Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome Mizue Anda1,2, Yoshiyuki Ohtsubo1, Takashi Okubo, Masayuki Sugawara, Yuji Nagata, Masataka Tsuda, Kiwamu Minamisawa3, and Hisayuki Mitsui3 Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, Katahira, Aoba-ku, Sendai 980-8577, Japan Edited by E. Peter Greenberg, University of Washington, Seattle, WA, and approved October 15, 2015 (received for review July 21, 2015) rRNA is essential for life because of its functional importance in protein (5.2 Mb in total) contains nine circular replicons: the main synthesis. The rRNA (rrn) operon encoding 16S, 23S, and 5S rRNAs is chromosome (3.7 Mb) and eight other replicons (designated locatedonthe“main” chromosome in all bacteria documented to date pAU20a to pAU20g and pAU20rrn)(Table1andFig. S1). The and is frequently used as a marker of chromosomes. Here, our genome five smallest replicons, pAU20d to pAU20g and pAU20rrn, analysis of a plant-associated alphaproteobacterium, Aureimonas sp. could be distinguished from the chromosome by their lower G+C rrn AU20, indicates that this strain has its sole operon on a small contents, suggesting that they had distinct evolutionary origins. The (9.4 kb), high-copy-number replicon. We designated this unusual repli- genome contained a single rrn operon, 55 tRNA genes, and 4,785 rrn rrn concarryingthe operon on the background of an -lacking chro- protein-coding genes (Table 1). Surprisingly, the rrn operon, which mosome (RLC) as the rrn-plasmid. Four of 12 strains close to AU20 also consisted of genes for 16S rRNA, tRNAIle,tRNAAla, 23S rRNA, had this RLC/rrn-plasmid organization. Phylogenetic analysis showed rrn and 5S rRNA, was located not on the chromosome but on the that those strains having the RLC/ -plasmid organization repre- Ile Aureimonas smallest replicon (9.4 kb), pAU20rrn. The tRNA gene on this sented one clade within the genus . Our finding introduces Ala a previously unaddressed viewpoint into studies of genetics, geno- replicon was also the only one in the genome. The tRNA gene Ala mics, and evolution in microbiology and biology in general. on pAU20rrn showed similarities to the tRNA gene and the tRNAThr gene on the chromosome (82% and 78% identical, ribosomal RNA operon | chromosome | plasmid | genome rearrangement | respectively; Fig. S2A), but the two replicons shared no other Aureimonas substantial similarities. Using the 16S rRNA gene as a probe, we MICROBIOLOGY performed Southern hybridization analysis of the genomic DNA ultipartite genomes containing more than one chromosome that was digested with the enzyme I-CeuI, an endonuclease Mare not unusual in bacteria. The presence of the main (i.e., whose cleavage site exists exclusively within the 23S rRNA gene largest) chromosome and the second chromosome(s) in one ge- (12) (Fig. 1A). The result showed a single 9-kb band, supporting nome is conserved among all members of some genera, such as the localization of the rrn operon on pAU20rrn. A single hy- Burkholderia and Vibrio. Chromosomes are distinguished from bridization band was also detected with EcoRI-digested DNA, plasmids by the localization of essential genes (1, 2). In general, consistent with the existence of a single rrn operon in the ge- main and second chromosomes are larger (>0.5 Mb) than co- nome. This rrn operon (with a size of 6 kb) had the typical core resident plasmids, have similar guanine+cytosine (G+C) contents promoter consisting of the putative UP element and −35 and −10 to each other, and are maintained by cell cycle-linked replication and active partitioning systems, whereas plasmids are diverse in Significance that some (i) can be conjugally mobilized, (ii) can replicate within one genus or within many genera (i.e., have a broad host range), (iii) are present in high copy numbers and are segregated sto- In bacterial genomes, chromosomes are distinguished from + plasmids by the localization of essential genes. It has been taken chastically, and (iv) have lower G C contents than their host rrn chromosomes (1–3). One of the practical ways of designating a for granted that fundamental genes such as the rRNA ( )op- eron should be transmitted faithfully on the chromosome. Here, replicon as a “chromosome” has been to examine the localization we found a striking exception: A plant-associated bacterium, of the rRNA (rrn) operon, which encodes rRNAs (16S, 23S, and 5S Aureimonas sp. AU20, and its close relatives harbor the rrn op- rRNAs), because of its functional importance in protein synthesis. eron only on a small, high-copy-number replicon but not on the Additional copies of the rrn operon are also sometimes found on chromosome. Our findings show the existence of novel genome the second chromosomes (e.g., in species of Rhodobacter, Brucella, organization in bacteria. Burkholderia,andVibrio)(4–7) or on plasmids (the 53.9-kb and 23- kb plasmids of Bacillus megaterium and Paracoccus species, re- Author contributions: M.A., Y.O., T.O., Y.N., K.M., and H.M. designed research; M.A., Y.O., spectively) (8, 9). However, so far, no documented bacterial strain and T.O. performed research; Y.O. led the AU20 genome finishing and developed compu- lacks the rrn operon on the “main” chromosome. tational tools; M.A., Y.O., T.O., and H.M. analyzed data; and M.A., Y.O., T.O., M.S., Y.N., M.T., Here, we report genome analysis of the genus Aureimonas, K.M., and H.M. wrote the paper. showing that one clade within this genus carries its sole rrn op- The authors declare no conflict of interest. eron on a small plasmid. This article is a PNAS Direct Submission. Data deposition: The sequences reported in this paper have been deposited in the Gen- Results and Discussion Bank database, www.ncbi.nlm.nih.gov/genbank/, and in the DNA Data Bank of Japan Plasmid Localization of the rrn Operon in Aureimonas sp. AU20. The database, www.ddbj.nig.ac.jp. A list of accession numbers is provided in Table 1 and Table S2. genus Aureimonas (family Aurantimonadaceae, order Rhizobiales, 1 class Alphaproteobacteria) contains three defined species, M.A. and Y.O. contributed equally to this work. 2 Aureimonas altamirensis, Aureimonas ureilytica,andAureimonas Present address: Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita, frigidaquae, and its members have been isolated from diverse Osaka 565-0871, Japan. environments (10). To characterize this bacterial group from a 3To whom correspondence may be addressed. Email: [email protected] or genomic viewpoint, we determined the complete genome se- [email protected]. quence of Aureimonas sp. AU20, an isolate from the stem of a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. soybean plant (11). The result showed that the AU20 genome 1073/pnas.1514326112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1514326112 PNAS Early Edition | 1of5 Downloaded by guest on September 28, 2021 A RLC clade RLC clade RLC clade ans e a cida lytica li a nganoxyd ei 0 r reilytica igidaquae 20 2 r U . manganoxydans f U12 U4 A AU40 AU20 AU40 D3 N4 A. ureilyticaA. altamirensisA. frigidaquaeA. coralicida A. manganoxydansF. pelagiAU22 AU12 AU 4 D3 N4 A. ur A. altamirensisA. frigidaquA. coralicida A F. pelagiAU22 AU12 AU4 AU AU40 D3 N4 A. u A. altamirensisA. A. co A. maF. pelagiAU22 A A (kb) (kb) 17.5 17.5 15 15 12.5 12.5 10 10 7.5 7.5 5 5 I-CeuI I-CeuI EcoRI E B RepA E E RepB RepA RepA RepA RepA RepA E E E Rep’ Rep’ Rep’ 5S 5S 5S 5S 5S pAU12rrn 5S RepC pAU20rrn pD3rrn pN4rrn pAUTrrn 13,404 bp pAU40rrn E E E E E 9,902 bp 9,969 bp E 9,393 bp 9,396 bp 9,912 bp E 23S 23S 16S 16S 23S 23S 23S 23S 16S 16S 16S 16S AU20 AU40 D3 N4 A. ureilytica AU12 Fig. 1. The rrn operons in members of the family Aurantimonadaceae. (A) Southern hybridization analysis. Total DNA was digested with I-CeuI, separated by electrophoresis (Left), transferred to a membrane, and probed with the 16S rRNA gene (Middle). (Right) EcoRI-digested total DNA was separated by electrophoresis, transferred, and probed with the same gene. One of the bands predictable from the genome sequence was not detected in the EcoRI-digested DNA of AU12 and AU4 (corresponding to a 34.3-kb band and a 48.2-kb band, respectively). (B)Mapsoftherrn-plasmids in strains AU20, AU40, D3, N4, and A. ureilyltica and another type of rrn-carrying replicon in AU12. Positions of EcoRI sites are marked with a red “E.” The rRNA and tRNA genes are shown in black. RepA and Rep′ encoded on the rrn-plasmids and RepC on pAU12rrn are homologs of different replication initiators. RepA and RepB on pAU12rrn are homologs of partitioning proteins. consensus hexamers (13) (Fig. S2B) and a putative rho-independent copies in the cell varied from 18 to 34 per chromosome equivalent terminator (Fig. S2C). throughout the growth phases (Fig. S4 C and D). The deduced The regions of the origin and terminus of replication of the amino acid sequences of the two ORFs present outside the rrn and chromosome (oriC and ter, respectively) were not unusual in repA/oriV regions did not have any motifs that are conserved among alphaproteobacteria (Fig. S3). In contrast, the replication system of proteins involved in active partitioning of replicons, suggesting the pAU20rrn was similar to the replication system of pPS10, a 10-kb stochastic segregation of pAU20rrn. high-copy-number plasmid isolated from Pseudomonas syringae that Taken together, pAU20rrn does not match the criteria of a has a stable host range confined to Pseudomonas species (14–16).