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Cloning, sequence analyses, expression, and distribution of ampC-ampR from Morganella morganii clinical isolates
L Poirel1, M Guibert, D Girlich
1Service de Bactériologie-Virologie, Hôpital de Bicêtre, Faculté de Médecine Paris-Sud, Le Kremlin-Bicêtre, France.
Abstract:
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.
Insights
This study identifies the ampC and ampR genes in Morganella morganii, revealing their role in cephalosporinase regulation. The findings suggest a chromosomal origin for plasmid-mediated cephalosporinases, impacting antibiotic resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Morganella morganii is known for high cephalosporinase expression.
- Cephalosporinases are crucial enzymes in bacterial antibiotic resistance.
- Understanding the genetic basis of cephalosporinase production is vital for combating antimicrobial resistance.
Purpose of the Study:
- To clone and characterize the genes responsible for cephalosporinase production in Morganella morganii.
- To elucidate the regulatory mechanisms of ampC gene expression in M. morganii.
- To investigate the role of ampR and ampD homologs in cephalosporinase regulation.
Main Methods:
- Shotgun cloning of M. morganii genomic DNA into pBKCMV vector.
- Gene sequencing and analysis of recombinant plasmids.
- Expression studies in E. coli using recombinant plasmids.
- Minimum Inhibitory Concentration (MIC) assays for beta-lactams.
- Transformation experiments with M. morganii and E. coli.
Main Results:
- A recombinant plasmid (pPON-1) containing ampC, ampR, hybF, and orf-1 genes was generated.
- The M. morganii AmpC beta-lactamase shares homology with other Enterobacteriaceae.
- M. morganii ampC expression is repressed by ampR and induced by beta-lactams.
- An AmpD-like protein is essential for inducible cephalosporinase production in M. morganii.
- The gene organization differs from C. freundii and E. cloacae.
Conclusions:
- The ampC and ampR genes in M. morganii are responsible for cephalosporinase production and exhibit inducible regulation.
- The findings support a chromosomal origin for plasmid-mediated cephalosporinases, exemplified by DHA-1.
- Understanding these genetic elements is critical for developing strategies against cephalosporinase-mediated resistance.