Transcriptome-Wide Identification of Hfq-Associated RNAs in Brucella suis by Deep Sequencing

Abstract

Recent breakthroughs in next-generation sequencing technologies have led to the identification of small noncoding RNAs (sRNAs) as a new important class of regulatory molecules. In prokaryotes, sRNAs are often bound to the chaperone protein Hfq, which allows them to interact with their partner mRNA(s). We screened the genome of the zoonotic and human pathogen Brucella suis 1330 for the presence of this class of RNAs. We designed a coimmunoprecipitation strategy that relies on the use of Hfq as a bait to enrich the sample with sRNAs and eventually their target mRNAs. By deep sequencing analysis of the Hfq-bound transcripts, we identified a number of mRNAs and 33 sRNA candidates associated with Hfq. The expression of 10 sRNAs in the early stationary growth phase was experimentally confirmed by Northern blotting and/or reverse transcriptase PCR.

Absence of the KhpA and KhpB (JAG/EloR) RNA‐binding proteins suppresses the requirement for PBP2b by overproduction of FtsA in Streptococcus pneumoniae D39

“…S11). We performed RIP‐Seq (RNA‐immunoprecipitation) experiments without crosslinking using KhpA‐L‐FLAG 3 or KhpB‐L‐FLAG 3 as bait to demonstrate that KhpA and KhpB indeed bind to RNA in cells (see Experimental procedures) (Saadeh et al ., ). We used the ratio of RNA reads obtained for each of the FLAG‐tagged strains compared to the untagged wild‐type strain as the criterion for RNA binding of RNA to KhpA or KhpB (Supporting Information Table S4).…”

“…Several general conclusions emerged from the RIP‐Seq experiments. With a fourfold enrichment as the cutoff (Saadeh et al ., ), KhpA or KhpB pulled down a remarkable overlapping set of ≈170 mRNA, tRNAs, intergenic RNAs and putative sRNAs (Fig. ; Supporting Information Table S4).…”

Small Noncoding RNA AbcR1 Addressing Multiple Target mRNAs From Transcriptional Factor and Two-Component Response Regulator of Brucella melitensis

“…Moreover, sRNAs can regulate gene expression and/or allow for rapid alterations to encounter stimuli and stress (30). While more than 200 sRNAs have been experimentally validated in bacteria, such as Salmonella enterica and E. coli, only 5 sRNAs from Brucella have been studied so far (31,32). In the present study, we identified and validated mRNA-AbcR sRNA interactions with B. melitensis target mRNAs (which belong to the transcription regulator family), as well as Two-component response regulators.…”

Comparative Transcriptome Analysis of Artificially Induced Rough-Mutant Brucella Strain RM57 and Its Parent Strain Brucella melitensis M1981

“…Hfq protein, encoded by the BMEI0872 gene is an important RNA chaperone involved in stress resistance and Brucella virulence in both macrophage and mice infection models (19). Furthermore, recent studies have demonstrated that this chaperone can regulate virB operon expression and plays a key role in the interaction between sRNAs and their target mRNAs (20,21). Downregulation of this gene in our mutant strain therefore indicates a reduction in virulence relative to the parental line.…”