Harnessing the intragenomic variability of rRNA operons to improve differentiation of Vibrio species.

Although the 16S rRNA gene is frequently used as a phylogenetic marker in analysis of environmental DNA, this marker often fails to distinguish closely related species, including those in the genus Vibrio. Here, we investigate whether inclusion and analysis of 23S rRNA sequence can help overcome the intrinsic weaknesses of 16S rRNA analyses for the differentiation of Vibrio species. We construct a maximum likelihood 16S rRNA gene tree to assess the use of this gene to identify clades of Vibrio species. Within the 16S rRNA tree, we identify the putative informative bases responsible for polyphyly, and demonstrate the association of these positions with tree topology. We demonstrate that concatenation of 16S and 23S rRNA genes increases the number of informative nucleotide positions, thereby overcoming ambiguities in 16S rRNA-based phylogenetic reconstructions. Finally, we experimentally demonstrate that this approach considerably improves the differentiation and identification of Vibrio species in environmental samples.

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.

RNA polymerase redistribution supports growth in E. coli strains with a minimal number of rRNA operons.

Bacterial transcription by RNA polymerase (RNAP) is spatially organized. RNAPs transcribing highly expressed genes locate in the nucleoid periphery, and form clusters in rich medium, with several studies linking RNAP clustering and transcription of rRNA (rrn). However, the nature of RNAP clusters and their association with rrn transcription remains unclear. Here we address these questions by using single-molecule tracking to monitor the subcellular distribution of mobile and immobile RNAP in strains with a heavily reduced number of chromosomal rrn operons (Δrrn strains). Strikingly, we find that the fraction of chromosome-associated RNAP (which is mainly engaged in transcription) is robust to deleting five or six of the seven chromosomal rrn operons. Spatial analysis in Δrrn strains showed substantial RNAP redistribution during moderate growth, with clustering increasing at cell endcaps, where the remaining rrn operons reside. These results support a model where RNAPs in Δrrn strains relocate to copies of the remaining rrn operons. In rich medium, Δrrn strains redistribute RNAP to minimize growth defects due to rrn deletions, with very high RNAP densities on rrn genes leading to genomic instability. Our study links RNAP clusters and rrn transcription, and offers insight into how bacteria maintain growth in the presence of only 1-2 rrn operons.

Organization and Characterization of the Promoter Elements of the rRNA Operons in the Slow-Growing Pathogen Mycobacterium kumamotonense.

The slow-growing, nontuberculous mycobacterium Mycobacterium kumamotonense possesses two rRNA operons, rrnA and rrnB, located downstream from the murA and tyrS genes, respectively. Here, we report the sequence and organization of the promoter regions of these two rrn operons. In the rrnA operon, transcription can be initiated from the two promoters, named P1 rrnA and PCL1, while in rrnB, transcription can only start from one, called P1 rrnB. Both rrn operons show a similar organization to the one described in Mycobacterium celatum and Mycobacterium smegmatis. Furthermore, by qRT-PCR analyses of the products generated from each promoter, we report that stress conditions such as starvation, hypoxia, and cellular infection affect the contribution of each operon to the synthesis of pre-rRNA. It was found that the products from the PCL1 promoter of rrnA play a pivotal role in rRNA synthesis during all stress conditions. Interestingly, the main participation of the products of transcription from the P1 promoter of rrnB was found during hypoxic conditions at the NRP1 phase. These results provide novel insights into pre-rRNA synthesis in mycobacteria, as well as the potential ability of M. kumamotonense to produce latent infections.

Short- and long-read metabarcoding of the eukaryotic rRNA operon: Evaluation of primers and comparison to shotgun metagenomics sequencing.

High-throughput sequencing-based analysis of microbial diversity has evolved vastly over the last decade. Currently, the go-to method for studying microbial eukaryotes is short-read metabarcoding of variable regions of the 18S rRNA gene with <500 bp amplicons. However, there is a growing interest in applying long-read sequencing of amplicons covering the rRNA operon for improving taxonomic resolution. For both methods, the choice of primers is crucial. It determines if community members are covered, if they can be identified at a satisfactory taxonomic level, and if the obtained community profile is representative. Here, we designed new primers targeting 18S and 28S rRNA based on 177,934 and 21,072 database sequences, respectively. The primers were evaluated in silico along with published primers on reference sequence databases and marine metagenomics data sets. We further evaluated a subset of the primers for short- and long-read sequencing on environmental samples in vitro and compared the obtained community profile with primer-unbiased metagenomic sequencing. Of the short-read pairs, a new V6-V8 pair and the V4_Balzano pair used with a simplified PCR protocol provided good results in silico and in vitro. Fewer differences were observed between the long-read primer pairs. The long-read amplicons and ITS1 alone provided higher taxonomic resolution than V4. Together, our results represent a reference and guide for selection of robust primers for research on and environmental monitoring of microbial eukaryotes.

Microbial Identification Using rRNA Operon Region: Database and Tool for Metataxonomics with Long-Read Sequence.

Recent development of long-read sequencing platforms has enabled researchers to explore bacterial community structure through analysis of full-length 16S rRNA gene (∼1,500 bp) or 16S-ITS-23S rRNA operon region (∼4,300 bp), resulting in higher taxonomic resolution than short-read sequencing platforms. Despite the potential of long-read sequencing in metagenomics, resources and protocols for this technology are scarce. Here, we describe MIrROR, the database and analysis tool for metataxonomics using the bacterial 16S-ITS-23S rRNA operon region. We collected 16S-ITS-23S rRNA operon sequences extracted from bacterial genomes from NCBI GenBank and performed curation. A total of 97,781 16S-ITS-23S rRNA operon sequences covering 9,485 species from 43,653 genomes were obtained. For user convenience, we provide an analysis tool based on a mapping strategy that can be used for taxonomic profiling with MIrROR database. To benchmark MIrROR, we compared performance against publicly available databases and tool with mock communities and simulated data sets. Our platform showed promising results in terms of the number of species covered and the accuracy of classification. To encourage active 16S-ITS-23S rRNA operon analysis in the field, BLAST function and taxonomic profiling results with 16S-ITS-23S rRNA operon studies, which have been reported as BioProject on NCBI are provided. MIrROR (http://mirror.egnome.co.kr/) will be a useful platform for researchers who want to perform high-resolution metagenome analysis with a cost-effective sequencer such as MinION from Oxford Nanopore Technologies. IMPORTANCE Metabarcoding is a powerful tool to investigate community diversity in an economic and efficient way by amplifying a specific gene marker region. With the advancement of long-read sequencing technologies, the field of metabarcoding has entered a new phase. The technologies have brought a need for development in several areas, including new markers that long-read can cover, database for the markers, tools that reflect long-read characteristics, and compatibility with downstream analysis tools. By constructing MIrROR, we met the need for a database and tools for the 16S-ITS-23S rRNA operon region, which has recently been shown to have sufficient resolution at the species level. Bacterial community analysis using the 16S-ITS-23S rRNA operon region with MIrROR will provide new insights from various research fields.

First next-generation sequencing data for Haploporidae (Digenea: Haploporata): characterization of complete mitochondrial genome and ribosomal operon for Parasaccocoelium mugili Zhukov, 1971.

The first data on a whole mitochondrial genome of Haploporidae, Parasaccocoelium mugili (Digenea: Haploporata: Haploporidae) was generated using the next-generation sequencing (NGS) approach. We sequenced the complete mitochondrial DNA (mtDNA) and ribosomal operon of Parasaccocoelium mugili, intestine parasite of mullet fish. The mtDNA of P. mugili contained 14,021 bp, including 12 protein-coding genes, two ribosomal genes, 22 tRNA genes, and non-coding region. The ribosomal operon of P. mugili was 8308 bp in length, including 18S rRNA gene (1981 bp), ITS1 rDNA (955 bp), 5.8S rRNA gene (157 bp), ITS2 rDNA (268 bp), 28S rRNA gene (4180 bp), and ETS (767 bp). We used the mtDNA protein-coding regions to make phylogenetic reconstructions of Haploporidae. Additionally, we performed the sequence cluster analysis based on codon usage bias of most of currently available mitochondrial genome data for trematodes. The observed gene arrangement in mtDNA sequence of P. mugili is identical to those of Plagiorchis maculosus (Rudolphi, 1802). Results of maximum likelihood (ML) phylogenetic analysis showed that P. mugili was closely related to Paragonimus species from the suborder Xiphidiata. The results of sequence cluster analysis based on codon usage bias showed that P. mugili has the highest similarity with Plagiorchis maculosus (Xiphidiata). Our results do not contradict to proposing a new suborder for Haploporoidea-Haploporata. On the basis of obtained results, the relationship between mitochondrial protein-coding gene rearrangements and synonymous nucleotide substitutions in mitochondrial genomes has been suggested.

Orthogonal translation enables heterologous ribosome engineering in E. coli.

The ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts. Heterologous ribosomes, comprising rRNAs and r-proteins derived from different microorganisms, may offer opportunities for novel translational functions. Such heterologous ribosomes have previously been evaluated in E. coli via complementation of a genomic ribosome deficiency, but this method fails to guide the engineering of refractory ribosomes. Here, we implement orthogonal ribosome binding site (RBS):antiRBS pairs, in which engineered ribosomes are directed to researcher-defined transcripts, to inform requirements for heterologous ribosome functionality. We discover that optimized rRNA processing and supplementation with cognate r-proteins enhances heterologous ribosome function for rRNAs derived from organisms with ≥76.1% 16S rRNA identity to E. coli. Additionally, some heterologous ribosomes undergo reduced subunit exchange with E. coli-derived subunits. Cumulatively, this work provides a general framework for heterologous ribosome engineering in living cells.

Unlinked rRNA genes are widespread among bacteria and archaea.

Ribosomes are essential to cellular life and the genes for their RNA components are the most conserved and transcribed genes in bacteria and archaea. Ribosomal RNA genes are typically organized into a single operon, an arrangement thought to facilitate gene regulation. In reality, some bacteria and archaea do not share this canonical rRNA arrangement-their 16S and 23S rRNA genes are separated across the genome and referred to as "unlinked". This rearrangement has previously been treated as an anomaly or a byproduct of genome degradation in intracellular bacteria. Here, we leverage complete genome and long-read metagenomic data to show that unlinked 16S and 23S rRNA genes are more common than previously thought. Unlinked rRNA genes occur in many phyla, most significantly within Deinococcus-Thermus, Chloroflexi, and Planctomycetes, and occur in differential frequencies across natural environments. We found that up to 41% of rRNA genes in soil were unlinked, in contrast to the human gut, where all sequenced rRNA genes were linked. The frequency of unlinked rRNA genes may reflect meaningful life history traits, as they tend to be associated with a mix of slow-growing free-living species and intracellular species. We speculate that unlinked rRNA genes may confer selective advantages in some environments, though the specific nature of these advantages remains undetermined and worthy of further investigation. More generally, the prevalence of unlinked rRNA genes in poorly-studied taxa serves as a reminder that paradigms derived from model organisms do not necessarily extend to the broader diversity of bacteria and archaea.

Targeting 16S rDNA for Stable Recombinant Gene Expression in Pseudomonas.

Ribosomal RNA (rRNA) operons have recently been identified as promising sites for chromosomal integration of genetic elements in Pseudomonas putida, a bacterium that has gained considerable popularity as a microbial cell factory. We have developed a tool for targeted integration of recombinant genes into the rRNA operons of various Pseudomonas strains, where the native context of the rRNA clusters enables effective transcription of heterologous genes. However, a sufficient translation of foreign mRNA  transcriptionally fused to rRNA required optimization of RNA secondary structures, which was achieved utilizing synthetic ribozymes and a bicistronic design. The generated tool further enabled the characterization of the six rRNA promoter units of P. putida S12 under different growth conditions. The presence of multiple, almost identical rRNA operons in Pseudomonas also allowed the integration of multiple copies of heterologous genetic elements. The integration of two expression cassettes and the resulting disruption of rRNA units only moderately affects growth rates, and the constructs were highly stable over more than 160 generations.

Structural Basis for Transcript Elongation Control by NusG Family Universal Regulators.

NusG/RfaH/Spt5 transcription elongation factors are the only transcription regulators conserved across all life. Bacterial NusG regulates RNA polymerase (RNAP) elongation complexes (ECs) across most genes, enhancing elongation by suppressing RNAP backtracking and coordinating ρ-dependent termination and translation. The NusG paralog RfaH engages the EC only at operon polarity suppressor (ops) sites and suppresses both backtrack and hairpin-stabilized pausing. We used single-particle cryoelectron microscopy (cryo-EM) to determine structures of ECs at ops with NusG or RfaH. Both factors chaperone base-pairing of the upstream duplex DNA to suppress backtracking, explaining stimulation of elongation genome-wide. The RfaH-opsEC structure reveals how RfaH confers operon specificity through specific recognition of an ops hairpin in the single-stranded nontemplate DNA and tighter binding to the EC to exclude NusG. Tight EC binding by RfaH sterically blocks the swiveled RNAP conformation necessary for hairpin-stabilized pausing. The universal conservation of NusG/RfaH/Spt5 suggests that the molecular mechanisms uncovered here are widespread.

Assembly of a complete genome sequence for Gemmata obscuriglobus reveals a novel prokaryotic rRNA operon gene architecture.

Gemmata obscuriglobus is a Gram-negative bacterium with several intriguing biological features. Here, we present a complete, de novo whole genome assembly for G. obscuriglobus which consists of a single, circular 9 Mb chromosome, with no plasmids detected. The genome was annotated using the NCBI Prokaryotic Genome Annotation pipeline to generate common gene annotations. Analysis of the rRNA genes revealed three interesting features for a bacterium. First, linked G. obscuriglobus rrn operons have a unique gene order, 23S-5S-16S, compared to typical prokaryotic rrn operons (16S-23S-5S). Second, G. obscuriglobus rrn operons can either be linked or unlinked (a 16S gene is in a separate genomic location from a 23S and 5S gene pair). Third, all of the 23S genes (5 in total) have unique polymorphisms. Genome analysis of a different Gemmata species (SH-PL17), revealed a similar 23S-5S-16S gene order in all of its linked rrn operons and the presence of an unlinked operon. Together, our findings show that unique and rare features in Gemmata rrn operons among prokaryotes provide a means to better define the evolutionary relatedness of Gemmata species and the divergence time for different Gemmata species. Additionally, these rrn operon differences provide important insights into the rrn operon architecture of common ancestors of the planctomycetes.

A real-time PCR assay for accurate quantification of the individual members of the Altered Schaedler Flora microbiota in gnotobiotic mice.

Changes in the gastrointestinal microbial community are frequently associated with chronic diseases such as Inflammatory Bowel Diseases. However, understanding the relationship of any individual taxon within the community to host physiology is made complex due to the diversity and individuality of the gut microbiota. Defined microbial communities such as the Altered Schaedler Flora (ASF) help alleviate the challenges of a diverse microbiota by allowing one to interrogate the relationship between individual bacterial species and host responses. An important aspect of studying these relationships with defined microbial communities is the ability to measure the population abundance and dynamics of each member. Herein, we describe the development of an improved ASF species-specific and sensitive real-time quantitative polymerase chain reaction (qPCR) for use with SYBR Green chemistry to accurately assess individual ASF member abundance. This approach targets hypervariable regions V1 through V3 of the 16S rRNA gene of each ASF taxon to enhance assay specificity. We demonstrate the reproducibility, sensitivity and application of this new method by quantifying each ASF bacterium in two inbred mouse lines. We also used it to assess changes in ASF member abundance before and after acute antibiotic perturbation of the community as well as in mice fed two different diets. Additionally, we describe a nested PCR assay for the detection of lowly abundant ASF members. Altogether, this improved qPCR method will facilitate gnotobiotic research involving the ASF community by allowing for reproducible quantification of its members under various physiological conditions.

Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli.

Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order) and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3' proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP) holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon), within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons are expressed independent of rRNA synthesis under specific conditions where further synthesis of ribosomes is not needed.

rRNA Operon Copy Number Can Explain the Distinct Epidemiology of Hospital-Associated Methicillin-Resistant Staphylococcus aureus.

The distinct epidemiology of original hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) and early community-associated MRSA (CA-MRSA) is largely unexplained. S. aureus carries either five or six rRNA operon copies. Evidence is provided for a scenario in which MRSA has adapted to the hospital environment by rRNA operon loss (six to five copies) due to antibiotic pressure. Early CA-MRSA, in contrast, results from wild-type methicillin-susceptible S. aureus (MSSA) that acquired mecA without loss of an rRNA operon. Of the HA-MRSA isolates (n = 77), 67.5% had five rRNA operon copies, compared to 23.2% of the CA-MRSA isolates (n = 69) and 7.7% of MSSA isolates (n = 195) (P < 0.001). In addition, 105 MSSA isolates from cystic fibrosis patients were tested, because these patients are repeatedly treated with antibiotics; 32.4% of these isolates had five rRNA operon copies. For all subsets, a correlation between resistance profile and rRNA copy number was found. Furthermore, we showed that in vitro antibiotic pressure may result in rRNA operon copy loss. We also showed that without antibiotic pressure, S. aureus isolates containing six rRNA copies are more fit than isolates with five copies. We conclude that HA-MRSA and cystic fibrosis isolates most likely have adapted to an environment with high antibiotic pressure by the loss of an rRNA operon copy. This loss has facilitated resistance development, which promoted survival in these niches. However, strain fitness decreased, which explains their lack of success in the community. In contrast, CA-MRSA isolates retained six rRNA operon copies, rendering them fitter and thereby able to survive and spread in the community.

Whole-genome optical mapping reveals a mis-assembly between two rRNA operons of Corynebacterium pseudotuberculosis strain 1002.

BACKGROUND: Studies have detected mis-assemblies in genomes of the species Corynebacterium pseudotuberculosis. These new discover have been possible due to the evolution of the Next-Generation Sequencing platforms, which have provided sequencing with accuracy and reduced costs. In addition, the improving of techniques for construction of high accuracy genomic maps, for example, Whole-genome mapping (WGM) (OpGen Inc), have allow high-resolution assembly that can detect large rearrangements. RESULTS: In this work, we present the resequencing of Corynebacterium pseudotuberculosis strain 1002 (Cp1002). Cp1002 was the first strain of this species sequenced in Brazil, and its genome has been used as model for several studies in silico of caseous lymphadenitis disease. The sequencing was performed using the platform Ion PGM and fragment library (200 bp kit). A restriction map was constructed, using the technique of WGM with the enzyme KpnI. After the new assembly process, using WGM as scaffolder, we detected a large inversion with size bigger than one-half of genome. A specific analysis using BLAST and NR database shows that the inversion occurs between two homology RNA ribosomal regions. CONCLUSION: In conclusion, the results showed by WGM could be used to detect mismatches in assemblies, providing genomic maps with high resolution and allow assemblies with more accuracy and completeness. The new assembly of C. pseudotuberculosis was deposited in GenBank under the accession no. CP012837.

Open complex scrunching before nucleotide addition accounts for the unusual transcription start site of E. coli ribosomal RNA promoters.

Most Escherichia coli promoters initiate transcription with a purine 7 or 8 nt downstream from the -10 hexamer, but some promoters, including the ribosomal RNA promoter rrnB P1, start 9 nt from the -10 element. We identified promoter and RNA polymerase determinants of this noncanonical rrnB P1 start site using biochemical and genetic approaches including mutational analysis of the promoter, Fe(2+) cleavage assays to monitor template strand positions near the active-site, and Bpa cross-linking to map the path of open complex DNA at amino acid and nucleotide resolution. We find that mutations in several promoter regions affect transcription start site (TSS) selection. In particular, we show that the absence of strong interactions between the discriminator region and σ region 1.2 and between the extended -10 element and σ region 3.0, identified previously as a determinant of proper regulation of rRNA promoters, is also required for the unusual TSS. We find that the DNA in the single-stranded transcription bubble of the rrnB P1 promoter complex expands and is "scrunched" into the active site channel of RNA polymerase, similar to the situation in initial transcribing complexes. However, in the rrnB P1 open complex, scrunching occurs before RNA synthesis begins. We find that the scrunched open complex exhibits reduced abortive product synthesis, suggesting that scrunching and unusual TSS selection contribute to the extraordinary transcriptional activity of rRNA promoters by increasing promoter escape, helping to offset the reduction in promoter activity that would result from the weak interactions with σ.

Decreases in average bacterial community rRNA operon copy number during succession.

Trait-based studies can help clarify the mechanisms driving patterns of microbial community assembly and coexistence. Here, we use a trait-based approach to explore the importance of rRNA operon copy number in microbial succession, building on prior evidence that organisms with higher copy numbers respond more rapidly to nutrient inputs. We set flasks of heterotrophic media into the environment and examined bacterial community assembly at seven time points. Communities were arrayed along a geographic gradient to introduce stochasticity via dispersal processes and were analyzed using 16 S rRNA gene pyrosequencing, and rRNA operon copy number was modeled using ancestral trait reconstruction. We found that taxonomic composition was similar between communities at the beginning of the experiment and then diverged through time; as well, phylogenetic clustering within communities decreased over time. The average rRNA operon copy number decreased over the experiment, and variance in rRNA operon copy number was lowest both early and late in succession. We then analyzed bacterial community data from other soil and sediment primary and secondary successional sequences from three markedly different ecosystem types. Our results demonstrate that decreases in average copy number are a consistent feature of communities across various drivers of ecological succession. Importantly, our work supports the scaling of the copy number trait over multiple levels of biological organization, ranging from cells to populations and communities, with implications for both microbial ecology and evolution.

pRNA: NoRC-associated RNA of rRNA operons.

Promoter-associated RNAs (pRNAs) are a family of ~90-100 nt-long divergent RNAs overlapping the promoter of the rRNA (rDNA) operon. pRNA transcripts interact with TIP5, a component of the chromatin remodeling complex NoRC, which recruits enzymes for heterochromatin formation and mediates silencing of rRNA genes. Here we present a comprehensive analysis of pRNA homologs, including different versions per species, as result of in silico studies in available metazoan genome assemblies. Comparative sequence analysis and secondary structure prediction ended up in two possible secondary structures, which let us assume a possible dual function of pRNAs for regulation of rRNA operons. Furthermore, we validated parts of our computational predictions experimentally by RT-PCR and sequencing. A representative seed alignment of the pRNA family, annotated with possible secondary structures was released to the Rfam database.

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.