Functional domains of the RhlR transcriptional regulator of Pseudomonas aeruginosa

J Bacteriol. 2003 Dec;185(24):7129-39. doi: 10.1128/JB.185.24.7129-7139.2003.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 4-Butyrolactone / analogs & derivatives*
  • 4-Butyrolactone / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Gene Expression Regulation, Bacterial
  • Mutagenesis, Site-Directed
  • Protein Structure, Tertiary
  • Pseudomonas aeruginosa / genetics*
  • Pseudomonas aeruginosa / metabolism
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / metabolism
  • Transcriptional Activation*

Substances

  • Bacterial Proteins
  • LexA protein, Bacteria
  • N-butyrylhomoserine lactone
  • RhlR protein, Pseudomonas aeruginosa
  • Serine Endopeptidases
  • 4-Butyrolactone