Sequence and secondary structure of the mitochondrial small-subunit rRNA V4, V6, and V9 domains reveal highly species-specific variations within the genus Agrocybe

A comparative study of variable domains V4, V6, and V9 of the mitochondrial small-subunit (SSU) rRNA was carried out with the genus Agrocybe by PCR amplification of 42 wild isolates belonging to 10 species, Agrocybe aegerita, Agrocybe dura, Agrocybe chaxingu, Agrocybe erebia, Agrocybe firma, Agrocybe praecox, Agrocybe paludosa, Agrocybe pediades, Agrocybe alnetorum, and Agrocybe vervacti. Sequencing of the PCR products showed that the three domains in the isolates belonging to the same species were the same length and had the same sequence, while variations were found among the 10 species. Alignment of the sequences showed that nucleotide motifs encountered in the smallest sequence of each variable domain were also found in the largest sequence, indicating that the sequences evolved by insertion-deletion events. Determination of the secondary structure of each domain revealed that the insertion-deletion events commonly occurred in regions not directly involved in the secondary structure (i.e., the loops). Moreover, conserved sequences ranging from 4 to 25 nucleotides long were found at the beginning and end of each domain and could constitute genus-specific sequences. Comparisons of the V4, V6, and V9 secondary structures resulted in identification of the following four groups: (i) group I, which was characterized by the presence of additional P23-1 and P23-3 helices in the V4 domain and the lack of the P49-1 helix in V9 and included A. aegerita, A. chaxingu, and A. erebia; (ii) group II, which had the P23-3 helix in V4 and the P49-1 helix in V9 and included A. pediades; (iii) group III, which did not have additional helices in V4, had the P49-1 helix in V9 and included A. paludosa, A. firma, A. alnetorum, and A. praecox; and (iv) group IV, which lacked both the V4 additional helices and the P49-1 helix in V9 and included A. vervacti and A. dura. This grouping of species was supported by the structure of a consensus tree based on the variable domain sequences. The conservation of the sequences of the V4, V6, and V9 domains of the mitochondrial SSU rRNA within species and the high degree of interspecific variation found in the Agrocybe species studied open the way for these sequences to be used as specific molecular markers of the Basidiomycota.     

Extreme differences in rates of molecular evolution of foraminifera revealed by comparison of ribosomal DNA sequences and the fossil record

Foraminifera have one of the best known fossil records among the unicellular eukaryotes. However, the origin and phylogenetic relationships of the extant foraminiferal lineages are poorly understood. To test the current paleontological hypotheses on evolution of foraminifera, we sequenced about 1,000 base pairs from the 3' end of the small subunit rRNA gene (SSU rDNA) in 22 species representing all major taxonomic groups. Phylogenies were derived using neighbor-joining, maximum-parsimony, and maximum-likelihood methods. All analyses confirm the monophyletic origin of foraminifera. Evolutionary relationships within foraminifera inferred from rDNA sequences, however, depend on the method of tree building and on the choice of analyzed sites. In particular, the position of planktonic foraminifera shows important variations. We have shown that these changes result from the extremely high rate of rDNA evolution in this group. By comparing the number of substitutions with the divergence times inferred from the fossil record, we have estimated that the rate of rDNA evolution in planktonic foraminifera is 50 to 100 times faster than in some benthic foraminifera. The use of the maximum-likelihood method and limitation of analyzed sites to the most conserved parts of the SSU rRNA molecule render molecular and paleontological data generally congruent.     

Molecular evolution of Mycoplasma capricolum subsp. capripneumoniae strains, based on polymorphisms in the 16S rRNA genes

Mycoplasma capricolum subsp. capripneumoniae belongs to the so-called Mycoplasma mycoides cluster and is the causal agent of contagious caprine pleuropneumonia (CCPP). All members of the M. mycoides cluster have two rRNA operons. The sequences of the 16S rRNA genes of both rRNA operons from 20 strains of M. capricolum subsp. capripneumoniae of different geographical origins in Africa and Asia were determined. Nucleotide differences which were present in only one of the two operons (polymorphisms) were detected in 24 positions. The polymorphisms were not randomly distributed in the 16S rRNA genes, and some of them were found in regions of low evolutionary variability. Interestingly, 11 polymorphisms were found in all the M. capricolum subsp. capripneumoniae strains, thus defining a putative ancestor. A sequence length difference between the 16S rRNA genes in a poly(A) region and 12 additional polymorphisms were found in only one or some of the strains. A phylogenetic tree was constructed by comparative analysis of the polymorphisms, and this tree revealed two distinct lines of descent. The nucleotide substitution rate of strains within line II was up to 50% higher than within line I. A tree was also constructed from individual operonal 16S rRNA sequences, and the sequences of the two operons were found to form two distinct clades. The topologies of both clades were strikingly similar, which supports the use of 16S rRNA sequence data from homologous operons for phylogenetic studies. The strain-specific polymorphism patterns of the 16S rRNA genes of M. capricolum subsp. capripneumoniae may be used as epidemiological markers for CCPP.     

Cell contraction caused by microtubule disruption is accompanied by shape changes and an increased elasticity measured by scanning acoustic microscopy

The relationships within the superorder Archonta, which contains the orders Dermoptera (flying lemurs), Scandentia (tree shrews), Chiroptera (bats), and Primates, were examined through the analysis of five newly derived and complete mitochondrial 12S rRNA sequences. The new data is combined with 83 additional known mammalian sequences to provide a full phylogenetic sampling. Phylogenetic hypotheses are generated using PAUP 3.1.1 (Swofford [1993] Illinois Natural History Survey, Champaign, IL) through analyses of all characters equally weighted, transversions only, and the effect of alignment gaps on phylogeny. The Parsimony Jackknifer (Farris et al. [1996] Cladistics 12:99-124) was used to assess the level of ambiguity present in the sequence data, and therefore the strength of the tree topologies. The conclusions of Springer and Douzery (1996, J. Mol. Evol. 43:357-373) which states that 12S rRNA is reliable to a time depth of 100 mya is unsupported by these analyses. The usefulness of 12S rRNA to aid in solving Archonta relationships and others of similar time depth is found to be suspect.     

Biodiversity at the molecular level: the domains, kingdoms and phyla of life

The results of comparative sequence analysis, mainly of small subunit (SSU) ribosomal (r)RNA sequences, have suggested that all of cellular life can be placed in one of three domains: the Archaea, Bacteria or Eucarya. There is some evidence that the Archaea may not be a monophyletic assemblage, but as yet this issue has not been resolved. Most of the lineages, and all of the deepest ones, in the tree based upon SSU rRNA sequences, are microbial. Traditional ideas of classification such as Whittaker's five kingdom scheme do not adequately describe life's diversity as revealed by sequence comparisons. There are many microbial groups that demonstrate much greater amounts of SSU rRNA sequence divergence than do members of the classical kingdoms, Animalia, Plantae and Fungi. The old microbial kingdoms Monera and Protista are clearly paraphyletic but as yet there is no consensus as to how they should be reorganized in taxonomic terms. New data from environmental analysis suggests that much of the microbial world is unknown. Every environment which has been analysed by molecular methods has revealed many previously unrecorded lineages. Some of these show great divergence from the sequences of cultured microorganisms suggesting that fundamentally new microbial groups remain to be isolated. The relationships of some of these new lineages may be expected to affect how the tree of life is organized into higher taxa, and to also influence which features will be recognized as synapomorphies. There is currently no objective measure whereby microbial diversity can be quantified and compared to the figures which are widely quoted for arthropods and other Metazoa.     

Products of a lagging DNA strand synthesis of nucleoprotein complexes harboring an extrachromosomal DNA closely related to avian myeloblastosis virus core-bound DNA

There are three group I introns in the nuclear small subunit ribosomal RNA gene (SSU rDNA) of the ballistoconidiogenous anamorphic yeast-like fungus Tilletiopsis flava JCM 5186. The size of these sequences were 325 nt (position 516), 335 nt (position 1199) and 437 nt (position 1506), respectively. The introns at position 516 (T.flav516) and position 1199 (T.flav1199) belonged to subgroup IB3, and that of position 1506 (T.flav1506) belonged to subgroup IC1. The results of comparison with other group I introns found in SSU rDNA of eucaryotes showed that the positions 516 and 1199 were common positions to IB3 group I introns of fungi and green algae, and that positions 943, 1506 and 1512 were those to IC1 group I introns of fungi, and green and red algae. It is indicated that the insertion position of introns have close relationship with the nature of the subgroup to which they belonged. For phylogenetic analysis, we employed 9 IB3 introns, in which 7 were at position 516 and 2 were at position 1199, and 25 IC1 introns. The maximum likelihood tree based on the conserved region alignment showed that group I introns of subgroup IB3 were phylogenetically distant from those of subgroup IC1. T.flav516 (basidiomycete) constituted a subcluster with R.dacr516 (basidiomycete) and M.albo516 (ascomycete). T. flav1199 was located at the closer position of C.chlo1199 (green alga) than other IB3 introns at position 516. T.flav1506 was located at the subcluster, which was constituted by the 1506 introns found in SSU rDNA of fungi (B.yama1506, P.cari1506, and P.inou1506) and those of green algae (C.elli1506, C.mira1506, G.spir1506, and M.sacl1506) with IC1 introns at the position 1512 (D.parv1512 and C.sacc1512). The analysis of flanking regions showed that both 5' and 3' flanking sequences were well conserved in each insertion site, and indicated that the ancestors of the intron at different site had been inherited from the different origin. Therefore, the two IB3 introns found positions 516 and 1199, T.flav516 and T.flav1199, were supposed to have the independent ancestors. Our results supported the theory of the diversity of group I introns that group I introns had been transferred horizontally to the distinct insertion site, and were inherited and diverged vertically.     

Sickness absence and ''working through'' illness: a comparison of two professional groups

The chloroplasts of euglenophytes and dinoflagellates have been suggested to be the vestiges of endosymbiotic algae acquired during the process of evolution. However, the evolutionary positions of these organisms are still inconclusive, and they have been tentatively classified as both algae and protozoa. A representative gene of the mitochondrial genome, cytochrome oxidase subunit I (coxI), was chosen and sequenced to clarify the phylogenetic positions of four dinoflagellates, two euglenophytes and one apicomplexan protist. This is the first report of mitochondrial DNA sequences for dinoflagellates and euglenophytes. Our COXI tree shows clearly that dinoflagellates are closely linked to apicomplexan parasites but not with algae. Euglenophytes and algae appear to be only remotely related, with euglenophytes sharing a possible evolutionary link with kinetoplastids. The COXI tree is in general agreement with the tree based on the nuclear encoded small subunit of ribosomal RNA (SSU rRNA) genes, but conflicts with that based on plastid genes. These results support the interpretation that chloroplasts present in euglenophytes and dinoflagellates were captured from algae through endosymbioses, while their mitochondria were inherited from the host cell. We suggest that dinoflagellates and euglenophytes were originally heterotrophic protists and that their chloroplasts are remnants of endosymbiotic algae.     

PCR amplification and comparison of nucleotide sequences from the groESL heat shock operon of Ehrlichia species

Degenerate PCR primers derived from conserved regions of the eubacterial groESL heat shock operon were used to amplify groESL sequences of Ehrlichia equi, Ehrlichia phagocytophila, the agent of human granulocytic ehrlichiosis (HGE), Ehrlichia canis, Bartonella henselae, and Rickettsia rickettsii. The groESL nucleotide sequences were less conserved than the previously determined 16S rRNA gene sequences of these bacteria. A phylogenetic tree derived from deduced GroEL amino acid sequences was similar to trees based on 16S rRNA gene sequences. Nucleotide sequences obtained from clinical samples containing E. equi, E. phagocytophila, or the HGE agent were very similar (99.9 to 99.0% identity), and the deduced amino acid sequences were identical. Some divergence was evident between nucleotide sequences amplified from samples originating from the United States (E. equi and the HGE agent) and sequences from the European species, E. phagocytophila. A single pair of PCR primers derived from these sequences was used to detect E. chaffeensis and HGE agent DNA in blood samples from human patients with ehrlichiosis.     

The presence of a dnaK (HSP70) multigene family in members of the orders Planctomycetales and Verrucomicrobiales

Sequences of the dnaK gene, coding for the 70-kDa heat shock protein (HSP70), were determined for six members of the order Planctomycetales, including representatives of three genera, and for the only cultivated member of the order Verrucomicrobiales, Verrucomicrobium spinosum. A fragment of the dnaK gene was amplified from these strains by PCR with oligonucleotide primers targeting regions of the dnaK gene that are conserved at the amino acid level, and the resulting PCR products were cloned into a plasmid vector. Sequence analysis of the cloned dnaK fragments revealed the presence of two different types of dnaK sequence in one of the planctomycete strains, Planctomyces maris, and in V. spinosum. Only one type of dnaK sequence was found for each of the remaining strains. Phylogenetic analysis of the partial sequence data suggested that the majority of planctomycete strains, including one of the Planctomyces maris sequences, form a coherent phylogenetic group branching adjacent to other main lines of descent within the domain Bacteria, as has been shown previously by 16S rRNA sequence analysis. One of the two V. spinosum dnaK sequences also appears to constitute a separate lineage within the gram-negative bacteria. Each of the remaining sequences from P. maris and V. spinosum, together with the single sequence obtained from Planctomyces limnophilus, appeared to be unrelated to the other planctomycete sequences and to occupy a position distant from that of other gram-negative bacteria. The phylogenetic diversity of dnaK sequences exhibited by P. maris and V. spinosum was comparable to that found in Synechococcus sp. strain PCC7942 and Escherichia coli, the only other prokaryotes for which a dnaK multigene family has been demonstrated.     

From the library of the Nederlands Tijdschrift voor Geneeskunde: a hygienist''s pearl, the manual of Ali Cohen (1872)

In an attempt to identify the taxonomic relationship between CAR bacillus and other bacteria, the SSU rRNA gene sequences of two CAR bacillus strains, CBM and CBR isolated from mice and rats respectively were used in the present studies. The SSU rRNA gene sequences, approximately 1.5 kb in size amplified from genomic DNAs from both strains, were determined and 96.8% homologies were found to exist between them. Those sequences were aligned to most eubacteria with a computer search showing high homology with those of Flavobacter/Flexibacter species especially closed to Fx. sancti and Fv. ferrugineum. Phylogenetic analysis indicated that CAR bacillus belongs to a species close to Fx. sancti and Fv. ferrugineum subdivision.     

Mitochondrial portraits of human populations using median networks

Analysis of variation in the hypervariable region of mitochondrial DNA (mtDNA) has emerged as an important tool for studying human evolution and migration. However, attempts to reconstruct optimal intraspecific mtDNA phylogenies frequently fail because parallel mutation events partly obscure the true evolutionary pathways. This makes it inadvisable to present a single phylogenetic tree at the expense of neglecting equally acceptable ones. As an alternative, we propose a novel network approach for portraying mtDNA relationships. For small sample sizes (< approximately 50), an unmodified median network contains all most parsimonious trees, displays graphically the full information content of the sequence data, and can easily be generated by hand. For larger sample sizes, we reduce the complexity of the network by identifying parallelisms. This reduction procedure is guided by a compatibility argument and an additional source of phylogenetic information: the frequencies of the mitochondrial haplotypes. As a spin-off, our approach can also assist in identifying sequencing errors, which manifest themselves in implausible network substructures. We illustrate the advantages of our approach with several examples from existing data sets.     

Humoral immune response to human papillomavirus infection

Nematodes are known to be a useful system for studies of comparative development. Here we perform a molecular phylogenetic analysis to allow for the independent interpretation of the developmental and morphological changes observed among a selected set of nematode species. Our molecular phylogenetic analysis is based on coding regions of the genes for RNA polymerase II, the small subunit rRNA and an expansion segment of the large subunit rRNA. Sequences were compared from five species in the family (Rhabditidae) that includes the developmental model organism Caenorhabditis elegans and from an outgroup taxon Aduncospiculum halicti (Diplogasterina). The phylogenetic analysis does not support the monophyly of the subfamily Mesorhabditinae and identifies the unnamed strain PS1010 as a sister taxon of C. elegans despite its morphologically divergent buccal capsule. On the basis of the inferred framework, we can begin to interpret the evolution of vulval development and of morphological differences among these nematode species.     

"Candidatus phytoplasma australiense," a new phytoplasma taxon associated with Australian grapevine yellows

A phytoplasma was detected in naturally diseased 'Chardonnay' grapevines exhibiting symptoms of Australian grapevine yellows disease. The use of PCR designed to amplify phytoplasma DNA resulted in detection of phytoplasma DNA in all of the diseased plants examined; no phytoplasma DNA was detected in healthy seedling grapevines. The collective restriction fragment length polymorphism (RFLP) patterns of amplified 16S ribosomal DNA differed from the patterns described previously for other phytoplamas. On the basis of the RFLP patterns, Australian grapevine yellows phytoplasma was classified as a representative of a new subgroup, designated subgroup 16SrI-J, in phytoplasma 16S rRNA group 16SrI (aster yellows and related phytoplasmas). A phylogenetic analysis in which parsimony of 16S rRNA gene sequences from this and other group 16SrI phytoplasmas was used identified the Australian grapevine yellows phytoplasma as a member of a distinct subclade (subclade xii) in the phytoplasma clade of the class Mollicutes. A phylogenetic tree constructed on the basis of 16S rRNA gene sequences was consistent with the hypothesis that there was divergent evolution of Australian grapevine yellows phytoplasma and its closet known relative, European stolbur phytoplasma (subgroup 16SrI-G), from a common ancestor. The unique properties of the DNA from the Australian grapevine yellows phytoplasma clearly establish that it represents a new taxon, "Candidatus Phytoplasma australiense."     

Phylogenetic analysis of Acinetobacter strains based on the nucleotide sequences of gyrB genes and on the amino acid sequences of their products

Partial nucleotide sequences of the gyrB genes (DNA gyrase B subunit genes) of 15 Acinetobacter strains, including the type and reference strains of genomic species 1 to 12 (A. calcoaceticus [genomic species 1], A. baumannii [genomic species 2], Acinetobacter genomic species 3, A. haemolyticus [genomic species 4], A. junii [genomic species 5], Acinetobacter genomic species 6, A. johnsonii [genomic species 7], A. lwoffii [genomic species 8], Acinetobacter genomic species 9, Acinetobacter genomic species 10, Acinetobacter genomic species 11, and A. radioresistens [genomic species 12]), were determined by sequencing the PCR-amplified fragments of gyrB. The gyrB sequence homology among these Acinetobacter strains ranged from 69.6 to 99.7%. A phylogenetic analysis, using the gyrB sequences, indicates that genomic species 1, 2, and 3 formed one cluster (87.3 to 90.3% identity), while genomic species 8 and 9 formed another cluster (99.7% identity). These results are consistent with those of DNA-DNA hybridization and of biochemical systematics. On the other hand, the topology of the published phylogenetic tree based on the 16S rRNA sequences of the Acinetobacter strains was quite different from that of the gyrB-based tree. The numbers of substitution in the 16S rRNA gene sequences were not high enough to construct a reliable phylogenetic tree. The gyrB-based analysis indicates that the genus Acinetobacter is highly diverse and that a reclassification of this genus would be required.     

Empirical estimation of the reliability of ribosomal RNA alignments

MOTIVATION: The automatic alignment of rRNA sequences can reproduce manual expert alignments with high, but not perfect, fidelity. We examine the use of empirical methods for the identification of regions of an alignment of a new sequence with an existing large alignment which can confidently be predicted to be correctly aligned. RESULTS: We show how to use a simple jack-knife procedure to derive an estimate of the reliability that is to be expected at each position of a large alignment of eukaryotic rRNA sequences. These reliabilities are then improved using measures that are specific to the input sequence. Regions where the sequence-specific reliability method performs particularly well are identified and seen to correspond with elements in the structure of the rRNA molecules that vary between species in the alignment. We also compare these reliability measures to an algorithmic alignment stability measure. AVAILABILITY: The software is available free of charge by sending an e-mail message to emmet@chah.ucc.ie. CONTACT: emmet@chah.ucc.ie     

Homology between the seed cytolysin enterolobin and bacterial aerolysins

Enterolobin, a 55-kDa cytolytic, inflammatory, and insecticidal protein isolated from seeds of the Brazilian tree Enterolobium contortisiliquum (Leguminosae-Mimosoideae) has been further purified and partially sequenced by using both manual and automated methods. A computational search of enterolobin partial amino acid sequence against the PIR database revealed possible sequence similarities with aerolysins, cytolytic proteins from Aeromonas species. An alignment of enterolobin partial sequence to the amino acid sequences of A. hydrophila and A. sobria aerolysins showed several similar regions with many residue identities. The seed protein enterolobin and the bacterial aerolysins may be homologous proteins despite the distant phylogenetic relationship.     

A proposal to revive the genus Kitasatospora (Omura, Takahashi, Iwai, and Tanaka 1982)

We determined almost complete 16S ribosomal DNA sequences for 12 actinomycete strains which were either previously classified as Kitasatospora strains or defined as Streptomyces strains but shown to contain major amounts of meso-diaminopimelic acid in their whole-cell hydrolysates. These sequences were subjected to phylogenetic analyses together with the sequences of 34 Streptomyces species. Phylogenetic trees were reconstructed by using both neighbor-joining and maximum-parsimony methods. The Kitasatospora species always formed a stable monophyletic clade. However, the genus Kitasatospora appeared to be either a sister taxon of the genus Streptomyces or a lineage that originated from within Streptomyces species, depending on the outgroup used. Phylogenetic trees were also constructed by using the sequences of the 16S-23S rRNA gene spacers. Streptomyces and Kitasatospora species were consistently recovered as two distinct clades independent of the outgroup used. On the basis of phylogenetic, chemotaxonomic, and phenotypic evidence, we propose that the genus Kitasatospora Omura et al. 1982 should be revived.     

Usefulness and limitation of phylogenetic analysis for hepatitis C virus core region: application to isolates from Egyptian and Yemeni patients

We report here the nucleotide sequences of the core region of HCV isolates from Egyptian and Yemeni patients and the method for classifying these HCV isolates by phylogenetic analysis. Sequence comparison suggested that the genotypes of these isolates were the same. Preliminary phylogenetic analysis of the HCV core region indicated that the genotypes of both isolates were 1c. However, an additional phylogenetic tree of the HCV core region constructed using a greater number of HCV isolates than that used in the preliminary analysis and on the basis of alignment of nucleotide sequences in an appropriate length indicated that the genotypes of these isolates were 4 and not 1c. For a more detailed analysis, the nucleotide sequences of the HCV E1 region as well as the core region for the same Yemeni patient were determined. A phylogenetic tree of the E1 region confirmed that the genotype of the HCV isolate from the Yemeni patient was 4. These data indicate that even when classifying HCV isolates using phylogenetic analysis, the misclassification would occur if care is not taken regarding the number and sequence lengths of the isolates included in the analysis.     

A qualitative evaluation of the published oligonucleotides specific for the 16S rRNA gene sequences of the ammonia-oxidizing bacteria

Over the past few years, there has been an increasing interest in making oligonucleotides specific for ammonia-oxidizing bacteria (AOB), in order to detect and monitor these slow growing bacteria in environmental samples, in enrichment cultures and in wastewater treatment plants. Based on 16S rDNA sequences, a broad selection of oligonucleotides have been designed, either encompassing all known AOB in the beta-subgroup of the Proteobacteria (beta AOB), or subclasses within beta AOB. Thirty different oligonucleotides have so far been published, with varying specificity. The first AOB-specific oligonucleotides published were obtained as a result of an alignment of only eleven 16S rDNA sequences from AOB. Including the present study, there are now forty nearly full length 16S rDNA sequences available from these bacteria, in addition to a number of partial sequences, so that an improved evaluation of the published oligonucleotides can be done. Two new 16S rRNA gene sequences from Nitrosospira are presented here, in a phylogenetic analysis containing every 16S rRNA gene sequences (> 1 kb) available from AOB. On the basis of an alignment of all these sequences, combined with searches in the nucleotide sequence databases, an evaluation of the thirty published oligonucleotides is presented. The analysis expose the strength and weakness of each oligonucleotide and discuss the use of oligonucleotides specific for 16S rRNA genes in future studies of AOB. The present work also identifies one new, broad range primer, specific for the AOB in the beta-subgroup of the Proteobacteria.