title: The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System.
abstract: The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence factor important for phagocytic avoidance, disruption of host cell signaling, and host cell cytotoxicity. ExsA is the master regulator of T3SS transcription. The expression, synthesis, and activity of ExsA is tightly regulated by both intrinsic and extrinsic factors. Intrinsic regulation consists of the well-characterized ExsECDA partner-switching cascade, while extrinsic factors include global regulators that alter exsA transcription and/or translation. To identify novel extrinsic regulators of ExsA, we conducted a transposon mutagenesis screen in the absence of intrinsic control. Transposon disruptions within gene PA2840, which encodes a homolog of the Escherichia coli RNA-helicase DeaD, significantly reduced T3SS gene expression. Recent studies indicate that E. coli DeaD can promote translation by relieving inhibitory secondary structures within target mRNAs. We report here that PA2840, renamed DeaD, stimulates ExsA synthesis at the posttranscriptional level. Genetic experiments demonstrate that the activity of an exsA translational fusion is reduced in a deaD mutant. In addition, exsA expression in trans fails to restore T3SS gene expression in a deaD mutant. We hypothesized that DeaD relaxes mRNA secondary structure to promote exsA translation and found that altering the mRNA sequence of exsA or the native exsA Shine-Dalgarno sequence relieved the requirement for DeaD in vivo. Finally, we show that purified DeaD promotes ExsA synthesis using in vitro translation assays. Together, these data reveal a novel regulatory mechanism for P. aeruginosa DeaD and add to the complexity of global regulation of T3SS.
  
  
  OBJECTIVE
  Although members of the DEAD box family of RNA helicases are appreciated for their roles in mRNA degradation and ribosome biogenesis, an additional role in gene regulation is now emerging in bacteria. By relaxing secondary structures in mRNAs, DEAD box helicases are now thought to promote translation by enhancing ribosomal recruitment. We identify here an RNA helicase that plays a critical role in promoting ExsA synthesis, the central regulator of the Pseudomonas aeruginosa type III secretion system, and provide additional evidence that DEAD box helicases directly stimulate translation of target genes. The finding that DeaD stimulates exsA translation adds to a growing list of transcriptional and posttranscriptional regulatory mechanisms that control type III gene expression.
authors.affiliation: Department of Microbiology, University of Iowa, Iowa City, Iowa, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Intile
authors.affiliation: Department of Microbiology, University of Iowa, Iowa City, Iowa, USA.
authors.forename: Grant J
authors.initials: GJ
authors.lastname: Balzer
authors.affiliation: Department of Microbiology and Immunology and Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina, USA.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Wolfgang
authors.affiliation: Department of Microbiology, University of Iowa, Iowa City, Iowa, USA tim-yahr@uiowa.edu.
authors.forename: Timothy L
authors.initials: TL
authors.lastname: Yahr
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D058947:Bacterial Secretion Systems; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D004251:DNA Transposable Elements; D015964:Gene Expression Regulation, Bacterial; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D037521:Virulence Factors / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D004251:DNA Transposable Elements; C095092:ExsA protein, bacteria; D012333:RNA, Messenger; D015534:Trans-Activators; D037521:Virulence Factors; D053487:DEAD-box RNA Helicases