Methanotrophic bacteria convert methane to methanol using methane monooxygenase (MMO) enzymes. In many strains, either an iron-containing soluble (sMMO) or a copper-containing particulate (pMMO) enzyme can be produced depending on copper availability; the mechanism of this copper switch has not been elucidated. A key player in methanotroph copper homeostasis is methanobactin (Mbn), a ribosomally produced, post-translationally modified natural product with a high affinity for copper. The Mbn precursor peptide is encoded within an operon that contains a range of putative transporters, regulators, and biosynthetic proteins, but the involvement of these genes in Mbn-related processes remains unclear. Extensive time-dependent qRT-PCR studies of Methylosinus trichosporium OB3b and the constitutive sMMO-producing mutant M. trichosporium OB3b PP358 show that the Mbn operon is indeed copper-regulated, providing experimental support for its bioinformatics-based identification. Moreover, the Mbn operon is co-regulated with the sMMO operon and reciprocally regulated with the pMMO operon. Within the Mbn and sMMO operons, a subset of regulatory genes exhibits a distinct and shared pattern of expression, consistent with their proposed functions as internal regulators. In addition, genome sequencing of the M. trichosporium OB3b PP358 mutant provides new evidence for the involvement of genes adjacent to the pMMO operon in methanotroph copper homeostasis.
