Spore-forming halophilic bacteria isolated from Arctic terrains: Implications for long-range transportation of microorganisms

Organisms living in the Arctic terrains such as Greenland have to deal with low temperature conditions. The mechanisms by which bacteria resist to low temperature are largely unknown; however, a well-known survival strategy of the microorganisms inhabiting the Arctic is spore forming. Moreover, halophilic bacteria are often resistant to various stresses. We have attempted isolation of spore-forming halophilic bacteria from Arctic terrains. We isolated 10 strains of spore-forming halophilic bacteria from the samples collected from a glacial moraine in Qaanaaq, Greenland in July 2007. Identification based on 16S rRNA gene sequence similarities showed that the isolates were closely related to the Oceanobacillus, Ornithinibacillus, Virgibacillus, Gracilibacillus, and Bacillus genera. In addition, the 16S rRNA sequences of some isolates were extremely similar to those of strains from the desert sand in China (100% identity, near full length), the source of the so-called “yellow dust.” Previous research indicated that yellow dust had been transported to Greenland by the wind. Our research implies the long-range transportation of these microorganisms to locations such as the Arctic.     

北极斯瓦尔巴群岛冰川流域可培养细菌的系统发育多样性及固氮菌初步研究

从北极斯瓦尔巴群岛冰川流域的上、中、下游采集了5种代表性土壤和冰川融水样品(MT、AT、TN、BT、MS),采用常规LB和严格无氮培养基低温培养方法,对其中的可培养细菌和潜在固氮细菌进行了总菌计数以及菌株的富集、分离与纯化。经菌落菌体形态观察、16S r RNA基因初步测序排重、固氮基因nif H的PCR检测,分别选取48株可培养分离细菌和31株潜在固氮菌进行了基于16S r RNA基因全序列测定的系统发育分析。目的是初步了解该特殊生境中可培养细菌(包括潜在固氮菌)的多样性及其分布状况,为进一步研究它们在此生境中的生态功能奠定基础。实验结果表明,可培养细菌在5个采集样品中的总菌数为BTTNATMT=MS,严格无氮培养基分离菌总菌数为BTATMTTNMS。48株可培养代表菌分属于Proteobacteria、Actinobacteria、Bacteroidetes、Firmicutes 4个门的7纲、14属之中,它们在5个样品中的多样性程度为BT(6纲、6属)MT(4纲、6属)AT、MS(均为3纲、3属)TN(2纲、3属),优势菌群主要分布在γ-proteobacteria纲的Pseudomonas属(20.8%,集中在AT、BT、MS样品中)和β-proteobacteria纲的Janthinobacterium属(35.4%,集中在MS样品中),表现出一定程度的系统发育多样性。潜在固氮菌实验结果表明,能在严格无氮培养基上生长且可检测到nif H的31株代表菌具有极为单一的系统发育多样性。其中90.32%菌株集中在γ-proteobacteria纲的Pseudomonas属,仅在MT和MS样品中有3株菌分布在α-proteobacteria纲的Rhizobium属、β-proteobacteria纲的Janthinobacterium属和Actinobacteria纲的Arthrobacter属,它们与分离自其他极端环境的相关标准菌株具有高度16S r RNA基因同源性。另外,本实验还发现有8株菌的16S r RNA基因序列与相关标准菌株的相似度低于98.50%,推测它们具有成为该特殊生境下新种的潜质。     

砾间接触氧化反应器中细菌多样性评价

采用分子生物学手段,通过构建16S rDNA基因文库,对新型剩余污泥减量化处理系统——砾间接触氧化反应器中细菌多样性进行了系统发育分析;通过多样性指数、丰富度指数、均匀度指数及优势度指数,对细菌多样性及优势菌种富集状况进行了系统评价。结果表明:共有80个克隆子,分别代表29个OTU,用于系统细菌分析。其中,石球外膜46个克隆子(17个OTU),石球内泥34个克隆子(12个OTU)。生物膜中细菌多样性及优势菌种富集状况均好于孔隙内泥中的细菌。     

Isolation,identification and characterization of highly tellurite-resistant,tellurite-reducing bacteria from Antarctica

The tellurium oxyanion, tellurite, is extremely noxious to most living organisms. Its toxicity has been mainly related to the generation of reactive oxygen species (ROS) as well as to an unbalancing of the thiol:redox buffering system. Nevertheless, a few bacteria are capable of thriving at high tellurite concentrations. One mechanism of resistance is the enzymatic and non-enzymatic reduction of tellurite to the less toxic elemental tellurium. This reduction generates nano- to micrometric tellurium crystals that display different shapes and sizes.To date, a very limited number of highly tellurite-resistant and tellurite-reducing bacterial species are available from international culture collections. In this work, we decided to look for tellurite-reducing bacteria from an extreme environment, Antarctica. This environment exhibits a combination of several extreme factors such as high UV-radiation and desiccation and freezing conditions that impact directly on the local biodiversity. Since, as does, all these factors induce ROS formation, we hypothesized that Antarctic bacteria could also exhibit tellurite-resistance. In this context, we isolated 123 tellurite-resistant bacteria, and characterized six new tellurite-resistant and tellurite-reducing bacterial strains from samples collected in Antarctica. These strains were identified according to their 16S rRNA gene sequence as Staphylococcus hameolyticus, Staphylococcus sciuri, Acinetobacter haemolyticus, Pseudomonas lini, and two strains of Psychrobacter immobilis.The isolates display tellurite-resistance about 35- to 500-fold higher than Escherichia coli (Te-sensitive organism), and a high level of tellurite reduction which might be interesting for an application in the field of bioremediation or nanoparticle biosynthesis.     

艾比湖原核微生物菌群结构演变与环境因子关系分析

用非培养法获得新疆维吾尔自治区艾比湖湖底沉积物原核微生物菌群组成,并与已有盐湖原核微生物菌群数据进行比对,分析湖泊由淡水湖向盐湖演替过程中原核微生物群落结构变化规律。实验获得艾比湖原核微生物16S rRNA基因序列,并从NCBI数据库下载赛里木湖、柴窝堡湖和顿巴斯他乌盐湖3个湖泊的非培养原核微生物16S rRNA基因序列数据。用不同盐湖细菌和古菌16S rRNA序列信息构建系统发育树并与其理化指标进行典型性相关分析。同源比对及聚类结果显示,艾比湖湖底沉积物中细菌包括4个门,拟杆菌门(Bacteroidetes)占克隆文库的64%,变形菌门(Proteobacteria)占9.4%,厚壁菌门(Firmicutes)占3.4%,放线菌门(Actinobacteria)占2.6%,此外含有未分类类群20.6%。古菌含有两个门,广古菌门(Euryarchaeota,98.3%)和盐纳古菌门(Nanohaloarchaeota,1.7%)。不同盐湖系统发育树结果显示,随盐度增加,盐湖细菌从变形菌门向拟杆菌门演替;古菌从奇古菌门和泉古菌门向广古菌门和盐纳古菌门演替。RDA结果显示,Na⁺、Cl-、SO₄2-和矿化度对盐湖原核微生物多样性结构起到决定性的作用,K⁺、Mg²⁺和Ca²⁺对艾比湖菌群结构影响作用最为显著。原核微生物群落会随着湖水盐浓度的增加和盐湖化学成分的不同而发生演替。     

Screening and molecular classification of low-temperature protease from Antarctic microorganism and its characterization

<正> 107 strains producing protease were screened from 260 strains of Ant-arctic psychrophilic bacteria,among which proteolytic activity of five strains wasmore than 45 U ml~(-1).The 16S rRNA gene sequences homology and phylogenet-ic analysis of five Antarctic psychrophilic bacteria showed that NJ276、NJ5-9、NJ16-70、NJ345 belonged to the described genus Pseudoalteromonas and NJ341belonged to the genus Colwellia.The growth and the protease characteristic offour Antarctic psychrophilic bacteria had been studied,and the result showedthat the optimal temperature for growth and protease-producing of four strainswas about 10℃.Their growth and protease-producing were still high during in-cubating 2-5 days.The maximum proteolytic activity occurred at pH 9 for fourAntarctic psychrophilic bacteria.The optimal temperature of protease action ofboth strains NJ276 and NJ5-9 was about 50℃,however,the optimal temperatureof protease action of both strains NJ341 and NJ345 was about 40℃,and theirproteolytic activity under 0℃ exhibited nearly 30% of the maximum activity,but their thermal stabilities were weaker.These results indicated that proteasesfrom NJ341 and NJ345 were low-temperature proteases.     

内蒙古河套灌区湿地芦苇根际可培养细菌多样性

以内蒙古河套灌区湿地植物芦苇(Phragmites australis)根际土壤为材料,采用分离培养法对天然湿地和退化湿地的芦苇根际细菌多样性进行了研究。16SrRNA基因系统发育分析的结果显示,分离自天然湿地芦苇根际的45株细菌分属于γ-变形菌纲(44.44%)、厚壁菌门(Firmicutes)(42.22%)和放线菌门(Actinobacteria)(13.33%)3大类群,其中,γ-变形菌纲为最优势类群,芽孢杆菌属(Bacillus)(42.2%)为最优势菌属;分离自退化湿地芦苇根际的44株细菌分属γ-变形菌纲(13.63%)、β-变形菌纲(6.82%)、厚壁菌门(47.73%)及放线菌门(Actino-bacteria)(31.82%)4大类群,其中厚壁菌门为最优势类群,芽孢杆菌属(40.9%)为最优势菌属。研究结果表明,天然湿地和退化湿地芦苇根际细菌都具有较丰富的多样性,但天然湿地与退化湿地芦苇根际细菌群落结构存在一定差异。     

南极普里兹湾可培养浮游细菌群落结构研究

针对南极普里兹湾内达恩利角附近海域的3个测站共12份海水样品,采用寡营养培养基分离培养极地浮游细菌,通过PCR方法获取其中95株细菌的16S rRNA基因序列。序列分析结果表明,分离获得的95株细菌分属α-变形杆菌纲(Alphaproteobactia)、γ-变形杆菌纲(Gammaproteobacteria)和拟杆菌门(Bacteroidetes)3个类群下的35个不同分类单元,其中α-变形杆菌纲为优势细菌,在不同水层中所占的比例为52.6%—62.5%。通过Shannon多样性指数分析发现,南极普里兹湾陆架区测站P2-14多样性指数较陆坡区两测站P2-11和P2-8的高,细菌多样性与浮游植物量和生产力水平呈现正相关。表层细菌Shannon多样性指数最低,50 m水层最高,100和200 m水层多样性指数变化不明显,真光层水体中细菌垂直分布与营养盐和溶解氧浓度存在关联。此外,采用寡营养陈海水培养基,还获得了一些与已报道的16S rRNA基因序列相似性较低的菌株,它们代表了微生物新物种,可能也是南极真光层特有的细菌。研究工作表明,南极普里兹湾真光层水体中蕴藏着丰富的浮游细菌资源,它们有待保护、开发和利用。     

南极长城站空气微生物的分子生态学分析

微生物是气溶胶的重要组成部分。本研究通过收集南极长城站附近气溶胶,构建气溶胶中空气微生物16S rRNA基因文库,发现南极空气微生物具有很高的多样性,93个克隆子包含了53个OTU(Operational Taxonomic Unit),Shannon指数为3.58,Simpson指数为0.04。南极长城站空气微生物的主要细菌类群为Actinobacteria、Cyanobacteria/Chloroplasts、Bacteroidetes、Firmicutes、Planctomycetes、Proteobacteria及Verrucomicrobia等7个门,Proteobacteria门的细菌是优势类群,占长城站空气微生物细菌克隆文库的65.7%。克隆文库中海源微生物序列及陆源微生物序列各占30.1%,说明长城站附近具有较强的海陆大气交换。我们发现有15个克隆子序列与人类活动相关,占总克隆子数的16.1%,说明人类活动已经影响到了长城站的空气微生物群落结构。     

南极罗斯海沉积物中可培养菌株的分离及胞外水解酶活性检测

研究了南极罗斯海地区沉积物中可培养微生物多样性,并检测了菌株胞外酶活性,为开发利用南极资源奠定基础。采用Zobell2216E培养基、土豆培养基(PDA)及稀释平板划线法分离并获得纯培养的细菌和真菌,分别通过16SrDNA、18S/ITS序列鉴定分析了可培养微生物的多样性和系统发育。从南极罗斯海沉积物样品中获得78株细菌和54株真菌,细菌、真菌的优势菌株分别为嗜冷杆菌属(Psychrobacter)、枝孢属(Cladosporium);78株细菌菌株中有2株与模式菌株的16SrDNA基因序列相似性小于97%,可能是潜在的细菌新物种。细菌API20NE生理生化和真菌产胞外酶活性结果表明,获得的细菌和真菌大多数可检测到胞外水解酶活性。本研究丰富了南极可培养微生物资源,为南极罗斯海地区可培养微生物的多样性研究以及南极微生物资源的应用研究奠定了基础。     

Phylogenetic analysis of cultivable bacteria isolated from Arctic sea-ice

Phylogenetic analysis based on 16S rDNA of 8 strains of cultivable bacteria isolated from Arctic sea-ice was studied.The results showed that strain BJ1 belonged to genus Planococcus,which was a genus of low mole percent G+C gram-positive bacteria;strain BJ6 belonged to genus Burkholderia of β-proteobacteria and the rest 6 strain all belonged to γ-proteobacteria,of which strain BJ8 was a species of Pseudoalteromonas,strain BJ2-BJ5 and BJ7 were members of genus Psychrobacter.Phylogenetic analysis also indicated that bacteria of genus Psychrobacter of the isolates formed a relatively independent phylogenetic cluster in comparison with other bacteria belonged to genus Psychrobacter.     

Isolation of novel psychrophilic bacteria from Arctic sea ice

The phylogenetic diversity of culturable psychrophilic bacteria associ ated with sea ice from the high latitude regions of Canadian Basin and Chukchi S ea,Arctic,was investigated.A total of 34 psychropilic strains were isolated u sing three methods of(Ⅰ)dilution plating(at 4 ℃),(Ⅱ)bath culturing(at-1 ℃)and dilution plating,and(Ⅲ)cold shock(-20 ℃ for 24 h),bath culturin g and dilution plating under aerobic conditions.Sea-ice samples were exposed to-20 ℃ for 24 h that might reduce the number of common microorganisms and encou rag e outgrowth of psychrophilic strains.This process might be able to be introduce d to isolation psychrophilic bacteria from other environmental samples in future study.16S rDNA nearly full-length sequence analysis revealed that psychrophil i c strains felled in two phylogenetic divisions,γ-proteobacteria(in the gen era Colwellia、Marinobacter、Shewanella、Glaciecola、Marinomonas and Pseudoalt eromon as) and Cytophaga-Flexibacter-Bacteroides(Flavobacterium and Psychrof lexus).Fi fteen of bacterial isolates quite likely represented novel species(16S rDNA seq uence similarity below 98%).One of strains(BSi20002)from Canadian Basin showe d 100% sequence similarity to that of Marinobacter sp.ANT8277 isolated from the Antarctic Weddell sea ice,suggesting bacteria may have a bipolar distribution at the species level.     

吉尔吉斯斯坦伊塞克湖一处土壤样品细菌多样性

采用纯培养方法从吉尔吉斯斯坦共和国伊塞克湖一处温泉旁的土壤中分离到32株细菌,通过ARDRA 分析后得到不同的类群,从各个类群中随机选取1个代表菌株进行16S rDNA 序列测定和系统发育的分析。结果表明：分离的菌株分布在假单胞菌属（Pseudomonas）、类芽孢杆菌属（Paenibacillus）、芽孢杆菌属（Bacillus）、寡养单胞菌属（Stenotrophomonas）、Lysinibacillus、溶杆菌属（Lysobacter）、短芽孢杆菌属（Brevibacillus）、根瘤菌属（Rhizobium）、变形杆菌属（Proteus）等9个属,且部分菌株的16S rDNA序列同源性低于97%,可能是潜在的新种。结果揭示了该样点可培养细菌种群的多样性及独特性。     

Phylogenetic analysis of bacteria in sea ice brine sampled from the Canada Basin,Arctic Ocean

Bacterial diversity in sea ice brine samples which collected from four stations located at the Canada Basin,Arctic Ocean was analyzed by PCR-DGGE.T wenty-three 16S rDNA sequences of bacteria obtained from DGGE bands were cloned and sequenced.Phylogenetic analysis clustered these sequences within γ-prote obacteria,Cytophaga-Flexibacter-Bacteroides(CFB)group,Firmicutes and A ctinobacteria.The phylotype of Pseudoalteromonas in the γ-proteobacteria was predominant and members of the CFB group and γ-proteobacteria were highly abundant in studied sea ice brine samples.     

北极黄河站植物根际土壤放线菌分离培养及其鉴定

通过稀释涂布平板法,对北极黄河站(挪威新奥尔松地区)7种常见植物根际土壤样品进行放线菌的分离培养。经扩增性rDNA限制性酶切片段分析(ARDRA)及16S rDNA序列分析确定放线菌53株,分别属于8个属,除了优势菌链霉菌属(Streptomyces)和节杆菌属(Arthrobacter)外,还发现了红球菌(Rhodococcus)、糖丝菌(Saccharothrix)、拉塞氏杆菌(Rathayibacter)、微球菌(Micrococcus)、诺卡氏菌(Nocardia)、克里贝拉(Kribbella)等6种稀有菌属。     

南海北部陆坡表层沉积物氨氧化古菌多样性初探

基于南海北部陆坡不同深度梯度3个站位表层沉积物古菌氨单加氧酶基因（amoA）文库,对3个站位氨氧化古菌进行多样性和系统发育学分析。多种方法构建的系统发育树表明：3个站位所有的amoA基因序列都隶属于奇古菌门中Group I分枝内的Group I.1a系群,且各站位之间氨氧化古菌多样性没有明显的差异。501站位黑色砂质沉积物中古菌amoA基因与该站位的16S rRNA基因的系统发育比对显示：这2种基因标记的系统发育树整体上具有潜在的对应关系,说明对样品的氨氧化古菌多样性分析比较全面且可靠;并进一步暗示该样品中氨的硝化作用主要由奇古菌门下的Group I.1a系群来执行。由此可以推测：Group I.1a系群可能在南海北部表层沉积物中氮素的生物地球化学循环过程中扮演重要的角色。     

海洋放线菌Y18的分子鉴定、生物活性及其抗菌物质发酵条件优化

从北冰洋楚科奇海海洋沉积物中分离到一株放线菌Y18,其良好生长需要海水。经16S rRNA基因序列分析确定该菌株属于拟诺卡氏菌属(Nocardiopsis),次级代谢产物合成酶基因及抗菌活性筛选发现,它含有聚酮类Ⅱ型(PKSⅡ)和非核糖体含硫多肽类(non ribosome-bound sulfur-containing peptides,NRPS)化合物合成酶基因,并且对枯草芽孢杆菌和白色念珠菌有抑制作用。以枯草芽孢杆菌为指示菌对Y18的发酵条件进行了初步优化,适合的发酵条件为:发酵培养基(蛋白胨2 g,酪素水解物2 g,酵母膏2 g,葡萄糖1 g,海水1 L),种子液接种量4%,发酵温度28℃,发酵培养基初始pH值8.0,培养时间6天,1 000 mL三角瓶装液量20%。并对Y18发酵液稳定性进行了测定,结果显示:发酵液对酸碱、热的稳定性都较强,4℃下贮藏发酵液的抗菌活性保留时间较长。     

南极菲尔德斯半岛表层土壤样品中细菌多样性的系统发育分析

通过PCR结合变性梯度凝胶电泳(DGGE)技术对从南极长城站附近表层土壤样品中获得16SrDNA序列特征片段V3区序列进行分离。对其中的主要12条DGGE条带进行胶回收,获得的DNA片段经测序以及计算机比对分析发现,它们分别属于β、γ、δ-变形细菌(Proteobac-teria)、噬纤维菌-屈挠杆菌-拟杆菌(Cytophaga-Flexibacter-Bacteroides,CFB)群细菌、放线细菌(Actinobacteria)、蓝细菌属(Cyanobacteria)、酸杆菌属(Acidobacteria)和绿屈挠菌属(Chlo-roflexi)等系统分类群。南极表层土壤样品中的大部分16S rDNA序列与从其他土壤或沉积物样品中直接获得的序列相似性较高(93%-100%)。     

南极菲尔德斯半岛土壤可培养细菌多样性分析

对中国第29次南极科学考察采自南极菲尔德斯半岛区域16个站位的土壤样品进行了细菌的分离培养。经细菌16S r DNA序列测定及系统发育分析,共鉴定得到3个门,13个属的67株细菌。其中优势门为放线菌门(Actinobacteria)和变形菌门(Proteobacteria),优势属为节杆菌属(Arthrobacter)和假单胞菌属(Pseudomonas)。对受到不同外界动物因素影响的4个典型站位的土壤理化性质进行了测定,初步分析了土壤理化因子对可培养细菌群落结构的影响。结果表明在菲尔德斯半岛人活动对土壤中可培养细菌群落的影响显著区别于企鹅和海豹。研究还对产淀粉酶的菌株进行了筛选,发现8株产淀粉酶的菌株。     

北极王湾夏季海水中DMSP降解基因的丰度及分布调查

作为海洋中最丰富的有机含硫化合物之一,二甲基巯基丙酸内盐(DMSP)不仅在浮游植物细胞内具有重要的生理生化及生态功能,同时也是海洋微生物的重要营养物,其分解产物二甲基硫(DMS)还是海洋中最重要的还原态挥发性生源有机硫化物,因此DMSP的代谢在全球硫循环中扮演着重要角色。细菌可通过脱甲基与裂解这两条途径降解海水中的DMSP,从而决定硫元素是以含硫蛋白形式进入微生物食物网还是以DMS形式进入大气。截至目前,在高纬度北极海域开展的有关细菌降解DMSP的相关研究报道很少。本文采用荧光定量PCR技术,首次对北极王湾夏季(2015及2016年)海水中涉及细菌降解DMSP的两条主要代谢途径中的脱甲基酶基因dmdA及裂解酶基因dddP的丰度及分布进行了检测。结果显示, 2015—2016年海水中DMSP降解酶基因(dmdA、dddP)与细菌16S rRNA基因的丰度比值的平均值分别为0.25%±0.31%、0.32%±0.58%。2015年水样中,除K5站位的dddP基因之外,表层水中dmdA及dddP的丰度相对值从湾口至湾内均呈递增趋势。但2016年的结果却显示出很大差异:湾口附近dmdA基因丰度相对值总体高于湾内;表层水中的dddP基因丰度相对值变化趋势不明显,而深层水中dddP基因丰度相对值从湾口到湾内总体呈递减趋势。本次研究的结果初步表明,含有DMSP降解基因的浮游细菌在王湾夏季水体中丰度很低,而且参与不同代谢途径的DMSP降解菌在海水中的时空分布存在很大变化。对于王湾水域中细菌参与DMSP分解代谢及其在当地硫元素循环中的生态地位的进一步认识,尚待后续研究工作的深入开展。