Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA

Abstract

Summary One of the prokaryotic post-transcriptional regulatory mechanisms involves the CsrA/RsmA family of proteins that act by modulating translation initiation at target mRNAs. In this study, we identified the regulon of RsmA of the Pseudomonas aeruginosa PAK strain by using cultures in the stationary phase of growth. The RsmA regulon includes over 500 genes, of which approximately one third were affected by an rsmA mutation negatively, while the rest were affected positively. By isolating RsmA/mRNA complexes, analyzing transcriptional and translational fusions, and performing gel-shift analyses, we identified 40 genes in six operons that are regulated by RsmA directly at the level of translation. All of these genes were affected by RsmA negatively and include genes encoding the type VI secretion system HSI-I, which has been implicated in the P. aeruginosa chronic infections. On the other hand, we were unable to demonstrate a direct interaction of RsmA with transcripts that are positively affected by this protein, including mRNAs encoding the type III secretion system and the type IV pili genes. Our work supports a model in which RsmA acts as a negative translational regulator, and where its positive effects are achieved indirectly by RsmA-mediated interference with translation of specific regulatory factors.

Sticky Situations: Key Components That Control Bacterial Surface Attachment

“…This intricate signaling system has been implicated as a switch between acute/planktonic and chronic/biofilm modes of growth by reciprocally regulating gene expression associated with type III secretion or type VI secretion and exopolysaccharide production. Ultimately, this system controls attachment and virulence via the CsrA homolog RsmA (18). While RsmA is thought to elevate type III secretion system (TTSS) transcript abundance, binding of RsmA to the 5= untranslated region of psl mRNA was recently shown to prevent ribosome access and protein translation (66).…”

FliW antagonizes CsrA RNA binding by a noncompetitive allosteric mechanism

“…Mutation of CsrA in these organisms has dramatic and highly pleiotropic defects in carbon metabolism, quorum sensing, biofilm formation, and motility (17)(18)(19)(20). CsrA-deficient bacterial pathogens are typically attenuated for virulence, which is likely a result of misregulation of target gene expression and the resulting inability to make critical physiological transitions during an infection (1,(21)(22)(23).…”

The Pseudomonas aeruginosa Two-Component Regulator AlgR Directly Activates rsmA Expression in a Phosphorylation-Independent Manner

“…The tssA1 gene encodes a portion of the T6SS and is directly regulated by RsmA (29,46). Previous studies have used a lacUV5 tssA1 construct containing 227 bp of tssA1 upstream sequence that had extremely low activity (28,29,31).…”

“…To ascertain whether AlgR control of rsmA was significant in a biological context, the direct RsmA targets hcnA and tssA1 were used to assess RsmA activity (29). A new integrating vector was constructed that contains the lacUV5 promoter and lacks a ribosome-binding site or a start codon for lacZ.…”

“…RsmA is considered a global regulator that controls the expression of many P. aeruginosa genes by binding to mRNAs (29). Two noncoding RNAs, RsmY and RsmZ, counteract RsmA (29,30).…”