INTRODUCTION

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Metallo--lactamases (molecular-class B, group 3 of the functional classification [7]) are monomeric or oligomeric proteins that require a metal cofactor (Zn²⁺ in native enzymes) for activity and are structurally and evolutionarily unrelated to active-site serine -lactamases (6, 13). The relevance of metallo--lactamases as resistance effectors is mostly due to their substrate specificity, which always includes carbapenems and often also cephalosporins and penicillins, and to their resistance to mechanism-based serine -lactamase inhibitors (6, 12, 19, 20, 26).

Several metallo--lactamases have been sequenced (4, 18, 26, 27, 29, 33). From a structural standpoint these enzymes appear to be clustered into three different evolutionary lineages: subclass B1 (including most known metallo--lactamases), subclass B2 (including the Aeromonas enzymes), and subclass B3 (including the highly divergent L1 enzyme from Stenotrophomonas maltophilia) (26). From a functional standpoint most of these enzymes exhibit a broad substrate specificity (subgroup 3a in the functional classification), whereas the Aeromonas enzymes behave as rather specific carbapenemases (subgroup 3b in the functional classification) (6, 26).

Additional metallo--lactamases have been purified and partially characterized from Myroides odoratus (formerly Flavobacterium odoratum) (30), Legionella (Fluoribacter) gormanii (14), and Burkholderia cepacia (3), but their definitive attribution to molecular-class B awaits the confirmation of sequence data. Among these, the enzyme of L. gormanii exhibits distinctive biochemical properties compared to other metallo--lactamases, with preferential activity for cephalosporin compounds (14). This warranted its inclusion in a further subgroup (subgroup 3c) of the functional classification (6, 26).

In this study we cloned and characterized a metallo--lactamase determinant from L. gormanii ATCC 33297^T, whose product overall resembles the enzyme that was previously purified from this strain (14). Sequencing revealed that the L. gormanii metallo--lactamase is a new member of the highly divergent subclass B3 lineage of class B -lactamases.

(These results were presented in part at the 39th Interscience Conference on Antimicrobial Agents and Chemotherapy, 1999.)

	MATERIALS AND METHODS

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Bacterial strains and genetic vectors. L. gormanii ATCC 33297^T was used as the donor strain for construction of the genomic library. Escherichia coli DH5 (Gibco-BRL, Bethesda, Md.) was used as the host for genetic vectors and recombinant plasmids. E. coli BL21(DE3)(pLysS) (Novagen Inc., Madison, Wis.) was used as a host for overexpression of the L. gormanii bla_FEZ-1 gene cloned in the expression vector pET-24. Plasmid pACYC184 (10) was used as the vector for construction of the L. gormanii genomic library. Plasmid pBC-SK (Stratagene, La Jolla, Calif.) was used for some subcloning steps. Plasmid pET-24 (Novagen) was used as a T7-based vector for tightly regulated overexpression of the bla_FEZ-1 gene in E. coli.

Antibiotics and -lactamase substrates. Antibiotics were obtained from Sigma Chemical Co. (St. Louis, Mo.) unless otherwise specified. Imipenem and cefoxitin were from Merck Research Laboratories (Rahway, N.J.), meropenem was from Zeneca Pharmaceuticals (Macclesfield, Cheshire, United Kingdom), cefuroxime and ceftazidime were from Glaxo-Wellcome (Verona, Italy), cefotaxime was from Hoechst-Marion-Roussel (Frankfurt, Germany), and aztreonam was from Bristol-Myers Squibb Co. (Wallingford, Conn.). Nitrocefin was from Unipath (Milan, Italy).

In vitro susceptibility testing. In vitro susceptibility of the E. coli clone carrying the cloned bla_FEZ-1 metallo--lactamase determinant was determined by a broth macrodilution method (22), using Mueller-Hinton medium (Difco Laboratories, Detroit, Mich.) and a bacterial inoculum of 10⁵ CFU per tube. Results were recorded after incubation at 28°C for 24 h.

-Lactamase assays. -Lactamase activity in crude E. coli cell extracts was assayed spectrophotometrically. Reactions were performed in phosphate-buffered saline (PBS), pH 7.3, at 25°C, in a total volume of 0.75 ml. The initial substrate concentrations were 100 µM for cephalosporins, 150 µM for carbapenems, and 1 mM for penicillins. For the indicated compounds, wavelengths and changes in the extinction coefficient, respectively, were as follows: imipenem, 299 nm and 9,000 M¹ cm¹; meropenem, 297 nm and 6,500 M¹ cm¹; nitrocefin, 482 nm and +15,000 M¹ cm¹; cefuroxime, 260 nm and 7,600 M¹ cm¹; cefotaxime, 260 nm and 7,500 M¹ cm¹; cefoxitin, 260 nm and 7,800 M¹ cm¹; penicillin G, 235 nm and 800 M¹ cm¹; and ampicillin, 235 nm and 820 M¹ cm¹. Inhibition of enzymatic activity by EDTA or dipicolinic acid was determined by measuring the residual activity after incubation of the crude extract, for 15 min at 25°C, in the presence of variable concentrations of each chelating agent. Crude cell extracts were prepared as follows. Cells were grown in Luria-Bertani broth (28) aerobically at 28°C, collected by centrifugation, resuspended in PBS (1/10 of the original culture volume), and disrupted by sonication (six times for 15 s each, at 50 W). The supernatant obtained after centrifugation at 10,000 × g for 10 min, to remove cell debris, represented the crude extract. Protein concentration in solution was determined with a commercial protein assay kit (Bio-Rad, Richmond, Calif.) with bovine serum albumin as a standard.

Protein analysis techniques. Analytical isoelectric focusing (IEF) of crude cell extracts was performed in precast 5% polyacrylamide gels containing ampholites (pH range, 3.5 to 9.5) (Ampholine PAGplate; Amersham Pharmacia Biotech, Uppsala, Sweden) using a Multiphor II Apparatus (Pharmacia). Gels were focused at 0.1 W/cm² for 2 h at 10°C. -Lactamases were detected as pink bands after overlaying the gel with filter paper soaked with a 0.25 mM nitrocefin solution in 30 mM ACES [N-(2-acetamido)-2-aminoethanesulfonic acid]-NaOH buffer, pH 7.0, supplemented with 2 mM ZnCl₂. Zymogram detection of -lactamase activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of protein preparations was performed essentially as previously described (21). After the initial renaturation treatment, the gel was equilibrated in PBS for 20 min at 37°C, and -lactamase activity was revealed by the appearance of pink bands after overlaying the gel with filter paper soaked with a 0.25 mM nitrocefin solution in PBS.

Recombinant DNA methodology. Basic recombinant DNA procedures were performed essentially as described by Sambrook et al. (28). Extraction of the genomic DNA from L. gormanii was performed as described previously (16) from cells grown on BCYE agar plates (Oxoid Ltd., Basingstoke, United Kingdom) at 37°C in an aerobic atmosphere enriched with 5% CO₂. For construction of the library, genomic DNA was partially digested with Sau3AI, and fragments in the 4- to 12-kb size range were purified by agarose gel electrophoresis using the Geneclean II kit (Bio-101, La Jolla, Calif.). The purified restriction fragments were ligated to BamHI-linearized and dephosphorylated pACYC184 vector, and the ligation mixture was used to transform E. coli DH5 by electroporation using a Gene Pulser apparatus (Bio-Rad) according to the manufacturer's instructions. The ratio of recombinant clones to those carrying an empty religated vector was >5, as shown by replica plating of transformants onto plates containing both chloramphenicol (85 µg/ml) and tetracycline (20 µg/ml). The system for overexpression of the bla_FEZ-1 gene in E. coli was prepared as follows. The bla_FEZ-1 open reading frame (ORF) and some downstream region were amplified by PCR using primers BLAFEZ-FN (5'-GGAATTCCATATGAAAAAAGTATTAAGTTTAAC), which added an NdeI linker overlapping the bla_FEZ-1 start codon (in boldface type), and BLAFEZ-RB (5'-GCGGATCCTTTGACCAATATG), which was designed 0.35 kb downstream of the bla_FEZ-1 stop codon and contained a BamHI linker. The 1.2-kb amplimer was digested with BamHI, cloned in plasmid pBC-SK digested with BamHI and EcoRV (the resulting recombinant plasmid was named pBLL/FEZ1), and subjected to confirmatory sequencing. The 1.2-kb NdeI-BamHI fragment of pBLL/FEZ1 was then subcloned in pET-24, and the resulting recombinant plasmid, named pET-24/FEZ1, was transformed into E. coli BL21(DE3)(pLysS). The expression experiments with E. coli BL21(DE3)(pLysS)(pET-24/FEZ1) were performed in duplicate. In these experiments the strain was grown aerobically at 28°C in Luria-Bertani medium containing chloramphenicol (70 µg/ml) and kanamycin (50 µg/ml). For induction, isopropyl--D-thiogalactopyranoside (IPTG) was added at a 0.5 mM final concentration when the culture turbidity achieved an A₆₀₀ of 0.8 to 0.9.

DNA sequencing and computer analysis of sequence data. DNA sequences were determined by the dideoxy-chain termination method using an automatic DNA sequencer (LICOR 4000; LI-COR Inc., Lincoln, Nebr.), the Thermosequenase DNA sequencing kit (Amersham), and IRD 800-labeled custom sequencing primers (MWG-Biotech, Munich, Germany). Sequences were determined on both strands, using denatured double-stranded DNA templates. Similarity searches against sequence databases were performed using an updated version of the BLAST program (1). Computer analysis of sequence data was performed using an updated version (8.1) of the Wisconsin Package (version 8.1; Genetics Computer Group Inc., Madison, Wis.) available at the Italian EMBL node of Bari. Multiple sequence alignments were generated with the help of the PILEUP program of the Genetics Computer Group package and manually refined considering the information available on the three-dimensional structures of the Bc-II, CcrA, and L1 enzymes (8, 9, 11, 31).

Gel permeation chromatography. Gel permeation chromatography was performed on a Superdex-75 column (1 by 30 cm; Pharmacia). The column was equilibrated and eluted in 10 mM sodium cacodylate buffer, pH 6.5, containing 0.2 M NaCl, and calibrated by determining the retention volume of different protein standards. The proteins were eluted at a flow rate of 1 ml/min, and the absorbance at 280 nm was recorded. A crude extract containing the FEZ-1 protein (300 µl), prepared from an induced culture of E. coli BL21(DE3)(pLysS)(pET-24/FEZ1), was eluted at the same flow rate. Fractions of 1 ml were collected, and the presence of the -lactamase activity was monitored by testing for cefuroxime hydrolysis.

Nucleotide sequence accession number. The nucleotide sequence reported in this paper has been submitted to the EMBL/GenBank/DDBJ sequence databases and assigned the accession no. Y17896.

	RESULTS

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Cloning of a metallo--lactamase determinant from L. gormanii ATCC 33297^T. A metallo--lactamase determinant was isolated from a genomic library of L. gormanii ATCC 33297^T constructed in the pACYC184 plasmid vector and transformed in E. coli DH5, by screening for clones showing a reduced susceptibility to imipenem. For this purpose, individual clones grown on a medium containing only chloramphenicol (for vector selection) were replica plated onto a medium containing chloramphenicol plus imipenem (5 µg/ml). One clone growing on this medium (clone 8AI) was isolated out of approximately 15,000 screened recombinants. Growth of this clone on the imipenem-containing medium was very slow, with colonies 1 mm in diameter being visible only after 4 to 5 days of incubation at 28°C.

View this table: [in this window] [in a new window]   TABLE 1.   -Lactamase activity in the crude extract of clone 8AI (DH5[pLLB-8AI]) with nitrocefin (NCF), imipenem (IMI), and meropenem (MEM)a

View larger version (50K): [in this window] [in a new window]   FIG. 1.   Results of zymogram analysis performed after renaturing SDS-PAGE using the chromogenic cephalosporin nitrocefin as the substrate for detection of -lactamase activity. Protein size standards (in kilodaltons) are indicated on the left. Lanes: 1, crude extract from E. coli DH5(pACYC184); 2, crude extract from E. coli DH5(pLLB-8AI).

View larger version (9K): [in this window] [in a new window]   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--lactamase activity (-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 blaFEZ-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.

Sequence of the cloned metallo--lactamase determinant. The 2.7-kb insert of plasmid pLLB-27B (Fig. 2) was sequenced. Analysis of sequence data revealed the presence of an 849-bp ORF (Fig. 3) encoding a protein which, in a BLAST search, showed the highest sequence similarity (29.3% identical residues) with the L1 enzyme of S. maltophilia and weaker similarities with other class B -lactamases. Results of subcloning experiments (Fig. 2) were consistent with the identification of this ORF as the metallo--lactamase determinant, which was named bla_FEZ-1. The bla_FEZ-1 ORF encodes a 282-amino-acid polypeptide whose amino-terminal sequence exhibits features typical of procaryotic signal peptides targeting protein secretion into the periplasmic space via the general secretory pathway (Fig. 3). According to known patterns (24), the cleavage site could be located after the Ser-17 residue. In this case the calculated molecular mass and pI value of the mature protein would be 29,567 Da and 7.71, respectively, which are in agreement with the results of analytical IEF and of zymograms performed after renaturing SDS-PAGE (see above). The low G+C content of the bla_FEZ-1 ORF (37.8%) is consistent with the average values reported for the genomes of Legionellaceae (5).

View larger version (46K): [in this window] [in a new window]   FIG. 3.   Nucleotide sequence of the blaFEZ-1 ORF and flanking regions. The initiation codon of the blaFEZ-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 blaFEZ-1 gene, possibly functioning as a transcriptional terminator, is overlined by arrows.

Comparison of the FEZ-1 enzyme with other metallo--lactamases. A multiple sequence alignment analysis with other class B -lactamases confirmed the closest similarity of the L. gormanii FEZ-1 enzyme with the S. maltophilia metallo--lactamase. FEZ-1 and L1 could be aligned over the entire sequence without introducing major gaps (Fig. 4), and the percent identity between the two (29.7%) was considerably higher than those between FEZ-1 and the other class B enzymes (Table 2). Compared to L1, the major differences of FEZ-1 are represented by the absence of the N-terminal 3₁₀ helix region that, in L1, is positioned prior to 1, and by an insertion within the C-terminal 5 helix which, in L1, is already considerably elongated in comparison with Bc-II and CcrA (31) (Fig. 4).

View larger version (47K): [in this window] [in a new window]   FIG. 4.   Comparison of the FEZ-1 amino acid sequence (in boldface type) with those of other molecular-class B -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 blaIMP 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, -lactamase II of Bacillus cereus 569/H (15); VIM-1, VIM-1 enzyme encoded by the blaVIM 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).

View this table: [in this window] [in a new window]   TABLE 2.   Pairwise comparison of percent amino acid sequence identity between class B -lactamasesa

Patterns of -lactam susceptibility of E. coli producing the L. gormanii metallo--lactamase. The susceptibility to several -lactams of E. coli DH5(pLLB-8AI), which carries the cloned bla_FEZ-1 determinant and produces the enzyme (Fig. 2), was compared to that of the same E. coli host carrying an empty vector.

View this table: [in this window] [in a new window]   TABLE 3.   In vitro susceptibility to various -lactams of E. coli DH5(pLLB-8AI), which contains the cloned blaFEZ-1 determinant, and of E. coli DH5 carrying the empty vector

Overexpression of the bla_FEZ-1 determinant in E. coli. A system for overexpression of the bla_FEZ-1 gene in E. coli was developed. In preliminary experiments, the use of expression systems based on the strong T7 phage promoter but characterized by relatively high basal levels of expression of the heterologous gene yielded unstable clones that eventually contained rearranged vectors and did not overproduce the enzyme. Therefore, a tightly regulated expression system was used in which the bla_FEZ-1 gene was cloned in the pET-24 vector and the resulting plasmid, pET-24/FEZ1, was introduced into the E. coli host BL21(DE3)(pLysS). Using this system, production of the FEZ-1 enzyme was detectable only upon induction with IPTG. In cell extracts prepared from induced cultures, the meropenem-hydrolyzing activity achieved a value of 377 ± 19 pmol/min/µg of protein at 2 h after induction, retained a similar value (383 ± 21 pmol/min/µg of protein) at 5 h after induction, and was found to be consistently decreased (107 ± 6 pmol/min/µg of protein) after 24 h, whereas in cell extracts prepared from uninduced cultures it remained lower than 5 pmol/min/µg of protein during the same time course. Interestingly, at 24 h after induction, a consistent amount of -lactamase activity was detected in the culture supernatant, revealing that a progressive leakage of the enzyme in the medium occurred during prolonged growth. In fact, the global amount of activity present in the culture was found to be maximal at 24 h after induction (Fig. 5). In analytical IEF and renaturing SDS-PAGE experiments, the -lactamase activity produced by E. coli BL21(DE3)(pLysS)(pET-24/FEZ1) was apparently identical to that produced by DH5(pLLB-8AI) (data not shown).

View larger version (20K): [in this window] [in a new window]   FIG. 5.   -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.

	DISCUSSION

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Results of this study showed that L. gormanii ATCC 33297^T carries an apparently resident class B -lactamase determinant whose product exhibits a distinctive preference for cephalosporin substrates. A similar feature remains as yet unique among class B enzymes (4, 6, 18, 26, 27) and reinforces the notion that considerable functional heterogeneity can be encountered among members of this class.

Sequencing of the cloned determinant revealed that its product is a new member of the highly divergent subclass B3 lineage, which thus far has included only one member, namely, the L1 enzyme of S. maltophilia (6, 26). The degree of sequence similarity between FEZ-1 and L1, which approaches 30% identical residues, suggests that the three-dimensional folding of the FEZ-1 molecule is probably very similar to that recently described for L1 (31). Moreover, the complete conservation of the residues that in L1 are known to be directly or indirectly involved in coordination of the two zinc ions suggests that the geometry of zinc coordination in the active site of FEZ-1 is the same as that in L1, being different from that observed in Bc-II and CcrA (8, 9, 11, 31). Since the two cysteine residues that in L1 (at positions 218 and 246) contribute an intramolecular disulfide bridge unique among class B enzymes (31) are conserved in FEZ-1, a similar disulfide bridge is likely to be retained also in the latter molecule. One of the major differences between FEZ-1 and L1 is represented by the absence, in the former, of the long N-terminal region which, in L1, plays a prominent role in providing the interactions between the A and C and between the B and D subunits (31). This difference could be responsible for the monomeric rather than oligomeric structure of the native FEZ-1 enzyme, although other differences found at positions that in L1 are known to participate in the subunit assembly (31) (Fig. 4) could also provide a contribution in this sense.

Results of this work also indicated that the bla_FEZ-1 gene can be expressed in E. coli, resulting in a functional product. Overproduction of the enzyme in E. coli, however, was found to be difficult using expression systems that are characterized by a high-level basal expression of the heterologous gene, and such overproduction could be obtained only with a tightly regulated expression system. This system could facilitate production of consistent amounts of the FEZ-1 enzyme, which, owing to its unique features, appears to be an interesting candidate for further structural and enzymological investigations. An analysis of the kinetic parameters of FEZ-1 with a broad array of -lactam substrates and with various chelating agents and other inhibitors is currently under way.