The location and environment of the acquiredbla IMP gene, which encodes the IMP-1 metallo-β-lactamase, were investigated in a JapanesePseudomonas 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 Tn402-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 anaacA4 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 addition of a poly-His C-terminal extension, designed to facilitate the purification of the protein, to the pMactamase of a thermophilic Bacillus liclteniformis strain modified the site of action of the signal peptidase. This resulted in the secretion of a protein with a different N-terminus, showing that this type of protein engineering might not always be as 'neutral' as generally assumed.

In the absence of penicillin, the ␤-lactamase encoding gene blaP of Bacillus licheniformis 749/I is negatively regulated by the transcriptional repressor BlaI. Three palindromic operator regions are recognized by BlaI: two in the blaP promoter (OP1 and OP2) and one (OP3) in the promoter of the blaI-blaR1 operon. In this study, the dissociation constant of the purified BlaI dimer was estimated at 25 M by equilibrium ultracentrifugation. Quantitative Western blot analysis indicates that the intracellular concentrations of BlaI in B. licheniformis 749/I and Bacillus subtilis transformed by a multicopy plasmid harboring the ␤-lactamase locus (blaP-blaIblaR1) were lower than (1.9 M) or in the same range as (75 M) the dissociation constant, respectively. This suggests that BlaI is partially dimeric in the cytoplasm of these strains and interacts in vivo with its operators as a preformed dimer. This hypothesis is supported by band shift assays on an operator containing a randomized half-operator sequence. The global dissociation constants of the operator-BlaI dimer complexes were measured by band shift assays and estimated as K dOP1 ‫؍‬ 1.7 ؎ 0.5 10 The blaP gene encodes the class A ␤-lactamase of Bacillus licheniformis 749/I. In the absence of ␤-lactams antibiotics, the BlaI repressor prevents the transcription of the blaP gene (1-3). Two additional genes, blaR1 and blaR2, are also involved in the induction of the ␤-lactamase synthesis (4, 5). The blaP, blaI, and blaR1 genes are clustered in a divergeon (bla divergeon) in which blaI and blaR1 form an operon (Fig. 1). The blaR1 gene encodes a transmembrane protein that acts as penicillin receptor (6 -8), and blaR2 is not identified yet and is not linked to the bla divergeon. The BlaR2 protein is essential for the inactivation of BlaI. In Staphylococcus aureus, the blaZ and mecA genes, encoding respectively a ␤-lactamase and the low affinity penicillinbinding protein 2Ј, are regulated by similar elements (9, 10).The blaI gene encodes a 128-residue protein characterized by two functional and separate domains (11). The DNA-binding domain, a helix-turn-helix recognition motif, is located in the N-terminal region, and the dimerization domain is in the Cterminal region. After deletion of the C-terminal domain, the repressor becomes unable to dimerize and to form a stable complex with its operators. DNase footprinting experiments and filter binding reactions revealed the presence of three regulatory regions that are recognized specifically by BlaI (11). These operators present a 23-bp-long dyad symmetry and show the deduced 5Ј-AAAGTATTACATATGTAACNTTT-3Ј consensus sequence (Fig. 1).In the presence of ␤-lactam antibiotics, the C-terminal domain of BlaR1 is acylated (6, 7). This event triggers the activation of the putative cytoplasmic metalloprotease motif of BlaR1 by self-proteolysis (8, 12). In S. aureus, the ␤-lactamase induction correlates with BlaI proteolysis between residues Asn 101 and Phe 102 , and it is postulated that the activated form of BlaR1 proteolyses BlaI (12-...

Abstract.Two new enzymes which hydrolyse D-alanyl-on the tripeptide L-Ala-Gly-Gly but it was not possible p-nitroanilide have been detected in Ochrobactrum an-to be certain that the same protein was responsible for thropi LMG7991 extracts. The first enzyme, DmpB, was both p-nitroanilide and peptide hydrolysing activities. purified to homogeneity and found to be homologous to The gene encoding the DmpA protein was cloned and sequenced. The deduced protein sequence exhibits varythe Dap protein produced by O. anthropi SCRC C1-38 (ATCC49237). The second enzyme, DmpA, exhibits a ing degrees of similarity with those corresponding to similar substrate profile when tested on p-nitroanilide several open reading frames found in the genomes of other prokaryotic organisms, including Mycobacteria. derivatives of glycine and L/D-alanine, but the amounts produced by the Ochrobactrum strain were not sufficient None of these gene products has been isolated or characterised, but a tentative relationship can be proposed with to allow complete purification. Interestingly, the DmpA preparation also exhibited an L-aminopeptidase activity the NylC amidase from Fla6obacterium sp. K172.Key words. Peptidase; stereospecificity; amidohydrolase.Aminopeptidases release the amino-terminal residue from peptide substrates. Most are L-aminopeptidases and exhibit a wide variety of molecular masses, quaternary structures, catalytic residues and specificity profiles [1 -10]. So far, the only aminopeptidase active on peptides containing N-terminal D-residues is that from Ochrobactrum anthropi SCRC C1-38. It has been characterized and named D-aminopeptidase (Dap) (E.C 3.4.11.19) [10,11]. Its sequence exhibits 25% identity with that of the Streptomyces R61 DD-carboxypeptidase and the residues most important for the catalytic mechanism of i-lactamases and DD-carboxypeptidases [11][12][13][14][15] appeared to be conserved. Here we report the isolation and purification of a similar D-aminopeptidase from O. anthropi LMG7991. Moreover, the same strain produces a second enzyme which hydrolyses D-alanyl-pnitroanilide but not its N-acetylated derivative, and thus represents a new potential member of the Daminopeptidase family. The study of D-aminopeptidases will contribute general information on this poorly characterized enzyme group and will help to elucidate the possible evolutionary relationship between D-aminopep-* Corresponding author.

The subclass B3 FEZ-1 ␤-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the ␤-lactam bond in penicillins, cephalosporins, and carbapenems. FEZ-1 has been extensively studied using kinetic, computational modeling and x-ray crystallography. In an effort to probe residues potentially involved in substrate binding and zinc binding, five site-directed mutants of FEZ-1 (H121A, Y156A, S221A, N225A, and Y228A) were prepared and characterized using metal analyses and steady state kinetics. The activity of H121A is dependent on zinc ion concentration. The H121A monozinc form is less active than the dizinc form, which exhibits an activity similar to that of the wild type enzyme. Metallo-␤-lactamases are bacterial enzymes that hydrolyze antibiotics of the ␤-lactam family. They are classified as class B (1) or group 3 (2) ␤-lactamases. In the last decade, the discovery of an increasing number of new metallo-␤-lactamases resulted in a subdivision into three molecular subclasses: B1, B2, and B3. Thereafter, a standard numbering scheme (3) was adopted that identifies conserved residues involved in the catalytic activity. Subclass B3 ␤-lactamases are broad spectrum enzymes that require one or two Zn (II) ions for activity (4). These enzymes are produced by various environmental species, of which some can cause opportunistic infections (such as S. maltophilia (5) and F. gormanii (6)), whereas others are not pathogenic such as Janthinobacterium lividum (7) and Caulobacter crescentus (8, 9). The structures of several subclass B1 ␤-lactamases have been solved by x-ray crystallography (BcII (10, 11), CcrA (12), BlaB (13), and IMP-1 (14)). To date, no structure of a subclass B2 enzyme is available. In subclass B3, the crystal structures of the metallo ␤-lactamases L1 from S. maltophilia (15) and FEZ-1 from F. gormanii (16) have been solved. Comparison of the tertiary structures of the different enzymes highlighted similar organizations of the secondary structure elements; they all contain an ␣␤␤␣ sandwich with two central ␤-sheets and ␣-helices on the external faces (10). The active site with the binuclear zinc center is located at the bottom of the ␤-sheet core. Zn1 is tetrahedrally coordinated by three histidines, His 116 , His 118 , His 196 , and a water molecule. In the subclass B1 enzymes, Zn2 is coordinated by His 263 , Asp 120 , Cys 221 , and two water molecules to form a trigonal bipyramid. In subclass B3 ␤-lactamases, a Ser residue replaces Cys 221 , and His 121 is the third ligand of Zn2. Our studies were performed on the FEZ-1 ␤-lactamase. FEZ-1 is a monomeric enzyme. The sequence of the mature protein is easily aligned with that of the L1 enzyme with 33% of isology (17). The two subclass B3 ␤-lactamases exhibit a broad activity spectrum against ␤-lactam antibiotics, but FEZ-1 shows a preference for cephalosporins (18), whereas the L1 ␤-lactamase seems to be more active against penicillins (19,20). Comparison of the x-ray structures reveals similar zinc binding sites in...