Inter- and intra-genomic heterogeneity of the intervening sequence in the 23S ribosomal RNA gene of Campylobacter jejuni and Campylobacter coli

An intervening sequence (IVS) can be present or absent in the 23S rRNA of Campylobacter jejuni and Campylobacter coli. As part of a survey, we used a polymerase chain reaction (PCR) assay to detect the presence of the IVS in 43 isolates of C. coli and 82 isolates of C. jejuni. An IVS was present in 40 (93.0%) of the C. coli and only 34 (41.5%) of the C. jejuni isolates. Twelve (27.9%) of the C. coli isolates and seven (8.5%) of the C. jejuni isolates resulted in two polymerase chain reaction products, indicating heterogeneity in the presence of the 23S rRNA IVS. Fourteen of the isolates with two products were evaluated by pulse-field gel electrophoresis; 13 different patterns were observed. The total band size of one isolate was substantially greater than the expected 1.7 Mb, possibly indicating a mixed culture. Southern blot analyses demonstrated the expected three rRNA operons in all tested isolates. Nested PCR reactions with operon-specific primers followed by primers for the IVS confirmed that the strains of interest contained either one or two operons carrying the IVS and the remaining operon(s) did not. Sequence analysis of the IVS and flanking regions of the 23S rRNA genes did not discriminate C. jejuni and C. coli as distinct populations. These results indicate horizontal transfer of 23S rRNA genes or portions of the genes between C. jejuni and C. coli. Also, data showing sequence polymorphisms between the three 23S rRNA loci outside of the IVS region suggest that the isolates with intra-genomic heterogeneity appear to be members of clones that have an ancient defect in gene conversion mechanisms needed for concerted evolution of the ribosomal operons.     

Functional Escherichia coli 23S rRNAs containing processed and unprocessed intervening sequences from Salmonella typhimurium.

We have introduced the intervening sequence (IVS) from 23S rRNA of the rrnD operon of Salmonella typhimurium into the equivalent position of Escherichia coli 23S rRNA. Salmonella typhimurium 23S rRNA is fragmented due to the RNase III-dependent removal of the approximately 100 nt stem-loop structure that comprises the IVS. In this study, we have found that insertion of the S. typhimurium IVS into E. coli 23S rRNA causes fragmentation of the RNA but does not affect ribosome function. Cells expressing the fragmented 23S rRNA exhibited wild-type growth rates. Fragmented RNA was found in the actively translating polysome pool and did not alter the sedimentation profile of ribosomal subunits, 70S ribosomes or polysomes. Finally, hybrid 23S rRNA carrying the A2058G mutation conferred high level erythromycin resistance indistinguishable from that of intact 23S rRNA carrying this mutation. These observations indicate that the presence of this IVS and its removal are phenotypically silent. As observed in an RNase III-deficient strain, processing of the IVS was not required for the production of functional ribosomes.     

Characterization of the 23S and 5S rRNA genes of Coxiella burnetii and identification of an intervening sequence within the 23S rRNA gene.

Characterization of the rRNA operon from the obligate intracellular bacterium Coxiella burnetii has determined the order of the rRNA genes to be 16S-23S-5S. A 444-bp intervening sequence (IVS) was identified to interrupt the 23S rRNA gene beginning at position 1176. The IVS is predicted to form a stem-loop structure formed by flanking inverted repeats, and the absence of intact 23S rRNA molecules suggests that the loop is removed. An open reading frame in the IVS has been identified that shows 70% similarity at the amino acid level to IVS open reading frames characterized from four species of Leptospira.     

Amplification and sequencing of variable regions in bacterial 23S ribosomal RNA genes with conserved primer sequences

Published bacterial 23S ribosomal RNA sequences were aligned, and universally conserved regions flanking highly variable regions were looked for. In strategically positioned conserved regions, six oligonucleotides suitable for polymerase chain reaction (PCR) and sequencing were designed, allowing fast sequencing of four of the most variable 23S rRNA regions. Two other primers were designed for PCR amplification of nearly complete 23S rRNA genes. All these primers successfully amplified fragments of 23S rRNA genes from seven unrelated bacteria. Four primers were used to determine 938 bp of sequence forCampylobacter jejuni subsp.jejuni. These results indicate that the oligonucleotide sequences presented here are useful for PCR amplification and sequence determination of variable 23S rRNA regions for a broad variety of eubacterial species.     

Cloning and characterization of genes coding for ribosomal RNA inCampylobacter jejuni

The DNA fragments coding for ribosomal RNA inCampylobacter jejuni have been cloned from a genomic library ofC. jejuni constructed inEscherichia coli. Clones carrying DNA Sequences for rRNA were identified by hybridization of 5-end-labeled rRNA fromC. jejuni to colony blots of transformants from this gene library. Cloned DNA sequences homologous to each of 5S, 16S, and 23S rRNA were idenfified by hybridization of labeled plasmid DNA to Northern blots of rRNA. The gene coding for 23S rRNA was found to be located on a 5.5kb HindIII fragment, while the 5S and 16S rRNA genes were on HindIII fragments of 1.65 and 1.7 kb, respecitively. The DNA fragment containing the 16S rRNA gene was characterized by restriction endonuclease mapping, and the location of the 16S rRNA gene on this fragment was determined by hybridization of 5-end-labeled rRNA to restriction fragments and also by DNA sequence determination. It appears that the major portion of the coding region for 16S rRNA is located on the 1.7-kb HindIII fragment, while a small portion is carried on an adjacent HindIII fragment of 7.5 kb. Cloned rRNA genes fromC. jejuni were used to study the organization of the rDNA inC. jejuni and other members of the genùsCampylobacter.     

Major differences between the rrnA operons of two strains of Agrobacterium vitis

The sequence of the rrnA operon and its flanking regions was determined for the Agrobacterium vitis type strain NCPPB3554. Compared to the earlier obtained rrnA sequence of A. vitis strain S4, several important differences were noted: the sequences diverged at the 5′-flanking region, within the 16S–23S intergenic region, and within the 23S rRNA sequence. The B8 stem-loop structure at the 5′-end of the 23S rRNA of strain NCPPB3554 was 142 nt shorter than that of strain S4. These findings have important consequences for the use of ribosomal RNA gene sequences in phylogenetic comparisons. Received: 16 February 1996 / Accepted: 26 April 1996     

Identification and characterization of an intervening sequence within the 23S ribosomal RNA genes of Edwardsiella ictaluri

Comparison of the 23S rRNA gene sequences of Edwardsiella tarda and Edw. ictaluri confirmed a close phylogenetic relationship between these two fish pathogen species and a distant relation with the 'core' members of the Enterobacteriaceae family. Analysis of the rrl gene for 23S rRNA in Edw. ictaluri revealed the presence of an intervening sequence (IVS) in helix-45. This new 98bp IVS shared 97% nucleotide identity with Salmonella typhimurium helix-45 IVS. Edw. ictaluri helix-45 IVS was present in all Edw. ictaluri strains analyzed and in at least six rrl operons within each cell. Fragmentation of 23S rRNA due to IVS excision by RNase III was observed by methylene blue staining of ribosomal RNA extracted from Edw. ictaluri isolates. This is the first report of an IVS in the 23S rRNA gene of the genus Edwardsiella.     

Fragmentation heterogeneity of 23S ribosomal RNA in Haemophilus species

The fragmentation of 23S rRNA of 22 Haemophilus influenzae strains and eight strains belonging to other Haemophilus species was investigated. Instead of intact molecules, the 23S rRNA molecules were found to be cleaved into two to five smaller conserved fragments in most strains examined, especially in H. influenzae type b (5/6) and nontypeable strains (5/5). One or two conserved potential cleavage sites were identified by PCR analysis of the strains showing a fragmented 23S rRNA pattern. The relevant nucleotide sequences were determined and compared to H. influenzae Rd, which contains intact 23S rRNA molecules. An identical 112 bp long intervening sequence (IVS) at position 542 and a conserved 121–123 bp IVS sequence at position 1171 were found in two H. influenzae type b strains and one nontypeable strain. Among the strains with fragmented 23S rRNA, nearly half showed a heterogeneous cleavage pattern due to the dispersion of IVSs among different 23S rRNA operons. The localization of the conserved H. influenzae IVSs coincided well with the extensively studied IVSs among other bacteria, but differed in nucleotide sequence from any other reported IVSs. Therefore, the IVSs of Haemophilus 23S rRNA may originate from a common source that is independent of other bacteria.     

Evolution of the plastid ribosomal RNA operon in a nongreen parasitic plant: Accelerated sequence evolution,altered promoter structure,and tRNA pseudogenes

The nucleotide sequence of a 7.4 kb region containing the entire plastid ribosomal RNA operon of the nongreen parasitic plant Epifagus virginiana has been determined. Analysis of the sequence indicates that all four rRNA genes are intact and almost certainly functional. In contrast, the split genes for tRNA^Ile and tRNA^Ala present in the 16S-23S rRNA spacer region have become pseudogenes, and deletion upstream of the 16S rRNA gene has removed a tRNA^Val gene and most of the promoter region for the rRNA operon. The rate of nucleotide substitution in 16S and 23S rRNAs is several times higher in Epifagus than in tobacco, a related photosynthetic plant. Possible reasons for this, including relaxed translational constraints, are discussed.     

Characterization of a separate small domain derived from the 5' end of 23S rRNA of an alpha-proteobacterium.

We demonstrate the presence of a separate processed domain derived from the 5' end of 23S rRNA in ribosomes of Rhodopseudomonas palustris, a member of the alpha-++proteobacteria. Previous sequencing studies predicted intervening sequences (IVS) at homologous positions within the 23S rRNA genes of several alpha-proteobacteria, including R.palustris, and we find a processed 23S rRNA 5' domain in unfractionated RNA from several species. 5.8S rRNA from eukaryotic cytoplasmic large subunit ribosomes and the bacterial processed 23S rRNA 5' domain share homology, possess similar structures and are both derived by processing of large precursors. However, the internal transcribed spacer regions or IVSs separating them from the main large subunit rRNAs are evolutionarily unrelated. Consistent with the difference in sequence, we find that the site and mechanism of IVS processing also differs. Rhodopseudomonas palustris IVS-containing RNA precursors are cleaved in vitro by Escherichia coli RNase III or a similar activity present in R.palustris extracts at a processing site distinct from that found in eukaryotic systems and this results in only partial processing of the IVS. Surprisingly, in a reaction unlike characterized cases of eubacterial IVS processing, an RNA segment larger than the corresponding DNA insertion is removed which contains conserved sequences. These sequences, by analogy, serve to link the 23S rRNA 5' rRNA domains or 5.8S rRNAs to the main portion of other prokaryotic 23S rRNAs or to eukaryotic 28S rRNAs, respectively.     

Identification of nine sequence types of the 16S rRNA genes of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp. <Emphasis Type="Italic">jejuni</Emphasis> isolated from broilers

Background  

Campylobacter is the most commonly reported bacterial cause of enteritis in humans in the EU Member States and other industrialized countries. One significant source of infection is broilers and consumption of undercooked broiler meat. Campylobacter jejuni is the Campylobacter sp. predominantly found in infected humans and colonized broilers. Sequence analysis of the 16S rRNA gene is very useful for identification of bacteria to genus and species level. The objectives in this study were to determine the degree of intraspecific variation in the 16S rRNA genes of C. jejuni and C. coli and to determine whether the 16S rRNA sequence types correlated with genotypes generated by PFGE analysis of Sma I restricted genomic DNA of the strains.     

Precise characterization of rDNA genes by intraspecies and inter-loci comparison of rDNA sequences and biochemical analysis of ribosomal RNA molecules in Agrobacterium tumefaciens

Annotation of rRNA genes has been incomplete in Agrobacterium species although a number of Agrobacterial rDNA fragments have been sequenced. In this study, precise characterization of rRNA operons (rrn) was carried out in two biovar 1 strains, C58 and MAFF301001. Complete DNA sequencing of four rrns in MAFF301001 indicated that each operon codes for 16S, 23S and 5S rRNA as well as three tRNAs, trn(Ile), trn(Ala) and trn(Met). The genes and 16S-23S ITS of a given locus were exactly identical with those in the other three loci, except for a T-base loss in the 23S rRNA gene of rrnA and in the 5S rRNA gene of rrnB. Comparison with the four C58 rDNAs available in the DNA database indicated extensive sequence and size variations in the 23S rRNA gene, suggesting the presence of an intervening sequence (IVS). Biochemical RNA analysis, including Northern hybridization and 5' end mapping, in MAFF301001 revealed 2886-base and 2571-base precursors, two 1.3-kb major fragments, a 150-base fragment and removal of an IVS for 23S rRNA. We confirmed similar biochemical characteristics in the C58 strain. The features of rDNA detected here enable correction of previously reported information about Agrobacterial rRNAs and rRNA genes and should be useful for phylogenetic considerations.     

Sequence requirements for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA.

The sequence requirements for splicing of the Tetrahymena pre-rRNA have been examined by altering the rRNA gene to produce versions that contain insertions and deletions within the intervening sequence (IVS). The altered genes were transcribed and the RNA tested for self-splicing in vitro. A number of insertions (8-54 nucleotides) at three locations had no effect on self-splicing activity. Two of these insertions, located at a site 5 nucleotides preceding the 3'-end of the IVS, did not alter the choice of the 3' splice site. Thus the 3' splice site is not chosen by its distance from a fixed point within the IVS. Analysis of deletions constructed at two sites revealed two structures, a hairpin loop and a stem-loop, that are entirely dispensable for IVS excision in vitro. Three other regions were found to be necessary. The regions that are important for self-splicing are not restricted to the conserved sequence elements that define this class of intervening sequences. The requirement for structures within the IVS for pre-rRNA splicing is in sharp contrast to the very limited role of IVS structure in nuclear pre-mRNA splicing.     

Composite structure of the chloroplast 23 S ribosomal RNA genes of Chlamydomonas reinhardii: Evolutionary and functional implications

The chloroplast ribosomal unit of Chlamydomonas reinhardii displays two features which are not shared by other chloroplast ribosomal units. These include the presence of an intron in the 23 S ribosomal RNA gene and of two small genes coding for 3 S and 7 S rRNA in the spacer between the 16 S and 23 S rRNA genes (Rochaix & Malnoë, 1978). Sequencing of the 7 S and 3 S rRNAs as well as their genes and neighbouring regions has shown that: (1) the 7 S and 3 S rRNA genes are 282 and 47 base-pairs long, respectively, and are separated by a 23 base-pair A + T-rich spacer. (2) A sequence microheterogeneity exists within the 3 S RNA genes. (3) The sequences of the 7 S and 3 S rRNAs are homologous to the 5′ termini of prokaryotic and other chloroplast 23 S rRNAs, indicating that the C. reinhardii counterparts of 23 S rRNA have a composite structure. (4) The sequences of the 7 S and 3 S rRNAs are related to that of cytoplasmic 5.8 S rRNA, suggesting that these RNAs may perform similar functions in the ribosome. (5) Partial nucleotide sequence complementarity is observed between the 5′ ends of the 7 S and 3 S RNAs on one hand and the 23 S rRNA sequences which flank the ribosomal intron on the other. These data are compatible with the idea that these small rRNAs may play a role in the processing of the 23 S rRNA precursor.     

Cloning and characterization of the rDNA repeat unit of Podospora anserina

Summary DNA coding for ribosomal RNA in Podospora anserina has been cloned and was found as a tandemly repeated 8.3 kb sequence. The cloned rDNA was characterized by restriction endonuclease mapping. The location of 5.8S, 18S and 28S rRNA coding regions was established by DNA-RNA hybridization and S1 nuclease mapping. The organization of P. anserina rRNA genes is similar to that of Neurospora crassa and Aspergillus nidulans. The rDNA unit does not contain the sequence coding for 5S RNA.