Molecular and Kinetic Characterization of MOX-9, a Plasmid-Mediated Enzyme Representative of a Novel Sublineage of MOX-Type Class C β-Lactamases

Abstract

The MOX lineage of β-lactamases includes a group of molecular class C enzymes (AmpCs) encoded by genes mobilized from the chromosomes of Aeromonas spp. to plasmids. MOX-9, previously identified as a plasmid-encoded enzyme from a Citrobacter freundii isolate, belongs to a novel sublineage of MOX enzymes, derived from the resident Aeromonas media AmpC. The bla_MOX-9 gene was found to be carried on a transposon, named Tn7469, likely responsible for its mobilization to plasmidic context. MOX-9 was overexpressed in Escherichia coli, purified, and subjected to biochemical characterization. Kinetic analysis showed a relatively narrow-spectrum profile with strong preference for cephalosporin substrates, with some differences compared with MOX-1 and MOX-2. MOX-9 was not inhibited by clavulanate and sulbactam, while both tazobactam and avibactam acted as inhibitors in the micromolar range.

Keywords: MOX; enzyme kineticss; β-lactamases.

FIG 1

Phylogenetic tree showing the relationship of enzymes of the MOX lineage (of which some are named CMY). Sequences have been aligned with the MAFFT webserver (https://mafft.cbrc.jp/alignment/server/) and the tree has been built by using the neighbor-joining method and 1,000 bootstraps. The tree was rooted on the CMY-2 sequence used as an outgroup. Sequences were retrieved from the NCBI reference gene catalog (https://www.ncbi.nlm.nih.gov/pathogens/refgene).

FIG 2

Genetic context of the bla_MOX-9 gene in C. freundii Cfr-FI-07 and its comparison to segments of the A. media R1-26 genome (region 3,313,893.3,318,039; accession number CP043579) and to the A. caviae pKAM376_5 plasmid. The Tn7469 transposon is indicated with a brace and its target site duplications are shown with yellow diamonds flanking the transposon. The ISKpn9-like element involved in the mobilization of bla_MOX-9 is boxed. Regions showing ≥99% nucleotide sequence identity are connected by blue areas. Hypothetical proteins are depicted in gray, while the IS5 element disrupted by Tn7469 transposition is in red.