Helicobacter pylori 23S rRNA gene A2142G, A2143G, T2182C, and C2195T mutations associated with clarithromycin resistance detected in Sudanese patients.

BACKGROUND: Clarithromycin resistant Helicobacter pylori (H. pylori) strains represent a worldwide health problem. These stains are usually carrying mutations within the 23S rRNA gene associated with clarithromycin resistance. This study aimed to detect H. pylori and clarithromycin resistant associated mutations from Sudanese patients with gastritis symptoms. MATERIALS AND METHODS: Two hundred and eighty-eight gastric biopsies were collected using gastrointestinal endoscopy from patients with gastritis symptoms in different hospitals in Khartoum state. H. pylori was detected by PCR using primer targeting 16S rRNA. Then allele-specific PCR and DNA sequencing were used to screen for the presence of A2142G and A2143G point mutations. RESULTS: Out of 288 samples, H. pylori was detected in 88 (~ 30.6%) samples by 16 s RNA. Allele-specific PCR detected the variant A2142G in 9/53 (~ 17%) sample, while A2143G mutation was not found in any sample. The DNA sequencing revealed the presence of mutations associated with clarithromycin-resistance in 36% (9/25) of samples; the A2142G was present in one sample, A2143G in 5 samples and T2182C in 4 samples. Additionally, another point mutation (C2195T) was detected in 3 samples. There was no association of 23S rRNA gene point mutations with gender, age group, and patients' geographical distribution. CONCLUSION: This study revealed a high frequency (36%) of mutations associated with clarithromycin resistance using DNA sequencing of the 23S rRNA gene's V domain. This information should be taken into consideration to avoid eradication therapy failing.

Multiple conversion between the genes encoding bacterial class-I release factors.

Bacteria require two class-I release factors, RF1 and RF2, that recognize stop codons and promote peptide release from the ribosome. RF1 and RF2 were most likely established through gene duplication followed by altering their stop codon specificities in the common ancestor of extant bacteria. This scenario expects that the two RF gene families have taken independent evolutionary trajectories after the ancestral gene duplication event. However, we here report two independent cases of conversion between RF1 and RF2 genes (RF1-RF2 gene conversion), which were severely examined by procedures incorporating the maximum-likelihood phylogenetic method. In both cases, RF1-RF2 gene conversion was predicted to occur in the region encoding nearly entire domain 3, of which functions are common between RF paralogues. Nevertheless, the 'direction' of gene conversion appeared to be opposite from one another-from RF2 gene to RF1 gene in one case, while from RF1 gene to RF2 gene in the other. The two cases of RF1-RF2 gene conversion prompt us to propose two novel aspects in the evolution of bacterial class-I release factors: (i) domain 3 is interchangeable between RF paralogues, and (ii) RF1-RF2 gene conversion have occurred frequently in bacterial genome evolution.

Systematics of a kleptoplastidal dinoflagellate, Gymnodinium eucyaneum Hu (Dinophyceae), and its cryptomonad endosymbiont.

New specimens of the kleptoplastidal dinoflagellate Gymnodinium eucyaneum Hu were collected in China. We investigated the systematics of the dinoflagellate and the origin of its endosymbiont based on light morphology and phylogenetic analyses using multiple DNA sequences. Cells were dorsoventrally flattened with a sharply acute hypocone and a hemispherical epicone. The confusion between G. eucyaneum and G. acidotum Nygaard still needs to be resolved. We found that the hypocone was conspicuously larger than the epicone in most G. eucyaneum cells, which differed from G. acidotum, but there were a few cells whose hypocone and epicone were of nearly the same size. In addition, there was only one site difference in the partial nuclear LSU rDNA sequences of a sample from Japan given the name G. acidotum and G. eucyaneum in the present study, which suggest that G. eucyaneum may be a synonym of G. acidotum. Spectroscopic analyses and phylogenetic analyses based on nucleomorph SSU rDNA sequences and chloroplast 23 s rDNA sequences suggested that the endosymbiont of G. eucyaneum was derived from Chroomonas (Cryptophyta), and that it was most closely related to C. coerulea Skuja. Moreover, the newly reported kleptoplastidal dinoflagellates G. myriopyrenoides and G. eucyaneum in our study were very similar, and the taxonomy of kleptoplastidal dinoflagellates was discussed.

Topological classification of RNA structures.

We present a novel topological classification of RNA secondary structures with pseudoknots. It is based on the topological genus of the circular diagram associated to the RNA base-pair structure. The genus is a positive integer number whose value quantifies the topological complexity of the folded RNA structure. In such a representation, planar diagrams correspond to pure RNA secondary structures and have zero genus, whereas non-planar diagrams correspond to pseudoknotted structures and have higher genus. The topological genus allows for the definition of topological folding motifs, similar in spirit to those introduced and commonly used in protein folding. We analyze real RNA structures from the databases Worldwide Protein Data Bank and Pseudobase and classify them according to their topological genus. For simplicity, we limit our analysis by considering only Watson-Crick complementary base pairs and G-U wobble base pairs. We compare the results of our statistical survey with existing theoretical and numerical models. We also discuss possible applications of this classification and show how it can be used for identifying new RNA structural motifs.

Study of bacterial communities in Antarctic coastal waters by a combination of 16S rRNA and 16S rDNA sequencing.

An ecological study on distribution of Antarctic bacterial communities was determined by 16S-based phylogenetic analyses of clone libraries derived from RNA and DNA extracted from two different marine areas and compared between each other. Superficial seawater samples were collected from four stations in Ross Sea, three of them located in Rod Bay and one in Evans Cove; for each station two clone libraries (16S rDNA and 16S rRNA) were prepared and evident divergences between DNA and RNA libraries of each site were obtained. Of all phylotypes 93.6% were found in RNA libraries; in contrast, only 31 phylotypes (70.5%) were retrieved from total microbial community (DNA libraries). DNA and RNA sequences related to gamma-Proteobacteria and Bacteroidetes groups, typical for Antarctic sea-ice bacterial communities, were detected in analysed sites. 16S rDNA and rRNA libraries derived from the two different areas were enriched by picophytoplanktonic 16S sequences of plastid and mitochondrion origins, reflecting that the algal blooms occurred during sampling (Antarctic summer 2003). The finding in Rod Bay libraries of high percentage of DNA clones apparently affiliated with beta-Proteobacteria typical for activated sludges and well water could be explained by the presence of a sewage depuration system at this site. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA gene sequencing is preferred approach to have a more reliable vision on the composition of microbial communities.

IWoCS: analyzing ribosomal intergenic transcribed spacers configuration and taxonomic relationships.

MOTIVATION: Lately the use of 16S-23S Intergenic Transcribed Spacer (ITS) sequences for bacterial typing purposes has increased. The presence of conserved regions like tRNA genes or boxes together with hypervariable regions allows performing intraspecific discrimination of very close bacterial strains. On the other hand this mosaic of variability makes the ITS a sequence difficult to analyze and compare. RESULTS: A software to study ITSs by a Word Count based System (IWoCS) is proposed. A large dataset of ITS was created (comprising 7355 sequences). A database indicating all the occurrences of possible n-mers (tags), describing each ITS sequence, was created (with n going from 5 to 13) including 32 061 819 entries. The database allows to analyze ITS sequences submitted by users using a web-based interface. The abundance in the database of each n-mer is given in a one-base sliding frame. A dominance plot reflects how common the tags are within different taxonomic levels. The obtained profile identifies highly repeated tags as evolutionarily conserved regions (like tRNA or boxes) or low frequency tags as regions specifically associated to taxonomic groups. The study of the dominance and abundance profiles combined with the taxonomy reports provides a novel tool for the use of the ITS in bacteria typing and identification. AVAILABILITY: The database is freely accessible at http://egg.umh.es/iwocs/.

Intraspecific diversity of the 23S rRNA gene and the spacer region downstream in Escherichia coli.

The molecular microevolution of the 23S rRNA gene (rrl) plus the spacer downstream has been studied by sequencing of different operons from some representative strains of the Escherichia coli ECOR collection. The rrl gene was fully sequenced in six strains showing a total of 67 polymorphic sites, a level of variation per nucleotide similar to that found for the 16S rRNA gene (rrs) in a previous study. The size of the gene was highly conserved (2902 to 2905 nucleotides). Most polymorphic sites were clustered in five secondary-structure helices. Those regions in a large number of operons were sequenced, and several variations were found. Sequences of the same helix from two different strains were often widely divergent, and no intermediate forms existed. Intercistronic variability was detected, although it seemed to be lower than for the rrs gene. The presence of two characteristic sequences was determined by PCR analysis throughout all of the strains of the ECOR collection, and some correlations with the multilocus enzyme electrophoresis clusters were detected. The mode of variation of the rrl gene seems to be quite similar to that of the rrs gene. Homogenization of the gene families and transfer of sequences from different clonal lines could explain this pattern of variation detected; perhaps these factors are more relevant to evolution than single mutation. The spacer region between the 23S and 5S rRNA genes exhibited a highly polymorphic region, particularly at the 3' end.

A common motif organizes the structure of multi-helix loops in 16 S and 23 S ribosomal RNAs.

Phylogenetic and chemical probing data indicate that a modular RNA motif, common to loop E of eucaryotic 5 S ribosomal RNA (rRNA) and the alpha-sarcin/ricin loop of 23 S rRNA, organizes the structure of multi-helix loops in 16 S and 23 S ribosomal RNAs. The motif occurs in the 3' domain of 16 S rRNA at positions 1345-1350/1372-1376 (Escherichia coli numbering), within the three-way junction loop, which binds ribosomal protein S7, and which contains nucleotides that help to form the binding site for P-site tRNA in the ribosome. The motif also helps to structure a three-way junction within domain I of 23 S, which contains many universally conserved bases and which lies close in the primary and secondary structure to the binding site of r-protein L24. Several other highly conserved hairpin, internal, and multi-helix loops in 16 S and 23 S rRNA contain the motif, including the core junction loop of 23 S and helix 27 in the core of 16 S rRNA. Sequence conservation and range of variation in bacteria, archaea, and eucaryotes as well as chemical probing and cross-linking data, provide support for the recurrent and autonomous existence of the motif in ribosomal RNAs. Besides its presence in the hairpin ribozyme, the loop E motif is also apparent in helix P10 of bacterial RNase P, in domain P7 of one sub-group of group I introns, and in domain 3 of one subgroup of group II introns.

Clostridium ljungdahlii sp. nov., an acetogenic species in clostridial rRNA homology group I.

Clostridium ljungdahlii sp. nov. strain ATCC 49587T (T = type strain) was isolated from chicken yard waste for its ability to produce ethanol from synthesis gas. This gram-positive, motile, sporeforming rod's metabolism was primarily acetogenic. C. ljungdahlii grew with carbon monoxide, hydrogen and carbon dioxide, ethanol, pyruvate, arabinose, xylose, fructose, or glucose. Methanol, ferulic acid, lactate, galactose, and mannose did not support growth. The G+C content was 22 to 23 mol%. C. ljungdahlii is the first acetogen in clostridial 23S rRNA homology group I.

Archaeal phylogeny: reexamination of the phylogenetic position of Archaeoglobus fulgidus in light of certain composition-induced artifacts.

A major and too little recognized source of artifact in phylogenetic analysis of molecular sequence data is compositional difference among sequences. The problem becomes particularly acute when alignments contain ribosomal RNAs from both mesophilic and thermophilic species. Among prokaryotes the latter are considerably higher in G + C content than the former, which often results in artificial clustering of thermophilic lineages and their being placed artificially deep in phylogenetic trees. In this communication we review archaeal phylogeny in the light of this consideration, focusing in particular on the phylogenetic position of the sulfate reducing species Archaeoglobus fulgidus, using both 16S rRNA and 23S rRNA sequences. The analysis shows clearly that the previously reported deep branching of the A. fulgidus lineage (very near the base of the euryarchaeal side of the archaeal tree) is incorrect, and that the lineage actually groups with a previously recognized unit that comprises the Methanomicrobiales and extreme halophiles.

The sequence of Methanospirillum hungatei 23S rRNA confirms the specific relationship between the extreme halophiles and the Methanomicrobiales.

We have determined the sequence of the 23S rRNA from the methanogenic archaeon Methanospirillum hungatei. This is the first such sequence from a member of the Methanomicrobiales. Moreover, it brings additional evidence to bear on the possible specific relationship between this particular group of methanogens and the extreme halophiles. Such evidence is critical in that several new (and relatively untested) methods of phylogenetic inference have lead to the controversial conclusion that the extreme halophiles are either not related to the archaea, or are only peripherally so. Analysis of the Methanospirillum hungatei 23S rRNA sequence shows the Methanomicrobiales are indeed a sister group of the extreme halophiles, further strengthening the conclusions reached from analysis of 16S rRNA sequences.