied from that of an oblate spheroid on mineral salts medium alone, to discshaped, irregular or triangular cells on the same medium amended with yeast extract and tryptone. To our knowledge, this is the first unequivocal demonstration of the ability of an Archacon to grow by mineralization of aromatic substrates, and it adds a new dimension to our appreciation of the physiological diversity of this group of prokaryotes.Abbreviations Ha. Haloarcula - Hf. Haloferax     

Diversity of halophilic microorganisms: Environments,phylogeny, physiology,and applications

The phylogenetic diversity of microorganisms living at high salt concentrations is surprising. Halophiles are found in each of the three domains: Archaea, Bacteria, and Eucarya. The metabolic diversity of halophiles is great as well: they include oxygenic and anoxygenic phototrophs, aerobic heterotrophs, fermenters, denitrifiers, sulfate reducers, and methanogens. The diversity of metabolic types encountered decreases with salinity. The upper salinity limit at which each dissimilatory process takes place is correlated with the amount of energy generated and the energetic cost of osmotic adaptation. Our understanding of the biodiversity in salt-saturated environments has increased greatly in recent years. Using a combination of culture techniques, molecular biological methods, and chemotaxonomic studies, we have obtained information on the nature of the halophilic Archaea as well as the halophilic Bacteria that inhabit saltern crystallizer ponds. Several halophilic microorganisms are being exploited in biotechnology. In some cases, such as the production of ectoine, the product is directly related to the halophilic behavior of the producing microorganism. In other cases, such as the extraction of β-carotene from Dunaliella or the potential use of Haloferax species for the production of poly-β-hydroxyalkanoate or extracellular polysaccharides, similar products can be obtained from non-halophiles, but halophilic microorganisms may present advantages over the use of non-halophilic counterparts. Journal of Industrial Microbiology & Biotechnology (2002) 28, 56–63 DOI: 10.1038/sj/jim/7000176 Received 20 May 2001/ Accepted in revised form 20 June 2001