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

ABSTRACTMethanobactin, 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 asmbnT, which encodes a TonB-dependent transporter hypothesized to be responsible for methanobactin uptake. To determine ifmbnTis truly responsible for methanobactin uptake, a knockout was constructed inMethylosinus trichosporiumOB3b using marker exchange mutagenesis. The resultingM. trichosporiummbnT::Gmrmutant 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 ofmmoXandpmoA, 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-typeM. trichosporiumOB3b, indicating that the TonB-dependent transporter encoded bymbnTis responsible for methanobactin uptake and that methanobactin is a key mechanism used by methanotrophs for copper uptake. When thembnT::Gmrmutant 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-typeM. trichosporiumOB3b, indicating that this methanotroph has multiple mechanisms for copper uptake.

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

Applied and Environmental Microbiology ◽

10.1128/aem.03981-14 ◽

2015 ◽

Vol 81 (7) ◽

pp. 2466-2473 ◽

Cited By ~ 11

Author(s):

Muhammad Farhan Ul-Haque ◽

Bhagyalakshmi Kalidass ◽

Alexey Vorobev ◽

Bipin S. Baral ◽

Alan A. DiSpirito ◽

...

Keyword(s):

Gene Expression ◽

Methane Monooxygenase ◽

Particulate Methane Monooxygenase ◽

Methylosinus Trichosporium Ob3b ◽

Methylosinus Trichosporium ◽

Monooxygenase Activity ◽

Content Type ◽

Soluble Methane Monooxygenase ◽

A Subunit ◽

Membrane Bound

ABSTRACTMethanotrophs 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 byMethylosinus trichosporiumOB3b 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. WhenMethylosinus trichosporiumOB3b was grown in the presence of 1 μM CuCl2, expression ofmmoX, encoding a subunit of the hydroxylase component of sMMO, was very low.mmoXexpression increased, however, when methanobactin fromMethylocystissp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated withM. trichosporiumOB3b. IfM. trichosporiumOB3b was grown in the absence of CuCl2, themmoXexpression 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 ofMethylosinus trichosporiumOB3b to SB2-Mb had no effect on expression ofmbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl2.mbnAexpression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl2. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin “piracy” may be commonplace.

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Cerium Regulates Expression of Alternative Methanol Dehydrogenases in Methylosinus trichosporium OB3b

Applied and Environmental Microbiology ◽

10.1128/aem.02542-15 ◽

2015 ◽

Vol 81 (21) ◽

pp. 7546-7552 ◽

Cited By ~ 42

Author(s):

Muhammad Farhan Ul Haque ◽

Bhagyalakshmi Kalidass ◽

Nathan Bandow ◽

Erick A. Turpin ◽

Alan A. DiSpirito ◽

...

Keyword(s):

Active Site ◽

Methane Monooxygenase ◽

Particulate Methane Monooxygenase ◽

Methylosinus Trichosporium Ob3b ◽

Methylosinus Trichosporium ◽

Content Type ◽

Expression Levels ◽

Soluble Methane Monooxygenase ◽

Discernible Effect ◽

Expression And Activity

ABSTRACTMethanotrophs have multiple methane monooxygenases that are well known to be regulated by copper, i.e., a “copper switch.” At low copper/biomass ratios the soluble methane monooxygenase (sMMO) is expressed while expression and activity of the particulate methane monooxygenase (pMMO) increases with increasing availability of copper. In many methanotrophs there are also multiple methanol dehydrogenases (MeDHs), one based on Mxa and another based on Xox. Mxa-MeDH is known to have calcium in its active site, while Xox-MeDHs have been shown to have rare earth elements in their active site. We show here that the expression levels of Mxa-MeDH and Xox-MeDH inMethylosinus trichosporiumOB3b significantly decreased and increased, respectively, when grown in the presence of cerium but the absence of copper compared to the absence of both metals. Expression of sMMO and pMMO was not affected. In the presence of copper, the effect of cerium on gene expression was less significant, i.e., expression of Mxa-MeDH in the presence of copper and cerium was slightly lower than in the presence of copper alone, but Xox-MeDH was again found to increase significantly. As expected, the addition of copper caused sMMO and pMMO expression levels to significantly decrease and increase, respectively, but the simultaneous addition of cerium had no discernible effect on MMO expression. As a result, it appears Mxa-MeDH can be uncoupled from methane oxidation by sMMO inM. trichosporiumOB3b but not from pMMO.

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Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

Applied and Environmental Microbiology ◽

10.1128/aem.03151-14 ◽

2014 ◽

Vol 81 (3) ◽

pp. 1024-1031 ◽

Cited By ~ 16

Author(s):

Bhagyalakshmi Kalidass ◽

Muhammad Farhan Ul-Haque ◽

Bipin S. Baral ◽

Alan A. DiSpirito ◽

Jeremy D. Semrau

Keyword(s):

Methane Monooxygenase ◽

High Specificity ◽

Copper Uptake ◽

Content Type ◽

Soluble Methane Monooxygenase ◽

Sds Page ◽

Genes Encoding ◽

Key Factor ◽

Membrane Bound

ABSTRACTIt is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that inMethylosinus trichosporiumOB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced byM. trichosporiumOB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and activein situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin.

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Marker Exchange Mutagenesis ofmxaF, Encoding the Large Subunit of the Mxa Methanol Dehydrogenase, in Methylosinus trichosporium OB3b

Applied and Environmental Microbiology ◽

10.1128/aem.03615-15 ◽

2015 ◽

Vol 82 (5) ◽

pp. 1549-1555 ◽

Cited By ~ 17

Author(s):

Muhammad Farhan Ul Haque ◽

Wenyu Gu ◽

Alan A. DiSpirito ◽

Jeremy D. Semrau

Keyword(s):

Large Subunit ◽

Methane Monooxygenase ◽

Methanol Dehydrogenase ◽

Methylosinus Trichosporium Ob3b ◽

Initial Oxidation ◽

Methylosinus Trichosporium ◽

Content Type ◽

Marker Exchange ◽

Oxidation Of Methane ◽

Soluble Methane Monooxygenase

ABSTRACTMethanotrophs have remarkable redundancy in multiple steps of the central pathway of methane oxidation to carbon dioxide. For example, it has been known for over 30 years that two forms of methane monooxygenase, responsible for oxidizing methane to methanol, exist in methanotrophs, i.e., soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), and that expression of these two forms is controlled by the availability of copper. Specifically, sMMO expression occurs in the absence of copper, while pMMO expression increases with increasing copper concentrations. More recently, it was discovered that multiple forms of methanol dehydrogenase (MeDH), Mxa MeDH and Xox MeDH, also exist in methanotrophs and that the expression of these alternative forms is regulated by the availability of cerium. That is, expression of Xox MeDH increases in the presence of cerium, while Mxa MeDH expression decreases in the presence of cerium. As it had been earlier concluded that pMMO and Mxa MeDH form a supercomplex in which electrons from Mxa MeDH are back donated to pMMO to drive the initial oxidation of methane, we speculated that Mxa MeDH could be rendered inactive through marker-exchange mutagenesis but growth on methane could still be possible if cerium was added to increase the expression of Xox MeDH under sMMO-expressing conditions. Here we report thatmxaF, encoding the large subunit of Mxa MeDH, could indeed be knocked out inMethylosinus trichosporiumOB3b, yet growth on methane was still possible, so long as cerium was added. Interestingly, growth of this mutant occurred in both the presence and the absence of copper, suggesting that Xox MeDH can replace Mxa MeDH regardless of the form of MMO expressed.

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Isolation of Copper Biochelates fromMethylosinus trichosporium OB3b and Soluble Methane Monooxygenase Mutants

Applied and Environmental Microbiology ◽

10.1128/aem.64.3.1115-1122.1998 ◽

1998 ◽

Vol 64 (3) ◽

pp. 1115-1122 ◽

Cited By ~ 43

Author(s):

Carlos M. Téllez ◽

Kristen P. Gaus ◽

David W. Graham ◽

Robert G. Arnold ◽

Roberto Z. Guzman

Keyword(s):

High Performance ◽

Methane Monooxygenase ◽

Size Exclusion ◽

Copper Binding ◽

Copper Uptake ◽

Wild Type ◽

Methylosinus Trichosporium ◽

Soluble Methane Monooxygenase ◽

Steady Level ◽

Presence And Absence

ABSTRACT Methylosinus trichosporium OB3b produces an extracellular copper-binding ligand (CBL) with high affinity for copper. Wild-type cells and mutants that express soluble methane monooxygenase (sMMO) in the presence and absence of copper (sMMOc) were used to obtain cell exudates that were separated and analyzed by size exclusion high-performance liquid chromatography. A single chromatographic peak, when present, contained most of the aqueous-phase Cu(II) present in the culture medium. In mutant cultures that were unable to acquire copper, extracellular CBL accumulated to high levels both in the presence and in the absence of copper. Conversely, in wild-type cultures containing 5 μM Cu(II), extracellular CBL was maintained at a low, steady level during exponential growth, after which the external ligand was rapidly consumed. When Cu(II) was omitted from the growth medium, the wild-type organism produced the CBL at a rate that was proportional to cell density. After copper was added to this previously Cu-deprived culture, the CBL and copper concentrations in the medium decreased at approximately the same rate. Apparently, the extracellular CBL was produced throughout the period of cell growth, in the presence and absence of Cu(II), by both the mutant and wild-type cultures and was reinternalized or otherwise utilized by the wild-type cultures when it was bound to copper. CBL produced by the mutant strain facilitated copper uptake by wild-type cells, indicating that the extracellular CBLs produced by the mutant and wild-type organisms are functionally indistinguishable. CBL from the wild-type strain did not promote copper uptake by the mutant. The molecular weight of the CBL was estimated to be 500, and its association constant with copper was 1.4 × 1016 M−1. CBL exhibited a preference for copper, even in the presence of 20-fold higher concentrations of nickel. External complexation may play a role in normal copper acquisition by M. trichosporium OB3b. The sMMOcphenotype is probably related to the mutant’s inability to take up CBL-complexed copper, not to a defective CBL structure.

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rpoN, mmoR and mmoG, genes involved in regulating the expression of soluble methane monooxygenase in Methylosinus trichosporium OB3b

Microbiology ◽

10.1099/mic.0.26060-0 ◽

2003 ◽

Vol 149 (7) ◽

pp. 1771-1784 ◽

Cited By ~ 48

Author(s):

Graham P. Stafford ◽

Julie Scanlan ◽

Ian R. McDonald ◽

J. Colin Murrell

Keyword(s):

Methane Monooxygenase ◽

Regulatory Gene ◽

Soluble Form ◽

Gene Encoding ◽

Independent Manner ◽

Methylosinus Trichosporium ◽

Marker Exchange ◽

Soluble Methane Monooxygenase ◽

Genes Encoding

The methanotrophic bacterium Methylosinus trichosporium OB3b converts methane to methanol using two distinct forms of methane monooxygenase (MMO) enzyme: a cytoplasmic soluble form (sMMO) and a membrane-bound form (pMMO). The transcription of these two operons is known to proceed in a reciprocal fashion with sMMO expressed at low copper-to-biomass ratios and pMMO at high copper-to-biomass ratios. Transcription of the smmo operon is initiated from a σ N promoter 5′ of mmoX. In this study the genes encoding σ N (rpoN) and a typical σ N-dependent transcriptional activator (mmoR) were cloned and sequenced. mmoR, a regulatory gene, and mmoG, a gene encoding a GroEL homologue, lie 5′ of the structural genes for the sMMO enzyme. Subsequent mutation of rpoN and mmoR by marker-exchange mutagenesis resulted in strains Gm1 and JS1, which were unable to express functional sMMO or initiate transcription of mmoX. An rpoN mutant was also unable to fix nitrogen or use nitrate as sole nitrogen source, indicating that σ N plays a role in both nitrogen and carbon metabolism in Ms. trichosporium OB3b. The data also indicate that mmoG is transcribed in a σ N- and MmoR-independent manner. Marker-exchange mutagenesis of mmoG revealed that MmoG is necessary for smmo gene transcription and activity and may be an MmoR-specific chaperone required for functional assembly of transcriptionally competent MmoR in vivo. The data presented allow the proposal of a more complete model for copper-mediated regulation of smmo gene expression.

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