Characterization of the role of ribonucleases in Salmonella small RNA decay

Abstract

In pathogenic bacteria, a large number of sRNAs coordinate adaptation to stress and expression of virulence genes. To better understand the turnover of regulatory sRNAs in the model pathogen, Salmonella typhimurium, we have constructed mutants for several ribonucleases (RNase E, RNase G, RNase III, PNPase) and Poly(A) Polymerase I. The expression profiles of four sRNAs conserved among many enterobacteria, CsrB, CsrC, MicA and SraL, were analysed and the processing and stability of these sRNAs was studied in the constructed strains. The degradosome was a common feature involved in the turnover of these four sRNAs. PAPI-mediated polyadenylation was the major factor governing SraL degradation. RNase III was revealed to strongly affect MicA decay. PNPase was shown to be important in the decay of these four sRNAs. The stability of CsrB and CsrC seemed to be independent of the RNA chaperone, Hfq, whereas the decay of SraL and MicA was Hfq-dependent. Taken together, the results of this study provide initial insight into the mechanisms of sRNA decay in Salmonella, and indicate specific contributions of the RNA decay machinery components to the turnover of individual sRNAs.

The influence of Hfq and ribonucleases on the stability of the small non-coding RNA OxyS and its targetrpoSinE. coliis growth phase dependent

“…It is known for some sRNAs that their turn-over is catalyzed by the endoribunucleases E or III. [23][24][25] Base pairing to the target RNA (B) stability determination of Oxys sRNA in strain lacking pNpase-RNase II (sK5726) and in a strain lacking pNpase-RNase II-RNase e ts (SK5704) and the isogenic wild type background MG1693. OxyS RNA half-lives were calculated from at least 3 independent biological repeats and the standard deviation is given.…”

“…We also analyzed strains lacking the RNA binding protein Hfq, which is known to affect RNA stability. 22,23 Since we found that DsrA is also expressed at high levels in the presence of hydrogen peroxide and we considered an influence of DsrA on OxyS stability, we also constructed and analyzed mutants lacking DsrA. Our data reveal a complex interplay of the different RNases, Hfq and DsrA in OxyS processing, which is strongly dependent on growth phase.…”

Regulation ofSalmonella entericaPathogenicity Island 1 by DNA Adenine Methylation

“…The oligonucleotides used for disruption (labeled ''UP'' and ''DO'') are listed in supporting information, Table S1, together with the oligonucleotides (labeled ''E'') used for allele verification by the polymerase chain reaction. Disruption of the rne gene, which encodes ribonuclease E, was performed with primers that eliminate the C-terminal region (Viegas et al 2007). For the construction of transcriptional and translational lac fusions in the Salmonella chromosome, FRT sites generated by excision of Km r cassettes (Datsenko and Wanner 2000) were used to integrate either plasmid pCE37 or pCE40 (Ellermeier et al 2002).…”

“…Ribonuclease E had been previously described as a SPI-1 regulator (Fahlen et al 2000). For construction of an Rne À mutant, only a portion at the 39 end of the rne coding sequence was eliminated (Viegas et al 2007). Analysis of hilD mRNA content was performed in two sets of experiments.…”

Discovery and Validation of New Regulatory RNA Elements in Chlamydia trachomatis

“…CTIG270 may be playing a role in the lack of bacterial cell division. C. trachomatis, although lacking Hfq and RNase E, encodes a homolog of RNase III which, among other functions, can degrade dsRNA (Viegas et al, 2007). To test this phenomenon we may need to genetically control the expression of ftsI inside Chlamydia and measure the effect on CTIG270 transcript half life at various time points.…”