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PubMed2004

Intragenomic heterogeneity and intergenomic recombination among haloarchaeal rRNA genes.

More than one copy of rRNA operons, which code for both the small-subunit (SSU) and large-subunit (LSU) rRNA, are often found in prokaryotes. It is generally assumed that all rRNA operons within a single cell are almost identical. A notable exception is the extremely halophilic archaeal genus Haloarcula, most species of which are known to harbor highly divergent rRNA operons that differ at approximately 5% of the nucleotide positions in the SSU gene and at 1 to 2% of the nucleotide positions in the LSU gene. We report that such intragenomic heterogeneity is not unique to Haloarcula, as high levels of intragenomic sequence variation have been observed for the SSU genes of two other genera of extreme halophiles, Halosimplex and Natrinema. To investigate this in detail, the two rRNA operons of Halosimplex carlsbadense and the four operons of Natrinema sp. strain XA3-1 were cloned and completely sequenced. The SSU and LSU genes of H. carlsbadense show the highest levels of intragenomic heterogeneity observed so far in archaea (6.7 and 2.6%). The operons of Natrinema sp. strain XA3-1 have additional unusual characteristics, such as identical internal transcribed spacers, while one of four SSU genes is 5% divergent and all LSU genes differ from each other by 0.9 to 1.9%. The heterogeneity among the Natrinema sp. strain XA3-1 LSU genes is localized in hot spots, and one of these regions is shown to be the result of a recombination event with a distantly related halophile. This is the first example of interspecies recombination between rRNA genes in archaea, and the recombination occurred over one of the largest phylogenetic distances ever reported for such an event. We suggest that intragenomic heterogeneity of rRNA operons is an ancient and stable trait in several lineages of the Halobacteriales. The impact of this phenomenon on the taxonomy of extremely halophilic archaea is discussed.

Base Sequence↗

PubMed1997

Strategies used by pathogenic and nonpathogenic mycobacteria to synthesize rRNA.

One rRNA operon of all mycobacteria studied so far is located downstream from a gene thought to code for the enzyme UDP-N-acetylglucosamine carboxyvinyl transferase (UNAcGCT), which is important to cell wall synthesis. This operon has been designated rrnAf for fast-growing mycobacteria and rrnAs for slow growers. We have investigated the upstream sequences and promoter activities of rrnA operons of typical fast growers which also possess a second rrn (rrnBf) operon and of the rrnA operons of the fast growers Mycobacterium abscessus and Mycobacterium chelonae, which each have a single rrn operon per genome. These fast growers have a common strategy for increasing the efficiency of transcription of their rrnA operons, thereby increasing the cells' potential for ribosome synthesis. This strategy involves the use of multiple (three to five) promoters which may have arisen through successive duplication events. Thus we have identified a hypervariable multiple promoter region (HMPR) located between the UNAcGCT gene and the 16S rRNA coding region. Two promoters, P1 and PCL1, appear to play pivotal roles in mycobacterial rRNA synthesis; they are present in all of the species examined and are the only promoters used for rRNA synthesis by the pathogenic slow growers. P1 is located within the coding region of the UNAcGCT gene, and PCL1 has a characteristic sequence that is related to but distinct from that of the additional promoters. In fast-growing species, P1 and PCL1 produce less than 10% of rRNA transcripts, so the additional promoters found in the HMPR are important in increasing the potential for rRNA synthesis during rapid growth. In contrast, rrnB operons appear to be regulated by a single promoter; because less divergence has taken place, rrnB appears to be younger than rrnA.

Base Sequence↗

PubMed1998

Molecular evolution of Mycoplasma capricolum subsp. capripneumoniae strains, based on polymorphisms in the 16S rRNA genes.

Mycoplasma capricolum subsp. capripneumoniae belongs to the so-called Mycoplasma mycoides cluster and is the causal agent of contagious caprine pleuropneumonia (CCPP). All members of the M. mycoides cluster have two rRNA operons. The sequences of the 16S rRNA genes of both rRNA operons from 20 strains of M. capricolum subsp. capripneumoniae of different geographical origins in Africa and Asia were determined. Nucleotide differences which were present in only one of the two operons (polymorphisms) were detected in 24 positions. The polymorphisms were not randomly distributed in the 16S rRNA genes, and some of them were found in regions of low evolutionary variability. Interestingly, 11 polymorphisms were found in all the M. capricolum subsp. capripneumoniae strains, thus defining a putative ancestor. A sequence length difference between the 16S rRNA genes in a poly(A) region and 12 additional polymorphisms were found in only one or some of the strains. A phylogenetic tree was constructed by comparative analysis of the polymorphisms, and this tree revealed two distinct lines of descent. The nucleotide substitution rate of strains within line II was up to 50% higher than within line I. A tree was also constructed from individual operonal 16S rRNA sequences, and the sequences of the two operons were found to form two distinct clades. The topologies of both clades were strikingly similar, which supports the use of 16S rRNA sequence data from homologous operons for phylogenetic studies. The strain-specific polymorphism patterns of the 16S rRNA genes of M. capricolum subsp. capripneumoniae may be used as epidemiological markers for CCPP.

Africa↗

PubMed1998

Chromosomal rearrangements in enteric bacteria.

Early genetic studies showed conservation of gene order in the enteric bacteria. Two recent methods using pulsed field gel electrophoresis (PFGE) to determine the physical map of the genome are: (i) partial digestion with the endonuclease I-CeuI, which digests the DNA of bacteria in the rrn operon for rRNA (ribosomal RNA), thus establishing the "rrn genomic skeleton" (the size in kbp of the intervals between rRNA operons); (ii) analysis of XbaI and B1nI sites within Tn10 insertions in the chromosome. The order of I-CeuI fragments, which is ABCDEFG in S. typhimurium LT2 and E. coli K-12, was found to be conserved in most Salmonella species, most of which grow in many hosts (host-generalists). However, in S. typhi, S. paratyphi C, S. gallinarum, and S. pullorum, species which are host-specialized, these fragments are rearranged, due to homologous recombination between the rrn operons, resulting in translocations and inversions. Inversions and translocations not involving the rrn operons are seldom detected except for inversions over the TER (termination of replication) region. Additive genetic changes (due to lateral transfer resulting in insertion of nonhomologous DNA) have resulted in "loops" containing blocks of DNA which provide new genes to specific strains, thus driving rapid evolution of new traits.

Chromosomes, Bacterial↗

PubMed1997

The role of ribosomal RNAs in macrolide resistance.

Macrolides are bacteriostatic antibiotics which interfere with the peptidyltransfer function of the ribosome. We have investigated the molecular mechanisms underlying macrolide resistance in Mycobacterium smegmatis, an eubacterium carrying two rRNA operons. Surprisingly, drug resistance was associated not with alterations in ribosomal proteins, but with a single point mutation in the peptidyltransferase region of one of the two 23S RNA genes, i.e. A2058-->G or A2059-->G. This mutation resulted in a heterozygous organism with a mutated and a wild-type rRNA operon respectively. Reverse transcriptase sequencing indicated the expression of both wild-type and mutated rRNAs. The mutated operon was introduced into genetically engineered rrn- strains of M. smegmatis carrying a single functional rRNA operon and into parental M. smegmatis with two chromosomal rRNA operons, using gene transfer as well as gene replacement techniques. The results obtained demonstrate the dominant nature of resistance. As exemplified in our results on macrolide resistance, a complete set of genetic tools is now available, which allows questions of dominance vs. recessivity and gene dosage effects in eubacterial ribosomal nucleic acids to be addressed experimentally in vivo.

Alleles↗

PubMed1999

Structure and organization of the rrnD operon of 'Brevibacterium lactofermentum': analysis of the 16S rRNA gene.

Five rRNA operons (rrn) were found by hybridization in the genome of 'Brevibacterium lactofermentum' ATCC 13869 and Corynebacterium glutamicum ATCC 13032. 'B. lactofermentum' DSM 20412 differed from the other corynebacteria tested in showing six hybridizing BamHI bands. Two of the rrn operons (rrnD and rrnE) were located in a single cosmid. Sequencing of the rrnD operon showed that it contains a complete 16S rRNA-23S RNA-5S rRNA gene cluster. Phylogenetic studies using the complete 16S rRNA sequence showed that 'B. lactofermentum' is closely related to several species of the genus Corynebacterium but only distantly related to the type species Brevibacterium linens and the authors suggest that it should be reclassified as Corynebacterium lactofermentum. The 5' end of mature 16S rRNA was identified by primer extension. Sequence elements similar to those of mycobacteria implicated in transcription antitermination (Boxes A, B, C) and in processing of the pre-rRNA to 16S rRNA were identified. An open reading frame encoding an rpoD-like sigma factor (named SigC) different from the previously reported SigA and SigB proteins was found upstream of rrnD in the opposite orientation. Both rpoD and sigC seem to be expressed from a bidirectional promoter region.

Amino Acid Sequence↗

PubMed2003

Analysis of conserved non-rRNA genes of Tropheryma whipplei.

The causative agent of Whipple's disease, Tropheryma whipplei, is a slow-growing bacterium that remains poorly-understood. Genetic characterization of this organism has relied heavily upon rRNA sequence analysis. Pending completion of a complete genome sequencing effort, we have characterized several conserved non-rRNA genes from T. whipplei directly from infected tissue using broad-range PCR and a genome-walking strategy. Our goals were to evaluate its phylogenetic relationships, and to find ways to expand the strain typing scheme, based on rDNA sequence comparisons. The genes coding for the ATP synthase beta subunit (atpD), elongation factor Tu (tuf), heat shock protein GroEL (groEL), beta subunit of DNA-dependent RNA polymerase (rpoB), and RNase P RNA (rnpB) were analyzed, as well as the regions upstream and downstream of the rRNA operon. Phylogenetic analyses with all non-rRNA marker molecules consistently placed T. whipplei within the class, Actinobacteria. The arrangement of genes in the atpD and rpoB chromosomal regions was also consistent with other actinomycete genomes. Tandem sequence repeats were found upstream and downstream of the rRNA operon, and downstream of the groEL gene. These chromosomal sites and the 16S-23S rRNA intergenic spacer regions were examined in the specimens of 11 patients, and a unique combination of tandem repeat numbers and spacer polymorphisms was found in each patient. These data provide the basis for a more discriminatory typing method for T. whipplei.

Actinomycetales↗

PubMed2001

Organization of ribosomal RNA genes from a Loofah witches' broom phytoplasma.

Using the technique of integrative mapping with three vectors carrying chromosomal rDNA sequences, one of two rRNA operons of loofah witches' broom (LfWB) phytoplasma was constructed. This is the first complete rRNA operon of a phytoplasma to be reported. The operon has a context of 5'-16S-23S-5S-3' with a tRNA(Ile) gene in the ITS and tRNA(Val) and tRNA(Asn) genes downstream from the 5S rRNA gene. Although the other operon has not been cloned, the DNA sequence of a PCR-amplified product shows that it has no tRNA(Ile) gene in the ITS region. The complete nucleotide sequences of 16S, 23S, and 5S rDNA are 1538, 2864, and 113 bp, respectively. Five -10-like sequences, but no -35 sequences, were found within a 494-bp leader region. There was a TG dinucleotide two nucleotides upstream from each -10-like sequence. The existence of a TG dinucleotide at this position has been reported to enhance the efficiency of a promoter without a -35 region. The regions immediately flanking the 5' and 3' ends of 16S and 23S rDNA can form long basepaired stems that contain sites for processing by RNase III. No obvious sequence for a rho-dependent or rho-independent termination site was found downstream from the tRNA(Asn) gene. The transcription may stop within a pyrimidine-rich region, as has been reported for several polypeptide-encoding genes and rRNA operons of archaeobacteria. The presence of the tRNA genes downstream from the 5S rRNA gene in the rRNA operon of LfWB phytoplasma further supports the hypothesis that phytoplasmas are phylogenetically closer to acholeplasmas than to mycoplasmas. The phylogenetic relatedness of LfWB phytoplasma to other phytoplasmas is discussed on the basis of the nucleotide sequence of rRNA genes and ITS.

3' Untranslated Regions↗

PubMed1995

Effects of the antiterminator BoxA on transcription elongation kinetics and ppGpp inhibition of transcription elongation in Escherichia coli.

It has been shown previously that two different mRNA chains (lacZ and infB) are elongated at a rate of approximately 40 nucleotides (nt)/s during steady state growth on minimal medium and that the rate of mRNA chain elongation is inhibited by ppGpp in vivo. On the other hand, it was found that a truncated ribosomal RNA chain was elongated at a rate of approximately 80 nt/s, independent of growth condition (Vogel, U., and Jensen, K. F. (1994) J. Biol. Chem. 269, 16236-16241). We reasoned that the different transcriptional behavior of mRNA genes and rRNA operons might be caused by the antiterminator sequences present in the rRNA operons. To test this possibility, we have (a) inserted the minimal antiterminator boxA sequence between the promoter and the lacZ and infB genes and (b) deleted the antiterminator sequences from the rRNA transcription unit and measured transcription elongation rates in vivo on the resulting hybrid genes. We found that insertion of boxA in front of the coding region of lacZ increased the transcription elongation rate from 42 nt/s to 69 nt/s during steady state growth and that it eliminated the ppGpp-dependent decrease in the transcription elongation rate during the stringent response. On the other hand, deletion of the antiterminator sequences from the rRNA operon resulted in a reduced transcription elongation rate, but the elongation rate was still insensitive to changes in the ppGpp pool. These results are consistent with the hypothesis that the antiterminator boxA is a primary determinant of the rate of transcription elongation rate.

Base Sequence↗

PubMed1994

Ribotyping as an epidemiologic tool for Escherichia coli.

Restriction fragment length polymorphism of ribosomal RNA genes was analysed among 133 Escherichia coli strains predominantly from blood and urine, including 21 isolates from faeces of healthy persons. The strains had also been characterized for their O:K:H serotypes, for the presence of P, S and type 1C fimbriae, non-P, non-S mannose-resistant haemagglutinins and haemolysin production. Hind III-digested genomic DNA was subjected to Southern blot analysis with either plasmid pKK3535 containing E. coli rRNA operon or purified rRNA as a probe. Among the 133 strains 20 ribotypes were obtained. The distribution of strains into different ribotypes generally correlated with their O:K:H serotype. Ribotype variation within serotypes was mainly seen among strains with the K5 capsule. The origin of the strains or the presence of virulence-associated factors did not correlate with the ribotype. In conclusion, ribotyping appears to be a valuable method in epidemiologic studies especially when the serotyping methods are not available.

Bacterial Adhesion↗

PubMed1998

Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3.

The complete sequence of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3, has been determined by assembling the sequences of the physical map-based contigs of fosmid clones and of long polymerase chain reaction (PCR) products which were used for gap-filling. The entire length of the genome was 1,738,505 bp. The authenticity of the entire genome sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA. As the potential protein-coding regions, a total of 2061 open reading frames (ORFs) were assigned, and by similarity search against public databases, 406 (19.7%) were related to genes with putative function and 453 (22.0%) to the sequences registered but with unknown function. The remaining 1202 ORFs (58.3%) did not show any significant similarity to the sequences in the databases. Sequence comparison among the assigned ORFs in the genome provided evidence that a considerable number of ORFs were generated by sequence duplication. By similarity search, 11 ORFs were assumed to contain the intein elements. The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 46 tRNA genes including two with the intron structure. All the assigned ORFs and RNA coding regions occupied 91.25% of the whole genome. The data presented in this paper are available on the internet at http:@www.nite.go.jp.

Chromosomes, Archaeal↗

PubMed1984

Level of rRNA, not tRNA, synthesis controls transcription of rRNA and tRNA operons in Escherichia coli.

We have recently proposed a model for the negative feedback control of rRNA and tRNA synthesis in Escherichia coli by products of rRNA operons or their derivatives (e.g., nontranslating ribosomes) (S. Jinks-Robertson, R.L. Gourse, and M. Nomura, Cell 33:865-876, 1983). In this paper, we examined the following questions. (i) Are the spacer tRNAs carried within rRNA operons the products responsible for the regulation of rRNA and tRNA transcription? (ii) Are tRNAs capable of regulating their own syntheses? We measured tRNA accumulations in cells containing plasmids with intact or defective rRNA operons or with tRNA operons. From the results obtained, we conclude that neither the tRNAs encoded within rRNA operons nor the tRNAs encoded in non-rRNA operons are capable of controlling rRNA or tRNA transcription. Therefore, the products responsible for the initial step leading to rRNA and tRNA regulation are rRNAs (or their derivatives).

Aspartate-tRNA Ligase↗

PubMed2001

Is the 16S-23S rRNA internal transcribed spacer region a good tool for use in molecular systematics and population genetics? A case study in cyanobacteria.

We amplified, TA-cloned, and sequenced the 16S-23S internal transcribed spacer (ITS) regions from single isolates of several cyanobacterial species, Calothrix parietina, Scytonema hyalinum, Coelodesmium wrangelii, Tolypothrix distorta, and a putative new genus (isolates SRS6 and SRS70), to investigate the potential of this DNA sequence for phylogenetic and population genetic studies. All isolates carried ITS regions containing the sequences coding for two tRNA molecules (tRNA and tRNA). We retrieved additional sequences without tRNA features from both C. parietina and S. hyalinum. Furthermore, in S. hyalinum, we found two of these non-tRNA-encoding regions to be identical in length but different in sequence. This is the first report of ITS regions from a single cyanobacterial isolate not only different in configuration, but also, within one configuration, different in sequence. The potential of the ITS region as a tool for studying molecular systematics and population genetics is significant, but the presence of multiple nonidentical rRNA operons poses problems. Multiple nonidentical rRNA operons may impact both studies that depend on comparisons of phylogenetically homologous sequences and those that employ restriction enzyme digests of PCR products. We review current knowledge of the numbers and kinds of 16S-23S ITS regions present across bacterial groups and plastids, and we discuss broad patterns congruent with higher-level systematics of prokaryotes.

Base Sequence↗

PubMed1995

Mapping of genes involved in macromolecular synthesis on the chromosome of Streptomyces coelicolor A3(2).

The genes for the beta, beta', and seven sigma factor subunits of RNA polymerase, for elongation factors EF-Tu1 and EF-Tu3, and for six rRNA operons were mapped on the combined genetic and physical map of the Streptomyces coelicolor chromosome. Like the previously mapped tRNA genes, the RNA polymerase and rRNA genes map to scattered positions. The lack of rRNA operons in the immediate vicinity of the origin of replication (oriC) and the absence of tRNA genes in any of the rRNA operons are novel features of the Streptomyces chromosome.

Chromosome Mapping↗

PubMed2006

A genetic model to investigate drug-target interactions at the ribosomal decoding site.

Recent advances in X-ray crystallography have greatly contributed to the understanding of the structural interactions between aminoglycosides and the ribosomal decoding site. Efforts to genetically probe the functional relevance of proposed drug-nucleotide contacts have in part been hampered by the presence of multiple rRNA operons in most bacteria. A derivative of the Gram-positive Mycobacterium smegmatis was rendered single rRNA operon allelic by means of gene inactivation techniques. In this system, genetic manipulation of the single chromosomal rRNA operon results in cells carrying homogeneous populations of mutant ribosomes. An exhaustive mutagenesis study of the ribosomal A site has been performed to define the importance of individual drug-nucleotide contacts. Mutational alterations in the M. smegmatis decoding site are discussed here, comparing the results with those obtained in other organisms. Implications for the selectivity of antimicrobial agents and for the fitness cost of resistance mutations are addressed.

Anti-Bacterial Agents↗

PubMed1984

Mycoplasmas (Mollicutes) have a low number of rRNA genes.

DNA from Mycoplasma, Ureaplasma, Acholeplasma, and Spiroplasma species digested by restriction endonucleases was hybridized with probes consisting of portions of the rrnB rRNA operon of Escherichia coli and the rRNA operon of Mycoplasma capricolum. The results indicate the presence of only one or two sets of rRNA genes in the genome of Mollicutes linked in the procaryotic fashion, 16S-23S-5S.

Acholeplasma↗

PubMed1995

Effects of different growth conditions on the in vivo activity of the tandem Escherichia coli ribosomal RNA promoters P1 and P2.

We have analyzed the relative activities of the Escherichia coli ribosomal RNA promoters P1 and P2 in vivo under different physiological conditions. Promoter efficiencies were determined by quantitative comparison of the transcript-specific primer extension products obtained from total RNA preparations. Cells were analyzed at different stages of the growth cycle, at different growth rates, and under conditions of stringent control. In addition, the rRNA gene dosage was altered by transformation with plasmids containing additional rrnD or rrnB transcription units, or rRNA operons in which one of the tandem promoters (P1) had been deleted. Under conditions of amino acid starvation (stringent control) we observed the expected strong reduction in P1-directed transcription. In contrast to the previous assumption that the P2 promoter is not regulated, we simultaneously noticed a smaller but significant repression of P2-directed transcription. In strains in which the rRNA gene dosage was increased by transformation with plasmids bearing rRNA transcription units, a similar degree of repression was observed. Repression of the P1 promoter activity was increased, however, when cells contained extra rRNA operons with P2 promoters only. As demonstrated under stringent control conditions, changes in the growth cycle also affected the activity of promoters P1 and P2. A greater proportion of P2-derived transcripts was observed when cells changed from exponential to stationary growth or if cultures were grown in minimal medium. Under steady-state, slow growth conditions (minimal medium) we obtained evidence showing that the ratio of P1/P2 transcription products is much lower for cells with extra rrnB as compared to extra rrnD operons or cells lacking extra rRNA operons, implying an operon-specific regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗

PubMed1982

Chromosomal locations of the genes for rRNA in Escherichia coli K-12.

Chromosomal locations of the seven rRNA operons in Escherichia coli K-12 were studied by digesting DNA from various merodiploid strains with SalI restriction enzyme followed by Southern gel analysis with 32P-labeled 23S rRNA as a probe. The seven unique SalI DNA fragments revealed in the autoradiograms were first correlated to the seven rRNA operons previously isolated as hybrid plasmids or transducing phages. The chromosomal locations of six (rrnA, B, C, D, E, and G) of the seven isolated operons were confirmed by increased gene dosage demonstrated in autoradiograms after Southern gel analysis of DNA from relevant merodiploid strains. The gene dosage analysis showed that the location of the remaining operon (now called rrnH) is between metD and proA. No evidence was obtained for the presence of rrnF, which was previously reported to map between aroB and malA. The chromosomal location of rrnH was confirmed by P1 transduction in the following way: a DNA fragment adjacent to rrnH was cloned into pBR322; the resulting hybrid plasmid was integrated at the homologous region of the chromosome of a polA mutant; and the ampicillin resistance marker originally carried by pBR322 was then used for mapping of the nearby rrnH by P1 transduction. A close linkage of rrnH to metD (about 60% cotransduction) was observed, and the data were consistent with the order metD-rrnH-proA. Thus, mapping of all seven rRNA operons has been completed. The present study has also determined the orientation of rrnG and rrnH and demonstrated that the direction of transcription of all the rRNA operons is identical to that of DNA replication.

Chromosome Mapping↗