Journal
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
Volume 1790, Issue 10, Pages 1307-1313Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbagen.2009.07.006
Keywords
Microcin; Reactive oxygen species; 2,4 DNP; Peptide uptake; E. coli
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Funding
- Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET, Argentina)
- Agencia de Promocien Cientificay Tecnolegica (FONCYT) [PICTO 843/04, PAE 22642]
- Secretaria de Ciencia y Tecnica de la Universiclad Nacional de Tucuman [Secretaria de Ciencia y Tecnica de la Universiclad Nacional de Tucuman (CILINT, Grant D313]
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Background: Microcin J25 targets the RNA polymerase as well as bacterial membranes. Because there is scarce information on the relationship between the uptake and the activity, a fluorescent microcin J25-derivative was used to further characterize its mechanism of action. Methods: MccJ25 I13K was labeled with FITC and its uptake by sensitive cells was assessed by fluorescence measurements from supernatants of MccJ25-Escherichia coli suspensions. The interaction of the peptide with bacterial membranes was investigated by fluorescence resonance energy transfer. Oxygen consumption was measured with Clark-type electrode. RNA synthesis was evaluated in vivo by incorporation of [H-3]uridine. ROS production was assayed by measuring the fluorescence emission of the ROS-sensitive probe 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate. Results: The protonophore 2,4-dinitrophenol decreased 80% of the MccJ25 uptake and prevented inhibition of transcriptional activity, the antibiotic intracellular target. On the other hand, peptide binding to bacterial membranes was not affected and antibacterial activity remained nearly unchanged. Proton gradient dissipation by protonophore accelerated cell oxygen consumption rates and enhanced MccJ25-related reactive oxygen species overproduction. General significance: The deleterious reactive oxygen species would be produced as a consequence of the minor fraction of MccJ25 that interacts with the bacterial plasma membrane from the periplasmic side. These results show the first evidence of the mechanism underlying ROS production in sensitive bacteria. (C) 2009 Elsevier B.V. All rights reserved.
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