Heteroduplex structures in 16S-23S rRNA intergenic transcribed spacer PCR products reveal ribosomal interoperonic polymorphisms within single Frankia strains

AIMS: Detection of polymorphisms in intergenic transcribed spacer (ITS) 16S-23S rRNA within single Frankia strains. METHODS AND RESULTS: Polymorphisms in the 16S-23S rRNA ITS were investigated in single-colony subcultures of seven Frankia isolates. Multiple ITS-polymerase chain reaction (PCR) bands were detected solely in isolates BMG5.5 and BMG5.11. The slow-migrating bands in the ITS-PCR agarose gel electrophoresis profiles of the isolates were revealed to be heteroduplexes on the basis of their migration shift in different electrophoretic matrices, southern hybridization and the single-strand DNA mung bean endonuclease digestion. Laser-scanned capillary electrophoresis detected two ITS-PCR fragments differing in length by three and six nucleotide insertions/deletions in strains BMG5.5 and BMG5.11, respectively. Sequence analysis of the cloned ITS showed that in strain BMG5.5 the two ITS differed by the presence of three to four copies of the 3-bp tandem repeat 5'-TGG-3'. In strain BMG5.11, the two ITS differed by the presence of two to three copies of the 6-bp tandem repeat 5'-CTTGGG-3'. CONCLUSIONS: We demonstrate the occurrence of ITS 16S-23S rRNa polymorphisms within single Frankia strains. SIGNIFICANCE AND IMPACT OF THE STUDY: We reported the occurrence of ITS 16S-23S rRNA polymorphisms within single Frankia strains from Elaeagnus host group recognized as the more flexible strains within Frankia genus. Furthermore, we underscored the applied interest of strains BMG5.11 and BMG5.5 in future ecological studies using ITS 16S-23S rRNA as molecular marker.     

Characterization of Paenibacillus popilliae rRNA operons

The terminal 39 nucleotides on the 3' end of the 16S rRNA gene, along with the complete DNA sequences of the 5S rRNA, 23S rRNA, tRNA(Ile), and tRNA(Ala) genes were determined for Paenibacillus popilliae using strains NRRL B-2309 and Dutky 1. Southern hybridization analysis with a 16S rDNA hybridization probe and restriction-digested genomic DNA demonstrated 8 copies of the 16S rRNA gene in P. popilliae strains KLN 3 and Dutky 1. Additionally, the 23S rRNA gene in P. popilliae strains NRRL B-2309, KLN 3, and Dutky 1 was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to occur as 8 copies. It was concluded that these 3 P. popilliae strains contained 8 rrn operons. The 8 operon copies were preferentially located on approximately one-half of the chromosome and were organized into 3 different patterns of genes, as follows: 16S-23S-5S, 16S-ala-23S-5S, and 16S-5S-ile-ala-23S-5S. This is the first report to identify a 5S rRNA gene between the 16S and 23S rRNA genes of a bacterial rrn operon. Comparative analysis of the nucleotides on the 3' end of the 16S rRNA gene suggests that translation of P. popilliae mRNA may occur in Bacillus subtilis and Escherichia coli.     

我国南北大豆产区慢生大豆根瘤菌的遗传多样性和系统发育研究

利用16S rRNA基因RFLP、16S rRNA基因序列分析以及16S-23S rRNA IGS PCR RFLP技术对分离自我国南北大豆产区的慢生大豆根瘤菌进行了群体遗传多样性和系统发育研究。16S rRNA基因PCR RFLP分析以及16S rRNA基因序列分析结果表明:所有供试慢生大豆根瘤菌可分为B.japonicum和B.elkanii两个类群,其中属于B.japonicum的为优势种群,占供试菌株的91%,属于B.elkanii的仅占9%,多样性水平较低。16S-23S rRNA IGS PCRRFLP研究结果表明:属于B.japonicum的慢生根瘤菌具有较丰富的遗传多样性,在69%的相似性水平上可分为群Ⅰ和群Ⅱ两大类群。群I的菌株以分离自黑龙江和河北等北部区域的菌株为代表,群Ⅱ的菌株以分离自广西和江苏等南部地域的菌株为代表,反映出明显的地域特征。两群菌株在系统发育上均与USDA6、USDA110和USDA122等B.japonicum的模式或代表菌株有差异。     

Diversity and origin of Desulfovibrio species: phylogenetic definition of a family

The different nutritional properties of several Desulfovibrio desulfuricans strains suggest that either the strains are misclassified or there is a high degree of phenotypic diversity within the genus Desulfovibrio. The results of partial 16S rRNA and 23S rRNA sequence determinations demonstrated that Desulfovibrio desulfuricans ATCC 27774 and "Desulfovibrio multispirans" are closely related to the type strain (strain Essex 6) and that strains ATCC 7757, Norway 4, and El Agheila Z are not. Therefore, these latter three strains of Desulfovibrio desulfuricans are apparently misclassified. A comparative analysis of nearly complete 16S rRNA sequences in which we used a least-squares analysis method for evolutionary distances, an unweighted pair group method, a signature analysis method, and maximum parsimony was undertaken to further investigate the phylogeny of Desulfovibrio species. The species analyzed were resolved into two branches with origins deep within the delta subdivision of the purple photosynthetic bacteria. One branch contained five deep lineages, which were represented by (i) Desulfovibrio salexigens and Desulfovibrio desulfuricans El Agheila Z; (ii) Desulfovibrio africanus; (iii) Desulfovibrio desulfuricans ATCC 27774, Desulfomonas pigra, and Desulfovibrio vulgaris; (iv) Desulfovibrio gigas; and (v) Desulfomicrobium baculatus (Desulfovibrio baculatus) and Desulfovibrio desulfuricans Norway 4. A correlation between 16S rRNA sequence similarity and percentage of DNA relatedness showed that these five deep lineages are related at levels below the minimum genus level suggested by Johnson (in Bergey's Manual of Systematic Bacteriology, vol. 1, 1984). We propose that this branch should be grouped into a single family, the Desulfovibrionaceae. The other branch includes other genera of sulfate-reducing bacteria (e.g., Desulfobacter and Desulfococcus) and contains Desulfovibrio sapovorans and Desulfovibrio baarsii as separate, distantly related lineages.     

A pseudogene cluster in the leader region of the Euglena chloroplast 16S-23S rRNA genes.

The nucleotide sequence of a region (leader region) preceding the 5'-end of 16S-23S rRNA gene region of Euglena gracilis chloroplast DNA was compared with the homologous sequences that code for the 16S-23S rRNA operons of Euglena and E. coli. The leader region shows close homology in sequence to the 16S-23S rRNA gene region of Euglena (Orozco et al. (1980) J. Biol.Chem. 255, 10997-11003) as well as to the rrnD operon of E. coli, suggesting that it was derived from the 16S-23S rRNA gene region by gene duplication. It was shown that the leader region had accumulated nucleotide substitutions at an extremely rapid rate in its entirety, similar to the rate of tRNAIle pseudogene identified in the leader region. In addition, the leader region shows an unique base content which is quite distinct from those of 16S-23S rRNA gene regions of Euglena and E. coli, but again is similar to that of the tRNAIle pseudogene. The above two results strongly suggest that the leader region contains a pseudogene cluster which was derived from a gene cluster coding for the functional 16S-23S rRNA operon possibly by imperfect duplication during evolution of Euglena chloroplast DNA.     

Use of 16S-23S rRNA spacer-region (SR)-PCR for identification of intestinal clostridia

The suitability of a species identification technique based on PCR analysis of 16S-23S rRNA spacer region (SR) polymorphism for human intestinal Clostridium species was evaluated. This SR-PCR based technique is highly reproducible and successfully differentiated the strains tested, which included 17 ATCC type strains of Clostridium and 152 human stool Clostridium isolates, at the species or intraspecies level. Ninety-eight of 152 stool isolates, including C. bifermentans, C. butyricum, C. cadaveris, C. orbiscindens, C. paraputrificum, C. pefringens, C. ramosum, C. scindens, C. spiroforme, C. symbiosum and C. tertium, were identified to species level by SR-PCR patterns that were identical to those of their corresponding ATCC type strains. The other 54 stool isolates distributed among ten SR-PCR patterns that are unique and possibly represent ten novel Clostridium species or subspecies. The species identification obtained by SR-PCR pattern analysis completely agreed with that obtained by 16S rRNA sequencing, and led to identification that clearly differed from that obtained by cellular fatty acid analysis for 23/152 strains (15%). These results indicate that SR-PCR provides an accurate and rapid molecular method for the identification of human intestinal Clostridium species.     

多项分类方法鉴定西藏地区藏灵菇中的乳酸球菌

【目的】采用多项分类法对16株分离自藏灵菇中的乳酸球菌进行准确鉴定。【方法】首先应用传统的生理生化试验,之后采用16S-23S rRNA间区序列多态性分析和变性梯度凝胶电泳(DGGE)进行了鉴定,最后,通过16S rRNA基因序列分析进行验证。【结果】将16株菌株初步鉴定为3个菌群:片球菌群、乳球菌群和肠球菌群,进一步鉴定为14株耐久肠球菌,1株乳酸片球菌,1株乳酸乳球菌乳酸亚种,16S rRNA基因序列分析验证的结果与前3种试验方法的结果相一致。【结论】试验结果表明传统的生理生化鉴定和16S-23S rRNA间区序列多态性分析和变性梯度凝胶电泳(DGGE)相结合的多项分类方法有利于乳酸球菌种间的准确鉴定。     

黄土高原地区大豆根瘤菌的遗传多样性和系统发育

【目的】研究黄土高原地区大豆根瘤菌的遗传多样性和系统发育。【方法】采用BOX-PCR、16S rDNAPCR-RFLP、16S-23S IGS PCR-RFLP和16S rRNA基因序列分析方法对分离自我国黄土高原地区4个省的15个地区的130株大豆根瘤菌及部分参比菌株进行了遗传多样性和系统发育分析。【结果】BOX-PCR反映的菌株多样性最丰富,形成的遗传群最多,16S rDNA PCR-RFLP方法在属、种水平上聚群较好,16S-23S IGSPCR RFLP反映的多样性介于BOX-PCR和16S rDNA PCR-RFLP之间,能够较好地反映出属、种和亲缘关系很近的菌株间的差异,3种方法聚类分析结果基本一致,可将所有供试菌株分为两大类群,中华根瘤菌属(Sinorhizobium)和慢生根瘤菌属(Bradyrhizobium)。从系统发育来看,供试的快生大豆根瘤菌为费氏中华根瘤菌(Sinorhizobium fredii),慢生大豆根瘤菌为日本慢生大豆根瘤菌(Bradyrhizobium japonicum)和辽宁慢生根瘤菌(Bradyrhizobium liaoningense)。【结论】我国黄土高原地区大豆根瘤菌具有较丰富的遗传多样性,S.fredii优势种,慢生大豆根瘤菌仅占10%,同时,分离到2株B.liaoningense。     

Gluconobacter sphaericus (Ameyama 1975) comb. nov., a brown pigment-producing acetic acid bacterium in the Alphaproteobacteria

Strain NBRC 12467(T )was examined genetically, phylogenetically, phenotypically, and chemotaxonomically. The DNA G+C content of the strain was 59.5 mol%. The strain represented low levels of DNA-DNA hybridization of 49-9% to the type strains of eight Gluconobacter species. The strain formed a cluster along with the type strains of G. albidus and G. kondonii in phylogenetic trees based on 16S rRNA gene sequences. In a phylogenetic tree based on 16S-23S rRNA gene ITS sequences, however, the strain formed an independent cluster from the type strains of the eight Gluconobacter species. Such phylogenetic relationships were supported by the calculated pair-wise 16S rRNA gene and 16S-23S rRNA gene ITS sequence similarities. The strain was distinguished from the type strains of the eight Gluconobacter species by 16S-23S rRNA gene ITS restriction analysis using five restriction endonucleases. The strain produced a water-soluble brown pigment and 2,5-diketo-D-gluconate from D-glucose, differing from the type strains of the eight Gluconobacter species, and acid from meso-erythritol very weakly, differing from the type strains of the remaining seven Gluconobacter species except for the type strain of G. roseus, but not from maltose, differing from the type strain of G. oxydans, and had Q-10. For the strain, which was once classified as G. oxydans subsp. sphaericus, Gluconobacter sphaericus (Ameyama 1975) comb. nov. is proposed. The type strain is NBRC 12467(T), which is also deposited as BCC 14448(T).     

16S-23S rDNA intergenic spacer region polymorphism of Lactococcus garvieae,Lactococcus raffinolactis and Lactococcus lactis as revealed by PCR and nucleotide sequence analysis

The intergenic spacer region (ISR) between the 16S and 23S rRNA genes was tested as a tool for differentiating lactococci commonly isolated in a dairy environment. 17 reference strains, representing 11 different species belonging to the genera Lactococcus, Streptococcus, Lactobacillus, Enterococcus and Leuconostoc, and 127 wild streptococcal strains isolated during the whole fermentation process of "Fior di Latte" cheese were analyzed. After 16S-23S rDNA ISR amplification by PCR, species or genus-specific patterns were obtained for most of the reference strains tested. Moreover, results obtained after nucleotide analysis show that the 16S-23S rDNA ISR sequences vary greatly, in size and sequence, among Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus lactis as well as other streptococci from dairy environments. Because of the high degree of inter-specific polymorphism observed, 16S-23S rDNA ISR can be considered a good potential target for selecting species-specific molecular assays, such as PCR primer or probes, for a rapid and extremely reliable differentiation of dairy lactococcal isolates.     

Rapid identification,differentiation, and proposed new taxonomic classification of Bifidobacterium lactis

Identification of Bifidobacterium lactis and Bifidobacterium animalis is problematic because of phenotypic and genetic homogeneities and has raised the question of whether they belong to one unique taxon. Analysis of the 16S-23S internally transcribed spacer region of B. lactis DSM10140(T), B. animalis ATCC 25527(T), and six potential B. lactis strains suggested two distinct clusters. Two specific 16S-23S spacer rRNA gene-targeted primers have been developed for specific detection of B. animalis. All of the molecular techniques used (B. lactis or B. animalis PCR primers, enterobacterial repetitive intergenic consensus PCR) demonstrated that B. lactis and B. animalis form two main groups and suggest a revision of the strains assigned to B. animalis. We propose that B. lactis should be separated from B. animalis at the subspecies level.     

Organization of the ribosomal RNA genes in Treponema phagedenis and Treponema pallidum.

The genomic DNA fragment which contains ribosomal RNA (rRNA) genes for Treponema phagedenis was cloned into bacteriophage vector lambda EMBL3. A restriction map of the fragment was constructed and the organization of the rRNA genes was determined. The fragment contained at least one copy of the 16S, 23S and 5S sequences and the genes are arranged in the order 16S-23S-5S. Southern hybridization using radiolabeled rRNA gene probes to genomic DNA from T. phagedenis strain Reiter and T. pallidum strain Nichols showed that these organisms have two radioactive fragments which hybridize to the probes in their genome. These results suggest that both pathogenic and non-pathogenic strains of Treponema may carry at least two sets of rRNA genes on their chromosomes.     

我国主要生态区域绿豆慢生根瘤菌的遗传多样性和系统发育研究

利用16SrRNAPCR-RFLP、16SrRNA序列分析以及16S-23SrRNAIGS(IntergeneticSpacer)PCR-RFLP技术对分离自中国主要生态区域的44株慢生型绿豆根瘤菌和5株参比菌株进行了遗传多样性和系统发育研究。16SrRNAPCR-RFLP分析表明:在76%的相似水平上,所有供试菌株可分为三大类群:群I由LYG1等13株慢生根瘤菌组成,该群在系统发育上与B.japonicum和B.liaoningense的参比菌株存在一定的差异;群Ⅱ由XJ1等21株供试菌株、B.japonicum和B.liaoningense的代表菌株组成;群Ⅲ由10株来自广东和广西的菌株和B.elkanii的代表菌株组成。16S-23SrRNAIGSPCR-RFLP分析将供试菌株分为A、B两大群。群A由34株供试菌株、B.japonicum和B.liaoningense的代表菌株组成。在85%的相似性水平上,可再分为AⅠ、AⅡ和AⅢ3个亚群。群B由10株分离自广西和广东的菌株和B.elkanii的代表菌株组成。在85%的相似性水平上,可再分为BI和BⅡ两亚群,表现出一定的多样性。与16SrRNAPCR-RFLP相比,16S-23SrRNAIGSPCR-RFLP具有更高的解析度,供试菌株表现出更加丰富的遗传多样性。分离自中国新疆、广东和广西等地的菌株在分群上具有较为明显的地域特征。     

Paenibacillus larvae 16S-23S rDNA intergenic transcribed spacer (ITS) regions: DNA fingerprinting and characterization

Paenibacillus larvae is the causative agent of American foulbrood in honey bee (Apis mellifera) larvae. PCR amplification of the 16S-23S ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) regions, and agarose gel electrophoresis of the amplified DNA, was performed using genomic DNA collected from 134 P. larvae strains isolated in Connecticut, six Northern Regional Research Laboratory stock strains, four strains isolated in Argentina, and one strain isolated in Chile. Following electrophoresis of amplified DNA, all isolates exhibited a common migratory profile (i.e., ITS-PCR fingerprint pattern) of six DNA bands. This profile represented a unique ITS-PCR DNA fingerprint that was useful as a fast, simple, and accurate procedure for identification of P. larvae. Digestion of ITS-PCR amplified DNA, using mung bean nuclease prior to electrophoresis, characterized only three of the six electrophoresis bands as homoduplex DNA and indicating three true ITS regions. These three ITS regions, DNA migratory band sizes of 915, 1010, and 1474 bp, signify a minimum of three types of rrn operons within P. larvae. DNA sequence analysis of ITS region DNA, using P. larvae NRRL B-3553, identified the 3' terminal nucleotides of the 16S rRNA gene, 5' terminal nucleotides of the 23S rRNA gene, and the complete DNA sequences of the 5S rRNA, tRNA(ala), and tRNA(ile) genes. Gene organization within the three rrn operon types was 16S-23S, 16S-tRNA(ala)-23S, and l6S-5S-tRNA(ile)-tRNA(ala)-23S and these operons were named rrnA, rrnF, and rrnG, respectively. The 23S rRNA gene was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to be present as seven copies. This was suggestive of seven rrn operon copies within the P. larvae genome. Investigation of the 16S-23S rDNA regions of this bacterium has aided the development of a diagnostic procedure and has helped genomic mapping investigations via characterization of the ITS regions.     

Genetic diversity of root-nodulating bacteria isolated from pea (Pisum sativum) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA inter-genic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S–23S IGS RFLP pat-terns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. legumino-sarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

Precise characterization of rDNA genes by intraspecies and inter-loci comparison of rDNA sequences and biochemical analysis of ribosomal RNA molecules in Agrobacterium tumefaciens

Annotation of rRNA genes has been incomplete in Agrobacterium species although a number of Agrobacterial rDNA fragments have been sequenced. In this study, precise characterization of rRNA operons (rrn) was carried out in two biovar 1 strains, C58 and MAFF301001. Complete DNA sequencing of four rrns in MAFF301001 indicated that each operon codes for 16S, 23S and 5S rRNA as well as three tRNAs, trn(Ile), trn(Ala) and trn(Met). The genes and 16S-23S ITS of a given locus were exactly identical with those in the other three loci, except for a T-base loss in the 23S rRNA gene of rrnA and in the 5S rRNA gene of rrnB. Comparison with the four C58 rDNAs available in the DNA database indicated extensive sequence and size variations in the 23S rRNA gene, suggesting the presence of an intervening sequence (IVS). Biochemical RNA analysis, including Northern hybridization and 5' end mapping, in MAFF301001 revealed 2886-base and 2571-base precursors, two 1.3-kb major fragments, a 150-base fragment and removal of an IVS for 23S rRNA. We confirmed similar biochemical characteristics in the C58 strain. The features of rDNA detected here enable correction of previously reported information about Agrobacterial rRNAs and rRNA genes and should be useful for phylogenetic considerations.     

6种区分乳酸乳球菌乳酸亚种和乳酸乳球菌乳脂亚种的分子生物学方法比较

【目的】比较并评价6种分子生物学技术对乳酸乳球菌乳酸亚种(Lactococcus lactis subsp.lactis)和乳酸乳球菌乳脂亚种(Lactococcus lactis subsp.cremoris)的区分效果。【方法】采用16S rRNA基因序列分析技术,16S-23S rRNA间区序列多态性分析技术,变性梯度凝胶电泳技术(DGGE),随机扩增多态性分析技术(RAPD),重复基因外回文序列分析技术(rep-PCR)和限制性酶切片段多态性分析技术(RFLP)对4株Lactococcus lactis subsp.lactis和Lactococcus lactis subsp.cremoris参考菌株进行了区分,并对这6种方法的区分效果进行了比较评价。【结果】16S rRNA基因序列分析技术,16S-23S rRNA间区序列多态性分析技术无法区分Lactococcus lactis subsp.lactis和Lactococcus lactis subsp.cremoris,而其余4种技术可以实现区分。【结论】变性梯度凝胶电泳(DGGE),随机扩增多态性分析技术(RAPD)耗时短,操作简单,试验结果准确稳定,更适合Lactococcus lactis subsp.lactis和Lactococcus lactis subsp.cremoris的快速准确区分。     

Molecular cloning and comparative sequence analyses of rRNA operons in Streptomyces nodosus ATCC 14899.

The genome of Streptomyces nodosus contains six ribosomal RNA (rRNA) operons. Four of the rRNA operons; rrnB, rrnD, rrnE and rrnF were cloned. We have completely sequenced all four operons, including a region 750 base pairs (bp) upstream of the 16S rRNA gene. The three rRNA genes present in each operon were closely linked in the order 16S-23S-5S. A sequence comparison of the four operons showed more than 99% sequence similarity between the corresponding 16S and 23S rRNA genes, and more than 97% similarity between 5S rRNA genes. The sequence differences observed between 23S rRNA genes appeared to be localized in two specific regions. Substantial sequence differences were found in the region upstream of the 16S rRNA gene as well as in the internal transcribed spacers. No tRNA gene was found in the 16S-23S spacer regions.