, 2002). Macomber et al. (2007) demonstrated that intracellular Cu failed to catalyse the formation of oxidative DNA damage. Indeed, excessive intracellular Cu suppressed iron-mediated oxidative killing (Macomber et al., 2007). More recently, experimental data suggest that the iron–sulphur clusters of dehydratase enzymes are the primary intracellular targets of Cu toxicity in E. coli (Macomber Fluorouracil & Imlay, 2009). The mechanisms for Cu toxicity in Xanthomonas spp. are not yet fully elucidated, even though Cu-based biocides containing copper hydroxide (Cu(OH)2), copper sulphate (CuSO4), and copper oxychloride are widely used in agricultural settings to limit the spread
of phytopathogenic fungi and bacteria (Hopkins, 2004). Cu-based bactericides are effective
control measures for plant diseases caused by X. campestris (McGuire, 1988). Here, we show experimentally that the presence of Cu synergistically increased the killing effects of H2O2 and organic hydroperoxide. Xanthomonas campestris pv. campestris (Xcc) was grown aerobically in Silva-Buddenhagen (SB) medium (0.5% sucrose, 0.5% yeast extract, 0.5% peptone, and 0.1% glutamic acid, pH 7.0) (Chauvatcharin et al., 2005) at 28 °C. Overnight cultures were inoculated into a fresh SB medium to yield an OD600 nm of 0.1. Exponential-phase cells (OD600 nm of 0.5 after 4 h) were used in all the experiments. General molecular techniques for bacterial genomic and JQ1 chemical structure plasmid DNA preparations, PCR, restriction endonuclease digestion, DNA ligation, transformation of E. coli, gel electrophoresis, and Southern blotting analysis were performed using standard protocols (Sambrook & Russell, 2001). The transformation of Xcc was performed using electroporation. Competent cells were prepared from an exponential-phase culture in SB medium. Cells were harvested, washed
once with 10% (v/v) glycerol, and resuspended Thiamet G in this same solution. Electroporation was conducted in a 0.2-cm electrode gap cuvette with a Gene Pulser electroporator (Bio-Rad) using the following settings: 2.5 kV, 200 Ω, and 25 μF. DNA sequencing was performed using an automated sequencer (ABI 310, Applied Biosystems). Oxidant killing experiments were performed as described previously (Banjerdkij et al., 2005). Bacterial cultures were grown to the exponential phase before aliquots of cells were removed and treated for 30 min with lethal concentrations of H2O2 (50 mM) or t-butyl hydroperoxide (tBOOH) (50 mM) that would reduce bacterial survival by 10- to 100-fold. Treatments with oxidant plus Cu included the addition of CuSO4, at a final concentration of 100 μM, to the killing mixture. In antioxidant protection tests, ROS scavengers, i.e., 0.4 M dimethyl sulphoxide (DMSO), 1.0 M glycerol, or 1 mM α-tocopherol, were added to bacterial cultures 10 min before the addition of oxidants (Mongkolsuk et al., 1998; Vattanaviboon & Mongkolsuk, 1998).