title: Tet-Trap, a genetic approach to the identification of bacterial RNA thermometers: application to Pseudomonas aeruginosa.
abstract: Modulation of mRNA translatability either by trans-acting factors (proteins or sRNAs) or by in cis-acting riboregulators is widespread in bacteria and controls relevant phenotypic traits. Unfortunately, global identification of post-transcriptionally regulated genes is complicated by poor structural and functional conservation of regulatory elements and by the limitations of proteomic approaches in protein quantification. We devised a genetic system for the identification of post-transcriptionally regulated genes and we applied this system to search for Pseudomonas aeruginosa RNA thermometers, a class of regulatory RNA that modulates gene translation in response to temperature changes. As P. aeruginosa is able to thrive in a broad range of environmental conditions, genes differentially expressed at 37 °C versus lower temperatures may be involved in infection and survival in the human host. We prepared a plasmid vector library with translational fusions of P. aeruginosa DNA fragments (PaDNA) inserted upstream of TIP2, a short peptide able to inactivate the Tet repressor (TetR) upon expression. The library was assayed in a streptomycin-resistant merodiploid rpsL(+)/rpsL31 Escherichia coli strain in which the dominant rpsL(+) allele, which confers streptomycin sensitivity, was repressed by TetR. PaDNA fragments conferring thermosensitive streptomycin resistance (i.e., expressing PaDNA-TIP2 fusions at 37°C, but not at 28°C) were sequenced. We identified four new putative thermosensors. Two of them were validated with conventional reporter systems in E. coli and P. aeruginosa. Interestingly, one regulates the expression of ptxS, a gene implicated in P. aeruginosa pathogenesis.
authors.affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, 20133 Milano, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Delvillani
authors.affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, 20133 Milano, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Sciandrone
authors.affiliation: Istituto di Tecnologie Biomediche, CNR, 20090 Segrate, Italy.
authors.forename: Clelia
authors.initials: C
authors.lastname: Peano
authors.affiliation: Istituto di Tecnologie Biomediche, CNR, 20090 Segrate, Italy Doctoral Program of Molecular and Translational Medicine, Università degli Studi di Milano, 20133 Milano, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Petiti
authors.affiliation: Department Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91052 Erlangen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Berens
authors.affiliation: Department Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91052 Erlangen, Germany.
authors.forename: Christiane
authors.initials: C
authors.lastname: Georgi
authors.affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, 20133 Milano, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Ferrara
authors.affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, 20133 Milano, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Bertoni
authors.affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, 20133 Milano, Italy.
authors.forename: Maria Enrica
authors.initials: ME
authors.lastname: Pasini
authors.affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, 20133 Milano, Italy.
authors.forename: Gianni
authors.initials: G
authors.lastname: Dehò
authors.affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, 20133 Milano, Italy federica.briani@unimi.it.
authors.forename: Federica
authors.initials: F
authors.lastname: Briani
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D018869:Heat-Shock Response / Q000235:genetics; D006801:Humans; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000235:genetics*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000254:growth & development; D012329:RNA, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D000091243:Ribosomal Protein S9; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C113064:PtxS protein, Pseudomonas aeruginosa; D012329:RNA, Bacterial; D012333:RNA, Messenger; D000091243:Ribosomal Protein S9; C000717190:RpsI protein, E coli; D014157:Transcription Factors; C553096:LpxT protein, E coli; D017856:Phosphotransferases (Phosphate Group Acceptor)
keywords: Escherichia coli; PA5194; Pseudomonas aeruginosa; bacterial riboregulators; lpxT; ptxS