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PubMed2000

Comparative analysis of the whole set of rRNA operons between an enterohemorrhagic Escherichia coli O157:H7 Sakai strain and an Escherichia coli K-12 strain MG1655.

Two primer sets for direct sequence determination of all seven rRNA operons (rrn) of Escherichia coli have been developed; one is for specific-amplification of each rrn operon and the other is for direct sequencing of the amplified operons. Using these primer sets, we determined the nucleotide sequences of seven rrn operons, including promoter and terminator regions, of an enterohemorrhagic E. coli (EHEC) O157:H7 Sakai strain. To elucidate the intercistronic or intraspecific variation of rrn operons, their sequences were compared with those for the K-12 rrn operons. The rrn genes and the internal transcribed spacer regions showed a higher similarity to each other in each strain than between the corresponding operons of the two strains. However, the degree of intercistronic homogeneity was much higher in the EHEC strain than in K-12. In contrast, promoter and terminator regions in each operons were conserved between the corresponding operons of the two strains, which exceeded intercistronic similarity.

Base Sequence↗
PubMed1986

3'-End formation of transcripts from the yeast rRNA operon.

Deletion analysis of artificial rRNA minigenes transformed into Saccharomyces cerevisiae revealed that a 110 bp long fragment corresponding to positions -36 to +74 relative to the 3'-end of the 26S rRNA gene, is both necessary and sufficient for obtaining transcripts whose 3'-termini are identical to those of 26S and 37S (pre-)rRNA. These termini are produced via processing of longer transcripts because in an rna 82.1 mutant the majority of the minigene transcripts extend further downstream. Since the rna 82.1 mutation inactivates an endonuclease involved in the 3'-processing of 5S pre-rRNA it is concluded that the maturation of 37S- and that of 5S pre-rRNA requires a common factor. Comparison of the spacer sequences between Saccharomyces carlsbergensis, Saccharomyces rosei and Hansenula wingei revealed several conserved sequence blocks within the region between +10 and +55. These conserved sequence tracts, which are part of a longer region showing dyad symmetry, are supposed to be involved in the interaction with the processing component(s). Deletion of the sequences required for the formation of the 3'-ends of 26S rRNA and 37S pre-rRNA revealed a putative terminator for transcription by RNA polymerase I situated at position +210. This site maps within a DNA fragment that also contains the enhancing element for rDNA transcription by RNA polymerase I.

Enhancer Elements, Genetic↗
PubMed1995

Characterization of a putative 23S-5S rRNA operon of Buchnera aphidicola (endosymbiont of aphids) unlinked to the 16S rRNA-encoding gene.

Buchnera aphidicola (Ba) is an endosymbiont of the aphid Schizaphis graminum. In order to obtain information on highly expressed genes, we have chosen to study Ba genes coding for rRNAs. Previously, the single-copy rrs gene was cloned and sequenced [Munson et al., Gene 137 (1993) 171-178], and found to constitute a single transcription unit unlinked to rrl and rrf. In the present study, a 6.1-kb Ba DNA fragment containing rrl was cloned into Escherichia coli (Ec) and sequenced. Based on sequence similarity to Ec, the following genes were identified: aroE-tRNA(Glu)-rrl-rrf-cysS. AroE and CysS had 48 and 54% amino acid (aa) identity, respectively, to the corresponding Ec proteins; tRNA(Glu), rrl and rrf had 80-90% nucleotide (nt) identity with the corresponding genes of Ec rrnB. Ba tRNA(Glu)-rrl-rrs appears to be part of a single transcriptional unit; a putative promoter and a Rho-independent terminator were identified. Comparisons of sequences of aroE-rrl from endosymbionts of seven additional species of aphids indicated conservation of the -35 (TTGACT) and -10 (TGTAA/TT) promoter regions, and boxA, tRNA(Glu) and boxC. Secondary structure analysis indicated that the Ba tRNA(Glu)-rrl-rrf operon resembled the homologous region of Ec rrnB. The results of this and previous studies indicate that Ba differs from most bacteria in having the single-copy rRNA genes organized into two transcription units.

Amino Acid Sequence↗
PubMed2002

tRNA gene clusters at the 3' end of rRNA operons are specific to virulent subgroups of Streptococcus agalactiae strains, as demonstrated by molecular differential analysis at the population level.

The aim of this work was to characterize a 2.4 kb randomly amplified polymorphic DNA (RAPD) fragment described as a marker for a phylogenetic group of Streptococcus agalactiae strains significantly associated with neonatal meningitis. This fragment was analysed by cloning and sequencing, and showed that two types of tRNA gene cluster flank the 3' end of the rRNA operons in S. agalactiae strains. Both types of tRNA gene cluster act as markers for phylogenetic subgroups of strains within the species. One type could be used to distinguish two of the three virulent intraspecies subgroups to which most of the S. agalactiae strains able to invade the central nervous system of neonates belong. This raises the possibility that there is a link between these tRNA genes and the virulence of the bacterium. Based on this analysis, PCR primers were designed to determine whether S. agalactiae strains are likely to belong to lineages of organisms in which most of the highly virulent strains isolated from cerebrospinal fluid cluster. In addition, this work demonstrated that RAPD can be used to detect novel particularities within intraspecies variants of pathogens.

Bacterial Typing Techniques↗
PubMed1991

Physical map of the chromosome of Lactococcus lactis subsp. lactis DL11 and localization of six putative rRNA operons.

A physical map of the chromosome of Lactococcus lactis subsp. lactis DL11 was constructed by using the contour-clamped homogeneous electric field mode of pulsed-field gel electrophoresis in one- and two-dimensional separations to analyze restriction digests of high-molecular-weight genomic DNA. The map, which shows all the observed NotI and SmaI sites (six and 21, respectively) and 8 of approximately 30 SalI sites, is circular and yields a total size of 2.58 megabase pairs for the L. lactis subsp. lactis DL11 chromosome. By using rDNA from Mycoplasma capricolum to probe Southern blots of pulsed-and fixed-field digestion patterns, six putative rRNA operons were identified in L. lactis subsp. lactis DL11 and placed on the map of the chromosome. Five of these loci are clustered in a region representing only 20% of the chromosome. The presence of a SmaI site in each of the putative operons allowed the direction of transcription of each operon to be deduced.

Blotting, Southern↗
PubMed2002

Variation in rRNA operon number as revealed by ribotyping of Bacillus anthracis strains.

Ribotyping of various Bacillus strains with one restriction enzyme (AccI) revealed significant similarity between Bacillus anthracis strains, Bacillus thuringiensis and Bacillus cereus strains, which are all members of the Bacillus cereus group. A further ribotyping study of 10 virulent and 8 attenuated B. anthracis strains, using 4 endonucleases and both 23S and 16S probes independently, was performed. The discrimination index D of Hunter and Gaston showed that the best combination for future large-scale ribotyping studies would be either the combination of AccI and 23S, or that of EcoRI and 16S. Depending on the B. anthracis strain analyzed 10 or 11 rRNA operons were found. In all cases, many strains were grouped into 2 to 3 patterns. Attenuated strains, including a laboratory-cured strain, yielded aberrant patterns.

Bacillus anthracis↗
PubMed2003

Genotypes at the internal transcribed spacers of the nuclear rRNA operon of Pneumocystis jiroveci in nonimmunosuppressed infants without severe pneumonia.

The frequency of Pneumocystis jiroveci (human-derived Pneumocystis) in immunocompetent infants developing acute respiratory syndromes has recently been evaluated and has been shown to be close to 25%. Until now, there have been no data on the genomic characteristics of the fungus in these patients, while molecular typing of P. jiroveci organisms was mostly performed with samples from immunosuppressed patients with pneumocystosis (Pneumocystis carinii pneumonia [PCP]). The present report describes the genotypes of P. jiroveci organisms in 26 nonimmunosuppressed infants developing a mild Pneumocystis infection contemporaneously with an episode of bronchioloalveolitis. The typing was based on sequence analysis of internal transcribed spacers (ITSs) 1 and 2 of the rRNA operon, followed by the use of two typing scores. By use of the first score, 11 P. jiroveci ITS types were identified: 10 were previously reported in immunosuppressed patients with PCP, while 1 was newly described. By use of the second score, 13 types were identified, of which 2 were newly described. The most frequent type was identified as type B(1)a(3) (first score), which corresponds to type Eg (second score). Mixed infections were diagnosed in three infants. The occurrence of such diversity of P. jiroveci ITS types, an identical main type, and mixed infections has previously been reported in immunosuppressed patients with PCP. Thus, the P. jiroveci ITS genotypes detected in immunocompetent infants and immunosuppressed patients developing different forms of Pneumocystis infection share characteristics, suggesting that both groups of individuals make up a common human reservoir for the fungus. Finally, the frequency of P. jiroveci in nonimmunosuppressed infants with acute respiratory syndromes and the genotyping results provide evidence that this infant population is an important reservoir for the fungus.

Ascomycota↗
PubMed1998

Transcription from heterologous rRNA operon promoters in chloroplasts reveals requirement for specific activating factors.

The plastid rRNA (rrn) operon in chloroplasts of tobacco (Nicotiana tabacum), maize, and pea is transcribed by the plastid-encoded plastid RNA polymerase from a sigma70-type promoter (P1). In contrast, the rrn operon in spinach (Spinacia oleracea) and mustard chloroplasts is transcribed from the distinct Pc promoter, probably also by the plastid-encoded plastid RNA polymerase. Primer-extension analysis reported here indicates that in Arabidopsis both promoters may be active. To understand promoter selection in the plastid rrn operon in the different species, we have tested transcription from the spinach rrn promoter in transplastomic tobacco and from the tobacco rrn promoter in transplastomic Arabidopsis. Our data suggest that transcription of the rrn operon depends on species-specific factors that facilitate transcription initiation by the general transcription machinery.

Arabidopsis↗
PubMed1996

Genus- and species-specific detection of Listeria monocytogenes using polymerase chain reaction assays targeting the 16S/23S intergenic spacer region of the rRNA operon.

In this study, the 16S/23S rRNA intergenic spacer (IGS) regions of six Listeria species were examined. DNA bands of 590 and 340 bp were observed following polymerase chain reaction (PCR) amplification of DNA from Listeria monocytogenes, Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria ivanovii strains with generic rRNA IGS oligonucleotide primers. For strains of Listeria grayi subspp. grayi and murrayi, DNA band sizes of 550 and 340 bp were observed with this primer pair. DNA bands of these sizes were not observed for other Gram-negative or- positive bacteria in this PCR assay. Four RsaI digestion profiles were noted for the Listeria PCR products. Listeria monocytogenes strains had one profile; L. innocua strains had a second; L seeligeri, L. welshimeri, and L ivanovii strains had a third; and L. grayi strains had a fourth. The small and large 16S/23S rRNA IGSs of L. monocytogenes ATCC 15313 were identical in the first 58 5' and the last 169 3' nucleotides. However, the large rRNA IGS contained a central 267-bp region with tRNA(Ile) and tRNA(Ala) genes. Large rRNA 16S/23S IGS nucleotide sequence data has not been previously reported. This data was used to develop novel Listeria genus-specific and L.monocytogenes species-specific PCR assays.

Base Sequence↗
PubMed1995

Characterization of the unlinked 16S rDNA and 23S-5S rRNA operon of Wolbachia pipientis, a prokaryotic parasite of insect gonads.

The rRNA-encoding genes (rDNAs) have been cloned and characterized from Wolbachia pipientis (Wp), the gonadial bacteria-like parasite of the mosquito Culex pipiens (Cp) and the moth Ephestia cautella (Ec). In Wp from both insect species the rDNAs are organized in a way which appears to be very unusual. The rRNAs are encoded by two unlinked transcription units, each present in a single copy per genome. One contains the 16S rDNA only, while the other is an operon encoding both the 23S and 5S rDNAs. Each transcription unit contains two putative upstream promoters, and downstream a Rho-independent terminator. The 16S rDNA, as well as the 23S-5S rRNA operon are not linked to any tRNA-encoding sequence and lack the antitermination boxes which are usually present immediately downstream from eubacterial promoters of rDNAs. Wp infecting Ec and Cp are highly similar taking as criteria the rDNAs and their flanking sequences. However, it clearly appears that each insect species harbours a different and specific Wp strain, or even subspecies. Phylogenetic relationships deduced from the complete sequences of their rDNAs undoubtedly confirm that Wp from Cp and Ec belong to the alpha-group of Proteobacteria, and are closely related to the Rickettsia.

Animals↗
PubMed1986

Analysis of transcription and processing signals of the 16S-23S rRNA operon of Mycoplasma hyopneumoniae.

The 16S and 23S rRNA genes of Mycoplasma hyopneumoniae are closely spaced in one operon. The two genes are separated by a spacer region of 500 bp which shows no sequence homology to bacterial tRNA genes. Within this operon seven 5' and five 3' ends of various rRNA species were mapped and the corresponding DNA was sequenced. The results are consistent with the following model for synthesis of rRNAs: Transcription of the operon is initiated from either of two tandemly arranged promoters leading to a large precursor RNA consisting of both 16S and 23S rRNAs. This primary transcript is first cleaved within stem structures surrounding the two rRNAs to yield premature 16S and 23S rRNAs. By further processing events the mature 5' and 3' ends are generated. The promoter sequences of this operon differ from those of other eubacterial promoters in lacking the typical -35 region. The putative termination site at the 3' end of the operon is reminiscent of rho-independent terminators in Escherichia coli.

Base Sequence↗
PubMed1994

Separate pathways for excision and processing of 16S and 23S rRNA from the primary rRNA operon transcript from the hyperthermophilic archaebacterium Sulfolobus acidocaldarius: similarities to eukaryotic rRNA processing.

In the hyperthermophilic archaebacterium Sulfolobus acidocaldarius, the mature 16S and 23S rRNA are generated by processing of a 5000-nucleotide transcript. Analysis of intermediates that accumulate in vivo indicates that the transcript contains 11 separate processing sites. The processing and maturation of 23S rRNA appears to follow the typical archaebacterial pathway, utilizing a bulge-helix-bulge motif within the 23S processing helix as the substrate for an excision endonuclease. The precursor 23S rRNA that is released is trimmed at its 5' and 3' ends to generate the mature 23S rRNA found in 50S ribosomal subunits. The pathway for processing and maturation of 16S rRNA is distinctive and does not use the bulge-helix-bulge motif in the 16S processing stem. Instead, the transcript is cleaved at several novel positions in the 5' leader and in the 3' intercistronic sequence. The excised precursor 16S is trimmed at the 5' end but an extra 60 nucleotides of what is normally spacer sequence is retained at the 3' end. The elongated 16S rRNA is present in active 30S subunits. An in vitro processing system for the 16S rRNA has been established. The RNA substrate containing the entire 144-nucleotide 5' leader and the first 72 nucleotides of 16S sequence is cleaved at the same positions observed in vivo by an endonuclease activity present in cell extract. These results demonstrate (i) that the 16S processing helix is neither utilized nor required for leader processing, and (ii) that complete maturation to the 5' end of 16S rRNA can occur in the absence of concomitant ribosome assembly and in the absence of all but the first 72 nucleotides of the 16S rRNA sequence. The endonuclease activity responsible for cleavage of the 5' leader substrate is sensitive to nuclease digestion, suggesting that it contains an essential RNA component. The cleavage sites appear to be located within regions of irregular secondary structure and have a consensus sequence of GAUUCC.

Bacterial Proteins↗
PubMed2004

The pathogen Mycobacterium marinum, a faster growing close relative of Mycobacterium tuberculosis, has a single rRNA operon per genome.

Although Mycobacterium marinum and Mycobacterium tuberculosis are very closely related they differ significantly in their growth rates. The Type strain of M. marinum and one clinical isolate were investigated and, like M. tuberculosis, were found to have a single rRNA (rrn) operon per genome located downstream from murA gene and controlled by two promoters. No sequence differences were found that account for the difference in the growth rates of the two species. We infer that M. tuberculosis has the capacity to synthesize rRNA much faster than it actually does; and propose that the high number of insertion sequences in this species attenuate growth rate to lower values.

Base Sequence↗
PubMed1983

Differential stringent control of the tandem E. coli ribosomal RNA promoters from the rrnA operon expressed in vivo in multicopy plasmids.

The tandem P1, P2 promoter region of the rrnA ribosomal operon has been fused to the t1, t2 terminator region of the rrnB operon in pBR322 plasmid derivatives. This deletes most internal RNA structural elements ordinarily processed out of ribosomal operon transcripts. In vivo as well as in vitro transcripts arising from both promoters terminate predominantly in the t1 terminator region about 40 base pairs beyond the mature rrnB 5S RNA gene. Stringent control of the P1 and P2 promoted transcripts has been assessed in vivo. In these plasmid fusions, the upstream (P1) promoter activity was subject to stringent control, while the downstream (P2) promoter activity was inhibited by amino acid starvation in both stringent and relaxed hosts. A plasmid with an additional deletion of the P2 region also showed stringent regulation of the P1 promoter.

Amino Acids↗
PubMed1996

The rRNA operons of Mycobacterium smegmatis and Mycobacterium tuberculosis: comparison of promoter elements and of neighbouring upstream genes.

Mycobacterium smegmatis has two rRNA (rrn) operons designated rrnAf and rrnBf. Appropriate restriction fragments of genomic DNA containing sequences immediately upstream from the 16S rRNA genes were cloned. We now report the nucleotide sequence of 552 bp upstream from the 5'-end of the Box AL antitermination element of the leader region of the rrnAf operon. The 5'-end of this segment of DNA was found to comprise 113 codons of an ORF encoding a protein which is significantly similar to UDP-N-acetylglucosamine 1-carboxyvinyl-transferase (EC 2.5.1.7), which is important to cell wall synthesis. A homologous ORF is located immediately upstream from the single rrn (rrnAs) operons of Mycobacterium tuberculosis and Mycobacterium leprae. Primer-extension analysis of the RNA fraction of M. smegmatis revealed four products which were related to transcription start points; the rrnBf operon appears to have a single promoter whereas the rrnAf operon has three (P1, P2 and P3). Analysis of M. tuberculosis RNA revealed two products corresponding to transcripts directed by promoters homologous with P1 and P3 of the rrnAf of M. smegmatis. Thus, the promoter and upstream regions of the rrnAf operon of M. smegmatis and the rrnAs operon of M. tuberculosis are homologous. The presence of P2 in M. smegmatis and its absence from M. tuberculosis is attributable to insertions/deletions of 97 bp.

Amino Acid Sequence↗
PubMed2004

Characterization of Paenibacillus popilliae rRNA operons.

The terminal 39 nucleotides on the 3' end of the 16S rRNA gene, along with the complete DNA sequences of the 5S rRNA, 23S rRNA, tRNA(Ile), and tRNA(Ala) genes were determined for Paenibacillus popilliae using strains NRRL B-2309 and Dutky 1. Southern hybridization analysis with a 16S rDNA hybridization probe and restriction-digested genomic DNA demonstrated 8 copies of the 16S rRNA gene in P. popilliae strains KLN 3 and Dutky 1. Additionally, the 23S rRNA gene in P. popilliae strains NRRL B-2309, KLN 3, and Dutky 1 was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to occur as 8 copies. It was concluded that these 3 P. popilliae strains contained 8 rrn operons. The 8 operon copies were preferentially located on approximately one-half of the chromosome and were organized into 3 different patterns of genes, as follows: 16S-23S-5S, 16S-ala-23S-5S, and 16S-5S-ile-ala-23S-5S. This is the first report to identify a 5S rRNA gene between the 16S and 23S rRNA genes of a bacterial rrn operon. Comparative analysis of the nucleotides on the 3' end of the 16S rRNA gene suggests that translation of P. popilliae mRNA may occur in Bacillus subtilis and Escherichia coli.

Bacillus subtilis↗
PubMed2005

Construction of a combined physical and genetic map of the chromosome of Lactobacillus acidophilus ATCC 4356 and characterization of the rRNA operons.

The combination of PFGE and hybridization approaches was used to study the genome of Lactobacillus acidophilus neotype strain ATCC 4356. PFGE analysis of chromosomal DNA after digestion with each of the rare-cutting restriction enzymes I-CeuI, NotI, CspI, SmaI, ApaI and SgrAI allowed the size of the circular chromosome of L. acidophilus to be estimated at 2.061 Mbp. The physical map contained 86 restriction sites for the six enzymes employed, with intervals between the sites varying from 1 to 88 kbp (approximately 0.05-4.3 % of the chromosome). Based on the physical map, a genetic map was constructed via Southern blot analyses of L. acidophilus DNA using specific gene probes. A total of 73 probes representing key genes, including 12 rRNA (rrn) genes, were positioned on the latter map. Mapping analysis also indicated the presence of four rrn operons (rrnA-D) on the chromosome, each containing a single copy of each of the three rrn genes 16S (rrl), 23S (rrs) and 5S (rrf). Operon rrnD was inverted in orientation with respect to the others and contained a long 16S-23S intergenic spacer region with tRNAIle and tRNAAla genes, whereas the other operons contained a short spacer lacking any tRNA genes. The high-resolution physical/genetic map constructed in this study provides a platform for genomic and genetic studies of Lactobacillus species and for improving industrial and probiotic strains.

Chromosome Mapping↗