The Unusual 23S rRNA Gene of Coxiella burnetii : Two Self-Splicing Group I Introns Flank a 34-Base-Pair Exon, and One Element Lacks the Canonical ΩG

Abstract

We describe the presence and characteristics of two self-splicing group I introns in the sole 23S rRNA gene of Coxiella burnetii. The two group I introns, Cbu.L1917 and Cbu.L1951, are inserted at sites 1917 and 1951 (Escherichia coli numbering), respectively, in the 23S rRNA gene of C. burnetii. Both introns were found to be self-splicing in vivo and in vitro even though the terminal nucleotide of Cbu.L1917 is adenine and not the canonical conserved guanine, termed ⍀G, found in Cbu.L1951 and all other group I introns described to date. Predicted secondary structures for both introns were constructed and revealed that Cbu.L1917 and Cbu.L1951 were group IB2 and group IA3 introns, respectively. We analyzed strains belonging to eight genomic groups of C. burnetii to determine sequence variation and the presence or absence of the elements and found both introns to be highly conserved (>99%) among them. Although phylogenetic analysis did not identify the specific identities of donors, it indicates that the introns were likely acquired independently; Cbu.L1917 was acquired from other bacteria like Thermotoga subterranea and Cbu.L1951 from lower eukaryotes like Acanthamoeba castellanii. We also confirmed the fragmented nature of mature 23S rRNA in C. burnetii due to the presence of an intervening sequence. The presence of three selfish elements in C. burnetii's 23S rRNA gene is very unusual for an obligate intracellular bacterium and suggests a recent shift to its current lifestyle from a previous niche with greater opportunities for lateral gene transfer.Group I introns are a distinct class of catalytic RNAs (ribozymes) that are considered a legacy of a primordial RNA world (40). They are able to self-splice by means of a two-step transesterification reaction using a guanosine molecule as a cofactor. The catalytic structure that is conserved among all group I introns consists of a specific arrangement of about 10 paired elements (P) that are capped by loops and connected by junctions (J) (38). The core of each intron is comprised of two separately folding helical domains made up of P4-P5-P6 and P3-P7-P9. The P4-P6 domain structurally supports the P3-P9 domain, which contains the active site (17). Splice sites are determined by helix P1, which pairs with the 5Ј substrate strand (5Ј exon), and by helix P10, which pairs with the 3Ј substrate strand (3Ј exon). A conserved G · U wobble pair contributes to the recognition of the 5Ј splice site, whereas the 3Ј splice site is recognized in part by a conserved terminal guanine, termed ⍀G (23). Based on conserved secondary-structure characteristics, group I introns are further classified into 13 subgroups (23, 34). These selfish genetic elements are distributed widely in nature, albeit with a bias towards fungi, plants, and red or green algae (14). Although group I introns are abundant in mitochondria and chloroplasts of lower eukaryotes, they are relatively rare in bacteria and until recently were unknown in bacterial structural RNAs. Group I introns in 23S rRNA genes have been...

Multiple self-splicing introns in the 16S rRNA genes of giant sulfur bacteria

“…Furthermore, introns are preferentially found in conserved regions of the rRNA genes (21,22), which can explain why the insertion sites of introns analyzed in this study are homologous to locations of introns found in mitochondrial and nuclear rRNA genes of eukaryotes. This is consistent with the hypothesis that introns can be horizontally transferred over large phylogenetic distances (50,51). All ORFs in the introns described here encoded IEPs typical of a common family of homing endonucleases, suggesting that the introns spread into cognate sites as DNA elements via homing (20).…”

“…Thus, it is tempting to speculate that large sulfur bacteria are the only group of bacteria accumulating introns in their 16S rRNA genes. However, the presence of introns, even multiple ones, has been described for the 23S rRNA genes of several distantly related bacteria (39,50,51,64,65). This gene bears the same evolutionary importance and conservation as the 16S rRNA gene, which indicates that 16S introns might be at least as frequent as 23S introns.…”

Toxic Introns and Parasitic Intein in Coxiella burnetii : Legacies of a Promiscuous Past

“…These genetic elements spread efficiently into an intronless cognate site by a process called homing at the DNA level or by reverse splicing at the RNA level (60). The typical secondary structure of a group I intron consists of about 10 paired (P) elements (59,74). P1 and P10 are complementary to the 5Ј exon and 3Ј exon, respectively, and are collectively called the internal guide sequence (IGS), which the intron uses to locate its 5Ј and 3Ј splice sites (31).…”

“…These traits are conferred by a biphasic developmental cycle that alternates between a fragile, metabolically active large cell variant (LCV) and a durable, dormant small cell variant (SCV) (8). Even though C. burnetii occupies an acidic parasitophorous vacuole where the opportunity for HGT is likely minimal, its genome contains 29 insertion sequences strewn across the chromosome, two group I introns and an intervening sequence (IVS) within its sole 23S rRNA gene, and a putative intein in the C-terminal region of the replicative DNA helicase (dnaB) gene (59,66).Group I introns self-splice independently of proteins (ribozyme) and are considered a holdover from a primordial RNA world (77). These genetic elements spread efficiently into an intronless cognate site by a process called homing at the DNA level or by reverse splicing at the RNA level (60).…”

Caldimicrobium thiodismutans sp. nov., a sulfur-disproportionating bacterium isolated from a hot spring, and emended description of the genus Caldimicrobium

“…In the 23S rRNA gene, an insert region was identified. The insertion exhibited homology to self-splicing group I introns in the 23S rRNA gene of Coxiella burnetii, including a region encoding a homing endonuclease (Raghavan et al, 2007).…”