The Legionella (Fluoribacter) gormanii metallo-beta-lactamase: a new member of the highly divergent lineage of molecular-subclass B3 beta-lactamases

Abstract

A metallo-beta-lactamase determinant was cloned from a genomic library of Legionella (Fluoribacter) gormanii ATCC 33297(T) constructed in the plasmid vector pACYC184 and transformed into Escherichia coli DH5alpha, by screening for clones showing a reduced susceptibility to imipenem. The product of the cloned determinant, named FEZ-1, contains a 30-kDa polypeptide and exhibits an isoelectric pH of 7.6. Sequencing revealed that FEZ-1 is a molecular-class B beta-lactamase which shares the closest structural similarity (29.7% of identical residues) with the L1 enzyme of Stenotrophomonas maltophilia, being a new member of the highly divergent subclass B3 lineage. All the residues that in L1 are known to be directly or indirectly involved in coordination of the zinc ions were found to be conserved also in FEZ-1, suggesting that the geometry of zinc coordination in the active site of the latter enzyme is identical to that of L1. Unlike L1, however, FEZ-1 appeared to be monomeric in gel permeation chromatography experiments and exhibited a distinctive substrate specificity with a marked preference for cephalosporins and meropenem. The properties of FEZ-1 overall resembled those of a beta-lactamase previously purified from the same strain of L. gormanii (T. Fujii, K. Sato, K. Miyata, M. Inoue, and S. Mitsuhashi, Antimicrob. Agents Chemother. 29:925-926, 1986) and are as yet unique among class B enzymes, reinforcing the notion that considerable functional heterogeneity can be encountered among members of this class. A system for overexpression of the bla(FEZ-1) gene in E. coli, based on the T7 phage promoter, was also developed.

FIG. 1

Results of zymogram analysis performed after renaturing SDS-PAGE using the chromogenic cephalosporin nitrocefin as the substrate for detection of β-lactamase activity. Protein size standards (in kilodaltons) are indicated on the left. Lanes: 1, crude extract from E. coli DH5α(pACYC184); 2, crude extract from E. coli DH5α(pLLB-8AI).

FIG. 2

Physical map of the insert of plasmid pLLB-8AI and subcloning strategy. Thick lines represent cloned DNA, while thin lines correspond to vector sequences. Production of metallo-β-lactamase activity (β-lact.) was assayed on crude extracts prepared from cells collected in the late-exponential phase of growth (A₆₀₀, 1.5 to 1.8), using meropenem as the substrate. The location of the bla_FEZ-1 ORF is also indicated. The BamHI site labeled with an asterisk was generated after cloning of the Sau3AI genomic DNA fragment in the BamHI site of the plasmid vector and is not present in the Legionella chromosomal DNA, as indicated by the results of Southern blot experiments (see text). Abbreviations for restriction enzymes: B, BamHI; B/S, BamHI/Sau3AI junction; H, HindIII; Hc, HincII; Sa, SalI; V, EcoRV; X, XbaI.

FIG. 3

Nucleotide sequence of the bla_FEZ-1 ORF and flanking regions. The initiation codon of the bla_FEZ-1 ORF is indicated, and protein translation is reported below the sequence. The putative signal peptide for protein secretion is underlined. An inverted repeat overlapping the termination codon of the bla_FEZ-1 gene, possibly functioning as a transcriptional terminator, is overlined by arrows.

FIG. 4

Comparison of the FEZ-1 amino acid sequence (in boldface type) with those of other molecular-class B β-lactamases. The numbering scheme refers to the L1 enzyme (31). Identical residues are indicated by an asterisk. Residues of the L1 enzyme involved in binding of Zn²⁺ are indicated by a z, and those involved in inter-subunit interactions are underlined (31). Secondary structure elements of Bc-II (9) are also indicated, above the sequences. Abbreviations: IMP-1, IMP-1 enzyme encoded by the bla_IMP gene cassette found in Serratia marcescens TN9106 (23) and in other gram-negative strains (2, 17); CcrA, CcrA enzyme of Bacteroides fragilis TAL3636 (25); Bc-II, β-lactamase II of Bacillus cereus 569/H (15); VIM-1, VIM-1 enzyme encoded by the bla_VIM gene cassette found in Pseudomonas aeruginosa VR-143/97 (18); IND-1, IND-1 enzyme of Chryseobacterium indologenes 001 (4); BlaB, BlaB enzyme of Chryseobacterium meningosepticum CCUG4310 (27); CphA, CphA enzyme of Aeromonas hydrophila AE036 (21); L1, L1 enzyme of S. maltophilia IID 1275 (32).

FIG. 5

β-Lactamase activity measured in different fractions of a culture of E. coli BL21(DE3)(pLysS)(pET-24/FEZ1) at different times after induction with IPTG. Activities were assayed using meropenem as the substrate and are relative to culture volumes. ▄, activity in the cell fraction; , activity in the culture supernatant.