Updating rDNA Restriction Enzyme Maps of Tetrahymena Reveals Four New Intron-Containing Species1

The extrachromosomal rDNA molecules from a number of Tetrahymena strains wered racterized by restriction enzyme mapping using three different restriction enzymes combined with gel blotting and hybridization analysis. Strains from four out of six recently described species were found to contain an intron in the 26s rRNA coding region. The evolutionary relationship among the species of the T. pyriformis complex was examined on the basis of the rDNA maps with emphasis on similarities between two of the new species and the widely studied T. thermophila and T. pigmentosa. Examination of a large number of T. pigmentosa strains showed this species to exhibit an unusual polymorphism with respect to its rDNA. It is suggested that recombinational cross-over events play a role in the formation of new rDNA alleles in this species.     

Extrachromosomal ribosomal RNA genes in Tetrahymena: structure and evolution

The macronuclear ribosomal RNA genes from a number of strains within several species of Tetrahymena have been characterized. Restriction enzyme analysis revealed that individual strains all contained entirely homogeneous populations of extrachromosomal palindromic ribosomal DNA, varying in molecular size from 12 × 10⁶ to 14 × 10⁶ in different strains. Considering that the evolutionary distance among some of the species is estimated to be of the order of 10⁶ years, the rDNA from all the species exhibited a strikingly high similarity in the localization of their restriction sites. Nevertheless, differences both inside and outside the gene region were clearly detectable, showing that the rDNA sequences have diverged in all species.Genetic polymorphism with respect to rDNA structure exists in Tetrahymena, but seems to be rare. In only two out of five species examined (T. borealis and T. pigmentosa) interbreeding strains differing in rDNA structure were found. While the differences detected in the T. borealis rDNA were confined to a small size difference located at the non-coding ends of the molecule, several differences were detected in the rDNA from the T. pigmentosa strains. One of the differences was shown to be due to the presence of an intervening sequence within the structural gene for 26 S rRNA in some of the strains. An intervening sequence of similar size located at the same position within the 26 S gene region was found by R-loop mapping in all strains of the species T. thermophila. Restriction enzyme analysis indicates that the rDNA from two other species contains a similar intervening sequence, and we therefore suggest that the size and localization of the intervening sequence is evolutionarily stable. The two intervening sequences examined so far, however, are not identical, as revealed by restriction enzyme mapping.     

Inheritance of the group I rDNA intron in Tetrahymena pigmentosa.

We have previously argued from phylogenetic sequence data that the group I intron in the rRNA genes of Tetrahymena was acquired by different Tetrahymena species at different times during evolution. We have now approached the question of intron mobility experimentally by crossing intron+ and intron- strains looking for a strong polarity in the inheritance of the intron (intron homing). Based on the genetic analysis we find that the intron in T. pigmentosa is inherited as a neutral character and that intron+ and intron- alleles segregate in a Mendelian fashion with no sign of intron homing. In an analysis of vegetatively growing cells containing intron+ and intron- rDNA, initially in the same macronucleus, we similarly find no evidence of intron homing. During the course of this work, we observed to our surprise that progeny clones from some crosses contained three types of rDNA. One possible explanation is that T. pigmentosa has two rdn loci in contrast to the single locus found in T. thermophila. Some of the progeny clones from the genetic analysis were expanded for several hundred generations, and allelic assortment of the rDNA was demonstrated by subcloning analysis.     

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.     

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.     

Identification of Azospirillum strains by restriction fragment length polymorphism of the 16S rDNA and of the histidine operon

Abstract DNA fingerprints of several Azospirillum strains, belonging to the five known species A. amazonense, A. brasilense, A. halopraeferens, A. irakense and A. lipoferum , were obtained by restriction analysis of the amplified 16S rDNA and by restriction fragment length polymorphism of the histidine biosynthetic genes. Data obtained showed that amplified rDNA restriction analysis is an easy, fast, reproducible and reliable tool for identification of Azospirillum strains, mainly at the species level, whereas restriction fragment length polymorphism could, in some cases, differentiate strains belonging to the same species. Moreover, both analyses gave congruent results in grouping strains and in the assignment of new strains to a given species.     

四膜虫S1株——上海四膜虫,新种

四膜虫S1是一株自接型四膜虫,根据形态特征应属梨形四膜虫复合种。本种除克隆内接合外,和其它十二种四膜虫均不接合。本种的异柠檬酸脱氢酶,四唑氧化酶,谷氨酸脱氢酶和乙酸酯酶的同功酶谱和其它十二种四膜虫相比,差异明显。此外,金亦石等(1987)所提供的S1和其它十种四膜虫的rDNA分子的四种限制性内切酶图谱也说明,S1株与除T.australis以外的其它九种的酶切图谱是显著不同的。据此,作者认为本种应是梨形四膜虫复合种中一新种,并定名为上海四膜虫。     

A riboprinting scheme for identification of unknown Acanthamoeba isolates at species level

We describe a riboprinting scheme for identification of unknown Acanthamoeba isolates at the species level. It involved the use of PCR-RFLP of small subunit ribosomal RNA gene (riboprint) of 24 reference strains by 4 kinds of restriction enzymes. Seven strains in morphological group I and III were identified at species level with their unique sizes of PCR product and riboprint type by Rsa I. Unique RFCP of 17 strains in group II by Dde I, Taq I and Hae III were classified into: (1) four taxa that were identifiable at the species level, (2) a subgroup of 4 taxa and a pair of 2 taxa that were identical with each other, and (3) a species complex of 7 taxa assigned to A. castellanii complex that were closely related. These results were consistent with those obtained by 18s rDNA sequence analysis. This approach provides an alternative to the rDNA sequencing for rapid identification of a new clinical isolate or a large number of environmental isolates of Acanthamoeba.     

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.     

Phylogenetic characterization of a new thermoacidophilic bacterium isolated from hot springs in Japan

Three strains of thermophilic-acidophilic bacteria isolated previously from different hot springs in Japan were characterized by molecular genetic methods. The strategy taken involved PCR amplification, sequencing and restriction pattern analysis of 16S rDNA, 16S-23S rDNA spacer polymorphism analysis and genomic DNA-DNA hybridization. A phylogenetic analysis based on 16S rDNA sequences showed that the new thermoacidophilic isolates formed a genetically coherent group at the species level and fell into a major cluster together with members of the genera Alicyclobacillus and Sulfobacillus with A. acidocaldarius and A. acidoterrestris as their closest relatives. The levels of binary sequence similarity between the isolates and the two Alicyclobacillus species were 97.6 to 97.9%, values considered low enough to warrant placement of the isolates in a distinct species of the genus Alicyclobacillus. The 16S rDNA restriction pattern analysis, but not 16S-23S rDNA spacer polymorphism analysis, was useful for differentiating the isolates from the established Alicyclobacillus species. DNA-DNA hybridization assays demonstrated a distinct phylogenetic position of our isolates as a genospecies within the genus Alicyclobacillus. On the basis of these results, the thermoacidophilic isolates should be classified into a new species of Alicyclobacillus. The results of this study suggest that this new genospecies of Alicyclobacillus is widely distributed in hot springs in Japan.     

Identification and differentiation of Heterotardigrada and Eutardigrada species by riboprinting

In the last decades, the number of known tardigrade species has considerably increased to more than 960 species with new ones being discovered every year. However, the study of tardigrade species presents a general problem which is frequently encountered during the work with invertebrates: small size and remarkable degrees of phenotypic plasticity may sometimes not permit a definite identification of the species. In this investigation we have used riboprinting, a tool to study rDNA sequence variation, in order to distinguish tardigrade species from each other. The method combines a restriction site variation approach of ribotyping with amplified DNAs. In eight investigated species of heterotardigrades and eutardigrades we have amplified the genes for the small subunit ribosomal RNA (SSU; 18S) and subsequently sequenced the genes. Virtual riboprints were used for identification of restriction sites from ten already published 18S rDNA sequences and seven new 18S rDNA sequences. On the basis of the obtained sequences, diagnostic restriction fragment patterns can be predicted with only 11 restriction enzymes. The virtual digestion confirmed the obtained restriction fragment patterns and restriction sites of all amplified and digested tardigrade DNAs. We show that the variation in positions and number of restriction sites obtained by standard restriction fragment analysis on agarose gels can be used successfully for taxonomic identification at different taxonomic levels. The simple restriction fragment analysis provides a fast and convenient method of molecular barcoding for species identification in tardigrades.     

Molecular identification and differentiation of Brevibacterium species and strains

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.     

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.     

<Emphasis Type="Italic">Metschnikowia viticola</Emphasis> sp. nov., a new yeast species from grape

Two yeast strains, producing needle-shaped ascospores under suitable conditions, were isolated from grapes grown in Hungary. Based on these two strains, Metschnikowia viticola (type strain NCAIM Y.01705, CBS 9950, JCM 12561) is proposed as a new yeast species. Considering its phenotypic features, the restriction fragment patterns of 18S rDNA and the sequence of the D1/D2 domain of 26S rDNA, the proposed new species is closely related to Candida kofuensis.     

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.     

Molecular divergence of the soil yeasts Williopsis sensu stricto

Fifty-three strains of saturn-spored yeasts were analyzed by means of restriction analysis of the amplified fragment of rDNA which comprised the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. The use of endonucleases HaeIII and MspI enabled clear differentiation of yeast species Williopsis mucosa, W. salicorniae, Zygowilliopsis californica, Komagataea pratensis, and the Williopsis sensu stricto complex. Minisatellite primer M13 was proposed for the differentiation between twin species of Williopsis sensu stricto, which have identical restriction profiles. PCR with primer M13 enabled reidentification of a number of collection strains, species identification of saturn-spored isolates from the Far East, and detection of three strains affiliated to novel taxa. The latter have unique PCR profiles and differ in the nucleotide sequences of ITS1 and ITS2 fragments of rDNA. Possible variations in the results obtained with different molecular methods are discussed.     

四膜虫T.S1株rDNA分子的形态以及限制性内切酶图谱分析

用电镜以及Southern杂交技术测得从我国分离的四模膜虫T.S1株的rDNA(rRNA基因)分子为20kb(相当于12.5×10~6道尔顿)的回文二聚体结构。制定了该rDNA分子的四种限制性内切酶图谱,并与已知的四膜虫10个物种的相应的限制酶图谱作了比较分析,发现T.S1株与其中9个物种的图谱显著地不同,但是与T.australis的rDNA的7种限制酶图谱相比较,竟有6种是一致的。两者的BglI图虽有差别,但也只是一个酶切位点之差。     

Geobacillus jurassicus sp. nov., a new thermophilic bacterium isolated from a high-temperature petroleum reservoir, and the validation of the Geobacillus species

Four thermophilic, spore-forming bacterial strains, DS1(T), DS2, 46 and 49, were isolated from the high-temperature Dagang oilfield, located in China. The strains were identified by using the polyphasic taxonomy approach. These were aerobic, gram-positive, rod-shaped, moderately thermophilic (with an optimum growth temperature of 60-65 degrees C), chemoorganotrophic bacteria capable of growing on various sugars, carboxylic acids and crude oil. Two strains, DS1(T) and DS2, were capable of growing on individual saturated hydrocarbons. The G + C content of the DNA of strains DS1(T) and DS2 was 54.5 and 53.8 mol%, respectively. The phylogenetic analysis of the 16S rDNA of strains DS1(T) and DS2 showed that they form a separate cluster within the genus Geobacillus. The cellular fatty acids of the isolates were dominated by iso-15:0, iso-16:0 and iso-17:0 acids, which are the typical fatty acids of bacteria from the genus Geobacillus. The DNA-DNA hybridization study and the comparative analysis of the morphological and chemotaxonomic characteristics of strains DS1(T) and DS2 showed that they differ from the previously described Geobacillus species and belong to a new species, which was called Geobacillus jurassicus. DS1(T) (=VKM B2301(T), = DSM 15726(T)) is the type strain of this species. According to both DNA-DNA reassociation studies and 16S rDNA sequence analysis, two other strains, 46 and 49, were assigned to the species G. stearothermophilus. In this paper, we provide evidence that the new combinations G. stearothermophilus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans may be considered to be valid.