The Sterne and Ames strains of Bacillus anthracis carry chromosomal genes bla1 and bla2, which confer -lactam resistance when expressed in Escherichia coli. MIC measurements and steady-state kinetic analyses indicate that Bla1 possesses penicillinase activity while Bla2 possesses penicillinase, cephalosporinase, and carbapenem-hydrolyzing activities.Anthrax, caused by Bacillus anthracis, can be treated with antibiotics such as tetracyclines, macrolides, quinolones, and -lactams. However, recent studies have shown that naturally occurring B. anthracis isolates can show variable sensitivity to multiple classes of antibiotics (5,7,20,23 (1,14), present in group 2 -lactamases. Bla2 shares 92% amino acid identity with the group 3 B. cereus 569H enzyme (17), shows conservation of the zinc-chelating motif common to group 3 enzymes (15), and is EDTA susceptible (data not shown).The bla1 and bla2 genes were previously cloned from the B. anthracis Sterne strain and expressed in Escherichia coli (8). However, the -lactamases were poorly expressed in the original constructs. In this work, a high-level expression system was modified to overexpress these enzymes as ompA-bla1 and ompA-bla2 gene fusion products (25). The gene for mature Bla1 or Bla2 enzyme was fused to the leader sequence of the E. coli OmpA protein, cloned into pET24a(ϩ), and transformed into E. coli BL21Star(DE3). pET24a(ϩ) contains an IPTG (isopropyl--D-thiogalactopyranoside)-inducible T7lacUV5 promoter for large-scale protein expression and a Kan r marker. Overlap extension PCR was employed as previously described (12) to construct pCMOB1 and pCMOB2. Briefly, four primers, ompA-bla1-bot, ompA-bla1-top, bla1-BamHI, and pET-SphI (Table 1), were used to PCR amplify two DNA fragments with overlapping ends from vectors pET24a(ϩ)-TEM1 (containing the ompA signal sequence) and pUTE523 (containing bla1) (25). The two overlapping PCR products were combined in a single PCR and amplified by using the external primers bla1-BamHI and pET-SphI to generate the fusion product, ompA-bla1. The fusion product was digested with BamHI and XbaI and ligated into pET-24a(ϩ). Ligation products were transformed into E. coli BL21Star (DE3), and plasmid DNA was isolated from a set of transformants. The sequence of the ompA-bla1 fusion gene was determined and confirmed by ABI3100 automated sequencing. The same procedure was used to construct pCMOB2. The four primers, ompA-bla2-bot, ompA-bla2-top, bla2-BamHI, and pETSphI (Table 1), were used to PCR amplify two DNA fragments with overlapping ends from vectors pET24a(ϩ)-TEM1 and pUTE490 (containing bla2) (25).The MICs of -lactam antibiotics were determined by using twofold dilutions in Luria-Bertani-kanamycin broth (LB-Kan; 25 g/ml) ( Table 2). Kanamycin was added to maintain the plasmid. In the absence of IPTG, expression of the cloned genes still occurs; therefore, IPTG was not added to the LBKan broth. An inoculum of 10 5 E. coli BL21Star(DE3) cells/ml containing pCMOB1, pCMOB2, or the pET24a(ϩ) religated vector was used. The MIC...
IMP-1 -lactamase is a zinc metallo-enzyme encoded by the transferable bla IMP-1 gene, which confers resistance to virtually all -lactam antibiotics including carbapenems. To understand how IMP-1 recognizes and hydrolyzes -lactam antibiotics it is important to determine which amino acid residues are critical for catalysis and which residues control substrate specificity. We randomized 27 individual codons in the bla IMP-1 gene to create libraries that contain all possible amino acid substitutions at residue positions in and near the active site of IMP-1. Mutants from the random libraries were selected for the ability to confer ampicillin resistance to Escherichia coli. Of the positions randomized, >50% do not tolerate amino acid substitutions, suggesting they are essential for IMP-1 function. The remaining positions tolerate amino acid substitutions and may influence the substrate specificity of the enzyme. Interestingly, kinetic studies for one of the functional mutants, Asn233Ala, indicate that an alanine substitution at this position significantly increases catalytic efficiency as compared with the wild-type enzyme.
IMP-1 beta-lactamase is a zinc metallo-enzyme encoded by the transferable bla(IMP-1) gene, which confers resistance to virtually all beta-lactam antibiotics including carbapenems. To understand how IMP-1 recognizes and hydrolyzes beta-lactam antibiotics it is important to determine which amino acid residues are critical for catalysis and which residues control substrate specificity. We randomized 27 individual codons in the bla(IMP-1) gene to create libraries that contain all possible amino acid substitutions at residue positions in and near the active site of IMP-1. Mutants from the random libraries were selected for the ability to confer ampicillin resistance to Escherichia coli. Of the positions randomized, >50% do not tolerate amino acid substitutions, suggesting they are essential for IMP-1 function. The remaining positions tolerate amino acid substitutions and may influence the substrate specificity of the enzyme. Interestingly, kinetic studies for one of the functional mutants, Asn233Ala, indicate that an alanine substitution at this position significantly increases catalytic efficiency as compared with the wild-type enzyme.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.