Bacillus amyloliquefaciens FZB42 is a Gram-positive plant growth-promoting bacterium with an impressive capacity to synthesize nonribosomal secondary metabolites with antimicrobial activity. Here we report on a novel circular bacteriocin which is ribosomally synthesized by FZB42. The compound displayed high antibacterial activity against closely related Gram-positive bacteria. Transposon mutagenesis and subsequent site-specific mutagenesis combined with matrix-assisted laser desorption ionization-time of flight mass spectroscopy revealed that a cluster of six genes covering 4,490 bp was responsible for the production, modification, and export of and immunity to an antibacterial compound, here designated amylocyclicin, with a molecular mass of 6,381 Da. Peptide sequencing of the fragments obtained after tryptic digestion of the purified peptide revealed posttranslational cleavage of an N-terminal extension and head-to-tail circularization of the novel bacteriocin. Homology to other putative circular bacteriocins in related bacteria let us assume that this type of peptide is widespread among the Bacillus/Paenibacillus taxon.
The first application of microchip electrophoresis with laser-induced fluorescence (MCE-LIF) detection to simultaneously determine glutathione (GSH) and hydrogen peroxide (H(2)O(2)) in mitochondria was described. Organoselenium probe Rh-Se-2 and bis(p-methylbenzenesulfonate)dichlorofluorescein (FS) synthesized in our laboratory were utilized as fluorescent probes for GSH and H(2)O(2), respectively. Rh-Se-2, which is nonfluorescent, reacts with GSH to produce rhodamine 110 (Rh110) with high quantum yield. Similarly, nonfluorescent FS reacts with H(2)O(2) and produces dichlorofluorescein (DCF) accompanied by drastic fluorescence enhancement. Both probes exhibit good sensitivity toward their respective target molecule determination. Fast, simple, and sensitive determination of GSH and H(2)O(2) was realized within 37 s using a running buffer of 50 mM mannitol, 40 mM HEPES (pH 7.4), and an electric field of 360 V/cm for separation. The linear ranges of the method were 3.3 x 10(-9)-1.0 x 10(-7) M/2.9 x 10(-7)-1.0 x 10(-4) M and 2.7 x 10(-9)-4.0 x 10(-7) M with detection limits (signal-to-noise ratio = 3) of 1.3 nM (0.16 amol) and 1.0 nM (0.12 amol) for GSH and H(2)O(2), respectively. The relative standard deviations (RSDs) of migration time and peak area were less than 1.0% and 4.0%, respectively. The MCE-LIF assay was utilized to investigate the levels of GSH and H(2)O(2) in mitochondria isolated from HepG2 cells and were found to be 2.01 +/- 0.21 mM and 5.36 +/- 0.45 microM, respectively. The method was further extended to observe situations of the two species in mitochondria of HepG2 cells experiencing cell apoptosis that were induced by doxorubicin and photodynamic therapy (PDT).
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