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PubMed1992

FIS-dependent trans activation of stable RNA operons of Escherichia coli under various growth conditions.

In Escherichia coli transcription of the tRNA operon thrU (tufB) and the rRNA operon rrnB is trans-activated by the protein FIS. This protein, which stimulates the inversion of various viral DNA segments, binds specifically to a cis-acting sequence (designated UAS) upstream of the promoter of thrU (tufB) and the P1 promoter of the rrnB operon. There are indications that this type of regulation is representative for the regulation of more stable RNA operons. In the present investigation we have studied UAS-dependent transcription activation of the thrU (tufB) operon in the presence and absence of FIS during a normal bacterial growth cycle and after a nutritional shift-up. In early log phase the expression of the operon rises steeply in wild-type cells, whereafter it declines. Concomitantly, a peak of the cellular FIS concentration is observed. Cells in the stationary phase are depleted of FIS. The rather abrupt increase of transcription activation depends on the nutritional quality of the medium. It is not seen in minimal medium. After a shift from minimal to rich medium, a peak of transcription activation and of FIS concentration is measured. This peak gets higher as the medium gets more strongly enriched. We conclude that a correlation between changes of the UAS-dependent activation of the thrU (tufB) operon and changes of the cellular FIS concentration under a variety of experimental conditions exists. This correlation strongly suggests that the production of FIS responds to environmental signals, thereby trans-activating the operon. Cells unable to produce FIS (fis cells) also show an increase of operon transcription in the early log phase and after a nutritional shift-up, albeit less pronounced than that wild-type cells. Presumably it is controlled by the ribosome feedback regulatory system. cis activation of the operon by the upstream activator sequence is apparent in the absence of FIS. This activation is constant throughout the entire growth cycle and is independent of nutritional factors. The well-known growth rate-dependent control, displayed by exponentially growing cells studied under various nutritional conditions, is governed by two regulatory mechanisms: repression, presumably by ribosome feedback inhibition, and stimulation by trans activation. FIS allows very fast bacterial growth.

Carrier Proteins↗

PubMedDate not supplied

Transfer of Nosema locustae (Microsporidia) to Antonospora locustae n. comb. based on molecular and ultrastructural data.

Nosema locustae is a microsporidian parasite of grasshopper pests that is used as a biological control agent, and is one of the emerging model systems for microsporidia. Due largely to its diplokaryotic nuclei, N. locustae has been classified in the genus Nosema, a large genus with members that infect a wide variety of insects. However, some molecular studies have cast doubt on the validity of certain Nosema species, and on the taxonomic position of N. locustae. To clarify the affinities of this important insect parasite we sequenced part of the rRNA operon of N. locustae and conducted a phylogenetic analysis using the complete small subunit rRNA gene. Nosema locustae is only distantly related to the nominotypic N. bombycis, and is instead closely related to Antonospora scoticae, a recently described parasite of bees. We examined the ultrastructure of mature N. locustae spores, and found the spore wall to differ from true Nosema species in having a multi-layered exospore resembling that of Antonospora (one of the distinguishing features of that genus). Based on both molecular and morphological evidence, therefore, we propose transferring N. locustae to the genus Antonospora, as Antonospora locustae n. comb.

Animals↗

PubMed1982

A pseudogene cluster in the leader region of the Euglena chloroplast 16S-23S rRNA genes.

The nucleotide sequence of a region (leader region) preceding the 5'-end of 16S-23S rRNA gene region of Euglena gracilis chloroplast DNA was compared with the homologous sequences that code for the 16S-23S rRNA operons of Euglena and E. coli. The leader region shows close homology in sequence to the 16S-23S rRNA gene region of Euglena (Orozco et al. (1980) J. Biol.Chem. 255, 10997-11003) as well as to the rrnD operon of E. coli, suggesting that it was derived from the 16S-23S rRNA gene region by gene duplication. It was shown that the leader region had accumulated nucleotide substitutions at an extremely rapid rate in its entirety, similar to the rate of tRNAIle pseudogene identified in the leader region. In addition, the leader region shows an unique base content which is quite distinct from those of 16S-23S rRNA gene regions of Euglena and E. coli, but again is similar to that of the tRNAIle pseudogene. The above two results strongly suggest that the leader region contains a pseudogene cluster which was derived from a gene cluster coding for the functional 16S-23S rRNA operon possibly by imperfect duplication during evolution of Euglena chloroplast DNA.

Base Sequence↗

PubMed1984

Mycoplasmal ribosomal RNA genes and their use as probes for detection and identification of Mollicutes.

The number and organization of ribosomal RNA (rRNA) genes in the genome of Mycoplasma, Ureaplasma, Acholeplasma and Spiroplasma species were studied by the Southern hybridization technique. Restriction endonuclease-digested DNAs of the organisms were hybridized with nick-translated probes consisting of defined portions of the rrnB rRNA operon of Escherichia coli and with a recombinant plasmid pMC5 constructed of pBR325 and an insert containing M. capricolum genes for 23S, 5S and most of the 16S rRNA gene. The hybridization data indicate the presence of only one or two sets of rRNA genes in the mollicutes tested, a number lower than in eubacteria. The rRNA genes in mollicutes appear to be organized as clusters (acting apparently as operons) in the typical prokaryotic fashion, 5'-16S-23S-5S-3'. Despite the marked sequence homology shared by the rRNA operons of the different mollicutes and of E. coli, the operons are not identical. Thus, there is an EcoRI restriction site in the 16S rRNA genes in only 8 of the 13 species tested. The recombinant plasmid pMC5 has provided a sensitive probe for detection and identification of mollicutes in contaminated cell cultures. The purified DNA of the tested cell culture, or its supernatant fluid, was digested by EcoRI, and Southern blot hybridization of the products was performed with nick-translated pMC5. The probe did not hybridize with eukaryotic DNA. Each of the mollicutes species examinated exhibited a species-specific hybridization pattern. The hybridization tests enabled the identification of the four most prevalent mycoplasma contaminants of cell cultures, M. orale, M. hyorhinis, M. arginini and A. laidlawii. The test is capable of detecting 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 10(5) mycoplasmas. The possibility of using this approach for detection and identification of noncultivable mycoplasmas in plant and insect tissues is under investigation.

Animals↗

PubMed2001

Genetic diversity and evolution of Mycoplasma capricolum subsp. capripneumoniae strains from eastern Africa assessed by 16S rDNA sequence analysis.

Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae), the causal agent of contagious caprine pleuropneumonia (CCPP), is a member of the so-called Mycoplasma mycoides cluster. These mycoplasmas have two rRNA operons in which intraspecific variations have been demonstrated. The sequences of the 16S rRNA genes of both operons from 13 field strains of M. capripneumoniae from three neighbouring African countries (Kenya, Ethiopia, and Tanzania) were determined. Four new and unique polymorphism patterns reflecting the intraspecific variations were found. Two of these patterns included length differences between the rrnA and rrnB operons. The length difference in one of the patterns was caused by a two-nucleotide insert (TG) in the rrnB operon and the length difference in the other pattern was due to a three-nucleotide deletion, also in the rrnB operon. Another pattern was characterised by a polymorphic position caused by a mutation that is known to cause streptomycin resistance in other bacterial species. The strain with this pattern was also found to be resistant to streptomycin. Streptomycin resistant clones were selected from four M. capripneumoniae strains to further investigate the correlation of this mutation to streptomycin resistance. Mutations in the 16S rRNA genes had occurred in two of these strains. The fourth pattern included a new polymorphism in position 1059. The results show that polymorphisms in M. capripneumoniae strains can be used as epidemiological markers for CCPP in smaller geographical areas and to study the molecular evolution of this species.

Animals↗

PubMed2000

Mutations at position A960 of E. coli 23 S ribosomal RNA influence the structure of 5 S ribosomal RNA and the peptidyltransferase region of 23 S ribosomal RNA.

The proximity of loop D of 5 S rRNA to two regions of 23 S rRNA, domain II involved in translocation and domain V involved in peptide bond formation, is known from previous cross-linking experiments. Here, we have used site-directed mutagenesis and chemical probing to further define these contacts and possible sites of communication between 5 S and 23 S rRNA. Three different mutants were constructed at position A960, a highly conserved nucleotide in domain II previously crosslinked to 5 S rRNA, and the mutant rRNAs were expressed from plasmids as homogeneous populations of ribosomes in Escherichia coli deficient in all seven chromosomal copies of the rRNA operon. Mutations A960U, A960G and, particularly, A960C caused structural rearrangements in the loop D of 5 S rRNA and in the peptidyltransferase region of domain V, as well as in the 960 loop itself. These observations support the proposal that loop D of 5 S rRNA participates in signal transmission between the ribosome centers responsible for peptide bond formation and translocation.

Aldehydes↗

PubMed1998

Studies on the large subunit rRNA genes and their flanking regions of Leuconostocs.

The 16S-23S (spacer-1) and 23S-5S (spacer-2) rRNA intergenic spacer regions of Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc mesenteroides subsp. dextranicum, and Leuconostoc mesenteroides subsp. cremoris were amplified by polymerase chain reactions and sequenced. The 23S rRNA genes of Leuconostoc lactis, Leuconostoc mesenteroides, and Leuconostoc mesenteroides subsp. dextranicum were also sequenced. The RNase III-like and RNase E processing sites, as well as putative antitermination signals, were identified within the spacer regions. A single tRNA(Ala) gene without the 3'-terminal CCA sequence was found in spacer-1 regions. Secondary structure models are proposed showing interactions between the two spacer regions of leuconostocs. For all strains studied, spacer-1 and spacer-2 were highly conserved and therefore could not be directly used for strain typing. Sequence information on 23S rRNA genes from Leuconostoc species allowed the determination of regions that can be used as targets for diagnostic probes and amplification primers. Secondary structures of variable helical elements of leuconostocs 23S rRNA were constructed and their primary structures were compared with those of several Gram-positive bacteria with low G+C contents. Comparative analysis revealed that restriction analysis of 23S rRNA variable regions appeared to be sufficient for the search for species-specific signatures. Our experimental observations revealed that one form of the rRNA operons was present in leuconostocs. We have also demonstrated the direct linkage between the three species of rRNA genes, which are organized as follows: 5'-16S rRNA-spacer-1-tRNA(Ala)-23S rRNA-spacer-2-5S rRNA-3'.

Base Sequence↗

PubMed1997

The actinomycete Thermobispora bispora contains two distinct types of transcriptionally active 16S rRNA genes.

Here we present the first description of the presence of two distinct types of 16S rRNA genes in the genome of a (eu)bacterium, Thermobispora bispora. We cloned and determined the nucleotide sequences of all four rRNA operons of T. bispora. Sequence comparisons revealed that the genome of T. bispora contains two distinct types of 16S rRNA genes, each type consisting of two identical or nearly identical copies, and three identical copies of the 23S RNA gene. The nucleotide sequences of the two types of 16S rRNA genes differ at 98 nucleotide positions (6.4% of total nucleotides) together with six regions of deletion-insertions. None of the base substitutions or insertion-deletions corresponds to any of the approximately 600 evolutionarily invariable or rarely variable nucleotides, indicating that both genes are functional. Both types of 16S rRNA genes are transcribed and processed as determined by Northern (RNA) hybridization and reverse transcriptase-mediated PCR.

Actinomycetales↗

PubMed1998

Homologous recombination between rrn operons rearranges the chromosome in host-specialized species of Salmonella.

Partial digestion with I-CeuI, which digests bacterial DNA at the gene coding for the large subunit rRNA, established the rrn genomic skeleton (the distance in kb between rRNA operons) in 56 strains of Salmonella, from Salmonella Reference B (SARB) set. All had seven I-CeuI sites, indicating seven rrn operons. The order of I-CeuI fragments was ABCDEFG in S. typhimurium LT2 and in 31 other species, mostly host-generalists; in S. typhi, S. paratyphi C, S. gallinarum, and S. pullorum (host-specialized species), these fragments are rearranged, due to homologous recombination between the rrn operons. Rearrangements, such as inversions and translocations not involving the rrn operons, are rare. I-CeuI fragments of some species are larger than the norm, suggesting the insertion of unique blocks of DNA by lateral transfer from other species.

Chromosomes, Bacterial↗

PubMed2000

A sensitive method to detect initiation of growth in Streptococcus gordonii using ribosomal RNA operon-reporter gene fusions.

A system for studying the early growth response of Streptococcus gordonii to environmental stimuli has been developed. A reporter gene, encoding alpha-amylase, has been integrated into an rRNA operon to monitor changes in cellular physiology associated with the initiation of growth. Two such strains with single integrants have been characterized during the transition from lag phase to exponential growth. Synthesis of the reporter is correlated to growth initiation in both strains, and the reporter enzyme is detectable with sufficient sensitivity. Comparison of the expression profiles of the two rrn operons containing the reporter gene suggests that they are differentially expressed over the course of growth.

Artificial Gene Fusion↗

PubMed1990

Localization and structural analysis of the ribosomal RNA operons of Rhodobacter sphaeroides.

We have identified, cloned and sequenced the three ribosomal RNA (rRNA) operons (rrn) present in the facultative photoheterotroph Rhodobacter sphaeroides. DNA sequence analysis has identified the 16S, 23S, and 5S rRNAs, two tRNAs (ile and ala) in the spacer region between the 16S and 23S rRNAs, and an f-met tRNA immediately following the 5S rRNA gene of all three operons. Physical mapping, genetic analysis, and Southern hybridization data indicate that rrnA is contained on a large chromosome and rrnB and rrnC are contained on a second smaller chromosome. These findings are discussed in relation to the origins of diploidy.

Base Sequence↗

PubMed1989

Genes encoding 5S rRNA and tRNAs in the extremely thermophilic archaebacterium Methanothermus fervidus.

Methanothermus fervidus was shown to have two 5S rRNA-encoding genes linked in rRNA operons to 16S and 23S rRNA-encoding genes. Sequencing of a cloned 5S rRNA gene confirmed that M. fervidus is a member of the Methanobacteriales, although its 5S rRNA is also similar in both primary sequence and predicted secondary structure to the 5S rRNA of the non-methanogenic, but also extremely thermophilic archaebacterium, Thermococcus celer. Two clusters of tRNA genes have also been cloned and sequenced form M. fervidus. The smaller cluster, cloned in pET5401, is composed of 5'-tRNA(UGUThr)-tRNA(UGGPro)-tRNA(GUCAsp)-tRNA(UUUL ys)-3' and the larger cluster, cloned in pET5475, is composed of 5'-tRNA(GUUAsn)-tRNA(CAUMet)-tRNA(UUCGlu)-tRNA(UAGL eu)-tRNA(GUGHis)-3'. The encoded tRNAs, with the exception of the tRNA(Leu), translate abundant codons in M. fervidus. The tRNA genes do not contain introns or encode 3'-terminal CCA residues. Homologous clusters of tRNA genes have been sequenced from Methanococcus vannielii and Methanococcus voltae, so that comparisons of transcription signals, gene organizations and primary sequences can be made and features possibly related to thermostability identified. During evolution, a 5S rRNA gene appears to have been incorporated into the cluster of tRNA genes in the methanococci but not in M. fervidus.

Amino Acid Sequence↗

PubMed1997

Copy number of the 16S rRNA gene in Coxiella burnetii.

Coxiella burnetii is an obligate intracellular bacterium with a doubling time of 5-7 hours. Chromosomal DNA from C. burnetii was digested with various restriction enzymes previously determined to not cut within the genomic 16S rRNA gene, or with a combination of these noncutting enzymes in conjunction with AflIII, a restriction enzyme that cuts twice within the 16S rRNA gene. Restriction fragments were resolved electrophoretically and probed with a radiolabeled DNA fragment containing the 3' AflIII portion of the C. burnetii 16S rRNA gene. Only a single DNA fragment in these digests hybridized to the probe, indicating that there is a single genomic copy of the 16S rRNA gene in C. burnetii and thus only a single copy of the rRNA operon.

Coxiella burnetii↗

PubMed1994

The ribosomal RNA (rrn) operons of fast-growing mycobacteria: primary and secondary structures and their relation to rrn operons of pathogenic slow-growers.

The two ribosomal RNA (rrn) operons (rrnA and rrnB) of Mycobacterium smegmatis were investigated. The leader regions, part of the 16S rRNA genes, the spacer-1 regions, part of the 23S rRNA genes, and the spacer-2 regions were amplified by PCR or by inverse PCR and the products were cloned and sequenced. No differences in the sequences of the two operons were detected downstream from the Box A antitermination element of the leader region. Upstream from Box A a slow-grower-like Box B antitermination element was found in rrnA but not in rrnB. Primer extension experiments revealed that the start of transcription lies at least 370 nucleotides upstream from the 5'-end of the 16S rRNA gene and an RNase processing site near to the Box A element. Secondary structures were deduced for pre-16S rRNA and pre-23S rRNA which are distinct from, but closely related to, the corresponding structures of slow-growing mycobacteria. On the basis of these results it is proposed that the emergence of the slow-growers from the main mycobacterial line was coincident with the deletion of a segment of DNA spanning an rrnB-like operon, leaving an rrnA-like operon as the sole source of rRNA. An explanation is also proposed for the need for two Box A motifs in the transcription of an rrn operon based on competition between the polymerase and the nascent 30S subunit for either protein S10 and/or Box A sequences.

Base Sequence↗

PubMed2003

Fis stabilizes the interaction between RNA polymerase and the ribosomal promoter rrnB P1, leading to transcriptional activation.

It has been shown that Fis activates transcription of the ribosomal promoter rrnB P1; however, the mechanism by which Fis activates rrnB P1 transcription is not fully understood. Paradoxically, although Fis activates transcription of rrnB P1 in vitro, transcription from the promoter containing Fis sites (as measured from rrnB P1-lacZ fusions) is not reduced in a fis null mutant strain. In this study, we further investigated the mechanism by which Fis activates transcription of the rrnB P1 promoter and the role of Fis in rRNA synthesis and cell growth in Escherichia coli. Like all other stringent promoters investigated so far, open complex of rrnB P1 has been shown to be intrinsically unstable, making open complex stability a potential regulatory step in transcription of this class of promoters. Our results show that Fis acts at this regulatory step by stabilizing the interaction between RNA polymerase and rrnB P1 in the absence of NTPs. Mutational analysis of the Fis protein demonstrates that there is a complete correlation between Fis-mediated transcriptional activation of rrnB P1 and Fis-mediated stabilization of preinitiation complexes of the promoter. Thus, our study indicates that Fis-mediated stabilization of RNA polymerase-rrnB P1 preinitiation complexes, presumably at the open complex step, contributes prominently to transcriptional activation. Furthermore, our in vivo results show that rRNA synthesis from the P1 promoters of several rRNA operons are reduced 2-fold in a fis null mutant compared with the wild type strain, indicating that Fis plays an important role in the establishment of robust rRNA synthesis when E. coli cells are emerging from a growth-arrested phase to a rapid growth phase. Thus, our results resolve an apparent paradox of the role of Fis in vitro and in vivo in the field.

Cell Division↗

PubMed2002

Relatedness of chromosomal and plasmid DNAs of Erwinia pyrifoliae and Erwinia amylovora.

The plant pathogen Erwinia pyrifoliae has been classified as a separate species from Erwinia amylovora based in part on differences in molecular properties. In this study, these and other molecular properties were examined for E. pyrifoliae and for additional strains of E. amylovora, including strains from brambles (Rubus spp.). The nucleotide composition of the internal transcribed spacer (ITS) region was determined for six of the seven 16S-23S rRNA operons detected in these species with a 16S rRNA gene probe. Each species contained four operons with a tRNA(Glu) gene and two with tRNA(Ile) and tRNA(Ala) genes, and analysis of the operons from five strains of E. amylovora indicated a high degree of ITS variability among them. One tRNA(Glu)-containing operon from E. pyrifoliae Ep1/96 was identical to one in E. amylovora Ea110, but three tRNA(Glu) operons and two tRNA(Ile) and tRNA(Ala) operons from E. pyrifoliae contained unique nucleotide changes. When groEL sequences were used for species-specific identification, E. pyrifoliae and E. amylovora were the closest phylogenetic relatives among a set of 12 bacterial species. The placement of E. pyrifoliae distinct from E. amylovora corroborated molecular hybridization data indicating low DNA-DNA similarity between them. Determination of the nucleotide sequence of plasmid pEP36 from E. pyrifoliae Ep1/96 revealed a number of presumptive genes that matched genes previously found in pEA29 from E. amylovora and similar organization for the genes and origins of replication. Also, pEP36 and pEA29 were incompatible with clones containing the reciprocal origin regions. Finally, the ColE1-like plasmid pEP2.6 from strain Ep1/96 contained sequences found in small plasmids in E. amylovora strains IL-5 and IH3-1.

Chaperonin 60↗

PubMed2001

Structure, expression and products of the ribosomal RNA operons of Rhodopseudomonas palustris No. 7.

Rhodopseudomonas palustris strains carry one or two ribosomal rRNA operons, and those with duplicated rrn operons grow faster. The two rrn operons in R. palustris No. 7 are virtually identical over a 54,70-bp stretch containing the genes for 16S rRNA, tRNAile, tRNAala, 23S rRNA and 5S rRNA, as well as the intergenic spacers and part of the extragenic spacer. In R. palustris, unlike most bacteria with multiple rrn operons, the putative promoter sequences of the two operons are highly diverged, suggesting possible functional differentiation. By simultaneous primer-extension analysis of both pre-rRNAs, we detected a two-fold higher level of expression from rrnA under photoautotrophic conditions. Alteration of the conditions of growth leads to changes in the relative levels of expression of the two operons. Within the 5,470-bp segment, only two sequence differences are found between the 23S rRNA genes; one is at the center of the 23S rRNA molecule and affects a site of unknown function, and the other is within or immediately adjacent to sequences involved in processing of the 5' 23S rRNA IVS. In vitro processing of 5' IVS-containing 23S rRNA precursors from each operon does not reveal any detectable difference between them. The 5' ends of the mature 16S, 23S, and 5S rRNAs were determined by primer-extension analysis, and the 3' end of 23S rRNA was determined by RNA linker ligation-mediated cDNA cloning. The 5' and 3' ends of the R. palustris 23S rRNA molecule are extensively processed, suggesting that, unlike the situation in the established eubacterial model, these ends cannot basepair.

Base Sequence↗

PubMed2003

Products transcribed from rearranged rrn genes of Escherichia coli can assemble to form functional ribosomes.

To examine the flexibility of rRNA operons with respect to fundamental organization, transcription, processing, and assembly of ribosomes, operon variations were introduced by a plasmid into an Escherichia coli strain that has deletions of all chromosomal copies of rRNA genes. In the reconstructed operons, a Salmonella intervening sequence (IVS) from 23S helix 45 was introduced into the E. coli 23S gene at the same position. Three different constructs of the E. coli 16S gene were then placed wholly within the IVS sequence, and the 16S gene was deleted from its normal position. The resulting plasmids thus had the normal operon promoters and the leader region followed by the 5' one-third of the 23S gene, the entire 16S gene within the IVS, the last two-thirds of the 23S gene, and the normal end of the operon. The three constructs differed in the amount of 16S leader and spacer regions they contained. Only two of the three constructs, those with redundant leader and spacer antiterminator signals, resulted in viable cultures of the rrn deletion strain. Electron micrographs of the variant operon suggest that the 23S rRNA is made in two separate parts which then must form subassemblies before assembling into a functional 50S subunit. Cells containing only the reshuffled genes were debilitated in their growth properties and ribosome contents. The fact that such out of the ordinary manipulation of rRNA sequences in E. coli is possible paves the way for detailed analysis of ribosome assembly and evolution.

DNA, Ribosomal Spacer↗