Functional domains of the RhlR transcriptional regulator of Pseudomonas aeruginosa.
The RhlR transcriptional regulator of Pseudomonas aeruginosa, along with its cognate autoinducer, N-butyryl homoserine lactone (C(4)-HSL), regulates gene expression in response to cell density. With an Escherichia coli LexA-based protein interaction system, we demonstrated that RhlR multimerized and that the degree of multimerization was dependent on the C(4)-HSL concentration. Studies with an E. coli lasB::lacZ lysogen demonstrated that RhlR multimerization was necessary for it to function as a transcriptional activator. Deletion analysis of RhlR indicated that the N-terminal domain of the protein is necessary for C(4)-HSL binding. Single amino acid substitutions in the C-terminal domain of RhlR generated mutant RhlR proteins that had the ability to bind C(4)-HSL and multimerize but were unable to activate lasB expression, demonstrating that the C-terminal domain is important for target gene activation. Single amino acid substitutions in both the N-terminal and C-terminal domains of RhlR demonstrated that both domains possess residues involved in multimerization. RhlR with a C-terminal deletion and an RhlR site-specific mutant form that possessed multimerization but not transcriptional activation capabilities were able to inhibit the ability of wild-type RhlR to activate rhlA expression in P. aeruginosa. We conclude that C(4)-HSL binding is necessary for RhlR multimerization and that RhlR functions as a multimer in P. aeruginosa.
Lamb
Janet R
JR
University of Rochester, Rochester, New York 14642, USA.
Patel
Hetal
H
Montminy
Timothy
T
Wagner
Victoria E
VE
Iglewski
Barbara H
BH
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D000099160:LexA Repressor Protein
D015107:4-Butyrolactone; D001426:Bacterial Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein; C092312:N-butyrylhomoserine lactone; C086515:RhlR protein, Pseudomonas aeruginosa