腾冲热海七株高温厌氧菌的分离及鉴定

摘要：【目的】了解中国云南腾冲热海环境中厌氧菌的分类学特征及生理生化特性。【方法】利用Hungate厌氧操作技术,从云南腾冲热海80-93℃温泉中分离出7株高温厌氧菌,对其进行形态、生长特征及16S rRNA基因序列分析,确定菌株的系统发育地位。【结果】7株菌株细胞形态均为杆状,不产芽孢,革兰氏阴性,严格厌氧,在70℃生长良好。其中较典型的菌株RH0802能在55-80℃温度范围内生长,最适生长温度为70℃;生长pH值范围为5.5-8.5,最适pH值为7.0,能利用葡萄糖、淀粉、甘露醇、甘露糖、核糖、麦芽糖、纤维二糖、木糖、果糖、半乳糖、木聚糖、甘油,不能利用蔗糖、丙酮酸。16S rRNA基因序列相似性的比较分析表明,其中5株菌与Caldanaerobacter属菌株的最高相似性均在98%以上,而RH0804与RH0806分别为96%和93%。菌株RH0802-RH0808的序列登录号分别为FJ748766、FJ748762、FJ748761、FJ748763、FJ748765、FJ748764和FJ748767,在中国普通微生物菌种保藏中心的保藏号分别为CGMCC1.5134-1.5140。【结论】分离自腾冲热海的7株嗜热厌氧菌与Caldanaerobacter属菌株有较高相似性,可将这7株菌株鉴定为Caldanaerobacter属菌株（Caldanaerobacter. sp）,其中菌株RH0804和RH0806有成为新种的可能。     

利用16S rRNA基因同源性分析鉴定两株明串珠菌

从酸马奶中分离出2株明串珠菌KLDS 5.0301和KLDS 5.0302,对2株菌的16S rRNA基因经PCR扩增测序,将测序结果同该属内菌株的16S rRNA序列作多序列比较,并建立明串珠菌属的系统发育树.结果表明,KLDS 5.0301的16S rRNA序列同L. garlicum的同源性百分比为100%.KLDS 5.0302的16S rRNA序列同L.mesenteroides LM2菌株的16S rRNA序列的同源性百分比为99.9%.根据系统发育树的结果,将KLDS5.0301鉴定为L.garlicum,KLDS 5.0302鉴定为L.mesenteroides.菌株KLDS 5.0301和KLDS 5.0302的16SrRNA序列已经在GeneBank申请国际序列注册号,分别为DQ239691和DQ297412.     

一株海洋产电菌Shewanella sp. S2的筛选和产电分析

以厦门白城海域的潮间带表面沉积物为菌种来源筛选得到一株具有电催化活性的菌株S2,该菌株的16S rRNA和gyrB基因发育树与Shewanella oneidensis MR-1同支,相似性分别为98.5%和87%,葡萄糖、木糖、半乳糖等碳源利用及最佳生长的NaCl浓度与S.oneidensis MR-1有显著差别,因此初步鉴定为Shewanella属菌株,命名为Shewanella sp.S2。初步研究了菌株S2产电活性,在以乳酸作为碳源产电时,电压最高为150mV,相应的电流密度为66.1mA/m2。     

一株高效降解芘的细菌分离、鉴定及其降解效果

摘要：【目的】获得高效降解高分子量多环芳烃的细菌,并研究其对多环芳烃的降解能力。【方法】利用富集培养和芘升华平板方法,从焦化厂污染土壤中分离多环芳烃降解细菌,对分离菌株通过形态特征、16S rRNA基因和gyrb基因序列相似性分析进行鉴定,并研究该菌对高分子量多环芳烃（HMW-PAHs）的降解效果。【结果】筛选到一株能以芘、苯并蒽、屈、苯并芘、茚并芘、苯并苝、荧恩为碳源和能源生长并降解这些底物的菌株HBS1,该菌株的16S rRNA基因和gyrb基因序列与Gordonia amicalis的相应基因的相似     

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。     

腾冲热海一株栖热菌裂解性噬菌体的分离及其特征

【目的】Thermus(栖热菌)属是嗜热细菌中比较古老的类群,本研究探索从腾冲热海热泉分离栖热菌噬菌体,并初步分析其特征。【方法】采用"双层平板法"从云南腾冲热海碱性热泉中分离纯化栖热菌噬菌体;对噬菌体及其宿主进行电镜形态观察,并进行噬菌体基因组限制性酶切片段多态性分析、噬菌体生理特征及蛋白组成分析。【结果】从腾冲热海热泉分离获得1株裂解性噬菌体,其宿主菌TC10通过16S rRNA基因序列分析鉴定为Thermus属菌株。此噬菌体为长尾型,头部直径67nm,尾管长837nm、宽10nm,最适感染温度为65℃-70℃,最适感染pH值为7.6,对氯仿不敏感,其形态与分离自俄罗斯勘察加半岛热泉的栖热菌噬菌体P23-45和P74-26有一定的差异,蛋白组成差异显著,为一株新的栖热菌噬菌体,命名为TTSP10(Tengchong Thermus Siphoviridae Bacteriophage)。     

对胞必佳生产菌株的重新鉴定

主要是从形态学观察、菌株脂肪酸成分和16S rRNA基因全序列3个方面出发,重新对胞必佳生产菌株红色诺卡氏菌(Nocardia rubra)进行鉴定。结果表明,该菌株并非诺卡氏菌属中的红色诺卡氏菌,而属于红球菌属。16S rRNA序列相似性比较和系统进化树进一步说明,该菌株与Rhodococcus ruber(AY114117.1)的同源性最高,是1株红色红球菌。     

八门湾红树林土壤小单孢菌的分离及其多样性

采用平板稀释法从海南八门湾红树林潮间带、木果榄和海莲红树根际土壤样品中分离和筛选小单孢菌科放线菌,对其分离方法进行了评价和比较,并通过16S rRNA基因测序探索了八门湾红树林小单孢菌科放线菌的多样性。采用4种预处理方法和5种分离培养基,经过排除重复菌株后共得到115株放线菌。16S rRNA基因测序结果显示,这些放线菌分布在5个科、9个属。其中小单孢菌科的菌株约占全部菌株的90%(105株),主要分布于3个属:Micromonospora(85%),Jishengella(9.5%)和Verrucosispora(5.5%),与其亲缘关系最近的菌株的相似性分布于98.5%~100%之间。     

一株耐盐弧菌Vibrio sp.K1-L的分离与鉴定

从山西运城盐湖水样中分离出一株细菌K1-L,能在2%-10%的MGM固体培养基上出现群游现象。该菌株为革兰氏阴性菌,周生鞭毛,电镜下大小约为0.4μm×(1-2)μm,其菌落形态为圆形,灰色。菌株K1-L的最适生长盐浓度、温度和pH值的范围分别为2%-6%、30-40℃和6.0-7.0,GC含量为47.06%。通过对其形态学、生理生化试验,16S rRNA基因序列及系统发育学的分析,发现其与Vibrio diabolicus HE800T和Vibrio rotiferianus LMG 21460T等细菌的16S rRNA有高度同源性,最大的相似性均为99%,确定该菌株K1-L为弧菌属(Vibrio)。     

1株甲基杆菌Methylobacterium sp.WGM16的鉴定及降解甲醇的最佳培养条件

从水稻根部土壤中筛选到1株粉红色、需氧的兼性甲基营养型菌株WGM16,该菌为革兰阴性杆菌。根据菌株16S rRNA基因序列比对分析及结合菌株常规形态特征、生理生化性状的鉴定,将该菌初步鉴定为Methylobacterium sp.PCR扩增到菌株WGM16编码甲醇脱氢酶α-亚基的mxaF基因,表明菌株WGM16中存在甲基营养代谢途径。在培养温度为32℃、以1%的甲醇作为碳源、pH值为8.0的培养条件下,其甲醇降解率可达75%。     

<Emphasis Type="Italic">Sulfolobus tokodaii</Emphasis> sp. nov. (f.<Emphasis Type="Italic"> Sulfolobus</Emphasis> sp. strain 7), a new member of the genus<Emphasis Type="Italic"> Sulfolobus</Emphasis> isolated from Beppu Hot Springs,Japan

The taxonomic position of a thermoacidophilic crenarchaeote Sulfolobus sp. strain 7, previously isolated from the Beppu Hot Springs in the geothermal area of Kyushu Island, Japan, was investigated by cloning and sequencing, by phylogenetic analysis of the 16S rRNA gene sequence, by DNA-DNA homology with similar species, and by biochemical characterization of the isolate. This isolate is an obligate aerobe and grows optimally at 80 degrees C and pH2.5-3 under aerobic and chemoheterotrophic growth conditions by aerobic respiration rather than simple fermentation. In conjunction with the phenotypic properties, the present phylogenetic analysis based on the 16S rRNA gene sequence and DNA-DNA hybridization experiments indicate that this isolate is related to the described Sulfolobus taxon and should be considered a novel species of the genus. We propose that this isolate is a novel species of the genus Sulfolobus that we name Sulfolobus tokodaii sp. nov. The type strain is strain 7 (JCM 10545).     

Nonomuraea maheshkhaliensis sp. nov., a novel actinomycete isolated from mangrove rhizosphere mud

A strain of Nonomuraea was isolated from Maheshkhali, Cox's Bazar, an unexplored region of Bangladesh. Strain 16-5-14(T) is a Gram-positive, aerobic, non-motile actinomycete that formed branched substrate and aerial mycelia. On the basis of 16S rRNA gene sequence similarity studies, strain 16-5-14(T) was shown to belong to the genus Nonomuraea, being most closely related to Nonomuraea kuesteri. Chemotaxonomic data supported allocation of the strain as a member of the genus Nonomuraea. The strain 16-5-14(T) contained MK-9(H(4)) as the major menaquinone, the polar lipid was phosphatidylethanolamine and major cellular fatty acids were observed as C(16 : 0 )(15.5%), iso-C(16 : 0) (13.8%) and 10-methyl C(17 : 0) (9.6%). Results of DNA-DNA hybridization and physiological tests allowed genotypic and phenotypic differentiation of strain 16-5-14(T) from closely related species N. kuesteri. Thus 16-5-14(T) represents a novel species of the genus Nonomuraea. On the basis of evaluation of the morphological, physiological and chemotaxonomic characteristics, 16S rRNA gene sequence comparisons and DNA-DNA hybridization, Nonomuraea maheshkhaliensis sp. nov. (type strain, 16-5-14(T)=JCM 13929(T)=MTCC 8545(T)) is proposed.     

Selenihalanaerobacter shriftii gen. nov., sp. nov., a halophilic anaerobe from Dead Sea sediments that respires selenate

We isolated an obligately anaerobic halophilic bacterium from the Dead Sea that grew by respiration of selenate. The isolate, designated strain DSSe-1, was a gram-negative, non-motile rod. It oxidized glycerol or glucose to acetate + CO2 with concomitant reduction of selenate to selenite plus elemental selenium. Other electron acceptors that supported anaerobic growth on glycerol were nitrate and trimethylamine-N-oxide; nitrite, arsenate, fumarate, dimethylsulfoxide, thiosulfate, elemental sulfur, sulfite or sulfate could not serve as electron acceptors. Growth on glycerol in the presence of nitrate occurred over a salinity range from 100 to 240 g/l, with an optimum at 210 g/l. Analysis of the 16S rRNA gene sequence suggests that strain DSSe-1 belongs to the order Halanaerobiales, an order of halophilic anaerobes with a fermentative or homoacetogenic metabolism, in which anaerobic respiratory metabolism has never been documented. The highest 16S rRNA sequence similarity (90%) was found with Acetohalobium arabaticum (X89077). On the basis of physiological properties as well as the relatively low homology of 16S rRNA from strain DSSe-1 with known genera, classification in a new genus within the order Halanaerobiales, family Halobacteroidaceae is warranted. We propose the name Selenihalanaerobacter shriftii. Type strain is strain DSSe-1 (ATCC accession number BAA-73).     

具抗肿瘤活性放线菌菌株YIM 90022的分离和系统发育分析

从青海盐碱土壤样品中分离到一株兼性嗜碱放线菌YIM 90022,该菌株的发酵产物具有很强的体外抗胃癌、肺癌、乳腺癌、皮肤癌、肾癌和子宫癌肿瘤细胞株活性。基于16S rRNA基因序列的系统发育分析表明,菌株YIM90022属于拟诺卡氏属(Nocardiopsis)的成员,与该属的4个有效发表种N.exhalansDSM 44407T,N.prasinaDSM43845T,N.metallicusDSM 44598T和N.listeriDSM 40297T系统发育关系最密切,与其分别以98.8%,98.5%,98.4%和97.8%的16S rRNA基因核苷酸序列相似性聚为一簇。但菌株YIM 90022不与这4个有效种中任何一个单独相聚,形成了一个独立亚分枝。结合形态特征、生理生化特性、细胞化学分类特征,以及rep-PCR基因指纹分析等方面的研究结果,菌株YIM 90022可能为拟诺卡氏菌属的一个潜在新种。菌株YIM 90022在大多数培养基上生长良好,气生菌丝和基内菌丝丰富,在酵母膏麦芽膏琼脂、燕麦片琼脂等培养基中产生可溶性色素。生长pH范围6.0~12.0,最适pH 8.5;能在含0~15%NaCl(W/V)的培养基上生长。     

Isolation and characterization of Bacillus thioparus sp. nov., chemolithoautotrophic, thiosulfate-oxidizing bacterium

A novel bacterium, strain BMP-1(T), was isolated from a continuous wastewater treatment culture system operating with a bacterial consortium. Cells of the isolate were Gram-variable, aerobic, moderately halotolerant, motile and endospore-forming rods. Strain BMP-1(T) grew chemolithoautotrophically by oxidation of thiosulfate to sulfate with a growth yield of 1.07 g protein mol(-1) of thiosulfate consumed. DNA G+C content was 43.8 mol%. Its cell wall had peptidoglycan based on m-diaminopimelic acid, and the major component of fatty acid was C(15 : 0). The 16S rRNA gene analysis showed that strain belongs to the genus Bacillus, sharing a 99.5% of sequence similarity with Bacillus jeotgali CCM 7133(T). DNA-DNA hybridization between the isolate of this study and this strain was 44%. Thus, the inclusion of strain BMP-1(T) in the genus Bacillus is suggested as a novel species and the name Bacillus thioparus sp. nov. (Type strain BMP-1(T)=BM-B-436(T)=CECT 7196(T)) is proposed. The sequence of the 16S rRNA gene has been deposited in GenBank with accession number DQ371431.     

Description of a novel actinobacterium Kocuria assamensis sp. nov., isolated from a water sample collected from the river Brahmaputra, Assam, India

A Gram-positive, pale yellow pigmented actinobacterium, strain S9-65(T) was isolated from a water sample collected from the river Brahmaputra, Assam, India and subjected to a polyphasic taxonomic study. The physiological and biochemical properties, major fatty acids (anteiso-C15:0 and anteiso-C17:0), estimated DNA G+C content (69.2 mol %) and 16S rRNA gene sequence analysis showed that strain S9-65(T) belonged to the genus Kocuria. Strain S9-65(T) exhibited highest 16S rRNA gene sequence similarity with Kocuria palustris (99.1%); however, the DNA-DNA relatedness value between strain S9-65(T) and K. palustris was 20.6%. On the basis of differential phenotypic characteristics and genotypic distinctiveness, strain S9-65(T) should be classified as representative of a novel species Kocuria, for which the name Kocuria assamensis is proposed. The type strain is S9-65(T) (=MTCC 10622(T) = DSM 23999(T)).     

含奥氏酮嗜盐紫色硫细菌的分离鉴定及系统发育分析

[目的]为挖掘我国紫色硫细菌物种和光合蛋白基因资源.[方法]采用Pfennig紫色硫细菌无机选择性培养基和琼脂稀释法.[结果]从青岛东风盐场分离获得一株含奥氏酮、耐高浓度硫化物、嗜盐耐碱紫色硫细菌菌株283-1.该菌株能氧化硫化物产生硫粒储存在细胞内、嗜盐、细胞含有奥氏酮类胡萝卜素、细菌叶绿素a强吸收峰位于830 nm处、运动、不产生气囊,表明属于Marichromatium属.16S rDNA序列同源性比较和系统发育分析也表明这一点.但该菌株能在1%～15%NaCl、7.5 mmol/L 高浓度硫化物、45℃、5000lux、pH9.0条件下生长良好,能很好的光同化C3和C4有机酸和葡萄糖酸钠等特性,与Marichromatium属4个种有明显不同.[结论]菌株283-1是Marichromatium属一个新分离物,编号 Marichromatium sp.283-1.     

Azospirillum himalayense sp. nov., a nifH bacterium isolated from Himalayan valley soil,India

Gram-negative, free-living bacterial strain ptl-3^T was isolated from Himalayan valley soil, India. Polyphasic taxonomy was performed including morphological characterization, fatty acid analysis, biochemical tests, 16S rRNA and nifH gene sequence analyses. 16S rRNA gene sequence analysis showed that the strain ptl-3^T belonged to the genus Azospirillum and was closely related to A. brasilense (98.7 % similarity) and A. rugosum (97 % similarity). 16S rRNA gene sequence similarity (96–95 %) was shown with other members of the genus Azospirillum. Major fatty acid 18:1ω7c was also similar to the genus Azospirillum. DNA–DNA relatedness value between strain ptl-3^T and A. brasilense was found to be 47 %. Various biochemical tests showed that the strain ptl-3^T differed from its closely related species A. brasilense. On the basis of phenotypic, chemotaxonomic and molecular genetics evidence, a bacterium with the type strain ptl-3^T is proposed as a novel species of the genus Azospirillum. The name of bacterial strain ptl-3^T has been proposed as Azospirillum himalayense sp. nov. The type strain of ptl-3^T (CCUG 58760^T, KCTC 23189^T) has been submitted to two culture collection centres. The accession numbers for 16S rRNA and nifH gene are GQ 284588 and GQ 249665. respectively.     

Hymenobacter perfusus sp. nov., Hymenobacter flocculans sp. nov. and Hymenobacter metalli sp. nov. three new species isolated from an uranium mine waste water treatment system

Three red-pink pigmented strains, designated A1-12(T), A2-50A(T) and A2-91(T), were recovered from two different sites in a uranium mine. For all strains, the optimum growth temperature was 25°C, the optimum pH was 6.0-6.5 and the DNA G+C contents were between 60 and 63.4 mol%. The major respiratory quinone was menaquinone 7 (MK-7) and the fatty acid profiles contained iso- and anteiso-branched C15 fatty acids, summed feature 3 (16:1 ω6c and/or ω7c and/or 15:0 iso 2-OH), summed feature 4 (17:1 anteiso B and/or iso I) and the unsaturated fatty acid 16:1 ω5c as the major components. Phylogenetic analysis of the 16S rRNA gene sequences showed that these organisms represented three distinct branches within the family Flexibacteraceae most closely related to the members of the genus Hymenobacter. Strain A1-12(T) formed a distinct phylogenetic line along with H. rigui KCTC 12533(T) and they shared approximately 98.9% 16S rRNA gene sequence similarity. However, these two strains shared only 14.7% pairwise similarity in their genomic DNA. Strains A2-50A(T) and A2-91(T) formed two distinct lineages, related to the species H. soli KCTC 12607(T), sharing about 95.5% 16S rRNA gene sequence similarity between themselves, and 88.3 and 92.0% with other members of the genus Hymenobacter. Based on the phylogenetic analysis and physiological and biochemical characteristics, these isolates were considered to represent three novel species for which we propose the names Hymenobacter perfusus for strain A1-12(T) (=CIP 110166=LMG 26000), Hymenobacter flocculans for strain A2-50A(T) (=CIP 110139=LMG 25699) and Hymenobacter metalli for strain A2-91(T) (=CIP 110140=LMG 25700).