The Surface-Associated and Secreted MopE Protein of
            <i>Methylococcus capsulatus</i>
            (Bath) Responds to Changes in the Concentration of Copper in the Growth Medium

bMethanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. When Methylosinus trichosporium OB3b was grown in the presence of 1 M CuCl 2 , expression of mmoX, encoding a subunit of the hydroxylase component of sMMO, was very low. mmoX expression increased, however, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated with M. trichosporium OB3b. If M. trichosporium OB3b was grown in the absence of CuCl 2 , the mmoX expression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb had no effect on expression of mbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl 2 . mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl 2 . These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin "piracy" may be commonplace. Methanotrophs are distinguished from other microorganisms by their ability to utilize methane as a sole carbon and energy source yet are phylogenetically and physiologically diverse. Microbial methane oxidation can be coupled to a variety of terminal electron acceptors, including oxygen, sulfate, nitrate, and nitrite (1-4). The aerobic methanotrophs are typically mesophilic and group phylogenetically within the Gammaproteobacteria and Alphaproteobacteria (1). Thermo-and meso-acidophilic aerobic methanotrophs, however, that grow at pH Ͻ3 and at optimal temperatures ranging from 35°C to greater than 50°C have also been discovered in the phylum Verrucomicrobia (5-9). Further, novel oxygenic methanotrophs that couple methane oxidation to nitrite reduction have been reported, e.g., "Candidatus Methylomirabilis oxyfera" that generates oxygen from a unique denitrification pathway, which is then used for methane oxidation (2). Aerobic methanotrophs are found in many environments, e.g., freshwater and marine sediments, bogs, forest, and agricultural soils, among other locations (1, 2, 5-11).These microorganisms have been extensively studied for many different reasons, including the fact that they play a key role in the global carbon cycle. All aerobic methanotrophs employ the enzyme methane monooxygenase (MMO) to co...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Methanobactin from Methylocystis sp. Strain SB2 Affects Gene Expression and Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

Haque

Kalidass

Vorobev

et al. 2015

“…That is, it has been shown that in M. album BG8, there exists an outer membrane protein, CorA, that is copper repressible and may serve to bind copper (44). Further, it has been found that M. capsulatus Bath synthesizes a similar outer membrane protein, MopE, as well as a secreted truncated form, MopE*, both of which bind Cu(II) (45)(46)(47). A gene encoding a protein similar to CorA and MopE, mbnP, is adjacent to the methanobactin gene cluster in M. trichosporium OB3b (36), and it may be that this serves as an alternative copper uptake mechanism in M. trichosporium OB3b.…”

Section: Discussionmentioning

confidence: 99%

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

Haque

Baral

et al. 2016

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.

“…The first step in methane metabolism, the oxidation of methane to methanol, is catalyzed by methane monooxygenase (MMO) metalloenzymes by either a copper-dependent membranebound particulate MMO (pMMO) (9) or an iron-dependent, soluble MMO (sMMO) (18). Therefore, metals are necessary for the growth of methanotrophic bacteria (12,13). Despite the importance of metals in methanotroph physiology, uptake of metal ions by methanotrophic bacteria is not well understood.…”

mentioning

confidence: 99%

Secretion of Flavins by Three Species of Methanotrophic Bacteria

Balasubramanian

Levinson

Rosenzweig

2010

We detected flavins in the growth medium of the methanotrophic bacterium Methylocystis species strain M. Flavin secretion correlates with growth stage and increases under iron starvation conditions. Two other methanotrophs, Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), secrete flavins, suggesting that flavin secretion may be common to many methanotrophic bacteria.Methanotrophic bacteria utilize methane as their sole carbon source and play a key role in the global carbon cycle. The first step in methane metabolism, the oxidation of methane to methanol, is catalyzed by methane monooxygenase (MMO) metalloenzymes by either a copper-dependent membranebound particulate MMO (pMMO) (9) or an iron-dependent, soluble MMO (sMMO) (18). Therefore, metals are necessary for the growth of methanotrophic bacteria (12,13). Despite the importance of metals in methanotroph physiology, uptake of metal ions by methanotrophic bacteria is not well understood. To study metal acquisition, we purified and characterized components of the spent growth medium from Methylocystis species strain M. Unexpectedly, we detected extracellular flavins (riboflavin and flavin mononucleotide [FMN]).To perform these experiments, the type II methanotroph Methylocystis sp. strain M was grown using a procedure similar to that described for Methylosinus trichosporium OB3b (7, 8, 10) (see the supplemental material). The spent growth medium from Methylocystis sp. strain M was purified by fast protein liquid chromatography (FPLC) using a Discovery DSC-18 reverse-phase column. The eluted fractions exhibit UV-visible spectral features that suggest the presence of flavins (Fig. 1A). When the fractions were purified further using a Supelco C 18 column, the purified flavins exhibited absorption features that were identical to those of commercial riboflavin (see Fig. S1 in the supplemental material). Upon excitation at 441 nm, the fluorescence emission spectrum of the purified fractions exhibited a broad peak between 450 and 700 nm, with a maximum at 523 nm (Fig. 1B). This feature is characteristic of the isoalloxazine ring in flavins (11,21).To characterize the amount of extracellular flavins secreted throughout growth, the cells were grown under three metal availability conditions: default growth ( Fig. S2 of the supplemental material. The growth rates, from an average of biological triplicates, determined for default, iron-starved, and copper-starved growth conditions between 12 and 28 h of growth are 0.043 Ϯ 0.003 h Ϫ1 , 0.014 Ϯ 0.003 h Ϫ1 , and 0.052 Ϯ 0.002 h Ϫ1 , respectively. These values imply that the availability of iron affects the growth of Methylocystis sp. strain M.We then characterized the amount of extracellular flavins in the spent medium isolated from three different stages of growth. The samples used for quantitation were normalized based on optical density measured at 600 nm. The dry weight and cell length, normalized to the optical density, remained unaffected by growth under different metal concentrations (see Fig. S4). ...