Characterization of a Functional Metallo-β-Lactamase from the Marine Bacterium Haliangium ochraceum

Background: Acquired metallo-β-lactamases (MBLs) are worrisome resistance determinants conferring broad-spectrum β-lactam resistance in clinically-relevant pathogens such as Enterobacteriaceae, Pseudomonas and Acinetobacter. However, the origin of these acquired determinants is still poorly understood. Exploiting the increasing number of microbial genome sequences available in public databases, we specifically investigated the presence of genes encoding for proteins showing homology with acquired subclass B1 enzymes in the genomes of environmental bacteria belonging to the class of δ-Proteobacteria. Methods: Using genome data mining (with the protein sequence of VIM-2 as the search template and the tblastn algorithm available at the NCBI web site), a gene encoding a subclass B1 MBL homologue (locus tag, Hoch_6144) was identified in the genome of Haliangium ochraceum DSMZ 14365. The Hoch_6144 ORF was cloned in plasmid vector pET-9a. The resulting plasmid (pET-HAL-1) were electroporated in E. coli BL21(DE3). The MBL was purified by chromatography and its functional properties investigated by means of kinetic analysis. Results: The MBL homologue encoded by gene Hoch_6144, named HAL-1, shared the closest ancestry with FIM-1, NDM-1 and VIM-2 acquired MBLs (identity range, 41-46 %). Significant β-lactam-hydrolyzing activity was found in crude extracts of E. coli BL21(DE3)/pET-HAL-1, that was inhibited >95% in the presence of 5 mM EDTA. HAL-1 was successfully produced and purified (>95%) using three chromatography steps. Purified HAL-1 exhibited a broad-spectrum of activity, with catalytic efficiencies for the hydrolysis of β-lactams comparable to that of VIM-2. Interestingly, HAL-1 showed low Km values for most tested substrates, at the exception of ceftazidime. Strikingly, doripenem (the latest carbapenem antibiotic to receive FDA approval) was hydrolyzed 45-fold more efficiently by HAL-1 than by the acquired VIM-2 MBL. Conclusions: In this work, we described a new functional MBL identified in a marine bacterium of the class δ-Proteobacteria, Haliangium ochraceum. HAL-1 exhibits functional properties very close to that of clinically-relevant subclass B1 enzymes and highlights the potential of δ-Proteobacteria as a reservoir of functional subclass B1 MBL determinants.