Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer

Abstract The 16S rRNA gene sequences of 19 strains covering 97% of the molecules were determined for the members of the family Rhizobiaceae and related bacteria by PCR and DNA sequencer. The three biovars of Agrobacterium were located separately, whereas Agrobacterium rubi clustered with A. tumefaciens . Phylogenetic locations for the species of the genera Rhizobium, Sinorhizobium, Agrobacterium, Phylobacterium, Mycoplana (M. dimorpha), Ochrobactrum, Brucella and Rochalimaea (a rickettsia) were intermingled with each other with the similarity values higher than 92%. The family Rhizobiaceae should be redefined including the above-mentioned genera despite the ability for plant association and nitrogen fixation. Bradyrhizobium japonicum and Mycoplana bullata were far remote from the other species and should be excluded from this family.     

Restriction fragment length polymorphisms of 16S rRNA genes in the differentiation of fast-growing mycobacterial species

Abstract DNA from several species of fast growing mycobacteria displayed a characteristic restriction fragment length polymorphism (RFLP) pattern when hybridizated to a Mycobacterium fortuitum 16S rRNA gene fragment. The resulting patterns were identical when comparing different strains belonging to the same species. The RFLP results were consistent with those obtained by DNA-DNA hybridization studies. Using this approach, we have been able to identify the number of copies for 16S rRNA genes in several fast-growing mycobacteria.     

Species of Borrelia distinguished by restriction site polymorphisms in 16S rRNA genes

Abstract Three phyletic groups of Borrelia associated with Lyme disease, B. burgdorferi, B. garinii and group VS461 can be distinguished from each other and other species of Borrelia by Bfa I restriction site polymorphisms in PCR amplified 16S rRNA genes. One strain isolated from an Ixodes pacificus tick in California that was previously unclassifiable was distinguishable from B. burgdorferi by an Mnl I restriction site polymorphism.     

DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities

A protocol was devised which permitted the extraction of DNA from deep marine sediments up to 503 m below the sea floor. These sediments have been laid down over the last 3 million years. 16S rRNA gene sequences were amplified from the DNA by the polymerase chain reaction. The details of the successful extraction and polymerase chain reaction methodology varied between samples from different depths. This emphasizes the attention to detail required to allow the diversity of bacteria in these deep sediments to be studied.     

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.     

Mycoplasma gallisepticum 16S rRNA genes

Abstract The genome of Mycoplasma gallisepticum A5969 contains a truncated pseudogene for 16S rRNA in addition to a single unsplit rRNA-operon and a second discontinuous set of rRNA genes. Other M. gallisepticum strains tested do not posses the truncated gene. This gene is almost identical to full-size isolated 16S rRNA gene starting from at least 500 nucleotides upstream of the coding sequence and ending at the 977th nucleotide within the structural part of 16S rRNA.     

Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a popular high-throughput fingerprinting technique used to monitor changes in the structure and composition of microbial communities. This approach is widely used because it offers a compromise between the information gained and labor intensity. In this review, we discuss the progress made in T-RFLP analysis of 16S rRNA genes and functional genes over the last 10 years and evaluate the performance of this technique when used in conjunction with different statistical methods. Web-based tools designed to perform virtual polymerase chain reaction and restriction enzyme digests greatly facilitate the choice of primers and restriction enzymes for T-RFLP analysis. Significant improvements have also been made in the statistical analysis of T-RFLP profiles such as the introduction of objective procedures to distinguish between signal and noise, the alignment of T-RFLP peaks between profiles, and the use of multivariate statistical methods to detect changes in the structure and composition of microbial communities due to spatial and temporal variation or treatment effects. The progress made in T-RFLP analysis of 16S rRNA and genes allows researchers to make methodological and statistical choices appropriate for the hypotheses of their studies.     

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.     

Sequence analysis of mitochondrial 16S ribosomal RNA gene fragment from seven mosquito species

Mosquitoes are vectors for the transmission of many human pathogens that include viruses, nematodes and protozoa. For the understanding of their vectorial capacity, identification of disease carrying and refractory strains is essential. Recently, molecular taxonomic techniques have been utilized for this purpose. Sequence analysis of the mitochondrial 16S rRNA gene has been used for molecular taxonomy in many insects. In this paper, we have analysed a 450 bp hypervariable region of the mitochondrial 16S rRNA gene in three major genera of mosquitoes,Aedes, Anopheles andCulex. The sequence was found to be unusually A + T rich and in substitutions the rate of transversions was higher than the transition rate. A phylogenetic tree was constructed with these sequences. An interesting feature of the sequences was a stretch of Ts that distinguished betweenAedes andCulex on the one hand, andAnopheles on the other. This is the first report of mitochondrial rRNA sequences from these medically important genera of mosquitoes.     

16S rRNA PCR鉴定脆弱类杆菌

目的:应用16SrRNA序列设计PCR引物鉴别脆弱类杆菌。方法:通过脆弱类杆菌16SrRNA序列特异性位点设计引物,对4株脆弱类杆菌及大肠杆菌、乳酸杆菌、嗜热链球菌等进行PCR扩增。应用琼脂糖电泳法对PCR扩增产物进行特异性检测。结果:脆弱类杆菌在176bp左右出现特异性条带,而其他细菌均未出现特异性条带。结论:通过16SrRNA序列中特异位点设计引物进行PCR,可特异性鉴定脆弱类杆菌。     

Fluorescent Pseudomonas species categorized by using polymerase chain reaction (PCR)/restriction fragment analysis of 16S rDNA

A rapid procedure for the identification of fluorescent pseudomonads, based on the polymerase chain reaction (PCR) and restriction fragment analysis of 16S rDNA genes is described. Thirty-one strains belonging to 10 different Pseudomonas species of the Pseudomonas fluorescens rRNA branch were characterized. Amplified rDNA was digested with 13 different restriction endonucleases. The combined data from restriction analysis enabled the definition of 17 different 16S rDNA genotypes. All type strains belonging to different species were differentiated. The good correlation between grouping obtained using restriction analysis with other molecular classification criteria demonstrates the value of the described method to characterize rapidly fluorescent Pseudomonas strains at the species level.     

嗜盐菌HBCC-2的16S rRNA基因测序分析及其培养特性

从连云港台南盐场海盐生产区中分离纯化到一株嗜盐古菌HBCC-2,该菌株经PCR扩增后,测定其16S rRNA基因序列,采用BLAST软件对基因库中基因序列进行同源性比较,选取其相似性序列,采用Clustalx1.8和MEGA3.1软件对其16S rDNA序列进行了系统发育分析研究,结果表明HBCC-2菌株与菌株Halorubrum sp.GSL5.48的相似性达99%,结合其形态观察及生理生化反应特性,初步确定该菌株属于嗜盐红菌属(Halorubrum),菌株HBCC-2的16S rDNA序列已登陆到GenBank,其序列号为EF687739.通过比较不同NaCl浓度、pH和培养温度对该菌株生长的影响情况,研究了该菌株的生长特性,结果表明NaCl浓度为4mol/L、温度为35℃和pH为7.0的培养条件下其生长最佳.     

双歧杆菌地高辛标记寡核苷酸基因探针制备和应用研究

目的探讨地高辛标记寡核苷酸基因探针应用于微生态研究的可行性和实用性。方法制备双歧杆菌属和部分种的地高辛标记16S rRNA寡核苷酸探针,初步应用于微生态制剂鉴定和临床肠道微生态检测,评价寡核苷酸探针杂交在肠道微生态研究和检测中的应用价值。结果地高辛标记寡核苷酸探针具有较好的特异性与灵敏度：地高辛标记的双歧杆菌属和种的共6种寡核苷酸基因探针与标准菌株杂交后灵敏度和特异度分别为属探针95％、75％,青春双歧87．5％、90％,两歧双歧87．5％、87．5％,短双歧87．5％、92．5％,婴儿双歧75％、95％,长双歧75％、100％。结论寡核苷酸基因探针用于肠道细菌的鉴定显示出一定前景,加大探针的种类与扩大调查范围有可能使该技术替代现有细菌培养技术。     

Bacterial phylogeny based on 16S and 23S rRNA sequence analysis

Abstract: Molecular phylogeny increasingly supports the understanding of organismal relationships and provides the basis for the classification of microorganisms according to their natural affiliations. Comparative sequence analysis of ribosomal RNAs or the corresponding genes currently is the most widely used approach for the reconstruction of microbial phylogeny. The highly and less conserved primary and higher order structure elements of rRNAs document the history of microbial evolution and are informative for definite phylogenetic levels. An optimal alignment of the primary structures and a careful data selection are prerequisites for reliable phylogenetic conclusions. rRNA based phylogenetic trees can be reconstructed and the significance of their topologies evaluated by applying distance, maximum parsimony and maximum likelihood methods of phylogeny inference in comparison, and by fortuitous or directed resampling of the data set. Phylogenetic trees based on almost equivalent data sets of bacterial 23S and 16S rRNAs are in good agreement and their overall topologies are supported by alternative phylogenetic markers such as elongation factors and ATPase subunits. Besides their phylogenetic information content, the differently conserved primary structure regions of rRNAs provide target sites for specific hybridization probes which have been proven to be powerful tools for the identification of microbes on the basis of their phylogenetic relationships.     

Molecular identification of pufferfish species using PCR amplification and restriction analysis of a segment of the 16S rRNA gene

This study amplified the mitochondrial 16S rRNA gene using polymerase chain reaction (PCR) with a template of total DNA from muscle tissues of nine pufferfish species collected from the coastal area of Okinawa Islands in Japan: Pleuranacanthus sceleratus, Triodon macropterus, Chelonodon patoca, Sphoeroides pachygaster, Arothron hispidus, A. stellatus, A. manilensis, A. mappa, and A. nigropunctatus. Then nucleotide sequence encoding a partial region of the 16S rRNA gene was compared among species. The sequenced fragment was also used to select restriction enzymes, yielding species-specific restriction fragment length polymorphisms (RFLP). The sequence of the segment of the 16S rRNA gene consisted of about 615 nucleotides and showed interspecies variations in the targeted region. After calculation of corresponding RFLP-patterns of nine species investigated with suitable restriction enzymes, three restriction enzymes – BanII, DdeI, and NlaIII – were found to be sufficient for identification of all nine species. Successful testing of this methodology in frozen and heated food samples suggests its utility for pufferfish species authentication in food products.     

从线粒体基因探讨中国大头蛙群的分类及其属内地位

Partial sequences of the mitochondrial 12S rRNA and 16S rRNA gene were determined for 8 populations of three species of Chinese Limnonectes, and aligned with the published sequences of Limnonectes from other parts of the world. When Nanorana parker, Paa boulengeri, Fejervarya limnocharis and Hoplobatrachus rugulosus was used as outgroup taxa (Accession Nos. AY158705, AY313685, AF206111, AF206491, AY322311). The sequences of the 12S rRNA and 16S rRNA genes totaled 950 nueleotide positions with gaps including 510 variable sites. We reconstructed phylogenetie trees using Clustal X 1.8, Mega 2.1 and PHYLIP 3.5e software, and using the maximum parsimony and maximum likelihood methods, respectively. Our analyses suggest that these fanged frogs from China are another monophyletie group in addition to the four monophyletie groups identified by previous studies. The Chinese Limnonectes were grouped into three elades (BCL 55% ). The first elade contains one species (BCL 100% ), from a population of Limnoneetes fragilis from Hainan Province. The second contains four individuals (BCL 100% ), i. e. two populations of Limnonectes kuhlii from Yunnan Province. The third contains one species (BCL 100% ), i. e. five populations of Limnonectes fujianensis from Fujian Province and 1 from Taiwan Province. The resulted phylogenetie trees indicate L. fragilis is basal to L. kuhlii L. fujianensis 。