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In the April issue ofAntimicrobial Agents and Chemotherapy (6), Proenca et al. described the cloning and sequencing of a 1.1-kb DNA fragment reportedly containing the penA gene encoding the chromosomal 1-lactamase of Pseudomonas cepacia 249 and deduced the primary structure of an unprocessed protein of 313 residues which was declared to belong to the AmpC family of ,B-lactamases, i.e., to structural class C (2). However, two important sequence elements which characterize these enzymes [Y150(A)SN and H314KTG] surprisingly were not found, and the putative active site serine at position 190 was much closer to the C terminus than it was in the other class C ,-lactamases. If the proposed sequence is coltrect, the penicillinase of P. cepacia must be considered a repre entative of a new original class of ,B-lactamases, quite distinct from all other active site serine ,B-lactamases identified so far and constructed on a pattern dramatically different from t at of all the presently known enzymes of this superfamily. A careful analysis of the published sequence yielded the following findings. (i) A search of the complete DNA sequence failed to identify the XmuI and PstI sites reported to be present in the sequenced EcoRI-EcoRI fragment, and the HincII site was found between 400 and 600 rather than between 200 and 400 bp. (ii) DNA sequence comparisons (EMBL data bank) revealed a very high similarity of nucleotides 426 to 827 with phage M13mpl8 nucleotides 5811 to 6224 (9), corresponding to a portion of the lacI gene. Interestingly, two stretches of extremely high similarity were interrupted by a short, 60nucleotide sequence (nucleotides 638 to 697) coding for a 20-residue peptide around the putative active serine, i.e., Ile-174-Lys-193. This is the only peptide in the "PenA" protein which exhibits a clear analogy with the active serine containing peptides of class C enzymes. If this DNA stretch is not included in the comparison, a 93.4% identity is found between nucleotides 426 to 637 and 698 to 827 of the penA gene and nucleotides 5811 to 6033 and 6093 to 6224 of the phage vector. Strikingly, most of the differences are insertions or deletions near or in multiplets. (iii) A simple search of the protein sequences in the SWISSPROT data bank revealed a much stronger similarity of PenA with Escherichia coli isoleucyl-tRNA synthetase (IleS, SWISSPROT: Syi E coli [8]) than with any of the known class C 1-lactamases. Indeed, for these ,B-lactamases, similarities were strictly restricted to the 20-residue peptide described above. The high similarity with the E. coli enzyme extends from residue 1 to 43 of the "PenA" protein with only three different aminio acids. A closer examination involving the six reading frames of the "penA" gene indicated that the similarity could be extended to a much longer portion of the gene product if four frameshifts were allowed. Under these conditions, 108 out of 112 residues could be identified to those of the Ile-tRNA synthetase. Furthermore, the sequence of nucleotides 7 to 69 of the putative penA gene was nearly identical to the corresponding part of the Ile-tRNA synthetase gene, and again, most of the differences as well as all of the frameshifts were due to insertions or deletions near multiplets. (iv) Similarly, nucleotides 828 to 1133 were translated in the six reading frames and the peptide sequences thus obtained

Analysis of the penA gene of Pseudomonas cepacia 249