A simple assay for screening microorganisms for chalkophore production.

Recently, methanotrophs were found to exude a chalkophore, that is, a metal ligand with great affinity and specificity to copper. A rapid screening method for chalkophore expression was developed by adopting the chrome azurol S (CAS) assay originally used for detecting siderophore production in diverse groups of bacteria and fungi. In this assay, iron(III) chloride was replaced with copper(II) chloride. Both liquid and agar plate versions of the Cu-CAS assay can be used to examine the activity of either isolated methanobactin or to screen organisms for production of a chalkophore. Although here we describe the use of this assay to screen methanotrophs for chalkophore production, it can be modified as necessary to screen other organisms for chalkophore production as well. Many siderophores can also bind copper in the presence of CAS. Therefore, this assay should be done in conjunction with the original iron-CAS assay to determine if any positive Cu-CAS assay results are due to nonspecific binding of copper by a siderophore. This inexpensive assay may also aid in analyses of the genetics of chalkophore synthesis.