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

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.

FIG. 1

Restriction endonuclease maps of the inserts from recombinant plasmid pPON-1, which codes for the ampR and ampC genes from M. morganii 1, and of pAC-1 (ampR, ampC) and pAC-2 (ampC) pACYC184 derivatives. The thick lines represent the cloned inserts from M. morganii 1, the thin lines indicate vector pBKCMV, and the dotted lines represent vector pACYC184. The five ORFs found in the 5,914-bp sequenced are indicated, as are their translation directions.

FIG. 2

Map of pPON-1 insert from M. morganii 1 and the primers used to PCR amplify the indicated genes from 15 M. morganii clinical isolates: primers 1 and 4 for hybF, ampR, and ampC; primers 2 and 4 for ampR and ampC; primers 3 and 4 for ampC; and primers 3 and 5 for ampC and orf-1. Cloning of ampR-ampC genes or of the ampC gene into pACYC184, giving recombinant plasmids pAC-1 or pAC-2, respectively, used similarly PCR-amplified fragments.

FIG. 3

Nucleotide sequence of the 2,126-bp fragment of pPON-1 containing the ampC- and ampR-coding regions. The deduced amino acid sequences are designated in single-letter code. The putative promoter sequences are represented by −35 and −10 regions (boxed). The start and stop codons of these genes are underlined.

FIG. 4

Comparison of amino acid sequences of AmpC from M. morganii SLM01 and DHA-1 from M. morganii 1. Dotted lines indicate identical amino acids. The boldface amino acids are characteristic either of serine β-lactamases (S-V-S-K) or of class C β-lactamases.

FIG. 5

Alignment of the intercistronic region of ampC-ampR from M. morganii, C. freundii, Y. enterocolitica, S. marcescens, and E. cloacae. The start codons and the −35 and −10 regions of the promoters are shown below the DNA sequences for ampR and above for the DNA sequences for ampC. The +1 sign indicates the putative mRNA transcription start site. The sequences marked Region 1 and Region 2 correspond to those conserved among ampC-ampR intercistronic regions. Region 1 and region 2 are the two components of the putative AmpR binding site. Only region 1 contains a LysR binding motif (T-N₁₁-A).

FIG. 6

Multiple-sequence alignment of amino acid sequences of AmpR regulating cephalosporinase expression. The origins of AmpR are as follows: M. morganii 1 (Mor str. 1), C. freundii OS60 (Cit OS60), Y. enterocolitica IP97 (Yer IP97), S. marcescens SR50 (Ser SR50), and E. cloacae MHN-1 (Ent MHN-1). Identical amino acids are boxed. The predicted helix-turn-helix DNA-binding motif of the LysR family is shown (HTH).

FIG. 7

Organization of the sequences surrounding ampC ampC in various enterobacterial species. The positions and directions of the fumarate operon (frdABCD), hybF, hybE, orf-1, ampC, and ampR genes are indicated with arrows. Also indicated are the locations of the putative promoters.