Contribution of teg49 Small RNA in the 5′ Upstream Transcriptional Region of sarA to Virulence in Staphylococcus aureus

Abstract

c High-throughput RNA sequencing technology has found the 5= untranslated region of sarA to contain two putative small RNAs (sRNAs), designated teg49 and teg48. Northern blot analysis disclosed that teg49 and teg48 were detectable within the P3-P1 and P1 sarA promoter regions, respectively. Focusing on teg49, we found that this sRNA, consisting of 196 nucleotides, is transcribed in the same direction as the sarA P3 transcript. The expression of both P3 and teg49 transcripts is dependent on sigB and cshA, which encodes a DEAD box RNA helicase. Within the sRNA teg49, there are two putative hairpin-loop structures, HP1 and HP2. Transversion mutation of the HP1 loop produced a smaller amount of sarA P3 and P2 transcripts and SarA protein than the corresponding HP1 stem and the HP2 stem and loop mutations, leading to lower RNAII transcription and derepression of aur transcription. The HP1 loop mutant also exhibited less biofilm formation than the parental and complemented strains. Complementation with shuttle plasmid pEPSA5 carrying teg49 was able to reestablish sarA P3 and P2 transcription and augment RNAII expression in the HP1 loop mutant. We thus conclude that teg49, embedded within the extended promoter regions of sarA, is modulated by sigB and cshA and plays an important trans-acting role in modulating the transcription and ensuing expression of sarA.

Characterization of RNA Helicase CshA and Its Role in Protecting mRNAs and Small RNAs of Staphylococcus aureus Strain Newman

“…Of interest are teg049 and teg048, both of which are found within the 5= untranscribed region (UTR) of sarA (Fig. 4A) (23). The sarA P1 and P3 transcripts were also protected by CshA upon MazF sa induction, concordant with the Northern blot data (Fig.…”

“…Cells were incubated for 30 min at 37°C and harvested at serial time points at 0, 1, 2, 5, and 10 min after rifampin treatment (200 g/ml). Bacterial pellets were resuspended in 1 ml of TRIzol (Invitrogen) with 0.1-mm glass/zirconia beads, and RNA was extracted as described previously (23). Total RNA (10 or 25 g) of each sample was applied to each lane of the RNA gel for Northern blotting as described previously (23).…”

“…Isolation of RNA and Northern blot hybridization were performed as previously described (23). In brief, overnight S. aureus cultures diluted 1:1,000 in TSB were grown at 37°C and induced with 1 mM IPTG to activate MazF sa expression at an OD 650 of ϳ0.4.…”

“…Both the cshA mutant and the complemented mutant of ALC6094, a Newman derivative, were created as described previously (23).…”

A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae

“…As a result, an ever-growing library of virulence-related regulatory RNAs is being established in this relatively young field of life science. As recent specific examples, there are: the sRNA RyhB, which participates in the regulation of the production of siderophores in E. coli (Porcheron and Dozois 2015); the Staphylococcus aureus sRNA teg49, whose lack hampers biofilm formation (Kim et al 2014), the sRNA NrsZ, which modulates Pseudomonas aeruginosa motility (Wenner et al 2014); and four novel sRNAs in Enterococcus faecalis, whose deletions affected bacterial virulence and stress tolerance when compared to the wild-type strains (Michaux et al 2014a). CRISPR elements are usually considered as the bacterial innate immune system to face FIGURE 6.…”