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.
Subscribe to Journal
Get full journal access for 1 year
only $53.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
All genomes used in this study were downloaded from NCBI, with assembly IDs listed in Supplementary Dataset S1. All Nanopore data are available at the Sequence Read Archive (SRA) under Bioproject ID PRJNA553237 or the European Nucleotide Archive (ENA) under PRJEB33278. All Moleculo data has been published previously, with publications listed in methods. Classifications and details of both the complete genome and long-read datasets are included in Supplementary Dataset S1 and S2, respectively.
Raoult D, Forterre P. Redefining viruses: lessons from Mimivirus. Nat Rev Microbiol. 2008;6:315–9.
Srivastava AK, Schlessinger D. Mechanism and regulation of bacterial ribosomal RNA processing. Annu Rev Microbiol. 1990;44:105–29.
Condon C, Squires C, Squires CL. Control of rRNA transcription in Escherichia coli. Microbiol Rev. 1995;59:623–45.
Gourse RL, Gaal T, Bartlett MS, Appleman JA, Ross W. rRNA transcription and growth rate–dependent regulation of ribosome synthesis in Escherichia coli. Annu Rev Microbiol. 1996;50:645–77.
Klappenbach JA, Dunbar JM, Schmidt TM. rRNA operon copy number reflects ecological strategies of bacteria. Appl Environ Microbiol. 2000;66:1328–33.
Hartmann RK, Ulbrich N, Erdmann VA. An unusual rRNA operon constellation: in Thermus thermophilus HB8 the 23S/5S rRNA operon is a separate entity from the 16S rRNA operon. Biochimie. 1987;69:1097–104.
Liesack W, Stackebrandt E. Evidence for unlinked rrn operons in the Planctomycete Pirellula marina. J Bacteriol. 1989;171:5025–30.
Munson MA, Baumann L, Baumann P. Buchnera aphidicola (a prokaryotic endosymbiont of aphids) contains a putative 16S rRNA operon unlinked to the 23s rRNA-encoding gene: sequence determination, and promoter and terminator analysis. Gene. 1993;137:171–8.
Andersson SGE, Zomorodipour A, Winkler HH, Kurland CG. Unusual organization of the rRNA genes in Rickettsia prowazekii. J Bacteriol. 1995;177:4171–5.
Rurangirwa FR, Brayton KA, McGuire TC, Knowles DP, Palmer GH. Conservation of the unique rickettsial rRNA gene arrangement in Anaplasma. Int J Syst Evolut Microbiol. 2002;52:1405–9.
Merhej V, Royer-Carenzi M, Pontarotti P, Raoult D. Massive comparative genomic analysis reveals convergent evolution of specialized bacteria. Biol Direct. 2009;4:13–25.
Andersson JO, Andersson SGE. Genome degradation is an ongoing process in Rickettsia. Mol Biol Evol. 1999;16:1178–91.
Zhi X-Y, Zhao W, Li W-J, Zhao G-P. Prokaryotic systematics in the genomics era. Antonie van Leeuwenhoek. 2012;101:21–34.
O’Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R, et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 2016;44:D733–45.
R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria; 2018.
Kuleshov V, Xie D, Chen R, Pushkarev D, Ma Z, Blauwkamp T, et al. Whole-genome haplotyping using long reads and statistical methods. Nat Biotechnol. 2014;32:261–6.
Kuleshov V, Jiang C, Zhou W, Jahanbani F, Batzoglou S, Snyder M. Synthetic long-read sequencing reveals intraspecies diversity in the human microbiome. Nat Biotechnol. 2016;34:64–9.
White RA, Bottos EM, Roy Chowdhury T, Zucker JD, Brislawn CJ, Nicora CD, et al. Moleculo long-read sequencing facilitates assembly and genomic binning from complex soil metagenomes. Am Soc Microbiol J. 2016;1:309–15.
Sharon I, Kertesz M, Hug LA, Pushkarev D, Blauwkamp TA, Castelle CJ, et al. Accurate, multi-kb reads resolve complex populations and detect rare microorganisms. Genome Res. 2015;25:534–43.
Flynn TM, Koval JC, Greenwald SM, Owens SM, Kemner KM, Antonopoulos DA. Parallelized, aerobic, single carbon-source enrichments from different natural environments contain divergent microbial communities. Front Microbiol. 2017;8:1540–14.
Bengtsson-Palme J, Hartmann M, Eriksson KM, Pal C, Thorell K, Larsson DGJ, et al. Metaxa2: improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data. Mol Ecol Resour. 2015;15:1403–14.
Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol. 2007;73:5261–7.
Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2012;41:D590–6.
Brown CT, Hug LA, Thomas BC, Sharon I, Castelle CJ, Singh A, et al. Unusual biology across a group comprising more than 15% of domain Bacteria. Nature. 2015;523:208–11.
Pei A, Nossa CW, Chokshi P, Blaser MJ, Yang L, Rosmarin DM, et al. Diversity of 23S rRNA genes within individual prokaryotic genomes. PLoS ONE. 2009;4:1–9.
Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010;26:2460–1.
Caporaso JG, Bittinger K, Bushman FD, DeSantis TZ, Andersen GL, Knight R. PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics. 2010;26:266–7.
Price MN, Dehal PS, Arkin AP. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol. 2009;26:1641–50.
Letunic I, Bork P. Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 2016;44:W242–5.
Pagel M. Inferring the historical patterns of biological evolution. Nature. 1999;401:877–84.
Revell LJ. phytools: an R package for phylogenetic comparative biology (and other things). Methods Ecol Evol. 2011;3:217–23.
Tung HoLS, Ané C. A linear-time algorithm for gaussian and non-gaussian trait evolution models. Syst Biol. 2014;63:397–408.
Novembre JA. Accounting for background nucleotide composition when measuring codon usage bias. Mol Biol Evol. 2002;19:1390–4.
Rocha E. Codon usage bias from tRNA’s point of view: redundancy, specialization, and efficient decoding for translation optimization. Genome Res. 2004;14:2279–86.
Vieira-Silva S, Rocha E. The systemic imprint of growth and its uses in ecological (meta)genomics. PLOS Genet. 2009;6:1–15.
Eddy SR. Accelerated profile HMM searches. PLoS Comput Biol. 2011;7:e1002195–16.
Moreno-Hagelsieb G, Collado-Vides J. A powerful non-homology method for the prediction of operons in prokaryotes. Bioinformatics. 2002;18:S329–36.
Shepherd J, Ibba M. Bacterial transfer RNAs. FEMS Microbiol Rev. 2015;39:280–300.
Parks DH, Chuvochina M, Waite DW, Rinke C, Skarshewski A, Chaumeil P-A, et al. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nat Biotechnol. 2018;36:996–1004.
Yuan C, Lei J, Cole J, Sun Y. Reconstructing 16S rRNA genes in metagenomic data. Bioinformatics. 2015;31:i35–i43.
Durand S, Gilet L, Condon C. The Essential Function of B. subtilis RNase III is to silence foreign toxin genes. PLOS Genet. 2012;8:e1003181–11.
Brewer TE, Handley KM, Carini P, Gilbert JA, Fierer N. Genome reduction in an abundant and ubiquitous soil bacterium “Candidatus Udaeobacter copiosus.” Nat Microbiol. 2016;2:16198.
Vartoukian SR, Palmer RM, Wade WG. Strategies for culture of “unculturable” bacteria. FEMS Microbiol Lett. 2010;309:1–7.
Garcia-Martinez J, Acinas SG, Anton AI, Rodriguez-Valera F. Use of the 16S-23S ribosomal genes spacer region in studies of prokaryotic diversity. J Microbiol Methods. 1999;36:55–64.
Zeng YH, Koblížek M, Li YX, Liu YP, Feng FY, Ji JD, et al. Long PCR-RFLP of 16S-ITS-23S rRNA genes: a high-resolution molecular tool for bacterial genotyping. J Appl Microbiol. 2012;114:433–47.
Cuscó A, Catozzi C, Viñes J, Sanchez A, Francino O. Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon. F1000Res. 2018;7:1755–25.
Brown CT, Olm MR, Thomas BC, Banfield JF. Measurement of bacterial replication rates in microbial communities. Nat Biotechnol. 2016;34:1256–63.
Roller BRK, Stoddard SF, Schmidt TM. Exploiting rRNA operon copy number to investigate bacterial reproductive strategies. Nat Microbiol. 2016;1:1–7.
Siehnel RJ, Morgan EA. Unbalanced rRNA gene dosage and its effects on rRNA and ribosomal-protein synthesis. J Bacteriol. 1985;163:476–86.
Moran NA. Microbial minimalism: genome reduction in bacterial pathogens. Cell. 2002;108:583–6.
Nelson WC, Stegen JC. The reduced genomes of Parcubacteria (OD1) contain signatures of a symbiotic lifestyle. Front Microbiol. 2015;6:693–14.
Burstein D, Sun CL, Brown CT, Sharon I, Anantharaman K, Probst AJ, et al. Major bacterial lineages are essentially devoid of CRISPR-Cas viral defence systems. Nat Commun. 2016;7:1–8.
Holman DB, Brunelle BW, Trachsel J, Allen HK. Meta-analysis to define a core microbiota in the swine gut. mSystems. 2017;2:676–14.
Anacker ML, Drecktrah D, LeCoultre RD, Lybecker M, Samuels DS. RNase III processing of rRNA in the Lyme disease Spirochete Borrelia burgdorferi. Journal of Bacteriology. Am Soc Microbiol J. 2018;200:1–11.
Iost I, Chabas S, Darfeuille F. Maturation of atypical ribosomal RNA precursors in Helicobacter pylori. Nucleic Acids Res. 2019;47:5906–21.
Gone S, Alfonso-Prieto M, Paudyal S, Nicholson AW. Mechanism of ribonuclease III catalytic regulation by serine phosphorylation. Nature. 2016;6:1–9.
Wilcon HR, Yu D, Peters HK III, Zhou J-G, Court DL. The global regulator RNase III modulates translation repression by the transcription elongation factor N. EMBO J. 2002;21:4154–61.
Hagen FS, Young ET. Effect of RNase III on efficiency of translation of bacteriophage T7 lysozyme mRNA. J Virol. 1978;26:793–804.
Bohannan BJM, Lenski RE. Linking genetic change to community evolution: insights from studies of bacteria and bacteriophage. Ecol Lett. 2000;3:362–77.
Song W, Joo M, Yeom J-H, Shin E, Lee M, Choi H-K, et al. Divergent rRNAs as regulators of gene expression at the ribosome level. Nat Microbiol. 2019;4:515–26.
Holland AD, Rothfuss HM, Lidstrom ME. Development of a defined medium supporting rapid growth for Deinococcus radiodurans and analysis of metabolic capacities. Appl Microbiol Biotechnol. 2006;72:1074–82.
Devos DP. Gemmata obscuriglobus. Curr Biol. 2013;23:R705–7.
This research was supported in part by the Chateaubriand Fellowship awarded to TEB from the Office for Science & Technology of the Embassy of France in the United States and a grant to NF from the U.S. National Science Foundation (EAR1331828). MA was supported by a research grant (15510) from Villum Fonden. AE gratefully acknowledges the support of a Leverhulme Trust Research Fellowship (RF-2017–652\2). ER was supported by the INCEPTION project (PIA/ANR-16-CONV-0005). We thank Will Trimble for assistance tracking down publicly available Moleculo sequences, Michael Engel for figure design input, and Eric Johnston for early discussions on unlinked rRNA genes.
Conflict of interest
MA and RK own a portion of the company DNASense.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Brewer, T.E., Albertsen, M., Edwards, A. et al. Unlinked rRNA genes are widespread among bacteria and archaea. ISME J (2019) doi:10.1038/s41396-019-0552-3