Utility of internally transcribed 16S-23S rDNA spacer regions for the definition of Pseudomonas stutzeri genomovars and other Pseudomonas species.
Bacteria identified and classified as Pseudomonas stutzeri, on the basis of traditional criteria, are recognized to be markedly heterogeneous, such that a systematic phenotypic characterization has not been correlated with genotypic groupings (i.e. genomovars) based upon DNA-DNA similarities. The internally transcribed 16S-23S rDNA spacer (ITS1) regions of P. stutzeri were analysed with respect to the ability of these nucleic acid regions to differentiate and identify the genomic groups (i.e. genomovars) of P. stutzeri. The ITS1s of 34 strains of P. stutzeri were amplified by PCR and the PCR product was subjected to RFLP analysis, which allowed the differentiation and identification of the strains to their respective genomovars. Sequence determination and analysis of ITS1s supported further the results obtained by RFLP, i.e. nucleotide signatures were identified in strains belonging to different genomovars. The ITS1s of all strains of P. stutzeri contained the tandem tRNA(Ile)/tRNA(Ala) genes and did not exhibit distinct sequence heterogeneity between different operons of a strain. Phylogenetically informative variable sites were located, exclusively, in non-coding regions. The results of the RFLP and sequence analysis of ITS1s supported and correlated with the phylogenetic relationships estimated from 16S rRNA gene sequence comparisons and DNA-DNA hybridizations, offering an alternative tool for genomovar and species differentiation. (+info)
High intraindividual variation in internal transcibed spacer sequences in Aeschynanthus (Gesneriaceae): implications for phylogenetics.
Aeschynanthus (Gesneriaceae) is a large genus of tropical epiphytes that is widely distributed from the Himalayas and China throughout South-East Asia to New Guinea and the Solomon Islands. Polymerase chain reaction (PCR) consensus sequences of the internal transcribed spacers (ITS) of Aeschynanthus nuclear ribosomal DNA showed sequence polymorphism that was difficult to interpret. Cloning individual sequences from the PCR product generated a phylogenetic tree of 23 Aeschynanthus species (two clones per species). The intraindividual clone pairs varied from 0 to 5.01%. We suggest that the high intraindividual sequence variation results from low molecular drive in the ITS of Aeschynanthus. However, this study shows that, despite the variation found within some individuals, it is still possible to use these data to reconstruct phylogenetic relationships of the species, suggesting that clone variation, although persistent, does not pre-date the divergence of Aeschynanthus species. The Aeschynanthus analysis revealed two major clades with different but overlapping geographic distributions and reflected classification based on morphology (particularly seed hair type). (+info)
High-resolution phylogenetic analysis of NO2--oxidizing Nitrobacter species using the rrs-rrl IGS sequence and rrl genes.
A high-resolution phylogenetic analysis of Nitrobacter strains and their neighbours was made using the rrs-rrl intergenic spacer sequence and the hypervariable part of the rrl gene. The phylogenetic tree obtained was consistent with that which was obtained previously but was much more discriminating, permitting the design of genus-specific primers. (+info)
Three new species in the Saccharomyces sensu stricto complex: Saccharomyces cariocanus, Saccharomyces kudriavzevii and Saccharomyces mikatae.
On the basis of genetic analysis, molecular karyotyping and sequence analyses of the 18S rRNA and internal transcribed spacer (ITS) region, three new Saccharomyces species are described, Saccharomyces cariocanus (with type strain NCYC 2890T), Saccharomyces kudriavzevii (with type strain NCYC 2889T) and Saccharomyces mikatae (with type strain NCYC 2888T). Genetic and molecular analyses did not confirm the previously observed conspecificity of Saccharomyces paradoxus and S. cariocanus. The latter species exhibits postzygotic isolation from representative strains from all known geographical populations of S. paradoxus: European, Far-East Asian, North American and Hawaiian. (+info)
Molecular systematics of European Hyalodaphnia: the role of contemporary hybridization in ancient species.
We examined phylogenetic relationships among Daphnia using mitochondrial DNA (mtDNA) sequences from the small subunit ribosomal RNA (12S), cytochrome c oxidase subunit I and nuclear DNA sequences from the first and second internal transcribed spacer representing 1612 base positions. Phylogenetic analyses using several species of the three main Daphnia subgenera, Ctenodaphnia, Hyalodaphnia and Daphnia, revealed that the Hyalodaphnia are a monophyletic sister group of the Daphnia. Most Hyalodaphnia species occur on one continent, whereas only three are found in North America and Europe. Endemicity of species is associated with variation in thermal tolerance and habitat differentiation. Although many species of the Hyalodaphnia are known to hybridize in nature, mtDNA divergence is relatively high ca. 9%) compared to other hybridizing arthropods (ca. 3%). Reproductive isolation in Daphnia seems to evolve significantly slower than genetic isolation. We related these findings to what is known about the ecology and genetics of Daphnia in order to better understand the evolutionary diversification of lineages. The relationship of these data to phylogenetic patterns is discussed in the context of speciation processes in Daphnia. (+info)
Detection and identification of mycobacteria by amplification of the internal transcribed spacer regions with genus- and species-specific PCR primers.
We evaluated the usefulness of PCR assays that target the internal transcribed spacer (ITS) region for identifying mycobacteria at the species level. The conservative and species-specific ITS sequences of 33 species of mycobacteria were analyzed in a multialignment analysis. One pair of panmycobacterial primers and seven pairs of mycobacterial species-specific primers were designed. All PCRs were performed under the same conditions. The specificities of the primers were tested with type strains of 20 mycobacterial species from the American Type Culture Collection; 205 clinical isolates of mycobacteria, including 118 Mycobacterium tuberculosis isolates and 87 isolates of nontuberculous mycobacteria from 10 species; and 76 clinical isolates of 28 nonmycobacterial pathogenic bacterial species. PCR with the panmycobacterial primers amplified fragments of approximately 270 to 400 bp in all mycobacteria. PCR with the M. tuberculosis complex-specific primers amplified an approximately 120-bp fragment only for the M. tuberculosis complex. Multiplex PCR with the panmycobacterial primers and the M. tuberculosis complex-specific primers amplified two fragments that were specific for all mycobacteria and the M. tuberculosis complex, respectively. PCR with M. avium complex-, M. fortuitum-, M. chelonae-, M. gordonae-, M. scrofulaceum-, and M. szulgai-specific primers amplified specific fragments only for the respective target organisms. These novel primers can be used to detect and identify mycobacteria simultaneously under the same PCR conditions. Furthermore, this protocol facilitates early and accurate diagnosis of mycobacteriosis. (+info)
Divergent mechanisms of 5' 23S rRNA IVS processing in the alpha-proteobacteria.
Widespread occurrence of a separate small RNA derived from the 5'-end of 23S rRNA and of an intervening sequence (IVS) which separates this domain from the main segment of 23S rRNA in the alpha-proteobacteria implies that processing reactions which act to excise the IVS are also maintained in this group. We previously characterized the first example of processing of this IVS in Rhodopseudomonas palustris, which is classified with the Bradyrhizobia In this case, IVS excision occurs by a multistep process and RNase III appears to act at an early step. Here, we characterize in vivo and in vitro IVS processing in two other related, but phenotypically distinct, Bradyrhizobia We also examine in vivo and in vitro processing of rRNA precursors from a more distantly related alpha-proteobacterium, Rhodobacter sphaeroides which produces a separate 5' 23S rRNA domain but has different sequences in the 5' 23S rRNA IVS. The details of the in vivo processing of all of the Bradyrhizobial rRNAs closely resemble the R. palustris example and in vitro studies suggest that all of the Bradyrhizobia utilize RNase III in the first step of IVS cleavage. Remarkably, in vivo and in vitro studies with R.sphaeroides indicate that initial IVS cleavage uses a different mechanism. While the mechanism of IVS cleavage differs among these alpha-proteobacteria, in all of these cases the limits of the internal segments processed in vivo are almost identical and occur far beyond the initial cleavage sites within the IVSs. We propose that these bacteria possess common secondary maturation pathways which enable them to generate similarly processed 23S rRNA 5'- and 3'-ends. (+info)
Strain identification of Trichophyton rubrum by specific amplification of subrepeat elements in the ribosomal DNA nontranscribed spacer.
Trichophyton rubrum is the commonest cause of dermatophytosis of skin and nail tissue. Molecular characterization of the T. rubrum ribosomal DNA nontranscribed-spacer region revealed two novel tandemly repetitive subelements (TRSs): TRS-1, containing a 27-bp palindromic sequence, and TRS-2. Specific amplification of TRS-1 produced strain-characteristic banding patterns (PCR types), with 21 TRS-1 PCR types recognized from 101 clinical isolates. Four simple patterns representing 1 to 4 copies of TRS-1 accounted for 75 (75%) of all 101 strains, whereas more complex patterns were observed for 21 (20%) of the 101 isolates. The copy number of TRS-2 was 0 to 3 repeats per cistron, with a majority of isolates having two copies of this element. Eleven isolates were polymorphic for TRS-2, and in combination, 23 separate PCR types were recognized by amplification of both TRS-1 and TRS-2. The PCR patterns from both elements were stable and reproducible. Elements with homology to TRS-1 were present in three phylogenetically related species, Trichophyton violaceum, Trichophyton gourvilii, and Trichophyton soudanense, but these elements were not identified in other dermatophyte taxa. There was no clear correlation of PCR type with specimen (skin or nail tissue), but certain PCR types appeared to show a bias in geographic distribution. This new method of typing T. rubrum will enable important questions about pathogenesis and epidemiology of this fungus to be addressed. (+info)