1型CRISPR位点间区序列分析在嗜热链球菌菌株分型中的应用

为了建立适用于嗜热链球菌菌株资源多样性调查的菌株分型方法,尝试将1型CRISPR位点间区序列分析用于嗜热链球菌的菌株分型,并与常用ERIC-PCR指纹图谱方法进行了比较。结果表明,1型CRISPR位点间区序列分析可以把30株从三个不同样品中分离的嗜热链球菌分成三种差异明显的类型:不同类型菌株之间没有相同的间区序列;而同一类型菌株之间,间区序列的组成和排列则基本一致,并且上述分型的结果与用ERIC-PCR指纹图谱技术获得的结果完全一致。因此,1型CRISPR位点间区序列分析是嗜热链球菌分型鉴定的可靠方法,并适用于大量菌株的分型鉴定和多样性调查。     

副溶血性弧菌肠细菌基因间共有重复序列-PCR分子分型和耐药性、血清型研究

目的对副溶血性弧菌进行ERIC-PCR分子分型、耐药性和血清型相关性研究。方法肠细菌基因间共有重复序列（ERIC）为引物,对40株菌株基因组DNA进行扩增,得到DNA指纹图谱,并利用SPSS13.0统计软件对DNA扩增图谱进行分析,做出聚类图从而分型,并与菌株血清型、耐药性比较分析。结果40株菌用ERIC-PCR分为5个型,分辨力指数为（DI）为0.5;血清分型分为4个型;对8种抗生素中的萘啶酸、头孢噻亏、头孢西丁出现了不同程度的耐药。耐药菌株均出现在ERIC-PCR方法分型A型和血清分型O3型中。结论研究显示ERIC-PCR方法可以用于该菌分型分析,具有较好的分型能力。血清分型与ERIC-PCR方法分型一致。通过ERIC-PCR分型的树状图和血清分型结果推断,血清型O3群菌株很可能起源于血清型O1群菌株,血清型O3群和O1群密切相关。     

污染土壤中有机磷农药降解菌的分离及其多样性

采用添加有机磷农药的选择性培养基,在长期受有机磷农药污染的土壤中分离到7株有机磷农药降解菌,经生理生化鉴定和系统发育分析,菌株mp-1鉴定为Pseudaminobactersp.,菌株mp-2为Alcaligenessp.,菌株mp-7为Brucellasp.,其他菌株为Ochrobactrumsp.。16SrDNA序列同源性比较、系统发育分析和染色体ERIC-PCR指纹图谱扩增表明有机磷农药长期污染的土壤中有机磷农药降解菌具有丰富的多样性。     

两个黄芪根瘤菌新类群代表菌株的16SrNDA序列分析

对黄芪根瘤菌两个新类群中心菌株CA8561和JL84进行了16SrNDA全序列测定,并进行了系统发育学分析。结果表明,CA8561位于Rhizobium分支中,JL84位于Sinorhizobium分支中。     

产蓝色素放线菌细胞化学组分及16S rDNA序列分析

对分离自南京土壤中的一株高产水溶性蓝色素的放线菌菌株Streptomyces sp.进行了细胞化学组分及16S rDNA序列分析。细胞化学组分分析结果表明待定菌株的细胞壁含LL－DAP及甘氨酸,全细胞水解物含葡萄糖及核糖,全细胞脂肪酸组分主要为14－17碳的脂肪酸,其中anteiso-15和iso-16为主要组分,应归入链霉菌属。基于16S rDNA序列的聚类结果表明所选菌株基本分成9个分支,能够产生可溶性蓝色素的菌株聚成其中两个分支,即以S.coelicolor为代表的分支和以S.cyaneus为代表的分支,待定菌株与Streptomyces in-digocolor的相似性为99.4%,属于S.cyaneus分支。     

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。     

两个黄芪根瘤菌新类群代表菌株的16S rDNA序列分析

对黄芪根瘤菌两个新类群中心菌株CA8561和JL84进行了16S rDNA全序列测定,并进行了系统发育学分析。结果表明,CA8561位于Rhizobium分支中,JL84位于Sinorhizobium分支中。     

培养和非培养法分析冷藏鸡肉胴体中的细菌多样性

运用纯培养和非培养法对冷藏鸡肉胴体上细菌多样性进行了对比研究。采用培养法从鸡肉胴体中初步分离到45株细菌菌株,16S rDNA-ARDRA分析得到9株代表性细菌,其16S rDNA序列系统发育分析表明,这些菌株隶属于Bacillus sp.、Shigella sp.、Pseudomonas sp.、Citrobacter sp.、Klebsiella sp.和Escherichia sp.6个属。16S rDNA-ARDRA联合PAGE和16S rDNA全序列分析的非培养法结果表明,冷藏鸡肉中细菌主要属于Acinetobacter sp.、Bacillus sp.、Acidovorax sp.、Brochothrix thermosphacta、Lactococcus garvieae和Leuconostoc lactis等16个属。非培养法揭示的细菌多样性比培养法丰富,但二者结合使用能让肉品中微生物多样性得到更全面的展示。     

ERIC-PCR鉴别苏云金芽胞杆菌与蜡状芽胞杆菌的研究

利用ERIC-PCR技术对苏云金芽孢杆菌(Bt)、蜡状芽孢杆菌(Bc)和对照菌基因组DNA进行扩增,回收、标记BtPCR扩增片段,分别与各菌株的基因组DNA进行斑点杂交和Southern杂交,筛选Bt标识序列。结果显示:Bt各菌株均可扩增得到250bp的特异片段;Bt和Bc均可得到600bp的共有扩增片段;以筛选得到的569bp片段为探针,可特异性地与Bt基因组DNA杂交;ERIC-PCR技术可以在DNA指纹图谱水平区分鉴别Bt与Bc菌,正确反映出两者的亲缘关系。结果表明ERIC-PCR技术在Bt的检测及在Bt与Bc的鉴定中具有较强的实用性。     

甘草根瘤菌的16S rDNA全序列测定及系统进化分析

通过对西北干旱半干旱地区68株甘草根瘤菌的表型多样性和抗逆性分离研究,发现1个新类群和1个具有较高抗逆性的菌株。对其中心菌株CCNWGX022和高抗性菌株CCNWGX035进行16S rDNA全序列测定及系统进化研究。结果表明,CCNWGX022和CCNWGX035与中慢生根瘤菌属内参比菌株的16S rDNA相似性分别大于96．8％和98．3％,因此它们均属于中慢生根瘤菌属。     

Evaluation of four molecular typing methodologies as tools for determining taxonomy relations and for identifying species among Yersinia isolates

In the last few decades, molecular typing has become an important tool in taxonomic, phylogenetic and identification studies of numerous groups of bacteria, including the yersiniae. In this study, Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed-Field Gel Electrophoresis (PFGE), 16S rRNA gene sequencing and Multilocus Sequence Analysis (MLSA) were performed to determine the ability of these techniques to be used in taxonomy and identification of Yersinia strains. A total of 60 Yersinia strains were genotyped by ERIC-PCR and PFGE. Moreover, an in silico analysis was carried out for 16S rRNA gene sequencing and MLSA, using 68 and 49 Yersinia strains, respectively. A phylogenetic tree constructed from the ERIC-PCR, 16S rRNA gene sequencing and MLSA data grouped most of the Yersinia species into distinct species-specific clusters. In the PFGE assay these clusters were not observed. On this basis, ERIC-PCR, 16S rRNA gene sequencing and MLSA seem to be valuable techniques for use in taxonomic and identification studies of the genus Yersinia, whereas PFGE does not. Furthermore, ERIC-PCR has the advantage of being a cheaper, easier and faster assay than 16S rRNA gene sequencing or MLSA, and for these reasons can be considerate an alternative tool in taxonomic studies of yersiniae.     

先锋牧草-香根草联合固氮菌多样性

[目的]香根草(Vetiver zizanioides)是一种多年生禾本科草本植物,具有极强的生态适应性和抗逆能力,可作饲料和水土保持用.通过研究香根草联合固氮菌多样性,为进一步研究和应用打下基础.[方法]采用无氮培养基,首次从香根草中分离到47株联合固氮菌,分别应用SDS-PAGE全细胞蛋白质电泳、DNA指纹图谱、唯一碳源和16S rDNA全序列测定等方法,进行聚类和多样性分析.[结果]SDS-PAGE、IS-PCR和Bio-BIQA碳源利用的聚类结果基本一致,将供试菌株分为6个类群和4个单菌株;16S rDNA序列测定表明,从香根草中分离的菌株包括了佛莱辛草螺菌(Herbaspirillum frisingense)、中型假食酸菌(Pseudacidovorax intermedius)、恶臭假单胞菌(Pseudomonas putida)、荧光假单胞菌(Pseudomonas fluorescens)、越南伯克氏菌(Burkholderia vietnamiensis)、阴沟肠杆菌(Enterobacter cloacae)、路德维希肠杆菌(Enterobacter ludwigii)和松江壳聚糖降解菌(Mitsuaria chitosanitabida)等不同菌种.[结论]香根草联合固氮菌具有较大的资源多样性,对固氮菌资源的扩展和将来牧草上的应用具有重要意义.     

先锋牧草-香根草联合固氮菌多样性研究

摘要：【目的】香根草（Vetiver zizanioides）是一种多年生禾本科草本植物,具有极强的生态适应性和抗逆能力,可作饲料和水土保持用。通过研究香根草联合固氮菌多样性,为进一步研究和应用打下基础。【方法】采用无氮培养基,首次从香根草中分离到47株联合固氮菌,分别应用SDS-PAGE全细胞蛋白质电泳、DNA指纹图谱、唯一碳源和16S rDNA全序列测定等方法,进行聚类和多样性分析。【结果】SDS-PAGE、IS-PCR和Bio-BIQA碳源利用的聚类结果基本一致,将供试菌株分为6个类群和4个单菌株;     

Characterisation of wild legume nodulating bacteria (LNB) in the infra-arid zone of Tunisia

We report on the isolation and the characterization of nitrogen-fixing root nodule bacteria isolated from natural legumes in a region of South Tunisia corresponding to the infra-arid climatic zone. A collection of 60 new bacterial root nodule isolates were obtained from 19 legume species belonging to the genera Acacia, Anthyllis, Argyrolobium, Astragalus, Calycotome, Coronilla, Ebenus, Genista, Hedysarum, Hippocrepis, Lathyrus, Lotus, Medicago, Ononis. The isolates were characterised by (1) comparative 16S ARDRA using 7 enzymes, (2) total cell protein SDS-PAGE analysis and (3) 16S rDNA sequencing. The results show that these isolates are diverse and belong to the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium. Bradyrhizobium were further characterised by 16S-23S rDNA IGS sequencing. Surprisingly strains nodulating Astragalus cruciatus, Lotus creticus and Anthyllis henoniana were identified as Rhizobium galegae, a species recorded only as endosymbiont of Galega officinalis and G. orientalis in northern regions so far.     

Genetic diversity of bradyrhizobial populations from diverse geographic origins that nodulate Lupinus spp. and Ornithopus spp

The genetic diversity of 45 bradyrhizobial isolates that nodulate several Lupinus and Ornithopus species in different geographic locations was investigated by 16S rDNA PCR-RFLP and sequence analysis, 16S-23S rDNA intergenic spacer (IGS) PCR-RFLP analysis, and ERIC-PCR genomic fingerprinting. Reference strains of Bradyrhizobium japonicum, B. liaoningense and B. elkanii and some Canarian isolates from endemic woody legumes in the tribe Genisteae were also included. The 16S rDNA-RFLP analysis resolved 9 genotypes of lupin isolates, a group of fourteen isolates presented restriction-genotypes identical or very similar to B. japonicum, while another two main groups of isolates (69%) presented genotypes that clearly separated them from the reference species of soybean. 16S rDNA sequencing of representative strains largely agreed with restriction analysis, except for a group of six isolates, and showed that all the lupin isolates are relatives of B. japonicum, but different lineages were observed. The 16S-23S IGS-RFLP analysis showed a high resolution level, resolving 19 distinct genotypes among 30 strains analysed, and so demonstrating the heterogeneity of the 16S-RFLP groups. ERIC-PCR fingerprint analysis showed an enormous genetic diversity producing a different pattern for each but two of the isolates. Phylogeny of nodC gene was independent from the 16S rRNA phylogeny, and showed a tight relationship in the symbiotic region of the lupin isolates with isolates from Canarian genistoid woody legumes, and in concordance, cross-nodulation was found. We conclude that Lupinus is a promiscuous host legume that is nodulated by rhizobia with very different chromosomal genotypes, which could even belong to several species of Bradyrhizobium. No correlation among genomic background, original host plant and geographic location was found, so, different chromosomal genotypes could be detected at a single site and in a same plant species, on the contrary, an identical genotype was detected in very different geographical locations and plants.     

Genetic and symbiotic diversity of nitrogen-fixing bacteria isolated from agricultural soils in the Western Amazon by using cowpea as the trap plant

Cowpea is a legume of great agronomic importance that establishes symbiotic relationships with nitrogen-fixing bacteria. However, little is known about the genetic and symbiotic diversity of these bacteria in distinct ecosystems. Our study evaluated the genetic diversity and symbiotic efficiencies of 119 bacterial strains isolated from agriculture soils in the western Amazon using cowpea as a trap plant. These strains were clustered into 11 cultural groups according to growth rate and pH. The 57 nonnodulating strains were predominantly fast growing and acidifying, indicating a high incidence of endophytic strains in the nodules. The other 62 strains, authenticated as nodulating bacteria, exhibited various symbiotic efficiencies, with 68% of strains promoting a significant increase in shoot dry matter of cowpea compared with the control with no inoculation and low levels of mineral nitrogen. Fifty genotypes with 70% similarity and 21 genotypes with 30% similarity were obtained through repetitive DNA sequence (BOX element)-based PCR (BOX-PCR) clustering. The 16S rRNA gene sequencing of strains representative of BOX-PCR clusters showed a predominance of bacteria from the genus Bradyrhizobium but with high species diversity. Rhizobium, Burkholderia, and Achromobacter species were also identified. These results support observations of cowpea promiscuity and demonstrate the high symbiotic and genetic diversity of rhizobia species in areas under cultivation in the western Amazon.     

PCR-酶切技术快速鉴定军团菌

[目的]建立一种新型的军团菌鉴定方法,并探讨该法在鉴定环境水源和临床标本军团菌菌株中的应用价值.[方法]根据军团菌16S rRNA基因保守序列设计引物,以分离培养得到的可疑军团菌菌株作为模板,采用PCR法对模板扩增,并用限制性内切酶对PCR产物进行酶切分析,建立一种嗜肺军团菌及非嗜肺军团菌的鉴定方法.对16株嗜肺军团菌、22株非嗜肺军团菌及12株其他细菌标准菌株进行检测,验证该方法的可靠性,最后用该法检测广州地区分离的169株可疑军团菌菌株并进行基因测序.[结果]该PCR方法检测嗜肺军团菌及非嗜肺军团菌所有标准菌株均为阳性,非军团菌检测结果均为阴性;进一步的Hinf Ⅰ酶切分析可准确的区分嗜肺军团菌标准菌株;广州地区分离的169株可疑军团菌菌株经该法检测发现160株为军团菌,其中79株为嗜肺军团菌,与基因测序检测结果一致.[结论]PCR-酶切技术可快速、特异地检测军团菌及嗜肺军团菌,适用于环境水源和临床标本可疑军团菌菌株的检测.     

Genetic Diversity among Thermophilic Bacteria Isolated from Geothermal Sites by Using Two PCR Typing Methods

Microbial communities thriving at two hot springs, Hammam Pharaon (Pharaoh's Bath) and Oyoun Mossa (Moses springs), in Egypt was studied by cultural and molecular methods. Thirteen morphologically distinct strains of facultative anaerobic thermophilic bacterial isolates have been characterized and identified using phenotypic and genotypic characters including RAPD-PCR, ERIC-PCR typing, plasmid analysis and 16S rRNA sequencing. All isolates produced plasmid DNA with various sizes ranging from 0.7 kb to a larger plasmid 7.2 kb. The bacterial strains could tolerate a temperature range between 45 to 85°C and a pH between 4–11. Also, sulphate-reducing bacteria (SRB) in the thermal springs were investigated with combined biochemical and molecular approaches. A sulphate-reducing bacteria medium containing lactate was used for enrichment and isolation, which yielded Gram negative, rod shaped, anaerobic, non-spore-forming and motile bacteria capable of reducing sulphate to sulphide. These grew at temperatures ranging from 30 to 50°C and could use pyruvate, lactate and ethanol as electron donors. The dissimilatory sulphite reductase (DSR) gene sequences of eleven representative isolates revealed that the strains belonged to the sulphur reducing bacterial species Desulfovibrio vulgaris. 16S rRNA gene partial sequence results indicated the presence of novel or existing species of Bacillus (one species), Anoxybacillus (four species) and Geobacillus (eight species). In this study phenotypic and genotypic diversity were applied for the first time to differentiate thermophilic bacteria of such geothermal sites in Sinai, Egypt.     

Diverse endophytic nitrogen-fixing bacteria isolated from wild rice Oryza rufipogon and description of Phytobacter diazotrophicus gen. nov. sp. nov.

Twenty-three nitrogen-fixing bacteria were isolated from surface-sterilized stems and roots of wild rice Oryza rufipogon. Four clusters were defined among these bacteria by SDS-PAGE protein patterns and further confirmed by IS-PCR finger-printing analysis. Phylogenetic analysis of 16S rRNA gene sequences showed that the representative strains LS 8 and LS 18 of cluster II formed a monophyletic group sharing 94.0-97.3% similarities with defined enterobacterial species within the genera Salmonella, Citrobacter, Pantoea, Klebsiella, and Enterobacter. DNA-DNA hybridization, physiological, biochemical tests, and cell morphology also revealed that these strains could be differentiated from the related enterobacterial species. Based upon these results, we propose Phytobacter diazotrophicus gen. nov., sp. nov. to the bacterial group represented by strains LS 8 and LS 18. The type strain is LS 8(T) (=DSM 17806(T) = LMG 23328(T) = CGMCC 1.5339(T)). The DNA G+C content of strain LS 8(T) is 58.6 +/- 0.5 mol%.     

Characterization of nitrogen-fixing Paenibacillus species by polymerase chain reaction-restriction fragment length polymorphism analysis of part of genes encoding 16S rRNA and 23S rRNA and by multilocus enzyme electrophoresis

Forty-two strains representing the eight recognized nitrogen-fixing Paenibacillus species and 12 non-identified strains were examined by restriction fragment length polymorphism (RFLP) analysis of part of 16S and 23S rRNA genes amplified by polymerase chain reaction (PCR). Eleven different 16S rDNA genotypes were obtained from the combined data of RFLP analysis with four endonucleases and they were in agreement with the established taxonomic classification. Only one group of unclassified strains (Group I) was assigned in a separate genotype, suggesting they belong to a new species. Using the 23S PCR-RFLP method only six genotypes were detected, showing that this method is less discriminative than the 16S PCR-RFLP. Using the multilocus enzyme electrophoresis (MLEE) assay, the 48 strains tested could be classified into 35 zymovars. The seven enzymatic loci tested were polymorphic and the different profiles obtained among strains allowed the grouping of strains into 10 clusters. The PCR-RFLP methods together with the MLEE assay provide a rapid tool for the characterization and the establishment of the taxonomic position of isolates belonging to this nitrogen-fixing group, which shows a great potentiality in promoting plant growth.