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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.
Bacteria usually have at least one rRNA operon on the chromosome, suggesting that the exclusive presence of rRNA operons on a plasmid is rare and unlikely to be stably maintained. Here, Anda et al. find that at least four bacterial clades in different phyla lost their chromosomal rRNA operons independently, and one of the clades has maintained this peculiar genome organization for hundreds of millions of years.