Characterization of Thermophilic Proteolytic Spore-Forming Bacteria from a Geothermal Site in Lithuania Based on 16S rDNA RFLP and ITS-PCR Analyses1

Forty-two strains of gram-positive, aerobic, heterotrophic, obligately thermophilic, spore-forming bacteria were isolated from a geothermal site near the Baltic Sea in Lithuania. All of the strains were able to hydrolyze collagen and/or casein. Since characteristics of proteolytic activity are correlated with taxonomic positions of bacteria, the strains were grouped on the basis of molecular biological analyses. On the basis of RFLP patterns of 16S rDNA and 16S–23S rDNA ITS-PCR analysis, the strains were subdivided into nine groups.     

新疆地区盐湖的中度嗜盐菌16S rDNA全序列及DNA同源性分析

通过数值分类和16S rDNA PCR-RFLP分析,对分离自新疆地区的中度嗜盐革兰氏阴性菌进行研究,发现了一个新类群。在此基础上,进行了中心株AI－3的16S rDNA全序列分析,并与中度嗜盐菌已知种和相关种进行比较,得到系统发育树状图。在此树状图中,大多数参比菌株聚在一起,其16S rDNA全序列的同源性在96％以上,而AI－3与参比菌株的16S rDNA全序列相比,其相似性低于75％。但是,AI－3与Alcanivorax borkumensis^[1]的16S rDNA全序列的相似性为96％,与Halobacillus litoralis的16S rDNA全序列的相似性为99％,三者构成一个独立的发育分支。这说明在系统发育上,AI－3与参比菌株属于不同的分支,是一个新的类群。在新类群内,菌株之间的DNA同源性大于70％,而中心株AI－3与标准菌株伸长盐单胞菌（Halomonas elongata)的DNA同源性为44％,表明新分离的菌株可能构成一个新种群。     

Molecular characterization of Serratia marcescens strains by RFLP and sequencing of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer

AIMS: To establish the specific DNA patterns in 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) regions from different kinds of Serratia marcescens strains using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequences analysis. METHODS AND RESULTS: Two pairs of primers based on the 16S rDNA and 16S-23S rDNA IGS were applied to amplify the rrn operons of two kinds of S. marcescens strains. About 1500 bp for 16S rDNA and four fragments of different sizes for 16S-23S rDNA IGS were obtained. PCR-amplified fragments were analysed by RFLP and sequence analysis. Two distinct restriction patterns revealing three to five bands between two kinds of strains were detected with each specific enzyme. According to the sequence analysis, two kinds of strains showed approximately 97% sequence homology of 16S rDNA. However, there was much difference in the sequences of IGS between the two kinds of strains. Intercistronic tRNA of strains H3010 and A3 demonstrated an order of tRNA of 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', but strain B17 harboured the tRNA of 5'-16S-tRNA(Glu)-tRNA(Ile)-23S-3'. CONCLUSIONS: The method was specific, sensitive and accurate, providing a new technique for differentiating different strains from the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provided the first molecular characterization of 16S rDNA and 16S-23S rDNA IGS from S. marcescens strains.     

斜茎黄芪根瘤菌的16SrDNA和23SrDNAPCR—RFLP比较分析

在表型性状数值分析和ＡＦＬＰ指纹图谱分析的基础上,选取５４株斜茎黄芪根瘤菌的代表菌株及已知根瘤菌参比菌株,进行１６ＳｒＤＮＡ和２３ＳｒＤＮＡ的ＰＣＲ－ＲＦＬＰ比较分析。结果表明斜茎黄芪根瘤菌具有极大的系统发育多样性,分别具有２４个１６ＳｒＤＮＡ遗传图谱类型和２２个２３ＳｒＤＮＡ遗传图谱类型,１６ＳｒＤＮＡ与２３ＳｒＤＮＡＰＣＲ－ＲＦＬＰ聚类分析树状图谱有较好的一致性,但也存在一些差异。在对较大类群的划分上,它们的结果与表型性状数值分析结果有较好的一致性。将１６ＳｒＤＮＡ和２３ＳｒＤＮＡＰＣＲ－ＲＦＬＰ分析数据合并在一起进行分析时,得出２６个综合遗传图谱类型和１个综合聚类分析树状图谱。很明显,１６ＳｒＤＮＡ与２３ＳｒＤＮＡ的合并,能够得出更可靠的系统发育结论。     

油菜内生细菌16S核糖体DNA的RFLP分析

植物内生细菌定殖在植物组织内部但不引起明显的病害症状。从健康油菜植株的不同器官中分离到大量内生细菌,这些细菌菌落形态存在明显的差异,表明油菜组织中存在大量内生细菌,且类群丰富。分离到的122株内生细菌根据菌落形态可以划分为35类;利用细菌通用引物对16S核糖体DNA进行扩增, 获得约15 kb片段,分别用内切酶HaeⅢ和MspⅠ对扩增产物进行限制性酶切,产生不同的酶切图谱,根据酶切图谱聚类分析结果,所有供试菌株被归为39类,这一结果从遗传上显示油菜内生细菌类群的多样性。两种方法归类结果比较发现菌落特征所反映的信息量有限,只能作为初步的参考指标,核糖体DNA的限制性片段长度多态性分析快速、准确,可以作为油菜内生细菌多样性分析的一种有效方法     

Cloning and sequencing of 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC)

AIMS: To clone and sequence the 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC). METHODS AND RESULTS: The primer sets for 16S rDNA and 16S-23S rDNA ISR amplified almost the full length of 16S rDNA and 16S-23S rDNA ISR. About 1500 bp for 16S rDNA and about 720 bp for 16S-23S rDNA ISR of the rrn operon of four strains of UPTC were identified after molecular cloning and sequencing. CONCLUSIONS: The four strains and CCUG18267 of UPTC showed approximately 99% sequence homology of 16S rDNA to each other, 96-97% to Camp. coli, 97-98% to Camp. jejuni and 97-98% to Camp. lari. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, the nucleotide sequence of 16S-23S rDNA ISR of UPTC has been analysed. The sequence of ISR was almost identical among the four strains of UPTC. It is interesting that the UPTC intercistronic tRNAs demonstrated an order of tRNA of 5'-16S-tRNAAla-tRNAIle-23S-3' in the organisms.     

16S rDNA序列分析在鉴定布鲁氏菌中的应用

[目的]建立布鲁氏菌的16S rDNA序列分析方法,评价该方法鉴定布鲁氏菌的特异性和实用性.[方法]用PCR扩增布鲁氏菌的16S rDNA片段,将扩增的产物纯化后测序,从GenBank下载与布鲁氏菌易发生血清学交叉反应的细菌的16S rDNA序列.使用DNAMAN软件进16S rDNA序列相似性分析.[结果]在布鲁氏菌中16S rDNA核苷酸序列相似性达到了99.74％,而与其他有血清型交叉反应的菌株相比较,16S rDNA序列间有显著差异.[结论]16S rDNA序列分析是一种快速、简便、特异的鉴定布鲁氏菌的方法之一.     

16S rDNA sequence analysis of Xylella fastidiosa strains

The 16S rDNA encoding the small subunit ribosomal RNA were amplified by PCR, cloned, and sequenced from 16 strains of Xylella fastidiosa originating from nine different hosts. In pair-wise comparisons, X. fastidiosa strains showed a maximum variation of 1.0% or 14 nucleotide positions. When all 16 sequences were considered as a set, 54 variable positions were found. Analysis of the sequence data indicated that the X. fastdiosa strains formed three rDNA groups. Group one includes Pierce's disease and mulberry leaf scorch strains; Group two, periwinkle wilt, plum leaf scald, phony peach, oak leaf scorch, and elm leaf scorch strains; and Group three, citrus variegated chlorosis and coffee leaf scorch strains. All X. fastidiosa strains exhibited significantly higher levels of sequence heterogeneity (63 to 83 nucleotide positions) when compared to species from Xanthomonas and Stenotrophomonas. Our data demonstrate that 16S rDNA sequence data could provide valuable information for future classification of X. fastidiosa at the sub-species level.     

Phenotypic characterization of Astragalus glycyphyllos symbionts and their phylogeny based on the 16S rDNA sequences and RFLP of 16S rRNA gene

In this study, the nitrogen fixing Astragalus glycyphyllos symbionts were characterized by phenotypic properties, restriction fragment length polymorphism (RFLP), and sequences of 16S rDNA. The generation time of A. glycyphyllos rhizobia in yeast extract mannitol medium was in the range 4–6 h. The studied isolates exhibited a low resistance to antibiotics, a moderate tolerance to NaCl, assimilated di- and trisaccharides, and produced acid in medium containing mannitol as a sole carbon source. In the cluster analysis, based on 86 phenotypic properties of A. glycyphyllos symbionts and the reference rhizobia, examined isolates and the genus Mesorhizobium strains were placed on a single branch, clearly distinct from other lineages of rhizobial genera. By the comparative analysis of 16S rRNA gene sequences and 16S rDNA–RFLP, A. glycyphyllos nodulators were also identified as the members of the genus Mesorhizobium. On the 16S rDNA sequence phylogram, the representatives of A. glycyphyllos nodule isolates formed a robust, monophyletic cluster together with the Mesorhizobium species at 16S rDNA sequence similarity of these bacteria between 95 and 99 %. Similarly, the cluster analysis of the combined RFLP–16S rDNA patterns, obtained with seven restriction endonucleases, showed that A. glycyphyllos rhizobia are closely related to the genus Mesorhizobium bacteria. The taxonomic approaches used in this paper allowed us to classify the studied bacteria into the genus Mesorhizobium.     

16S rDNA sequence analysis of culturable marine biofilm forming bacteria from a ship's hull

Marine bacteria from the hull of a ship in the form of biofilms or microfouling were isolated, cultured, and identified by phylogenetic analysis using 16S rDNA sequences. With an average length of 946 bp, all the 16 sequences were classified using the Ribosomal database project (RDP) and were submitted to the National Center for Biotechnology Information. Phylogenetic analysis using 16S rDNA sequences indicated that the 16 strains belonged to the Firmicutes (IK-MB6 Exiguobacterium aurantiacum, IK-MB7 Exiguobacterium arabatum, IK-MB8 Exiguobacterium arabatum, IK-MB9 Jeotgalibacillus alimentarius, IK-MB10 Bacillus megaterium, IK-MB11 Bacillus pumilus, IK-MB12 Bacillus pumilus, IK-MB13 Bacillus pumilus, IK-MB14 Bacillus megaterium), High GC, Gram-positive bacteria (IK-MB2 Micrococcus luteus, IK-MB5 Micrococcus luteus, IK-MB16 Arthrobacter mysorens), G-Proteobacteria (IK-MB3 Halomonas aquamarina, IK-MB15 Halotalea alkalilenta), CFB group bacteria (IK-MB1 Myroides odoratimimus), and Enterobacteria (IK-MB4 Proteus mirabilis). Among the 16 strains, representatives of the Firmicutes were dominant (56.25%) compared to the high GC, Gram-positive bacteria (18.75%), G-Proteobacteria (12.5%), CFB group bacteria (6.25%), and Enterobacteria (6.25%). Analysis revealed that majority of marine species found in marine biofilm are of anthropogenic origin.     

Phylogeny of green sulfur bacteria on the basis of gene sequences of 16S rRNA and of the Fenna-Matthews-Olson protein

The phylogeny of green sulfur bacteria was studied on the basis of gene sequences of the 16S rRNA and of the Fenna-Matthews-Olson (FMO) protein. Representative and type strains (31 strains total) of most of the known species were analyzed. On the basis of fmoA gene sequences from Chlorobium tepidum ATCC 49652(T) and Chlorobium limicola DSM 249(T) available from the EMBL database, primers were constructed that allowed sequence analysis of a major part of the fmoAgene. The largely congruent phylogenetic relationship of sequences of the fmoA gene and of 16S rDNA gives considerable support to the phylogeny of green sulfur bacteria previously suggested on the basis of 16S rDNA sequences. Distinct groups of strains were recognized on the basis of 16S rDNA and FMO sequences and supported by characteristic signature amino acids of FMO. Marine strains formed clusters separate from freshwater strains. The resulting phylogenetic grouping and relationship of the green sulfur bacteria do not correlate with their current taxonomic classification.     

16S和23S rDNA基因序列分析分类鉴定中国衣原体流行株

通过分析比较部分16S/23S rDNA序列,对现有保存的9株国内衣原体流行株进行了分子遗传学鉴定。虽然这些分离株分离自不同的动物,但它们的16S/23S扩增部分完全相同,经16S/23S rDNA序列同源性比较可以一致鉴定国内流行株为鹦鹉热嗜衣原体。     

Phylogenetic analysis of cecal microbiota in chicken by the use of 16S rDNA clone libraries

The chicken cecum contains a great many bacteria, most of which are strict anaerobes. A strictly anaerobe culture-based method was used in the present study, in conjunction with the 16S rDNA clone library, to elucidate bacterial diversity and the phylogenetic relationship of cecal microbiota in the chicken. A comparative 16S rDNA sequence analysis of cultivated strains and retrieved clones from cecal contents was performed. Approximately 90% of the bacterial cells detected by microscopy did not form colonies on a medium 10 in plate-in-bottle. The 19 isolated strains yielded 11 distinct rDNA sequences, 58% of which were classified as low G + C gram-positive bacteria, 26% were related to Bacteroides spp., and 16% were classified as Proteobacteria. Based on the sequence analysis of 164 clones, 24% were identified to belong to 8 known species and 76% were considered to be 65 novel phylotypes. Approximately 94% of cloned sequences were classified into low G + C gram-positive bacteria, 4% were related to Bacteroides spp., and 2% were classified into Proteobacteria. Clostridium subcluster XIVa (38%), Clostridium cluster IV (13%), Lactobacillus spp. (24%), and Bacteroides spp. (4%) were the major groups constituting the cecal microbiota in chicken, in which the Clostridium subcluster XIVa was the most phylogenetically diverse group in chicken cecum. The 16S rDNA sequences of Lactobacillus acidophilus, L. crispatus, L. salivarius, and L. reuteri were the most frequently found in the Lactobacillus group in chicken cecum.     

Evolutionary relationships among Magnetospirillum strains inferred from phylogenetic analysis of 16S rDNA sequences.

We have investigated the evolutionary relationships between two facultatively anaerobic Magnetospirillum strains (AMB-1 and MGT-1) and fastidious, obligately microaerophilic species, such as Magnetospirillum magnetotacticum, using a molecular phylogenetic approach. Genomic DNA from strains MGT-1 and AMB-1 was used as a template for amplification of the genes coding for 16S rRNA (16S rDNA) by the polymerase chain reaction. Amplified DNA fragments were sequenced (1,424 bp) and compared with sequences for M. magnetotacticum MS-1 and Magnetospirillum gryphiswaldense MSR-1. Phylogenetic analysis of the aligned 16S rDNA sequences indicated that the two new magnetic spirilla, AMB-1 and MGT-1, lie within the alpha subdivision (alpha-1) of the eubacterial group Proteobacteria and are closely related to Rhodospirillum fulvum and to several endosymbiotic bacteria. Strains AMB-1, MGT-1, and MS-1 formed a cluster, termed group I, in which they were more closely related to each other than to group II, which contained M. gryphiswaldense MSR-1. Group I strains were also physiologically distinct from strain MSR-1. Sequence alignment studies allowed elucidation of genus-specific regions of the 16S rDNA, and oligonucleotide primers complementary to two of these regions were used to develop a specific polymerase chain reaction assay for detection of magnetic spirilla in natural samples.     

Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.

The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis.     

Total bacterial and species-specific 16S rDNA micro-array quantification of complex samples

AIMS: We describe a novel DNA-micro-array-based method that targets 16S rDNA to quantify changes in both the total bacterial DNA and the species-specific DNA composition. METHODS AND RESULTS: Quantifications were achieved by combining competitive PCR for quantifying total bacterial DNA with quantification of species-specific DNA composition based on signature 16S rDNA sequences. We constructed 11 different probes, which were evaluated on 21 different strains, in addition to complex samples. The signals obtained with sequence-specific labelling of the probes corresponded well with what should be expected based on 16S rDNA phylogenetic reconstruction. The quantification of species-specific DNA composition showed that the micro-array approach could be used to accurately determine differential growth of bacteria in mixed samples. We analysed samples containing mixtures of Lactococcus lactis and different species of propionibacteria during a 2-week incubation period. Lactococcus lactis grew fast, reaching a maximum after 12 h, Propionibacterium acidipropionici and Propionibacterium freudenreichii reached a maximum after 48 h, whereas Propionibacterium jensenii showed a slow increase during the whole growth period. The 16S rDNA total bacterial DNA quantification was compared with real-time PCR, absorbance measurements (ABS600) and colony forming units (CFU). CONCLUSION: The accuracy of the array approach was in the same range or better than the alternative techniques. The potential of the 16S rDNA micro-array method was further demonstrated using a liquid cheese model. SIGNIFICANCE AND IMPACT OF THE STUDY: This is to our knowledge the first time quantification of the total bacterial DNA and the species-specific DNA compositions of mixed populations have been achieved in the same assay.     

香蕉束顶病植原体16S rDNA片段的RFLP和序列分析

香蕉束顶病（ＢＢＴ）是一种发生在蕉类作物的严重病害。从带有典型香蕉束顶病症状的香蕉植株中按照检测植原体的方法提取ＤＮＡ,扩增患病植株中植原体１６ＳｒＤＮＡ片段,证明香蕉束顶病中有植原体存在。对此扩增片段进行限制性酶切片段长度多态性（ＲＦＬＰ）分析和核酸序列分析,并与已知植原体的序列进行同源性比较,构建进化树。结果显示该片段与Ｇｒ１的亲缘关系最近。     

Combining denaturing gradient gel electrophoresis of 16S rDNA V3 region and 16S-23S rDNA spacer region polymorphism analyses for the identification of staphylococci from Italian fermented sausages

Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (PCR-DGGE) and 16S-23S rDNA intergenic spacer region polymorphism (ISR-PCR) analyses were tested as tool for differentiation of staphylococcal strains commonly isolated from fermented sausages. Variable V3 regions of 25 staphylococcal reference strains and 96 wild strains of species belonging to the genera Staphylococcus, Micrococcus and Kocuria were analyzed. PCR-DGGE profiles obtained were species-specific for S. sciuri, S. haemolyticus, S. hominis, S. auricularis, S. condimenti, S. kloosi, S. vitulus, S. succinus, S. pasteuri, S. capitis and S. (Macrococcus) caseolyticus. Moreover, 7 groups could be distinguished gathering the remaining species as result of the separation of the V3 rDNA amplicons in DGGE. Furthermore, the combination of the results obtained by PCR-DGGE and ISR-PCR analyses allowed a clear differentiation of all the staphylococcal species analysed, with exception of the pairs S. equorum-S. cohnii and S. carnosus-S. schleiferi. The suitability of both molecular techniques and of the combination their results for the identification of staphylococci was validated analysing partial nucleotide sequence of the 16S rDNA of a representative number of wild strains.