Cloning, sequence analyses, expression, and distribution of ampC-ampR fromMorganella morganii clinical isolates

Shotgun cloning experiments with restriction enzyme-digested genomic DNA fr om Morganella morganii 1 which expresses high levels of cephalosporinase, i nto the pBKCMV cloning vector gave a recombinant plasmid, pPON-1, which enc oded four entire genes: ampC, ampR, an hybF family gene, and orf-1 of unkno wn function. The deduced AmpC beta-lactamase of pI 7.6 shared structural an d functional homologies with AmpC from Citrobacter freundii, Escherichia co li, Yersinia enterocolitica, Enterobacter cloacac, and Serratia marcescens. The overlapping promoter organization of ampC and ampR, although much shor ter in M. morganii than in the other enterobacterial species, suggested sim ilar AmpR regulatory properties. The MICs of beta-lactams for E. coli MC410 0 (ampC: mutant) harboring recombinant plasmid pACYC184 containing either a mpC and ampR (pAC-1) or ampC (pAC-2) and induction experiments showed that the ampC gene of M. morganii 1 was repressed in the presence of ampR and wa s activated when a beta-lactam inducer was added. Moreover, transformation of M. morganii 1 or of E. coli JRG582 (Delta ampDE) harboring ampC and ampR with a recombinant plasmid containing ampD from E. cloacae resulted in a d ecrease in the beta-lactam MICs and an inducible phenotype for M. morganii 1, thus underlining the role of an AmpD-like protein in the regulation of t he dl. morganii cephalosporinase, Fifteen other M. morganii clinical isolat es with phenotypes of either low-level inducible cephalosporinase expressio n or high-level constitutive cephalosporinase expression harbored the same ampC-ampR organization, with the hybF and orf-1 genes surrounding them; the organization of these genes thus differed from those of ampC-ampR genes in C.freundii and E, cloacac, which are located downstream from the fumarate operon, Finally, an identical AmpC beta-lactamase (DHA-1) was recently iden tified as being plasmid encoded in Salmonella enteritidis, and this is conf irmatory evidence of a chromosomal origin of the plasmid-mediated cephalosp orinases.