Analysis of random and site-directed mutations in rhII, a Pseudomonas aeruginosa gene encoding an acylhomoserine lactone synthase.
The opportunistic human pathogen Pseudomonas aeruginosa possesses two cell density-dependent genetic regulatory systems that control expression of a number of secreted virulence factors. These two systems, the lasI-lasR and rhII-rhIR gene pairs, are members of the luxI-luxR family of quorum-sensing signal generators and signal receptors. The rhII gene in P. aeruginosa encodes a 201-amino-acid protein that catalyses the synthesis of an autoinducer, butyrylhomoserine lactone. Through a programme of random and site-specific mutagenesis of rhII we have gained a better understanding of how its protein product functions. Eight residues critical to butyrylhomoserine lactone synthesis by RhiI were identified by random mutagenesis, and all mapped to a conserved region that spans residues 24-104. Seven of the eight residues were charged amino acids and the other was a glycine. By using site-specific mutagenesis we showed that an active-site cysteine or serine was not required for butyrylhomoserine lactone synthesis, and that two conserved aromatic amino acids in the postulated active site region could be altered without complete loss of RhII activity. Furthermore, two residues towards the C-terminus that align with critical residues in LuxI can be altered in RhII without loss of activity. These studies suggest that as opposed to the current models for acyl substrate binding to quorum-sensing signal generators, charged amino acid residues participate directly in the catalysis of butyrylhomoserine lactone synthesis rather than cysteines, serines or hydrophobic amino acids.
Parsek
M R
MR
Department of Microbiology, University of Iowa, Iowa City 52242, USA.
Schaefer
A L
AL
Greenberg
E P
EP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D005798:Genes, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics*
D001426:Bacterial Proteins; C085427:LuxI protein, Bacteria; D014157:Transcription Factors