The Intervening Sequence of Coxiella burnetii: Characterization and Evolution.

Indu Warrier,Rahul Raghavan,Mathias C Walter,Linda D Hicks,Dimitrios Frangoulidis,Michael F Minnick

doi:10.3389/fcimb.2016.00083

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.

Highlights

Coxiella burnetii, the etiological agent of Q fever in humans, is an obligate intracellular pathogen that replicates in a parasitophorous vacuole (PV; Voth and Heinzen, 2007)
C. burnetii was inoculated at a concentration of 1.6 × 104 genome equivalents per ml (GE/ml) quantified by quantitative PCR with a primer set specific to the C. burnetii rpoS gene (Table S1; Coleman et al, 2004)
intervening sequence (IVS) elements of other bacteria are associated with 23S rRNA genes and excised by RNase III (Burgin et al, 1990; Ralph and McClelland, 1993; EvguenievaHackenberg and Klug, 2000); an endoribonuclease that cleaves rRNA precursors during maturation (Dunn and Studier, 1973)

Summary

Introduction

The etiological agent of Q (query) fever in humans, is an obligate intracellular pathogen that replicates in a parasitophorous vacuole (PV; Voth and Heinzen, 2007). IVSs have been identified in several bacteria, including Salmonella (Burgin et al, 1990), Leptospira (Ralph and McClelland, 1993), Yersinia (Skurnik and Toivanen, 1991), Campylobacter (Konkel et al, 1994), Proteus and Providencia (Miller et al, 2000) Within these bacteria, IVSs occur only in certain isolates, and not all rrn operons contain the IVS in a given bacterial strain (Burgin et al, 1990; Hsu et al, 1992; Mattatall and Sanderson, 1996; Miller et al, 2000). Conservation of IVS insertion sites across species indicates that continuity of the 23S rRNA at these positions is not necessary for ribosome function

Objectives

Methods

Results