Purification and properties of an inducible cephalosporinase from Pseudomonas maltophilia GN12873

Saino, Yushi; Inoue, Matsuhisa; Mitsuhashi, Susumu

doi:10.1128/aac.25.3.362

Cited by 86 publications

(61 citation statements)

References 19 publications

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“…(This procedure was performed by Affiniti Research Products Ltd.) Serum was separated and assayed using the above ELISA technique. The maximum working dilution of this polyclonal serum in ELISA assays was 1:10 6 . Peak fractions containing L1 protein were pooled and used for further analysis.…”

Section: Methodsmentioning

confidence: 99%

“…S. maltophilia is inherently resistant to most antibacterial drugs, including most, if not all, ␤-lactams (3,4). ␤-Lactam resistance in this organism is mainly mediated by the inducible expression of two ␤-lactamases (designated L1 and L2) (5,6). These are bacterial enzymes that hydrolyze ␤-lactam antibiotics, rendering them ineffective as inhibitors of bacterial cell wall synthesis (7).…”

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confidence: 99%

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Characterization of Monomeric L1 Metallo-β-lactamase and the Role of the N-terminal Extension in Negative Cooperativity and Antibiotic Hydrolysis

Simm¹,

Higgins²,

Carenbauer³

et al. 2002

Journal of Biological Chemistry

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The L1 metallo-␤-lactamase from Stenotrophomonas maltophilia is unique among this class of enzymes because it is tetrameric. Previous work predicted that the two regions of important intersubunit interaction were the residue Met-140 and the N-terminal extensions of each subunit. The N-terminal extension was also implicated in ␤-lactam binding. Mutation of methionine 140 to aspartic acid results in a monomeric L1 ␤-lactamase with a greatly altered substrate specificity profile. A 20-amino acid N-terminal deletion mutant enzyme (NDel) could be isolated in a tetrameric form but demonstrated greatly reduced rates of ␤-lactam hydrolysis and different substrate profiles compared with that of the parent enzyme. Specific site-directed mutations of individual N terminus residues were made (Y11S, W17S, and a double mutant L5A/L8A). All N-terminal mutant enzymes were tetramers and all showed higher K m values for ampicillin and nitrocefin, hydrolyzed ceftazidime poorly, and hydrolyzed imipenem more efficiently than ampicillin in contrast to wild-type L1. Nitrocefin turnover was significantly increased, probably because of an increased rate of breakdown of the intermediate species due to a lack of stabilizing forces. K m values for monomeric L1 were greatly increased for all antibiotics tested. A model of a highly mobile N-terminal extension in the monomeric enzyme is proposed to explain these findings. Tetrameric L1 shows negative cooperativity, which is not present in either the monomer or N-terminal deletion enzymes, suggesting that the cooperative effect is mediated via N-terminal intersubunit interactions. These data indicate that while the N terminus of L1 is not essential for ␤-lactam hydrolysis, it is clearly important to its activity and substrate specificity.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Characterization of Monomeric L1 Metallo-β-lactamase and the Role of the N-terminal Extension in Negative Cooperativity and Antibiotic Hydrolysis

Simm¹,

Higgins²,

Carenbauer³

et al. 2002

Journal of Biological Chemistry

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“…The substrate profile and zinc requirement identify this B. fragilis enzyme as a class B P-lactamase (1). The Bacillus cereus type II 3-lactamases of strains 569/H/9 (16,17) and 5/B/6 (19) are the most extensively studied enzymes of this class, which may also include the Li P-lactamase of Pseudomonas maltophilia GN12873 (29) and the 3-lactamase of Flavobacterium odoratum GN14053 (31).…”

mentioning

confidence: 99%

Sequencing the gene for an imipenem-cefoxitin-hydrolyzing enzyme (CfiA) from Bacteroides fragilis TAL2480 reveals strong similarity between CfiA and Bacillus cereus beta-lactamase II

1990

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Using a newly constructed Bacteroides fragilis-Escherichia coli cloning shuttle vector, pJST61, we have cloned the cefoxitin (FOX)-imipenem (IMP) resistance determinant from B. fragilis TAL2480. FOX-IMP resistance in this strain results from the production of a periplasmic, Zn2+-containing j-lactamase which hydrolyzes carbapenems and cephamycins and whose activity is resistant to clavulanic acid but sensitive to Zn2+-binding reagents, including EDTA. The pJST61 vector permits efficient library construction in E. coli and allows for the transfer of the library to B. fragilis recipients for the screening or selection of specific phenotypes. The library clone containing the FOX-IMP resistance gene was detected after transfer to B. fragilis TM4000 (Fox-Imps) selecting for Foxr. One of the isolates carrying plasmid pJST241 is resistant to FOX and IMP and synthesizes a periplasmic protein with substrate and inhibitor properties identical to those of strain TAL2480. On the basis of deletion analysis, Tn1000 insertion mutations, and DNA sequencing, we have defined the 747-base cfi4 (FOX-IMP resistance) gene within the 3.6-kilobase cloned insert in pJST241. The MATERIALS AND METHODSMedia and growth conditions. ML broth or Luria agar was used as the nutrient source for all E. coli strains; brain heart infusion (BBL Microbiology Systems, Cockeysville, Md.) broth (BHIS) supplemented with 5 jig of hemin per ml and 5 mg of yeast extract per ml was used for all B. fragilis cultures. Bacto-Agar (Difco Laboratories, Detroit, Mich.) was added at 1.5% for plates. Antibiotics were added when required in the following concentrations (jig/ml), unless

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“…With enterobacteria and P . aeruginosa, only high-level constitutive ("stably de-repressed") producers of Class I enzymes showed this behaviour.13* l 6 Nevertheless, mutant strains were obtained with high-and low-level constitutive P-lactamase expression from all three strains. Mutant strains of these types (table I) were designated by the suffixes -con and -def, respectively.…”

Section: Susceptibility Testsmentioning

confidence: 99%

“…Unlike L1 enzyme, it is inhibited by clavulanate and sulbactam. 6 In view of the wide substrate range of this combination of P-lactamases, their contribution to resistance has been assumed to be of major importance. However, this view has not been confirmed by comparative susceptibility tests on strains with mutations in the genes controlling B-lactamase production, except that mutant strains with high-level constitutive expression of the enzymes have been reported to show slightly increased resistance, compared with their /?-lactamase-inducible parent strains.…”

Section: Introductionmentioning

confidence: 99%

Susceptibility to -lactam antibiotics of mutant strains of Xanthomonas maltophilia with high- and low-level constitutive expression of L1 and L2 -lactamases

Akova

Bonfiglio²,

Livermore³

1991

Journal of Medical Microbiology

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Summary. Xanthomonas maltophilia produces two inducible P-lactamases, L 1 and L2, and resists the antimicrobial activity of P-lactam antibiotics, including carbapenems. L1 is a zinc-metaloenzyme with carbapenemase activity; L2 is an unusual cephalosporinase. Mutant strains with high-and low-level constitutive expression of these enzymes were derived from three reference strains of X. maltophilia. With a single exception, the mutant strains had altered expression of both enzymes, indicating that these P-lactamases share regulatory components. The exception was a mutant strain that had low-level constitutive (basal) expression of L1 enzyme but remained inducible for L2. A parent strain with low-level Blactamase inducibility was more susceptible to penicillins, cephalosporins and carbapenems than were those in which higher levels of enzyme activity were inducible. Mutations that caused high-level constitutive P-lactamase expression increased resistance to penicillins and newer cephalosporins. P-Lactamase basal mutant strains, including the one that remained inducible for L2 enzyme, were more susceptible than inducible strains to these drugs. Organisms with inducible or high-level constitutive P-lactamase expression were equally resistant to meropenem and imipenem but basal mutant strains, including the one that remained inducible for L2 enzyme, were more susceptible to meropenem than imipenem. Minimal inhibitory concentrations of meropenem, penicillins and cephalosporins, but not imipenem, were greater on Mueller Hinton agar than on IsoSensitest or Diagnostic Sensitivity Test agars. This behaviour was independent of P-lactamase inducibility, and may reflect permeability differences between cells grown on different media.

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Purification and properties of an inducible cephalosporinase from Pseudomonas maltophilia GN12873

Cited by 86 publications

References 19 publications

Characterization of Monomeric L1 Metallo-β-lactamase and the Role of the N-terminal Extension in Negative Cooperativity and Antibiotic Hydrolysis

Characterization of Monomeric L1 Metallo-β-lactamase and the Role of the N-terminal Extension in Negative Cooperativity and Antibiotic Hydrolysis

Sequencing the gene for an imipenem-cefoxitin-hydrolyzing enzyme (CfiA) from Bacteroides fragilis TAL2480 reveals strong similarity between CfiA and Bacillus cereus beta-lactamase II

Susceptibility to -lactam antibiotics of mutant strains of Xanthomonas maltophilia with high- and low-level constitutive expression of L1 and L2 -lactamases

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