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.     

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.     

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.     

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.     

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.     

Discordant phylogenies within the rrn loci of Rhizobia

It is evident from complete genome sequencing results that lateral gene transfer and recombination are essential components in the evolutionary process of bacterial genomes. Since this has important implications for bacterial systematics, the primary objective of this study was to compare estimated evolutionary relationships among a representative set of alpha-Proteobacteria by sequencing analysis of three loci within their rrn operons. Tree topologies generated with 16S rRNA gene sequences were significantly different from corresponding trees assembled with 23S rRNA gene and internally transcribed space region sequences. Besides the incongruence in tree topologies, evidence that distinct segments along the 16S rRNA gene sequences of bacteria currently classified within the genera Bradyrhizobium, Mesorhizobium and Sinorhizobium have a reticulate evolutionary history was also obtained. Our data have important implications for bacterial taxonomy, because currently most taxonomic decisions are based on comparative 16S rRNA gene sequence analysis. Since phylogenetic placement based on 16S rRNA gene sequence divergence perhaps is questionable, we suggest that the proposals of bacterial nomenclature or changes in their taxonomy that have been made may not necessarily be warranted. Accordingly, a more conservative approach should be taken in the future, in which taxonomic decisions are based on the analysis of a wider variety of loci and comparative analytical methods are used to estimate phylogenetic relationships among the genomes under consideration.     

The organization of two rRNA (rrn) operons of the slow-growing pathogen Mycobacterium celatum provides key insights into mycobacterial evolution

The slow-growing Mycobacterium celatum is known to have two different 16S rRNA gene sequences. This study confirms the presence of two rrn operons and describes their organization. One operon (rrnA) was found to be located downstream from murA and the other (rrnB) was found downstream from tyrS. The promoter regions were sequenced, and also the intergenic transcribed spacer (ITS1 and ITS2) regions separating the 16S rRNA, 23S rRNA and 5S rRNA gene coding regions. Analysis of the RNA fraction revealed that rrnA is regulated by two (P1 and PCL1) promoters and rrnB is regulated by one (P1). These data show that the two rrn operons of M. celatum are organized in the same way as the two rrn operons of classical fast-growing mycobacteria. This information was incorporated into a phylogenetic analysis of the genus based on both 16S rRNA gene sequences and (where possible) the number of rrn operons per genome. The results suggest that the ancestral Mycobacterium possessed two (rrnA and rrnB) operons per genome and that subsequently, on two separate occasions, an operon (rrnB) was lost, leading to two clusters of species having a single operon (rrnA); one cluster includes the classical pathogens and the other includes Mycobacterium abscessus and Mycobacterium chelonae.     

Characterization of Paenibacillus popilliae rRNA operons

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

Distinct Types of rRNA Operons Exist in the Genome of the Actinomycete Thermomonospora chromogena and Evidence for Horizontal Transfer of an Entire rRNA Operon

We describe here the presence of two distinct types of rRNA operons in the genome of a thermophilic actinomycete Thermomonospora chromogena. The genome of T. chromogena contains six rRNA operons (rrn), of which four complete and two incomplete ones were cloned and sequenced. Comparative analysis revealed that the operon rrnB exhibits high levels of sequence variations to the other five nearly identical ones throughout the entire length of the operon. The coding sequences for the 16S and 23S rRNA genes differ by approximately 6 and 10%, respectively, between the two types of operons. Normal functionality of rrnB is concluded on the basis of the nonrandom distribution of nucleotide substitutions, the presence of compensating nucleotide covariations, the preservation of secondary and tertiary rRNA structures, and the detection of correctly processed rRNAs in the cell. Comparative sequence analysis also revealed a close evolutionary relationship between rrnB operon of T. chromogena and rrnA operon of another thermophilic actinomycete Thermobispora bispora. We propose that T. chromogena acquired rrnB operon from T. bispora or a related organism via horizontal gene transfer.     

A Brevibacterium lactofermentum 16S rRNA gene used as target site for homologous recombination

Genes for rRNA are highly conserved and present in multiple copies in most prokaryotic organisms increasing the number of theoretical sites for homologous recombination. They might be targets for integration events between unrelated microorganisms providing that an efficient genetic transfer is present. We have used a plasmid containing a portion of the 16S rRNA gene from the rrnD operon of Brevibacterium lactofermentum to transform the same strain resulting in non-essential inactivation of various rrn operons. Integration of the transforming DNA occurs in all cases. The system may be used to test possible gene transfer at least among closely related strains and is of great interest for integration of foreign DNA and for mapping.     

Variation of 16S-23S rRNA intergenic spacer regions (ISRs) in Acinetobacter baylyi (strain B2) isolated from activated sludge

To determine the variability of the 16S-23S rRNA intergenic spacer region (ISR) of the newly described Acinetobacter baylyi, 88 clones containing ISR amplicons were screened and 14 chosen for further analysis. Two different sized 16S-23S rRNA ISRs were distinguished comprising five variable and four conserved nucleotide blocks. The major regions of heterogeneity between the different sized ISRs were due to blocks of substitutions with unique secondary structures interspersed with nucleotide substitutions, rather than differences caused by presence or absence of tRNA genes, which is often the case. Recombination events causing shuffling of nucleotide blocks are considered the most likely explanation for the mosaic structure observed between the different copies of the ISR. Single base differences present in the long ISR (LISR) were then exploited in attempts to detect possible heterogeneity between rrn copies in Acinetobacter baylyi but variability was not detected by RFLP analysis of LISR-specific PCR products. These primers were shown to be highly specific for 3 Acinetobacter baylyi strains based on LISR sequence homogeneity.     

Sequence heterogeneities among 16S ribosomal RNA sequences, and their effect on phylogenetic analyses at the species level

We have analyzed what phylogenetic signal can be derived by small subunit rRNA comparison for bacteria of different but closely related genera (enterobacteria) and for different species or strains within a single genus (Escherichia or Salmonella), and finally how similar are the ribosomal operons within a single organism (Escherichia coli). These sequences have been analyzed by neighbor-joining, maximum likelihood, and parsimony. The robustness of each topology was assessed by bootstrap. Sequences were obtained for the seven rrn operons of E. coli strain PK3. These data demonstrated differences located in three highly variable domains. Their nature and localization suggest that since the divergence of E. coli and Salmonella typhimurium, most point mutations that occurred within each gene have been propagated among the gene family by conversions involving short domains, and that homogenization by conversions may not have affected the entire sequence of each gene. We show that the differences that exist between the different operons are ignored when sequences are obtained either after cloning of a single operon or directly from polymerase chain reaction (PCR) products. Direct sequencing of PCR products produces a mean sequence in which mutations present in the most variable domains become hidden. Cloning a single operon results in a sequence that differs from that of the other operons and of the mean sequence by several point mutations. For identification of unknown bacteria at the species level or below, a mean sequence or the sequence of a single nonidentified operon should therefore be avoided. Taking into account the seven operons and therefore mutations that accumulate in the most variable domains would perhaps increase tree resolution. However, if gene conversions that homogenize the rRNA multigene family are rare events, some nodes in phylogenetic trees will reflect these recombination events and these trees may therefore be gene trees rather than organismal trees.      

Identification of cold shock gene loci in Sinorhizobium meliloti by using a luxAB reporter transposon

Using a luxAB reporter transposon, seven mutants of Sinorhizobium meliloti were identified as containing insertions in four cold shock loci. LuxAB activity was strongly induced (25- to 160-fold) after a temperature shift from 30 to 15 degrees C. The transposon and flanking host DNA from each mutant was cloned, and the nucleic acid sequence of the insertion site was determined. Unexpectedly, five of the seven luxAB mutants contained transposon insertions in the 16S and 23S rRNA genes of two of the three rrn operons of S. meliloti. Directed insertion of luxAB genes into each of the three rrn operons revealed that all three operons were similarly affected by cold shock. Two other insertions were found to be located downstream of a homolog of the major Escherichia coli cold shock gene, cspA. Although the cold shock loci were highly induced in response to a shift to low temperature, none of the insertions resulted in a statistically significant decrease in growth rate at 15 degrees C.     

Horizontal transfer of segments of the 16S rRNA genes between species of the Streptococcus anginosus group

The nature in variation of the 16S rRNA gene of members of the Streptococcus anginosus group was investigated by hybridization and DNA sequencing. A collection of 708 strains was analyzed by reverse line blot hybridization. This revealed the presence of distinct reaction patterns representing 11 different hybridization groups. The 16S rRNA genes of two strains of each hybridization group were sequenced to near-completion, and the sequence data confirmed the reverse line blot hybridization results. Closer inspection of the sequences revealed mosaic-like structures, strongly suggesting horizontal transfer of segments of the 16S rRNA gene between different species belonging to the Streptococcus anginosus group. Southern blot hybridization further showed that within a single strain all copies of the 16S rRNA gene had the same composition, indicating that the apparent mosaic structures were not PCR-induced artifacts. These findings indicate that the highly conserved rRNA genes are also subject to recombination and that these events may be fixed in the population. Such recombination may lead to the construction of incorrect phylogenetic trees based on the 16S rRNA genes.     

Intraspecific diversity of the 23S rRNA gene and the spacer region downstream in Escherichia coli

The molecular microevolution of the 23S rRNA gene (rrl) plus the spacer downstream has been studied by sequencing of different operons from some representative strains of the Escherichia coli ECOR collection. The rrl gene was fully sequenced in six strains showing a total of 67 polymorphic sites, a level of variation per nucleotide similar to that found for the 16S rRNA gene (rrs) in a previous study. The size of the gene was highly conserved (2902 to 2905 nucleotides). Most polymorphic sites were clustered in five secondary-structure helices. Those regions in a large number of operons were sequenced, and several variations were found. Sequences of the same helix from two different strains were often widely divergent, and no intermediate forms existed. Intercistronic variability was detected, although it seemed to be lower than for the rrs gene. The presence of two characteristic sequences was determined by PCR analysis throughout all of the strains of the ECOR collection, and some correlations with the multilocus enzyme electrophoresis clusters were detected. The mode of variation of the rrl gene seems to be quite similar to that of the rrs gene. Homogenization of the gene families and transfer of sequences from different clonal lines could explain this pattern of variation detected; perhaps these factors are more relevant to evolution than single mutation. The spacer region between the 23S and 5S rRNA genes exhibited a highly polymorphic region, particularly at the 3' end.     

Ribotyping of 16S and 23S rRNA genes and organization of rrn operons in members of the bacterial genera Gemmata,Planctomyces, Thermotoga,Thermus, and Verrucomicrobium

The number of organization of rrn genes of two members of the order Planctomycetales, Planctomyces limnophilus and Gemmata obscuriglobus, as well as three species from other bacterial phyla, namely Thermotoga maritima, Thermus aquaticus and Verrucomicrobium spinosum were examined by Southern blot hybridization analysis of restricted DNA with labeled 16S- and 23S rRNAs. Ribotyping analysis revealed that two species contain unlinked 16S- and 23S rRNA genes. Planctomyces limnophilus possessed two unlinked rrn genes which were separated from each other by at least 4.3 kb, and Thermus aquaticus had to unlinked 16S and 23S rRNA genes, separated from each other by at least 2.5 kb. Gemmata obscuriglobus exhibited five genes for which the organization could as yet not be determined because of the complex hybridization patterns. In the other two species, rrn genes clustered in operons. Thermotoga maritima had a single gene for each rRNA species which were separated by not more than 1.5 kb, while Verrucomicrobium spinosum had four copies of probably linked 16S and 23S rRNA genes with a maximal distance between 16S and 23S rRNA genes of 1.3 kb.