拟诺卡氏菌16S rRNA,gyrB,sod和rpoB基因的系统发育分析

为了更好地了解拟诺卡氏菌属(Nocardiopsis)各物种间的系统发育关系,该属现有有效描述种的gyrB,sod和rpoB基因的部分序列被测定,结合16S rRNA基因,对拟诺卡氏菌属进行了系统发育重建。研究发现拟诺卡氏菌属gyrB,sod和rpoB基因的平均相似性分别为87.7%、87.3%和94.1%,而16S rRNA基因的平均相似性则达到96.65%,3个看家基因均比16S rRNA具有更高的分歧度。比较基于不同基因的系统树发现,由gyrB基因得到的系统树拓扑结构与16S rRNA得到的结构在亚群上基本一致。因此,gyrB基因在拟诺卡氏菌属的系统分类上比16S rRNA基因更具优越性。     

拟诺卡氏菌16S rRNA,gyrB,sod和rpoB基因的系统发育分析

为了更好地了解拟诺卡氏菌属(Nocardiopsis)各物种间的系统发育关系,该属现有有效描述种的gyrB,sod和rpoB基因的部分序列被测定,结合16S rRNA基因,对拟诺卡氏菌属进行了系统发育重建。研究发现拟诺卡氏菌属gyrB,sod和rpoB基因的平均相似性分别为87.7%、87.3%和94.1%,而16S rRNA基因的平均相似性则达到96.65%,3个看家基因均比16S rRNA具有更高的分歧度。比较基于不同基因的系统树发现,由gyrB基因得到的系统树拓扑结构与16S rRNA得到的结构在亚群上基本一致。因此,gyrB基因在拟诺卡氏菌属的系统分类上比16S rRNA基因更具优越性。     

根结线虫放线菌及其生物防治活性研究

从感染植物根部的根结线虫卵和雌虫中,分离得到放线菌20株。形态、细胞壁氨基酸组分和16S rRNA基因序列分析表明:分离菌株分属于链霉菌属(Streptomyces)、诺卡氏菌属(Nocardia)和假诺卡氏菌属(Pseudonocardia),其中链霉菌占80.0%。分离菌株对根结线虫Meloidogynespp.卵的平均寄生率、卵的孵化率、幼虫死亡率分别为54.1%、40.4%和26.2%。根据体外测试的结果,选择具有高致病力的3株链霉菌(1-17,2-6,9-47)和1株诺卡氏菌(5-1)进行温室番茄防效测试,其生防效率分别为31.4%、37.7%、56.4%和42.4%。     

养殖乌鳢诺卡氏菌病及其病原研究

2006年6月至7月,浙江萧山的部分乌鳢养殖池塘出现大规模死鱼现象,发病率达35%,死亡率100%。病死鱼鱼龄多在18个月左右,体长30－35 cm。症状主要表现为体表、鳍条充血,肝、脾、肾等内脏器官出现直径1－5 mm的乳白色结节。从病鱼的肝脏和肾脏中分离到菌株W060622,经光镜、电镜观察,在形态上均表现为长或短的分支状杆菌。回归感染试验证实W060622即引起此次乌鳢结节病的病原菌。通过16S rRNA基因的全序列测定,并与Genbank中相关序列进行比对,发现W060622与鰤鱼诺卡氏菌Nocardia seriolea JCM3360T (Z36925)的16S rRNA基因序列相似性达99.9%。结合菌株回归感染、形态学和生物学特征及16S rRNA分子鉴定,确定W060622为鰤鱼诺卡氏菌。     

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.     

罗布泊盐湖可培养放线菌多样性及PKS Ⅱ功能基因筛选

目的:挖掘新疆罗布泊盐湖可培养放线菌的资源和探索该环境放线菌产生Ⅱ型聚酮合成酶(PKS)基因的潜能。方法:从新疆罗布泊盐湖采集到10份样品,采用不同盐浓度的9种选择性培养基分离放线菌,并通过PCR技术扩增其16S r DNA序列、聚酮合成酶(PKS)基因分子筛选,测序后进行系统发育分析和BLAST比较研究。结果:试验结果共得42株放线菌,包括6个属,链霉菌属(Streptomyces)和拟诺卡氏菌属(Nocardiopsis)的类群最多,糖霉菌属(Glycomyces)、放线多孢菌属(Actinopolyspora)、微球菌(Micrococcus)和栖白蚁菌属(Isoptericola)的菌株相对较少。研究获得新的放线菌分类单元,其16S r DNA基因序列与已知菌株的序列相似性低于97%。42株放线菌中共有29株产生PKSⅡ基因,主要是链霉菌属和拟诺卡氏菌属。结论:研究结果表明罗布泊盐湖放线菌具有丰富的资源多样性和较强合成抗生素的潜在能力。     

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。     

青海盐碱环境中具抗肿瘤活性放线菌的筛选和多样性研究

从我国青海省采集盐碱土样或泥样,用添加1.0~3.0mol/L NaCl的GPY琼脂培养基和ISP2琼脂培养基分离到145株典型放线菌菌株。采用6种肿瘤细胞株对分离菌株的发酵产物进行体外筛选,得到26株抗肿瘤活性阳性菌株(17.9%),19株为拟诺卡氏属(Nocardiopsis)菌株,7株为链霉菌属(Streptomyces)菌株。在抗肿瘤活性、形态特征、生理生化特性和全细胞水解物氨基酸组分分析等实验结果的基础上,选取差异较大的8株抗肿瘤活性阳性菌株进行16S rRNA基因序列测定和系统发育多样性分析。结果表明,2株属于链霉菌属(Streptomyces)的1个已知物种和1个潜在新种;6株属于拟诺卡氏菌属(Nocardiopsis),可能代表该属的4个新种。研究表明青海盐碱环境中存在产生抗肿瘤活性物质的重要放线菌资源,也提示其中蕴藏着较丰富的微生物多样性。     

三江源地区不同退化程度草地的土壤放线菌区系

用不同分离方法,对三江源地区不同退化程度草地土壤放线菌的数量和多样性进行了比较.从5份土样中共分离放线茵178株,根据表型特征和16S rRNA基因序列分析,分别归入7个已知属:小单孢菌属(Micromonospora)、原小单孢菌属(Promicromonospora)、诺卡氏菌属(Nocardia)、假诺卡氏菌属(Pseudonocardia)、游动放线菌属(Actinoplanes)、韩国生工茵属(Kribbella)和链霉菌属(Streptomyces).其中链霉菌属分离菌株可归入7个表型类群.发现轻度退化高寒草原的土壤放线菌数量,丰度和多样性高于重度退化高寒草原;针茅高寒草原的土壤放线菌数量和多样性高于蒿草高寒草甸,而其中链霉菌的种类低于后者.表明高寒草地的退化程度与其中土壤放线茵的数量和多样性呈负相关.     

热带药用植物根际放线菌的分离、鉴定及生物活性分析

从海南热带植物园采集12种药用植物的根际土样,采用选择性分离方法,分离得到400株根际放线菌。使用5种活性筛选模型对分离菌株进行生物活性评价,154株放线菌在一个或多个活性筛选模型中显示为阳性,菌株初筛阳性率达38.5%;根据菌株形态特征并结合代谢产物的生物活性,从中挑选出28株菌进行16S rRNA基因序列分析,发现其分属于链霉菌属、诺卡氏菌属、小单孢菌属和野野村菌属。     

对诺卡氏菌AS4.1186和AS4.1187保藏菌株的重新分类研究

重新对念球状诺卡氏菌 (Nocardianostocoides)AS4 1 1 86和鲑色诺卡氏菌桔橙变种(Nocardiasalmonicolorvar.aurantiaca)AS4 1 1 87进行的多相分类研究表明 ,菌株AS4 1 1 86与德克萨斯糖丝菌 (Saccharothrixtexasensis)NRRLB 1 6 1 3 4 T 关系密切 ,它们的 1 6SrDNA序列相似性为 99 3 % ,DNA同源性为 77 6 % ;菌株AS4 1 1 87与赤红球菌 (Rhodococcusruber)DSM 43 3 3 8T 之间有着密切的关系 ,其 1 6SrDNA序列相似性为 99 5 % ,DNA同源性为 82 9%。胞壁组分、枝菌酸、甲基萘醌、磷酸类脂和DNAG +Cmol%测定等化学分类结果支持了上述结论。在形态和生理生化特性上 ,菌株AS4 1 1 86与NRRLB 1 6 1 3 4 T,菌株AS4 1 1 87与DSM 43 3 3 8T 之间也表现出非常相似的性状。根据系统发育分析、DNA同源性值、化学分类、形态和生理生化特性等研究结果 ,我们对菌株AS4 1 1 86和…     

Rhodococcus cercidiphylli sp. nov., a new endophytic actinobacterium isolated from a Cercidiphyllum japonicum leaf

An endophytic actinobacterium, designated YIM 65003(T), was isolated from a surface sterilized leaf sample of Cercidiphyllum japonicum collected from Yunnan province, south-west China. The morphological and chemotaxonomic properties of the isolate were typical of members of the genus Rhodococcus. Analysis of the 16S rRNA gene sequence revealed that the isolate was most closely related to Rhodococcus fascians DSM 20669(T) (99.6%) and Rhodococcus yunnanensis YIM 70056(T) (99.0%). DNA-DNA hybridization with the above microorganisms (46.3% and 48.8%, respectively), in combination with differences in the biochemical and physiological properties, suggested that strain YIM 65003(T) should be classified within a novel species of the genus Rhodococcus, for which the name Rhodococcus cercidiphylli sp. nov. is proposed, with YIM 65003(T) (=CCTCC AB 207160(T)=DSM 45141(T)) as the type strain. The 16S rRNA gene sequence of strain YIM 65003(T) has been deposited in GenBank under the accession number EU325542.     

Characterization of the ribosomal rrnD operon of the cephamycin-producer 'Nocardia lactamdurans' shows that this actinomycete belongs to the genus Amycolatopsis

The cephamycin producer strain 'Nocardia lactamdurans' contains four ribosomal RNA (rrn) operons. One of them (rrnD) was cloned from a DNA library in the bifunctional cosmid pJAR4. A 2229 bp region of rrnD has been sequenced. The 'N. lactamdurans' rrnD operon maintains the canonical order 5'-16S-23S-5S-3'. Four of the consensus Gürtler-Stanisch sequences were found in the 16S rRNA gene and a fifth one in the sequenced 5' region of the 23S rRNA gene. The anti Shine-Dalgarno sequence of 'N. lactamdurans' (located in the 3'-end of the 16S rRNA gene) was found to be 5'-CCUCCUUUCU-3' and is identical to that of Corynebacterium lactofermentum and Mycobacterium tuberculosis. A phylogenetic analysis of 'N. lactamdurans' by the neighbor-joining method using the entire 16S rRNA nucleotide sequence revealed that this actinomycete is closely related to Amlycolatopsis orientalis subsp orientalis, Amycolatopsis coloradensis, Amycolatopsis alba, Amycolatopsis sulphurea and other Amycolatopsis sp. but only distantly related to species of the genus Nocardia. The cephamycin producer 'N. lactamdurans' NRRL 3802 should be, therefore, classified as Amycolatopsis lactamdurans. The deduced secondary structure of the 16S rRNA is very similar to that of A. colorandensis and A. alba but different from those of species of the Nocardia genus supporting the incorporation of 'N. lactamdurans' into the genus Amycolatopsis.     

Ribosomal ribonucleic acid similarities in the classification of Rhodococcus and related taxa

Duplexes were prepared between 14C-labelled rRNA from both Rhodococcus equi C7 and Rhodococcus rhodochrous N54 and DNA from 16 actinomycetes representing the genera Rhodococcus, Mycobacterium, Nocardia, Saccharopolyspora and Streptomyces. The relationships between the organisms were determined by plotting the temperature at which 50% of the duplex was denatured (Tm(e)) against the percentage of rRNA binding (microgram 14C-labelled rRNA duplexed per 100 micrograms filter-bound DNA). All of the strains formed stable duplexes but each organism occupied a definite area on the rRNA similarity map. All of the organisms share a close phylogenetic relationship but representatives of the genera Rhodococcus, Mycobacterium, Nocardia and Streptomyces fell into four recognizable clusters on the similarity map. These data support and extend current trends in the classification of Rhodococcus and allied taxa. The guanine plus cytosine content of the DNA from the test strains was within the range 69.3 to 76.9 mol %.     

Isolation and Characterization of New Cyclohexylacetic Acid-Degrading Bacteria

Six cyclohexylacetic acid-degrading strains were isolated from soil samples in Japan and identified as members of the genera Cupriavidus (strain KUA-1), Rhodococcus, and Dietzia by 16S rRNA gene sequence analysis. For the first time members of these genera were shown to be capable of degrading cyclohexylacetic acid. A selected strain, KUA-1, which is the first reported Gram-negative organism capable of growth on cyclohexylacetic acid, was identified as a Cupriavidus metallidurans, based on morphologic and physiologic characteristics and its 16S rRNA gene sequence. Metabolite analysis by HPLC-MS indicated that 1-cyclohexenylacetic acid is an intermediate of cyclohexaneacetic acid metabolism in strain KUA-1.     

First human case of nocardiosis caused by Nocardia pseudobrasiliensis in Japan

Nocardia sp. IFM 0896, an actinomycete with biochemical characteristics that differed from Nocardia brasiliensis, was isolated from a 71-year-old Japanese man with a history of tuberculosis and cancer. Although the isolate was tentatively identified as N. brasiliensis, the morphological and physiological characteristics of strain IFM 0896 were different from those of N. brasiliensis IFM 0236T. The results of 16S rRNA gene sequence phylogenies and PCR-RFLP analysis of a heat shock protein revealed that Nocardia sp. IFM 0896 belongs to the species N. pseudobrasiliensis. This is the first clinical isolation report of N. pseudobrasiliensis in Japan.     

Identification of Escherichia coli through analysis of 16S rRNA and 16S-23S rRNA internal transcribed spacer region sequences

A bacterial strain, designated BzDS03 was isolated from water sample, collected from Dal Lake Srinagar. The strain was characterized by using 16S ribosomal RNA gene and 16S-23S rRNA internal transcribed spacer region sequences. Phylogenetic analysis showed that 16S rRNA sequence of the isolate formed a monophyletic clade with genera Escherichia. The closest phylogenetic relative was Escherichia coli with 99% 16S rRNA gene sequence similarity. The result of Ribosomal database project's classifier tool revealed that the strain BzDS03 belongs to genera Escherichia.16S rRNA sequence of isolate was deposited in GenBank with accession number FJ961336. Further analysis of 16S-23S rRNA sequence of isolate confirms that the identified strain BzDS03 be assigned as the type strain of Escherichia coli with 98% 16S-23S rRNA sequence similarity. The GenBank accession number allotted for 16S-23S rRNA intergenic spacer sequence of isolate is FJ961337.     

一株乙腈降解菌的鉴定及其生物学特性

【目的】明确乙腈降解菌BX2的分类地位及生物学特性,评价其处理含乙腈废水的可行性。【方法】通过形态特征、生理生化特性以及系统发育分析对菌株BX2进行鉴定。考察温度、初始pH及接种量等因素对菌株生长的影响,确定菌株的最佳生长条件及在该条件下的乙腈降解能力。测定菌株BX2对NaCl的耐受能力。【结果】乙腈降解菌BX2的形态特征及生理生化特性与紫红红球菌(Rhodococcus rhodochrous)最相近。其16S rRNA、gyrB、secA1基因序列与紫红红球菌的相似性分别为99.37%、99.29%、97.87%。最佳生长条件为35℃,初始pH 7.5,接种量1%。此条件下,菌株BX2在16 h内对浓度为800 mg/L乙腈的降解率为95.87%。菌株BX2在NaCl含量高于6%的培养基中无法生长。【结论】菌株BX2被鉴定为紫红红球菌。该菌株有较强的环境适应能力,可降解高浓度乙腈,在含氰废水的生物修复中有很好的应用前景。     

Biodegradation of di-n-butyl phthalate by Rhodococcus sp. JDC-11 and molecular detection of 3, 4-phthalate dioxygenase gene

Rhodococcus sp. JDC-11, capable of utilizing di-n-butyl phthalate (DBP) as the sole source of carbon and energy, was isolated from sewage sludge and confirmed mainly based on 16S rRNA gene sequence analysis. The optimum pH, temperature, and agitation rate for DBP degradation by Rhodococcus sp. JDC-11 was 8.0, 30 degrees C, and 175 rpm, respectively. In addition, the effect of glucose concentration on DBP degradation indicated that low concentration of glucose inhibited the degradation of DBP while high concentrations of glucose increased its degradation. Meanwhile, the substrates utilization test showed that JDC-11 could also utilize other phthalates. Furthermore, the major metabolites of DBP degradation were identified as mono-butyl phthalate and phthalic acid by gas chromatography-mass spectrometry and the metabolic pathway of DBP degradation by Rhodococcus sp. JDC-11 was tentatively speculated. Using a set of new degenerate primer, partial sequence of the 3, 4-phthalate dioxygenase gene was obtained from the strain. Sequence analysis revealed that the phthalate dioxygenase gene of JDC-11 was highly homologous to the large subunit of phthalate dioxygenase from Rhodococcus coprophilus strain G9.