Biochemical Characterization of β-Lactamases Bla1 and Bla2 from Bacillus anthracis

Abstract

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...

MICs of Selected Antibiotics forBacillus anthracis,Bacillus cereus,Bacillus thuringiensis, andBacillus mycoidesfrom a Range of Clinical and Environmental Sources as Determined by the Etest

“…The latter events led to published statements that penicillins, at least alone, are not recommended for the treatment of anthrax (5,6). The B. anthracis genome sequence shows that this organism encodes two ␤-lactamases, a penicillinase and a cephalosporinase (6,7,25,31). These ␤-lactamases are two examples of a significant number of genes (including those for motility, for example) that are shared with the closely related B. cereus which, though present, are not expressed as a result of a truncation in the plcR positive regulator gene (31,34).…”

Carbapenemases: the Versatile β-Lactamases

“…In fact, carbapenems and olivanic acids were some of the most potent naturally occurring ␤-lactams to be identified from diverse sources in early natural product screening programs (24). Because of the prevalence of these molecules in the soil, it is only logical to expect that enzymes capable of degrading these ␤-lactams would be produced by environmental organisms such as Bacillus cereus and Bacillus anthracis, bacteria with well-characterized metallo-␤-lactamases that would provide a selective advantage for growth of these environmental species (98,128). These chromosomal carbapenemases may have evolved initially as a mechanism for bacteria to protect themselves from external threats to their cell wall (20), but in addition these ␤-lactamases may also play a role in the regulation of cell wall synthesis (151).…”

Determination of Antibiotic Efficacy against Bacillus anthracis in a Mouse Aerosol Challenge Model

“…For example, although penicillin has long been considered the treatment of choice for anthrax, numerous reports of ␤-lactamase-producing strains and treatment failures have appeared in the literature (4,14,22,27). Additionally, two open reading frames coding for ␤-lactamases in the B. anthracis genome have been identified (10,29). More recently, several reports of B. anthracis resistance to ciprofloxacin, macrolides, and tetracyclines have appeared in the literature (5,11,36).…”