Dual Pathways for Copper Uptake by Methanotrophic Bacteria

Abstract

Methanobactin (Mb), a 1217-Da copper chelator produced by the methanotroph Methylosinus trichosporium OB3b, is hypothesized to mediate copper acquisition from the environment, particularly from insoluble copper mineral sources. Although indirect evidence suggests that Mb provides copper for the regulation and activity of methane monooxygenase enzymes, experimental data for direct uptake of copper loaded Mb (Cu-Mb) are lacking. Uptake of intact Cu-Mb by M. trichosporium OB3b was demonstrated by isotopic and fluorescent labeling experiments. Confocal microscopy data indicate that Cu-Mb is localized in the cytoplasm. Both Cu-Mb and unchelated Cu are taken up by M. trichosporium OB3b, but by different mechanisms. Uptake of unchelated Cu is inhibited by spermine, suggesting a porin-dependent passive transport process. By contrast, uptake of Cu-Mb is inhibited by the uncoupling agents carbonyl cyanide m-chlorophenylhydrazone and methylamine, but not by spermine, consistent with an active transport process. Cu-Mb from M. trichosporium OB3b can also be internalized by other strains of methanotroph, but not by Escherichia coli, suggesting that Cu-Mb uptake is specific to methanotrophic bacteria. These findings are consistent with a key role for Cu-Mb in copper acquisition by methanotrophs and have important implications for further investigation of the copper uptake machinery. Methanobactin (Mb), a 1217-Da copper chelator produced by the methanotroph Methylosinus trichosporium OB3b, is hypothesized to mediate copper acquisition from the environment, particularly from insoluble copper mineral sources. Although indirect evidence suggests that Mb provides copper for the regulation and activity of methane monooxygenase enzymes, experimental data for direct uptake of copper loaded Mb (Cu-Mb) are lacking. Uptake of intact Cu-Mb by M. trichosporium OB3b was demonstrated by isotopic and fluorescent labeling experiments. Confocal microscopy data indicate that Cu-Mb is localized in the cytoplasm. Both Cu-Mb and unchelated Cu are taken up by M. trichosporium OB3b, but by different mechanisms. Uptake of unchelated Cu is inhibited by spermine, suggesting a porin-dependent passive transport process. By contrast, uptake of Cu-Mb is inhibited by the uncoupling agents carbonyl cyanide m-chlorophenylhydrazone and methylamine, but not by spermine, consistent with an active transport process. Cu-Mb from M. trichosporium OB3b can also be internalized by other strains of methanotroph, but not by Escherichia coli, suggesting that Cu-Mb uptake is specific to methanotrophic bacteria. These findings are consistent with a key role for Cu-Mb in copper acquisition by methanotrophs and have important implications for further investigation of the copper uptake machinery.