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Antimicrobial Agents and Chemotherapy, May 1998, p. 1245-1248, Vol. 42, No. 5
Microbiologie Moléculaire et
Génie des Protéines, Sciences de la Vie et de la
Santé, Pavillon Charles-Eugène Marchand et Faculté de
Médecine, Université Laval, Ste-Foy, Québec, Canada
G1K 7P4
Received 8 July 1997/Returned for modification 21 October
1997/Accepted 10 February 1998
We have determined the nucleotide sequence of the blaS
gene encoding the carbapenem-hydrolyzing L-1 Two We have reported the cloning in Escherichia coli of the
blaS gene producing the chromosomal L-1 The bacterial strains and plasmids used in this study are described in
Table 1. E. coli DH5
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Molecular Heterogeneity of the L-1
Metallo-
-Lactamase Family from Stenotrophomonas
maltophilia
ABSTRACT
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Abstract
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References
-lactamase from
Stenotrophomonas maltophilia GN12873. Analysis of the DNA
and deduced amino acid sequences identified a product of 290 amino
acids. Comparisons of the L-1 amino acid sequence with those of other
zinc -lactamases showed 88.6% identity with the L-1 enzyme from
S. maltophilia IID1275 and less than 20% identity with
other class B metalloenzymes.
TEXT
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Abstract
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-lactamases, L-1 and L2,
cause -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--lactamase enzymes (18).
-lactamase from
S. 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.
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).
TABLE 1.
Bacterial strains and plasmids used in this study
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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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--lactamase from strain GN12873 was similar to the value
obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(1, 14).
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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 the
blaS 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
-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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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 -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.
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The alignment obtained for class B -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 and
Bacteroides fragilis (CcrA3) -lactamases (3,
4) and using the numbering scheme of CcrA3. The alignment
obtained was similar to what others have proposed for class B
-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).
Nucleotide sequence accession number. The sequence reported here has been assigned GenBank accession no. AF010282.
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ACKNOWLEDGMENTS |
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We thank Y. Saino, Gunma University, Maebashi, Japan, for S. maltophilia GN12873; J. Renaud, Université Laval, for technical assistance in nucleotide sequencing; O. Herzberg, CARB, University of Maryland, College Park, for comments regarding the L-1 structure; and J.-M. Frère, Université de Liège, Liège, Belgium, for confirmation of the L-1 sequence reported here.
This study was supported by grants to R.C.L. from the Canadian Center of Excellence as a member of the Canadian Bacterial Diseases Network. R.C.L. is a Research Scholar of Exceptional Merit of and J.D. obtained a studentship from Le Fonds de la Recherche en Santé du Québec.
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FOOTNOTES |
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* Corresponding author. Mailing address: Microbiologie Moléculaire et Génie des Proteines, Sciences de la Vie et de la Santé, Pavillon Charles-Eugène Marchand et Faculté de Médecine, Université Laval, Ste-Foy, Québec, Canada G1K 7P4. Phone: (418) 656-3070. Fax: (418) 656-7176. E-mail: rclevesq{at}rsvs.ulaval.ca.
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