Culturable halophilic archaeal diversity of Ayakekumu salt lake located in Xinjiang, China

Molecular diversity of halophilic archaea from Ayakekumu salt lake was investigated by the polymerase chain reaction (PCR) amplification and culture methods. 19 water samples and 15 soil samples were taken from 19 sites within Ayakekumu salt lake in winter and spring. Under aerobic culture conditions, some halophilic microorganisms were isolated by five different media. The 16S rRNA gene sequences of 62 red strains were amplified by using PCR, determined by the DNA sequencer and analyzed through the BLASTn program subsequently. Results revealed that all sequences belonged to six genera grouped within the Halobacteriaceae. Mostly 16S rRNA gene sequences related to the genera Halorubrum (47%) and Natrinema (24%) were detected. Subsequent analysis by using Shannon index indicated that cultured halophilic archaeal diversities are not significantly different between winter and spring samplings in Ayakekumu salt lake. Similarity values of haloarchaeal 16S rRNA gene sequences to known sequences were less than 97%, suggesting the presence of two novel taxa. In addition, taxonomic characteristics of Natrinema altunense and Halobiforma lacisalsi isolated from Ayakekumu salt lake had been described previously. The discovery of the novel species provides new opportunity to further examine the diversity of these halophilic microorganisms in Ayakekumu salt lake.     

新疆阿牙克库木湖可培养嗜盐古菌的种群结构

从新疆南部的阿牙克库木湖采集了19个水样和15个土样,分离培养嗜盐微生物。采用PCR方法获取其中62株嗜盐古菌16S rRNA基因序列。序列分析结果表明,分离到的菌株分属6个属,占已报道嗜盐古菌属总数的27%,其中以Halorubrum和Natrinema属的菌株为优势菌株。通过Shannon多样性指数分析发现,阿牙克库木湖冬春两季嗜盐古菌多样性差异不明显。研究还发现4个嗜盐古菌新物种,表明阿牙克库木湖蕴藏着具有地域特点的嗜盐古菌资源。     

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。     

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。     

新疆艾比湖嗜盐菌的细菌视紫红质和16S rRNA基因序列的研究

为了研究分析嗜盐古生菌物种与细菌视紫红质(BR)蛋白基因资源,从40份土壤、湖水及淤泥样品中分离出148株嗜盐菌,对其中6株菌采用聚合酶链式反应(PCR)方法对其编码螺旋C至螺旋G的蛋白基因片段和16SrRNA基因进行了扩增,并测定了基因的核苷酸序列。与已报道的相应片段进行对比,ABDH10,ABDH1I和ABDH40中的螺旋C至螺旋G的蛋白与其他菌株差异显著。基于16SrRNA序列的同源性比较以及系统发育学研究表明,ABDH10和ABDH40是Natronorubrum属下的新成员和Natrinema属下的新成员,ABDH40的16SrRNA序列已登录到GenBank,其序列号为AY989910。ABDH11中的螺旋C至螺旋G的蛋白与其他菌株差异显著。     

Isolation and characterization of culturable halophilic microorganisms of salt ponds in Lianyungang,China

The Lianyungang salt ponds are an extreme saline environment, and their microbial communities have not been characterized. A typical extreme halophilic archaeon strain designated as HBCC-2 (GenBank accession number: EF687739) was isolated from the salt ponds of Lianyungang in Jiangsu Province, P. R. China, using conventional microbial culture methods. The other halotolerant bacterial strain designated as HBCC-3 (GenBank accession number: EU377478) was isolated from the same sampling sites. The morphological and physiological characteristics of HBCC-2 and HBCC-3 were observed and examined. G+C content of HBCC-2 and HBCC-3 were determined using high-performance liquid chromatography. The cellular phospholipid fatty acids were analyzed using gas chromatography-mass spectrometry. The 16S rRNA gene sequences of the strains HBCC-2 and HBCC-3 were amplified by PCR using archaeal primers and bacterial primers, respectively. Homology of 16S rRNA gene sequences of the strains HBCC-2 and HBCC-3 were compared with the other similar sequences obtained from GenBank using the BLAST program. Phylogenetic analysis was performed using the software MEGA 4.0 after multiple alignments of sequence data using software CLUSTALW 1.8. The evolutional distances (by Kimura’s model) were calculated and the clusters were performed with the neighbor-joining method. The results showed that the 16S rRNA gene sequences of the strains HBCC-2 and HBCC-3 are related to the genera Halorubrum and Alkalibacillus, respectively. Two phylogenetic trees were constructed by phylogenetic analysis based on the 16S rRNA gene sequence. Based on the above results, the strains HBCC-2 and HBCC-3 were finally identified. The discovery of the two species provides an opportunity to further study these halophilic microorganisms in the Lianyungang salt ponds.     

新疆达坂盐湖沉积土壤嗜盐细菌的定向富集与多样性分析

采集新疆达坂盐湖的沉积土壤样品,以选择性富集培养获得的嗜盐细菌基因组DNA为模板,扩增16SrRNA基因,在此基础上构建嗜盐细菌的16SrRNA基因文库,随机挑选文库中的100个阳性克隆子进行群落结构多样性分析。16SrRNA基因序列分析结果表明:100个克隆分属于细菌域9个属的27个种,其中芽孢杆菌属(Bacillus)为优势菌群(48%),喜盐芽孢杆菌属(Halobacillus)(14%)、盐单胞菌属(Halomonas)(13%)为次优势菌群。分析的阳性克隆子中,10个克隆子与GenBank中已报道16SrRNA基因序列的相似性在88.80%到96.90%之间,可能代表新属或新种。研究结果表明,新疆达坂盐湖沉积土壤的富集培养物中存在种类较为丰富的嗜盐细菌。     

Diversity of halophilic archaea from six hypersaline environments in Turkey

The diversity of archaeal strains from six hypersaline environments in Turkey was analyzed by comparing their phenotypic characteristics and 16S rDNA sequences. Thirty-three isolates were characterized in terms of their phenotypic properties including morphological and biochemical characteristics, susceptibility to different antibiotics, and total lipid and plasmid contents, and finally compared by 16S rDNA gene sequences. The results showed that all isolates belong to the family Halobacteriaceae. Phylogenetic analyses using approximately 1,388 bp comparisions of 16S rDNA sequences demonstrated that all isolates clustered closely to species belonging to 9 genera, namely Halorubrum (8 isolates), Natrinema (5 isolates), Haloarcula (4 isolates), Natronococcus (4 isolates), Natrialba (4 isolates), Haloferax (3 isolates), Haloterrigena (3 isolates), Halalkalicoccus (1 isolate), and Halomicrobium (1 isolate). The results revealed a high diversity among the isolated halophilic strains and indicated that some of these strains constitute new taxa of extremely halophilic archaea.     

连云港台北和盐城三圩盐田土壤嗜盐菌多样性研究

盐田土壤嗜盐微生物对盐田生态系统的良性循环和盐的生产至关重要。本文对江苏连云港台北盐田土壤和盐城三圩盐田土壤的嗜盐细菌和古菌的多样性进行了研究, 结果表明两地盐土嗜盐细菌和古菌的分布具有相似性和独特性。采用培养法从两地盐土中共分离到17株嗜盐细菌, 其中Halomonas为两地盐土共有的嗜盐细菌, 而Halobacillus和Pontibacillus仅在三圩盐土中发现。通过非培养的16S rDNA 基因文库法从两地盐土中发现了13种嗜盐古菌, 台北盐土有Halobacterium 和 Haloplanus, 三圩盐土有Halobacterium, Natronobacterium, Halogeometricum 和 Haloarcula。10个嗜盐古菌的16S rDNA和GenBank已知序列的同源性为92%~97%, 可能为这些属中的新种。该研究为盐田环境嗜盐微生物资源的开发和利用奠定了基础。     

连云港台北和盐城三圩盐田土壤嗜盐菌多样性研究

盐田土壤嗜盐微生物对盐田生态系统的良性循环和盐的生产至关重要.本文对江苏连云港台北盐田土壤和盐城三圩盐田土壤的嗜盐细菌和古菌的多样性进行了研究,结果表明两地盐土嗜盐细菌和古菌的分布具有相似性和独特性.采用培养法从两地盐土中共分离到17株嗜盐细菌,其中Halomonas为两地盐土共有的嗜盐细菌,而Halobacillus和Pontibacillus仅在三圩盐土中发现.通过非培养的16S rDNA基因文库法从两地盐土中发现了13种嗜盐古菌,台北盐土有Halobacterium和Haloplanus,三圩盐土有Halobacterium, Natronobacterium, Halogeometricum和Haloarcula. 10个嗜盐古菌的16S rDNA和GenBank已知序列的同源性为92%～97%.可能为这些属中的新该研究为盐田环境嗜盐微生物资源的开发和利用奠定了基础.     

青藏高原察尔汗盐湖地区可培养中度嗜盐菌的群落结构与多样性

【目的】研究青海察尔汗盐湖地区的可培养中度嗜盐菌的群落结构及多样性。【方法】采用多种选择性培养基进行中度嗜盐菌的分离、培养;通过16S r RNA基因序列扩增、测定,根据序列信息,进行系统进化树构建、群落结构组成分析及多样性指数计算。【结果】从察尔汗盐湖卤水及湖泥中分离到中度嗜盐菌421株,合并重复菌株后共83株中度嗜盐菌。菌株16S rRNA基因序列信息显示,4株中度嗜盐菌为潜在的新分类单元。83株嗜盐细菌分布于3个门的6个科16个属。其中,Bacillus属、Oceanobacillus属和Halomonas属为优势属。多样性结果显示,水样中的菌株多样性高于泥样,而泥样中的菌株优势度高于水样。【结论】察尔汗盐湖中度嗜盐菌具有丰富的遗传多样性,种群种类丰富,优势菌群集中,该盐湖地区存在可分离培养的中度嗜盐菌的疑似新物种。     

盐地碱蓬内生中度嗜盐菌的分离与系统发育多样性分析

为了了解东营滨海盐地碱蓬植株内生中度嗜盐菌的多样性,采用传统分离鉴定技术和基于16S rRNA序列分析对样品中可培养细菌的多样性进行研究。根据其生理生化特征、16S rRNA序列测定和系统发育分析,分离获得的15株内生菌可分为4个类群,涉及Halomonadaceae科的Chromohalobacter属、Kushneria属、Halomonas属以及Bacillaceae科的Bacillus属。类群I中4菌株的16S rRNA序列与Chromohalobacter israelensis的最高相似性为95%。类群II共7株菌,归属于Kushneria属,是碱蓬内生中度嗜盐菌中的优势类群。类群III菌株的16S rRNA序列与一株尚无明确分类地位的Gammaproteobacteria亚门耐盐固氮细菌Haererehalobacter sp.JG11的相似性为99%。类群IV中的芽孢杆菌的16S rRNA序列与已知细菌的相似性为96%,很可能代表了Bacillus属的新种。各种水解酶类的分析表明,在分离的15株菌中有3株菌产蛋白酶,14株产酯酶,8株产DNA酶,11株产半乳糖苷酶,14株产脲酶。研究结果揭示,盐地碱蓬中存在较为丰富的中度嗜盐菌多样性和系统发育多样性,并且潜藏着较多的新的微生物类群。     

基于高通量测序研究青藏高原茶卡盐湖微生物多样性

【目的】茶卡盐湖(Chaka Salt Lake,CSL)是青藏高原有名的天然结晶盐湖,具有独特的石盐盐湖矿床,盛产青盐。盐湖卤水环境中存在丰富的嗜盐菌资源和潜在的新种,细菌和古菌的群落结构特征和物种多样性尚不明确。【方法】采用Illumina高通量测序平台对茶卡盐湖水样和底泥混合物中的细菌和古菌群落进行16S r RNA基因(V3-V5区)高通量测序,检测4个样本的群落结构差异和微生物多样性。【结果】获得细菌和古菌总有效序列分别为117 192和110 571条。结果分析表明细菌和古菌的物种注释(Operational taxonomic unit,OTU)数目分别为421和317,获得分类地位明确的细菌种类为14门28纲170属,古菌为5门4纲34属。细菌的优势类群是厚壁菌门(Firmicutes),所占比例为68.37%,其次为变形菌门Proteobacteria(20.49%);优势种属依次为芽孢杆菌属Bacillus(41.94%)、海洋芽孢杆菌属Oceanobacillus(8.03%)、假单胞菌属Pseudomonas(7.67%)、盐厌氧菌属Halanaerobium(7.42%)和乳球菌属Lactococcus(7.38%);古菌的优势类群以广古菌门(Euryarchaeota)盐杆菌纲(Halobacteria)为主,优势菌是Halonotius(17.21%)和盐红菌属Halorubrum(16.23%)。【结论】揭示了茶卡盐湖中细菌和古菌的群落结构及物种多样性,为嗜盐菌的开发及后续微生物资源的挖掘提供了理论依据。     

新疆塔里木盆地可培养嗜盐放线菌系统发育多样性

应用纯培养手段和基于16S rRNA基因序列的系统发育分析,对从塔里木盆地高盐环境土壤样品中分离的18株可培养嗜盐放线菌多样性进行了研究.实验结果表明,18株嗜盐放线菌可3个(GlycomycetaceaePseudonocardineae和Nocardiopsaceae),在有效发表的5个属的嗜盐放线菌中有4个属的嗜盐放线菌被分离到.多数菌株属于Actinopolyspora属(38.9%),Nocardiopsis属(27.8%)和Streptomonospora属(22.2%),是塔里木盆地高盐环境中嗜盐放线菌的优势类群.这些分离菌株中,菌株YIM 92370与最近种的相似性为92%,在Glycomycetaceae科内形成一个独立的分支,极有可能代表Glycomycetaceae科的一个新属.研究结果表明塔里木盆地高盐环境中存在有较为丰富的嗜盐放线菌系统发育多样性,并且潜藏着新类型的放线菌资源.     

高盐盐湖可分离嗜盐耐盐菌的种群多样性及四氢嘧啶产量评价

为了解柴达木盆地茶卡盐湖、柯柯盐湖和小柴旦盐湖等三大硫酸镁亚型高盐盐湖可分离嗜盐耐盐菌的种群多样性,采用RM中、高盐培养基筛选分离可培养的嗜盐菌和耐盐菌,扩增16S rRNA基因序列进行种属鉴定和环境因子典范对应分析(CCA),选取优势菌属构建系统发育树,并采用高效液相色谱法(HPLC)检测次级代谢产物四氢嘧啶(Ect...     

河南叶县岩盐可培养中度嗜盐菌的多样性

【背景】嗜盐微生物因为独特的生理和代谢特征而对高盐环境有着良好的适应能力,在环境污染治理、酶制剂等领域具有很高的应用和研究价值,是一类重要的极端环境微生物资源。【目的】为了更好地认识我国岩盐微生物的多样性,开发和利用嗜盐微生物资源,积累丰富的微生物菌种资源。【方法】在5%和10%的盐度下,使用Alkaline oligotrophic medium (AOM)、Neutral haloarchaeal medium (NHM)、diluted modified marine agar (dmMA)和ISP3 medium (ISP3)四种培养基,分离和纯化河南叶县岩盐矿的卤水和盐土中的嗜盐菌,使用细菌通用引物27F和1492R扩增和测序纯化菌株的16S rRNA基因,使用EzBioCloud和NCBI上的Blast比对进行分子鉴定,使用Mega 5.0进行遗传进化分析。【结果】从河南叶县岩盐卤水和盐土中一共分离和纯化到78株细菌,菌株16S rRNA基因序列显示它们来自3个门：厚壁菌门(Firmicutes)的Bacillus 26株、Halobacillus 30株、Oceanobacillus 10株和Staphylococcus 1株;变形菌门(Proteobacteria)的Sphingomonas 3株和Halomonas 5株;放线菌门(Actinobacteria)的Brevibacterium 3株。Halobacillus和Bacillus的细菌在叶县岩盐可培养中度嗜盐菌中具有较高的丰度。AOM培养基分离出了最多5个属的细菌,并且仅从AOM培养基分离出了Sphingomonas和Brevibacterium的细菌;另外,仅从ISP3分离出了Oceanobacillus的细菌;4种培养基都培养出了Halobacillus和Bacillus的细菌。来自卤水的有Bacillus、Halobacillus、Oceanobacillus、Sphingomonas和Staphylococcus;来自盐土有Bacillus、Brevibacterium、Halomonas。另外,5%和10%两个盐度下都纯化出Bacillus、Brevibacterium、Halobacillus和Sphingomonas;仅在10%的盐度下纯化到Halomonas和Oceanobacillus的细菌;仅在5%的盐度下培养出一株Staphylococcus。【结论】揭示了河南叶县岩盐可培养中度嗜盐菌的多样性,发现Halobacillus和Bacillus在可分离培养的中度嗜盐菌中具有较高的丰度,为深入开展岩盐嗜盐微生物研究积累了较为丰富的微生物菌株资源。     

Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods

A combination of culture-dependent and culture-independent methodologies (Bacteria and Archaea 16S rRNA gene clone library analyses) was used to determine the microbial diversity present within a geographically distinct high Arctic permafrost sample. Culturable Bacteria isolates, identified by 16S rRNA gene sequencing, belonged to the phyla Firmicutes, Actinobacteria and Proteobacteria with spore-forming Firmicutes being the most abundant; the majority of the isolates (19/23) were psychrotolerant, some (11/23) were halotolerant, and three isolates grew at -5 degrees C. A Bacteria 16S rRNA gene library containing 101 clones was composed of 42 phylotypes related to diverse phylogenetic groups including the Actinobacteria, Proteobacteria, Firmicutes, Cytophaga - Flavobacteria - Bacteroides, Planctomyces and Gemmatimonadetes; the bacterial 16S rRNA gene phylotypes were dominated by Actinobacteria- and Proteobacteria-related sequences. An Archaea 16S rRNA gene clone library containing 56 clones was made up of 11 phylotypes and contained sequences related to both of the major Archaea domains (Euryarchaeota and Crenarchaeota); the majority of sequences in the Archaea library were related to halophilic Archaea. Characterization of the microbial diversity existing within permafrost environments is important as it will lead to a better understanding of how microorganisms function and survive in such extreme cryoenvironments.     

Archaeal Microbial Diversity of Hypersaline Lake Acıgöl,Denizli, Turkey

Archaeal diversity in Lake Ac?göl, a closed-basin, alkaline, hypersaline lake located at the northern edge of western Tourides in southwest Anatolia, was investigated using culture-independent methods. Microbial mat samples were collected from six different points. Archaeal 16S rRNA gene libraries were generated using domain specific oligonucleotide primers, and 16S rRNA gene sequences of clone libraries were analyzed phylogenetically. Denaturing gradient gel electrophoresis of 16S rRNA genes showed a variance in diversity with spatial differences. Archaeal diversity of Ac?göl is dominated by the members of family Halobacteriaceae which requires both high salt concentration and high pH for growth. Sequence analysis of archaeal 16s rRNA genes indicates the presence of the phylotypes affiliated with the genera Halorubrum, Halosimplex, Halorhabdus, Haloterrigena and Natronococcus in the analyzed samples.     

Combined use of cultivation-dependent and cultivation-independent methods indicates that members of most haloarchaeal groups in an Australian crystallizer pond are cultivable

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCR-amplified 16S rRNA genes and by cultivation. High viable counts (10(6) CFU/ml) were obtained on solid media. Long incubation times (> or =8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.     

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。