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A carbapenem-resistant Pseudomonas aeruginosa strain (PA41437) susceptible to expanded-spectrum cephalosporins was recovered from several consecutive lower-respiratory-tract specimens of a patient who developed a ventilator-associated pneumonia while hospitalized in an intensive care unit. Cloning experiments identified OXA-198, a new class D β-lactamase which was weakly related (less than 45% amino acid identity) to other class D β-lactamases. Expression in Escherichia coli TOP10 and in P. aeruginosa PAO1 led to transformants that were resistant to ticarcillin and showed reduced susceptibility to carbapenems and cefepime. The bla OXA-198 gene was harbored by a class 1 integron carried by a ca. 46-kb nontypeable plasmid. This study describes a novel class D β-lactamase involved in carbapenem resistance in P. aeruginosa .

Indoleamine 2,3-dioxygenase (IDO) is a heme dioxygenase which has been shown to be involved in the pathological immune escape of diseases such as cancer. The synthesis and structure-activity relationships (SAR) of a novel series of IDO inhibitors based on the indol-2-yl ethanone scaffold is described. In vitro and in vivo biological activities have been evaluated, leading to compounds with IC 50 values in the micromolar range in both tests. Introduction of small substituents in the 5- and 6-positions of the indole ring, indole N-methylation and variations of the aromatic side chain are all well tolerated. An iron coordinating group on the linker is a prerequisite for biological activity, thus corroborating the virtual screening results. © 2011 Elsevier Ltd. All rights reserved.

The metallo-β-lactamase (MBL) GOB-1 was expressed via a T7 expression system in Escherichia coli BL21(DE3). The MBL was purified to homogeneity and shown to exhibit a broad substrate profile, hydrolyzing all the tested β-lactam compounds efficiently. The GOB enzymes are unique among MBLs due to the presence of a glutamine residue at position 116, a zinc-binding residue in all known class B1 and B3 MBL structures. Here we produced and studied the Q116A, Q116N and Q116H mutants. The substrate profiles were similar for each mutant, but with significantly reduced activity compared with that of the wild-type. In contrast to the Q116H enzyme, which bound two zinc ions just like the wild-type, only one zinc ion is present in Q116A and Q116N. These results suggest that the Q116 residue plays a role in the binding of the zinc ion in the QHH site.

A beta-lactamase produced by Pseudomonas stutzeri was purified to protein homogeneity, and its physicochemical and catalytic properties were determined. Its profile was unusual since, in addition to penicillins, the enzyme hydrolysed second- and third-generation ‘beta-lactamase-stable’ cephalosporins and monobactams with similar efficiencies. On the basis of the characteristics of the interaction with beta-iodopenicillanic acid, the enzyme could be classified as a class-A beta-lactamase. However, when compared with most class-A beta-lactamases, it exhibited significantly lower kcat./Km values for the compounds usually considered to be the best substrates of these enzymes.

9G4H9, a catalytic antibody displaying β-lactamase-like activity, has been developed by the anti-idiotypic approach using β-lactamase as the first antigen. Thus 9G4H9 represents the 'internal image' of β-lactamase. We selected a cyclic peptide anchored to a bacteriophage M13 library using 9G4H9 as the target. Pep90 is a cyclic heptapeptide enclosed between two cysteine residues. We showed that Pep90 could inhibit both TEM-1 β-lactamase (K(i) = 333 μm) and several penicillin-binding proteins (IC₅₀ values ranging from 6-62 μm). We determined that the tryptophan residue of Pep90 is of crucial importance for its inhibitory activity. Using Pep90 as a scaffold, we generated a new class of peptidomimetics that retained inhibitory activity towards TEM-1 β-lactamase.

While TEM- and SHV-derived extended-spectrum β-lactamases (ESBLs) arose by point mutations in genes encoding broad-spectrum variants that were extensively disseminated into mobile elements, CTX-M β-lactamases are derived from natural (chromosomal) counterparts that already have oxyimino-cephalosporinase activity (11). To date, the CTX-M/KLU β-lactamases account for nearly 110 representatives clustered in five main groups (http://www.lahey.org/studies/webt.asp): CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25. A hypothetical chromosomal counterpart for the CTX-M-25 subfamily is still missing. Kluyvera georgiana 14751 was isolated from a bloodstream infection in a 62-year-old male patient through the SENTRY Antimicrobial Surveillance Program in Louisville, KY, on 2 December 2002. Biochemical classification, ambiguous between Kluyvera georgiana and Kluyvera ascorbata, was resolved by 16S rRNA gene sequencing (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AM933755","term_id":"193227311","term_text":"AM933755"}}AM933755), displaying 99.7% identity with the sole available sequence from K. georgiana ATCC 51603 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF047186","term_id":"3273703","term_text":"AF047186"}}AF047186). Identity to all deposited K. ascorbata sequences ranged from 97.4 to 98.5% (6). The strain was resistant to ampicillin, gentamicin, nalidixic acid (Table ​(Table1)1) and trimethoprim-sulfamethoxazole (TMP-SMX) (by disc diffusion). Conjugation to Escherichia coli {"type":"entrez-protein","attrs":{"text":"CAG12177","term_id":"47220029","term_text":"CAG12177"}}CAG12177 resulted in E. coli CK10 harboring an IncFII group conjugative plasmid of ca. 30 kb (pTC10; partial sequence deposited in EMBL under accession no. {"type":"entrez-nucleotide","attrs":{"text":"FN568351","term_id":"290964838","term_text":"FN568351"}}FN568351), responsible for TMP-SMX and aminoglycoside resistance (Table ​(Table1)1) by a class 1 integron harboring dfrA17 and aadA5 gene cassettes (previously reported individually in different Kluyvera intermedia isolates; EMBL accession no. {"type":"entrez-nucleotide","attrs":{"text":"EU523051","term_id":"171188351","term_text":"EU523051"}}EU523051 and {"type":"entrez-nucleotide","attrs":{"text":"EU523052","term_id":"171188353","term_text":"EU523052"}}EU523052). Upstream of the integron we found a blaTEM-1b gene followed by a region, including the Tn3-tnpR, the first 91 bp of Tn3-tnpA, and an IS26 which is also found in plasmids carrying some blaCTX-M genes. However, no CTX-M could be recovered from the transferred plasmid. TABLE 1. MICs of antibiotics for Kluyvera georgiana 14751, the recipient strain, and the derived recombinant clone Partially EcoRI-digested chromosomal DNA from K. georgiana 14751 was cloned in a pK19 vector (Kanr) and transformed into E. coli Top10F′ (E. coli TKE14751-1KA2), yielding pTKE-1KA2 with an 8-kb insert (Fig. ​(Fig.1)1) harboring a bla gene (876 bp) which encodes a novel CTX-M-78 (EMBL accession no. {"type":"entrez-nucleotide","attrs":{"text":"AM982522","term_id":"209413817","term_text":"AM982522"}}AM982522) closely related to CTX-M-39 (96.2% amino acid identity) (1) and clustered in the CTX-M-25 subgroup. As seen in Table ​Table1,1, the MIC increases for cefotaxime, while that of ceftazidime is almost unaffected, correlating with the expected (and experimental [data not shown]) kinetics for most CTX-M enzymes. FIG. 1. Schematic representation of the insert of pTKE-1KA2 from Kluyvera georgiana 14751 (top) and comparison with available sequences harboring blaCTX-M-25 (middle) and blaCTX-M-26 (bottom). As in other chromosome-encoded blaCTX-M/KLU, a 1,227-bp att-like gene encoding a putative aspartate aminotransferase is located upstream, with at least 96% identity with other entries from Kluyvera. Downstream, an orf3-like gene similar to a putative ttrR response regulator from Kluyvera georgiana (5) and similar to the orf3 version from the complex class 1 integrons associated with blaCTX-M-2 (found as a fusion gene orf3::qacEΔ1) (7), and an orf4-like gene encoding a putative autotransporter in Kluyvera ascorbata (2), were also found. In contrast to other kluyveras, in which sequences similar to IRr-ISEcp1 were found downstream of blaCTX-M/KLU (8), we did not observe any sequence that could serve as putative right inverted repeats (IRrs) there (Fig. ​(Fig.1).1). A 35-bp sequence seems to represent the boundary between the chromosome-derived information and the plasmid-acquired information. In blaCTX-M-25 and blaCTX-M-26 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF518567","term_id":"37789822","term_text":"AF518567"}}AF518567 and {"type":"entrez-nucleotide","attrs":{"text":"AY455830","term_id":"38373216","term_text":"AY455830"}}AY455830, respectively) (4), the immediate upstream region is occupied by ISEcp1, also associated with other blaCTX-M genes (3). A putative 5-bp target site for ISEcp1B (AATAC) was found immediately upstream of the 35-bp sequence in the chromosomal DNA from K. georgiana 14751. CTX-M-78 possesses high similarity with members of the CTX-M-25 subgroup, making this enzyme the closest representative to be considered one of the probable progenitors of the subfamily.

International audience; Although commercialized inhibitors of active site serine β-lactamases are currently used in coadministration with antibiotic therapy, no clinically useful inhibitors of metallo-β-lactamases (MBLs) have yet been discovered. In this paper, we investigated the inhibitory effect of mercaptophosphonate derivatives against the three subclasses of MBLs (B1, B2, and B3). All 14 tested mercaptophosphonates, with the exception of 1a, behaved as competitive inhibitors for the three subclasses. Apart from 13 and 21, all the mercapto-phosphonates tested exhibit a good inhibitory effect on the subclass B2 MBL CphA with low inhibition constants (Ki < 15 μM). Interestingly, compound 18 turned out to be a potent broad spectrum MBL inhibitor. The crystallographic structures of the CphA−10a and CphA−18 complexes indicated that the sulfur atom of 10a and the phosphonato group of 18 interact with the Zn2+ ion, respectively. Molecular modeling studies of the interactions between compounds 10a and 18 and the VIM-4 (B1), CphA (B2), and FEZ-1 (B3) enzymes brought to light different binding modes depending on the enzyme and the inhibitor, consistent with the crystallographic structures.

Objectives A natural variant of the AmpC enzyme from Escherichia coli HKY28 with a tripeptide deletion (Gly-286/Ser-287/Asp-288) was recently described. The isolate produced an inhibitor-sensitive AmpC beta-lactamase variant that also conferred higher than usual levels of resistance to ceftazidime in the E. coli host. To demonstrate whether this is true in other class C beta-lactamase enzymes, we deleted the equivalent tripeptide in the FOX-4 plasmid-mediated class C beta-lactamase. Methods By site-directed mutagenesis, we deleted the tripeptide Gly-306/Asn-307/Ser-308 of FOX-4, thus generating FOX-4(DeltaGNS). The enzymes (FOX-4 wild-type and DeltaGNS) were purified and kinetic parameters (kcat, Km, kcat/Km) as well as IC50 values of several beta-lactams were assessed. Modelling studies were also performed. Results FOX-4(DeltaGNS) did not increase the catalytic efficiency towards ceftazidime, although it conferred a slight increase in the susceptibility to beta-lactamase inhibitors. There was also a noteworthy decrease in the cefoxitin MIC with the FOX-4(DeltaGNS) mutant (from 512 to 16 mg/L) as well as a 10-fold decrease in kcat/Km towards imipenem, which revealed specific structural features. Conclusions Although deletions in the R2-loop are able to extend the substrate spectrum of class C enzymes, the present results do not confirm this hypothesis in FOX-4. The FOX-4 R2 site would already be wide enough to accommodate antibiotic molecules, and thus any amino acid replacement or deletion at this location would not affect the hydrolytic efficiency towards beta-lactams and would have a less drastic effect on the susceptibility to beta-lactamase inhibitors.

Background Papain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes. Methods To have a better insight into the mechanism of activation of this protease family, we compared the pH unfolding of the zymogen and the mature form of the mite cysteine protease Der p 1. Results We showed that the presence of the propeptide does not significantly influence the pH-induced unfolding of the catalytic domain but does affect its fluorescence properties by modifying the exposure of the tryptophan 192 to the solvent. In addition, we demonstrated that the propeptide displays weaker pH stability than the protease domain confirming that the unfolding of the propeptide is the key event in the activation process of the zymogen. General significance Finally, we show, using thermal denaturation and enzymatic activity measurements, that whatever the pH value, the propeptide does not stabilize the structure of the catalytic domain but very interestingly, prevents its autolysis.

Klebsiella pneumoniae ML1708 exhibited a multiresistance phenotype, including resistance to all beta-lactams tested, chloramphenicol, ciprofloxacin, nalidixic acid, tetracycline, and streptomycin. The double-disk synergy test was positive. ML1708 harbored a 50 kb conjugative plasmid that encoded a beta-lactamase of pI 5.5. The corresponding bla gene was identified by polymerase chain reaction and sequencing as a bla(TEM) gene. The deduced protein sequence revealed a new variant of TEM-1 beta-lactamase designated TEM-164. TEM-164 contains the unusual following mutations: L40V and I279T. These modifications may result in a change of the pI to 5.5 and hydrolyze cefotaxime and ceftazidime.

Bacteria can defend themselves against β-lactam antibiotics through the expression of class B β-lactamases, which cleave the β-lactam amide bond and render the molecule harmless. There are three subclasses of class B β-lactamases (B1, B2, and B3), all of which require Zn 2+ for activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo-β-lactamases are most active as dizinc enzymes, subclass B2 enzymes, such as Aeromonas hydrophila CphA, are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals allowed us to solve the crystal structures of the dizinc forms of the wild-type enzyme and N220G mutant. The first zinc ion binds in the cysteine site, as previously determined for the monozinc form of the enzyme. The second zinc ion occupies a slightly modified histidine site, where the conserved His118 and His196 residues act as metal ligands. This atypical coordination sphere probably explains the rather high dissociation constant for the second zinc ion compared to those observed with enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results from immobilization of the catalytically important His118 and His196 residues, as well as the folding of the Gly232-Asn233 loop into a position that covers the active site.

In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carboxymethyl cellulose as a substrate. Moreover, the study of pH and the thermal dependence of the hydrolytic activity shows that RBcel1 was active from pH 6 to pH 9 and remained significantly active when temperature decreased (18% of activity at 10 degrees C). It is interesting that RBcel1 was able to synthetize non-reticulated cellulose using cellobiose as a substrate. Moreover, by a combination of bioinformatics and enzyme analysis, the physiological relevance of the RBcel1 protein and its mesophilic homologous Pst_2494 protein from P. stutzeri, A1501, was established as the key enzymes involved in the production of cellulose by bacteria. In addition, RBcel1 and Pst_2494 are the two primary enzymes belonging to the GH5 family involved in this process.

Background: In effort to identify novel bacterial agents, this study was initiated to evaluate the antimicrobial properties of 17 crude extracts from 12 medicinal plants against beta-lactam-resistant bacteria. Methodology: The antimicrobial activities of plant extracts were evaluated against clinically proved beta-lactam-resistant bacteria ( Klebsiella pneumoniae , Klebsiella oxytoca , Enterobacter cloacae , Serratia marcescens , Acinetobacter baumannii , Staphylococcus aureus and Enterococcus sp.) and reference strains of bacteria ( Escherichia coli ATCC 35218, Enterobacter aerogenes ATCC 29751, E. aerogenes ATCC 13048, Pseudomonas aeruginosa ATCC 27853 and Enterococcus hirae ATCC 9790) by using disc-diffusion and agar-dilution assays. Results: The crude plant extracts demonstrated broad spectrum activity against all bacteria tested with inhibition zones in the range of 8-30 mm. The minimal inhibitory concentration (MIC) values of different plant extracts against the tested bacteria were found to range from ≤ 0.3 to ≥ 10 mg ml -1 . The most active plant extracts were from Dortenia picta and Bridelia micrantha (MIC: 1.25-10 mg ml -1 ) on beta-lactam-resistant Gram-negative bacilli and the extracts from B. micrantha , Mallotus oppositifolius , Garcinia lucida , Garcinia. kola , Campylospermum densiflorum (leaves) and C. zenkeri (root) on beta-lactam-resistant Gram-positive cocci (MIC: ≤ 0.3-5 mg ml -1 ). Conclusion: Of the 17 plant extracts studied, seven showed good antimicrobial activity against the tested bacteria. The stem bark of B. micrantha and the leaves of D. picta were most active towards beta-lactamase producing Gram-negative bacilli. This study shows that medicinal plants could be sources of compounds which can be used to fight against beta-lactam resistant bacteria. Background: In efforts to fight against bacteria resistance to antibiotics, this study was initiated to evaluate the antimicrobial properties of 17 crude extracts from 12 medicinal plants against β-lactam-resistant bacteria. Methodology: The antimicrobial activities of plants extracts were evaluated against β-lactam-resistant clinical ( Klebsiella pneumoniae , Kl. Oxytoca , Enterobacter cloacae , Serratia marcescens , Acinetobacter baumannii , Staphylococcus aureus and Enterococcus sp.) and reference ( Escherichia coli ATCC 35218, Enterobacter aerogenes ATCC 29751, E. aerogenes ATCC 13048, Pseudomonas aeruginosa ATCC 27853 and Enterococcus hirae ATCC 9790) strains by using disc-diffusion and agar-dilution assays. Results: The crude plant extracts demonstrated broad spectrum activity against all bacteria tested with inhibition zones in the range of 8-30 mm. The minimal inhibitory concentration (MIC) values of different plant extracts against the tested bacteria were found to range from ≤ 0.3 to ≥ 10 mg ml -1 . The most active plant extracts were from Dortenia picta and Bridelia micrantha (MIC: 1.25-10 mg ml -1 ) on β-lactam-resistant Gram-negative bacilli and the extracts from B. micrantha , Mallotus oppositifolius , Garcinia lucida , G. kola , Campylospermum densiflorum (leaves) and C. zenkeri (root) on β-lactam-resistant Gram-positive cocci (MIC: ≤ 0.3-5 mg ml -1 ). Conclusion: Of the 17 plant extracts studied, seven showed good antimicrobial activity against the tested bacteria. The stem bark of B. micrantha and the leaves of D. picta were most active towards β-lactamase producing Gram-negative bacilli. This study shows that medicinal plants could be sources of compounds which can be used to fight against β-lactam resistant bacteria.

Objectives: The aim of the study was the biochemical characterization of a new variant of the metallob-lactamase, IMP-22. Moreover, the genetic environment of the blaIMP-22 gene was investigated in Pseudomonas fluorescens and Pseudomonas aeruginosa collected from urban wastewater and a teaching hospital in L’Aquila, Italy. Methods: Molecular characterization of genetic elements was carried out by PCR and DNA sequencing methods. The new enzyme was purified from recombinant Escherichia coli BL21(DE)Rosetta/pBC-SK/ IMP-22. Steady-state kinetic parameters (Km and Vmax) were determined for a large pattern of substrates. Results: A new IMP metallo-b-lactamase gene was found in a class 1 integron and in one case, in a plasmid of Pseudomonas spp. The blaIMP-22 encodes for a pre-protein of 246 amino acids and the N-terminus of the mature b-lactamase (NH2-PDLK) was also determined. The molecular mass and pI were 24930 Da and 6.2, respectively. On the basis of the kinetic parameters calculated (Km and Vmax), IMP-22 was found to hydrolyse narrow- and extended-spectrum b-lactams. Enzyme activity was found to be inhibited by metal chelators such as EDTA, 1,10-o-phenathroline and dipicolinic acid with an IC50 of 800, 750 and 300 mM, respectively. Conclusions: The finding of the blaIMP-22 gene in P. fluorescens environmental strains and P. aeruginosa clinical isolate suggests the ongoing spread of blaMBL genes in several bacterial species and in different environments.

In order to test the use of a subunit recombinant vaccine for its capacity to induce antibodies against the nonimmunogenic heat-stable enterotoxin STa from Escherichia coli and the TEM-1 beta-lactamase, cattle were immunized with a hybrid protein created by insertion of the STa sequence at position 197 of the TEM-1 beta-lactamase. Specific anti-STa IgG and IgG1 antibodies were detected at low levels, while no IgG2 antibodies were detected. In contrast, high levels of the different anti-TEM IgG subtypes were detected in cattle sera. In addition, beta-lactamase activity was inhibited by the sera. The presence of antibodies against STa and TEM-1 beta-lactamase was assessed in sera from 366 cattle taken from the field. No significant level of IgGs against the toxin or the TEM-1 was detected. A comparison of the antibody level between the immunized and the nonimmunized animals clearly demonstrated that STa was not able to induce a significant level of antibodies in the vaccinated animals. In contrast, a strong antibody response against TEM-1 beta-lactamase was demonstrated.

Two AmpC variants harboring the S287N substitution were obtained by mutagenesis from cephalosporinases representative of the phylogenetic groups A and B2 of Escherichia coli . Their biochemical characterization revealed that the S287N replacement led to an important increase in the catalytic efficiency toward extended-spectrum cephalosporins in the AmpC β-lactamase of group A only.

Insertion of heterologous peptide sequences into a protein carrier may impose structural constraints that could help the peptide to adopt a proper fold. This concept could be the starting point for the development of a new generation of safe subunit vaccines based on the expression of poorly immunogenic epitopes. In the present study, we characterized the tolerance of the TEM-1 class A beta-lactamase to the insertion of two different peptides, the V3 loop of the gp120 protein of HIV, and the thermostable STa enterotoxin produced by enterotoxic Escherichia coli. Insertion of the V3 loop of the HIV gp120 protein into the TEM-1 scaffold yielded insoluble and poorly produced proteins. By contrast, four hybrid beta-lactamases carrying the STa peptide were efficiently produced and purified. Immunization of BALB/c mice with these hybrid proteins produced high levels of TEM-1-specific antibodies, together with significant levels of neutralizing antibodies against STa.

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) beta-lactamase from Aeromonas hydrophila is a Zn(2+)-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His(118), Asp(120), His(196) and His(263)) and in substrate specificity (Val(67), Thr(157), Lys(224) and Lys(226)), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp(120) and His(263), and thus is located in the 'cysteine' zinc-binding site. His(118) and His(196) residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val(67) plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val(67) also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys(224) in the binding of substrate. Lys(226) is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding.

The system described here allows the expression of protein fragments into a solvent-exposed loop of a carrier protein, the beta-lactamase BlaP. When using Escherichia coli constitutive expression vectors, a positive selection of antibioresistant bacteria expressing functional hybrid beta-lactamases is achieved in the presence of beta-lactams making further screening of correctly folded and secreted hybrid beta-lactamases easier. Protease-specific recognition sites have been engineered on both sides of the beta-lactamase permissive loop in order to cleave off the exogenous protein fragment from the carrier protein by an original two-step procedure. According to our data, this approach constitutes a suitable alternative for production of difficult to express protein domains. This work demonstrates that the use of BlaP as a carrier protein does not alter the biochemical activity and the native disulphide bridge formation of the inserted chitin binding domain of the human macrophage chitotriosidase. We also report that the beta-lactamase activity of the hybrid protein can be used to monitor interactions between the inserted protein fragments and its ligands and to screen neutralizing molecules.

The crystallographic structure of ACT-1, which is the first plasmid-mediated AmpC-type beta-lactamase to have been completely analyzed in terms of nucleotide sequence and which has a high degree of sequence similarity to the chromosomal AmpC enzymes of Enterobacter cloacae and the plasmid-encoded MIR-1, has been solved at 2.4 A resolution. The overall structure of ACT-1 is similar to those of other class C beta-lactamases, such as the AmpC enzymes from E. cloacae P99 and Escherichia coli.

In efforts to find new bioactive β-lactamase inhibitors, this study investigated 16 Cameroonian plants belonging to 10 families which were evaluated for anti-β-lactamase activity. The investigation showed that extracts 2, 6, 3 and 5 of the 16 plants investigated presented interesting in vitroβ-lactamase inhibition (over 90%), respectively, of the β-lactamases TEM-1, OXA-10, IMP-1 and P99. These extracts were from Mammea africana (all β-lactamases), Garcinia lucida, G. kola (OXA-10, IMP-1 and P99), Bridelia micrantha (OXA-10, P99), Ochna afzelii (OXA-10, P99), Prunus africana (IMP-1) and Adenia lobata (TEM-1). After elimina tion of tannins (according to the European Pharmacopoeia) the extracts from B. micrantha, G. lucida and M. africana were tested further for their anti-β-lactamase activity. The extracts from B. micrantha and G. lucida exhibited potent inhibitory activity, respectively, of β-lactamase OXA-10 (IC50 = 0.02 mg/mL) and P99 (IC50 = 0.01 mg/mL). The anti-β-lactamase activity of M. africana extract was weak. The isolation and the structural elucidation of the active constituents of G. lucida and B. micrantha will provide useful leads in the development of β-lactamase inhibitors. Copyright © 2007 John Wiley & Sons, Ltd.

Mapping of epitopes is a crucial step for the study of immune pathways, the engineering of vaccines and the development of immunoassays. In this work, the Bacillus licheniformis beta-lactamase BlaP has been engineered to display heterologous polypeptides in a permissive and solvent-exposed loop. When combined with phage display, this modified enzyme can be used for epitope mapping by cloning random gene fragments. The procedure presented in this paper allows the selection of large infectious phage libraries with high diversity and efficient beta-lactamase activities. A useful aspect of the proposed technique results from the possibility of using the beta-lactamase activity carried by phages to evaluate the proportion of immobilised phages during the successive enrichment steps of the library or competition experiments with the selected phages. Another advantage of the technique derives from the fact that the epitope is selected as a bifunctional hybrid protein, which can be overproduced and purified. The resulting recombinant protein associates an epitope with a specific and efficient enzymatic activity. This constitutes an original tool for immunoassay development. A virus influenza hemagglutinin (HA1)-gene fragment library has been generated with this system and used to identify a linear epitope.

A novel natural TEM β-lactamase with extended-spectrum activity, TEM-138, was identified in a ceftazidime-resistant clinical isolate of Salmonella enterica serovar Infantis. Compared to TEM-1, TEM-138 contains the following mutations: E104K, N175I, and G238S. The bla TEM-138 gene was located on a 50-kb transferable plasmid. Expression studies with Escherichia coli revealed efficient ceftazidimase and cefotaximase activities for TEM-138.

Cefotaxime-resistant Escherichia coli TUM1121 was isolated from an abscess of an 83-year-old patient. The CTX-M-14 gene was located on a 70kb plasmid. The enzyme was purified and its activity was analysed. CTX-M-14 was poorly active against ceftazidime and aztreonam. Aztreonam behaved as a competitive inhibitor. Among the tested suicide substrates for class A β-lactamases, sulbactam was a rather good substrate. Tazobactam and clavulanic acid behaved as inactivators. The interactions between clavulanic acid and CTX-M-14 were characterised by progressive inactivation of the β-lactamase. Carbapenems such as imipenem, meropenem or doripenem did not behave as inactivators of CTX-M-14, however very small k cat values were observed. This result shows that CTX-M-14 is able to hydrolyse carbapenems.

Morganella morganii produces an inducible, chromosomally encoded AmpC beta-lactamase. We describe in this study three new variants of AmpC within this species with apparent pIs of 6.6 (M19 from M. morganii strain PP19), 7.4 (M29 from M. morganii strain PP29), and 7.8 (M37 from M. morganii strain PP37). After gene sequencing, deduced amino acid sequences displayed one to six substitutions when compared to the available Morganella AmpC sequences. An AmpR-encoding gene was also found upstream of ampC, including the LysR regulators´ helix-turn-helix DNA-binding domain and the putative T-N11-A-protected region in the ampR-ampC intercistronic sequence. All three AmpC variants were purified from in vitro-generated derepressed mutants and showed overall similar kinetic parameters. None of the observed amino acid changes, occurring at the surface of the protein, appear to have a major influence in their catalytic properties. Morganella AmpCs exhibit the highest catalytic efficiencies (k(cat)/K(m)) on classical penicillins, cefoxitin, narrow-spectrum cephalosporins, and cefotaxime. Cefotaxime was more effectively hydrolyzed than other oxyimino-cephalosporins, whereas cefepime was 3 log-fold less efficiently hydrolyzed than other cephalosporins such as cephalothin. Several differences with other AmpC beta-lactamases were found. Ampicillin was more efficiently hydrolyzed than benzylpenicillin. High k(cat)/K(m) values were observed for oxacillin and piperacillin, which are usually poor substrates for AmpC. A fairly efficient hydrolysis of imipenem was detected as well. Aztreonam, carbenicillin, and tazobactam were effective transient inactivators of these variants. Morganella morganii produces an inducible, chromosomally encoded AmpC beta-lactamase. We describe in this study three new variants of AmpC within this species with apparent pIs of 6.6 (M19 from M. morganii strain PP19), 7.4 (M29 from M. morganii strain PP29), and 7.8 (M37 from M. morganii strain PP37). After gene sequencing, deduced amino acid sequences displayed one to six substitutions when compared to the available Morganella AmpC sequences. An AmpR-encoding gene was also found upstream of ampC, including the LysR regulators´ helix-turn-helix DNA-binding domain and the putative T-N11-A-protected region in the ampR-ampC intercistronic sequence. All three AmpC variants were purified from in vitro-generated derepressed mutants and showed overall similar kinetic parameters. None of the observed amino acid changes, occurring at the surface of the protein, appear to have a major influence in their catalytic properties. Morganella AmpCs exhibit the highest catalytic efficiencies (k(cat)/K(m)) on classical penicillins, cefoxitin, narrow-spectrum cephalosporins, and cefotaxime. Cefotaxime was more effectively hydrolyzed than other oxyimino-cephalosporins, whereas cefepime was 3 log-fold less efficiently hydrolyzed than other cephalosporins such as cephalothin. Several differences with other AmpC beta-lactamases were found. Ampicillin was more efficiently hydrolyzed than benzylpenicillin. High k(cat)/K(m) values were observed for oxacillin and piperacillin, which are usually poor substrates for AmpC. A fairly efficient hydrolysis of imipenem was detected as well. Aztreonam, carbenicillin, and tazobactam were effective transient inactivators of these variants. Fil: Power, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentina Fil: Galleni, Moreno. Université de Liège; Bélgica Fil: Ayala, Juan A. Consejo Superior de Investigaciones Científicas; España Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentina

A commercially available poly(ethylene-co-butylene) copolymer, endcapped by a short polyisoprene block and a hydroxyl group (PI-b-PEB-OH), has been derivatized into a macroinitiator for atom transfer radical polymerization (ATRP) by esterification of the hydroxyl end-group by an activated bromidecontaining acyl bromide. Two types of triblock copolymers, PI-b-PEB-b-poly- (dimethylaminoethyl methacrylate) (PDMAEMA) and PI-b-PEB-b-poly[2-(tert-butylamino) ethyl methacrylate] (PTBAEMA), have been synthesized and used to coat polystyrene particles. These coatings have been permanently immobilized by UV cross-linking of the isoprene units. They exhibit a biocidal activity against Gramnegative bacteria either intrinsically in case of the PTBAEMA block or upon quaternization of the PDMAEMA block by octyl bromide. The antimicrobial activity is directly related to the concentration of coated PS particles in the medium.

The complete understanding of the morphological differentiation of streptomycetes is an ambitious challenge as diverse sensors and pathways sensitive to various environmental stimuli control the process. Germination occupies a particular position in the life cycle as the good achievement of the process depends on events occurring both during the preceding sporulation and during germination per se. The cyclic AMP receptor protein (crp) null mutant of Streptomyces coelicolor, affected in both sporulation and germination, was therefore presented as a privileged candidate to highlight new proteins involved in the shift from dormant to germinating spores. Our multidisciplinary approach-combining in vivo data, the analysis of spores morphological properties, and a proteome study-has shown that Crp is a central regulatory protein of the life cycle in S. coelicolor; and has identified spores proteins with statistically significant increased or decreased expression that should be listed as priority targets for further investigations on proteins that trigger both ends of the life cycle.

Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein.

β-Lactamases represent the major cause of bacterial resistance against β-lactam antibiotics, and they have been divided into four classes (A to D) on the basis of their amino acid sequences (21). The class B enzymes have no sequence or structural similarity to the active-site serine enzymes of classes A, C, and D (6); require a bivalent metal ion (Zn2+) for activity; and constitute group 3 in the Bush-Jacoby-Medeiros functional classification (2). The identification of Zn-β-lactamase-producing pathogenic strains of Aeromonas, Bacteroides, Flavobacterium, Legionella, Serratia, and Stenotrophomonas has greatly increased interest in this class of enzymes (2). The fact that they hydrolyze almost all β-lactam antibiotics, including carbapenems, underlines their clinical relevance. In consequence, the potential spreading of these enzymes among pathogenic bacteria is a frightening possibility, which emphasizes the importance of understanding their properties. On the basis of the sequences, three subclasses of class B β-lactamases (B1 to B3) were identified, and a standard numbering scheme (BBL numbering) was proposed (13) by analogy to the ABL numbering scheme which has been widely used for class A β-lactamases. Due to the general low degree of identity between subclass sequences (

In order to test the use of DNA vaccination for its capacity to induce antibodies against the non-immunogenic heat-stable enterotoxin STa from Escherichia coli, BALB/c mice were immunized with plasmid DNA encoding hybrid proteins made by the insertion of wild type STa or insertion of the Cys6Ala, Cys17Ala and Cys6Ala-Cys17Ala STa mutants at positions 195 or 216 of the TEM-1 beta-lactamase. No STa specific antibodies could be detected after three plasmid injections, but a subsequent boost with native STa peptide was capable of inducing low levels of neutralizing antibodies, as tested in the suckling mouse assay. Highest STa specific responses were found in mice primed with the double mutated STa inserted in position 195. This plasmid induced highest T-cell responses to the TEM-1 protein, indicating that priming of helper T-cell responses to the carrier protein was essential. Mixed IgG1/IgG2a isotypes also reflected this T helper 1 type priming. Moreover, insertion into loop A of the TEM-1 carrier may be more suitable than insertion into loop B, because of reduced competition between carrier and hapten B cell responses.

Increased resistance to beta-lactam antibiotics is mainly due to beta-lactamases. X-ray structures of zinc beta-lactamases unraveled the coordination of the metal ions, but their mode of action remains unclear. Recently, enzymes in which one of the zinc ligands was mutated have been characterized and their catalytic activity against several beta-lactam antibiotics measured. A molecular modeling study of these enzymes was performed here to explain the catalytic activity of the mutants. Coordination around the zinc ions influences the way the tetrahedral intermediate is bound; any modification influences the first recognition of the substrate by the enzyme. For all the studied mutants, at least one of the interactions fails, inducing a loss of catalytic efficiency compared to the wild type. The present studies show that the enzyme cavity is a structure of high plasticity both structurally and mechanistically and that local modifications may propagate its effects far from the mutated amino acid.

The beta-lactamases are involved in bacterial resistance to penicillin and related compounds. Members of the metallo-enzyme class are now found in many pathogenic bacteria and are thus becoming of major clinical importance. The structures of the Zn-beta-lactamase from Fluoribacter gormanii (FEZ-1) in the native and in the complex form are reported here. FEZ-1 is a monomeric enzyme, which possesses two zinc-binding sites. These structures are discussed in comparison with those of the tetrameric L1 enzyme produced by Stenotrophomonas maltophilia. From this analysis, amino acids involved in the oligomerization of L1 are clearly identified. Despite the similarity in fold, the active site of FEZ-1 was found to be significantly different. Two residues, which were previously implicated in function, are not present in L1 or in FEZ-1. The broad-spectrum substrate profile of Zn-beta-lactamases arises from the rather wide active-site cleft, where various beta-lactam compounds can be accommodated.

The VIM metallo-beta-lactamases are emerging resistance determinants, encoded by mobile genetic elements, that have recently been detected in multidrug-resistant nosocomial isolates of Pseudomonas aeruginosa and other Gram-negative pathogens. In this work a T7-based expression system for overproduction of the VIM-2 enzyme by Escherichia coli was developed, which yielded approximately 80 mg of protein per litre of culture. The enzyme was mostly released into the medium, from which it was recovered at >99% purity by an initial ammonium sulphate precipitation followed by two chromatography steps, with almost 80% efficiency. Determination of kinetic parameters of VIM-2 under the same experimental conditions previously used for VIM-1 (the first VIM-type enzyme detected in clinical isolates, which is 93% identical to VIM-2) revealed significant differences in K(m) values and/or turnover rates with several substrates, including penicillins, cephalosporins and carbapenems. Compared with VIM-1, VIM-2 is more susceptible to inactivation by chelators, indicating that the zinc ions of the latter are probably more loosely bound. These data indicated that at least some of the amino acid differences between the two proteins have functional significance. Molecular modelling of the two enzymes identified some amino acid substitutions, including those at positions 223, 224 and 228 (in the BBL numbering), that could be relevant to the changes in catalytic behaviour.

The CphA metallo-beta-lactamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results in a drastic decrease in the hydrolytic activity. In this study, we analyzed the role of Asn116 and Cys221, two residues of the active site. These residues were replaced by site-directed mutagenesis and the different mutants were characterized. The C221S and C221A mutants were seriously impaired in their ability to bind the first, catalytic zinc ion and were nearly completely inactive, indicating a major role for Cys221 in the binding of the catalytic metal ion. By contrast, the binding of the second zinc ion was only slightly affected, at least for the C221S mutant. Mutation of Asn116 did not lead to a drastic decrease in the hydrolytic activity, indicating that this residue does not play a key role in the catalytic mechanism. However, the substitution of Asn116 by a Cys or His residue resulted in an approximately fivefold increase in the affinity for the second, inhibitory zinc ion. Together, these data suggested that the first zinc ion is located in the binding site involving the Cys221 and that the second zinc ion binds in the binding site involving Asn116 and, presumably, His118 and His196.

The sequenced chromosome ofCaulobacter crescentusCB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-β-lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned inEscherichia colifromC. crescentustype strain DSM4727. Expression studies confirmed the metallo-β-lactamase activity of its product, CAU-1. The enzyme produced inE. coliwas purified by two ion-exchange chromatography steps. CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (>9), and unlike the L1 enzyme ofStenotrophomonas maltophilia, the native form is monomeric. Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed. Affinities for the various β-lactams were poor overall (Kmvalues were always >100 μM and often >400 μM). The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3. InC. crescentus, the CAU-1 enzyme is produced independently of β-lactam exposure and, interestingly, theblaCAUdeterminant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure. The CAU-1 enzyme is the first example of a metallo-β-lactamase in a member of the α subdivision of the classProteobacteria.

We have investigated the influence of substrate binding on the zinc ion affinity of representatives from the three metallo-beta-lactamase subclasses, B1 (BcII from Bacillus cereus and BlaB from Chryseobacterium meningosepticum), B2 (CphA from Aeromonas hydrophila), and B3 (L1 from Stenotrophomonas maltophilia). By competition experiments with metal-free apoenzymes and chromophoric zinc chelators or EDTA, we determined the dissociation constants in the absence and presence of substrates. For the formation of the monozinc enzymes we determined constants of 1.8, 5.1, 0.007, and 2.6 nm in the absence and 13.6, 1.8, 1.2, and 5.7 pm in the presence of substrates for BcII, BlaB, CphA, and L1, respectively. A second zinc ion binds in the absence (presence) of substrates with considerably higher dissociation constants, namely 1.8 (0.8), 0.007 (0.025), 50 (1.9), and 0.006 (0.12) microm for BcII, BlaB, CphA, and L1, respectively. We have concluded that the apo form might be the prevailing state of most of the metallo-beta-lactamases under physiological conditions in the absence of substrates. Substrate availability induces a spontaneous self-activation due to a drastic decrease of the dissociation constants, resulting in the formation of active mononuclear enzymes already at picomolar free zinc ion concentrations. In the presence of substrates, the binuclear state of the enzymes only exists at unphysiologic high zinc concentrations and might be of no biological relevance. From the competition experiments with EDTA it is further concluded that the reactivation rate does not depend on the pool of free zinc ions but proceeds via the EDTA-Zn(II)-enzyme ternary complexes.

A new method for the identification and quantification of penicillin-binding proteins is described which uses fluorescein-coupled penicillins. It allows the rapid detection of 0.2 pmol with the naked eye and 2 fmol with the help of an A.L.F. automatic DNA sequencer. Direct labelling can also be performed on whole bacterial cells.

A detailed kinetic study of the interaction between two ethylidene derivatives of tricyclic carbapenems, Lek 156 and Lek 157, and representative β-lactamases andd-alanyl–d-alanine peptidases (dd-peptidases) is presented. Both compounds are very efficient inactivators of theEnterobacter cloacae908R β-lactamase, which is usually resistant to inhibition. Preliminary experiments indicate that various extended-spectrum class C β-lactamases (ACT-1, CMY-1, and MIR-1) are also inactivated. With theE. cloacae908R enzyme, complete inactivation occurs with a second-order rate constant,k2/K′, of 2 × 104to 4 × 104M−1s−1, and reactivation is very slow, with a half-life of >1 h. Accordingly, Lek 157 significantly decreases the MIC of ampicillin forE. cloacaeP99, a constitutive class C β-lactamase overproducer. With the other serine β-lactamases tested, the covalent adducts exhibit a wide range of stabilities, with half-lives ranging from long (>4 h with the TEM-1 class A enzyme), to medium (10 to 20 min with the OXA-10 class D enzyme), to short (0.2 to 0.4 s with the NmcA class A β-lactamase). By contrast, both carbapenems behave as good substrates of theBacillus cereusmetallo-β-lactamase (class B). TheStreptomycessp. strain R61 and K15 extracellulardd-peptidases exhibit low levels of sensitivity to both compounds.

Several cysteinyl peptides have been synthesised and shown to be reversible competitive inhibitors of the Bacillus cereus metallo-beta-lactamase. The pH dependence of pKi indicates that the thiol anion displaces hydroxide ion from the active site zinc(II). D,D-Peptides bind to the enzyme better than other diastereoisomers, which is compatible with the predicted stereochemistry of the active site.

VIM-1 is a new group 3 metallo-β-lactamase recently detected in carbapenem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Mediterranean area. In this work, VIM-1 was purified from an Escherichia coli strain carrying the cloned bla VIM-1 gene by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme exhibited a molecular mass of 26 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an acidic pI of 5.1 in analytical isoelectric focusing. Amino-terminal sequencing showed that mature VIM-1 results from the removal of a 26-amino-acid signal peptide from the precursor. VIM-1 hydrolyzes a broad array of β-lactam compounds, including penicillins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanism-based serine-β-lactamase inactivators. Only monobactams escape hydrolysis. The highest catalytic constant/ K m ratios (>10 6 M −1 · s −1 ) were observed with carbenicillin, azlocillin, some cephalosporins (cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome), imipenem, and biapenem. Kinetic parameters showed remarkable variability with different β-lactams and also within the various penam, cephem, and carbapenem compounds, resulting in no clear preference of the enzyme for any of these β-lactam subfamilies. Significant differences were observed with some substrates between the kinetic parameters of VIM-1 and those of other metallo-β-lactamases. Inactivation assays carried out with various chelating agents (EDTA, 1,10- o -phenanthroline, and pyridine-2,6-dicarboxylic acid) indicated that formation of a ternary enzyme-metal-chelator complex precedes metal removal from the zinc center of the protein and revealed notable differences in the inactivation parameters of VIM-1 with different agents.

A metallo-β-lactamase determinant was cloned from a genomic library of Legionella ( Fluoribacter ) gormanii ATCC 33297 T constructed in the plasmid vector pACYC184 and transformed into Escherichia coli DH5α, by screening for clones showing a reduced susceptibility to imipenem. The product of the cloned determinant, named FEZ-1, contains a 30-kDa polypeptide and exhibits an isoelectric pH of 7.6. Sequencing revealed that FEZ-1 is a molecular-class B β-lactamase which shares the closest structural similarity (29.7% of identical residues) with the L1 enzyme of Stenotrophomonas maltophilia , being a new member of the highly divergent subclass B3 lineage. All the residues that in L1 are known to be directly or indirectly involved in coordination of the zinc ions were found to be conserved also in FEZ-1, suggesting that the geometry of zinc coordination in the active site of the latter enzyme is identical to that of L1. Unlike L1, however, FEZ-1 appeared to be monomeric in gel permeation chromatography experiments and exhibited a distinctive substrate specificity with a marked preference for cephalosporins and meropenem. The properties of FEZ-1 overall resembled those of a β-lactamase previously purified from the same strain of L. gormanii (T. Fujii, K. Sato, K. Miyata, M. Inoue, and S. Mitsuhashi, Antimicrob. Agents Chemother. 29:925–926, 1986) and are as yet unique among class B enzymes, reinforcing the notion that considerable functional heterogeneity can be encountered among members of this class. A system for overexpression of the bla FEZ-1 gene in E. coli , based on the T7 phage promoter, was also developed.

Two metal ion binding sites are conserved in metallo-β-lactamase from Aeromonas hydrophila. The ligands of a first zinc ion bound with picomolar dissociation constant were identified by EXAFS spectroscopy as one Cys, two His and one additional N/O donor. Sulfur-to-metal charge transfer bands are observed for all mono- and di-metal species substituted with Cu(II) or Co(II) due to ligation of the single conserved cysteine residue. Binding of a second metal ion results in non-competitive inhibition which might be explained by an alternative kinetic mechanism. A possible partition of metal ions between the two binding sites is discussed.

Cd-substituted forms of the Bacillus cereus metallo-β-lactamases (BCII) were studied by perturbed angular correlation of γ-rays (PAC) spectroscopy. At very low [Cd]:[apo-β-lactamase] ratios, two nuclear quadrupole interactions (NQI) were detected. For [Cd]:[apo-β-lactamase] ratios between 0.8 and 3.0, two new NQIs appear, and the spectra show that up to 2 cadmium ions can be bound per molecule of apoenzyme. These results show the existence of two interacting Cd-binding sites in BCII. The relative populations of the two NQIs found at low [Cd]:[apo-β-lactamase] ratios yielded a 1:3 ratio for the microscopic dissociation constants of the two different metal sites (when only one cadmium ion is bound). X-ray diffraction data at pH 7.5 demonstrate that also for Zn(II) two binding sites exist, which may be bridged by a solvent molecule. The measured NQIs could be assigned to the site with three histidines as metal ligands (three-His site) and to the site with histidine, cysteine, and aspartic acid as metal ligands (Cys site), respectively, by PAC measurements on the Cys168Ala mutant enzyme. This assignment shows that cadmium ions preferentially bind to the Cys site. This is in contrast to the preference of Zn(II) in the hybrid Zn(II)Cd(II) enzyme, where an analysis of the corresponding PAC spectrum showed that Cd(II) occupied the Cys site, whereby Zn(II) occupied the site with three histidines. The difference between Zn(II) and Cd(II) in affinity for the two sites is combined with the kinetics of hydrolysis of nitrocefin for different metal ion substitutions (Zn2E, ZnE, Cd2E, CdE, and ZnCdE) to study the function of the two metal ion binding sites.

The location and environment of the acquired bla IMP gene, which encodes the IMP-1 metallo-β-lactamase, were investigated in a Japanese Pseudomonas aeruginosa clinical isolate (isolate 101/1477) that produced the enzyme. In this isolate, bla IMP was carried on a 36-kb plasmid, and similar to the identical alleles found in Serratia marcescens and Klebsiella pneumoniae clinical isolates, it was located on a mobile gene cassette inserted into an integron. The entire structure of this integron, named In31, was determined. In31 is a class 1 element belonging to the same group of defective transposon derivatives that originated from Tn 402 -like ancestors such as In0, In2, and In5. The general structure of In31 appeared to be most closely related to that of In5 from pSCH884, suggesting a recent common phylogeny for these two elements. In In31, the bla IMP cassette is the first of an array of five gene cassettes that also includes an aacA4 cassette and three original cassettes that have never been described in other integrons. The novel cassettes carry, respectively, (i) a new chloramphenicol acetyltransferase-encoding allele of the catB family, (ii) a qac allele encoding a new member of the small multidrug resistance family of proteins, and (iii) an open reading frame encoding a protein of unknown function. All the resistance genes carried on cassettes inserted in In31 were found to be functional in decreasing the in vitro susceptibilities of host strains to the corresponding antimicrobial agents.

The bla IMP gene coding for the IMP-1 metallo-β-lactamase produced by a Pseudomonas aeruginosa clinical isolate (isolate 101/1477) was overexpressed via a T7 expression system in Escherichia coli BL21(DE3), and its product was purified to homogeneity with a final yield of 35 mg/liter of culture. The structural and functional properties of the enzyme purified from E. coli were identical to those of the enzyme produced by P. aeruginosa . The IMP-1 metallo-β-lactamase exhibits a broad-spectrum activity profile that includes activity against penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems. Only monobactams escape its action. The enzyme activity was inhibited by metal chelators, of which 1,10- o -phenanthroline and dipicolinic acid were the most efficient. Two zinc-binding sites were found. The zinc content of the P. aeruginosa 101/1477 metallo-β-lactamase was not pH dependent.

The most widely used inactivators of active-site serine beta-lactamases behave as substrates of four class B metallo-beta-lactamases, but the efficiency of the catalytic process can vary by several orders of magnitude. A comparison of the kinetic parameters for the alpha and beta isomers of 6-iodopenicillanic acid shows that there is no general preference for the alpha isomer and that the efficient hydrolysis of imipenem by these enzymes must rest on other factors.

Amino acid derived hydroxamates were synthesized and tested for inhibitory activity against different metallo- B -lactamases. Several compounds inhibited the clinically relevant enzyme from Aeromonas hydrophila AE036.