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PubMed1988

Molecular cloning and characterization of an rRNA operon in Streptomyces lividans TK21.

The number of rRNA genes in Streptomyces lividans was examined by Southern hybridization. Randomly labeled 23 and 16S rRNAs were hybridized with BamHI, BglII, PstI, SalI, or XhoI digests of S. lividans TK21 DNA. BamHi, BglII, SalI and XhoI digests yielded six radioactive bands each for the 23 and 16S rRNAs, whereas PstI digests gave one band for the 23S rRNA and one high-intensity band and six low-density bands for the 16S rRNA. The 7.4-kilobase-pair BamHI fragment containing one of the rRNA gene clusters was cloned into plasmid pBR322. The hybrid plasmid, pSLTK1, was characterized by physical mapping, Southern hybridization, and electron microscopic analysis of the R loops formed between pSLTK1 and the 23 and 16S rRNAs. There were at least six rRNA genes in S. lividans TK21. The 16 and 23S rRNA genes were estimated to be about 1.40 and 3.17 kilobase pairs, respectively. The genes for the rRNAs were aligned in the sequence 16S-23S-5S. tRNA genes were not found in the spacer region or in the context of the rRNA genes. The G + C content of the spacer region was calculated to be approximately 58%, in contrast to 73% for the chromosome as a whole.

Base Sequence↗
PubMed2000

rRNA operon copy number reflects ecological strategies of bacteria.

Although natural selection appears to favor the elimination of gene redundancy in prokaryotes, multiple copies of each rRNA-encoding gene are common on bacterial chromosomes. Despite this conspicuous deviation from single-copy genes, no phenotype has been consistently associated with rRNA gene copy number. We found that the number of rRNA genes correlates with the rate at which phylogenetically diverse bacteria respond to resource availability. Soil bacteria that formed colonies rapidly upon exposure to a nutritionally complex medium contained an average of 5.5 copies of the small subunit rRNA gene, whereas bacteria that responded slowly contained an average of 1.4 copies. In soil microcosms pulsed with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), indigenous populations of 2,4-D-degrading bacteria with multiple rRNA genes ( = 5.4) became dominant, whereas populations with fewer rRNA genes ( = 2.7) were favored in unamended controls. These findings demonstrate phenotypic effects associated with rRNA gene copy number that are indicative of ecological strategies influencing the structure of natural microbial communities.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2005

Comparative and functional analysis of the rRNA-operons and their tRNA gene complement in different lactic acid bacteria.

The complete genome sequences of the lactic acid bacteria (LAB), Lactobacillus plantarum, Lactococcus lactis, and Lactobacillus johnsonii were used to compare location, sequence, organisation, and regulation of the ribosomal RNA (rrn) operons. All rrn operons of the examined LAB diverge from the origin of replication, which is compatible with their efficient expression. All operons show a common organisation of 5'-16S-23S-5S-3' structure, but differ in the number, location and specificity of the tRNA genes. In the 16S-23S intergenic spacer region, two of the five rrn operons of Lb. plantarum and three of the six of Lb. johnsonii contain tRNA-ala and tRNA-ile genes, while L. lactis has a tRNA-ala gene in all six operons. The number of tRNA genes following the 5S rRNA gene ranges up to 14, 16, and 21 for L. lactis, Lb. johnsonii and Lb. plantarum, respectively. The tRNA gene complements are similar to each other and to those of other bacteria. Micro-heterogeneity was found within the rRNA structural genes and spacer regions of each strain. In the rrn operon promoter regions of Lb. plantarum and L. lactis marked differences were found, while the promoter regions of Lb. johnsonii showed a similar tandem promoter structure in all operons. The rrn promoters of L. lactis show either a single or a tandem promoter structure. All promoters of Lb. plantarum contain two or three -10 and -35 regions, of which either zero to two were followed by an UP-element. The Lb. plantarum rrnA, rrnB, and rrnC promoter regions display similarity to the rrn promoter structure of Esherichia coli. Differences in regulation between the five Lb. plantarum promoters were studied using a low copy promoter-probe plasmid. Taking copy number and growth rate into account, a differential expression over time was shown. Although all five Lb. plantarum rrn promoters are significantly different, this study shows that their activity was very similar under the circumstances tested. An active promoter was also identified within the Lb. plantarum rrnC operon preceding a cluster of 17 tRNA genes.

Amino Acid Sequence↗
PubMed1997

Partial sequence of the rRNA operon of Pneumocystis carinii isolates from Puerto Rico.

Several reports indicate geographic variation of isolates of Pneumocystis carinii hominis. We have sequenced the internal transcribed spacer (ITS) region and large subunit Group I intron of rRNA genes from P. carinii DNA obtained from two patients from Puerto Rico. Both can be subclassified as Type II, according to the sequence of the ITS region. A system capable of identifying individual isolates will be an essential tool for epidemiological studies of the organism. The amplification of DNA from fixed tissues may facilitate the processing of a large number of samples.

Base Sequence↗
PubMed1985

Identification of an rRNA operon promoter from Zea mays chloroplasts which excludes the proximal tRNAValGAC from the primary transcript.

The transcriptional start site of the tRNA operon from Zea mays chloroplasts has ben identified by a combination of S1 mapping and Southern hybridization with in vitro capped chloroplast RNA as radioactive probe. This is the first example in which the transcriptional start site of a chloroplast gene has ben established by identification of the triphosphate-bearing RNA terminus. The start site is located at position -117 proximal to the 16S rRNA gene and is preceded by -10 and -35 sequences homologous to prokaryotic promoters. The primary transcript directed by this promoter does not include tRNAValGAC sequences which are coded further upstream between positions -302 and -231. A major processing site of the primary rRNA transcript was identified at position -30 which is embedded in a secondary structure typical for prokaryotic RNase III processing sites. Several putative processing and start sites of the tRNAValGAC transcripts have been mapped by primer extension with reverse transcriptase.

Base Sequence↗
PubMed2002

Sequence diversity of the internal transcribed spacer (ITS) region of the rRNA operons among different serogroups of Legionella pneumophila isolates.

The genus Legionella is represented by 48 species and Legionella pneumophila includes 15 serogroups. In this work, we have studied the intergenic 16S-23S spacer region (ITS) in L. pneumophila to determine the feasability of using amplification polymorphisms in this region, to establish intraspecies differences and to discriminate Legionella species. The amplification of this region, using 16S14F and 23S0R primers, and the analysis of amplicons by the analysis of fragments technique showed that all the L. pneumophila serogroups studied presented the same electrophoretic pattern. Moreover, the analysis of different Legionella species showed that the amplification polymorphisms obtained were species-specific. In order to study the sequence variability of this region, the existence in L. pneumophila of three ribosomal operons was determined by pulsed field gel eletrophoresis (PFGE). Two of the 16S-23S rRNA ITS presented a tRNA Ala and the third one a tRNA Ile. Nevertheless, the variability expected in this region of the operon was not found and the rest of the ITS contained only punctual mutations.

Base Sequence↗
PubMed1999

Physical and genetic map of Streptococcus mutans GS-5 and localization of five rRNA operons.

The physical map of the 2.1 megabase chromosome of Streptococcus mutans GS-5 has been refined by including all ApaI and SmaI fragments of 5 kbp or greater, and by positioning the fragments generated by the endonuclease I-CeuI. Sixty-three new genetic loci have been added to the map, so that it now contains 90 loci. The new loci include those for 35 cloned streptococcal genes of established function and for 23 S. mutans genes of putative function. In addition, five rrn operons were identified and placed on the map of the chromosome. The presence of a SmaI site in each of the rrn operons allowed the direction of transcription of each operon to be deduced. The orientation of the rrn loci indicates that their transcription is directed away from a small region of the chromosome, identifying a possible region for the initiation of chromosome replication.

Blotting, Southern↗
PubMed1998

Recombination between rRNA operons created most of the ribotype variation observed in the seventh pandemic clone of Vibrio cholerae.

Individual rrn operons and their flanking regions have been analysed in a study of the molecular basis of ribotype variation in the seventh pandemic clone of Vibrio cholerae. The genome of an early isolate of the seventh pandemic clone had nine rrn operons of which two were in tandem with other rrn operons. The site for BglI, the most discriminatory enzyme used for ribotyping, was found to be present in the 16S sequence of three of the operons of the earliest isolate. This site was observed to be gained or lost in specific operons in many later isolates, presumably by recombination, and this gave most of the ribotype variation. Additional rrn recombination events were uncovered by analysis of the 16S-23S intergenic spacers associated with each operon. Spacers of 431, 509, 607 and 711 bp were found. A total of at least eight rrn recombination events were detected. Three rrn loci were primarily involved in this recombination, with four new forms generated from that in the early strains for operon B and two new forms each for operons C and G. In addition there was variation due to deletion of tandem operons. The frequency of recombination between rrn operons was very high as there were nine new ribotypes found among 47 isolates sampled over the 33 year period of study. This means that any variation could undergo precise reversion by the same recombination event within the time frame covered by the study. Recombination between rrn operons may be a factor in ribotype variation in all systems. The recombination observed is thought to be that which results in concerted evolution and the data give an indication of the rate involved.

Bacterial Typing Techniques↗
PubMed2001

Ralstonia paucula (Formerly CDC group IV c-2): unsuccessful strain differentiation with PCR-based methods, study of the 16S-23S spacer of the rRNA operon, and comparison with other Ralstonia species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum).

Ralstonia paucula (formerly CDC group IV c-2) can cause serious human infections. Confronted in 1995 with five cases of nosocomial bacteremia, we found that pulsed-field gel electrophoresis could not distinguish between the isolates and that randomly amplified polymorphic DNA analysis was poorly discriminatory. In this study, we used PCR-ribotyping and PCR-restriction fragment length polymorphism analysis of the spacer 16S-23S ribosomal DNA (rDNA); both methods were unable to differentiate R. paucula isolates. Eighteen strains belonging to other Ralstonia species (one R. eutropha strain, six R. pickettii strains, three R. solanacearum strains, and eight R. gilardii strains) were also tested by PCR-ribotyping, which failed to distinguish between the four species. The 16S-23S rDNA intergenic spacer of R. paucula contains the tRNA(Ile) and tRNA(Ala) genes, which are identical to genes described for R. pickettii and R. solanacearum.

Bacterial Typing Techniques↗
PubMed1983

Structure and organization of rRNA operons in the region of the replication origin of the Bacillus subtilis chromosome.

Structure and organization of two complete ribosomal RNA (rRNA) gene sets, rrnO and rrnA, were determined for the first time in Bacillus subtilis. They are located at the region of the replication origin of the chromosome. Each set constitutes a single operon of: two tandem promoters - leader sequence - 16S rRNA gene - Ile-tRNA gene - Ala-tRNA gene - 23S rRNA gene - 5S rRNA gene - termination signal. The first promoter (P1) of rrnO differs from that of rrnA in sequence and function. P1 of rrnO was used very little for transcription either in vivo or in vitro while P1 was predominantly used in rrnA. A putative transcript of the entire operon was determined and constructed into a secondary structure. Analysis of in vivo transcripts by S1 mapping revealed primary processing sites at the loop and stem structure of 16S rRNA in rrnO and rrnA. A unique sequence in the leader region of rrnO can be formed into a highly complexed secondary structure and affects processing of mature 16S rRNA. The sequences of the two spacer tRNA genes are highly conserved between B. subtilis and Escherichia coli.

Bacillus subtilis↗
PubMed1997

Inter- and intraspecies comparison of the 16S-23S rRNA operon intergenic spacer regions of six Listeria spp.

The 16S-23S rRNA intergenic spacer (IGS) regions found in six Listeria species were characterized. PCR amplification of the 16S-23S IGS with a "generic primer" set generated products of about 340 bp (small) and 550 to 590 bp (large) with DNA from all Listeria strains tested. Seven Listeria monocytogenes serotype 4b strains and one L. monocytogenes serotype 4d strain also had an additional PCR product of ca. 360 bp. The 360-bp PCR product from one of these L. monocytogenes serotype 4b strains was identical in nucleotide sequence to the small 340-bp IGS, except that it contained an 18-bp tandem repeat. The small rRNA IGSs of L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi were 83 to 99% homologous to that of L. monocytogenes. The large rRNA IGS of L. monocytogenes was 81 to 96% homologous to those of the other Listeria species and agreed with current taxonomic division among these species. The nucleotide sequences of the central 274 bp of the large rRNA IGS of strains from seven different L. monocytogenes serotypes were highly homologous; however, serotype-specific differences were noted, and four groups were identified within L. monocytogenes based on this analysis.

Animals↗
PubMed1988

Chromosomal organization of rRNA operons in Bacillus subtilis.

Integrative mapping with vectors containing ribosomal DNA sequences were used to complete the mapping of the 10 rRNA gene sets in the endospore forming bacterium Bacillus subtilis. Southern hybridizations allowed the assignment of nine operons to distinct BclI restriction fragments and their genetic locus identified by transductional crosses. Nine of the ten rRNA gene sets are located between 0 and 70 degrees on the genomic map. In the region surrounding cysA14, two sets of closely spaced tandem clusters are present. The first (rrnJ and rrnW) is located between purA16 and cysA14 closely linked to the latter; the second (rrnI, rrnH and rrnG) previously mapped within this area is located between attSPO2 and glpT6. The operons at or near the origin of replication (rrnO,rrnA and rrnJ,rrnW) represent "hot spots" of plasmid insertion.

Bacillus subtilis↗
PubMed2005

Transcriptional polarity in rRNA operons of Escherichia coli nusA and nusB mutant strains.

Synthesis of ribosomes in Escherichia coli requires an antitermination system that modifies RNA polymerase to achieve efficient transcription of the genes specifying 16S, 23S, and 5S rRNA. This modification requires nucleotide signals in the RNA and specific transcription factors, such as NusA and NusB. Transcription of rrn operons in strains lacking the ability to produce either NusA or NusB was examined by electron microscopy. The distribution and numbers of RNA polymerase molecules on rrn operons were determined for each mutant. Compared to the wild type, the 16S gene in the nusB mutant strain had an equivalent number of RNA polymerase molecules, but the number of RNA polymerase molecules was reduced 1.4-fold for the nusA mutant. For both mutant strains, there were twofold-fewer RNA polymerase molecules on the 23S RNA gene than for the wild type. Overall, the mutant strains each had 1.6-fold-fewer RNA polymerase molecules on their rrn operons than did the wild type. To determine if decreased transcription of the 23S gene observed by electron microscopy also affected the 30S/50S ribosomal subunit ratio, ribosome profiles were examined by sucrose gradient analysis. The 30S/50S ratio increased 2.5- to 3-fold for the nus mutant strains over that for wild-type cells. Thus, strains carrying either a nusA mutation or a nusB mutation have defects in transcription of 23S rRNA.

Centrifugation, Density Gradient↗
PubMed1990

Potential binding sites of the trans-activator FIS are present upstream of all rRNA operons and of many but not all tRNA operons.

FIS, the Escherichia coli protein that stimulates the inversion of various DNA segments by binding to a recombinational enhancer, trans-activates a number of stable RNA operons and binds to the upstream activator sequence (UAS) of these operons (Nilsson et al. (1990) EMBO J. 9, 727). In a search for potential FIS-binding sites we have compared UASs of other stable RNA operons with a consensus FIS-binding sequence, compiled by comparing recombinational enhancers. Such sites can thus be recognized upstream of all rRNA and 13 tRNA operons. Matching with the consensus sequence varied, suggesting that the affinity of FIS for the sites differed. Accordingly, FIS binding to an upstream sequence of the metY(nusA) operon was found to be weaker than that to the UAS of the thrU(tufB) operon. No FIS binding sites were found upstream three tRNA operons.

Anticodon↗
PubMed1992

'boxA'-like sequence between the 16 S/23 S spacer in rRNA operon of mycoplasmas.

We have found that a boxA-like sequence is conserved in the 16 S and 23 S rRNA intergenic spacer regions of mycoplasmas, and that it always locates on loop regions of the hypothetical secondary stem-loop structures. A nucleotide sequence similar to the '-10' box of prokaryotic promoters was identified at upstream sites of the boxA-like sequence in the 16 S/23 S spacer regions. These structures may represent an internal promoter between the 16 S and 23 S rRNA genes in mycoplasmas.

Base Sequence↗
PubMed1998

Sequence diversity in the 16S-23S intergenic spacer region (ISR) of the rRNA operons in representatives of the Escherichia coli ECOR collection.

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

Base Sequence↗