Rrn Loci Research Articles

Clustering of rRNA operons in E. coli is disrupted by σH.

Chromosomal organization in E. coli as examined by Hi-C methodology indicates that long-range interactions are sparse. Yet, spatial co-localization or 'clustering' of 6/7 ribosomal RNA (rrn) operons distributed over half the 4.6 Mbp genome has been captured by two other methodologies - fluorescence microscopy and Mu transposition. Our current understanding of the mechanism of clustering is limited to mapping essential cis elements. To identify trans elements, we resorted to perturbing the system by chemical and physical means and observed that heat shock disrupts clustering. Levels of σH are known to rise as a cellular response to the shock. We show that elevated expression of σH alone is sufficient to disrupt clustering, independent of heat stress. The anti-clustering activity of σH does not depend on its transcriptional activity but requires core-RNAP interaction and DNA-binding activities. This activity of σH is suppressed by ectopic expression of σD suggesting a competition for core-RNAP. A query of the other five known σ factors of E. coli found that elevated expression of FecI, the ECF σ factor that controls iron citrate transport, also perturbs clustering and is also suppressed by σD. We discuss a possible scenario for how these membrane-associated σ factors participate in clustering of distant rrn loci.

Bacteria can maintain rRNA operons solely on plasmids for hundreds of millions of years

It is generally assumed that all bacteria must have at least one rRNA operon (rrn operon) on the chromosome, but some strains of the genera Aureimonas and Oecophyllibacter carry their sole rrn operon on a plasmid. However, other related strains and species have chromosomal rrn loci, suggesting that the exclusive presence of rrn operons on a plasmid is rare and unlikely to be stably maintained over long evolutionary periods. Here, we report the results of a systematic search for additional bacteria without chromosomal rrn operons. We find that at least four bacterial clades in the phyla Bacteroidota, Spirochaetota, and Pseudomonadota (Proteobacteria) lost chromosomal rrn operons independently. Remarkably, Persicobacteraceae have apparently maintained this peculiar genome organization for hundreds of millions of years. In our study, all the rrn-carrying plasmids in bacteria lacking chromosomal rrn loci possess replication initiator genes of the Rep_3 family. Furthermore, the lack of chromosomal rrn operons is associated with differences in copy numbers of rrn operons, plasmids, and chromosomal tRNA genes. Thus, our findings indicate that the absence of rrn loci in bacterial chromosomes can be stably maintained over long evolutionary periods.

Genotyping of Treponema pallidum in Cuba (2018-2019): Increased Circulation of Recombinant Genotype and No New Treponema pallidum Subspecies endemicum Infection Among Syphilis Patients.

This study aimed to determine the allelic profiles of Treponema pallidum in patients confirmed with syphilis in Cuba (2018-2019) and to explore mutations leading to macrolide and tetracycline resistance. Multilocus sequence typing and polymerase chain reaction of rrn loci (23S and 16S rDNA), followed by Sanger sequencing, were used to define the allelic profile of TPA and resistance mutations, respectively. Allelic profile 1.3.1 and the recombinant profile were identified, with 15.7.3 having an increased frequency. We did not detect the presence of the T. pallidum subspecies endemicum among syphilis patients, as in previous reports. A high frequency of macrolide-resistant strains and the absence of mutations potentially causing tetracycline resistance were found. Understanding the current status of treponemal infection in Cuban patients provides insights into the syphilis epidemiology.

High Prevalence of Diverse Insertion Sequences within the rRNA Operons of Mycoplasma bovis.

Data presented in this study show a high prevalence of diverse ISs within the M. bovis rrn locus resulting in intraspecies variability and diversity. Such abundance of IS elements near or within the rrn locus may offer a selective advantage to M. bovis Moreover, the fact that expression of the rrs genes as well as the number of viable cells increased in the group of strains with IS element insertion within a putative promoter -35 sequence (configuration F) in comparison to that in strains with one or two rrn operons that do not have ISs may serve as a basis for understanding the possible role of M. bovis IS elements in fundamental biological processes such as regulation of ribosome synthesis and function.

Rapid Acquisition of Linezolid Resistance in Methicillin-Resistant Staphylococcus aureus: Role of Hypermutation and Homologous Recombination.

BackgroundWe previously reported the case of a 64-year-old man with mediastinitis caused by Staphylococcus aureus in which the infecting bacterium acquired linezolid resistance after only 14 days treatment with linezolid. We therefore investigated relevant clinical isolates for possible mechanisms of this rapid acquisition of linezolid resistance.MethodsUsing clinical S. aureus isolates, we assessed the in vitro mutation rate and performed stepwise selection for linezolid resistance. To investigate homologous recombination, sequences were determined for each of the 23S ribosomal RNA (23S rRNA) loci; analyzed sequences spanned the entirety of each 23S rRNA gene, including domain V, as well as the 16S-23S intergenic spacer regions. We additionally performed next-generation sequencing on clinical strains to identify single-nucleotide polymorphisms compared to the N315 genome.ResultsStrains isolated from the patient prior to linezolid exposure (M5-M7) showed higher-level linezolid resistance than N315, and the pre-exposure strain (M2) exhibited more rapid acquisition of linezolid resistance than did N315. However, the mutation rates of these and contemporaneous clinical isolates were similar to those of N315, and the isolates did not exhibit any mutations in hypermutation-related genes. Sequences of the 23S rRNA genes and 16S-23S intergenic spacer regions were identical among the pre- and post-exposure clinical strains. Notably, all of the pre-exposure isolates harbored a recQ missense mutation (Glu69Asp) with respect to N315; such a lesion may have affected short sequence recombination (facilitating, for example, recombination among rrn loci). We hypothesize that this mechanism contributed to rapid acquisition of linezolid resistance.ConclusionsHypermutation and homologous recombination of the ribosomal RNA genes, including 23S rRNA genes, appear not to have been sources of the accelerated acquisition of linezolid resistance observed in our clinical case. Increased frequency of short sequence recombination may have resulted from a recQ variant in the infecting organism.

Recombination and Annealing Pathways Compete for Substrates in Making rrn Duplications in Salmonella enterica

Tandem genetic duplications arise frequently between the seven directly repeated 5.5-kb rrn loci that encode ribosomal RNAs in Salmonella enterica. The closest rrn genes, rrnB and rrnE, flank a 40-kb region that includes the purHD operon. Duplications of purHD arise by exchanges between rrn loci and form at a high rate (10(-3)/cell/division) that remains high in strains blocked for early steps in recombination (recA, recB, and/or recF), but drops 30-fold in mutants blocked for later Holliday junction resolution (ruvC recG). The duplication defect of a ruvC recG mutant was fully corrected by an added mutation in any one of the recA, recB, or recF genes. To explain these results, we propose that early recombination defects activate an alternative single-strand annealing pathway for duplication formation. In wild-type cells, rrn duplications form primarily by the action of RecFORA on single-strand gaps. Double-strand breaks cannot initiate rrn duplications because rrn loci lack Chi sites, which are essential for recombination between two separated rrn sequences. A recA or recF mutation allows unrepaired gaps to accumulate such that different rrn loci can provide single-strand rrn sequences that lack the RecA coating that normally inhibits annealing. A recB mutation activates annealing by allowing double-strand ends within rrn to avoid digestion by RecBCD and provide a new source of rrn ends for use in annealing. The equivalent high rates of rrn duplication by recombination and annealing pathways may reflect a limiting economy of gaps and breaks arising in heavily transcribed, palindrome-rich rrn sequences.

Sequestering from host and characterization of sequence of a ribosomal RNA operon ( rrn) from “ Candidatus Liberibacter asiaticus”

A 5005 bp rrn DNA sequence of “ Candidatus Liberibacter asiaticus” was obtained by PCR using primers conserved to Rhizobiaceae of alpha-proteobacteria. The rrn locus consisted of a 16S rRNA gene ( rrs), an intergenic transcribed spacer (ITS) containing two genes of tRNA Ile and tRNA Ala, a 23S rRNA gene ( rrl), an ITS without tRNA gene and a 5S rRNA gene ( rrf). Like rrs, rrl and rrf were also related (89–90%) to those of the members of Rhizobiaceae. Interestingly, the genes of tRNA Ile and tRNA Ala were more related to those of non- Rhizobiaceae alpha-proteobacteria. The non-tRNA gene regions of the 16S-23S ITS and 23S-5S rRNA ITS did not share significant similarity to any known non-Liberibacter DNA sequences, and could be used for specific and efficient detection of “ Ca. L. asiaticus”.

The rrn locus and gyrB genotyping confirm the existence of two clonal groups in strains of Yersinia enterocolitica subspecies palearctica biovar 1A

Eighty-one strains of Yersinia enterocolitica biovar 1A representing several serotypes isolated from India, France, Germany and the USA were analyzed using ribotyping, 16S–23S rDNA intergenic spacer length polymorphism analysis (PCR-ribotyping) and gyrB restriction fragment length polymorphism. Ribotyping with BglI, NciI and EcoRV distinguished 81 strains into 4, 3 and 2 ribotypes respectively. BglI- NciI combination gave the highest Simpson's diversity index (DI = 0.43). Strains with identical ribotypes were further differentiated by PCR-ribotyping. The combination of BglI- NciI ribotyping with PCR ribotyping increased DI to 0.72. This suggested that the combination of the two may be used for molecular epidemiological studies of Y. enterocolitica biovar 1A. This approach clearly resolved the strains into two clonal groups, each comprising strains isolated from humans, swine, pork and wastewater. PCR-RFLP of the gyrB gene using three enzymes ( AluI, MspI and HinfI) distinguished strains into seven types and confirmed the existence of two clonal groups. Thus, assessment of heterogeneity based on chromosomal restriction analysis (ribotyping), rRNA spacer length polymorphism (PCR-ribotyping) and gyrB gene analysis were in concordance and provided unequivocal evidence for the presence of two groups amongst strains of Y. enterocolitica biovar 1A despite their diverse geographic origins. These data also grouped clinical and non-clinical strains of serotype O:6,30–6,31 into discrete subgroups.

Physical and genetic map of theStaphylococcus xylosusC2a chromosome

Staphylococcus xylosus is a ubiquitous bacterium frequently isolated from mammalian skin and occurring naturally on meat and dairy products. A physical and genetic map of the S. xylosus C2a chromosome was constructed by pulsed-field gel electrophoresis analysis after digestion with AscI, ApaI, I-CeuI, SfiI and SmaI enzymes and hybridization analysis. The chromosome size was estimated to be 2868+/-10 kb. Thirty-three genetic markers were mapped. The probable origin of replication (oriC) was positioned. Six rrn loci were located, and their orientation was determined. The chromosomes of six additional S. xylosus strains were also analysed by I-CeuI digestion, and an intraspecies diversity of the chromosome size and the number of rrn operons was shown.

Physical and genetic map of the Weissella paramesenteroides DSMZ 20288T chromosome and characterization of different rrn operons by ITS analysis

The Weissella paramesenteroides DSMZ 20288T chromosome was analysed by pulsed-field gel electrophoresis, enabling the construction of a physical and genetic map. A total of 21 recognition sites of the restriction enzymes AscI, I-CeuI, NotI and SfiI were mapped on the chromosome, which was found to be circular with an estimated size of 2026 kb. This is believed to constitute the first study into the genomic organization of a strain of this genus, addressing the localization of important chromosomal regions such as oriC and terC. A total of 23 genetic markers were mapped, including eight rrn operons that were precisely assigned in 37 % of the W. paramesenteroides chromosome. The transcription direction of rrn loci was determined and three different rrn clusters were recognized regarding the presence/absence of tRNA genes in ITS regions.

Physical and genetic map of the Lactobacillus sakei 23K chromosome.

The Lactobacillus sakei 23K chromosome was analysed by pulsed-field gel electrophoresis after digestion with the restriction enzymes AscI, NotI and SfiI. The chromosome size was estimated to be 1845+/-80 kb. The use of I-CeuI, specific for rrn genes encoding 23S rRNAs, showed that seven rrn loci were present, on 40% of the chromosome. The seven rrn clusters were mapped and their orientation was determined, allowing the position of the replication origin to be estimated. Partial I-CeuI digestions were used to construct a backbone and the different restriction fragments obtained with AscI, NotI and SfiI were assembled to a physical map by Southern hybridization. Eleven L. sakei gene clusters previously identified were mapped, as well as 25 new loci located randomly on the chromosome and 11 regions flanking the rrn gene clusters. A total of 47 clusters were thus mapped on L. sakei chromosome. The new loci were sequenced, allowing the identification of 73 complete or incomplete coding sequences. Among these 73 new genes of L. sakei, the function of 36 could be deduced from their similarity to known genes described in databases. However, 10 genes had no homologues, 10 encoded proteins similar to proteins of unknown function and 17 were similar to hypothetical proteins.

Molecular changes at Rrn loci in barley (Hordeum vulgare L.) hybrids with H. bulbosum (L.)

Southern blots of restriction fragments of genomic DNAs from Hordeum vulgare (L.), H. bulbosum (L.), and interspecific hybrids and their derivatives were hybridized with rDNA probe to identify locus-specific modifications at Rrn loci. H. bulbosum rDNA revealed a single EcoRV site per repeat compared with two sites in H. vulgare rDNA repeats. H. bulbosum accessions possessed at least two rDNA repeat lengths, indicating heterozygosity at the Rrn locus. Hybrids possessed both H. vulgare and H. bulbosum rDNA repeats. Two of the hybrid derivatives possessed bulbosum-specific Sau3AI and HaeIII rDNA fragments, while amphiploid and doubled haploid derivatives lacked H. bulbosum rDNA repeat units and (or) fragments. Two hybrid derivatives, one amphiploid and a doubled haploid derived from the same parental combination, lacked the vulgare Rrn2-specific 9.0-kb rDNA repeat. This is the first conclusive evidence for the elimination of vulgare genetic material in vulgare-bulbosum hybrids. The ratios of 9.0- to 9.9-kb vulgare repeats and H. vulgare to H. bulbosum rDNA repeats indicate partial loss of the vulgare-specific 9.0-kb rDNA repeat among the hybrids. Differences in MboI and Sau3AI fragments and the ratios of 9.0 to 9.9 kb vulgare rDNA repeats revealed differential methylation at Rrn1 and Rrn2 loci. Hybrids and derivatives showed differential distribution of methylation of EcoRI, BglII, and SacI sites at the Rrn1 locus. Two of the hybrid derivatives exhibited extensive CpG-biased methylation. Data presented here are indicative of the differences in the onset of events triggered by the interaction of the component genomes and enabled detection of differential methylation among Rrn loci, loss of H. vulgare genetic material, and development of doubled haploids with the Rrn1 locus.

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.

Cloning and sequencing of a 16S/23S ribosomal spacer from Haemophilus parainfluenzae reveals an invariant, mosaic-like organisation of sequence blocks

A 16S/23S ribosomal spacer from a Haemophilus parainfluenzae rrn locus was cloned and sequenced. Analysis of PCR-amplified genomic fragments showed that this region is strongly conserved among unrelated isolates; computer analysis of database homologies showed that the spacer consists of sequence blocks, arranged in a mosaic-like structure, with strong homologies with analogous blocks present in the spacer regions of Haemophilus influenzae, Haemophilus ducreyi and Actinobacillus spp. It also contains a tRNA Glu gene, which is highly homologous to tRNA Glu genes found in spacers of other species. These data strongly support the hypothesis that recombination events are involved in the organisation of the sequence of the spacer, as a result of horizontal gene transfer.

Cloning and sequencing of a 16S/23S ribosomal spacer from Haemophilus parainfluenzae reveals an invariant, mosaic-like organisation of sequence blocks.

A 16S/23S ribosomal spacer from a Haemophilus parainfluenzae rrn locus was cloned and sequenced. Analysis of PCR-amplified genomic fragments showed that this region is strongly conserved among unrelated isolates; computer analysis of database homologies showed that the spacer consists of sequence blocks, arranged in a mosaic-like structure, with strong homologies with analogous blocks present in the spacer regions of Haemophilus influenzae, Haemophilus ducreyi and Actinobacillus spp. It also contains a tRNA(Glu) gene, which is highly homologous to tRNA(Glu) genes found in spacers of other species. These data strongly support the hypothesis that recombination events are involved in the organisation of the sequence of the spacer, as a result of horizontal gene transfer.

Physical map of the genome of Oenococcus oeni PSU-1 and localization of genetic markers.

A physical map of the chromosome of Oenococcus oeni PSU-1 was constructed. This represents the first map for a strain of this species. A total of 37 restriction sites for the rare-cutting endonucleases Ascl, Fsel, Notl and Sfil were mapped on the chromosome, which was found to be circular with an estimated size of 1857 kb. Fragment order was determined using several approaches: analysis of partial and double digestions, two-dimensional pulsed-field gel electrophoresis, isolation of linking clones, and Southern hybridization with labelled restriction fragments both from PSU-1 and from O. oeni strain GM. Oenococcal genes alsS/alsD, mleA and mir, two phage attachment sites and recurrent sequences such as IS1165-like elements and rrn loci were located on the physical map. Specific fragments hybridizing with gene probes from Lactococcus lactis, Leuconostoc mesenteroides and Bacillus subtilis were also identified. The two ribosomal operons have been precisely located and their transcription direction determined.

Characterization of the rrnB operon of the plant pathogen Rhodococcus fascians and targeted integrations of exogenous genes at rrn loci.

A 6.0-kb SalI DNA fragment containing an entire rRNA operon (rrnB) was cloned from a cosmid gene bank of the phytopathogenic strain Rhodococcus fascians D188. The nucleotide sequence of the 6-kb fragment was determined and had the organization 16S rRNA-spacer-23S rRNA-spacer-5S rRNA without tRNA-encoding genes in the spacer regions. The 5' and 3' ends of the mature 16S, 23S, and 5S rRNAs were determined by alignment with the rrn operons of Bacillus subtilis and other gram-positive bacteria. Four copies of the rrn operons were identified by hybridization with an rrnB probe in R. fascians type strain ATCC 12974 and in the virulent strain R. fascians D188. However, another isolate, CECT 3001 (= NRRL B15096), also classified as R. fascians, produced five rrn-hybridizing bands. An integrative vector containing a 2.5-kb DNA fragment internal to rrnB was constructed for targeted integration of exogenous genes at the rrn loci. Transformants carrying the exogenous chloramphenicol resistance gene (cmr) integrated in different rrn operons were obtained. These transformants had normal growth rates in complex medium and minimal medium and were fully stable for the integrated marker.

Differences in chromosome number and genome rearrangements in the genus Brucella.

We have studied the genomic structure and constructed the SpeI, PacI and I-CeuI restriction maps of the four biovars of the pathogenic bacterium Brucella suis. B. suis biovar 1 has two chromosomes of 2.1 Mb and 1.15 Mb, similar to those of the other Brucella species: B. melitensis, B. abortus, B. ovis and B. neotomae. Two chromosomes were also observed in the genome of B. suis biovars 2 and 4, but with sizes of 1.85 Mb and 1.35 Mb, whereas only one chromosome with a size of 3.1 Mb was found in B. suis biovar 3. We show that the differences in chromosome size and number can be explained by rearrangements at chromosomal regions containing the three rrn genes. The location and orientation of these genes confirmed that these rearrangements are due to homologous recombination at the rrn loci. This observation allows us to propose a scheme for the evolution of the genus Brucella in which the two chromosome-containing strains can emerge from an hypothetical ancestor with a single chromosome, which is probably similar to that of B. suis biovar 3. As the genus Brucella is certainly monospecific, this is the first time that differences in chromosome number have been observed in strains of the same bacterial species.

Physical and genetic map of the Clostridium acetobutylicum ATCC 824 chromosome.

A physical and genetic map of the Clostridium acetobutylicum ATCC 824 chromosome was constructed. The macrorestriction map for CeuI, EagI, and SstII was created by ordering the 38 restriction sites by one- and two-dimensional pulsed-field gel electrophoresis (PFGE) and by using an original strategy based on the CeuI enzyme and indirect end labelling by hybridization on both sides of the CeuI sites with rrs (16S RNA) and 3' rrl (23S RNA) probes. The circular chromosome was estimated to be 4.15 Mb in size, and the average resolution of the physical map is 110 kb. The chromosome contains 11 rrn loci, which are localized on 44% of the chromosome in a divergent transcriptional orientation regarding the presumed location of the replication origin. In addition to these 11 rrn operons, a total of 40 identified genes were mapped by hybridization experiments with genes from C. acetobutylicum and from various other clostridia as probes. The genetic map of C. acetobutylicum was compared to that of the three other endospore-forming bacteria characterized so far: Bacillus subtilis, Clostridium beijerinckii, and Clostridium perfringens. Parodoxically, the chromosomal backbone of C. acetobutylicum showed more similarity to that of B. subtilis than to those of the clostridia.

Mapping of the ribosomal operons on the linear chromosomal DNA of Streptomyces ambofaciens DSM40697.

Streptomyces ambofaciens DSM40697 possess six rRNA gene clusters which can be distinguished by probing BfrI-digested total DNA with the PCR-amplified l6S-23S ribosomal genet internal transcribed spacer. The six rrn loci of S. ambofaciens were cloned as recombinant cosmids and located on the AseI-Dŕal physical map of the linear chromosomal DNA. For five of the six ribosomal gene sets, the transcriptional orientation was determined relative to the physical map and was shown to be divergent away from an oriC-like locus.