In the course of surveying for the carbapenem-hydrolyzing metallo-␤-lactamase gene bla IMP in pathogenic bacteria by the PCR method, we detected a gene encoding a variant metallo-␤-lactamase, designated IMP-3, which differed from IMP-1 by having low hydrolyzing activity for penicillins and carbapenems. PCR product direct sequencing of a 2.2-kb segment revealed that the gene bla IMP-3 was located on a cassette inserted within a class I integron in the pMS390 plasmid. The 741-bp nucleotide sequence of bla IMP-3 was identical to that of bla IMP-1 , except for seven base substitutions. Among these were two, at nucleotide positions 314 and 640, which caused amino acid alterations. Hybrid bla genes were constructed from bla IMP-3 and bla IMP-1 by recombinant DNA techniques, and ␤-lactamases encoded by these genes were compared with those of the parents IMP-3 and IMP-1 under the same experimental conditions. The kinetic parameters indicated that the inefficient hydrolysis of benzylpenicillin, ampicillin, imipenem, and ceftazidime by IMP-3 was due to the substitution of glycine for serine at amino acid residue 196 in the mature enzyme. This alteration corresponded to the presence of guanine instead of an adenine at nucleotide position 640 of the bla IMP-3 gene. This indicated that extension of the substrate profile in the metallo-␤-lactamase IMP-1 compared to IMP-3 is the result of a one-step single-base mutation, suggesting that the gene bla IMP-3 is an ancestor of bla IMP-1 .␤-Lactamases are enzymes that hydrolyze ␤-lactam antibiotics, conferring resistance to a variety of these antibiotics for most pathogenic bacteria. These enzymes have been classified phylogenetically based on their functional and molecular characteristics (5).Molecular class B metallo-␤-lactamases belonging to functional group 3a subclass B1 are characteristic in their broad substrate spectrum, which extends to most ␤-lactam antibiotics, except for monobactams, and have activities as penicillinases, cephalosporinases, and carbapenemases (5,15,23). They have been reported in Bacillus cereus, alkalophilic Bacillus sp., Bacteroides fragilis, Pseudomonas aeruginosa, Serratia marcescens, and Klebsiella pneumoniae (23). Among this group of metallo-␤-lactamases, ESP from P. aeruginosa GN17203, IMP-1 from S. marcescens TN9106, and DK4 from K. pneumoniae are all plasmid mediated and were found to be the same enzyme because the nucleotide sequences of their genes are identical (14, 22; GenBank accession number D29636).Genes bla ESP and bla IMP , respectively encoding ESP and IMP-1 ␤-lactamase, were identified in the cassettes inserted in the integrons on plasmids (24). Both cassettes had the same nucleotide sequence, but they were found to be inserted into different integrons, class 1 integron 0 (In0) for the bla ESP cassette (2, 14) and the class 3 integron for the bla IMP cassette (1). This fact suggested that the bla IMP (bla ESP ) cassette has been disseminated among different integrons. Since In0 is reported to be widespread among clinical isolates of ...

A class C beta-lactamase from a clinical isolate of Enterobacter cloacae strain GC1 with improved hydrolytic activity for oxyimino beta-lactam antibiotics has been analyzed by X-ray crystallography to 1.8 A resolution. Relative to the wild-type P99 beta-lactamase, this natural mutant contains a highly unique tandem repeat Ala211-Val212-Arg213 [Nugaka et al. (1995) J. Biol. Chem. 270, 5729-5735]. The 39.4 kDa chromosomal beta-lactamase crystallizes from poly(ethylene glycol) 8000 in potassium phosphate in space group P2(1)2(1)2 with cell dimensions a = 78.0 A, b = 69.5 A, and c = 63.1 A. The crystal structure was solved by the molecular replacement method, and the model has been refined to an R-factor of 0.20 for all nonzero data from 8 to 1.8 A. Deviations of model bonds and angles from ideal values are 0.008 A and 1.4 degrees, respectively. Overlay of alpha-carbon atoms in the GC1 and P99 beta-lactamases results in an rms deviation of 0.6 A. Largest deviations occur in a loop containing Gln120 and in the Omega loop region (200-218) where the three residues 213-215 are disordered. Possibly as a result of this disorder, the width of the opening to the substrate binding cavity, as measured from the 318-324 beta-strand to two loops containing Gln120 and Tyr150 on the other side, is 0.6-1.4 A wider than in P99. It is suggested that conformational flexibility in the expanded Omega loop, and its influence on adjacent protein structure, may facilitate hydrolysis of oxyimino beta-lactams by making the acyl intermediate more open to attack by water. Nevertheless, backbone atoms in core catalytic site residues Ser64, Lys67, Tyr150, Asn152, Lys318, and Ser321 deviate only 0.4 A (rmsd) from atoms in P99. A rotation of a potential catalytic base, Tyr150, relative to P99 at pH 8, is consistent with the requirement for a lower than normal pK(a) for this residue.

Mutant strains lacking outer membrane protein(s) were isolated from Escherichia coli, Proteus mirabilis, and Enterobacter cloacae. The outer membrane protein(s) of P. mirabilis and E. cloacae corresponding to E. coli porin were identified on the basis of their function, namely, their ability to allow the permeation of glucose as demonstrated by [14C]glucose uptake by intact cells. P. mirabilis has only one outer membrane pore protein (molecular weight, 40,000), but E. cloacae has at least two such proteins (molecular weights, 37,000 and 39,000 to 40,000). When the bacteria lost these proteins or porin, the outer membrane permeation of cefazolin was found to be greatly reduced in these three species. Such a change in the outer membrane permeation closely correlated with a significant decrease in the bacterial susceptibility to cephalosporins, including cefoxitin. These results suggested that the main pathway for cephalosporin permeation is the pore made up of these proteins. The 39,000- to 40,000-molecular-weight protein in E. cloacae was also assumed to play an important role in the outer membrane permeation of tetracycline and chloramphenicol. On the other hand, the permeation route of penicillins was obscure. The susceptibility to penicillins, except in some cases, was little influenced by the absence of the proteins. Ampicillin was found to pass through the outer membrane via the same route as the cephalosporins, but the possibility that ampicillin and other penicillins possess another unknown route for outer membrane permeation could not be ruled out.

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