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.     

Mapping and spacer identification of rRNA operons of Salmonella typhimurium.

The rRNA operons of Salmonella typhimurium have been characterized with respect to their map position, orientation, and type of tRNA spacer. One of the seven rrn operons was found to be linked to pheA and another was found to be linked to aroE. This information, together with published information about the other five rrn operons, shows that S. typhimurium and Escherichia coli are essentially identical in terms of the number, the map position, and the orientation of all seven operons. S. typhimurium and E. coli were also similar in that four of the rrn spacer regions code for tRNAGlu2 and three code for tRNAAla1B. However, the two species differed in that rrnD coded for tRNAGlu2 and rrnB coded for tRNAAla1B in S. typhimurium. This is the opposite of the arrangement in E. coli. We have tabulated the coordinates of the BamHI and PstI sites flanking six of the S. typhimurium rrn genes and present revisions for the coordinates of some of the E. coli sites.     

Exchange of Spacer Regions between Rrna Operons in Escherichia Coli

The Escherichia coli rRNA operons each have one of two types of spacer separating the 16S and 23S coding regions. The spacers of four operons encode tRNA(Glu2) and the other three encode both tRNA(Ile) and tRNA(Ala1B). We have prepared a series of mutants in which the spacer region of a particular rrn operon has been replaced by the opposite type. Included among these were a mutant retaining only a single copy of the tRNA(Glu2) spacer (at rrnG) and another retaining only a single copy of the tRNA(Ile)-tRNA(Ala1B) spacer (at rrnA). While both mutants grew more slowly than controls, the mutant deficient in tRNA(Glu2) spacers was more severely affected. At a frequency of 6 X 10(-5), these mutants phenotypically reverted to faster growing types by increasing the copy number of the deficient spacer. In most of these phenotypic revertants, the deficient spacer type appeared in a rrn operon which previously contained the surplus type, bringing the ratio of spacer types closer to normal. In a few cases, these spacer changes were accompanied by an inversion of the chromosomal material between the donor and recipient rrn operons. Two examples of inversion of one-half of the E. coli chromosome between rrnG and rrnH were observed. The correlation of spacer change with inversion indicated that, in these particular cases, the change was due to an intrachromatid gene conversion event accompanied by a reciprocal crossover rather than reciprocal exchange between sister chromatids.     

Loss of the spacer loop sequence from the rrnB operon in the Escherichia coli K-12 subline that bears the relA1 mutation.

A polymorphism affecting the spacer region of the rrnB rRNA operon is described. Strains from a major Escherichia coli K-12 subbranch are missing a 106-nucleotide portion of the rrnB 16S-to-23S spacer, and a 20-nucleotide sequence is found in its place. We have called this mutant operon rrnB2. The rrnB2 spacer was most probably derived from either rrnC or rrnE. This alteration of rrnB may have occurred by a recombinational exchange or by gene conversion. In the genealogy of E. coli K-12 strains, the appearance of rrnB2 is associated with the spontaneous occurrence of the first relaxed mutation, but attempts to show a selective relationship between the two mutational events have had negative results. The sequences of the rrnG and rrnC 16S-to-23S spacers have also been determined and their comparisons to the other rrn operons encoding tRNAGlu2 are presented.     

First complete nucleotide sequence and heterologous gene organization of the two rRNA operons in the phytoplasma genome

Phytoplasmas are cell-wallless Gram-positive low G + C bacteria belonging to the Mollicutes that inhabit the cytoplasm of plants and insects. Although phytoplasmas possess two ribosomal RNA (rrn) operons, only one has been fully sequenced. Here, we determined the complete nucleotide sequence of both rrn operons (designated rrnA and rrnB) of onion yellows (OY) phytoplasma. Both operons have rRNA genes organized as 5'-16S-23S-5S-3' with very highly conserved sequences; the 16S, 23S, and 5S rRNA genes are 99.9, 99.8, and 99.1% identical between the two operons. However, the organization of tRNA genes in the upstream region from 16S rRNA gene and in the downstream region from 5S rRNA gene differs markedly. Several promoter candidates were detected upstream from both operons, which suggests that both operons are functional. Interestingly, both have a tRNA(Ile) gene in the 16S-23S spacer region, while the reported rrnB operon of loofah witches' broom phytoplasma does not, indicating heterogenous gene organization of rrnB within phytoplasmas. The phytoplasma tRNA gene organization is similar to that of acholeplasmas, a closely related mollicute, and different from that of mycoplasmas, another mollicute. Moreover, the organization suggests that the rrn operons were derived from that of a related nonmollicute bacterium, Bacillus subtilis. This data should shed light on the evolutionary relationships and phylogeny of the mollicutes.     

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.     

Conservation of the mosaic structure of the four internal transcribed spacers and localisation of the rrn operons on the Streptococcus pneumoniae genome

The detection of heterogeneity of the 16S-23S ribosomal intergenic transcribed spacer (ITS) region has become rather common over the past years for identification and typing purposes of bacteria. The ITS not only varies in sequence and length, but also in number of alleles per genome and in their position on the chromosome together with the ribosomal clusters. The ITS characterisation has allowed discrimination of several species within a genus and variation in ITS sequences between the multiple rrn operons present within a genome may be as high or greater than between strains of the same species or subspecies. It is important to understand the variability of ITS sequences in a given genome to gain insights into bacterial physiology and taxonomy. The present study describes the possibility to type Streptococcus pneumoniae by PCR-ribotyping of the spacer region, the determination of the molecular structure of the ITS, and the determination of the number and localisation of rrn operons in this microorganism. Our results show that the genome of S. pneumoniae contains four ribosomal operons, showing the same genomic organisation among strains, each containing a single ITS allele of 270 bp. The ITS sequence presents a mosaic organisation of blocks highly conserved intra- and inter-species within the genus Streptococcus, giving no possibility for variations to arise.     

Rates and consequences of recombination between rRNA operons

A mutant strain of Escherichia coli was created by inserting a cassette encoding sucrose sensitivity and neomycin resistance (sacB-neo) into the small-subunit rRNA-encoding gene rrs in the rrnB operon. During growth in a complex medium, the cassette was lost from the population, and a complete rrs gene was restored at a rate of 5 x 10(-9) per cell division. Repair of this lesion required flanking regions of DNA that were similar to the six remaining intact rRNA operons and reestablished the full complement of seven rRNA operons. The relative fitness of strains with restored rrnB operons was 1 to 2% higher than that of the mutant strain. The rrnB operon normally contains a spacer region between the 16S and 23S rRNA-encoding genes that is similar in length and tRNA gene content to the spacer in rrnC, -E, and -G. In 2 of the 14 strains in which rrnB was restored, the spacer region had the same length as the spacer region in rrnA, -D, and -H. The requirement for flanking regions of nearly identical DNA and the replication of the spacer region from other rRNA operons during the repair of rrnB suggest that the restoration was accomplished via gene conversion. The rate of gene conversion was 10-fold less than the fixation of point mutations in the same region of the chromosome but was apparently sufficient to homogenize the sequences of rRNA genes in E. coli. These findings are discussed in the context of a conceptual model describing the presence of sequence heterogeneity in coevolving rRNA genes.     

Transcriptional regulation by antitermination. Interaction of RNA with NusB protein and NusB/NusE protein complex of Escherichia coli

A recombinant heterodimeric NusB/NusE protein complex of Escherichia coli was expressed under the control of a synthetic mini operon. Surface plasmon resonance measurements showed that the heterodimer complex has substantially higher affinity for the boxA RNA sequence motif of the ribosomal RNA (rrn) operons of E.coli as compared to monomeric NusB protein. Single base exchanges in boxA RNA reduced the affinity of the protein complex up to 15-fold. The impact of base exchanges in the boxA RNA on the interaction with NusB protein was studied by (1)H,(15)N heterocorrelation NMR spectroscopy. Spectra obtained with modified RNA sequences were analysed by a novel generic algorithm. Replacement of bases in the terminal segments of the boxA RNA motif caused minor chemical shift changes as compared to base exchanges in the central part of the dodecameric boxA motif.     

Structure and rearrangements of rRNA genes in chloroplast DNA in two strains of Euglena gracilis

Summary The organisation of the rRNA genes in the chloroplast genomes of two strains of Euglena gracilis were analyzed and compared. It was previously shown that the bacillaris strain contains three complete rrn (rRNA) operons (7) and that the Z-S strain contains one operon (21). Using heteroduplex analysis it was found that the bacillaris strain contains, apart from the three complete rrn operons, an extra 16S rRNA gene, an extra partial 23S rRNA gene sequence and an inverted duplication of a stretch within the 5S–16S spacer. In addition a short (<100 bp) inverted repeat sequence (13) which forms a stem/loop structure in single-stranded cpDNA was located between the 3-end of the extra 16S rRNA gene and the partial 23 S rRNA sequence.The Z-S strain differs from the bacillaris strain by a deletion of two units of the complete rrn operons. The region upstream of the single complete rrn operon, including the inverted repeats, the partial 23S and the extra 16S rRNA sequences is identical with the bacillaris strain.The only non-homology found in heteroduplexes between the SalI fragments of B of the two strains is the deletion-insertion loop which represents the two rrn operons. A small deletion loop was found occasionally in hetero-and in homoduplexes of both strands in the region of variable size. Apart from the deletion/insertion of two rrn operons the two genomes appear to be colinear as can be seen from partial denaturation mapping. The organisation of the rRNA genes of the two strains is compared with those of the Z strain and the bacillaris-ATCC strain.     

The role of recombination and mutation in 16S-23S rDNA spacer rearrangements

The intragenomic heterogeneity of the bacterial intergenic (16S-23S rDNA) spacer region (ISR) was analysed from the following species in which sequences for the complete rRNA operon (rrn) set have been determined (rrn number): Enterococcus faecalis (6) and E. faecium (6), Bacillus subtilis (10), Staphylococcus aureus (9), Vibrio cholerae (4), Haemophilus influenzae (6) and Escherichia coli (7). It was found that some spacer sequence blocks were highly conserved between operons of a genome, whereas the presence of others was variable. When these variations were analysed using the program PLATO and partial likelihood phylogenies determined by DNAml for each operon set, three regions showed significant (Z>3.3) spatial variation [Region I was 78-184 nt long (2.14.4) possibly due to recombination or selection. Within Region I, there was sequence block variation in all operon sets [some operons contained tRNA genes (tRNAala, tRNAile or tRNAglu), whereas others had sequence blocks such as VS2 (S. aureus) or rsl (E. coli)]. Q Analysis of the ISR sequence from E. faecalis and E. faecium showed that there was more interspecies than intraspecies variation (both in DNA sequence and in the presence or absence of blocks). Dot matrix analysis of the sequence blocks in the nine rrn ISRs from S. aureus showed that there was significant homology between VS2 and VS5/VS6. Furthermore, repeat motifs with only A or T were present in higher copy numbers in VS5/VS6 than in VS2. Since these sequence blocks (VS2 and VS5-VS6) are related, intragenic evolution resulting in AT expansion may have occurred between these two regions. A model is proposed that postulates a role for recombination and AT-expansion in intra-genomic ISR variations. This process may represent a general mechanism of concerted evolution for bacterial ISR rearrangements.     

A rapid PCR procedure for the specific identification of Lactobacillus sanfranciscensis, based on the 16S-23S intergenic spacer regions

AIMS: The organization of ribosomal RNA (rrn) operons in Lactobacillus sanfranciscensis was studied in order to establish an easy-to-perform method for identification of L. sanfranciscensis strains, based on the length and sequence polymorphism of the 16S-23S rDNA intergenic spacer region (ISR). METHODS AND RESULTS: PCR amplification of the 16S-23S rDNA ISRs of L. sanfranciscensis gave three products distinguishing this micro-organism from the remaining Lactobacillus species. Sequence analysis revealed that two of the rrn operons were organized as in previously reported lactobacilli: large spacer (L-ISR), containing tRNA(Ile) and tRNA(Ala) genes; small spacer (S-ISR) without tRNA genes. The third described spacer (medium, M-ISR), original for L. sanfranciscensis, harboured a tRNA-like structure. An oligonucleotide sequence targeting the variable region between tDNA(Ile) and tDNA(Ala) of L. sanfranciscensis L-ISR was approved to be suitable in species-specific identification procedure. Analysis by pulse-field gel electrophoresis of the chromosomal digest with the enzyme I-CeuI showed the presence of seven rrn clusters. Lactobacillus sanfranciscensis genome size was estimated at c. 1.3 Mb. CONCLUSIONS: Direct amplification of 16S-23S ISRs or PCR with specific primer derived from L-ISR showed to be useful for specific typing of L. sanfranciscensis. This was due to the specific rrn operon organization of L. sanfranciscensis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: In this paper, we have reported a rapid procedure for L. sanfranciscensis identification based on specific structures found in its rrn operon.