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

Abstract

Methanobactin, 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.

FIG 1

Methanobactin gene cluster in Methylosinus trichosporium OB3b. ECF, extracytoplasmic function; Mb, methanobactin.

FIG 2

Verification of knockout of mbnT in M. trichosporium by PCR. M, molecular weight markers; lane 1, PCR of mbnT from the M. trichosporium OB3b mbnT::Gm^r mutant; lane 2, PCR of mbnT from wild-type M. trichosporium OB3b; lane 3, PCR of pK18mobsacB backbone in M. trichosporium OB3b mbnT::Gm^r; lane 4, PCR of pK18mobsacB backbone in pWG011.

FIG 3

Characterization of wild-type M. trichosporium OB3b (black bars) and the mbnT::Gm^r mutant (white bars) grown in the presence of various amounts of copper. (A) Copper associated with biomass; (B) RT-qPCR of pmoA; (C) RT-qPCR of mmoX; (D) RT-qPCR of mbnA. Error bars indicate standard deviations from at least duplicate biological replicates. Indicated P values are from one-way analysis of variance (ANOVA).

FIG 4

Immuno-blotting assays for location of methanobactin in wild-type M. trichosporium OB3b and in the mbnT::Gm^r mutant as a function of the concentration of copper in the growth medium (0.2, 5, 10, or 20 μM copper). Fifty nanomoles lysozyme (lys) and 50 nmol methanobactin (mb) were used as negative and positive controls, respectively.

FIG 5

Characterization of wild-type M. trichosporium OB3b (black bars) and the mbnT::Gm^r mutant (white bars) grown in the presence of 1 μM copper and various amounts of methanobactin (MB). (A) Copper associated with biomass; (B) RT-qPCR of pmoA; (C) RT-qPCR of mmoX; (D) RT-qPCR of mbnA. Error bars indicate standard deviations from at least duplicate biological replicates. Indicated P values are from one-way analysis of variance (ANOVA).