A Comparative Study of Eight Distinct  -Lactamases Synthesized by Gram-negative Bacteria

Jack, George W.; Richmond, M. H.

doi:10.1099/00221287-61-1-43

Cited by 129 publications

(66 citation statements)

References 29 publications

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“…It follows that the P-lactamase produced by strains carrying RP I cannot be the only factor influencing their penicillin resistance. Furthermore, no destruction of either benzylpenicillin or cephaloridine could be detected even after prolonged incubation with broken cell preparations (Jack & Richmond, 1970) of either P. aeruginosa or E. coli carrying the mutated R-factor. The partial resistance of these strains could not therefore be due to a penicillin or cephalosporin amidase.…”

mentioning

confidence: 94%

R-factor Mediated Resistance to Penicillins which does not Involve a -Lactamase

Curtis

Richmond

Stanisich³

1973

Journal of General Microbiology

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confidence: 94%

R-factor Mediated Resistance to Penicillins which does not Involve a -Lactamase

Curtis

Richmond

Stanisich³

1973

Journal of General Microbiology

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“…Although some chromosomally coded enzymes have been recognized in Escherichia coll (6), the enzymes in the enterobacteria and the pseudomonads are often coded for by resistance transfer plasmids. The enzymes vary among themselves in substrate profile (relative activity against a range of penicillins and cephalosporins), molecular weights, and other chemical properties (7,8), but without sequence studies it was not possible to decide if the enzymes were evolutionarily related to the proteins from the Gram-positive bacteria, or even among themselves.We chose to study the enzyme from the plasmid R6K [formerly known as RTEM (9)] expressed in E. coli strain W3310, because the yields were better than for other enterobicterial penicillinases considered. A method was developed (10) for the isolation of the protein in gram quantities in apparently homogeneous form, and structural and mechanistic studies were started (11).…”

mentioning

confidence: 99%

Partial amino acid sequence of penicillinase coded by Escherichia coli plasmid R6K.

Ambler¹,

Scott²

1978

Proc. Natl. Acad. Sci. U.S.A.

153

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ABSTRACr Direct studies of the amino acid sequence of an Escherichia coli plasmid-coded penicillinase (penicillin amido~lactamhydrolase, EC 3.5.2.6) are in complete agreement with results derived from the translation of the DNA sequence of a related plasmid, apart from a single amino acid substitution. This penicillinase from a Gram-negative bacterium shows 30-35 identity with functionally similar enzymes from Gram-positive bacteria. This paper should be read in con unction with the report of the DNA sequence of the gene [Steliffe, J. G. (1978) Proc. NadL Acad. Sci. USA 75, 3737-374LJ. Penicillinases are enzymes that inactivate the penicillin and cephalosporin antibiotics. They have a wide distribution in bacteria (1). Most of the enzymes act by hydrolyzing the ftlactam bond, and so they are often known as f3-lactamases, or more systematically as penicillin amido-fl-lactamhydrolases (EC 3.5.2.6). Several Gram-positive bacteria produce inducible extracellular penicillinases, and large amounts of pure enzyme can readily be prepared from constitutive mutants. The amino acid sequences of proteins from Staphylococcus aureus (2, 3) and Bacillus licheniformis (2, 4) and of one of the two distinct enzymes in Bacillus cereus (5) have been studied and are sufficiently similar to one other that a common evolutionary origin and mechanism of action is indicated. The normal enzymes from Gram-negative organisms are quite different in their physiology of production, because they are generally noninducible, cell-bound, and produced at relatively low levels. Although some chromosomally coded enzymes have been recognized in Escherichia coll (6), the enzymes in the enterobacteria and the pseudomonads are often coded for by resistance transfer plasmids. The enzymes vary among themselves in substrate profile (relative activity against a range of penicillins and cephalosporins), molecular weights, and other chemical properties (7,8), but without sequence studies it was not possible to decide if the enzymes were evolutionarily related to the proteins from the Gram-positive bacteria, or even among themselves.We chose to study the enzyme from the plasmid R6K [formerly known as RTEM (9)] expressed in E. coli strain W3310, because the yields were better than for other enterobicterial penicillinases considered. A method was developed (10) for the isolation of the protein in gram quantities in apparently homogeneous form, and structural and mechanistic studies were started (11). The same enzyme system is also being used by Knox et al. (12), for the study of the three-dimensional structure of a penicillinase by x-ray crystallography.The preliminary studies (11) produced much information on peptide compositions and sequence, and suggested a possible mechanism of action for the enzyme (13). Indications were obtained of a limited sequence similarity to the enzymes from the Gram-positive bacteria, but unfortunately the information was insufficient to allow the deduction of long lengths of sequence. Recently, as a result of the interest in the t...

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“…Enterobacter cloacae 214 [14], E. cloacae 53 [5], and E. coli W3110 (RTEM) which carries R-plasmid RTEM [I 51 were obtained from M. H. Richmond (University of Bristol, England). Other bacterial strains used were clinical isolates stocked in our laboratories.…”

Section: B-lactamase-producing Bacteriamentioning

confidence: 99%

Monoclonal antibodies against species-specific cephalosporinase of Pseudomonas aeruginosa

Murakami

Yoshida

1985

Eur J Biochem

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Ten hybridomas which produced monoclonal antibodies against species-specific cephalosporinase of Pseudomonas aeruginosa were constructed by somatic cell fusion of SP-2/0 myeloma cells with spleen cells from hyperimmunized BALB/c mice and intraperitoneally injected into mice. Monoclonal antibodies were partially purified from ascites fluids. All ten antibodies thus prepared were IgG immunoglobulins. In a solid-phase immunoassay the antibodies gave positive reactions which could be prevented by the presence of free cephalosporinase. When the antibodies were precipitated with anti-(mouse IgG), the cephalosporinase activity was co-precipitated. Also, these antibodies were co-eluted with cephalosporinase activity in gel filtration. Nine of the monoclonal antibody preparations elevated the cephalosporinase activity by about 6 -40% and only one inhibited it by about 50%. All the monoclonal antibodies were highly specific to P. aeruginosa cephalosporinase and showed no cross-reaction with nine cephalosporinases and four penicillinases of other gram-negative bacteria.The main mechanism of bacterial resistance to /3-lactam antibiotics is the production of /3-lactamase which cleaves the b-lactam rings of the antibiotics rendering them inactive [l, 21. They are produced in many bacterial species and differ from each other in their enzymological and physicochemical properties. Although several classifications of /3-lactamases have been proposed [2-41, the enzymes can be generally classified into the two groups of R-plasmid-mediated /3-lactamases and species-specific /3-lactamases. The /3-lactamase of Pseudomonas aeruginosa used in this study is a species-specific cephalosporinase which is more active against cephalosporins.Anti-serum against /3-lactamase was used to identify or classify /?-lactamases based on its specific reaction with the enzymes [5 -91. Monoclonal antibodies are useful probes for assessing the structural similarities between macromolecules such as /I-lactamases [lo]. Thus, a monoclonal antibody against /$-lactamase can be expected to reveal some interspecies similarities and intraspecies differences of the enzymes or to be a good reagent for detecting the enzyme. In the present investigation, we have prepared a set of monoclonal antibodies against the cephalosporinase of P. aeruginosa by the hybridoma technique [l 11 and characterized them. kindly supplied by T. Sawai (Chiba University, Japan). Enterobacter cloacae 214 [14], E. cloacae 53 [5], and E. coli W3110 (RTEM) which carries R-plasmid RTEM [I 51 were obtained from M. H. Richmond (University of Bristol, England). Other bacterial strains used were clinical isolates stocked in our laboratories. Pseudomonas aeruginosa SR24-12 produces cephalosporinase constitutively and is a mutant of strain SR24 which produces the enzyme inducibly. MATERIALS AND METHODS AntibioticsPurijkation of the cephalosporinase P. aeruginosa SR24-12 was cultured in 34 1 of heart infusion broth (Eiken, Tokyo, Japan) at 37°C for 6 h with aeration. About 69 g (wet weight) of cells we...

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A Comparative Study of Eight Distinct -Lactamases Synthesized by Gram-negative Bacteria

Cited by 129 publications

References 29 publications

R-factor Mediated Resistance to Penicillins which does not Involve a -Lactamase

R-factor Mediated Resistance to Penicillins which does not Involve a -Lactamase

Partial amino acid sequence of penicillinase coded by Escherichia coli plasmid R6K.

Monoclonal antibodies against species-specific cephalosporinase of Pseudomonas aeruginosa

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