Re-identification of Gluconobacter strains based on restriction analysis of 16S-23S rDNA internal transcribed spacer regions

Thirty Gluconobacter strains maintained at Culture Collection NBRC were re-identified at the species level on the basis of restriction analysis of 16S-23S rDNA internal transcribed spacer (ITS) regions by digestion with two restriction endonucleases MboII and Bsp1286I. The strains examined were divided into seven groups, designated as Group I and Group III-VIII, by the combination of the restriction patterns obtained with the two restriction endonucleases. Group I included seven strains, which gave "G. oxydans patterns" with the two restriction endonucleases and were re-identified as G. oxydans. Group III included 12 strains, which gave "G. frateurii patterns" and were re-identified as G. frateurii. Group IV included six strains, which gave "G. cerinus pattern" with MboII and "G. frateurii pattern" with Bsp1286I and were re-identified as G. frateurii. Group V included one strain (NBRC 3274), which gave respectively "G. frateurii pattern" and "G. cerinus pattern" and was re-identified as G. cerinus. Group VI included one strain (NBRC 3990), which gave respectively "G. oxydans pattern" and an unidentified restriction pattern and was re-identified temporarily as G. oxydans. Group VII included two strains (NBRC 3250 and NBRC 3273), which gave respectively an unidentified restriction pattern and "G. oxydans pattern." Group VIII included one strain (NBRC 3266), which gave unidentified restriction patterns. The three strains of Group VII and Group VIII were suggested to constitute new taxa by sequencing of 16S-23S rDNA ITS regions.     

Molecular authentication of ginseng cultivars by comparison of internal transcribed spacer and 5.8S rDNA sequences

Molecular authentication among three Panax species and within cultivars and accessions of P. ginseng was investigated using the DNA sequence in the ribosomal ITS1–5.8S–ITS2 region. Four single-nucleotide polymorphisms were identified between P. ginseng and other Panax species. In the electrophoresis profile, obtained after digestion with the enzyme TaqI, three fingerprinting patterns were obtained from cultivars and accessions of Panax species. Consequently, this authentication procedure based upon the restriction fragment length polymorphism in the ribosomal ITS1–5.8S–ITS2 region can now be utilized to differentiate these Panax species as well as major Korean cultivars such as Gopoong and Kumpoong from other cultivars and accessions in Panax species at the DNA level. O. T. Kim and K. H. Bang contributed equally to this paper.     

DNA fingerprinting of Paenibacillus popilliae and Paenibacillus lentimorbus using PCR-amplified 16S-23S rDNA intergenic transcribed spacer (ITS) regions

Failure to identify correctly the milky disease bacteria, Paenibacillus popilliae and Paenibacillus lentimorbus, has resulted in published research errors and commercial production problems. A DNA fingerprinting procedure, using PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS) regions, has been shown to easily and accurately identify isolates of milky disease bacteria. Using 34 P. popilliae and 15 P. lentimorbus strains, PCR amplification of different ITS regions produced three DNA fingerprints. For P. lentimorbus phylogenic group 2 strains and for all P. popilliae strains tested, electrophoresis of amplified DNA produced a migratory pattern (i.e., ITS-PCR fingerprint) exhibiting three DNA bands. P. lentimorbus group 1 strains also produced this ITS-PCR fingerprint. However, the fingerprint was phase-shifted toward larger DNA sizes. Alignment of the respective P. popilliae and P. lentimorbus group 1 ITS DNA sequences showed extensive homology, except for a 108 bp insert in all P. lentimorbus ITS regions. This insert occurred at the same location relative to the 23S rDNA and accounted for the phase-shift difference in P. lentimorbus group 1 DNA fingerprints. At present, there is no explanation for this 108 bp insert. The third ITS-PCR fingerprint, produced by P. lentimorbus group 3 strains, exhibited approximately eight DNA bands. Comparison of the three fingerprints of milky disease bacteria to the ITS-PCR fingerprints of other Paenibacillus species demonstrated uniqueness. ITS-PCR fingerprinting successfully identified eight unknown isolates as milky disease bacteria. Therefore, this procedure can serve as a standard protocol to identify P. popilliae and P. lentimorbus.     

A microarray for screening the variability of 16S-23S rRNA internal transcribed spacer in Pseudomonas syringae

The 16S-23S ribosomal internal transcribed spacer (ITS1) is often used as a subspecies or strain-specific molecular marker for various kinds of bacteria. However, the presence of different copies of ITS1 within a single genome has been reported. Such mosaicism may influence correct typing of many bacteria and therefore knowledge about exact configuration of this region in a particular genome is essential. In order to screen the variability of ITS1 among and within Pseudomonas syringae genomes, an oligonucleotide microarray targeting different configurations of ITS1 was developed. The microarray revealed seven distinct variants in 13 pathovars tested and detected mosaicism within the genomes of P. syringae pv. coronafaciens, pisi, syringae and tabaci. In addition, the findings presented here challenge the using of rRNA analysis for pathovar and strain determination.     

Variability of the 16S-23S rRNA gene internal transcribed spacer in Pseudomonas avellanae strains

The 16S-23S rRNA gene internal transcribed spacer region (ITS1) from 34 strains of Pseudomonas avellanae and some strains of Pseudomonas syringae pathovars was amplified and assessed by restriction fragment length polymorphism (RFLP) using 10 restriction enzymes. In addition, the ITS1 region of four representative P. avellanae strains was sequenced and compared by the neighbour-joining algorithm with that of P. syringae pathovars. Two main groups of P. avellanae strains were observed that did not correlate with their origin. The ITS1 region sequencing revealed a high similarity with the P. syringae complex. One group of P. avellanae strains showed high similarity to P. s. pv. actinidiae and P. s. pv. tomato; another group showed similarity with P. s. pv. tabaci and P. s. pv. glycinea. Two strains clustered with P. s. pv. pisi. The difficulties to unambiguously classify the strains associated with hazelnut decline in Greece and Italy are discussed.     

Analysis of 16S-23S rRNA internal transcribed spacer regions in Pasteurellaceae isolated from laboratory rodents

The internal transcribed spacer (ITS) regions of members of Pasteurellaceae isolated from rodents, including the [Pasteurella] pneumotropica biotypes Jawetz and Heyl, [Actinobacillus] muris, "Hemophilus influenzaemurium" and Bisgaard taxon 17 were studied and their feasibility to discriminate these species was analyzed. The reference strains of all species analyzed showed unique species-specific ITS patterns which were further present in 49 clinical isolates of [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris allowing their identification by comparison to the reference strains pattern. Sequence analysis of the amplified fragments revealed in all species, with exception of "H. influenzaemurium", a larger ITS(ile+ala) which contained the genes for tRNA(Ile(GAU)) and tRNA(Ala(UGC)) and a smaller ITS(glu) with the tRNA(Glu(UUC)) gene. "H. influenzaemurium" revealed two each of the larger and respectively the smaller ITS fragments. Both the length and the sequence of each ITS type were highly conserved within the [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris strains tested. On the contrary, ITS sequences revealed significant interspecies variations with identity levels ranging from 61.2 to 89.5% for ITS(ile+ala) and 56.5 to 86.8% for ITS(glu). Sequences regions with significant interspecies variation but highly conserved within the species were identified and might be used to design probes for the identification of rodent Pasteurellaceae to the species level.     

Phylogenetic relationships among Cucumis species based on the ribosomal internal transcribed spacer sequence and microsatellite markers

The phylogenetic relationships within the genus Cucumis (a total of 25 accessions belonging to 17 species) were studied using the nuclear ribosomal DNA internal transcribed spacer (ITS) region. The analysis included commercially important species such as melon (C. melo L.) and cucumber (C. sativus). Two additional cucurbit species, watermelon and zucchini, were also included as outgroups. The data obtained reflected the clustering of Cucumis species in four main groups, comprising accessions from cucumber, melon, C. metuliferus and the wild African species. Some of the species clustered in different positions from those reported in classifications previously described by other authors. The data obtained clearly identify a division between the 2n=2x = 14 species (C. sativus) and the 2n = 2x = 24 ones (C. melo and wild species). Within the wild species we identified a subgroup that included C. sagittatus and C. globosus. Oreosyce africana, also classified as Cucumis membranifolius, was shown to be nested within Cucumis. Three accessions previously classified as independent species were shown to be genotypes of Cucumis melo. A set of melon and cucumber SSRs were also used to analyse the Cucumis species and the results were compared with the ITS data. The differential amplification of the SSRs among the accessions made it possible to distinguish three main groups: melon, cucumber and the wild species, though with less detail than applying ITS. Some SSRs were shown to be specific for melon, but other SSRs were useful for producing PCR fragments in all species of the genus.We are grateful to NCRPIS, IPK in Gatersleben, Semillas Fitó S.A., Michel Pitrat and Fernando Nuez for providing seeds. We would also like to thank Vanessa Alfaro, Trinidad Martínez and Núria Galofré for their excellent technical assistance. This work was financed by project AGL2000-0360 of Spains Ministerio de Ciencia y Tecnología (MCYT). AJMs work was supported by a postdoctoral contract from Spains MCYT.     

16S-23S ribosomal DNA intergenic spacer regions in cellulolytic myxobacteria and differentiation of closely related strains

The diversity of 16S-23S rDNA intergenic spacer regions (ISR) among cellulolytic myxobacterial strains was assayed. Agarose gel electrophoresis of PCR amplification products from ten strains shows that there are at least four copies of rRNA operons in the genus Sorangium, based on their size and restriction enzymatic digest maps. There are two sequence organization patterns: tRNA(Ile)-tRNA(Ala)-containing ISR and tRNA-lacking ISR. The tRNA-containing ISRs are highly similar among strains and within a strain (more than 98% similarity) and contain the essential functional regions, such as a ribonuclease III recognition site and an antiterminator recognition site boxA. The tRNA-lacking ISR has no such functional sites that are important for yielding mature rRNA, which suggests that this type of rRNA operons might be degenerate. The tRNA-lacking ISR is divided into two types based on their sizes and sequences, which exhibits about 90% similarity within each type. Thus, the tRNA-lacking ISR polymorphisms can be used to discriminate among different strains of sorangial species.     

Intraspecific groups of Umbelopsis ramanniana inferred from nucleotide sequences of nuclear rDNA internal transcribed spacer regions and sporangiospore morphology

Umbelopsis ramanniana is a well-known species in this genus. A characteristic morphological feature of this fungus is the remarkable variation in the sporangiospore shape, which implies the genetic variations occur in the nucleotide sequences of the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA (nrDNA) in the U. ramanniana isolates. The relationship between the variations of the sequences of the nrDNA ITS regions and those of the sporangiospore morphology was investigated for 12 isolates of U. ramanniana collected in Europe. Neighbor-joining and parsimony analyses on the sequences suggested that these isolates split into three groups. Precise examination of the morphology showed that the isolates of those respective groups were different from each other in their sporangiospore shape. The present study implies at least three intraspecific groups exist in U. ramanniana and that the variations in the nucleotide sequences of the nrDNA ITS regions correlate well with those in the sporangiospore shape in these intraspecific groups.     

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.     

Identification of Escherichia coli through analysis of 16S rRNA and 16S-23S rRNA internal transcribed spacer region sequences

A bacterial strain, designated BzDS03 was isolated from water sample, collected from Dal Lake Srinagar. The strain was characterized by using 16S ribosomal RNA gene and 16S-23S rRNA internal transcribed spacer region sequences. Phylogenetic analysis showed that 16S rRNA sequence of the isolate formed a monophyletic clade with genera Escherichia. The closest phylogenetic relative was Escherichia coli with 99% 16S rRNA gene sequence similarity. The result of Ribosomal database project's classifier tool revealed that the strain BzDS03 belongs to genera Escherichia.16S rRNA sequence of isolate was deposited in GenBank with accession number FJ961336. Further analysis of 16S-23S rRNA sequence of isolate confirms that the identified strain BzDS03 be assigned as the type strain of Escherichia coli with 98% 16S-23S rRNA sequence similarity. The GenBank accession number allotted for 16S-23S rRNA intergenic spacer sequence of isolate is FJ961337.     

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.     

Nucleotide sequences of the 5.8S rRNA gene and internal transcribed spacer regions in carrot and broad bean ribosomal DNA

Summary Nucleotide sequences of the first and second internal transcribed spacers (ITS1 and ITS2, respectively) of ribosomal DNA (rDNA) from two dicot plants, carrot and broad bean, were determined. These sequences were compared with those of rice, a monocot plant, and other eukaryotic organisms. Both types of ITS region in some species of Angiospermae were the shortest among all eukaryotes so far examined and showed a wide range of variation in their G+C content, in contrast to a general trend toward very high G+C content in animals. Phylogenetic relationships of plants with animals and lower eukaryotes were considered using the nucleotide sequences of carrot and broad bean 5.8S rDNA that were determined in the present study, together with that of wheat 5.8S rRNA, which has been reported previously.     

Heterogeneity of strains assigned to Gluconobacter frateurii Mason and Claus 1989 based on restriction analysis of 16S-23S rDNA internal transcribed spacer regions

Twenty-three strains, which were assigned to Gluconobacter frateurii and maintained at Culture Collection NBRC, were re-identified at the species level on the basis of restriction analysis of 16S-23S rDNA ITS regions by digestion with six restriction endonucleases: Bsp1286I, MboII, AvaII, TaqI, BsoBI, and BstNI. The strains examined were divided into six groups, Group III-1, Group III-2, Group III-3, Group III-4, Group III-5, and Group IV. Group III-1 and Group III-4 respectively were divided into two subgroups, Subgroup III-1a, Subgroup III-1b and Subgroup III-4a, Subgroup III-4b. Gluconobacter frateurii NBRC 3264(T) was included in Group III-2, along with strains NBRC 3265 and NBRC 3270, and G. thailandicus BCC 14116(T) was included in Group III-3, along with strains NBRC 3254, NBRC 3256, NBRC 3258, NBRC 3255, and NBRC 3257. These groupings were supported by a phylogenetic tree based on 16S-23S rDNA ITS sequences. Strains of group III-2 and Group IV were unequivocally re-identified as G. frateurii, but strains of Group III-3, Group III-4, and Group III-5 were not necessarily re-identified as G. frateurii. The results obtained indicate that the 23 strains have a taxonomically heterogeneous nature, and they are referred to as the G. frateurii complex.     

Genetic heterogeneity in internal transcribed spacer genes of Balantidium coli (Litostomatea, Ciliophora)

The species Balantidium coli is the only ciliate that parasitizes humans. It has been described in other primates, and it has been proposed that the species B. suis from pigs and B. struthionis from ostriches are synonyms of B. coli. Previous genetic analysis of pig and ostrich Balantidium isolates found a genetic polymorphism in the ITS region but its taxonomic relevance was not established. We have extended the genetic analysis to Balantidium isolates of pig, gorilla, human and ostrich origin. We have PCR-amplified and sequenced the ITS region of individual Balantidium cells. The predicted ITS secondary structures of the sequences obtained were transferred by homology modelling to the sequences of other Trichostomatia ciliates (Isotricha, Troglodytella, Lacrymaria and Spathidium) and compared to determine the importance of the differences in the primary sequences. The results show that the ITS2 secondary structure of the species considered follows the general pattern of other ciliates, although with some deviations. There are at least two main types of ITS sequence variants in B. coli which could be present in the same cell and they are common to the mammal and avian hosts studied. These data do not support B. suis and B. struthionis as distinct species.     

Identification of strains assigned to the genus Gluconobacter Asai 1935 based on the sequence and the restriction analyses of the 16S-23S rDNA internal transcribed spacer regions

Thirteen reference strains, including the type strains of the type species of the genus Gluconobacter, Gluconobacter oxydans (NBRC 14819T), Gluconobacter cerinus (NBRC 3267T), and Gluconobacter frateurii (IFO 3264T) were examined for their species identification based on the sequence and the restriction analyses of the 16S-23S rDNA internal transcribed spacer (ITS) regions. A phylogenetic tree constructed by the neighbor-joining method represented three clusters corresponding respectively to the three species, G. oxydans, G. cerinus, and G. frateurii. The type strain of Gluconobacter asaii (NBRC 3276T), which is a junior subjective synonym of G. cerinus, was included completely in the G. cerinus cluster. Several restriction endonucleases discriminating the three species from one another were selected by computer analyses: Bsp1286I, MboII, SapI, Bpu10I, EarI, BsiHKAI, and FatI. On digestion of the PCR products with restriction endonucleases Bsp1286I and MboII, all the restriction patterns coincided with those of the type strains of the three species except for strain NBRC 3251. This strain gave a different pattern from the type strain of G. frateurii, when digested with MboII. However, strain 3251 was included phylogenetically in the G. frateurii cluster. All the reference strains were thus identified at the species level by the sequence and the restriction analyses of the 16S-23S rDNA ITS regions.