This approach has been complicated by the fact
that most strains of P. aeruginosa produce at least two
distinct extracellular proteases (25) and that alkaline prote-ase does not possess a unique or stringent substrate specific-ity (26). We have been unable to specifically inhibit the
activity of interfering protease (elastase) to facilitate detec-tion of alkaline protease mutants on skim milk agar plates
(unpublished data). To circumvent these problems, we chose
to isolate alkaline protease mutants in strain PA103, which
produces alkaline protease (4) but no detectable elastase
(28). Despite the fact that strain PA103 is serum sensitive
(41), it is virulent in a mouse eye model of corneal infection
(17, 27). We have isolated alkaline protease-deficient mu-tants of strain PA103 after ethyl methanesulfonate (EMS)
mutagenesis and screening of mutagenized colonies on skim
milk agar plates.