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.     

Molecular cloning and comparative sequence analyses of rRNA operons in Streptomyces nodosus ATCC 14899.

The genome of Streptomyces nodosus contains six ribosomal RNA (rRNA) operons. Four of the rRNA operons; rrnB, rrnD, rrnE and rrnF were cloned. We have completely sequenced all four operons, including a region 750 base pairs (bp) upstream of the 16S rRNA gene. The three rRNA genes present in each operon were closely linked in the order 16S-23S-5S. A sequence comparison of the four operons showed more than 99% sequence similarity between the corresponding 16S and 23S rRNA genes, and more than 97% similarity between 5S rRNA genes. The sequence differences observed between 23S rRNA genes appeared to be localized in two specific regions. Substantial sequence differences were found in the region upstream of the 16S rRNA gene as well as in the internal transcribed spacers. No tRNA gene was found in the 16S-23S spacer regions.     

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.     

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.     

Precise characterization of rDNA genes by intraspecies and inter-loci comparison of rDNA sequences and biochemical analysis of ribosomal RNA molecules in Agrobacterium tumefaciens

Annotation of rRNA genes has been incomplete in Agrobacterium species although a number of Agrobacterial rDNA fragments have been sequenced. In this study, precise characterization of rRNA operons (rrn) was carried out in two biovar 1 strains, C58 and MAFF301001. Complete DNA sequencing of four rrns in MAFF301001 indicated that each operon codes for 16S, 23S and 5S rRNA as well as three tRNAs, trn(Ile), trn(Ala) and trn(Met). The genes and 16S-23S ITS of a given locus were exactly identical with those in the other three loci, except for a T-base loss in the 23S rRNA gene of rrnA and in the 5S rRNA gene of rrnB. Comparison with the four C58 rDNAs available in the DNA database indicated extensive sequence and size variations in the 23S rRNA gene, suggesting the presence of an intervening sequence (IVS). Biochemical RNA analysis, including Northern hybridization and 5' end mapping, in MAFF301001 revealed 2886-base and 2571-base precursors, two 1.3-kb major fragments, a 150-base fragment and removal of an IVS for 23S rRNA. We confirmed similar biochemical characteristics in the C58 strain. The features of rDNA detected here enable correction of previously reported information about Agrobacterial rRNAs and rRNA genes and should be useful for phylogenetic considerations.     

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.     

Nucleotide sequence of the Bacillus subtilis ribosomal RNA operon, rrnB

The primary sequence of DNA covering a complete ribosomal RNA (rRNA) operon from Bacillus subtilis, designated rrnB has been elucidated. Following a set of tandem promoters, rrnB encodes: (i) a 16S and a 23S rRNA determinant with no tRNA spacer region in between; (ii) a 5S rRNA determinant; and (iii) 21 contiguous tRNA species; before (iv) two characteristic terminator hairpins are found. More than 7 kb are included within this operon, which maps between aroG and thr5 on the B. subtilis chromosome. This represents the first report of the entire sequence of an rRNA operon from B. subtilis or from any Gram-positive organism.     

Characterization of an rRNA operon (rrnB) of Mycobacterium fortuitum and other mycobacterial species: implications for the classification of mycobacteria.

Mycobacteria are thought to have either one or two rRNA operons per genome. All mycobacteria investigated to date have an operon, designated rrnA, located downstream from the murA gene. We report that Mycobacteriun fortuitum has a second rrn operon, designated rrnB, which is located downstream from the tyrS gene; tyrS is very close to the 3' end of a gene (3-mag) coding for 3-methylpurine-DNA-glycosylase. The second rrn operon of Mycobacterium smegmatis was shown to have a similar organization, namely, 5' 3-mag-tyrS-rrnB 3'. The rrnB operon of M. fortuitum was found to have a single dedicated promoter. During exponential growth in a rich medium, the rrnB and rrnA operons were the major and minor contributors, respectively, to pre-rRNA synthesis. Genomic DNA was isolated from eight other fast-growing mycobacterial species. Samples were investigated by Southern blot analysis using probes for murA, tyrS, and 16S rRNA sequences. The results revealed that both rrnA and rrnB operons were present in each species. The results form the basis for a proposed new scheme for the classification of mycobacteria. The approach, which is phylogenetic in concept, is based on particular properties of the rrn operons of a cell, namely, the number per genome and a feature of 16S rRNA gene sequences.     

Novel arrangement of rRNA genes in Mycoplasma gallisepticum: separation of the 16S gene of one set from the 23S and 5S genes.

Large restriction fragments from the DNA of Mycoplasma gallisepticum S6 and PG31, which were prepared by digestion with BglI, BssHII, SmaI, or XhoI and which were separated by pulsed-field electrophoresis, were hybridized with probes containing most, or different parts, of an rRNA operon of Mycoplasma capricolum. The results showed that the genomes contained three widely separated rRNA loci. One locus contained genes for all three rRNA species and another contained 23S and probably 5S rRNA genes, whereas the third appeared to have only a 16S rRNA gene.     

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.     

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.