RsmW, Pseudomonas aeruginosa small non-coding RsmA-binding RNA upregulated in biofilm versus planktonic growth conditions

Abstract

BackgroundBiofilm development, specifically the fundamentally adaptive switch from acute to chronic infection phenotypes, requires global regulators and small non-coding regulatory RNAs (sRNAs). This work utilized RNA-sequencing (RNA-seq) to detect sRNAs differentially expressed in Pseudomonas aeruginosa biofilm versus planktonic state.ResultsA computational algorithm was devised to detect and categorize sRNAs into 5 types: intergenic, intragenic, 5′-UTR, 3′-UTR, and antisense. Here we report a novel RsmY/RsmZ-type sRNA, termed RsmW, in P. aeruginosa up-transcribed in biofilm versus planktonic growth. RNA-Seq, 5’-RACE and Mfold predictions suggest RsmW has a secondary structure with 3 of 7 GGA motifs located on outer stem loops. Northern blot revealed two RsmW binding bands of 400 and 120 bases, suggesting RsmW is derived from the 3’-UTR of the upstream hypothetical gene, PA4570. RsmW expression is elevated in late stationary versus logarithmic growth phase in PB minimal media, at higher temperatures (37 °C versus 28 °C), and in both gacA and rhlR transposon mutants versus wild-type. RsmW specifically binds to RsmA protein in vitro and restores biofilm production and reduces swarming in an rsmY/rsmZ double mutant. PA4570 weakly resembles an RsmA/RsmN homolog having 49 % and 51 % similarity, and 16 % and 17 % identity to RsmA and RsmN amino acid sequences, respectively. PA4570 was unable to restore biofilm and swarming phenotypes in ΔrsmA deficient strains.ConclusionCollectively, our study reveals an interesting theme regarding another sRNA regulator of the Rsm system and further unravels the complexities regulating adaptive responses for Pseudomonas species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0771-y) contains supplementary material, which is available to authorized users.

Global transcriptome responses including small RNAs during mixed‐species interactions with methicillin‐resistant Staphylococcus aureus and Pseudomonas aeruginosa

“…A suicide vector was generated using In‐Fusion HD cloning kit (Clontech Laboratories), as described previously (Miller et al., ), which allowed for deletion of the pa1595 gene through allelic exchange with an antibiotic cassette, pucGM. Briefly, primers were designed according to the manufacturer's suggestion (Table S1) 1 kb upstream and downstream regions of the pa1595 gene were amplified by PCR from P. aeruginosa chromosome.…”

Broth versus Surface-Grown Cells: Differential Regulation of RsmY/Z Small RNAs in Pseudomonas aeruginosa by the Gac/HptB System

“…However, here we present a model where small RNA regulation is dependent on the conditions in which we study P. aeruginosa (i.e., broth versus surface) which favor two different bacterial lifestyles. Furthermore, RsmW a novel RNA under the control of the Gac system has been identified to be strongly upregulated in biofilm conditions compared to planktonic cultures which further strengthens the existence of differential genetic regulation depending on the selected growth conditions (Miller et al, 2016). Our data confirm that the GacS/GacA system is not the only one responsible for the control of rsmZ expression and implicates the presence of unidentified key factors that are important when cells are grown on a surface rather than in planktonic cultures, as exemplified by the use of swarming motility as a model for surface behavior ( Figure 5 ).…”

RsmV a small non-coding regulatory RNA in Pseudomonas aeruginosa that sequesters RsmA and RsmF from target mRNAs

“…Both of these findings are consistent with RsmV serving a role in RsmA/RsmF sequestration. When compared to the previously identified sequestering RNAs RsmW, RsmY, and RsmZ (13, 31), RsmV demonstrated activity comparable to RsmY when using the P tssA1’-‘lacZ reporter (Fig. 3B) and had the strongest inhibitory activity for the P exsD-lacZ reporter (Fig.…”

“…Transcription of rsmY and rsmZ is controlled by the GacAS two component system (20). A previous study found that GacAS does not control rsmW transcription (31). To determine whether rsmV transcription is regulated by GacA, a P rsmV-lacZ transcriptional reporter was integrated at the ΦCTX phage attachment site of wt cells and a ∆ gacA mutant.…”

“…The primary RsmA/RsmF sequestering RNAs in Pseudomonas aeruginosa are RsmY and RsmZ. In addition to RsmY/RsmZ, RsmW plays a smaller role in the sequestration of RsmA (31) and can also sequester RsmF (Fig. 2).…”