Suitability of partial 16S ribosomal RNA gene sequence analysis for the identification of dangerous bacterial pathogens

AIMS: In a bioterrorism event a rapid tool is needed to identify relevant dangerous bacteria. The aim of the study was to assess the usefulness of partial 16S rRNA gene sequence analysis and the suitability of diverse databases for identifying dangerous bacterial pathogens. METHODS AND RESULTS: For rapid identification purposes a 500-bp fragment of the 16S rRNA gene of 28 isolates comprising Bacillus anthracis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, and eight genus-related and unrelated control strains was amplified and sequenced. The obtained sequence data were submitted to three public and two commercial sequence databases for species identification. The most frequent reason for incorrect identification was the lack of the respective 16S rRNA gene sequences in the database. CONCLUSIONS: Sequence analysis of a 500-bp 16S rDNA fragment allows the rapid identification of dangerous bacterial species. However, for discrimination of closely related species sequencing of the entire 16S rRNA gene, additional sequencing of the 23S rRNA gene or sequencing of the 16S-23S rRNA intergenic spacer is essential. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides comprehensive information on the suitability of partial 16S rDNA analysis and diverse databases for rapid and accurate identification of dangerous bacterial pathogens.     

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.     

Comparative phylogenetic assignment of environmental sequences of genes encoding 16S rRNA and numerically abundant culturable bacteria from an anoxic rice paddy soil.

We used both cultivation and direct recovery of bacterial 16S rRNA gene (rDNA) sequences to investigate the structure of the bacterial community in anoxic rice paddy soil. Isolation and phenotypic characterization of 19 saccharolytic and cellulolytic strains are described in the accompanying paper (K.-J. Chin, D. Hahn, U. Hengstmann, W. Liesack, and P. H. Janssen, Appl. Environ. Microbiol. 65:5042-5049, 1999). Here we describe the phylogenetic positions of these strains in relation to 57 environmental 16S rDNA clone sequences. Close matches between the two data sets were obtained for isolates from the culturable populations determined by the most-probable-number counting method to be large (3 x 10(7) to 2.5 x 10(8) cells per g [dry weight] of soil). This included matches with 16S rDNA similarity values greater than 98% within distinct lineages of the division Verrucomicrobia (strain PB90-1) and the Cytophaga-Flavobacterium-Bacteroides group (strains XB45 and PB90-2), as well as matches with similarity values greater than 95% within distinct lines of descent of clostridial cluster XIVa (strain XB90) and the family Bacillaceae (strain SB45). In addition, close matches with similarity values greater than 95% were obtained for cloned 16S rDNA sequences and bacteria (strains DR1/8 and RPec1) isolated from the same type of rice paddy soil during previous investigations. The correspondence between culture methods and direct recovery of environmental 16S rDNA suggests that the isolates obtained are representative geno- and phenotypes of predominant bacterial groups which account for 5 to 52% of the total cells in the anoxic rice paddy soil. Furthermore, our findings clearly indicate that a dual approach results in a more objective view of the structural and functional composition of a soil bacterial community than either cultivation or direct recovery of 16S rDNA sequences alone.     

Sequence-based differentiation of strains in the Mycobacterium avium complex.

The complete 16S-23S rDNA internal transcribed spacer (ITS) was sequenced in 35 reference strains of the Mycobacterium avium complex. Twelve distinct ITS sequences were obtained, each of which defined a "sequevar"; a sequevar consists of the strain or strains which have a particular sequence. ITS sequences were identified which corresponded to M. avium (16 strains, four ITS sequevars) and Mycobacterium intracellulare (12 strains, one ITS sequevars). The other seven M. avium complex strains had ITS sequences which varied greatly from those of M. avium and M. intracellulare and from each other. The 16S-23S rDNA ITS was much more variable than 16S rDNA, which is widely used for genus and species identification. Phylogenetic trees based on the ITS were compatible with those based on 16S rDNA but were more detailed and had longer branches. The results of ITS sequencing were consistent with the results of hybridization with M. avium and M. intracellulare probes (Gen-Probe) for 30 of 31 strains tested. Serologic testing correlated poorly with ITS sequencing. Strains with the same sequence were different serovars, and those of the same serovar had different sequences. Sequencing of the 16S-23S rDNA ITS should be useful for species and strain differentiation for a wide variety of bacteria and should be applicable to studies of epidemiology, diagnosis, virulence, and taxonomy.     

Use of restriction fragment length polymorphism analysis to differentiate strains of psychrophilic and psychrotropic clostridia associated with blown pack' spoilage of vacuum-packed meats

Reference and meat strains of psychrophilic and psychrotrophic clostridia were differentiated using restriction fragment length polymorphism (RFLP) analysis of genomic DNA (DNA-RFLP) and the polymerase chain reaction-amplified 16S rDNA gene (PCR-RFLP). Groupings obtained with PCR-RFLP were confirmed with 16S rDNA gene sequencing. DNA-RFLP resolved 19 of the 22 meat strains into 11 groups. Three meat strains were untypable using this method. All reference strains representing different genotypic species could be distinguished by the restriction patterns of 16S rDNA genes. With PCR-RFLP, the 22 meat strains produced eight distinct genotypes. 16S rDNA gene sequencing confirmed that each genotype was represented by a distinct sequence. PCR-RFLP restriction patterns of 15 meat strains matched those of one of two of the seven reference strains used. Seven meat strains whose RFLP restriction patterns of 16S rDNA genes differed from those of any reference strains probably represent four previously undescribed species. Although RFLP analysis of the amplified 16S rDNA gene allowed differentiation of psychrophilic and psychrotrophic clostridia at the genotypic species level and below, comparison of PCR-RFLP patterns and 16S rDNA sequences of unknown clostridial isolates with patterns and sequences of reference strains may not effect ready identification of these micro-organisms. The results of this study will be useful in diagnosis of the cause of premature spoilage of chilled vacuum-packed meats and in tracing spoilage-causing clostridia to their source(s) in the abattoir.     

FastGroup: A program to dereplicate libraries of 16S rDNA sequences

Background

Ribosomal 16S DNA sequences are an essential tool for identifying and classifying microbes. High-throughput DNA sequencing now makes it economically possible to produce very large datasets of 16S rDNA sequences in short time periods, necessitating new computer tools for analyses. Here we describe FastGroup, a Java program designed to dereplicate libraries of 16S rDNA sequences. By dereplication we mean to: 1) compare all the sequences in a data set to each other, 2) group similar sequences together, and 3) output a representative sequence from each group. In this way, duplicate sequences are removed from a library.

Results

FastGroup was tested using a library of single-pass, bacterial 16S rDNA sequences cloned from coral-associated bacteria. We found that the optimal strategy for dereplicating these sequences was to: 1) trim ambiguous bases from the 5' end of the sequences and all sequence 3' of the conserved Bact517 site, 2) match the sequences from the 3' end, and 3) group sequences >=97% identical to each other.

Conclusions

The FastGroup program simplifies the dereplication of 16S rDNA sequence libraries and prepares the raw sequences for subsequent analyses.     

Genetic diversity of rhizobia associated with Desmodium species grown in China

AIMS: Desmodia are leguminous plants used as important forage and herbal medicine in China. Little information is available about the nodule bacteria of Desmodium species. To understand the genetic diversity of rhizobia associated with Desmodium species grown in China, isolates from temperate and subtropical regions were obtained and analysed. METHODS AND RESULTS: A total of 39 rhizobial strains isolated from 9 Desmodium species grown in China were characterized by PCR-based 16S rDNA gene and 16S-23S rDNA intergenic spacer gene restriction fragment length polymorphism (RFLP) and 16S rRNA gene sequencing. The results showed high diversity among rhizobia symbiotic with Desmodium species. Most microsymbionts of Desmodium species belonged to Bradyrhizobium closely related to Bradyrhizobium elkanii, Bradyrhizobium japonicum and Bradyrhizobium yuanmingense. Several small groups or single strain were related to Rhizobium, Sinorhizobium or Mesorhizobium. CONCLUSIONS: Desmodium species formed nodules with diverse rhizobia in Chinese soils. SIGNIFICANCE AND IMPACT OF THE STUDY: These results offered the first systematic information about the microsymbionts of desmodia grown in the temperate and subtropical regions of China.     

Use of rpoB gene analysis for identification of nitrogen-fixing Paenibacillus species as an alternative to the 16S rRNA gene

AIM: To avoid the limitations of 16S rRNA-based phylogenetic analysis for Paenibacillus species, the usefulness of the RNA polymerase beta-subunit encoding gene (rpoB) was investigated as an alternative to the 16S rRNA gene for taxonomic studies. METHODS AND RESULTS: Partial rpoB sequences were generated for the type strains of eight nitrogen-fixing Paenibacillus species. The presence of only one copy of rpoB in the genome of P. graminis strain RSA19(T) was demonstrated by denaturing gradient gel electrophoresis and hybridization assays. A comparative analysis of the sequences of the 16S rRNA and rpoB genes was performed and the eight species showed between 91.6-99.1% (16S rRNA) and 77.9-97.3% (rpoB) similarity, allowing a more accurate discrimination between the different species using the rpoB gene. Finally, 24 isolates from the rhizosphere of different cultivars of maize previously identified as Paenibacillus spp. were assigned correctly to one of the nitrogen-fixing species. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study indicate that rpoB is a powerful identification tool, which can be used for the correct discrimination of the nitrogen-fixing species of agricultural and industrial importance within the genus Paenibacillus.     

Structural analysis and genetic variation of the 16S-23S rDNA internal spacer region from Micrococcus luteus strains

AIMS: To clone and sequence the 16S-23S ribosomal DNA (rDNA) internal spacer region (ISR) from Micrococcus luteus. METHODS AND RESULTS: The primer pair for 16S-23S rDNA ISR amplified a fragment of about 850 bp in length for two strains, JCM3347 and JCM3348 and a fragment of about 790 bp for a strain, ATCC9341. After sequencing the ISRs were identified by the comparison of the ISRs and the flanking regions of ISR. CONCLUSIONS: Although the sequence difference of the ISR occurred at only one position between the two JCM strains, the highly variable length (440 and 370 bp) and sequence similarity (about 40%) were demonstrated between the ISRs of the two JCM strains and a ATCC strain. SIGNIFICANCE AND IMPACT OF THE STUDY: A CCTCCT sequence was first detected at the 3'-end of the 16S rDNA of the three strains. Moreover, highly similar sequence to the 21-bp region containing a putative rRNA processing site was observed in the ISR of the three strains. Interestingly, no intercistronic tRNAs were demonstrated in the ISRs from the three strains.     

金针菇rDNA序列结构特征分析

rDNA序列中的ITS作为DNA barcoding广泛应用于真菌的系统发育与物种辅助鉴定,IGS被认为可以用于种内水平不同菌株的鉴别。食用菌中还没有完整的rDNA序列的报道。本研究采用二代和三代测序技术分别对金针菇单核菌株“6-3”进行测序,用二代测序的数据对三代测序组装得到的基因组序列进行修正,得到一个在基因完整性、连续性和准确性均较好的基因组序列,对比Fibroporia vaillantii rDNA序列,获得金针菇完整的rDNA序列。金针菇rDNA序列结构分析表明,它有8个rDNA转录单元,长度均为5 903bp,有9个基因间隔区,其长度有较大差异,3 909-4 566bp。rDNA转录单元中,各元件的序列长度分别为：18S rDNA 1 796bp、ITS1 234bp、5.8S rDNA 173bp、ITS2 291bp、28S rDNA 3 410bp。基因间间隔区中,IGS1 1 351-1 399bp、5S rDNA 124bp、IGS2 2 435-3 092bp。金针菇的5S、5.8S、18S、28S rDNA序列准确性得到转录组数据的验证,也得到系统发育分析结果的支持。多序列比对发现,不同拷贝的基因间间隔区序列（IGS1和IGS2）存在丰富的多态性,多态性来源于SNP、InDel和TRS（串联重复序列）,而TRS来源于重复单元的类型和数量。9个基因间间隔区之间,IGS1只有少量的SNP和InDel,IGS2不仅有SNP和InDel,还有TRS。本研究结果提示,在应用IGS进行种内水平不同菌株之间的鉴别时,需要选取不同拷贝之间的保守IGS序列。     

Discrimination of Bacillus cereus and Bacillus thuringiensis with 16S rRNA and gyrB gene based PCR primers and sequencing of their annealing sites

AIMS: To evaluate the possibility for discrimination of Bacillus cereus and B. thuringiensis using 16S rRNA and gyrB gene based PCR methods, and to obtain the sequences of the primer annealing sites so that the PCR results may be explained. METHODS AND RESULTS: Based on the sequence difference in the variable region (V1) of 16S rRNA and in the gyrB gene between B. cereus and B. thuringiensis, PCR primers specific to these Bacillus spp. were designed. When these primers were used to discriminate B. cereus and B. thuringiensis, six of 82 B. cereus strains were identified as B. thuringiensis while 67 of 73 B. thuringiensis strains were identified as B. cereus. Sequence analysis of the primer annealing sites showed that there is no clear-cut difference in the V1 region of 16S rRNA, and in the gyrB gene, between B. cereus and B. thuringiensis strains. CONCLUSIONS: Although 16S rDNA based probes and gyrB gene based PCR primers have been suggested for the discrimination of B. cereus and B. thuringiensis strains, when a large number of Bacillus strains was tested, results showed that discrimination between B. cereus and B. thuringiensis is difficult. Therefore, to distinguish B. thuringiensis from B. cereus, a single feature, such as the presence of a parasporal crystal protein or cry gene, may sometimes be reliable. SIGNIFICANCE AND IMPACT OF THE STUDY: Discrimination between B. cereus and B. thuringiensis is a challenging debate to which this paper makes a contribution.     

Application of the partial 16S rDNA sequence as an index for rapid identification of species in the genus Bacillus

A comparison of 16S rRNA gene (rDNA) sequences was made among type strains of 69 Bacillus species approved in the International Journal of Systematic Bacteriology (IJSB) until 1998. The results suggested that 5' end region (approx. 275 bp) was the hypervariant region (HV region) in the gene and was highly specific for each type strain. Furthermore, a sequence analysis of the HV region of Bacillus strains revealed that this region was highly conserved within the species. These results indicate that the HV region is a useful index for the identification or grouping of Bacillus species.     

Diversity of Actinoplanes and Related Genera Isolated from an Italian Soil

Actinoplanes and related genera are good producers of bioactive secondary metabolites. However, many strains within these genera present similar morphological characteristics, and this prevents an effective discrimination of replicate strains during industrial isolation and screening programs. Using PCR-RFLP analysis of the 23S rDNA gene and of the 16S-23S intergenic spacer, we have analyzed 182 strains of Actinoplanes and related genera obtained through a selective isolation method from a single Italian soil. Combining the 23S and IGS data, 99 unique profiles were observed, and morphologically undistinguishable strains were discriminated. Further analyses on a restricted number of strains through 16S sequencing and hybridization to a probe for secondary metabolism established a good correlation between strain diversity seen by PCR-RFLP and that seen by the other methods. Overall, the data indicate the presence of a high diversity of Actinoplanes and related genera isolated from a single Italian soil.     

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.     

Comparative Phylogenetic Assignment of Environmental Sequences of Genes Encoding 16S rRNA and Numerically Abundant Culturable Bacteria from an Anoxic Rice Paddy Soil

We used both cultivation and direct recovery of bacterial 16S rRNA gene (rDNA) sequences to investigate the structure of the bacterial community in anoxic rice paddy soil. Isolation and phenotypic characterization of 19 saccharolytic and cellulolytic strains are described in the accompanying paper (K.-J. Chin, D. Hahn, U. Hengstmann, W. Liesack, and P. H. Janssen, Appl. Environ. Microbiol. 65:5042–5049, 1999). Here we describe the phylogenetic positions of these strains in relation to 57 environmental 16S rDNA clone sequences. Close matches between the two data sets were obtained for isolates from the culturable populations determined by the most-probable-number counting method to be large (3 × 10⁷ to 2.5 × 10⁸ cells per g [dry weight] of soil). This included matches with 16S rDNA similarity values greater than 98% within distinct lineages of the division Verrucomicrobia (strain PB90-1) and the Cytophaga-Flavobacterium-Bacteroides group (strains XB45 and PB90-2), as well as matches with similarity values greater than 95% within distinct lines of descent of clostridial cluster XIVa (strain XB90) and the family Bacillaceae (strain SB45). In addition, close matches with similarity values greater than 95% were obtained for cloned 16S rDNA sequences and bacteria (strains DR1/8 and RPec1) isolated from the same type of rice paddy soil during previous investigations. The correspondence between culture methods and direct recovery of environmental 16S rDNA suggests that the isolates obtained are representative geno- and phenotypes of predominant bacterial groups which account for 5 to 52% of the total cells in the anoxic rice paddy soil. Furthermore, our findings clearly indicate that a dual approach results in a more objective view of the structural and functional composition of a soil bacterial community than either cultivation or direct recovery of 16S rDNA sequences alone.     

埃莎霉素Ⅰ组分高含量、高产量基因工程菌WSJ-IA及其原株的鉴定

【目的】利用调节基因acyB2激活异戊酰基转移酶(ist)基因表达的特点,将ist与调节基因acyB2在异戊酰螺旋霉素(埃莎霉素)Ⅰ产生菌菌株中共表达,获得埃莎霉素Ⅰ单组分的高含量及高产量菌株WSJ-IA。对其及原始螺旋霉素产生菌菌株Streptomyces spiramyceticus F21进行了初步鉴定。【方法】从形态学、培养和生理生化特征、细胞壁化学组成、16S rRNA基因序列、5个看家基因(atpD、gyrB、rpoB、recA和trpB)蛋白分析和系统发育树构建等方面对该菌株及其原株进行了鉴定。【结果】两株菌在形态培养特征、生理生化特征、细胞壁化学组成、16S rRNA基因序列和5个看家基因蛋白水平基本一致,在系统发育树分析中同处在一个分支中。而在16S rRNA基因序列和5个看家基因蛋白水平在系统发育上它们均与已知相近菌株处于不同的分支上,并且与不同基因的相近菌株各有不同,其中无一报道产生螺旋霉素。【结论】Streptomyces spira-myceticus F21可能是一个产生螺旋霉素的链霉菌新种,16S rRNA基因序列和5个看家基因蛋白序列分析可以作为埃莎霉素Ⅰ基因工程菌生产过程中进行鉴别的分子标志。     

DNA-based subtyping of verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2 strains from human and raw meat sources

AIMS: To investigate subtyping methods for verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2. METHODS AND RESULTS: Eleven human and food strains isolated over a 15-year period were examined. All were intimin (eae)-negative, but all possessed enterohaemolysin and VT1-encoding sequences which in nine strains were vtx1c variant. Ten strains had VT2 genes which were all vtx2d. Plasmid profiles and randomly amplified polymorphic DNA-PCR were not discriminatory. Long-PCR restriction fragment length polymorphism of amplicons bound by the p gene and the VT2A subunit had screening potential. Pulsed field gel electrophoresis (PFGE) using XbaI gave fine discrimination although VT2 sequences were located on a 220 kbp fragment conserved in nine strains and on a 200 kbp fragment in the 10th. CONCLUSIONS: As a result of apparent clonality, PFGE proved essential for differentiation. Long-PCR has promise for screening but requires further evaluation of inter-strain variable sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: A combined phenotypic and genotypic screen, and PFGE for selected strains was effective.     

Variation of 16S-23S internally transcribed spacer sequence and intervening sequence in rDNA among the three major Agrobacterium species

We analyzed 16S-23S internally transcribed spacer (ITS) and neighboring sequences among 37 strains belonging to the three major pathogenic Agrobacterium species, in order to know variation in each species and to develop a simple discrimination method. Number of ITS size variation was 9, 4, and 7 in Agrobacterium tumefaciens, Agrobacterium vitis, and Agrobacterium rhizogenes, respectively. The ITS sequence of most strains in each species was distinguishable from that of the other two species. The region surrounded by 16S rRNA gene and trn(Ala) contained information to distinguish between the ITS variants and was easy for sequencing. Intervening sequences (IVSs) in 23S rRNA gene were classified into short and long types in each species. Some long-type IVSs of A. vitis were very similar to that of A. tumefaciens, while the other long-type IVSs of A. vitis were very similar to that of A. rhizogenes. Two A. vitis strains simultaneously contained both types of IVS. Similarly, the two exceptional A. vitis strains possessed A. tumefaciens-type ITS in addition to A. vitis-type ITS. These results suggest horizontal transfer of rDNA and subsequent recombination. Among the three species, A. tumefaciens was most variable based on 16S rRNA gene, ITS and IVS sequences.     

Differences between subcultures of the Mesorhizobium loti type strain from different culture collections

Because of differences in the reported 16S rRNA gene sequence of the Mesorhizobium loti type strain available from different culture collections, we collected different subcultures of this strain and compared them by 16S rDNA sequencing, SDS-PAGE of whole-cell protein extracts and RAPD-PCR. Our results indicate that the 16S rDNA sequence differences can be explained by the presence of two different organisms in one of the subcultures. In addition, even for subcultures of the type strain that had identical 16S rDNA sequences, small differences could be observed in the protein profiles and in the RAPD-PCR patterns. These latter observations indicate that maintenance procedures necessary for long-term preservation by freeze-drying can cause subcultures of the same original strain to undergo changes, effectively leading to different fingerprints even though 16S rDNA sequences remain identical.