Single Amino Acid Replacements at Positions Altered in Naturally ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1995, p. 145–149 0066-4804/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 39, No. 1

Single Amino Acid Replacements at Positions Altered in Naturally Occurring Extended-Spectrum TEM b-Lactamases JESUS BLAZQUEZ, MARIA-ISABEL MOROSINI, MARIA-CRISTINA NEGRI, MARINA GONZALEZ-LEIZA, AND FERNANDO BAQUERO* Servicio de Microbiologıá, Hospital Ramo ń y Cajal, Madrid, Spain Received 16 February 1994/Returned for modification 26 April 1994/Accepted 17 October 1994

By directed mutagenesis, we constructed a set of seven TEM-1 derivatives containing single replacements in each one of the amino acids substituted in naturally occurring extended-spectrum TEM b-lactamases. The exact contribution of each mutation to the resistance phenotype was determined. In addition, mutant enzyme production and stabilities were studied. Five of seven mutations determined to some extent variations in cephalosporin and/or monobactam activity. Dramatic changes in the hydrolysis of ceftazidime and aztreonam occurred when a serine was at position 164. Changes at positions 104, 238, and 240 showed more leaky variation in activity towards cephalosporins and aztreonam. Replacements at positions 237 and 265 caused no variation in susceptibility to cephalosporins. Interestingly, the change from Gln to Lys at position 39 found in TEM-2, classically considered a neutral change, slightly but consistently increased the MIC of ceftazidime and aztreonam. The in vitro construction of mutations appearing in naturally occurring TEM-b-lactamases, studied in the same genetic context, may help to understand the evolution of extended-spectrum b-lactamases. The production of TEM-type b-lactamases is the most prevalent mode of resistance to b-lactam antibiotics. TEM-1 blactamase is considered a broad-spectrum enzyme because it hydrolyzes both penicillins and cephalosporins (4). TEM-1, however, cannot efficiently inactivate new extended-spectrum cephalosporins, such as cefotaxime and ceftazidime, and monobactams, such as aztreonam. Molecular variants of TEM-1, termed extended-spectrum b-lactamases, emerged and disseminated probably as a consequence of the introduction of these extended-spectrum b-lactam antibiotics. After years of intensive study of molecular variations involved in substrate profile alterations of TEM enzymes, it is now known that clinically resistant variants are the result of amino acid substitutions in one of several well-defined positions or of combinations of two to five of these substitutions (for a review, see reference 11). Figure 1 shows the modified positions in these resistant variants and the amino acids by which they are substituted in naturally occurring TEM-type b-lactamases. For each position, the replacement is with a particular amino acid, the only exception being Arg-164, which can be replaced by either Ser or His. Several authors constructed TEM-1 derivatives containing some of the abovedescribed single mutations by either oligonucleotide-directed mutagenesis (10, 30) or subcloning of fragments containing those mutations (28). Biochemical and microbiological studies have been performed with some of these mutants. Nevertheless, none of these publications includes a comparative study of the variations in b-lactamase phenotype and stability resulting from all seven single mutations corresponding to the seven positions modified in naturally occurring b-lactamases. In addition, the microbiological activities were, in each one of these studies, measured in a different genetic context (promoter, plasmid, and strain), which precluded reliable phenotypic comparisons among the three studies. In this work, we describe the construction and characteriza-

tion of seven different mutants with changes in each one of the seven positions altered in clinical b-lactamases. In addition, we introduced these mutant derivatives into a strain lacking the outer membrane protein OmpF, as the interplay of TEM type b-lactamases and porin mutations leads to higher resistance levels (20, 24). It is expected that our study may serve to obtain new insights for understanding the selective process and the resulting evolution of extended-spectrum b-lactamases. To our knowledge, this is the first study including mutant TEM-1 derivatives with single mutations in all seven positions modified in naturally occurring extended-spectrum TEM-type b-lactamases. MATERIALS AND METHODS Escherichia coli strains and plasmids. The bacterial strains used for determination of MICs were RYC1000 (araD139 DlacU169 rpsL Drib7 thiA gyrA recA56) and MH621 [araD139 DlacU169 rpsL Drib7 thiA relA f(ompF-lacZ)16-21(Hyb)] (9). Strains for oligonucleotide-directed mutagenesis were CJ236 [dut-1 ung-1 thi-1 recA1/pCJ105 (F9, Cmr)] (13) and TG1 [D(lac-pro) supE thi hsdDS/F9 traD36 proA1B1 lacI lacZM15] (17). The hybrid phage M13mpVAp was constructed by cloning a BamHI-BamHI fragment from plasmid pKT254VAp (8) containing the blaTEM-1 gene from transposon Tn3, encoding the TEM-1 b-lactamase, into the BamHI site of phage M13mp19. An EcoRI-SalI fragment from the hybrid phage M13mpVAp was cloned in plasmid pBGS192 (31) digested with the same restriction enzymes. This new hybrid plasmid was named pBGTEM-1. The blaTEM-1 gene from this plasmid was sequenced in its entirety to verify that it was the previously described gene coding for TEM-1. The mutant derivatives, constructed by directed mutagenesis, were made by substituting the adequate mutated fragment from M13mpVAp, after mutagenesis and sequencing, for that of pBGTEM-1. Plasmids containing the mutant derivatives were named by adding the number of the b-lactamase (if previously described) or the amino acid change, as corresponded in each case, to the prefix pBGTEM-. Antibiotic susceptibility testing. Agar dilution assays were performed and interpreted according to the guidelines of the National Committee for Clinical Laboratory Standards (18). Standard antibiotic powders were kindly provided by the indicated pharmaceutical companies: amoxicillin and clavulanic acid, SmithKline Beecham Laboratories; cephaloridine, Eli Lilly and Co.; ceftazidime, Glaxo; cefotaxime, Hoechst Roussel Pharmaceuticals; aztreonam, Bristol-Myers Squibb; and meropenem, Zeneca Pharmaceuticals. The E-test (AB Biodisk, Solna, Sweden) was performed in Mueller-Hinton agar (Difco Laboratories, Detroit, Mich.) according to previously published methods (2). Recombinant DNA techniques. Isolation of plasmid DNA, transformation, restriction endonuclease digestion, ligation, agarose gel electrophoresis, and other standard recombinant techniques were performed as described before (25). Nucleotide sequence was determined by the dideoxynucleotide chain ter-

* Corresponding author. Mailing address: Servicio de Microbiologıá, Hospital Ramo ń y Cajal, Carretera de Colmenar Km 9,100, Madrid 28034, Spain. Fax: (34) 1-3368809. 145

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FIG. 1. Diagram of a TEM b-lactamase, showing the residues modified in extended-spectrum TEM enzymes. Arrows indicate the amino acids by which they are substituted in naturally occurring enzymes. At the bottom, a diagram of the blaTEM-1 gene, including internal relevant restriction sites, is shown. At the extremes, sites from both the vector (pBGS192) and cloned fragment, from pKT254VAp (8), are boxed. B, BamHI; E, EcoRI; H, HindIII; Hc, HincII; P, PstI; S, SalI; Sm, SmaI. The circle labelled P indicates the position of the bla natural promoter from Tn3. Numbering is according to Ambler (1). Nt and Ct, N terminus and C terminus, respectively.

mination method by using Sequenase (U.S. Biochemicals) and blaTEM-1-specific primers. Site-directed mutagenesis and selection of mutants. For all substitutions except Gly-2403Ser (TEM-19), site-directed mutagenesis was performed on M13mpVAp by the method of Kunkel et al. (13). The sequences of the oligonucleotides used in these mutagenesis experiments are shown in Table 1. Several phage plaques were selected, cultured overnight in 23TY medium, harvested, and, when a change in the isoelectric point (pI) was expected, sonicated. Proteins contained in the supernatant of these sonicate extracts were subjected to isoelectric focusing. Clones exhibiting the expected changes in b-lactamase pI were selected. For mutants with no expected change in pI, several phage plaques were also selected. In all cases, single-stranded DNA was extracted, and the appropriate segment of the bla gene, which theoretically contained the desired mutation, was sequenced. When the presence of the desired mutation but no others was demonstrated, the fragment was interchanged with that of pBGTEM-1. Plasmid pBGTEM-19 was constructed by substituting the BamHI-SmaI fragment from plasmid pAT264 (28), containing the 39 end of the mutant bla gene, for that of pBGTEM-1. The fragment from pAT264 contained the desired mutation. Extracts from each of the final constructions were checked to determine that the pI of each mutant enzyme was consistent with that expected. Pulse and chase experiments. Pulse and chase experiments were performed in maxicells of strain RYC1000 prepared as described before (26). Labelling with 35 [ S]methionine was done for 2 min at 378C. At this time, an excess of unlabelled methionine was added, and maxicells were incubated for 30 and 60 min at 378C. Labelled proteins at 0, 30, and 60 min were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) as described before (14). As a control to identify the TEM polypeptides, we used maxicells of strain RYC1000 containing the vector pBGS192, encoding an aminoglycoside-phosphotransferase but lacking the bla gene. The phosphotransferase also served as a control for protein stability because no degradation was found after 60 min of incubation with unlabelled methionine (3).

TABLE 1. Nucleotide sequences of the oligonucleotides used in directed mutagenesis Change

TEMa

Sequence

Gln-393Lys Glu-1043Lys Arg-1643Ser Ala-2373Thr Gly-2383Serb Glu-2403Lys Thr-2653Met

2 17 12 n.f. 19 n.f. n.f.

59-CTCGTGCACCCAACTTATCTTCAGC 59-GACTGGTGAGTATTTAACCAAGTCATT 59-CCGGTTCCCAACTATCAAGGCGA 59-CCCACGCTCACCGGTTCCAGATTTA — 59-GACCCACGCTTACCGGCTCCAG 59-CTCCCCGTCATGTAGATAACT

a Naturally occurring TEMs which contain only the indicated mutation. n.f., not found in clinical isolates as a single mutation. b Constructed by cloning the appropriate fragment from pAT264 (see text).

Isoelectric focusing. Analytical isoelectric focusing was performed in precast polyacrylamide gels (pH range, 3 to 9 and/or 4 to 6.5) by using a Pharmacia (Uppsala, Sweden) PhastSystem apparatus according to the instructions of the manufacturer. b-Lactamase activity was revealed by hydrolysis of the chromophore b-lactam nitrocefin (Oxoid). Preparation of crude enzyme extracts. Strain RYC1000 containing plasmid pBGTEM-1 or one of its seven mutant derivatives was grown in Luria-Bertani medium until the culture reached an optical density at 600 nm of 1.00. The cells were harvested by centrifugation and washed twice with 0.1 M potassium phosphate buffer (pH 7.5). For preparation of cell extracts, cells were ruptured by ultrasonic treatment at 48C. Cell debris was removed by centrifugation. The supernatant was used for kinetic tests. As a negative control, extracts from strain RYC1000 containing vector pBGS192 were used for the tests. Kinetics. b-Lactamase activities were determined by measuring the decrease in absorbance of cephaloridine at 275 nm. Kinetic studies were performed at a constant temperature of 258C in a UVIKON-940 spectrophotometer. One unit of b-lactamase activity was defined as the amount of enzyme that hydrolyzes 1 mmol of substrate in 1 min at 258C in 0.1 M potassium phosphate buffer (pH 7.5). For specific enzyme activities, protein concentrations were determined as described before (15). Kinetic parameters were obtained by linear plots of the initial steady-state velocities at different substrate concentrations (5).

RESULTS Microbiological activities. Tables 2 and 3 show the microbiological activities of some b-lactam antibiotics with strains RYC1000 and MH621 containing the wild-type (TEM-1) and mutant TEM derivatives. (i) Gln-39 to Lys (TEM-2). The Gln-39 to Lys mutation did not change the susceptibility pattern observed for the wild-type enzyme with amoxicillin, amoxicillin plus clavulanic acid, cefotaxime, aztreonam, and meropenem. Susceptibility to cephaloridine and ceftazidime was slightly (twofold) but consistently decreased in more than five independent experiments and in both the agar dilution and E tests. (ii) Glu-104 to Lys (TEM-17). Susceptibility to amoxicillin, amoxicillin plus clavulanic acid, and meropenem was not affected by this mutation. Susceptibility to ceftazidime and aztreonam was significantly decreased (four- to eightfold). (iii) Arg-164 to Ser (TEM-12). A decrease in susceptibility to cefotaxime and aztreonam, particularly dramatic in the case of ceftazidime (from 0.5 to 32 mg/ml in the OmpF2 strain), was detected. The amoxicillin MIC remained $2,048 mg/ml, and the meropenem MIC was not affected.

AMINO ACID CHANGES IN TEM b-LACTAMASES

VOL. 39, 1995 TABLE 2. Microbiological activities of b-lactam antibiotics with strain RYC1000 (OmpF1) producing mutant b-lactamases b-Lactamase

No TEMb TEM-1 Lys-39 Lys-104 Ser-164 Thr-237 Ser-238 Lys-240 Met-265

MICa (mg/ml) AMX

AM/AC

CER

CTX

CAZ

AZT

MEM

#32 $2,048 $2,048 $2,048 $2,048 2,048 2,048 $2,048 $2,048

2/1 16/8 16/8 16/8 8/4 16/8 4/2 16/8 16/8

4 32 64 16 16 32 32 64 64

0.03 0.03 0.03 0.06 0.12 0.03 0.06 0.03 0.03

#0.06 0.12 0.25 0.5 4 0.12 0.25 0.5 0.12

#0.015 0.06 0.06 0.25 0.25 0.06 0.12 0.25 0.06

0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003

a AMX, amoxicillin; AM/AC, amoxicillin plus clavulanate; CER, cephaloridine; CTX, cefotaxime; CAZ, ceftazidime; AZT, aztreonam; MEM, meropenem. b Strain RYC1000 containing plasmid pBGS192. All values in the table are the modal result of a minimum of three independent experiments.

(iv) Ala-237 to Thr. No significant variation in susceptibility was found for most of the b-lactams tested with this mutation. A slight decrease in the MIC of amoxicillin (in both OmpF1 and OmpF2 contexts) and amoxicillin plus clavulanate (in the OmpF2 context) was consistently detected in several independent experiments. (v) Glu-238 to Ser (TEM-19). The presence of this mutation originated a significant decrease in the MIC of amoxicillin plus clavulanate. On the other hand, an increase in the MIC of cefotaxime (only twofold in the OmpF1 but eightfold in the OmpF2 context) was regularly observed. (vi) Glu-240 to Lys. The Glu-240 to Lys change originated a significant increase in resistance to ceftazidime (from 0.5 to 4 mg/ml in the OmpF2 strain) and aztreonam (from 0.25 to 1 mg/ml). No changes in the activity of the other b-lactams were detected. (vii) Thr-265 to Met. The Thr-265 to Met mutation did not produce major variations in susceptibility to the b-lactam antibiotics tested in either the OmpF1 or OmpF2 context. With cephaloridine, a twofold increase was observed in the OmpF1 strain (Table 2). b-Lactamase activities. As shown in Table 4, the mutations Lys-39, Lys-104, Thr-237, and Met-265 did not alter the affinity to cephaloridine. However, mutations Ser-164 and Ser-238 significantly increased (about five times) the affinity of b-lactamase and, to a minor extent, so did Lys-240. The maximal

TABLE 3. Microbiological activities of b-lactam antibiotics with strain MH621 (OmpF2) producing mutant b-lactamases b-Lactamase

No TEM TEM-1 Lys-39 Lys-104 Ser-164 Thr-237 Ser-238 Lys-240 Met-265

b

MICa (mg/ml) AMX

#32 $2,048 $2,048 $2,048 $2,048 2,048 2,048 $2,048 $2,048

AM/AC CER

4/2 32/16 32/16 32/16 16/8 16/8 8/4 32/16 32/16

2 128 256 128 64 128 64 256 128

CTX

CAZ

AZT

MEM

0.06 0.12 0.12 0.12 0.25 0.12 1 0.12 0.12

#0.25 0.5 1 4 32 0.5 1 4 0.5

#0.12 0.25 0.5 1 2 0.25 0.5 1 0.25

0.006 0.006 0.006 0.006 0.006 0.006 0.006 0.006 0.006

a AMX, amoxicillin; AM/AC, amoxicillin plus clavulanate; CER, cephaloridine; CTX, cefotaxime; CAZ, ceftazidime; AZT, aztreonam; MEM, meropenem. b Strain MH621 containing plasmid pBGS192. All values in the table are the modal result of a minimum of three independent experiments.

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TABLE 4. Kinetic parameters of TEM-1 and mutant enzymes for cephaloridinea b-Lactamase

Kmb (mM)

Vmaxc

Vmax/Kmc

TEM-1 Lys-39 Lys-104 Ser-164 Thr-237 Ser-238 Lys-240 Met-265

550 (1.0) 550 (1.0) 500 (0.9) 100 (0.2) 500 (0.9) 80 (0.15) 385 (0.7) 550 (1.0)

1.0 1.5 0.25 0.01 0.9 0.005 0.7 1.0

1.0 1.5 0.28 0.05 1.0 0.03 1.0 1.0

a Values are the means for a minimum of three independent experiments. The activity of an extract from strain RYC1000(pBGS192) lacking TEM b-lactamase was undetectable. b Relative Km is indicated in parentheses. c Relative values.

relative rates of hydrolysis for cephaloridine decreased in most tested mutations, particularly Ser-164 and Ser-238 (100 to 200 times). Vmax was not altered in the Met-265 mutant and increased slightly for the Lys-39 mutation. The highest relative hydrolytic efficiency (Vmax/Km) was found for Lys-39 (1.5, compared with 1 for the wild-type TEM-1 enzyme). The same catalytic efficiencies as TEM-1 were found for the mutants Lys-240, Thr-237, and Met-265. Ser-164 and Ser-238 showed the lowest efficiencies for cephaloridine (20 to 30 times weaker than TEM-1). b-Lactamase production and stabilities. In order to know the possible implications of different mutations in a possible increase in b-lactamase stability, we performed pulse and chase experiments with maxicells transcribing and translating only plasmid-encoded proteins. Degradation is measured by the relative decrease in radioactive labelling of these proteins over time. When we examined RYC1000 maxicells carrying the mutant bla genes for protein synthesis, we found that all of them produced polypeptides (TEM b-lactamases) of the same size and in the same amounts as those carrying the wild-type gene. All TEM derivatives constructed in this work appeared to be almost completely degraded after 30 min and totally so after 60 min of incubation with unlabelled methionine. Nevertheless, as previously published (3), the aminoglycoside-phosphotransferase polypeptide encoded by pBGS192 showed no degradation after 30 and 60 min of incubation (data not shown). None of the described TEM mutants showed significant increases in stability after the cited periods of incubation; that does not exclude differences in stability at shorter times. DISCUSSION Some of the single mutants studied in this work have been previously obtained and characterized. Nevertheless, some of those mutants were constructed in different plasmids or genetic contexts. Indeed, the bla genes used for some of those constructions were not the original blaTEM-1 genes but derivatives with theoretically ‘‘neutral’’ mutations (11, 30). The aim of this work was to construct a collection of TEM-1 derivatives in the same genetic context (plasmid, promoter, or strain) and define the change in the enzyme properties (kinetics, amount, and stability) and in the phenotype conferred by each one of these mutants. The Lys-39 mutation (characterizing the TEM-2 enzyme) is frequent among extended-spectrum b-lactamases, being present in TEM-3, TEM-7, TEM-8, TEM-11, TEM-13, TEM-14, TEM-16, TEM-18, and TEM-24 (11, 19). Such frequency is higher than expected if the low frequency of TEM-2 among the

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natural E. coli isolates is considered (11). Our results suggests that the Lys-39 mutation by itself may provide the host strain with better survival at low concentrations of some cephalosporins or monobactams, thus increasing the possibilities for acquisition of new mutations. This effect could be increased by the higher promoter strength of the natural blaTEM-2 gene from Tn1 (7). The change Glu-1043Lys was identified previously in TEM-3, TEM-4, TEM-6, TEM-8, TEM-9, TEM-14, TEM-15, TEM-16, TEM-17, TEM-18, TEM-24, and TEM-26 (6, 11, 19), and the change Glu-2403Lys was identified in TEM-5, TEM10, and TEM-24 (6, 11). These changes at positions 104 and 240 consistently decreased susceptibility to ceftazidime and aztreonam. It has been sugested that enzymes with lysines at positions 104 and 240 could establish electrostatic interactions with the oxime acid group of ceftazidime and aztreonam (30). The change Arg-1643Ser has been found previously in TEM-5, TEM-7, TEM-8, TEM-9, TEM-10, TEM-12, TEM-24, and TEM-26 (6, 11, 19). Dramatic changes in the hydrolysis of ceftazidime and aztreonam occurred when we introduced the mutation Ser-164. The results with these changes are very striking if we consider that, in the structure of TEM-1, the side chain of Arg-164 is not in a position to interact directly with the substrate. Nevertheless, the Arg to Ser change could alter the conformation or orientation of an omega loop, affecting the substrate profile of the enzyme (12, 22). In general, our results with the mutations Lys-104, Ser-164, and Lys-240 are in accordance with those reported previously (28, 30). Sowek et al. (30) found a catalytic efficiency for cephaloridine in the Ser-164 mutant that was higher than ours, which can be related to the differences in the studied wild-type bla gene and/or in the enzyme preparation. These three mutations, particularly Lys-104 and Ser-164, are in fact more frequently found among the more active extended-spectrum b-lactamases. In our work, the strains harboring such mutations required higher MICs of ceftazidime and aztreonam. The Ala-2373Thr change, found in TEM-5 and TEM-24 (6, 29), is apparently neutral in relation to the tested antibiotics except for amoxicillin, which had a decreased MIC. This decrease of activity for penicillins has been shown previously by Healey et al. (10). The change Gly-2383Ser, found previously in TEM-3, TEM-4, TEM-8, TEM-14, TEM-15, and TEM-19 (11), produces a decrease in cefotaxime susceptibility. These results are consistent with those of Sougakoff et al. (28). Susceptibility to amoxicillin plus clavulanate is increased. Enzymatic activity for cephaloridine decreased with this mutation, although affinity to this substrate was increased sevenfold. Thr-2653Met was found in TEM-4, TEM-9, TEM-13, and TEM-14 (11); nevertheless, this change had never been obtained previously as a single mutation. It was suggested by others that this mutation, far away from the active-site pocket, could be a functionally silent substitution (16), and our data give support to this view. Modifications in the amino acid sequence of a b-lactamase may modify the stability of the protein, influencing the turnover of the enzyme and therefore the b-lactam/b-lactamase interplay and consequently the antibiotic activity. Our pulse and chase data show that none of the seven single mutations studied increases the amount or the stability of the TEM protein at the times tested, but we cannot exclude that differences could be detected at shorter time points. The five TEM-1 natural mutations determining different rates of decrease in susceptibility to b-lactam antibiotics are not the only ones that could have been selected in nature. Many other TEM mutations have been constructed that are

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able to increase resistance to extended-spectrum cephalosporins (21–23). It remains a puzzling fact that these mutations have not yet been recovered either in clinical isolates or in stepwise in vitro selection tests. The understanding of the evolution of extended-spectrum b-lactamases should be based on the data obtained by the in vitro reproduction of their evolutionary molecular intermediates. Since TEM-1 seemed to be the more widespread enzyme among plasmid-encoded b-lactamases, single point mutations leading to small decreases in the antimicrobial activity of the newest cephalosporins and monobactams could have been selected under the pressure of the low antibiotic concentrations obtained during clinical therapy. ACKNOWLEDGMENTS We thank P. Courvalin for the generous gift of plasmid pAT264, J. C. Pe´rez Dıáz for helpful discussions, and L. de Rafael for English correction. This research was supported by a grant from Zeneca Pharmaceuticals. REFERENCES 1. Ambler, R. P. 1979. Amino acid sequences of b-lactamases, p. 99–125. In J. M. T. Hamilton-Miller and J. T. Smith (ed.), Beta-lactamases. Academic Press, London. 2. Baquero, F., R. Cantoń, J. Martıńez-Beltra ń, and A. Bolmstro¨m. 1992. The E-test as an epidemiological tool. Diagn. Microbiol. Infect. Dis. 15:483–487. 3. Bla ´zquez, J., J. Davies, and F. Moreno. 1991. Mutations in the aphA-2 gene of transposon Tn5 mapping within the regions highly conserved in aminoglycoside-phosphotransferases strongly reduce aminoglycoside resistance. Mol. Microbiol. 5:1511–1518. 4. Bush, K. 1989. Classification of b-lactamases: groups 1, 2a, 2b, and 2b9. Antimicrob. Agents Chemother. 33:264–270. 5. Bush, K., J. Freudenberger, and R. B. Sykes. 1982. Interaction of azthreonam and related monobactams with b-lactamases from gram-negative bacteria. Antimicrob. Agents Chemother. 22:414–420. 6. Chanal, C., M.-C. Poupart, D. Sirot, R. Labia, J. Sirot, and R. Cluzel. 1992. Nucleotide sequences of CAZ-2, CAZ-6, and CAZ-7 b-lactamase genes. Antimicrob. Agents Chemother. 36:1817–1820. 7. Chen, S. T., and R. C. Clowes. 1987. Variations between the nucleotide sequences of Tn1, Tn2, and Tn3 and expression of b-lactamase in Pseudomonas aeruginosa and Escherichia coli. J. Bacteriol. 169:913–916. 8. Fellay, R., J. Frey, and H. Kirsch. 1987. Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gram-negative bacteria. Gene 52:147–154. 9. Hall, M. N., and T. J. Silhavy. 1981. The ompB locus and the regulation of the major outer membrane porin proteins of Escherichia coli. J. Mol. Biol. 146:23–43. 10. Healey, W. J., M. R. Labgold, and J. H. Richards. 1989. Substrate specificities in class A b-lactamases: preference for penams vs. cephems. The role of residue 237. Proteins Struct. Funct. Genet. 6:275–283. 11. Jacoby, G. A., and A. A. Medeiros. 1991. More extended-spectrum b-lactamases. Antimicrob. Agents Chemother. 35:1697–1704. 12. Jelsch, C., F. Lenfant, J. M. Masson, and J. P. Samama. 1992. b-Lactamase TEM-1 of E. coli: crystal structure determination at 2.5 Å resolution. FEBS Lett. 299:135–142. 13. Kunkel, T. A., J. D. Roberts, and R. A. Zakour. 1987. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154:367–382. 14. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–685. 15. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275. 16. Mabilat, C., and P. Courvalin. 1990. Development of ‘‘oligotyping’’ for characterization and molecular epidemiology of TEM b-lactamases in members of the family Enterobacteriaceae. Antimicrob. Agents Chemother. 34: 2210–2216. 17. Messing, J., R. Crea, and P. H. Seeburg. 1981. A system for shotgun DNA sequencing. Nucleic Acids Res. 9:309–321. 18. National Committee for Clinical Laboratory Standards. 1990. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A2. National Committee for Clinical Laboratory Standards, Villanova, Pa. 19. Naumovski, L., J. P. Quinn, D. Miyashiro, M. Patel, K. Bush, S. B. Singer, D. Graves, T. Palzkill, and A. M. Arvin. 1992. Outbreak of ceftazidime resistance due to a novel extended-spectrum b-lactamase in isolates from cancer patients. Antimicrob. Agents Chemother. 36:1991–1996.

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