Molecular Heterogeneity of the L-1 Metallo-β-Lactamase Family from Stenotrophomonas maltophilia

Two β-lactamases, L-1 and L2, cause β-lactam resistance in Stenotrophomonas maltophilia. The native L-1 metalloenzyme has an isoelectric point of 6.9 and a molecular mass of 118 kDa. The L-1 enzyme from S. maltophilia IID1275 has been sequenced and has low identity with all other known metallo-β-lactamase enzymes (18).

We have reported the cloning in Escherichia coli of theblaS gene producing the chromosomal L-1 β-lactamase fromS. maltophilia GN12873 having biochemical properties and an isoelectric point similar to those of the enzyme expressed in the parental strain (5). We present here the nucleotide sequence of the blaS gene and comparisons between the L-1 enzymes from strains IID1275 and GN12873, showing the heterogeneity of S. maltophilia enzymes. A multiple alignment defined the conserved amino acid boxes found in all class B metalloenzymes.

The bacterial strains and plasmids used in this study are described in Table 1. E. coli DH5α was the recipient strain used for construction, maintenance, and propagation of recombinant plasmids. Bacterial cells were routinely grown on tryptic soy agar (Difco Laboratories, Detroit, Mich.) containing appropriate antibiotics (ampicillin, 50 μg/ml; imipenem, 10 μg/ml; kanamycin, 50 μg/ml).

Large plasmid DNA preparations were done by using the Qiagen Maxi Kit (Qiagen, Chatsworth, Calif.). Restriction enzyme, T4 ligase, and DNA-modifying enzyme reactions were done as recommended by the manufacturer (New England Biolabs, Inc., Beverly, Mass.). Construction of recombinant molecules and transformation and selection of bacterial clones were done by standard procedures (15).

Nucleotide sequence determinations were done on an Applied Biosystems 373 DNA sequencer using ABI Prism dye terminator cycle sequencing ready reaction kits with the AmpliTaq DNA polymerase protocol as recommended by the manufacturer (Perkin-Elmer, Mississauga, Ontario, Canada). Sequencing primers were usually 21-mers selected from the last 50 nucleotides read from chromatograms and synthesized on a Beckman Oligo1000 DNA synthesizer.

Electrophoretograms were visualized by using Factura, Gene Navigator, and AutoAssembler software (ABI). DNA sequence analysis was done with the Genetics Computer Group software package (Wisconsin Package Version 9.0; Genetics Computer Group, Madison, Wis.). Molecular masses were predicted from amino acid sequences as previously described (2). Multiple alignments were done with PileUp and CLUSTAL W (16).

The physical map of encoding L-1-pMON13 is shown in Fig.1. Restriction endonuclease sites deduced from the DNA sequences matched the physical map previously reported (5), except for one PstI site not identified as two restriction sites separated by only 12 nucleotides.

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Fig. 1.

Physical map of recombinant plasmid pMON13. The open boxes represent the 1.4-kb BamHI-SalI DNA fragment from pMON01 cloned into pBGS18⁺. The shaded box represents the position of the blaS gene in the cloned moiety, and the arrow indicates the direction of transcription.

The complete nucleotide sequence obtained is shown in Fig.2 and is 1,425 bp long. Analysis of the nucleotide sequence revealed an open reading frame (ORF), selected by a BLAST search with the PIR database, long enough to encode the putative L-1 polypeptide. We determined that the G+C content was 56.6% for the 5′ upstream region of the ORF, 68.7% within the ORF, and 68.4% for the 3′ downstream region. A putative ribosome binding site was found 10 bp upstream of an ATG start codon (positions 100 to 102 in Fig. 2), but no putative promoter was identified. One possible terminator was localized at the end of the ORF (nucleotide positions 947 to 963 in Fig. 2), and a second terminator forming a loop structure was localized 260 bp downstream of the termination codon (positions 1229 to 1250). The deduced polypeptide was 290 amino acids long with a calculated putative signal peptide of 33 amino acids which agreed with the previously published N-terminal peptide sequence, except for 8 amino acids (1). The molecular weight of the L-1 metallo-β-lactamase from strain GN12873 was similar to the value obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1, 14).

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Fig. 2.

Nucleotide and deduced amino acid sequences ofblaS encoding the L-1 metallo-β-lactamase from S. maltophilia GN12873. The proposed Shine-Dalgarno ribosome binding site (RBS) is identified. The 33-amino-acid leader peptide underlined has been identified previously (1). The termination codon is indicated by an asterisk, and the putative terminators, ter1 and ter2, for secondary loop structures are underlined. Vertical arrows identify the cutting sites mapped for restriction endonucleases.

Curiously, 12 to 14% heterogeneity was observed between the nucleotide and amino acid sequences of the L-1 enzymes from strains GN12873 and IID1275. We found 88.7% DNA identity between both of theblaS structural genes (data not shown). Comparisons between the L-1 amino acid sequences showed 33 amino acid changes in a total of 290 amino acids, mostly in regions outside conserved amino acid boxes that we identified (Fig. 3). Such heterogeneity is unique to the family of L-1 chromosomal enzymes; other β-lactamase types, such as class C chromosomal enzymes, have not been shown to have such heterogeneity within the same species of bacteria.

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Fig. 3.

Alignment of the L-1 amino acid sequences with the sequences of eight class B metallo-β-lactamases. The black boxes identify amino acids conserved in four of the eight polypeptides, and the gray boxes indicate amino acids that are similar in four of the eight polypeptides. Amino acids that are conserved in seven of the eight metallo-β-lactamases and known to be important in the crystal structure available are represented as boxes I to IV. Numbering of amino acids follows that of the B. fragilis CcrA3 enzyme (4). Sequences: L-1 (GN12873), from S. maltophilia GN12873 (5; this work) (GenBank accession no. AF010282); L-1 (IID1275), from S. maltophiliaIID1275 (18) (GenBank accession no. S45349); 170, from alkalophilic Bacillus sp. strain 170 (7) (GenBank accession no. M15350); 569/H/9, from B. cereus 569/H/9 (6) (GenBank accession no. M11189); 5/6/B, from B. cereus 5/6/B (8) (GenBank accession no. M19530); CcrA3, from B. fragilis QMCN3 (12); IMP-1, from S. marcescens TN9106 (10) (GenBank accession no. D50438) (identical to the amino acid sequence of the K. pneumoniaemetallo-β-lactamase from plasmid pDK4-4 [GenBank accession no.D29636]); CphA, from A. hydrophila AE036 (23) (GenBank accession no. X57102).

Additional comparisons were done between the GN12873 L-1 enzyme and all other known class B metalloenzymes, and the results are summarized in Table 2. Amino acid identity between the family of L-1 β-lactamases and all other class B enzymes was low. CcrA (11; GenBank accession no. M63556), CfiA (17), CcrA3 (12), and CcrA4 (12) differed by zero to four amino acids (13). CphA2 (GenBank accession no. U60294) and CphA (9; GenBank accession no. X57102) shared greater than 95% amino acid identity (13). Thus, only CphA and CcrA3 were included in Table 2.

The alignment obtained for class B β-lactamases is shown in Fig. 3. Amino acids conserved in four of eight polypeptides are shaded black, and amino acids similar in four of eight polypeptides are shaded gray; these identified four regions conserved among seven of the eight metalloenzymes used in the multiple alignment. The multiple alignment was constructed by taking into consideration the three-dimensional structures of the Bacillus cereus 569/H/9 andBacteroides fragilis (CcrA3) β-lactamases (3,4) and using the numbering scheme of CcrA3. The alignment obtained was similar to what others have proposed for class B β-lactamases when considering active-site elements (3, 4,13). The difference in our alignment appeared as a gap in the C-terminal region and was introduced by the family of L-1 enzymes used in the alignment.

The alignment shown in Fig. 3 identified conserved features among class B metalloenzymes that can be correlated to structure and function. The blocks of amino acids conserved in box I contained the triad Leu, Val, and Ile-Asp that is found in all class B enzymes including Asp69, which is known to be buried in the structure and has been implicated in zinc ligand positioning (4). Box II contained the His Xaa His Xaa Asp consensus sequence which has been proposed as the ligand of Zn1 and Zn2 in the CcrA3 enzyme structure (4). Box III contained the consensus sequence Gly His162 Thr, which, along with His99 and His101, forms the ligand of Zn1. Box IV contained His223, which is the ligand of Zn2 (4).

• Copyright © 1998 American Society for Microbiology