The Intervening Sequence of Coxiella burnetii: Characterization and Evolution

Abstract

The intervening sequence (IVS) of Coxiella burnetii, the agent of Q fever, is a 428-nt selfish genetic element located in helix 45 of the precursor 23S rRNA. The IVS element, in turn, contains an ORF that encodes a hypothetical ribosomal S23 protein (S23p). Although S23p can be synthesized in vitro in the presence of an engineered E. coli promoter and ribosome binding site, results suggest that the protein is not synthesized in vivo. In spite of a high degree of IVS conservation among different strains of C. burnetii, the region immediately upstream of the S23p start codon is prone to change, and the S23p-encoding ORF is evidently undergoing reductive evolution. We determined that IVS excision from 23S rRNA was mediated by RNase III, and IVS RNA was rapidly degraded, thereafter. Levels of the resulting 23S rRNA fragments that flank the IVS, F1 (~1.2 kb) and F2 (~1.7 kb), were quantified over C. burnetii's logarithmic growth phase (1–5 d). Results showed that 23S F1 quantities were consistently higher than those of F2 and 16S rRNA. The disparity between levels of the two 23S rRNA fragments following excision of IVS is an interesting phenomenon of unknown significance. Based upon phylogenetic analyses, IVS was acquired through horizontal transfer after C. burnetii's divergence from an ancestral bacterium and has been subsequently maintained by vertical transfer. The widespread occurrence, maintenance and conservation of the IVS in C. burnetii imply that it plays an adaptive role or has a neutral effect on fitness.

Coxiella burnetiismall RNA 12 binds CsrA regulatory protein and transcripts for the CvpD type IV effector, regulates pyrimidine and methionine metabolism, and is necessary for optimal intracellular growth and vacuole formation during infection

“…RNase III Assay was performed as previously described [46]. Briefly, 200 nM substrate RNA was incubated for 30 minutes at 37°C with or without recombinant C. burnetii RNase III or E. coli RNase III.…”

“…Briefly, 200 nM substrate RNA was incubated for 30 minutes at 37°C with or without recombinant C. burnetii RNase III or E. coli RNase III. C. burnetii intervening sequence (IVS) RNA was used as a positive control substrate [46]. The resulting reactions were electrophoresed on a 7% denaturing polyacrylamide gels stained with 2 μg/mL acridine orange to visualize.…”

“…Briefly, biotin-labeled CbsR12 was synthesized in vitro as described above for RNA-RNA EMSAs. C. burnetii csrA-1 and csrA-2 genes were cloned into the expression vector pQE30, expressed, and natively purified as previously described [46]. 1 nM biotin-labeled CbsR12 diluted in TE buffer (10mM Tris-HCL, 1mM EDTA) was heated at 75°C for 3 minutes and equilibrated to room temperature for 10 minutes.…”

“…Recombinant Coxiella RNase III was expressed from a previously generated pQE30 construct and purified as before [46]. C. burnetii carA and metK genes were cloned in frame into the pQE30 expression vector (Qiagen) and the resulting N-terminal His 6 -tagged proteins were expressed and purified as previously described for the C. burnetii RNA helicase [90].…”

“…RNase III assay of in vitro- transcribed CbsR12 with the C. burnetii IVS RNA as a positive control substrate. Results from treatment with E. coli ( Ec ) RNase III (New England BioLabs), recombinant C. burnetii ( Cb ) RNase III or no-enzyme controls are shown and were done as previously described [46]. Arrows indicate RNase III-processed (blue) and un-processed (red) CbsR12 RNA.…”