ANALYSIS OF RANDOM AND SITE-DIRECTED MUTATIONS IN RHLL, 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 express ion of a number of secreted virulence factors. These two systems, the lasI-lasR and rhII-rhIR gene pairs, are members of the luxI-luxR famil y of quorum-sensing signal generators and signal receptors. The rhII g ene in P. aeruginosa encodes a 201-amino-acid protein that catalyses t he synthesis of an autoinducer, butyrylhomoserine lactone. Through a p rogramme of random and site-specific mutagenesis of rhII we have gaine d a better understanding of how its protein product functions. Eight r esidues critical to butyrylhomoserine lactone synthesis by RhII were i dentified by random mutagenesis, and all mapped to a conserved region that spans residues 24-104. Seven of the eight residues were charged a mino acids and the other was a glycine. By using site-specific mutagen esis we showed that an active-site cysteine or serine was not required for butyrylhomoserine lactone synthesis, and that two conserved aroma tic amino acids in the postulated active site region could be altered without complete loss of RhII activity. Furthermore, two residues towa rds the C-terminus that align with critical residues in LuxI can be al tered in RhII without loss of activity. These studies suggest that as opposed to the current models for acyl substrate binding to quorum-sen sing signal generators, charged amino acid residues participate direct ly in the catalysis of butyrylhomoserine lactone synthesis rather than cysteines, serines or hydrophobic amino acids.