[5] β-Lactamase assays

Ross, Gayle; O'Callaghan, Cynthia H.

doi:10.1016/0076-6879(75)43081-6

Cited by 104 publications

(57 citation statements)

References 28 publications

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“…Preliminary results (N. Curtis and M. Boxall, unpublished data) indicate that the defect does not involve major outer membrane proteins, which have been shown to determine the rate of entry of certain molecules into gram-negative bacteria (11,12,14,15) (19), and the PBP procedure can be used to study the inherent activity, or lack of it, of 8-lactam compounds against the target proteins in the absence of permeability barriers. Methods for assessing penetrability are, however, less than adequate (16,18).…”

Section: Resultsmentioning

confidence: 99%

Inhibition of Escherichia coli K-12 by β-Lactam Antibiotics With Poor Antibacterial Activity: Interaction of Permeability And Intrinsic Activity Against Penicillin-Binding Proteins

Curtis¹,

Brown²,

Boxall³

et al. 1979

Antimicrob Agents Chemother

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The effect of methicillin, cloxacillin, 1078/1/1, penicillin G, and cephaloridine upon the penicillin-binding proteins of a permeability mutant of Escherichia coli K-12 and its isogenic wild type have been investigated. Comparison of the 50% inhibition values for the antibiotics against the penicillin-binding proteins of the two strains with the minimal inhibitory concentrations for the same compounds indicates that methicillin, cloxacillin, 1078/1/1, and to a lesser extent penicillin G, owe their poor antibacterial activity to exclusion from the bacterial cell, whereas cephaloridine is not excluded and is equally active against both the mutant and its wild type. The results further suggest that the lesion in the permeability mutant E. coli DC2 allows free access of all the compounds tested to the inner membrane target proteins.

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Section: Resultsmentioning

confidence: 99%

Inhibition of Escherichia coli K-12 by β-Lactam Antibiotics With Poor Antibacterial Activity: Interaction of Permeability And Intrinsic Activity Against Penicillin-Binding Proteins

Curtis¹,

Brown²,

Boxall³

et al. 1979

Antimicrob Agents Chemother

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“…The bla gene encoding ␤-lactamase was carried by the plasmid pLY11, a derivative of pBR322. The ability of ␤-lactamase to hydrolyze the C-N bond in the ␤-lactam ring of penicillin G was measured in a kinetic assay (47) performed at room temperature. Aliquots (0.2 ml each) of the cell fractions were added to 1 ml of reaction mix (0.1 M phosphate buffer [pH 7.3] plus 0.5 mg of penicillin G per ml) in a quartz cuvette.…”

Section: Methodsmentioning

confidence: 99%

Topology analysis of the colicin V export protein CvaA in Escherichia coli

1995

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The antibacterial protein toxin colicin V is secreted from Escherichia coli cells by a dedicated export system that is a member of the multicomponent ATP-binding cassette (ABC) transporter family. At least three proteins, CvaA, CvaB, and TolC, are required for secretion via this signal sequence-independent pathway. In this study, the subcellular location and transmembrane organization of membrane fusion protein CvaA were investigated. First, a series of CvaA-alkaline phosphatase (AP) protein fusions was constructed. Inner and outer membrane fractionations of cells bearing these fusions indicated that CvaA is inner membrane associated. To localize the fusion junctions, the relative activities of the fusion proteins, i.e., the amounts of phosphatase activity normalized to the rate of synthesis of each protein, as well as the stability of each fusion, were determined. These results indicated that all of the fusion junctions occur on the same side of the inner membrane. In addition, the relative activities were compared with that of native AP, and the protease accessibility of the AP moieties in spheroplasts and whole cells was analyzed. The results of these experiments suggested that the fusion junctions occur within periplasmic regions of CvaA. We conclude that CvaA is an inner membrane protein with a single transmembrane domain near its N terminus; the large C-terminal region extends into the periplasm. This study demonstrates the application of AP fusion analysis to elucidate the topology of a membrane-associated protein having only a single transmembrane domain.

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“…Cells were lysed by treatment for 30 min at 0°C with lysozyme (2 mg/ml) in 20 mM Tris hydrochloride-10 mM EDTA (pH 7.3), followed by three freezethaw cycles. After centrifugation for 30 min at 4°C, Ilactamase activity in the lysate supernatant was determined from the rate of benzylpenicillin hydrolysis, which was monitored at 240 nm (46,47 (17), and the concentration of OmpA protein relative to OmpC was quantified by densitometry.…”

Section: Methodsmentioning

confidence: 99%

The ompA 5' untranslated RNA segment functions in Escherichia coli as a growth-rate-regulated mRNA stabilizer whose activity is unrelated to translational efficiency

1990

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The 5' untranslated region (UTR) of the long-lived Escherichia coli ompA message can function in vivo as an mRNA stabilizer. Substitution of this ompA mRNA segment for the corresponding segment of the labile bla gene transcripts prolongs their lifetime by a factor of 6. We show here that the function of this ompA mRNA stabilizer requires the presence of a 115-nucleotide ompA RNA segment that lies upstream of the ribosomebinding site. Although deletion of this segment reduced the half-life of the ompA transcript by a factor of 5, its absence had almost no effect on the translational efficiency of ompA mRNA. Like the ompA ranscript, but unlike bla mRNA, hybrid ompA-bla messages containing the complete ompA 5' UTR were significantly less stable under conditions of slow bacterial growth. We conclude that the stabilizing activity of the ompA 5' UTR is growth rate regulated and that the mechanism of mRNA stabilization by this RNA segment is not related to the spacing between translating ribosomes.

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[5] β-Lactamase assays

Cited by 104 publications

References 28 publications

Inhibition of Escherichia coli K-12 by β-Lactam Antibiotics With Poor Antibacterial Activity: Interaction of Permeability And Intrinsic Activity Against Penicillin-Binding Proteins

Inhibition of Escherichia coli K-12 by β-Lactam Antibiotics With Poor Antibacterial Activity: Interaction of Permeability And Intrinsic Activity Against Penicillin-Binding Proteins

Topology analysis of the colicin V export protein CvaA in Escherichia coli

The ompA 5' untranslated RNA segment functions in Escherichia coli as a growth-rate-regulated mRNA stabilizer whose activity is unrelated to translational efficiency

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