The Pseudomonas quorum-sensing regulator RsaL belongs to the tetrahelical superclass of H-T-H proteins.
In the opportunistic human pathogen Pseudomonas aeruginosa, quorum sensing (QS) is crucial for virulence. The RsaL protein directly represses the transcription of lasI, the synthase gene of the main QS signal molecule. On the basis of sequence homology, RsaL cannot be predicted to belong to any class of characterized DNA-binding proteins. In this study, an in silico model of the RsaL structure was inferred showing that RsaL belongs to the tetrahelical superclass of helix-turn-helix proteins. The overall structure of RsaL is very similar to the N-terminal domain of the lambda cI repressor and to the POU-specific domain of the mammalian transcription factor Oct-1 (Oct-1 POUs). Moreover, residues of Oct-1 POUs important for structural stability and/or DNA binding are conserved in the same positions in RsaL and in its homologs found in GenBank. These residues were independently replaced with Ala, and the activities of the mutated variants of RsaL were compared to that of the wild-type counterpart in vivo by complementation assays and in vitro by electrophoretic mobility shift assays. The results validated the RsaL in silico model and showed that residues Arg 20, Gln 38, Ser 42, Arg 43, and Glu 45 are important for RsaL function. Our data indicate that RsaL could be the founding member of a new protein family within the tetrahelical superclass of helix-turn-helix proteins. Finally, the minimum DNA sequence required for RsaL binding on the lasI promoter was determined, and our data support the hypothesis that RsaL binds DNA as a dimer.
Rampioni
Giordano
G
Department of Biology, University Roma Tre, Viale Marconi, 446, 00146, Rome, Italy.
Polticelli
Fabio
F
Bertani
Iris
I
Righetti
Karima
K
Venturi
Vittorio
V
Zennaro
Elisabetta
E
Leoni
Livia
L
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D019077:Helix-Turn-Helix Motifs; D008958:Models, Molecular; D008969:Molecular Sequence Data; D053038:Quorum Sensing; D012097:Repressor Proteins / Q000737:chemistry* / Q000502:physiology
D001426:Bacterial Proteins; D012097:Repressor Proteins; C118377:rsaL protein, Pseudomonas aeruginosa; D004247:DNA