Metallo-␤-lactamases (M␤Ls) are zinc-dependent enzymes able to hydrolyze and inactivate most ␤-lactam antibiotics. The large diversity of active site structures and metal content among M␤Ls from different sources has limited the design of a pan-M␤L inhibitor. Here we report the biochemical and biophysical characterization of a novel M␤L, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp 120 , His 121 , His 263 , and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear M␤Ls. Contrasting all other related M␤Ls, GOB-18 is fully active against a broad range of ␤-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of M␤Ls.The expression of ␤-lactam degrading enzymes (␤-lactamases) is the most common mechanism of antibiotic resistance among bacteria (1, 2). These enzymes have been grouped into four classes (A-D) according to sequence homology (3, 4). Class A, C, and D enzymes use an active site serine residue as a nucleophile, whereas class B lactamases (generically termed metallo-␤-lactamases, M␤Ls) 9 employ one or two Zn(II) ions to cleave the ␤-lactam ring.M␤Ls have particular importance in the clinical setting since they can hydrolyze a broader spectrum of ␤-lactam substrates than the serine-type enzymes and are resistant to most clinically employed inhibitors (5-11). The design of an efficient pan-M␤L inhibitor has been mostly limited by a striking diversity in the active site structures, catalytic features, and metal ion requirements for activity among different enzymes. Based on this heterogeneity, M␤Ls have been classified into three subclasses: B1, B2, and B3 (3, 6). Subclass B1 includes several chromosomally encoded enzymes such as BcII from Bacillus cereus (12-14), CcrA from Bacteroides fragilis (15-18), BlaB from Elizabethkingia meningoseptica (formerly, Chryseobacterium meningosepticum) (19), as well as the transferable VIM (20)-, IMP (21, 22)-, SPM (23, 24)-, and GIM-type enzymes. Subclass B2 includes the CphA (25, 26) and ImiS (27) lactamases from Aeromonas species. Subclass B3, originally represented only by L1 from Stenotrophomonas maltophilia (28 -30), now includes enzymes from other opportunistic pathogens like FEZ-1 from Legionella gormanii (31) and GOB from E. meningoseptica (32), as well as from environmental bacteria such as CAU-1 from Caulobacter crescentus (33) and THIN-B from Janthinobacterium lividum (34).Molecular structures of M␤Ls from the three subclasses have been solved by x-ray crystallography (12,14,15,25,31). Comparison of the tertiary structure of enzymes belonging to the different subclasses reveals a common ␣␤/␤␣ sandwich fold, in which different insertions and deletions have resulted in different loop topologies and, ultimately, in different zinc coordination environments and metal site occupancies among B1, B2, and B3 en...

Members of the genus Acinetobacter possess distinct plasmid types which provide effective platforms for the acquisition, evolution, and dissemination of antimicrobial resistance structures. Many plasmid-borne resistance structures are bordered by short DNA sequences providing potential recognition sites for the host XerC and XerD site-specific tyrosine recombinases (XerC/D-like sites). However, whether these sites are active in recombination and how they assist the mobilization of associated resistance structures is still poorly understood. Here we characterized the plasmids carried by Acinetobacter baumannii Ab242, a multidrug-resistant clinical strain belonging to the ST104 (Oxford scheme) which produces an OXA-58 carbapenem-hydrolyzing class-D β-lactamase (CHDL). Plasmid sequencing and characterization of replication, stability, and adaptive modules revealed the presence in Ab242 of three novel plasmids lacking self-transferability functions which were designated pAb242_9, pAb242_12, and pAb242_25, respectively. Among them, only pAb242_25 was found to carry an adaptive module encompassing an ISAba825-blaOXA-58 arrangement accompanied by a TnaphA6 transposon, the whole structure conferring simultaneous resistance to carbapenems and aminoglycosides. Ab242 plasmids harbor several XerC/D-like sites, with most sites found in pAb242_25 located in the vicinity or within the adaptive module described above. Electrotransformation of susceptible A. nosocomialis cells with Ab242 plasmids followed by imipenem selection indicated that the transforming plasmid form was a co-integrate resulting from the fusion of pAb242_25 and pAb242_12. Further characterization by cloning and sequencing studies indicated that a XerC/D site in pAb242_25 and another in pAb242_12 provided the active sister pair for the inter-molecular site-specific recombination reaction mediating the fusion of these two plasmids. Moreover, the resulting co-integrate was found also to undergo intra-molecular resolution at the new pair of XerC/D sites generated during fusion thus regenerating the original pAb242_25 and pAb242_12 plasmids. These observations provide the first evidence indicating that XerC/D-like sites in A. baumannii plasmids can provide active pairs for site-specific recombination mediating inter-molecular fusions and intra-molecular resolutions. The overall results shed light on the evolutionary dynamics of A. baumannii plasmids and the underlying mechanisms of dissemination of genetic structures responsible for carbapenem and other antibiotics resistance among the Acinetobacter clinical population.