Sequence organization of the chloroplast ribosomal spacer ofSpinacia oleracea including the 3′ end of the 16S rRNA and the 5′ end of the 23S rRNA

The 2201-bp spacer between the chloroplast ribosomal 16S and 23S genes ofSpinacia oleracea was sequenced. It contains the genes of the tRNA^Ile (GAU) and tRNA^Ala (UGC) which are both interrupted by introns of respectively 728 and 816 bp. These introns belong to the class II according to the classfication of Michel and Dujon [17]. Comparison of the rDNA spacer sequence of maize, tobacco and spinach indicates that no conserved polypeptide is encoded within the introns of the two tRNA genes and that the two main insertions/deletions between the three plants are located within two loops of the class II introns secondary structure, which is therefore conserved. Based on the sequence complementarity observed between the upstream and downstream parts, of the 16S and 23S rRNA genes, RNase III-like secondary structures involved in the processing of the rRNA precursor are proposed.     

Rapid Identification of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">acidophilus</Emphasis> by Restriction Analysis of the 16S–23S rRNA Intergenic Spacer Region and Flanking 23S rRNA Gene

A rapid molecular approach was developed for the initial identification of Lactobacillus acidophilus strains which are difficult to identify using a single biochemical test. The 16S–23S rRNA intergenic spacer regions and flanking 23S rRNA genes of 19 strains of lactobacilli were amplified and the nucleotide sequences and restriction site polymorphisms were analyzed. AluI was the most useful of the restriction enzymes analyzed and produced reproducible digestion profiles in the L. helveticus, L. plantarum, and L. casei groups, as well as in L. acidophilus. This restriction fragment length polymorphism method may be useful for the identification of L. acidophilus strains in dairy products.     

Mutational analysis of basic residues in the N-terminus of the rRNA:m<Superscript>6</Superscript>A methyltransferase ErmC′

Erm methyltransferases mediate the resistance to the macrolide-lincosamide-streptogramin B antibiotics via dimethylation of a specific adenine residue in 23S rRNA. The role of positively charged N-terminal residues of the ErmC' methyltransferase in RNA binding and/or catalysis was determined. Mutational analysis of amino acids K4 and K7 was performed and the mutants were characterized in in vivo and in vitro experiments. The K4 and K7 residues were suggested not to be essential for the enzyme activity but to provide a considerable support for the catalytic step of the reaction, probably by maintaining the optimum conformation of the transition state through interactions with the phosphate backbone of RNA.     

Complete cloning of the chloroplast genome of safflower in λ EMBL3 and mapping of 23S and 16S rRNA genes

Summary A recombinant DNA library was constructed from partial BamHI or MboI digests of safflower (Carthamus tinctorius L.) chloroplast DNA, in the BamHI site of EMBL3. Seventeen recombinants, selected by chromosome walking, were found to contain overlapping fragments of the entire chloroplast genome. These clones were mapped using single and double digests of BamHI, EcoRI and HindIII. cDNAs synthesized from isolated 16S and 23S chloroplast rRNAs were used to map the ribosomal RNA genes relative to physical maps of the above restriction enzymes. The mapped positions of the rRNA genes for the safflower chloroplast DNA are in good agreement with previously published data for tobacco, spinach and several other higher plants.     

Analysis of the Gut Microbiota by High-Throughput Sequencing of the V5–V6 Regions of the 16S rRNA Gene in Donkey

Considerable evidence suggests that the gut microbiota is complex in many mammals and gut bacteria communities are essential for maintaining gut homeostasis. To date the research on the gut microbiota of donkey is surprisingly scarce. Therefore, we performed high-throughput sequencing of the 16S rRNA genes V5–V6 hypervariable regions from gut fecal material to characterize the gut microbiota of healthy donkeys and compare the difference of gut microbiota between male and female donkeys. Sixty healthy donkeys (30 males and 30 females) were enrolled in the study, a total of 915,691 validated reads were obtained, and the bacteria found belonged to 21 phyla and 183 genera. At the phylum level, the bacterial community composition was similar for the male and female donkeys and predominated by Firmicutes (64 % males and 64 % females) and Bacteroidetes (23 % males and 21 % females), followed by Verrucomicrobia, Euryarchaeota, Spirochaetes, and Proteobacteria. At the genus level, Akkermansia was the most abundant genus (23 % males and 17 % females), followed by Sporobacter, Methanobrevibacter, and Treponema, detected in higher distribution proportion in males than in females. On the contrary, Acinetobacter and Lysinibacillus were lower in males than in females. In addition, six phyla and 15 genera were significantly different between the male and female donkeys for species abundance. These findings provide previously unknown information about the gut microbiota of donkeys and also provide a foundation for future investigations of gut bacterial factors that may influence the development and progression of gastrointestinal disease in donkey and other animals.     

Cleavage of the sarcin–ricin loop of 23S rRNA differentially affects EF-G and EF-Tu binding

Ribotoxins are potent inhibitors of protein biosynthesis and inactivate ribosomes from a variety of organisms. The ribotoxin α-sarcin cleaves the large 23S ribosomal RNA (rRNA) at the universally conserved sarcin–ricin loop (SRL) leading to complete inactivation of the ribosome and cellular death. The SRL interacts with translation factors that hydrolyze GTP, and it is important for their binding to the ribosome, but its precise role is not yet understood. We studied the effect of α-sarcin on defined steps of translation by the bacterial ribosome. α-Sarcin-treated ribosomes showed no defects in mRNA and tRNA binding, peptide-bond formation and sparsomycin-dependent translocation. Cleavage of SRL slightly affected binding of elongation factor Tu ternary complex (EF-Tu•GTP•tRNA) to the ribosome. In contrast, the activity of elongation factor G (EF-G) was strongly impaired in α-sarcin-treated ribosomes. Importantly, cleavage of SRL inhibited EF-G binding, and consequently GTP hydrolysis and mRNA–tRNA translocation. These results suggest that the SRL is more critical in EF-G than ternary complex binding to the ribosome implicating different requirements in this region of the ribosome during protein elongation.     

Sequence Diversity in the 16S–23S Intergenic Spacer Region (ISR) of the rRNA Operons in Representatives of the Escherichia coli ECOR Collection

The ribosomal RNA multigene family in Escherichia coli comprises seven rrn operons of similar, but not identical, sequence. Four operons (rrnC, B, G, and E) contain genes in the 16S–23S intergenic spacer region (ISR) for tRNA^Glu-2 and three (rrnA, D, and H) contain genes for tRNA^Ile-1 and tRNA^Ala-1B. To increase our understanding of their molecular evolution, we have determined the ISR sequence of the seven operons in a set of 12 strains from the ECOR collection. Each operon was specifically amplified using polymerase chain reaction primers designed from genes or open reading frames located upstream of the 16S rRNA genes in E. coli K12. With a single exception (ECOR 40), ISRs containing one or two tRNA genes were found at the same respective loci as those of strain K12. Intercistronic heterogeneity already found in K12 was representative of most variation among the strains studied and the location of polymorphic sites was the same. Dispersed nucleotide substitutions were very few but 21 variable sites were found grouped in a stem-loop, although the secondary structure was conserved. Some regions were found in which a stretch of nucleotides was substituted in block by one alternative, apparently unrelated, sequence (as illustrated by the known putative insertion of rsl in K12). Except for substitutions of different sizes and insertions/deletions found in the ISR, the pattern of nucleotide variation is very similar to that found for the 16S rRNA gene in E. coli. Strains K12 and ECOR 40 showed the highest intercistronic heterogeneity. Most strains showed a strong tendency to homogenization. Concerted evolution could explain the notorious conservation of this region that is supposed to have low functional restrictions. Received: 31 July 1997 / Accepted: 17 October 1997     

Binding of the S7 Protein with Fragment 926–986/1219–1393 of the 16S rRNA As a Key Step in the Assembly of the Small Subunit of Prokaryotic Ribosomes

Both structural and thermodynamic studies are necessary to understand the ribosome assembly. An initial step was made in studying the interaction between a 16S rRNA fragment and S7, a key protein in assembling the prokaryotic ribosome small subunit. The apparent dissociation constant was obtained for complexes of recombinant Escherichia coliandThermus thermophilusS7 with a fragment of the 3" domain of the E. coli16S rRNA. Both proteins showed high rRNA-binding activity, which was not observed earlier. Since RNA and proteins are conformationally labile, their folding must be considered to correctly describe the RNA–protein interactions.     

The conserved A-site finger of the 23S rRNA: just one of the intersubunit bridges or a part of the allosteric communication pathway?

During the translocation of tRNAs and mRNA relative to the ribosome, the B1a, B1b and B1c bridges undergo the most extensive conformational changes among the bridges between the large and the small ribosomal subunits. The B1a bridge, also called the "A-site finger" (ASF), is formed by the 23S rRNA helix 38, which is located right above the ribosomal A-site. Here, we deleted part of the ASF so that the B1a intersubunit bridge could not be formed (DeltaB1a). The mutation led to a less efficient subunit association. A number of functional activities of the DeltaB1a ribosomes, such as tRNA binding to the P and A-sites, translocation and EF-G-related GTPase reaction were preserved. A moderate decrease in EF-G-related GTPase stimulation by the P-site occupation by deacylated tRNA was observed. This suggests that the B1a bridge is not involved in the most basic steps of the elongation cycle, but rather in the fine-tuning of the ribosomal activity. Chemical probing of ribosomes carrying the ASF truncation revealed structural differences in the 5S rRNA and in the 23S rRNA helices located between the peptidyltransferase center and the binding site of the elongation factors. Interestingly, reactivity changes were found in the P-loop, an important functional region of the 23S rRNA. It is likely that the A-site finger, in addition to its role in subunit association, forms part of the system of allosteric signal exchanges between the small subunit decoding center and the functional centers on the large subunit.     

16S–23S rRNA Intergenic Spacer Region Variability in the Genus Frankia

16S–23S rRNA internally transcribed spacer (ITS) sequences from 53 Frankia strains were sequenced and sized from polymerase chain reaction amplification products and compiled with 14 selected 16S–23S ITS sequences from public database. Frankia genomes included two to three ITS copies lacking length polymorphism except for nine strains. No tRNA gene was encountered in this region. Frankia strains exhibited various lengths (369 to 452 nt) and a wide range of sequence similarity (35–100%) in the ITS region. The average pairwise distance varied from 0.368 (clusters 1 and 2) to 0.964 (clusters 3 and 4) and was 0.397, 0.138, 0.129, and 0.016, respectively, for cluster 4 (saprophytic non-infective/non-effective), clusters 1 and 3 (facultative symbiotic), and cluster 2 (obligate symbiotic). This suggests a gradual erosion of Frankia diversity concomitantly with a shift from saprophytic non-infective/non-effective to facultative and symbiotic lifestyle. Comparative sequence analyses of the 16S–23S rRNA intergenic spacer region of Frankia strains are not useful to assign them to their respective cluster or host infection group. Accurate assignment required the inclusion of the adjacent 16S and 23S rRNA gene fragments.     

Is the In Situ Accessibility of the 16S rRNA of Escherichia coli for Cy3-Labeled Oligonucleotide Probes Predicted by a Three-Dimensional Structure Model of the 30S Ribosomal Subunit?

Systematic studies on the hybridization of fluorescently labeled, rRNA-targeted oligonucleotides have shown strong variations in in situ accessibility. Reliable predictions of target site accessibility would contribute to more-rational design of probes for the identification of individual microbial cells in their natural environments. During the past 3 years, numerous studies of the higher-order structure of the ribosome have advanced our understanding of its spatial conformation. These studies range from the identification of rRNA-rRNA interactions based on covariation analyses to physical imaging of the ribosome for the identification of protein-rRNA interactions. Here we reevaluate our Escherichia coli 16S rRNA in situ accessibility data with regard to a tertiary-structure model of the small subunit of the ribosome. We localized target sequences of 176 oligonucleotides on a 3.0-Å-resolution three-dimensional (3D) model of the 30S ribosomal subunit. Little correlation was found between probe hybridization efficiency and the proximity of the probe target region to the surface of the 30S ribosomal subunit model. We attribute this to the fact that fluorescence in situ hybridization is performed on fixed cells containing denatured ribosomes, whereas 3D models of the ribosome are based on its native conformation. The effects of different fixation and hybridization protocols on the fluorescence signals conferred by a set of 10 representative probes were tested. The presence or absence of the strongly denaturing detergent sodium dodecyl sulfate had a much more pronounced effect than a change of fixative from paraformaldehyde to ethanol.     

Is the in situ accessibility of the 16S rRNA of Escherichia coli for Cy3-labeled oligonucleotide probes predicted by a three-dimensional structure model of the 30S ribosomal subunit?

Systematic studies on the hybridization of fluorescently labeled, rRNA-targeted oligonucleotides have shown strong variations in in situ accessibility. Reliable predictions of target site accessibility would contribute to more-rational design of probes for the identification of individual microbial cells in their natural environments. During the past 3 years, numerous studies of the higher-order structure of the ribosome have advanced our understanding of its spatial conformation. These studies range from the identification of rRNA-rRNA interactions based on covariation analyses to physical imaging of the ribosome for the identification of protein-rRNA interactions. Here we reevaluate our Escherichia coli 16S rRNA in situ accessibility data with regard to a tertiary-structure model of the small subunit of the ribosome. We localized target sequences of 176 oligonucleotides on a 3.0-A-resolution three-dimensional (3D) model of the 30S ribosomal subunit. Little correlation was found between probe hybridization efficiency and the proximity of the probe target region to the surface of the 30S ribosomal subunit model. We attribute this to the fact that fluorescence in situ hybridization is performed on fixed cells containing denatured ribosomes, whereas 3D models of the ribosome are based on its native conformation. The effects of different fixation and hybridization protocols on the fluorescence signals conferred by a set of 10 representative probes were tested. The presence or absence of the strongly denaturing detergent sodium dodecyl sulfate had a much more pronounced effect than a change of fixative from paraformaldehyde to ethanol.     

16S rRNA Gene-Based Oligonucleotide Microarray for Environmental Monitoring of the Betaproteobacterial Order “Rhodocyclales”

For simultaneous identification of members of the betaproteobacterial order “Rhodocyclales” in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the “Rhodocyclales.” The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent “Rhodocyclales” diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed “Rhodocyclales”-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of “Rhodocyclales” populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.     

The Thermus thermophilus5S rRNA–Protein Complex: Identification of Specific Binding Sites for Proteins L5 and L18 in the 5S rRNA

Three 5S rRNA-binding ribosomal proteins (L5, L18, TL5) of extremely thermophilic bacterium Thermus thermophilushave earlier been isolated. Structural analysis of their complexes with rRNA requires identification of their binding sites in the 5S rRNA. Previously, a TL5-binding site has been identified, a TL5–RNA complex crystallized, and its structure determined to 2.3 Å. The sites for L5 and L18 were characterized, and two corresponding 5S rRNA fragments constructed. Of these, a 34-nt fragment specifically interacted with L5, and a 55-nt fragment interacted with L5, L18, and with both proteins. The 34-nt fragment–L5 complex was crystallized; the crystals are suitable for high-resolution X-ray analysis.     

Cladistic analysis of 5S rRNA and 16S rRNA secondary and primary structure—The evolution of eukaryotes and their relation to archaebacteria

Summary The secondary structure of 5S rRNA has been elucidated by a cladistic analysis resulting in minimal models for eukaryotes, eubacteria, and halophilic-methanogenic archaebacteria, as well as for an ur-5S rRNA. This ancestor of all present-day 5S rRNA molecules is compared with an ur-tRNA and can be fitted into a tRNA-like structure allowing tertiary-structure interactions at the equivalent positions. A phylogenetic analysis of eukaryotic 5SrRNA and 16S rRNA sequences confirms particular monophyletic taxa: rhodophytes (red algae), chlorobionts (green algae and plants), metazoans (multicellular animals), euglenozoans (euglenids and trypanosomatids), a group of zygomycetes (excluding Kickxellales), a group of ascomycetes (excluding Protomycetales), two distinct groups of basidiomycetes, and a group consisting of phaeophyceans (brown algae) and oomycetes (water molds). The Euglenozoa show a distinct relation to the Eumycota (true fungi) and Metazoa. An analysis of archaebacterial sequences substantiates the paraphyletic nature of this third urkingdom defining the eubacteria as a sister group of the halophile-methanogens and defining the eukaryotes as a sister group of a particular lineage of the eocytes/sulfur-dependents. The latter fact implies that even the eocytes/sulfur-dependent archaebacteria are paraphyletic.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986Dedicated to the memory of Erik Huysmans who died on July 8, 1986, at the age of 29.     

Polyphasic taxonomic studies of lactic acid bacteria associated with Tunisian fermented meat based on the heterogeneity of the 16S–23S rRNA gene intergenic spacer region

The objective of this work was to investigate the structure and diversity of lactic acid bacteria (LAB) communities in traditionally fermented meat collected from different areas of Tunisia. A polyphasic study, which involves phenotypic tests and ribosomal DNA-based techniques, was used to identify Gram-positive and catalase-negative isolates. PCR amplification of the 16S–23S rDNA ISR of 102 isolates and other reference LAB strains gave (1) one type of rrn operon (M-ISR) for lactococci, (2) two types of rrn operon (S-ISR and M-ISR) for enterococci, (3) two types of rrn operon (S-ISR and L-ISR) for Lactobacilli, and (4) three PCR amplicons (S-ISR, M-ISR, and L-ISR) obtained for Pediococcus spp. and Weissella genus. The clustering and comparison of ISR–RFLP profiles given by the isolates with those given by reference LAB strains, allowed their identification as Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis, Enterococcus sanguinicola, Enterococcus hawaiiensis, Lactobacillus sakei, Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus alimentarius, Pediococcus pentosaceus, and Weissella confusa. Combined 16S–23S rDNA ISR and RFLP patterns can be considered as a good potential target for a rapid and reliable differentiation between isolates of LAB and provided further information on the organization of their rrn operons.