Characterization of a plasmid-borne and constitutively expressed bla_mox-1 gene encoding AmpC-type β-lactamase

A 1954-bp DNA fragment containing the bla_Mox-1 gene, identified on a large resident plasmid (pRMOX-1) of Klebsiella pneumoniae NU2936, was sequenced and an open reading frame (ORF) coding for a 390-amino-acid (aa) MOX-1 was found. The total deduced aa sequence of MOX-1 shared considerable homology with that of AmpC-type class C β-lactamases of Gram^- bacteria, especially of Pseudomonas aeruginosa PAO1 [51.3%; 63.8% at the nucleotide (nt) level]. However, the regulatory gene ampR and a 38-bp AmpR-binding region were not present upstream from bla_MOX-1 although the expression of P. aeruginosa ampC is directly regulated by AmpR. Possible - 35 and -10 regions, a ShineDalgarno (SD) sequence and terminators were identified which are peculiar to bla_Mox-1. On the other hand, a sequence highly homologous (91.6%) to the region upstream from dhfrX in the In7 integron carried by plasmid pDGO100 was found upstream from bla_MOX-1 at nt 1 to 488. No significant difference was detected between the promoter activities of bla_MOX-1 in ampD^- and ampD⁺ strains of Enterobacter cloacae, as measured by the chloramphenicol acetyltransferase (CAT) assay. These results clearly show that bla_MOX-1 belongs to the group of ampC-related bla genes and that it is expressed constitutively, independently of transcriptional regulators such as AmpR, AmpG and AmpD. Homology analysis among AmpC enzymes or ampC genes implied that integration of the chromosomal ampC gene into a large resident plasmid, followed by transconjugation, was involved in the evolution of bla_MOX-1.