Cloning, sequence analyses, expression, and distribution of ampC-ampR from Morganella morganii clinical isolates.
Shotgun cloning experiments with restriction enzyme-digested genomic DNA from Morganella morganii 1, which expresses high levels of cephalosporinase, into the pBKCMV cloning vector gave a recombinant plasmid, pPON-1, which encoded four entire genes: ampC, ampR, an hybF family gene, and orf-1 of unknown function. The deduced AmpC beta-lactamase of pI 7.6 shared structural and functional homologies with AmpC from Citrobacter freundii, Escherichia coli, Yersinia enterocolitica, Enterobacter cloacae, and Serratia marcescens. The overlapping promoter organization of ampC and ampR, although much shorter in M. morganii than in the other enterobacterial species, suggested similar AmpR regulatory properties. The MICs of beta-lactams for E. coli MC4100 (ampC mutant) harboring recombinant plasmid pACYC184 containing either ampC 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 was 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 decrease 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 the M. morganii cephalosporinase. Fifteen other M. morganii clinical isolates with phenotypes of either low-level inducible cephalosporinase expression 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. cloacae, which are located downstream from the fumarate operon. Finally, an identical AmpC beta-lactamase (DHA-1) was recently identified as being plasmid encoded in Salmonella enteritidis, and this is confirmatory evidence of a chromosomal origin of the plasmid-mediated cephalosporinases.
Poirel
L
L
Service de Bactériologie-Virologie, Hôpital de Bicêtre, Faculté de Médecine Paris-Sud, Le Kremlin-Bicêtre, France.
Guibert
M
M
Girlich
D
D
Naas
T
T
Nordmann
P
P
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D002510:Cephalosporinase / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004755:Enterobacteriaceae / Q000187:drug effects / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000502:physiology; D010641:Phenotype; D012477:Salmonella enteritidis / Q000187:drug effects / Q000235:genetics; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D001618:beta-Lactamases / Q000235:genetics*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; C071526:AmpR protein, Bacteria; C062460:AmpD protein, Bacteria; D009238:N-Acetylmuramoyl-L-alanine Amidase; D002510:Cephalosporinase; C048795:AmpC beta-lactamases; D001618:beta-Lactamases