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Antimicrob Agents Chemother. 2007 November; 51(11): 4009–4014. Published online 2007 September 10. doi: 10.1128/AAC.00537-07. | PMCID: PMC2151419 |
Copyright © 2007, American Society for Microbiology Molecular and Biochemical Characterization of the Chromosome-Encoded Class A β-Lactamase BCL-1 from Bacillus clausii![[down-pointing small open triangle]](/corehtml/pmc/pmcents/x25BF.gif) Delphine Girlich,1 Roland Leclercq,2 Thierry Naas,1 and Patrice Nordmann1* Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre,1 Service de Microbiologie, Hopital Côte de Nacre, Université de Caen, 14033 Caen Cedex, France2 Received April 23, 2007; Revised June 28, 2007; Accepted August 30, 2007. A chromosomal β-lactamase gene from Bacillus clausii NR, which is used as a probiotic, was cloned and expressed in Escherichia coli. It encodes a clavulanic acid-susceptible Ambler class A β-lactamase, BCL-1, with a pI of 5.5 and a molecular mass of ca. 32 kDa. It shares 91% and 62% amino acid identity with the chromosomally encoded PenP penicillinases from B. clausii KSM-K16 and Bacillus licheniformis, respectively. The hydrolytic profile of this β-lactamase includes penicillins, narrow-spectrum cephalosporins, and cefpirome. This chromosome-encoded enzyme was inducible in B. clausii, and its gene is likely related to upstream-located regulatory genes that share significant identity with those reported to be upstream of the penicillinase gene of B. licheniformis. The blaBCL-1 gene was located next to the known chromosomal aadD2 gene and the erm34 gene, which encode resistance to aminoglycosides and macrolides, respectively. Similar genes were found in a collection of B. clausii reference strains. Bacillus species are used as probiotics in both humans and animals for the prevention of infectious bacterial diarrhea ( 8, 10, 18, 28) or preneoplastic lesions ( 29). Enterogermina (Sanofi-Aventis, Milan, Italy) is a pharmaceutical preparation containing an aqueous suspension of a mix of viable antibiotic-resistant alkaliphilic Bacillus clausii strains (NR, OC, SIN, and T) ( 10), initially identified as Bacillus subtilis ( 37). B. clausii spores are able to germinate after exposure to acidic pH in the stomach and to grow in the presence of bile and with a limited amount of oxygen, consistent with the beneficial health effect of probiotics ( 8). In their vegetative form, B. clausii strains are able to induce an immune response ( 39), as recently described for “ Bacillus polyfermenticus” ( 22). Bacillus spores are used as probiotics, although they may carry antibiotic resistance determinants and express toxins ( 14, 19). Resistance to erythromycin is one of the reported features of B. clausii ( 3, 10), due to the presence of an erm-related gene ( 5). B. clausii strains are also resistant to penicillin G, cephalothin, and cefotaxime ( 4). Therefore, it was interesting to determine the resistance determinants responsible for resistance phenotype in B. clausii. The study was initiated with one of the strains, B. clausii NR, present in the Enterogermina mixture and with the B. clausii reference strain ATCC 21537. A β-lactamase with an uncommon hydrolytic profile that included cefpirome was characterized. Bacterial strains. B. clausii NR, SIN, T, OC, and DSM 8716 were from Sanofi-Aventis. B. clausii ATCC 21537 was used as a reference strain. Most of the work reported below was performed in parallel with B. clausii NR and ATCC 21537 strains. Escherichia coli DH10B and E. coli BL21(DE3) (Invitrogen, Life Technologies, Cergy-Pontoise, France) were used for cloning experiments and β-lactamase overexpression, respectively ( 17). Antimicrobial agents and MIC determinations. The antimicrobial agents and their sources are described elsewhere ( 32). Antibiotic disks (Bio-Rad, Marnes-La-Coquette, France) were used for routine antibiograms. MICs of β-lactams were determined by an agar dilution technique on Mueller-Hinton agar (Bio-Rad) as described elsewhere ( 32), according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) ( 11). Cloning experiments and analysis of recombinant plasmids. Whole-cell DNA of B. clausii strains NR and ATCC 21537 were extracted as described previously ( 6). EcoRI-restricted whole-cell DNA of B. clausii NR was ligated into the EcoRI site of plasmid pJIM2246, a cloning vector that replicates in E. coli and gram-positive bacteria ( 34, 37). Recombinant plasmids were transformed by electroporation (Gene Pulser II; Bio-Rad) into electrocompetent E. coli DH10B cells. Antibiotic-resistant colonies were selected on Trypticase soy (TS) agar plates containing ampicillin (100 μg/ml) and kanamycin (20 μg/ml). Recombinant plasmid DNA (pAK) was obtained from 10-ml TS broth cultures grown overnight in the presence of ampicillin (100 μg/ml) at 37°C. Plasmid DNAs were extracted and purified with a QIAGEN plasmid DNA maxikit (QIAGEN, Courtaboeuf, France). PFGE, Southern hybridization, and PCR experiments. The chromosomal location of the β-lactamase genes was determined in whole-cell DNAs of B. clausii NR and ATCC 21537 by using the I-CeuI technique ( 4, 24). Electrophoresis was performed with a CHEF-DRII apparatus (Bio-Rad) used for pulsed-field gel electrophoresis (PFGE), as described elsewhere ( 24), at 14°C and 5 V/cm with a 120° switch angle, with one linear switch ramp of 20 to 120 s for 15 h and a second one of 60 to 100 s for 12 h. The sizes of the I-CeuI-generated fragments were determined by comparison with those of E. coli K-12 ( 24). After Southern transfer ( 35) onto a nylon membrane (Hybond N +; Amersham Pharmacia Biotech, Orsay, France), the DNAs were hybridized with two probes: a 921-bp PCR-generated probe specific for blaBCL-1 (primers A and B) (Table 1) and a 1,504-bp PCR-generated probe for 16S and 23S rRNA genes, using universal primers (Table 1). Labeling and signal detection were carried out by using the nonradioactive enhanced chemiluminescence hybridization kit according to the instructions of the manufacturer (Amersham Pharmacia Biotech). | TABLE 1.Nucleotide sequences of primers used for amplification and sequence analysis |
β-Lactamase expression and purification. The blaBCL-1 gene was amplified by using primers A-NdeI and B-BamHI (Table 1) from pAK and cloned into the expression plasmid pET9a as described elsewhere ( 17). Recombinant plasmid pET-BCL-1, used for BCL-1 overexpression, was introduced into E. coli BL21(DE3) by electroporation as described elsewhere ( 17). A culture of E. coli BL21(DE3)(pET-BCL-1) was induced with 0.4 mM isopropyl-β-thiogalactopyranoside (IPTG) at 37°C for 5 h in TS broth with amoxicillin (30 μg/ml) and kanamycin (30 μg/ml). Two liters of this culture was pelleted and resuspended in 30 ml of 50 mM phosphate buffer (pH 6.5). The β-lactamase extract was obtained after sonification and purified as described elsewhere ( 17). It was dialyzed overnight against 50 mM phosphate buffer (pH 6.5) and loaded onto an S-Sepharose column (Amersham Biosciences, Orsay, France) preequilibrated with the same buffer. The β-lactamase activities, as determined qualitatively for each fraction by using nitrocefin hydrolysis (Oxoid, Dardilly, France), were recovered in the flowthrough and dialyzed overnight against 20 mM Tris HCl buffer (pH 9). The β-lactamase was loaded onto a Q-Sepharose column preequilibrated with the same buffer and eluted with a linear NaCl gradient (0 to 1 M). The fractions containing the highest β-lactamase activity were pooled and dialyzed against 100 mM phosphate buffer (pH 7.0) prior to 10-fold concentration (Vivaspin; molecular weight cutoff, 10,000; Sartorius, Göttingen, Germany). The protein content was measured by using the Bio-Rad DC protein assay (Bio-Rad). The protein purification rate and the molecular mass of BCL-1 β-lactamase were estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis ( 32) and Coomassie blue staining. IEF analysis and induction studies. Purified enzyme and β-lactamase extracts from 10-ml cultures of B. clausii NR were subjected to analytical isoelectric focusing (IEF), as previously described ( 30). Induction studies were performed with cultures of B. clausii NR and B. clausii ATCC 21537 with 1 to 5 μg of cefoxitin per ml as the β-lactam inducer ( 31) and 100 μM penicillin G as the substrate. NH2-terminal sequencing. The signal peptide cleavage site was identified as described elsewhere ( 17) by N-terminal Edman sequencing on an Applied Biosystems Procise 494HT sequencer with the reagents and by the methods recommended by the manufacturer. Kinetic measurements. Purified β-lactamase was used for kinetic measurements at 30°C in 100 mM sodium phosphate (pH 7) ( 31). The kcat and Km values were determined by analyzing β-lactam hydrolysis under initial-rate conditions with an Ultrospec 2000 UV spectrophotometer (Amersham Pharmacia Biotech) and were analyzed by computer with Swift II software (Amersham Pharmacia Biotech), as previously described ( 30). The 50% inhibitory concentrations of clavulanic acid, tazobactam, and sulbactam were determined ( 30) with cephalothin as the substrate. The specific activities of the protein extracts and purified β-lactamase from E. coli BL21(DE3)(pET-BCL-1) were determined with 100 μM cephalothin as the substrate ( 30). DNA sequencing and protein analysis. Both strands of the cloned DNA fragment of recombinant plasmid pAK were sequenced with an Applied Biosystems sequencer (ABI 377). The nucleotide and deduced protein sequences were analyzed with software available on the Internet at the National Center for Biotechnology Information website ( http://www.ncbi.nlm.nih.gov). Multiple protein sequence alignments were carried out by using the program ClustalW available online at the University of Cambridge ( http://www.ebi.ac.uk/clustalw/). Nucleotide sequence accession number. The nucleotide sequence of blaBCL-1 has been assigned to the GenBank nucleotide database under accession number EF540343. Cloning and sequence analysis of a β-lactamase gene from B. clausii NR. Cloning experiments gave E. coli DH10B recombinant clones on kanamycin- and ampicillin-containing TS agar plates. One of these recombinant plasmids, pAK, was retained for further analysis. Its 4.5-kb DNA insert was sequenced, and an open reading frame (ORF) of 924 bp was identified. The G+C content of this ORF was 46.6%, which is close to the G+C ratio of Bacillus licheniformis genes (46%) ( 33). Within the deduced protein of this ORF (307 amino acids), termed BCL-1 (for B. clausii), amino acid residues characteristic of Ambler class A and serine β-lactamases were identified ( 1, 21) (Fig. ). β-Lactamase BCL-1 from B. clausii NR shared 91% amino acid identity with PenP from B. clausii KSM-K16, recently available in GenBank (accession number YP_173768), 62% with the chromosomally encoded class A β-lactamase BlaP from B. licheniformis ATCC 14580 ( 33), 58% with the β-lactamase from Bacillus thuringiensis subsp. israelensis ATCC 35646 (GenBank accession no. ZP_00742695), 48% with that from Bacillus cereus ATCC 14579 ( 19), and 47% with Bla1 from Bacillus anthracis ( 9) (Fig. ). Identification of a similar β-lactamase in B. clausii strain KSM-K16 (91% amino acid identity) indicated a slight variability of this β-lactamase sequence in B. clausii. The bla gene was located downstream of the chromosomal aadD2 gene, which encodes aminoglycoside resistance. This feature is further evidence for the chromosomal location of this gene and intrinsic β-lactam resistance in B. clausii. The blaR1- and blaI-like genes were identified 200 bp upstream of blaBCL-1 and divergently transcribed, encoding BlaR1 and BlaI proteins that were 35% identical to the coinducer and repressor of the β-lactamase PenP of B. licheniformis ( 23, 33) (Fig. ). These proteins shared 89% amino acid identity with those reported from B. clausii KSM-K16 (GenBank accession no. YP_173769). Therefore, blaBCL-1 gene expression was likely inducible, as is that of blaP from B. licheniformis ( 23). Interestingly, the order of the regulatory genes is the same in B. clausii ( blaBCL-1- blaR1-blaI) and in Staphylococcus aureus ( blaZ- mecR1-mecI) ( 34) and reversed compared to that of B. licheniformis ( blaP- blaI-blaR1) ( 23). Further upstream of blaBCL-1, a gene encoding the aminoglycoside 4′-O-nucleotidyltransferase was identified, sharing 100% amino acid identity with that from B. clausii SIN ( 4) and 47% with the protein encoded by ant( 4′)- Ia, or aadD (GenBank accession no. V01282), of plasmid pUB110 from S. aureus ( 27) (Fig. ). After digestion with the enzyme I-CeuI, which recognizes a sequence specific for rRNA operons, total DNA of the B. clausii strains yielded six fragments (Fig. ). Southern hybridization showed that all fragments hybridized with an rrs probe specific for 16S and 23S rRNA genes. The blaBCL-1 probe hybridized to a single chromosomal fragment of both strains. The size of this fragment in B. clausii NR (ca. 700 kb) differed from that in B. clausii ATCC 21537 (ca. 800 kb) (Fig. ), suggesting genetic variability of these strains. Susceptibility testing. B. clausii NR and B. clausii ATCC 21537 were resistant to ceftazidime, cefotaxime, cefuroxime, cefepime, cefpirome, and aztreonam (Table 2). B. clausii strains SIN, T, OC, ATCC 21536, and DSM 8716 displayed the same β-lactam resistance profile (data not shown). Addition of clavulanic acid had little effect in terms of reducing MICs of most β-lactams except for cefpirome. | TABLE 2.MICs of β-lactams for BCL-1-producing and non-BCL-1-producing strains |
However, once cloned in pJIM2246 (pAK) or pET9a (pBCL-1) and expressed in E. coli DH10B or E. coli BL21(DE3), respectively, β-lactamase BCL-1 conferred a different resistance pattern, i.e., resistance or reduced susceptibility to penicillin G, amoxicillin, ticarcillin, and cephalothin (Table 2). Addition of clavulanic acid and tazobactam strongly lowered the MICs of several β-lactams (Table 2). These results indicated that BCL-1 is a narrow-spectrum β-lactamase that may contribute to a minor extent to the β-lactam resistance profile observed with B. clausii. The discrepancy between the β-lactam resistance profiles of B. clausii and E. coli(pBCL-1) may be due to the chromosomal and plasmid locations of blaBCL-1. The blaBCL-1 gene is much more strongly expressed in E. coli than in B. clausii, conferring higher MICs to penicillins. Conversely, resistance of B. clausii NR or ATCC 21537 to cephalosporins was most probably due to other nonenzymatic mechanism in B. clausii (e.g., penicillin-binding protein or affinity). The β-lactam pattern of BCL-1 resembled that of the chromosomally encoded β-lactamase Bla1 from B. anthracis once it was cloned and expressed in E. coli ( 9, 26). BCL-1 did not confer resistance to expanded-spectrum cephalosporins, aztreonam, moxalactam, or imipenem (Table 2). Biochemical analysis of BCL-1. The specific activity of purified BCL-1, measured with 100 μM of cephalothin as the substrate, was 35 U mg of protein−1 with a 26-fold purification factor. Its purity was estimated to be >90% by SDS-PAGE analysis (data not shown). Kinetic parameters of β-lactamase BCL-1 revealed its strong activity against penicillins and narrow-spectrum cephalosporins (Table 2). A significant hydrolytic activity against cefuroxime and cefpirome was also observed, whereas the activity for ceftazidime and cefepime could not be precisely determined because of its weak affinity ( Km > 1,000). Very little information about the kinetics of class A β-lactamases from Bacillus species is available. Partial biochemical characterization of class A β-lactamases of B. licheniformis, B. anthracis, and B. cereus is available ( 16, 25, 26). Nevertheless, catalytic efficiencies of BCL-1 were similar to those of PenP from B. licheniformis for cefuroxime, cefotaxime, and ceftazidime ( 25). Comparison of catalytic efficiencies of BCL-1 showed a 10-fold-lower efficiency than that of Bla1 from B. anthracis for penicillins and a 2-fold-higher catalytic efficiency for cephaloridine ( kcat/ Km value of 2.5 versus 1.1). In contrast to Bla1, BCL-1 preferentially hydrolyzed cephalosporins, as demonstrated by kcat values for cefepime, cefotaxime, and ceftazidime ( kcat of >40, 22, and >3.3 s −1, respectively, versus <0.2 s −1 for Bla1) ( 26). Kinetic parameters of BCL-1 were not comparable to those reported for the β-lactamase BcI from B. cereus ( 16). BcI hydrolyzed benzylpenicillin with a 200-fold-higher activity ( kcat, 2,200 s −1) than did BCL-1 ( kcat, 97 s −1), although with a weaker affinity ( Km of 65 μM versus 12 μM for BCL-1). In contrast, BCL-1 showed a higher activity for cephalothin, cefotaxime, cefuroxime (100-fold), and even cefpirome (500-fold) (Table 3) than did BcI for cephalosporin C ( kcat, 0.2 s −1) ( 16). In both cases, BCL-1 showed a better catalytic activity than other class A β-lactamases from Bacillus species for cephalosporins. | TABLE 3.Kinetic parameters of purified β-lactamase BCL-1a |
Inhibition studies showed that the 50% inhibitory concentrations of clavulanic acid, tazobactam, and sulbactam were low: 85, 4 and 75 nM, respectively. As reported for Bla1 of B. anthracis, tazobactam was a better inhibitor than clavulanic acid. Based on its kinetic parameters, β-lactamase BCL-1 belongs to group 2a of β-lactamases according to the Bush-Medeiros-Jacoby classification ( 7). IEF analysis showed that cultures of B. clausii NR gave multiple pIs ranging from 5.5 to 6, probably due to ragged amino termini, as shown for the β-lactamases produced by B. licheniformis 749/C (BL-βL) ( 38) and B. cereus 569/H (β-lactamase I) ( 13). IEF analysis of culture extracts of E. coli BL21(DE3)(pBCL-1) performed after 2 h of IPTG induction gave a major pI of 5.5. N-terminal amino acid sequencing of the mature protein revealed the cleavage site for the leader peptide between residues 15 and 16 (V-S) (Fig. ). The molecular mass of BCL-1, as determined by SDS-PAGE with the purified enzyme, was ca. 32 kDa (data not shown). Induction studies showed that BCL-1 expression was induced by cefoxitin (5 μg/ml) for B. clausii NR and ATCC 21537. Specific activity after induction was 500 U/mg of protein, versus 1 U/mg at the basal level. Standard deviations were within 10%. This β-lactamase was inducible, as is the naturally occurring class A β-lactamase BlaP from B. licheniformis ( 23). Conclusion. Our study indicates that the penicillin-susceptible B. clausii strain NR harbors a chromosomally encoded and functional β-lactamase. BCL-1 preferentially hydrolyzed penicillins and was inhibited by β-lactam inhibitors. Expression of blaBCL-1 was inducible in B. clausii. As for B. licheniformis and staphylococci, blaI and blaR1 were identified at a common chromosomal locus, forming an operon, and are probably involved in the regulation of the β-lactamase structural gene ( 15, 40). In addition to the class A BCL-type β-lactamase PenP, in silico analysis of the genome of B. clausii KSM-K16 (GenBank accession no. YP_173769) revealed the presence of two genes encoding class D β-lactamases sharing 54% and 35% amino acid identity with YbxI from B. subtilis ( 12). Nevertheless, the amino acid sequence of the first of these two sequences lacks the catalytic lysine residue of the classical SXXK motif. Further studies will be necessary to show if these class D β-lactamases significantly contribute to the overall β-lactam susceptibility pattern of B. clausii. Indeed, Colombo et al. reported that YbxI of B. subtilis had a hydrolytic activity intermediate between those of penicillin-binding proteins and β-lactamases ( 12). Genomic analysis of B. clausii KSM-K16 showed that it does not seem to contain additional chromosomally encoded metallo-β-lactamase in contrast to B. anthracis, which contains a gene encoding Bla2 (class B) in addition to a gene for Bla1 (class A) ( 9), or B. cereus, which contains a gene for BcII (class B) in addition to the BcI and BcIII (class A) genes ( 9, 13). Probiotics may play an important role in protecting the mucosal surfaces of the gastrointestinal tract ( 36). Oral administration of high numbers of multidrug-resistant microorganisms might be a cause for concern if clinically important resistance determinants happened to be located on transferable genetic elements. No such transferable β-lactamase gene was identified here. The blaBCL-1 and the previously identified erm34 genes ( 5) are chromosomal and probably species specific. 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