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

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 5′ substrate strand (5′ exon), and by helix P10, which pairs with the 3′ substrate strand (3′ exon). A conserved G · U wobble pair contributes to the recognition of the 5′ splice site, whereas the 3′ splice site is recognized in part by a conserved terminal guanine, termed ΩG (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 reported to occur in hyperthermophilic bacteria of the genus Thermotoga (26) and in Simkania negevensis, a Chlamydiales member (7). A putative group I intron was detected in the sole 23S rRNA gene of Coxiella burnetii (strain Nine Mile phase I RSA493) when the entire genome was sequenced (31); in addition, an intervening sequence (IVS) that produces a fragmented 23S rRNA had been reported earlier by Afseth et al. (1). C. burnetii, the etiological agent of human Q fever, is a gram-negative obligate intracellular bacterium. This category B select agent is distributed almost worldwide and has a broad range of susceptible hosts, including arthropods, fish, birds, and wild and domestic mammals (6). Human infection is acquired mainly through inhalation of aerosols of animal origin and can cause a wide array of conditions, ranging from asymptomatic infection to an acute self-limiting febrile illness or severe, chronic diseases like endocarditis and hepatitis (21). The aim of this study was to characterize the splicing properties of the intron reported by Seshadri et al. (31) in the 23S rRNA gene of C. burnetii, since both nonsplicing and self-splicing introns are known to occur in bacterial 23S rRNA (7, 26). During our investigation, we discovered that, in contrast to what was noted in the earlier report, two group I self-splicing introns flank a 34-bp exon and interrupt the 23S rRNA gene of C. burnetii. We also discovered that the terminal nucleotide of one of the introns was adenine and not the canonical ΩG. This is the first time that a group I intron without ΩG has been found in nature. This is significant because the ΩG is considered invariable and thought to play an essential role in RNA splicing (38, 40). This novel intron is also smaller than most group I introns, as it does not encode a homing endonuclease (HE), making it more difficult to identify in whole-genome sequences than relatively larger introns that code for an LAGLIDADG HE. Nevertheless, a nucleotide BLAST search against GenBank revealed a number of group I introns with conserved sequences. Secondary structures for both introns were also inferred by locating conserved sequences. In addition to sequencing the Nine Mile strain, we sequenced the introns of eight other strains of C. burnetii representing seven additional genomic groups (3, 15) and found them to be highly conserved (Table ​(Table1),1), implying a possible role in C. burnetii's biology. TABLE 1. C. burnetii 23S rRNA gene group I introns are highly conserved among eight genotypes