SYNTHESIS OF MULTIPLE EXOPRODUCTS IN PSEUDOMONAS-AERUGINOSA IS UNDER THE CONTROL OF RHLR-RHLI, ANOTHER SET OF REGULATORS IN STRAIN PAO1 WITH HOMOLOGY TO THE AUTOINDUCER-RESPONSIVE LUXR-LUXI FAMILY

Mutants of Pseudomonas aeruginosa PAO1 that were deficient in the abil ity to produce proteases that degrade casein were detected among the s urvivors of chemical mutagenesis, One such mutant (PDO31) showed reduc ed production of elastolytic activity, beta-hemolytic activity, and py ocyanin, A 4.3-kb EcoRI fragment from a gene bank of PAO1 that complem ented defects in PDO31 was found, Transposon mutagenesis and deletion derivatives of the clone were used in conjunction with complementation tests to determine the physical location of the gene of interest, Nuc leotide sequence analysis revealed an open reading frame (rhlR) encodi ng a putative 27.6-kDa protein (RhlR) with homology to autoinducer-res ponsive regulators of quorum sensing systems such as LuxR of Vibrio fi scheri and LasR of P. aeruginosa. Further sequence analysis downstream of rhlR revealed an independently transcribed gene (rhlI) that encode s a putative 22.2-kDa protein with homology to members of the family o f autoinducer synthetases, such as LuxI of V, fischeri and LasI of P. aeruginosa. The rhlRI sequences were also recently reported by others (U, A, Ochsner and J, Reiser, Proc, Natl, Acad, Sci, USA 92: 6424-6428 , 1995) as an autoinducer-mediated regulation mechanism for rhamnolipi d biosurfactant synthesis in P, aeruginosa PG201, Mutants with defects in rhlR or rhlI were constructed in PAO1 by gene replacement, using c lones modified by Tn501 insertion, Compared with the wild type, the rh lR and rhlI mutants both showed defects in the production of elastase, LasA protease, rhamnolipid, and pyocyanin, Transcription from the gen e for elastase, as measured with a lasB-cat fusion, demonstrated that production of elastase was subject to cell density-dependent gene acti vation in PAO1, However, transcription of lasB-cat in the rhlI mutant, which had lost the presumptive autoinducer synthetase (predicted to a ctivate RhlR), showed low basal activity and had lost all cell density -dependent transcription of lasB, Thus, RhlR-RhlI represent the second autoinducer-responsive regulatory mechanism found in P, aeruginosa th at controls expression of multiple virulence factor exoproducts, inclu ding elastase.