A TonB-Dependent Transporter Is Responsible for Methanobactin Uptake by Methylosinus trichosporium OB3b

Abstract

dMethanobactin, a small modified polypeptide synthesized by methanotrophs for copper uptake, has been found to be chromosomally encoded. The gene encoding the polypeptide precursor of methanobactin, mbnA, is part of a gene cluster that also includes several genes encoding proteins of unknown function (but speculated to be involved in methanobactin formation) as well as mbnT, which encodes a TonB-dependent transporter hypothesized to be responsible for methanobactin uptake. To determine if mbnT is truly responsible for methanobactin uptake, a knockout was constructed in Methylosinus trichosporium OB3b using marker exchange mutagenesis. The resulting M. trichosporium mbnT::Gm r mutant was found to be able to produce methanobactin but was unable to internalize it. Further, if this mutant was grown in the presence of copper and exogenous methanobactin, copper uptake was significantly reduced. Expression of mmoX and pmoA, encoding polypeptides of the soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), respectively, also changed significantly when methanobactin was added, which indicates that the mutant was unable to collect copper under these conditions. Copper uptake and gene expression, however, were not affected in wild-type M. trichosporium OB3b, indicating that the TonB-dependent transporter encoded by mbnT is responsible for methanobactin uptake and that methanobactin is a key mechanism used by methanotrophs for copper uptake. When the mbnT::Gm r mutant was grown under a range of copper concentrations in the absence of methanobactin, however, the phenotype of the mutant was indistinguishable from that of wild-type M. trichosporium OB3b, indicating that this methanotroph has multiple mechanisms for copper uptake.

Methanobactin transport machinery

“…2A), with the ΔmbnT strain exhibiting much lower levels of 65 Cu. This result supports the experiments reported recently by Gu and coworkers (29).…”

“…Recent quantitative RT-PCR (qRT-PCR) studies of Ms. trichosporium OB3b (32) revealed that these genes, including mbnE, are coregulated with mbnA and are all down-regulated in response to Cu addition. Taken together with the cessation of Mbn production in an mbnA deletion mutant (33) and the reported mbnT knockout strain (29), there is strong genetic evidence for the involvement of mbnT and mbnE in Mbn transport. However, the interaction of these proteins with Mbn has not been investigated biochemically.…”

“…One of the genes identified in most mbn operons is mbnT, which encodes a TBDT. Disruption of this gene abolishes Mbn uptake, indicating that it is the Mbn importer (29). Also present in most of these…”

Bacterial copper storage proteins

“…However, the observation that Csp3 is present alone in 22 methanotrophs (Csp1 is rarely present on its own) could indicate the function of this protein is independent of Csp1 and Mbn (see section about the functions of Csps). Much attention has been paid to the role of Mbn in Cu acquisition and utilization by methanotrophs (17,(50)(51)(52)(54)(55)(56)(57)(58)(59)(60). However, the currently identified Mbn operon is only found in just over 10% of sequenced methanotroph genomes, suggesting other systems are important for these processes.…”

“…1, A and B) that is part of a highly specialized Cu-uptake system, secreted to sequester Cu under limiting conditions (17,50). The mbnA gene, which codes for leader (cleaved) and core (modified) peptides has been identified in an operon along with proteins either shown or suggested to be involved in modification reactions, apo-Mbn export, and re-incorporation of Cu(I)-Mbn (17,51,(55)(56)(57)(58)(59). Related Mbn operons are present in some non-methanotrophic bacteria (17,51,59).…”