An assay for screening microbial cultures for chalkophore production

Abstract

Methanotrophs, bacteria that utilize methane as their sole carbon and energy source, are known to have high requirements for copper. These bacteria have recently been found to synthesize a copper-chelating agent, or chalkophore, termed methanobactin. To aid in screening methanobactin production by methanotrophs, a plate assay developed from the chrome azurol S (CAS) assay for siderophore production, was modified. In the typical CAS assay, a colour change from blue to orange in iron–CAS plates is observed as iron (III) ion weakly bound to CAS is sequestered by siderophores with higher affinities. In our modified assay, iron (III) chloride of the original CAS solution was substituted with copper (II) chloride, and removal of copper from CAS caused a colour change from blue to yellow. Assay results indicated that of the four tested methanotrophs ( Methylosinus trichosporium OB3b, Methylococcus capsulatus Bath, Methylomicrobium album BG8 and Methylocystis parvus OBBP), only M. trichosporium OB3b, M. capsulatus Bath and M. album BG8 produced chalkophores capable of competing with CAS for copper, while M. parvus OBBP did not or did not export sufficient concentrations of methanobactin for detection by this assay. It was also found using Fe–CAS plates that at least M. trichosporium OB3b and M. album BG8 produce siderophores. These results may be expanded for the detection of chalkophores in other microorganisms as well as for screening of putative mutants of chalkophore synthesis.