Loss of RNase R Induces Competence Development in Legionella pneumophila

Abstract

RNase R is a processive 3-5 exoribonuclease with a high degree of conservation in prokaryotes. Although some bacteria possess additional hydrolytic 3-5 exoribonucleases such as RNase II, RNase R was found to be the only predicted one in the facultative intracellular pathogen Legionella pneumophila. This provided a unique opportunity to study the role of RNase R in the absence of an additional RNase with similar enzymatic activity. We investigated the role of RNase R in the biology of Legionella pneumophila under various conditions and performed gene expression profiling using microarrays. At optimal growth temperature, the loss of RNase R had no major consequence on bacterial growth and had a moderate impact on normal gene regulation. However, at a lower temperature, the loss of RNase R had a significant impact on bacterial growth and resulted in the accumulation of structured RNA degradation products. Concurrently, gene regulation was affected and specifically resulted in an increased expression of the competence regulon. Loss of the exoribonuclease activity of RNase R was sufficient to induce competence development, a genetically programmed process normally triggered as a response to environmental stimuli. The temperature-dependent expression of competence genes in the rnr mutant was found to be independent of previously identified competence regulators in Legionella pneumophila. We suggest that a physiological role of RNase R is to eliminate structured RNA molecules that are stabilized by low temperature, which in turn may affect regulatory networks, compromising adaptation to cold and thus resulting in decreased viability.RNA molecules provide the dynamic link between DNAencoded information and protein synthesis. The unstable, labile nature of RNA is critical, as it allows a rapid adjustment of specific protein expression levels in response to environmental changes. In bacteria, RNA degradation occurs through an initial endoribonuclease cleavage followed by 3Ј-5Ј exoribonuclease digestion (6,14). In Escherichia coli, the latter reaction involves three processive 3Ј-5Ј exoribonucleases: PNPase, RNase R, and RNase II. PNPase is a phosphorolytic enzyme and part of the degradosome, a multiprotein complex also involving the RNA helicase RhlB, an enolase, and the endoribonuclease RNase E (27). RNase II and RNase R are hydrolytic exoribonucleases that share similar domain architectures and belong to the RNase II family of RNases (18,23,38). In contrast to RNase II, RNase R is able to degrade structured RNA in vitro without the aid of helicase activity (11) and leads to 2nt as the end product (1, 11). However, the in vivo function of RNase R is poorly understood. The enzyme has been reported to play a role in rRNA quality control and the decay of mRNA fragments with extensive secondary structures (10, 12). RNase R is emerging as an important component in adaptation to stress. In the ribosome rescue system, RNase R associates with SmpB and the tmRNA (21) and participates in the degradation of the mRNA on which tmRNA-...

Antibiotics and UV Radiation Induce Competence for Natural Transformation inLegionella pneumophila

“…After 24 h of growth in 1 ml of AYE, UV-irradiated cells or norfloxacin-exposed cells were collected by centrifugation, and the total RNA was extracted with 1 ml of TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Northern blot analyses were performed with denaturing Tris-borate-EDTA (TBE)-urea 6% acrylamide gel as previously described (6). Briefly, 2 g of total RNA in denaturing buffer was loaded per lane and run in TBE buffer.…”

“…However, the frequency of transformation can be increased after genetic alterations in the comR, proQ, and rnr genes, suggesting that L. pneumophila is capable of high transformability under conditions that have yet to be found (6,40). Transcriptional profiling of constitutively competent L. pneumophila mutants (comR, proQ, and rnr) revealed comEA as the most differentially expressed gene in the competence state (6). ComEA is a small DNA-binding periplasmic protein important for DNA uptake (7,35) and is indeed required for the natural transformation of L. pneumophila (6).…”

“…L. pneumophila has been shown to be naturally transformable at low frequency under microaerophilic conditions (41). However, the frequency of transformation can be increased after genetic alterations in the comR, proQ, and rnr genes, suggesting that L. pneumophila is capable of high transformability under conditions that have yet to be found (6,40). Transcriptional profiling of constitutively competent L. pneumophila mutants (comR, proQ, and rnr) revealed comEA as the most differentially expressed gene in the competence state (6).…”

“…Transcriptional profiling of constitutively competent L. pneumophila mutants (comR, proQ, and rnr) revealed comEA as the most differentially expressed gene in the competence state (6). ComEA is a small DNA-binding periplasmic protein important for DNA uptake (7,35) and is indeed required for the natural transformation of L. pneumophila (6).…”

σSControls Multiple Pathways Associated with Intracellular Multiplication ofLegionella pneumophila

“…The L. pneumophila strain Philadelphia-1 microarray consisting of 2,997 unique 70-mer oligonucleotides representing all of the L. pneumophila predicted open reading frames (ORFs) presented in duplicate was previously published (16). Oligonucleotides were resuspended to a concentration of 30 M in 50% dimethyl sulfoxide and printed on UltraGaps aminosilanecoated slides (Corning Life Sciences) using a SpotArray 72 Microarray Printing System spotter (Perkin Elmer).…”