We have studied the mechanism by which beta-lactam challenge leads to beta-lactamase induction in Aeromonas hydrophila through transposon-insertion mutagenesis. Disruption of the dd-carboxypeptidases/endopeptidases, penicillin-binding protein 4 or BlrY leads to elevated monomer-disaccharide-pentapeptide levels in A. hydrophila peptidoglycan and concomitant overproduction of beta-lactamase through activation of the BlrAB two-component regulatory system. During beta-lactam challenge, monomer-disaccharide-pentapeptide levels increase proportionately with beta-lactamase production and beta-lactamase induction is inhibited by vancomycin, which binds muro-pentapeptides. Taken together, these data strongly suggest that the Aeromonas spp. beta-lactamase regulatory sensor kinase, BlrB, responds to the concentration of monomer-disaccharide-pentapeptide in peptidoglycan.