Production, isolation, purification, and functional characterization of methanobactins

Aerobic methane-oxidizing bacteria (methanotrophs) have a high conditional need for copper because almost all known species express a copper-containing particulate methane monooxygenase for catalyzing the conversion of methane to methanol. This demands a copper homeostatic system that must both supply and satisfy adequate copper for elevated needs while also shielding the cells from copper toxicity. After considerable effort, it was discovered that some methanotrophs produce small peptidic molecules, called methanobactins, which bind copper, mediate copper transport into the cell, and reduce copper toxicity. Unfortunately, isolating, purifying, and proving the functionality of these molecules has been challenging. In fact, until very recently, only one complete structure had been reported for methanobactins. As such, there is a desperate need for more studies seeking such molecules. The purpose of this chapter is to describe methods used to isolate and purify the original methanobactin with a published complete structure, which is made by Methylosinus trichosporium OB3b. Methods are also included for assessing the function of such molecules under pseudonatural conditions such as growth on mineral copper sources. Special emphasis is placed on verifying that isolated molecules are "true" methanobactins, because recent work has shown that methanotrophs produce other small molecules that also bind metals in solution.