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Antimicrobial Agents and Chemotherapy, July 2008, p. 2473-2479, Vol. 52, No. 7
0066-4804/08/$08.00+0     doi:10.1128/AAC.01062-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Functional Diversity among Metallo-β-Lactamases: Characterization of the CAR-1 Enzyme of Erwinia carotovora

Magdalena Stoczko,¹ Jean-Marie Frère,² Gian Maria Rossolini,¹ and Jean-Denis Docquier^1,2*

Dipartimento di Biologia Molecolare, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi, Università di Siena, I-53100, Siena, Italy,¹ Centre d'Ingénierie des Protéines and Laboratoire d'Enzymologie, Université de Liège, B-4000 Liège, Belgium²

Received 12 August 2007/ Returned for modification 17 October 2007/ Accepted 23 April 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Metallo-β-lactamases (MBLs) are zinc-dependent bacterial enzymes characterized by an efficient hydrolysis of carbapenems and a lack of sensitivity to commercially available β-lactamase inactivators. Apart from the acquired subclass B1 enzymes, which exhibit increasing clinical importance and whose evolutionary origin remains unclear, most MBLs are encoded by resident genes found in the genomes of organisms belonging to at least three distinct phyla. Using genome database mining, we identified an open reading frame (ORF) (ECA2849) encoding an MBL-like protein in the sequenced genome of Erwinia carotovora, an important plant pathogen. Although no detectable β-lactamase activity could be found in E. carotovora, a recombinant Escherichia coli strain in which the ECA2849 ORF was cloned showed decreased susceptibility to several β-lactams, while carbapenem MICs were surprisingly poorly affected. The enzyme, named CAR-1, was purified by means of ion-exchange chromatography steps, and its characterization revealed unique structural and functional features. This new MBL was able to efficiently hydrolyze cephalothin, cefuroxime, and cefotaxime and, to a lesser extent, penicillins and the other cephalosporins but only poorly hydrolyzed meropenem, while imipenem was not recognized. CAR-1 is the first example of a functional naturally occurring MBL in the family Enterobacteriaceae (order Enterobacteriales) and highlights the extraordinary structural and functional diversity exhibited by MBLs.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Metallo-β-lactamases (MBLs), or Ambler's class B β-lactamases, are zinc-dependent enzymes involved in bacterial resistance (1, 33). In the clinical setting, the most important enzymes, belonging to five sublineages (IMP- and VIM-type MBLs, SPM-1, GIM-1, and SIM-1), are horizontally acquired and are encoded by mobile genetic elements (integrons and/or plasmids), which account for their current spread in important human opportunistic pathogens, such as Pseudomonas spp., Acinetobacter spp., and Enterobacteriaceae (20, 32, 33, 39). Apart from these acquired enzymes, most MBLs identified thus far are encoded by resident genes found in various bacterial species, most of which are not pathogenic (e.g., Caulobacter vibrioides or Shewanella spp.) or are occasionally found in the clinical setting (e.g., members of the genus Flavobacteria) (4, 12, 31, 34).

All known MBLs share common functional features, such as (i) the ability to hydrolyze carbapenem compounds with high catalytic efficiencies while (ii) not being sensitive to classical β-lactamase inactivators (e.g., clavulanate, tazobactam, and sulbactam) and (iii) being readily inactivated by metal chelators (33). However, they exhibit an important structural and functional heterogeneity, reflected in the nature of the residues coordinating the metal ions that represent the basis for their classification into three distinct subclasses (15) and rather large differences in substrate profiles, which might be very narrow in subclass B2 enzymes (e.g., Aeromonas sp. strain CphA and relatives) to exceedingly broad in other enzymes (e.g., most acquired subclass B1 enzymes) (reference 33 and references therein).

Thus far, resident enzymes have been identified in species belonging to three phyla, namely, Firmicutes, Bacteroidetes, and Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria) (reference 33 and references therein). From the perspective of investigating the evolutionary relationships of MBLs, the postgenomic approach has already proven to be a powerful tool in detecting new enzymes, some of which might exhibit peculiar functional properties and/or genetic locations (e.g., CAU-1 from C. vibrioides (12) and BJP-1 of Bradyrhizobium japonicum (36).

In this work, we report the identification of a new MBL encoded by the chromosome of Erwinia carotovora subsp. atroseptica DSM 30184 (also recently named Pectobacterium atrosepticum) (17) that exhibits the zinc-binding motifs of subclass B3 enzymes, although with very peculiar functional and structural features. This bacterium, an important plant pathogen causing soft rot and blackleg diseases (2), is also known to produce a carbapenem compound and belongs to the family Enterobacteriaceae, which also includes important human pathogens, among which no resident MBL genes have been identified. Therefore, E. carotovora subsp. atroseptica is an organism of great interest for investigating the potential production of a resident MBL.

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Bacterial strains and culture conditions. E. carotovora subsp. atroseptica DSM 30184 was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany) and was used as a source of the MBL gene (bla_CAR-1). Escherichia coli DH5 (Gibco Life Technologies, Gaithersburg, MD) was used as a host for recombinant plasmids, while E. coli BL21(DE3) (Stratagene, La Jolla, CA) was used for overproduction of the CAR-1 enzyme using a T7 promoter-based expression system. Bacteria were always grown aerobically. E. carotovora was cultured at 28°C in nutrient broth (Oxoid Ltd., Basingstoke, United Kingdom) supplemented, in β-lactamase induction experiments, with subinhibitory concentrations of ampicillin or imipenem and following appropriate biosafety procedures (8a). E. coli DH5 derivative strains were cultured at 37°C in Mueller-Hinton broth (Oxoid Ltd., Basingstoke, United Kingdom). ZYP-0.8G medium was used for routine propagation of E. coli BL21(DE3) derivatives, while ZYP-5052 medium was used for production of the recombinant protein (37).

Database search and sequence analysis. Database screening was performed using BLAST software (version 2.2.14) available from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST). DNA and protein sequence alignments and phylogenetic trees were created using ClustalX (version 1.83) and TreeView (version 1.6.1) (9, 30). Protein theoretical pIs and molecular masses were calculated with software running at the ExPASy proteomics server (http://www.expasy.org/). Signal peptide cleavage site was predicted using SignalP (version 3.0) (5). Putative promoter sequences and binding sites for regulatory proteins were performed using bprom software (Softberry, Inc.).

Molecular methods. Recombinant DNA procedures were carried out as described by Sambrook and Russel (35). The ECA2849 open reading frame (ORF) (identified as bla_CAR-1) was amplified by PCR with primers CAR-fwd (5'-CACCCATATGAAAAACCAACGTCTTACG), which added an NdeI restriction site (underlined) to the 5' end, and CAR-rev (5'-GCGGATCCTATTTTTGAATCCGTGCCTG), which added a BamHI restriction site (underlined) to the 3' end of the gene. PCR was performed with 5 U of the Expand High Fidelity PCR system DNA polymerase (Roche Biochemicals, Mannheim, Germany) in accordance with the manufacturer's instructions, using 200 µM deoxynucleoside triphosphates, 50 pmol of each primer, and 2 µl of boiled E. carotovora cells as the template in a total volume of 50 µl. The cycling conditions were as follows: after the initial denaturation step of 96°C for 3 min, 30 cycles of denaturation at 96°C for 40 s, annealing at 53°C for 40 s, and extension at 72°C for 2 min and a final extension step at 72°C for 20 min. The amplified DNA was cloned into vector pLB-II (a pBC-SK derivative [Stratagene, La Jolla, CA] modified in our laboratory) (7), yielding the recombinant plasmid pLBII-CAR-1. After confirmatory sequencing, the 1,029-bp NdeI-BamHI fragment was subcloned into the expression vector pET-9a (Novagen, Madison, WI) to obtain the recombinant plasmid pET9-CAR-1.

Antimicrobial susceptibility testing. The in vitro antimicrobial susceptibility profiles of E. carotovora and E. coli DH5 derivatives were determined by the broth microdilution method as recommended by the Clinical Laboratory Standards Institute (10), using nutrient broth and Mueller-Hinton broth, respectively, with a bacterial inoculum of 5 x 10⁴ CFU/well. MICs were recorded after 24 h of incubation at 28°C for E. carotovora and 18 h at 37°C for E. coli.

Preparation of crude extracts and β-lactamase assays. Crude extracts of E. carotovora subsp. atroseptica and of E. coli strains were prepared in 50 mM HEPES-NaOH buffer, pH 7.5, supplemented with 50 µM ZnSO₄ (HZN buffer) as described previously (36). β-Lactamase activity was assayed spectrophotometrically on a Cary UV-visible-light spectrophotometer (Varian, Walnut Creek, CA) using various β-lactam substrates, including representatives of penicillins, cephalosporins, carbapenems, and aztreonam (at an initial concentration ranging from 50 to 500 µM). Reactions were performed in HZN buffer at 30°C in a total volume of 500 µl. Inhibition of enzymatic activity by EDTA was determined by measuring the residual activity after incubation of crude extract with 5 mM EDTA for 20 min at 25°C.

Production and purification of CAR-1. The cloned MBL of E. carotovora was purified from a culture of E. coli BL21(DE3)/pET9-CAR-1 grown in 3 liters of ZYP-5052 medium supplemented with 50 µg/ml kanamycin for 48 h at 37°C. Cells were harvested by centrifugation (10,000 x g; 30 min; 4°C), resuspended in 50 ml of 20 mM Tris-HCl supplemented with 50 µM ZnSO₄ (pH 8.5) (TZN buffer), and lysed using a cell disruption system (Constant Systems Ltd., Daventry, United Kingdom). Cell debris was then removed by centrifugation (12,000 x g; 1 h; 4°C), and the clarified supernatant was loaded (flow rate, 2 ml/min) onto an XK16 column (GE Healthcare, Uppsala, Sweden) packed with 25 ml of DEAE-Sepharose (GE Healthcare) preequilibrated with TZN buffer. Elution was performed with an NaCl linear gradient (0 to 0.5 M in 325 ml; flow rate, 2 ml/min) in the same buffer. Fractions containing β-lactamase activity were pooled and concentrated by ultrafiltration, and the buffer was changed to 20 mM ethanolamine-HCl containing 50 µM ZnSO₄ (pH 9.5) (EZN buffer) using a HiPrep 26/10 desalting column (GE Healthcare). The sample was then loaded (0.8 ml/min) on a Mono Q 5/50 GL column (GE Healthcare) preequilibrated with EZN buffer and eluted with an NaCl linear gradient (0 to 25 mM in 50 ml) in the same buffer. Active fractions were pooled and stored in small aliquots at –80°C until they were used.

Protein analysis techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and determination of the protein concentration in solution were performed as described previously (36). The molecular mass of the native CAR-1 enzyme was estimated by size exclusion chromatography using a Superdex 75 HR 10/30 column (GE Healthcare) in HZN buffer supplemented with 150 mM NaCl as previously described (36). The molecular mass of the enzymatic preparation of CAR-1 was measured by electrospray mass spectrometry as described previously (12), using a Finnigan LTQ mass spectrometer equipped with an ion spray source (Thermo Electron Co., Shaumberg, IL).

Determination of kinetics parameters and inactivation by chelating agents. The hydrolysis of β-lactam substrates was monitored by measuring the variation in absorbance under the experimental conditions reported previously (21). All measurements were performed on a Cary 100 UV-visible-light spectrophotometer (Varian) at 30°C in HZN buffer in a total volume of 500 µl. The purified CAR-1 was diluted in HZN buffer supplemented with 20 µg/ml bovine serum albumin to prevent enzyme denaturation. Enzyme concentrations in the assays ranged between 1.9 and 3,800 nM. The steady-state kinetic parameters (k_cat and K_m) were calculated after direct fit of the Henri-Michaelis-Menten equation on the experimental data (initial rates versus substrate concentrations). The inactivation kinetics of CAR-1 by divalent ion chelators (EDTA and 1,10-o-phenanthroline and pyridine-2,6-dicarboxilic [dipicolinic] acid) was investigated in 50 mM HEPES, pH 7.5, at 30°C using 1 mM cephalothin as the reporter substrate, as previously described (6, 11, 27).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Identification of an MBL in E. carotovora. Postgenomic screening of available bacterial genomes, using MBL protein sequences as template queries, revealed the presence of an MBL homologue in the chromosome of an important plant pathogen, E. carotovora subsp. atroseptica SCRI1043 (accession number BX950851). The 1,029-bp MBL-like gene (locus tag, ECA2849) encodes a 342-residue protein exhibiting the best, although overall low, similarity with MBLs of subclass B3, with identity scores ranging from 19.2 (FEZ-1) to 27.5% (BJP-1).

Phenotypic properties of E. carotovora. Crude extracts obtained from E. carotovora subsp. atroseptica DSM 30184 grown in liquid medium did not show any β-lactamase activity against various β-lactam substrates (data not shown), although PCR experiments confirmed the presence of the ECA2849 ORF in the genome of the strain, whose sequence was identical to that of ECA2849 of strain SCRI1043. The same result was obtained with crude extracts prepared at different time intervals (up to 24 h) when the strain was grown in the presence of subinhibitory concentrations of ampicillin or imipenem as an inducer (final concentrations, 0.06 and 0.03 µg/ml, respectively) (data not shown). However, the susceptibility profile of E. carotovora showed rather high MICs for several antibiotics, especially cephalosporins, piperacillin, and aztreonam (Table 1), strongly suggesting the involvement of mechanisms of resistance other than production of β-lactamase (e.g., efflux pump, lowered permeability, or low affinity of penicillin-binding proteins).

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TABLE 1. In vitro susceptibility profile of E. carotovora subsp. atroseptica DSM 30184, E. coli DH5(pLBII-CAR-1) carrying the cloned blaCAR-1 gene, and E. coli DH5 carrying the empty plasmid

Genetic background of the MBL-like gene in Erwinia. The G+C content of the ECA2849 ORF (bla_CAR-1; 47.0%) is in good agreement with the value of the whole E. carotovora genome (51.0%) and suggests a natural occurrence of the gene in that species. The bla_CAR-1 gene was located downstream of a gene oriented in the opposite direction and encoding a LysR-type transcriptional regulator (ECA2848) that probably controls the expression of the β-lactamase gene in the original host. Interestingly, this protein was poorly related to other known enterobacterial β-lactamase transcriptional regulators, such as AmpR and NmcR (identity, <20%) (23, 28). These data might explain the lack of detectable β-lactamase activity in Erwinia, even in the presence of a β-lactam antibiotic in the culture. A typical arrangement of overlapping promoters was found between the two ORFs, and putative binding motifs for the ⁷⁰ and Lrp (leucine-responsive regulatory protein) (8) transcriptional factors were identified. The bla_CAR-1 gene is also close to the extremity of a horizontally acquired genomic island (HAI12) that includes 29 ORFs (mostly encoding proteins of unknown function) and an Rhs element (2, 19) (Fig. 1).

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FIG. 1. Genetic context of the MBL gene in the genome sequence of E. carotovora, showing the presence of a putative transcriptional regulator of the LysR family that likely controls its expression and its close relationship with a horizontally acquired genomic island.

Cloning and characterization of the MBL homologue. The ECA2849 ORF was amplified by PCR and cloned under the transcriptional control of the P_lac promoter in a high-copy-number plasmid (pLB-II) used to transform E. coli DH5. Expression of this putative β-lactamase gene in E. coli DH5(pLBII-CAR-1) was associated with a decrease in susceptibility to various cephalosporins (especially cephalothin, cefuroxime, and cefotaxime) and ampicillin, while the MICs for other tested antibiotics showed no variation or were only slightly affected (Table 1).

The resulting strain produced a detectable amount of β-lactamase activity, although only with some of the tested β-lactam substrates, that was inhibited >90% in the presence of 5 mM EDTA, which confirmed the production of a metalloenzyme. The highest activities were measured with cephalothin, cefuroxime, and cefotaxime (specific activities, 2,500 to 3,900 nmol/min·mg protein). Surprisingly, activity against carbapenems was extremely low and even undetectable with imipenem (specific activity, 0.8 nmol/min·mg protein). These data confirmed that the product of the ECA2849 ORF was a functional MBL, which was named CAR-1 (after E. carotovora), and were in agreement with the unusual susceptibility profile exhibited by the recombinant MBL-producing E. coli strain.

Sequence features of CAR-1. CAR-1 could be aligned with subclass B3 MBLs without introducing major gaps (Fig. 2). A striking feature of CAR-1 is its notably longer N terminus, accounting for an extra 35 to 54 residues in the mature protein, in comparison with other subclass B3 members. Among known MBLs, CAR-1 shows the highest identity scores with subclass B3 MBLs (19.2 to 27.5%), and particularly BJP-1, rather than with subclasses B1 (12 to 17%) and B2 (14 to 15%). However, the identity scores of CAR-1 with subclass B3 MBLs are overall lower than those calculated between the other members of this subclass (21 to 40%). A phylogenetic tree constructed on the basis of a multiple-protein sequence alignment with MBLs representative of all known sublineages confirmed that this putative protein would represent the most distant member of subclass B3, although it shares all the zinc-binding motifs typical of the subclass (Fig. 2 and 3). This is also reflected by the fact that, among the 18 strictly conserved residues shared by other subclass B3 members, CAR-1 shows substitutions at 4 (Thr57Ile, Ala117Gly, Gly149Asp, and Asp257Thr) (Fig. 2). From a structural standpoint, positions 57 and 257 are rather distant from the active site and likely do not bear any functional relevance. By contrast, substitutions at the two remaining positions might not be neutral. Indeed, position 117 is located between the two metal-binding histidines, where a hydrophobic residue is usually present (or a Ser in some B1 MBLs), whereas the glycine residue found in CAR-1 is unprecedented. Similarly, position 149 is located at the beginning of the active-site loop L2, and the nature of the corresponding residue might affect the orientation of the latter.

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FIG. 2. Amino acid sequence alignment of CAR-1 with subclass B3 MBLs. Secondary-structure elements (β, strands; 310 and , helices) of the L1 enzyme are indicated above the sequences (38). The N-terminal signal peptide is shown with light letters (underlined for CAR-1). Numbering is in accordance with the BBL scheme (15). Residues identical in all sequences are indicated by a black background, and residues identical in all sequences but CAR-1 are indicated by a gray background.

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FIG. 3. Unrooted tree showing the phylogenetic relationship of CAR-1 with other MBLs belonging to the three subclasses (dashed lines, subclass B1; dotted lines, subclass B2; solid lines, subclass B3). Only the first variant is shown for each sublineage. The accession numbers of sequences can be found in references 22, 31, and 33.

Purification and biophysical properties of CAR-1. The CAR-1 enzyme was overexpressed in E. coli using a T7-based system. The β-lactamase was purified from 3 liters of E. coli BL21(DE3)/pET9-CAR-1 culture grown in ZYP-5052 autoinducing medium by means of two steps of anion-exchange chromatography, yielding approximately 3 mg of pure enzyme per liter of culture (purification, 170-fold). Most of the contaminating proteins were removed by the first step (DEAE-Sepharose at pH 8.5), while the second step (MonoQ at pH 9.5) yielded >95% pure enzyme, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown).

The protein mass determined by electrospray mass spectrometry was 35,059.0 ± 8 Da, in perfect agreement with the theoretical mass of the mature protein (35,058.7 Da) obtained after cleavage of the 21-amino-acid leader peptide (1 amino acid shorter than predicted by SignalP software). The mass of CAR-1 determined by size exclusion chromatography yielded a molecular mass of 40 ± 6 kDa, suggesting that the native enzyme is monomeric in solution, like most of the subclass B3 MBLs (3, 11, 12, 27, 36) except L1, which is found as a homotetramer in the native state (6, 38).

The monomeric nature of CAR-1 is compatible with the lack of Met175, involved with tetramerization in L1 enzyme (16, 38). In CAR-1, as in GOB-1 and BJP-1, this residue is replaced by a lysine. The two cysteines that form a disulfide bridge located in the C-terminal domains of L1 and FEZ-1 (Cys256 and Cys290), and probably also of CAU-1 and BJP-1 (Cys200 and Cys220), are not present in CAR-1. However, two cysteine residues (positions 8 and 58) are present at the N-terminal end of the latter, with Cys58 exposed to the solvent and thus compatible with the formation of a disulfide bridge that might anchor the longer N-terminal extension to the globular domain of the protein.

Functional properties of CAR-1. The kinetic parameters of the purified CAR-1 were determined for a representative set of β-lactam antibiotics, including penicillins, narrow- and expanded-spectrum cephalosporins, carbapenems, and aztreonam, under the experimental conditions used for BJP-1 and THIN-B (12, 36) (Table 2). CAR-1 was able to hydrolyze most penicillins and cephalosporins (except temocillin and cefepime) but, surprisingly, showed a very poor hydrolysis of carbapenems. With meropenem, a pseudo-first-order kinetic reaction was observed, as the initial rate of hydrolysis was proportional to the substrate concentration up to almost 1 mM, indicating a rather low affinity for the substrate. More strikingly, hydrolysis of imipenem could not be detected with enzyme concentrations up to almost 4 µM. In addition, imipenem (up to 250 µM) did not affect the initial rate of hydrolysis of cephalothin in competition experiments. This underlines its very poor, if any, interaction with the enzyme.

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TABLE 2. Kinetic properties of the purified CAR-1 MBL with various β-lactam compoundsa

The best substrates of CAR-1 are cephalothin, cefuroxime, and cefotaxime, for which the highest turnover rates and catalytic efficiencies were observed (k_cat, >240 s^–1; k_cat/K_m, >10⁶ M^–1·s^–1). Cefoxitin, an -methoxy derivative, is hydrolyzed 500- to 1,000-fold less efficiently than the former substrates, and the enzyme apparently shows a poor affinity for the substrate. Oxyimino-cephalosporins showed very different behaviors; some were very good substrates (e.g., cefuroxime and cefotaxime), while others showed a very poor interaction with the enzyme (e.g., cefepime). This likely reflects the importance of the nature of the leaving group in hydrolysis, as the presence of a positive side chain (such as in ceftazidime and cefepime) is apparently detrimental to binding to the active site and/or catalysis.

Penicillin substrates are overall less efficiently hydrolyzed by CAR-1 than cephalosporins, as they show an increase of K_m values with a concomitant decrease of k_cat values. Similarly to cefoxitin, the presence of an -methoxy group in temocillin had an important effect on the enzyme activity, as no detectable hydrolysis could be observed. As observed for all other MBLs, aztreonam was not hydrolyzed by CAR-1 and did not affect the hydrolysis rates of other substrates in competition experiments. The catalytic efficiencies of representative subclass B3 enzymes for various β-lactams are compared in Table 2, showing the very unique substrate profile of CAR-1, which is strongly oriented toward cephalosporins, excluding cefepime.

All the tested chelating agents behaved as strong inactivators of CAR-1, and the measured inactivation rates were found to be independent of the chelator concentration and very similar for all three compounds (ranging from 2.5 x 10^–2 to 3.2 x 10^–2 s^–1). These data indicate that inhibition proceeds by scavenging the free metal, and the inactivation rates represent the rates of dissociation of the protein-zinc ion complex. This behavior is similar to what has been observed with most subclass B3 enzymes (L1, FEZ-1, and CAU-1) (6, 12, 27) except THIN-B (in which inactivation apparently occurs by formation of an enzyme-metal-chelator ternary complex) (11) and BJP-1 (which shows reduced sensitivity toward all chelating agents) (36).

Concluding remarks. Genome database mining represents a very interesting way to investigate the distribution of resident and functional MBLs in a wide range of microorganisms, providing useful data related to the evolution of MBLs. In this work, we identified and characterized a new member of the rapidly growing family of class B enzymes, which shows increasing heterogeneity. Indeed, although CAR-1 exhibits all the metal-binding signatures of subclass B3 MBLs, it also represents the most divergent enzyme identified thus far, and considering its unique structural and functional properties, it might belong to a new subclass (B4). This is also supported by the low identity scores of CAR-1 with other subclass B3 MBLs, which do not exceed 27.5%, while higher identities could be observed between enzymes belonging to two different subclasses (maximum B1-B2 identity, 28%).

As already observed with B. japonicum, the susceptibility profile of E. carotovora could not be easily correlated with the eventual production of a β-lactamase but is apparently dependent on other factors (36). CAR-1 is also the first MBL showing a very weak hydrolysis of carbapenems, which are good to excellent substrates for all previously reported enzymes. Its unusual and very peculiar functional properties make it a very interesting model for structure-function relationship studies and further support the hypothesis of different possible evolutionary pathways leading to β-lactamase activity in the superfamily of metal-dependent metallohydrolases, as suggested earlier (18).

As for the CAU-1 and BJP-1 MBLs (of C. vibrioides and B. japonicum, respectively), the real physiological roles of these enzymes in the original hosts are possibly different from antibiotic resistance, as suggested by the peculiar susceptibility profile and the absence of β-lactamase production in the presence of β-lactam antibiotics, and they remain enigmatic. We could not exclude the possibility that their roles might be related to the natural habitats of the organisms, where a potentially high selective pressure might be a consequence of modern agricultural techniques in which antibiotic usage might be rather high, and due to the presence of naturally occurring β-lactam producers in the soil (29), as far as, in this case, an important plant pathogen (E. carotovora) is concerned. In addition, the factors able to trigger the expression of the β-lactamase, likely not β-lactams, certainly will be interesting to identify.

More strikingly, some strains of E. carotovora have been reported to produce a β-lactam compound structurally related to carbapenems but without the typical -hydroxyethyl side chain. However, the β-lactam biosynthetic operon (carAH genes) is missing from the sequenced genome in which CAR-1 was identified, making its relationship with the β-lactam biosynthesis and/or protection system, in which the involvement of other proteins (CarF and CarG, which likely represent the natural resistance mechanism) has already been demonstrated, unlikely (although not impossible) (25, 26).

Finally, CAR-1 also represents the first example of a functional naturally occurring MBL encoded by the genome of a bacterium belonging to the family Enterobacteriaceae (order Enterobacteriales), thus increasing the number of orders and families in the phylum Proteobacteria in which naturally occurring MBLs have been detected. CAR-1 also highlights the extraordinary structural heterogeneity exhibited by enzymes found in bacterial species belonging to the gammaproteobacteria, a class in which representatives of the three subclasses were previously found (e.g., SLB-1, CphA, and L1) (6, 24, 31), underlining the complex and fascinating evolution of MBLs.

 
This work was supported by grants from the European Union (contracts HPRN-CT-2002-00264 and LSHN-CT-2003-503335) and by the FRS-FNRS (Brussels, Belgium). M.S. was supported by the European research network on MBLs (MEBEL) (contract no. HPRN-CT-2002-00264). J.-D.D. was supported by a postdoctoral grant from the Belgian Fonds National de la Recherche Scientifique.

 
* Corresponding author. Mailing address: Dipartimento di Biologia Molecolare, Università di Siena, Policlinico Le Scotte, I-53100 Siena, Italy. Phone: 39 0577 233134. Fax: 39 0577 233334. E-mail: jddocquier{at}unisi.it

Published ahead of print on 28 April 2008.

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Antimicrobial Agents and Chemotherapy, July 2008, p. 2473-2479, Vol. 52, No. 7
0066-4804/08/$08.00+0     doi:10.1128/AAC.01062-07
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