methanobactin and the link between copper and bacterial ...a second methanotroph, methylococcus...

Methanobactin and the Link between Copper and Bacterial MethaneOxidation

Alan A. DiSpirito,a Jeremy D. Semrau,b J. Colin Murrell,c Warren H. Gallagher,d Christopher Dennison,e Stéphane Vuilleumierf

Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa, USAa; Department of Civil and EnvironmentalEngineering, University of Michigan, Ann Arbor, Michigan, USAb; Earth and Life Systems Alliance, School of Environmental Sciences, University of East Anglia, Norwich,United Kingdomc; Department of Chemistry, University of Wisconsin—Eau Claire, Eau Claire, Wisconsin, USAd; Institute for Cell and Molecular Biosciences, Medical School,Newcastle University, Newcastle upon Tyne, United Kingdome; Department of Microbiology, Genomics and the Environment, UMR 7156 UNISTRA-CNRS, Université deStrasbourg, Strasbourg, Francef

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .387INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .388

Physiology and Biochemistry of Methanotrophs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .388The “Copper Switch” in Methanotrophs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .389Evidence Suggesting a Copper-Specific Uptake System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .390Initial Identification and Isolation of Methanobactin (a Copper-Binding Compound or “Chalkophore”) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .390Methanobactins as Chalkophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .390

STRUCTURAL PROPERTIES OF METHANOBACTINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .393Structural Diversity and Core Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .393Copper Coordination Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .394

METAL BINDING PROPERTIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .394Binding and Reduction of the Primary Metal, Copper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .394Binding of Other Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .395Metal Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .395

BIOSYNTHESIS OF METHANOBACTIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .395Identification of the Polypeptide Precursor of Methanobactin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .395Current Hypotheses on the Nature of the Methanobactin Biosynthesis Pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .397

REGULATION OF GENE EXPRESSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .397PHYSIOLOGICAL FUNCTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .399

Copper Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .399Do Methanobactins Have a Role as Signaling Molecules?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .400Membrane Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .400Detoxification of Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .400

MEDICAL, INDUSTRIAL, AND ENVIRONMENTAL IMPLICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .401Metal Mobilization or Immobilization for Remediation of Polluted Subsurface Environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .401Formation of Gold Nanoparticles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .401Possible Therapeutic Treatment of Human Ailments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .401

CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .402ACKNOWLEDGMENTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .402REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .402AUTHOR BIOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .408

SUMMARY

Methanobactins (mbs) are low-molecular-mass (�1,200 Da)copper-binding peptides, or chalkophores, produced by manymethane-oxidizing bacteria (methanotrophs). These moleculesexhibit similarities to certain iron-binding siderophores but areexpressed and secreted in response to copper limitation. Structur-ally, mbs are characterized by a pair of heterocyclic rings withassociated thioamide groups that form the copper coordinationsite. One of the rings is always an oxazolone and the second ring anoxazolone, an imidazolone, or a pyrazinedione moiety. The mbmolecule originates from a peptide precursor that undergoes aseries of posttranslational modifications, including (i) ring forma-tion, (ii) cleavage of a leader peptide sequence, and (iii) in somecases, addition of a sulfate group. Functionally, mbs represent theextracellular component of a copper acquisition system. Consis-tent with this role in copper acquisition, mbs have a high affinityfor copper ions. Following binding, mbs rapidly reduce Cu2� to

Cu1�. In addition to binding copper, mbs will bind most transi-tion metals and near-transition metals and protect the hostmethanotroph as well as other bacteria from toxic metals. Severalother physiological functions have been assigned to mbs, basedprimarily on their redox and metal-binding properties. In thisreview, we examine the current state of knowledge of this noveltype of metal-binding peptide. We also explore its potential appli-cations, how mbs may alter the bioavailability of multiple metals,

Published 16 March 2016

Citation DiSpirito AA, Semrau JD, Murrell JC, Gallagher WH, Dennison C,Vuilleumier S. 2016. Methanobactin and the link between copper and bacterialmethane oxidation. Microbiol Mol Biol Rev 80:387–409.doi:10.1128/MMBR.00058-15.

Address correspondence to Alan A. DiSpirito, [email protected].

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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and the many roles mbs may play in the physiology of metha-notrophs.

INTRODUCTION

Methanobactins (mbs) were first identified in aerobic meth-ane-oxidizing bacteria (methanotrophs). This remarkablegroup of bacteria can grow using methane as their sole source ofcarbon and energy. They are ubiquitous in environments whereoxygen and methane are available, and they play a major role inconsuming much of the methane produced in the biosphere, thusmitigating its effects in global warming (1–4). Due to their growthon an inexpensive, readily available, and, if generated via metha-nogenesis (5), renewable carbon source, methanotrophs also haveconsiderable potential for the production of bulk and fine chem-icals and for bioremediation of pollutants in the environment (2,6–8).

The first report of a bacterium growing on methane was bySöhngen, working in Beijerinck’s laboratory in Delft in The Neth-erlands, who in 1906 reported the isolation of Bacillus methanicusfrom aquatic plants and pond water (9). It was not until 50 yearslater that this microbe was reisolated and renamed Pseudomonasmethanica (10, 11). A second methanotroph, Methylococcus cap-sulatus (Texas strain), was isolated in 1966 (12). A landmark inmethanotroph biology came in 1970, when Whittenbury and col-leagues isolated, from a variety of terrestrial and freshwater envi-ronments, and described over 100 new aerobic methanotrophsgrowing on methane (13). They then devised a classificationscheme, i.e., type I versus type II, based on the ability of thesemethanotrophs to grow on methane, pathways of carbon assimi-lation, formation of resting stages (cysts and spores), morphology,the possession of complex intracytoplasmic membrane arrange-ments, and the moles percent G�C content of their DNA. Subse-quently, Bowman and colleagues isolated a similar number ofmethanotrophs from various environments and classified themaccording to the scheme of Whittenbury and colleagues and ac-cording to their 16S rRNA phylogeny (14, 15). It is remarkablethat though there was no DNA sequencing at the time, the overallclassification scheme of Whittenbury and colleagues still remainsa robust and convenient way of grouping methanotrophs today.

Accordingly, there are currently 15 genera of methanotrophswithin the family Methylococcaceae and 3 in the Methylother-maceae family of the Gammaproteobacteria class. Methylobacter,Methylocaldum, Methylococcus, Methylogaea, Methyloglobulus,Methylomagnum, Methylomarinum, Methylomicrobium, Methylo-monas, Methyloparacoccus, Methyloprofundus, Methylosoma,Methylosphaera, Methylosarcina, and Methylovulum are themethanotrophs in the Methylococcaceae family, and Methylohalo-bius, Methylomarinovum, and Methylothermus are the metha-notrophs in the Methylothermaceae family (16–21, 227). Withinthe class Alphaproteobacteria, the genera Methylosinus and Methy-locystis are found in the family Methylocystaceae and the generaMethylocella, Methyloferula, and Methylocapsa in the family Bei-jerinkiaceae. In the last 15 years, there have been increasing reportsof facultative methanotrophs within the Methylocella, Methylo-capsa, and Methylocystis genera that can use multicompounds forgrowth in addition to methane (22–26). Also known today arefilamentous methanotrophs from other genera, such as Creno-thrix and Clonothrix, and nonproteobacterial (verrucomicrobial)methanotrophs of the genus Methylacidiphilum growing at hightemperatures and low pH have also been discovered recently (27).

Finally, it was shown that “Candidatus Methylomirabilis oxyfer-ans,” a member of the NC10 phylum, generates dioxygen for theoxidation of methane despite being an obligate anaerobe (28, 29).Taken together, these data clearly illustrate the widespread natureof methanotrophic bacteria in most ecosystems of our planet.

Physiology and Biochemistry of Methanotrophs

Methanotrophs can use methane as an energy source and also toprovide carbon for all of their cellular constituents (6, 30, 31). Theinitial oxidation of methane to methanol is catalyzed by the en-zyme methane monooxygenase (MMO). There are two structur-ally and biochemically distinct forms of MMO, a membrane-as-sociated or particulate MMO (pMMO) and a cytoplasmic orsoluble MMO (sMMO), which represent evolutionarily indepen-dent solutions to the same molecular problem of methane oxida-tion (32–37). The sMMO is a three-component binuclear ironactive-center monooxygenase that belongs to a large group of bac-terial hydrocarbon oxygenases, known as soluble di-iron mo-nooxygenases (SDIMOs) (38), which are also homologous to theR2 subunit of class I ribonucleotide reductase. Two very similarsMMO systems, from Methylococcus capsulatus (Bath) (39–43)and Methylosinus trichosporium OB3b (44–47), have been studiedin detail. sMMO is encoded by a six-gene operon, mmoXYBZDC,and has three components: (i) a 250-kDa hydroxylase with an�2�2�2 structure in which the � subunits (MmoX) contain thebinuclear iron active center where substrate oxygenation occurs,(ii) a 39-kDa NAD(P)H-dependent reductase (MmoC) with fla-vin adenine dinucleotide (FAD) and Fe2S2 prosthetic groups, and(iii) a 16-kDa component (MmoB) known as protein B or cou-pling/gating protein that does not contain prosthetic groups ormetal ions (39, 48). There are X-ray crystal structures for the hy-droxylase component (49–52), nuclear magnetic resonance(NMR)-derived structures for protein B (39, 53, 54), and anNMR-derived structure for the flavin domain of the reductase(55). The complex formed by the three components has beenstudied structurally via small-angle X-ray scattering analysis andbiophysically by electron paramagnetic resonance spectroscopy,ultracentrifugation, and calorimetric analysis (56, 57). The cata-lytic cycle of sMMO has been extensively studied, and excellentprogress has been made toward understanding the mechanism ofoxygen and hydrocarbon activation at the binuclear iron center(45) (45, 58–62). Detailed reviews of the structure and catalyticmechanisms of sMMO are available (6, 37, 47, 63, 64).

The pMMO, in contrast, is a copper- and possibly iron-contain-ing, membrane-associated enzyme that is associated with unusualintracytoplasmic membranes that take the form of vesicular disksin type I methanotrophs and paired peripheral layers in type IIorganisms (65–75). Intracytoplasmic membranes are enriched inpMMO and can be physically separated from the cytoplasmicmembrane on the basis of sedimentation velocity in sucrose den-sity gradients (76). An understanding of the structure and mech-anism of pMMO has emerged more slowly than that for sMMObecause of losses of activity when the enzyme is solubilized.pMMO consists of three polypeptides of approximately 49, 27,and 22 kDa encoded by the genes pmoCAB (77). There are oftenmultiple copies of these pmo genes in methanotrophs (78, 79).Recent studies have shown that native pMMO forms a complexwith methanol dehydrogenase (MeDH), which may supply elec-trons to the pMMO (80, 81), similar to what has been found for

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the hydroxylamine oxidoreductase and ammonia monooxygen-ase redox couples (82–85).

Some methanotrophs, such as M. capsulatus (Bath) and M. tri-chosporium OB3b, can produce either form of MMO. Mostknown methanotrophs possess only pMMO, e.g., Methylomonasmethanica, Methylomicrobium album BG8, Methylocystis parvusOBBP, and the verrucomicrobial and NC10 methanotrophs. Onlya few methanotrophs within the Beijerinckiaceae family, e.g.,Methylocella silvestris and Methyloferula stellata, have sMMO butdo not possess pMMO (21, 86).

The methanol produced by MMO is oxidized to formaldehydeby a calcium or rare-earth-dependent pyrroloquinoline quinone(PQQ)-containing MeDH (87–91). Formaldehyde is an impor-tant branch point in the metabolism of methanotrophic metabo-lism and represents the point at which one-carbon (C1) interme-diates can be either oxidized to CO2 to derive energy or assimilatedinto biomass. Since formaldehyde is toxic, methanotrophs mustprotect themselves against accumulation of this metabolic inter-mediate. Multiple pathways for metabolism of formaldehyde arefound in methanotrophs (2, 26, 92–96). For example, oxidativedissimilation of formaldehyde can occur by its conjugation to tet-rahydromethanopterin (H4MPT) (97, 98), via dye-linked mem-brane-associated (93) or via NAD�-dependent (95, 96, 99) form-aldehyde dehydrogenases. Formate, resulting from the oxidationof formaldehyde by formaldehyde dehydrogenases, is further ox-idized to carbon dioxide by an NAD�-dependent formate dehy-drogenase which generates NADH, which is then available foroxidation of methane, biosynthetic reactions, and energy genera-tion within the cell (100–102). Methanotrophs also possess twopathways for fixation of formaldehyde into biomass, the serineand ribulose monophosphate (RuMP) cycles, which are active inalphaproteobacterial and gammaproteobacterial methanotrophs,respectively. The pathways of carbon fixation in methanotrophshave been reviewed extensively (see, e.g., reference 6).

The “Copper Switch” in Methanotrophs

Early attempts to characterize methane oxidation were compli-cated by different reports on the cellular location of the MMO.MMOs were described either as soluble or as membrane associ-ated, depending on the strain and, for some strains, on the report-ing laboratory. Several groups initially reported activity in the par-ticulate or membrane fraction (103, 104), whereas other groupsdetected activity in the soluble fraction (105, 106). Subsequentstudies showed that the cellular location varied with cultivationconditions. Oxygen limitation was reported to induce methaneoxidation in the soluble fraction in M. trichosporium OB3b (36,107). However, it was subsequently shown that oxygen was not theregulatory factor and that the switch between the membrane-as-sociated and soluble activities was related to biomass concentra-tion (108). The defining moment in the discovery of this “switch”was when Dalton and colleagues attempted to grow M. parvusOBBP to high cell densities in chemostat culture. They observedthat cultures of M. parvus OBBP, when supplied with methane,air, and a nitrate mineral salts (NMS) solution at relatively low celldensities, simply stopped growing. However, when additionaltrace element solution was added, the cultures immediatelystarted growing again. The “secret ingredient” in the trace elementsolution was narrowed down to copper ions (108). It was subse-quently realized that M. parvus OBBP contained only pMMO,resulting in the high requirements for copper ions, and did not

contain an sMMO which would have allowed it to grow to thesame high cell densities observed at the time with M. capsulatusBath and M. trichosporium OB3b under copper limitation. Thelatter strains, when confronted with the same conditions,switched to expression of the sMMO and carried on growing (35,109). Interestingly, copper had earlier been shown to enhancegrowth of Methanomonas margaritae, a methanotroph which doesnot contain sMMO, but these original observations were neverinvestigated further (110).

Dalton and colleagues followed up their observations in detailand established the existence of this “copper switch,” i.e., the reg-ulation of expression of the two different forms of MMO inmethanotrophs in response to the copper-to-biomass ratio of cul-tures of methanotrophs which possess both sMMO and pMMO.This allowed them to explain many of the earlier observations onmetal-dependent growth of methanotrophs. For instance, theyshowed that in M. capsulatus Bath, expression of the sMMO wasobserved only at high cell densities when copper ions in the me-dium were depleted, whereas additions of excess copper ions al-lowed this methanotroph to express active pMMO. Subsequently,Murrell and colleagues showed at the molecular level that undergrowth with low concentrations of copper ions, expression ofsMMO was initiated at a �54 promoter upstream of the sMMOgene cluster (mmoXYBZDC). Conversely, under high-coppergrowth conditions, expression of sMMO was repressed, and highlevels of expression of the genes encoding pMMO (pmoCAB) al-lowed both M. trichosporium OB3b and M. capsulatus Bath togrow using pMMO (34, 111–113).

Further research established that copper affects metha-notrophic physiology and gene expression much more broadly.For example, it was found that the intracytoplasmic membranecontent in methanotrophs increased with increasing copper in thegrowth medium (66, 109, 114). This was not totally unexpected,however: considering that the pMMO is localized in the intracy-toplasmic membranes, then greater expression and activity ofpMMO would logically require more of these membranes. Moresurprisingly, however, it was recently discovered that pMMO andthe PQQ-linked MeDH encoded by the mxa operon form a super-complex anchored in the intracytoplasmic membranes and thatelectron transfer from the PQQ-linked MeDH to pMMO in vivomay drive the oxidation of methane (80, 81). In support of thelatter finding, it was recently found that not only does expressionof pmo genes increase with increasing copper, but that of genes inthe mxa operon does so as well (91).

It has also been shown by proteomics that additional steps in theoxidation of methane to carbon dioxide are overexpressed withincreasing availability of copper, such as that of proteins involvedin lipid, cell wall, and membrane synthesis (66, 115). Conversely,the ability of methanotrophs to direct carbon from methane topoly-3-hydroxybutyrate increases with decreasing copper avail-ability (66, 116), suggesting that to some extent energy metabo-lism of methanotrophs is controlled by copper. Such a conclusionwas actually already reached earlier by Dalton and colleagues, whoshowed that the biomass yield and carbon conversion efficiency inmethanotrophs when grown on methane increased as copper in-creased, i.e., when methanotrophs switch from expressing sMMOto expressing pMMO (117). The “surfaceome,” or proteins on theouter surface of the outer membrane, is also controlled by copperavailability in some methanotrophs. The expression of severalmulti-c-type cytochromes and proteins believed to be involved in

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copper uptake also varies, and in most cases decreases, as copperavailability increases (118–123).

Additional insight into how methanotrophs handle copper hasbeen provided by the recent discovery of the Csps, a new family ofcopper storage proteins, in M. trichosporium OB3b (124). Thismethanotroph possesses three Csps: Csp1 and Csp2, which havepredicted twin arginine translocase-targeting signal peptides andare therefore thought to be exported after folding, as well as thecytosolic Csp3. Csp1 forms a tetramer of four helix bundles thatcan bind up to 52 Cu1� ions via Cys residues that point into thecore of the bundle. Switchover to sMMO is accelerated in the�csp1 csp2 mutant compared to the wild type, suggesting thatthese proteins play a role in storing copper for the pMMO andprovide an internal copper source when copper becomes limiting.Under such conditions, mb is produced, which can readily removeall Cu1� from Csp1 and therefore may play a role in helping toutilize Csp1-bound copper.

Evidence Suggesting a Copper-Specific Uptake System

The first evidence for a copper-specific uptake system and for theproduction of an extracellular copper-binding ligand came duringthe phenotypic characterization of constitutive sMMO mutants(sMMOC) of M. trichosporium OB3b (125, 126). Phelps et al. (126)isolated five sMMOC mutants by culturing M. trichosporiumOB3b in the presence of dichloromethane, acting as a mutagenfollowing its cometabolic transformation by methane monooxy-genase to formyl chloride. In addition to the sMMOC phenotype,sMMOC mutants were defective in copper acquisition (125, 127,128). A drastic increase in soluble versus insoluble copper in theculture medium was also observed and fostered speculation on theproduction of an extracellular Cu2�-complexing agent(s) analo-gous to Fe3�-complexing siderophores (127). Subsequent studiesrevealed the presence of a low-molecular-mass copper-bindingligand, although the identity of this compound was not deter-mined (125, 127).

Initial Identification and Isolation of Methanobactin (aCopper-Binding Compound or “Chalkophore”)

Somewhat paradoxically, the “copper-binding ligand” or “cop-per-binding compound” was first isolated from M. capsulatusBath during the purification of pMMO and thus under conditionsof high copper concentration (73). Separation of this copper-binding compound from the pMMO revealed a low-molecular-mass yellow-fluorescent copper-containing molecule. The colorand fluorescence properties of this molecule were similar to thoseof the water-soluble pigment observed in cells cultured in low-copper medium (73). Subsequent characterization of this water-soluble pigment revealed that it was identical to the copper-bind-ing compound that copurified with the pMMO (73, 128). Theproduction of water-soluble pigments by methanotrophs hadbeen noted during the initial isolation of many type strains over 40years ago but was associated with cells cultured in low-iron me-dium (13). The copper-binding compound was eventually termedmethanobactin (mb), based on the molecule’s antimicrobial ac-tivity toward Gram-positive bacteria (129, 130). Once identified,this copper-binding compound was isolated or identified in anumber of different methanotrophs, including Methylomicro-bium album BG8 (131), Methylocystis strain SB2 (132), Methylo-cystis rosea (strain SV97) (133), Methylocystis hirsuta CSC1 (133),and Methylocystis strain M (133). The crystal structures of the mbs

from M. trichosporium OB3b (134, 135), M. hirsuta CSC1 (133),and Methylocystis strain M (133) have been determined. Further-more, the chemical structures of the mbs from Methylocystis strainSB2 (132) and M. rosea (133) have been deduced. This review willfocus on these mbs that have allowed putative mbs to be identifiedfrom methanotrophs with sequenced genomes.

Methanobactins as Chalkophores

Functionally, mbs are similar to siderophores. Like siderophores,they are all low-molecular-mass (�1,200-Da) compounds pro-duced by bacteria under low-copper conditions (125, 126, 128,136–138). Following the nomenclature of siderophores, which isfrom the Greek for iron bearing or iron carrying, mbs are chalko-phores (copper bearing or copper carrying) (134, 139). mbs arecurrently the only known representatives of this group. Consistentwith their role as the extracellular component of a copper acqui-sition system, mbs have some of the highest known binding affin-ities for copper ions (2, 131, 135, 138, 140, 141) (Table 1).

Although chalkophores and siderophores share a number ofproperties, these two groups of metal-binding compounds can bedistinguished in a number of ways. With the exception of thephytosiderophore domoic acid (142), siderophores are expressedunder iron limitation, whereas chalkophores are expressed undercopper limitation. Many siderophores bind copper, and chalko-phores can bind iron (142–148); however, different metal bindingconstants characterize the two groups, and colorimetric assayshave been developed to distinguish between them based on thisdifference (138, 149, 150). Structurally, chalkophores differ fromsiderophores by their typical heterocyclic rings and associatedthioamide groups (Fig. 1 and 2). Different ring systems have char-acteristic UV-visible absorbance, circular dichroism (CD), andfluorescent spectral properties, which can be used for identifica-tion and to characterize the metal binding properties of the mol-ecule (131, 133–136, 138, 148, 151–153). Excitation energy trans-fer occurs between the two rings in mb (136, 154), as observed forthe chromophores of light-harvesting complexes (155–157), re-sulting in the fluorescent properties of mbs. For example, theemission intensity of mbs increases with selective hydrolysis of theone of the rings, and emission intensity often increases followingmetal addition (132, 136–138, 141, 148, 154). Again, this propertycan be used in both identification and characterization of mbs.

However, not all of the properties just described are sufficient toidentify mbs. For instance, Clostridium cellulolyticum produces acopper-binding secondary metabolite, closthioamide, with a mo-lecular mass similar to that of mbs (158–160), and like mbs, clos-thioamide has thioamide groups, will reduce Cu2� to Cu1�, andhas a high Cu1� binding affinity (1015 M1). Closthioamine willalso test positive with the copper-chrome azural S (Cu-CAS) as-say, a liquid or plate assay used to screen for mb production (138,149, 161). However, closthioamide can be distinguished spectro-scopically from mbs; e.g., closthioamide shows a single UV-visibleabsorption maximum at 270 nm arising from its two characteris-tic phenolic groups separated by six thioamide moieties. Further-more, closthioamide is able to form a dinuclear Cu1� complex.Also in contrast to that of mbs, closthioamide synthesis is notregulated by copper, and unlike for mbs, the molecule is believedto be produced by a polyketide synthase (see below).

Similarly, under low-copper conditions, Paracoccus denitrifi-cans also produces a low-molecular-mass 716.18-Da porphyrin,coproporphyrin III, that appears to be involved in copper acqui-

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TA

BLE

1M

etalbindin

gaffi

nities

(K)

ofmbs

fromM

.trichosporiumO

B3b,M

ethylocystisstrain

M,M

ethylocystishirsuta

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1,Methylocystis

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103

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TF

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�10

81.0

�10

52.6

�10

79.9

�10

9—

——

——

——

mb-O

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—6.5

�10

61.8

�10

54.7

�10

49.0

�10

56.8

�10

5—

——

——

—m

b4 -O

B3b

PT

6.8172

——

1.0�

105

8—

——

——

——

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B3b

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—1.0

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52

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25

——

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Com

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——

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12

d—

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Com

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133�

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102

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——

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——

——

—

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d—

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mb-C

SC1

Com

petitiontitration

sw

ithB

CS

7.5133

�8

�10

20

——

——

——

——

——

——

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106

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ompetition

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IEN

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27

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108

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106

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mb

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1.1�

107

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106

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105

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105

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104

aA

bbreviations,IT

C,isoth

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T,poten

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ITC

,displacemen

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b,meth

anobactin

;mb

2 ,mb

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b4 ,m

btetram

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CS,bath

ocuproin

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RIE

N,trieth

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residues.




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FIG 1 Chemical structures of full-length mb-OB3b (135, 151) (A), mb-M (133) (B), mb-CSC1 (133) (C), mb-rosea (133) (D), and mb-SB2 (132) (E). Aminoacids marked with asterisks are observed in some but not all samples.

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sition (162). Unfortunately, the copper binding properties werenot reported in the initial publication, and we are not aware of anyfollow-up studies. Coproporphyrin III has a typical heme UV-visible absorption spectrum and shows different �, �, and � max-ima depending on the metal coordinated by the heme group, al-lowing it to be distinguished from mbs based on this property.

STRUCTURAL PROPERTIES OF METHANOBACTINS

Structural Diversity and Core Features

The core structural features of mbs are shown in Fig. 1 and 2. Asmentioned above, mbs are modified peptides characterized by thepresence of one oxazolone ring and a second oxazolone, imida-zolone, or pyrazinedione ring, which are separated by 2 to 4 aminoacid residues (see Biosynthesis of Methanobactin below). Eachring has an adjacent thioamide group. Structurally, mbs can bedivided into two types (Fig. 3 and 4). One type (group I) is repre-sented by mb from M. trichosporium OB3b (mb-OB3b) (Fig. 1A,3A, and 4). In addition to these core properties, mb-OB3b alsocontains Cys residues in the mature peptide, which are linked byan intramolecular disulfide bond (Fig. 1A and 3A). Cu1�-mb-OB3b has a pyramid-like shape with the metal coordination site atthe base (133–135). The disulfide bond is present in both apo- andCu1�-mb-OB3b. mb-OB3b is structurally the most complex mbcharacterized so far and is currently the only structurally charac-terized representative of this mb type. However, based on se-quence similarity and alignments, the putative mbs from Methy-losinus sp. strain LW3 (mb-LW3), Methylosinus sp. strain LW5(mb-LW4), and Methylosinus sp. strain PW1 (mb-PW1) and oneof the two mbs from M. parvus OBBP [mb-OBBP(1)] would alsofall within this group (Fig. 4). In this group, the core peptide ispredicted to contain 2 or more Cys residues in the mature peptideand/or to contain an additional ring(s). If the Cys residues are notincorporated into a ring, the second Cys and either the third orfourth Cys are predicted to form an intermolecular disulfidebond.

The second group (group II) is represented by the isolated mbsfrom M. rosea (mb-rosea) and Methylocystis strain SB2 (mb-SB2),

which show high similarity to the structurally characterized (133)mbs from M. hirsuta CSC1 (mb-CSC1) and Methylocystis strain M(mb-M). In this group, mbs lack the Cys residues in the maturepeptide and are smaller and probably less rigid due to the absenceof the disulfide bond found in mb-OB3b. Heterocyclic rings areseparated by two or three amino acids. In contrast to members ofgroup II mbs from methanotrophic organisms, putative mbs fromnonmethanotrophs (mb-B-8, mb-14-3, mb-B510, and mb-21721) contain four Cys residues in the apo-protein. However,based on the location of Cys residues, we predict that all 4 Cysresidues are incorporated in the two heterocyclic rings. mbs fromthe structurally characterized members in this group contain asulfate group, which appears to aid in the formation of a tightbend in the molecule (Fig. 3B) by making a hydrogen bond withthe backbone amide of Ser2. The sulfate group also increases af-finity for copper ions (133). Overall, mbs from the Methylocystis

FIG 2 Core features of mbs. AA, amino acid(s). R-groups can be Arg, Ile, Met,or Pro.

FIG 3 Crystal structures of Cu1�-mb-OB3b (A), Cu1�-mb-M (B), and Cu1�-mb-CSC1 (C), with a Thr residue missing at the C terminus in panels B and Ccompared to the largest form isolated. The copper ions are represented as grayspheres, and the oxazolone (oxa) and pyrazinedione (pyra) rings are labeled, asare the coordinating atoms. Hydrogen-bonding interactions are shown inpanels B and C as dashed orange lines.




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strains display a hairpin-like shape (Fig. 3B and C) (133). Theconserved T/S adjacent to the putative C-terminal ring suggeststhat the other members of this group also contain a sulfate group(Fig. 4).

Truncated forms have been identified for all characterized mbs(133, 135, 138), which results from the loss of one or more C-ter-minal amino acid residues (Fig. 1 and 4). The different forms showvery similar copper binding properties (133, 135) but distinct re-duction potentials (133, 135) and spectral properties (138). Themechanism leading to the loss of C-terminal amino acids remainsan open question but does not appear to involve N-protonation asobserved for microcin B17 (163), since the C-terminal oxazolonering of mb remains intact. In microcin B16, this reaction results inautoproteolysis of the ring and protein splicing. In Methylocystisstrains, the sulfate group can also be lost, which does affect copperaffinity (133).

mbs thus have a number of unusual structural features. Modi-fications of two residues to form oxazolone or imidazolone ringsare rare but have been observed in several classes of ribosomallysynthesized and posttranslationally modified peptide (RiPP) sec-ondary metabolites (163–166). Pyrazinedione rings are even moreuncommon and have been detected only in the non-amino-acid-containing molecules selerominol (167) and flutamide (168) fromfungi. The presence of thioamide groups in natural products isalso rare and has been identified only in closthioamide from Clos-tridium cellulolyticum (153, 160) and thioviridamide from Strep-tomyces olivoviridis (169, 170). Although rare, O-sulfonation ofSer or Thr has been reported in eukaryotic proteins (171), but toour knowledge, mb is the only bacterial peptide that contains thisposttranslational modification and is ribosomally produced, asdescribed below.

Copper Coordination Site

In the three mbs whose crystal structures have been determined,Cu1� is coordinated in a similar manner by an N2S2 ligand set witha distorted tetrahedral geometry (Fig. 3) (133–135). The C-termi-nal S and N ligands in the mb-CSC1 and mb-M are switchedcompared to their position in mb-OB3b (133); however, the re-sulting coordination geometry is very similar. The coordinationsites are stabilized by the two 5-membered chelate rings formedupon ligation and a number of hydrogen bonding and interac-tions (Fig. 3), for example, between the backbone amide of Cys3and a coordinating sulfur in Cu1�-mb-OB3b and a -anion in-teraction between the sulfate and the pyrazinedione ring in mb-CSC1 and mb-M (133, 135).

METAL BINDING PROPERTIES

Binding and Reduction of the Primary Metal, Copper

mb binds both Cu2� and Cu1�, and the binding by mb appears todepend on pH (135, 172) and on the ratio of Cu2�/Cu1� to mb(136, 141, 172) (Table 1). As discussed below, mb is able to com-plex both soluble and insoluble forms of Cu1� and Cu2�. Ther-modynamic, spectral, and kinetic studies have been carried out onthe addition of Cu2� to mb-OB3b and mb-SB2 (136, 141). Thesestudies (136, 141, 172) are complicated by the fact that mb rapidlyreduces Cu2� to Cu1�. Since the oxidation state of copper respon-sible for the high bonding constant for experiments in which Cu2�

has been added to apo-mbs is not known, we will indicate that amixture of these oxidation states is present by using “Cu2�/Cu1�”from this point on. At low ratios of Cu2�/Cu1� to mb-OB3b,mb-OB3b initially binds Cu2�/Cu1� as an oligomer/tetramer(136, 141). Pre-steady-state kinetic data also suggest that the ini-tial binding of metal is on only one of the rings and its associatedthioamide. In mb-OB3b, initial Cu2�/Cu1� coordination is to theoxazolone A, followed by a short (8- to 10-ms) lag period and thencoordination to oxazolone B (141). At higher ratios of Cu2�/Cu1�

to mb-OB3b, mb-OB3b coordinates Cu2�/Cu1� as a dimer, fol-lowed by a monomer at Cu2�/Cu1�-to-mb-OB3b ratios above 0.5Cu2� per mb-OB3b. mb-SB2 appears to follow a similar tetramer-dimer-monomer binding sequence depending on the copper-to-mb ratio. For mb-SB2, the initial binding of Cu2�/Cu1� is tothe imidazolone ring, followed by coordination to the oxazolonering (154).

A number of different methods have been used to determinemetal binding affinity constants for mbs (Table 1). Affinities for

FIG 4 mb precursor peptides. Sequences were detected in bacteria with knowngenome sequences, in methanotrophs with characterized mbs as well as fromother methanotrophs, and from selected nonmethanotrophs, directly from theDNA sequence using Fuzztran in Mobyle (http://mobyle.pasteur.fr), with the op-timized mb sequence motif (Prosite format) [ILMV]-[AIKST](1,3)-[IV]-[KNRT]-[IV]-X-[AKQ]-[KRT]-X-[ILM]-X-[IV]-X-[GV]-R-X(2)-[AL]-X-C(1,2)-[GA](0,1)-[ST](0,2)-X(0,2)-C(1,2) as a query. Only sequences also featur-ing downstream mb biosynthesis cassette genes mbnB and mbnC (see the text andTable 2) are shown. Known and predicted leader sequences and sequences notobserved in the final product are shown in black, sequences detected in bacteria ofknown genome sequence from methanotrophs with mbs either whose crystalstructure has been determined (mb-OB3b) or whose primary structures were pre-dicted (mb-SB2 and mb-rosea) are shown in red, sequences detected in bacteria ofknown genome sequence from methanotrophs are shown in blue, and sequencesdetected in bacteria of known genome sequence from nonmethanotrophs areshown in green. Amino acids observed in some but not all samples are shown intan. Amino acids with a gray background represent the amino acid pair shown orpredicted to be posttranslationally modified into an oxazolone, imidazolone, orpyrazinedione group in structurally characterized mbs; underlined Cs representCys residues known or predicted to be present in the mature peptide. mbs werefrom M. trichosporium OB3b (mb-OB3b), Methylosinus sp. strains LW3 (mb-LW3), LW4 (mb-LW4), and PW1 (mb-PW1), M. parvus OBBP (mb-OBBP),M. rosea (mb-rosea), Methylocystis strains SB2 (mb-SB2), SC2 (mb-SC2), andLW5 (mb-LW5), Cupriavidus basiliensis B-8 (mb-B-8), Pseudomonas ex-tremaustralis 14-3 (mb-14-3), Azospirillum sp. strain B510 (mb-B510), Tis-trella mobilis KA081020-065 (mb-mobilis), Comamonas composti DSM 21721(mb-21721), and Gluconoacetobacter sp. strain SXCC (mb-SXCC). Numbersin parentheses after a strain name are used for species with more than one mbgene cluster. Stop codons are indicated by an asterisk and gaps in the sequencealignment by a hyphen.

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Cu1� can be determined from competition studies using a well-established approach (173–175) with chromophoric ligand suchas bathocuproine disulfonate. The measurement of the reductionpotential of the Cu-mb then allows the Cu2� affinity to be calcu-lated. The Cu1� affinity is �1021 M1 for all mbs analyzed usingthis approach (133, 135) and is one of the highest known forbiological systems. Such a high affinity raises the issue of howcopper is released from mb within a methanotroph. The reductionof the disulfide, in mb-OB3b (135), and removal of the sulfate, inmb-CSC1 (133), do decrease the Cu1� affinity by �2 orders ofmagnitude, but the values are still high. Given that the Cu2� af-finities are significantly weaker (Table 1), oxidation may be uti-lized for release of the metal, which may be more likely for thoseCu-mbs with lower reduction potentials, such as Cu-mb-CSC1(133).

Other copper-chelating compounds, primarily for Cu2�, havealso been used to estimate affinities (91, 125, 128, 133, 135), as wellas isothermal titration calorimetry (136, 141) and displacementisothermal titration calorimetry (136) (Table 1). mbs have beenshown to solubilize and bind insoluble forms of Cu1� under an-aerobic conditions (141) and to extract Cu from copper minerals(176), humic material (177), and glass (178). The ability of mbs toextract copper from copper-containing minerals as well as otherorganic molecules should be considered in modeling bioavailablesources of copper (179–183).

Binding of Other Metals

In addition to copper, mb-OB3b and mb-SB2 have been shown tobind a number of transition and near-transition metals (137, 148,154, 184) (Table 1). In general, the spectral properties followingbinding are unique for each metal and can be used in initial anal-ysis of metal binding and in metal competition studies (137, 154).The primary function of mbs appears to be copper acquisition,and the capacity to bind other metals appears to be an inadvertentconsequence of the Cu1� coordination site. The peptide backboneof mbs appears to influence the binding of other metals. For ex-ample, the order of metal binding preference by mb-OB3b isCu2�/Cu1� � Hg2� � Au3� Zn2� Cd2� Co2� Fe3� Mn2� Ni2�, whereas the metal binding preference for mb-SB2 is Au3� Hg2� Cu2�/Cu1� Zn2� Cd2� � Co2� �Fe3� Mn2� Ni2� (137, 154).

Although strong binding of other metals such as mercury bymbs may seem counterproductive for methanotrophic growth, itmay actually be of some benefit. Specifically, by strongly bindingmercury, mbs may significantly alter its speciation and bioavail-ability, thereby reducing its toxicity. Indeed, growth studiesshowed that in the presence of mercury, added as mercuric chlo-ride, methanotrophic growth was completely inhibited, butgrowth did occur when mb was also added (184). Interestingly,such a protective effect was seen in a variety of methanotrophswhen exposed to mercury and mb-OB3b. These results suggestthat mb secreted by any methanotroph could serve to protect thebroader microbial community from mercury toxicity (184).

Further indirect evidence of the importance of mb binding tometals other than copper was afforded by the observation that inM. trichosporium OB3b sMMO is expressed and active in thepresence of both gold and copper. This finding was unexpectedsince, as discussed above, copper strongly represses expression ofsMMO. Also, the amount of copper associated with biomass sig-nificantly decreased in the presence of gold, compared to when

gold was not added (91). This suggests that mb-OB3b is limited inits ability to bind copper in the presence of gold. Indeed, if mb waspreloaded with copper and added exogenously to M. trichospo-rium OB3b, sMMO expression was not observed, and copper lev-els associated with biomass increased (91). These findings suggestthat although methanotrophs synthesize mb for copper uptake,such uptake may be compromised if other metals such as gold arealso present due to their ability to compete effectively for bindingto mb. These observations suggest that in such situations, sMMOmay be expressed and active even in the presence of copper, whichmay alter the methanotrophic community structure as well as ac-tivity.

Metal Reduction

As mentioned above, another property exhibited by all mbs exam-ined so far is their ability to reduce Cu2� to Cu1� (133, 135, 136,141, 185). In addition, mb-OB3b and mb-SB2 have also beenshown to reduce Au3� and Ag� to Au0 and Ag0, respectively (137,148). In the case of gold, no other oxidation states were observed.In contrast, no change in the oxidation states of Zn2�, Cd2�,Co2�, Fe3�, Mn2�, and Ni2� was observed following binding bymb-OB3b and mb-SB2 (137, 148). Surprisingly, in the absence ofan external reductant, both mb-OB3b and mb-SB2 have beenshown to reduce multiple Cu2�and Au3� ions, which raises thequestion of the electron donor for this reaction.

BIOSYNTHESIS OF METHANOBACTIN

Identification of the Polypeptide Precursor ofMethanobactin

Although a potential peptide sequence precursor of mb-OB3b wasidentified following hydrolysis of the oxazolone rings (132), thequestion whether this peptide was assembled by a nonribosomalpeptide synthetase or encoded by DNA and synthesized on theribosome remained. Determination of the genome sequence of M.trichosporium OB3b helped resolve this question. BLAST searchesusing the predicted mb peptide precursor revealed a short openreading frame (ORF) with a perfect match at a location within theM. trichosporium OB3b genome sequence where no protein prod-uct had been predicted (Fig. 4). The genome region of the putativemb precursor matching sequence in M. trichosporium OB3b had anumber of distinctive and striking features (Fig. 5; Table 2). Theseincluded (i) a precursor peptide composed of leader and corepeptide sequences followed by a stop codon, as expected for post-translationally modified peptide natural products (164), (ii) a po-tential cleavage site between the leader and core peptide, sugges-tive of secretion, and (iii) genes upstream and downstream of themb precursor gene encoding protein sequences compatible withpossible roles in maturation of the mb precursor sequence, trans-port, and regulation of mb biosynthesis (Fig. 5, Table 2).

Elaboration on this initial search revealed a series of genomescontaining gene clusters with characteristics matching those of theM. trichosporium OB3b mb gene cluster, e.g., in M. parvus OBBP(186, 187) and Methylosinus sp. strain LW3 (186), as well as non-methanotrophs Azospirillum sp. strain B510 (132, 187), Azospiril-lum sp. strain B506 (186), Pseudomonas extremaustralis (187),Pseudomonas extremaustralis subsp. laumondii TT01 (186), Tis-trella mobilis (187), Gluconacetobacter sp. strain SXCC (132, 186).Gluconacetobacter oboediens (186, 187) Methylobacterium sp.strain B34 (186), Cupriavidus basilensis B-8 (186), Photorhabdus




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luminescens (186), and Vibrio caribbenthicus BAA-2122 (186). No-tably, this list includes not only methanotrophic strains known toproduce mb for which a genome sequence was available but alsogenomes of other strains, suggesting that such gene clusters mayencode peptide-derived products with more diverse functionsthan those associated with mbs.

Considering the mb precursor peptides identified so far, it isstriking that the leader peptides are much more strongly con-served than core peptide sequences (Fig. 4). It is also interesting tonote that sequence conservation does not follow taxonomic affil-iation and that identical precursor sequences are found in geneclusters of strains from different genera (Fig. 4). This stronglysuggests that horizontal gene transfer has contributed to dissem-

ination of mb gene clusters in the environment, and indeed, mo-bile genetic elements have often been identified in the sequencesimmediately adjacent to mb gene clusters (186).

The lack of sequence conservation in mb core peptides alsoimplies that designing sequence motifs to detect mb gene clustersis challenging and that database hits using sequence motifs basedon known mb precursor sequences need to be substantiated withother evidence. In our view, for a sequence hit to be consideredindicative of an mb precursor, additional evidence should include(i) the presence of the only conserved genes in the mb gene cluster(Fig. 5), i.e., the genes for mb biosynthesis cassette proteins MbnBand MbnC, in the immediate vicinity of the mb peptide precursorand (ii) the presence of 2 or more cysteines in the core mb peptide

FIG 5 mb gene clusters. Gene clusters of complete genomes of methanotrophs M. trichosporium OB3b (OB3b) and Methylocystis sp. strain SB2 and M. rosea SV97(SB2/SV97), which produce mbs whose chemical structures have been determined/deduced (Fig. 1). Shown are the gene for the mb peptide precursor MbnA(black) and associated genes for the mb biosynthesis cassette protein MbnB (red), mb biosynthesis cassette protein MbnC (orange), MATE efflux pump MbnM(dark brown), aminotransferase MbnN (yellow), diheme cytochrome c peroxidase MbnH (dark green) and associated MbnP of unknown function (light green),FAD-dependent oxidoreductase MbnF (violet), sulfotransferase MbnS (dark blue), TonB-dependent receptor domain MbnT (light brown), FecI-like RNApolymerase sigma-70 domain MbnI (light blue), and FecR-like membrane sensor MbnR (light purple). Other genes of unknown function are shown in white, andgenes flanking the clusters with predicted functions not thought to be associated with mb maturation or transport are shown in gray.

TABLE 2 Genes in the methanobactin gene cluster

Gene Proposed annotation Characteristics/comments InterProScan ID

mbnA mb peptide precursor Short ORF with N-terminal leader peptide NAmbnB mb biosynthesis cassette protein MbnB TIM barrel enzyme (aldolase/isomerase type) IPR026432, IPR0078801mbnC mb biosynthesis cassette protein MbnC Less well defined than MbnB IPR023973mbnE FAD-dependent oxidoreductase Often also found when mbnH and mbnP are missing IPR003042mbnH Di-heme cytochrome c peroxidase IPR023929, IPR004852mbnI RNA polymerase sigma 70 domain Putative �70 factor; in some cases (e.g., strains SB2, SC2, SV97), methanobactin

precursor peptide is the C-terminal peptide of MbnImbnM MATE efflux pump Multiantimicrobial extrusion protein IPR004839, IPR005814mbnN Aminotransferase Rarely found (e.g., strains OB3b, LW4), pyridoxal-phosphate dependent IPR023977mbnP Partner of mbnH Conserved protein of unknown functionmbnR FecR-like, putative sigma factor activator IPR000863mbnS Sulfotransferase Rarely found (e.g., strains SB2, SC2, SV97); sulfonation of Thr? IPR012373mbnT TonB-dependent receptor Plug domain, large membrane protein family IPR000531

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sequence (Fig. 4; Table 2). Regarding the latter, it is striking thatmb peptide precursors identified so far contain either 2 or at least4 cysteines, just as in the two types of mbs characterized so far.Nevertheless, the spacing between cysteine residues in the mbpeptide precursor sequence appears to be quite variable, with 1 or2 residues between the first and the second cysteines and, if pres-ent, 2 to 4 residues between the second and the third cysteines and1 to 3 residues between the third and the fourth cysteines, respec-tively (Fig. 4). It is also striking to note that “doublets” of twocysteine residues immediately adjacent to each other have so farbeen found only in putative mb precursor peptides of presumablynonmethanotrophic strains (Fig. 4).

The overall structure of mb gene clusters and related gene clus-ters is beyond the scope of this review, but their analysis, using acombination of sequence-based, motif-based, and gene synteny-based searches, will undoubtedly yield many interesting findingsin the future, as more mb gene clusters are identified in the in-creasing number of microbial genomes that are being sequenced.Summarizing the quite large diversity of mb gene clusters thathave already been detected (see, e.g., reference 186), two majortypes of mb gene clusters seem to emerge with respect to the lo-calization of the mbnA gene for the mb precursor peptide (Fig. 5).Whereas mbnA is always directly upstream of and in the sameorientation as mbnB and mbnC, it may be found as a short ORF, asexemplified for strain OB3b (Fig. 5), or actually represent the 3=end of regulatory gene mbnI, as in Methylocystis strain SB2 and M.rosea (strain SV97) (Fig. 5). The implications of the latter localiza-tion of the mbnA gene, notably in terms of regulation of mb ex-pression in strains with this gene arrangement (also see Regulationof Gene Expression below), are completely unknown at present.Currently, the cytochrome c peroxidase MbnH (Fig. 5; Table 2), aswell as the FAD-dependent oxidoreductase MbnF, present insteador sometimes in addition to MbnH in methanotroph gene clusters(186, 187), are likely candidates to be involved in the oxidationsteps required for ring formation (see below) (Fig. 6). In addition,the aminotransferase MbnN, found in the mb-OB3b but not themb-SB2/mb-rosea gene cluster (Fig. 5), may be involved in for-mation of the N-terminal keto-isopropyl group from Ile1 in thepeptide precursor, and the sulfotransferase MbnS, found in themb-SB2 and mb-rosea but not the mb-OB3b gene cluster, maycatalyze sulfonation of the threonine (Fig. 1 and 5). Two othergene products, TonB receptor and multidrug and toxin extrusion(MATE) protein) (Fig. 5; Table 2) have been suggested (186, 187)to be involved in uptake and secretion of mature mbs, respec-tively. See a recent review by Kenney and Rosenzweig (186) for adetailed description and phylogenetic analysis of several mb geneclusters.

Current Hypotheses on the Nature of the MethanobactinBiosynthesis Pathway

Despite the identification of the genetic determinants of mb pro-duction, the precise mechanism involved in maturation of mbsfrom peptide precursors still is an open question. The presence ofheterocyclic rings would suggest a pathway similar to that of otherpostribosomal peptide synthesis (PRPS) proteins (163–166, 188).For instance, in the PRPS protein microcin B17, McbB (cyclode-hydrase), McbC (flavin mononucleotide [FMN] dehydrogenase),and McbD convert Gly-Cys and Gly-Ser dipeptide sequences intothiazole and oxazole rings, respectively (163). In this reaction se-quence, the cyclodehydrase catalyzes ring formation via the amine

bond with the thiol or alcohol, followed by oxidation by an FMNdehydrogenase (163–166). In mb, ring formation is initiated froman X-Cys dipeptide sequence, resulting in formation of either anoxazolone, imidazolone, or pyrazinedione ring with a neighbor-ing thioamide group. If the catalytic sequence in ring formation inmb followed the microcin B17 example, the thiol group on Cysfrom mb would have to be replaced by a hydroxyl group, possiblywith an amine intermediate. The thioamide group then wouldhave to be introduced via an amide-to-thioamide replacement, asproposed for two other natural products with thioamide groups(153), closthioamide from Clostridium cellulolyticum (160) andthioviridamide from Streptomyces olivoviridis (169, 170). As analternative, the thioamide group of mbs was proposed to originatefrom the Cys thiol, as hydrolysis of the oxazolone rings resulted inan X-Gly-thioamide sequence (132). Based on the precursor pep-tide sequences that have now been identified, the latter reactionmechanism now appears highly likely.

The absence of a cyclodehydrase-like gene in mb gene clustersalso suggest that ring formation differs from that of other naturalproducts in other aspects as well. The simplest and shortest reac-tion sequence in oxazolone ring formation in mbs would involvean oxidation followed by a rearrangement that changes the con-nectivity of the peptide backbone (Fig. 6). In such a scheme, Cysthiols would likely need to be protected against oxidation (bluecircle), as protein-linked thioesters, or alternatively as disulfides(as shown in Fig. 6), possibly via glutathionylation as observedwith MetE in Escherichia coli (189). This reaction may occur dur-ing the initial reaction of peroxidase, which is one of the proteinsof the mb gene cluster (Table 2). The reaction sequence couldoperate during formation of both oxazolone rings of mb-OB3b,from Leu1 and Cys2 and from Pro7 and Cys8 of the precursorpeptide, respectively. Methylocystis strain SB2 and M. rosea sharethe same mb gene clusters and identical precursor peptides (Fig. 4and 5), but the mature mb-SB2 (132) as characterized is suggestedto contain an imidazolone ring, whereas mb-rosea is predicted tohave a pyrazinedione ring that is also present in mb-CSC1 andmb-M (133). However, imidazolone and pyrazinedione rings areactually isomeric and can be interconverted by a simple sequenceof hydration, rearrangement, and dehydration reactions (190).Nevertheless, the mechanism of formation of this ring is likely tobe more complex than that of oxazolone ring formation (Fig. 6).One possible sequence would replace the transamination reac-tion, proposed for mb-OB3b, with an oxidative deamination ofthe N-terminal amine, which would produce ammonia that couldbe used for subsequent aminolysis of the oxazolone ring (Fig. 6). Acondensation reaction would then lead to cyclization and the for-mation of the imidazolone or, with an intervening rearrangementstep, to the pyrazinedione. It should be noted that the Methylocys-tis species lack the aminotransferase found in the mb-OB3b genecluster. Alternate reaction schemes would involve two changes ofconnectivity in the peptide backbone and not just one as proposedfor oxazolone ring formation (Fig. 6). Clearly, the formation ofthe different types of heterocyclic ring systems in mbs now needsto be addressed experimentally. The production and characteriza-tion of mutants will hopefully help in this respect, and such studieshave now been initiated (187).

REGULATION OF GENE EXPRESSION

The discovery of the mb biosynthesis gene cluster raised two mainquestions. First, is mb biosynthesis regulated with respect to




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copper? Second, what is the role of mb in the “copper switch”controlling the expression of the two forms of MMO? The firstquestion was easily answered through the use of reverse transcrip-tion-quantitative PCR (RT-qPCR) using primers specific to somegene(s) of the mbn operon. Initial studies found that in M. tricho-sporium OB3b, expression of mbnA, the gene encoding the poly-peptide precursor of mb, did indeed vary with respect to copper,with expression dropping over three orders of magnitude when

copper concentrations increased from zero (no amendment) to 1�M copper, and expression was largely invariant at higher copperconcentrations (140). Such a decrease in expression was reflectedin the finding that mb in the spent medium was highest at copperconcentrations less than 1 �M and dropped �5-fold when thecopper concentration was increased to 5 �M (136, 138, 141).Given that the two forms of MMO are also regulated by copper, itwas possible that mb was directly involved in this copper switch.

FIG 6 Proposed reaction schemes for biosynthesis of the oxazoline rings with associated thioamide groups via a tandem two-step sequence of peroxidation anddehydration reactions. Cysteine thiols are likely protected against oxidation, possibly as disulfides involving one of the proteins of the mb gene cluster (bluecircles). For imidazolone and pyrazinedione ring formation, oxazolone rings are modified via a transamination/deamination step followed by an aminolysis stepto open the oxazolone ring followed by ring formation and dehydration.

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Further genetic analyses, however, found that it is not (187). RT-qPCR of mmoX and pmoA from both wild-type M. trichosporiumOB3b and an mbnA mutant where a gentamicin cassette was in-serted, knocking out mbnA, showed that mmoX and pmoA expres-sion followed similar patterns in both strains as the copper/bio-mass ratio varied but that the magnitudes of such changes werelower in the mbnA::Gmr mutant than in the wild-type strain (187).To answer the second question above, a mutant in which the mmooperon was deleted was constructed. In this mutant, the “copperswitch” was inverted; i.e., pmoA expression was greatest in theabsence of copper and dropped �2 orders of magnitude whencopper concentrations increased (187). Further, expression ofmbnA was largely invariant in this mutant with respect to copper.Since all deleted genes in the mmo operon with the exception ofmmoD are known to encode polypeptides of sMMO and sinceMmoD is not required for sMMO activity (32, 51, 63), these ex-pression data suggest that MmoD is a key component of the cop-per switch, with MmoD serving to regulate expression of mb andmb then amplifying the magnitude of the response in the copperswitch.

On the basis of these findings, a model for the regulation ofexpression of the mmo and pmo operons by mb and MmoD wasproposed (187). During growth at low copper/biomass ratios,MmoD protein represses expression of the pmo operon and alsoupregulates expression of the mmo operon, including that ofmmoR and mmoG, which were shown previously to play key rolesin regulating mmo expression (113). Further, MmoD is postulatedto enhance expression of mb, which serves to amplify expressionof the mmo operon. During growth with high copper concentra-tions, mb binds copper and can no longer enhance the expressionof the mmo operon. Under these conditions, it is presumed thatMmoD also binds copper and no longer represses expression ofthe pmo operon or induces expression of sMMO or mb.

There are, however, a number of issues with the model as pro-posed in Fig. 7. For such a model to be accurate, mmoD would berequired to be constitutively expressed with respect to copper inorder for sMMO and mb expression to occur after copper is re-moved. This version of the model implies, however, that as thecopper/biomass ratio increases, expression of the entire mmooperon no longer occurs. Recent qRT-PCR analysis, however,

shows that mmoD, unlike mmoX, is constitutively expressed withrespect to copper (J. D. Semrau, unpublished data). Nevertheless,expression of mmoD, like that of mmoX, increases with increasingaddition of exogenous mb, suggesting that expression of mmoD isat least partly coregulated with that of the entire mmo operon.

In addition, this model, although explaining the genetic basisfor the copper switch in methanotrophs, does not explicitly con-sider the role of two genes known to play roles in the regulation ofthe mmo operon, i.e., mmoR and mmoG upstream of mmoX (113,191). Based on available data, it appears that mmoR and mmoG donot play a significant role in the copper switch; i.e., they do notappear to have any control over expression of the pmo operon. It ispossible, however, that regulation of the mmo operon is the resultof a concerted interaction of mb and/or MmoD with these geneproducts.

This model also ignores the possibility that the presence ofputative regulatory genes (e.g., mbnI and mbnR [Fig. 5]) in mbgene clusters may play a role in regulating gene expression, partic-ularly the mb gene cluster and/or the mmo operon. Given this, arevised model of the copper switch in methanotrophs is now pro-posed in Fig. 8. Here MmoD is again postulated to be a key com-ponent of the copper switch, but MbnI is proposed to be respon-sible for inducing expression of the mb gene cluster, as well asmmoR and mmoG. Collectively, mb, MmoR, and MmoG inter-act to induce expression from the �N promoter upstream ofmmoX, while MbnI binds to the �70 promoter upstream ofmmoY. In this updated and revised current model, mmoD isconstitutively expressed but associates with copper when it ispresent, preventing repression of the pmo operon or expressionof the mb gene clusters. Further experiments will show whetherthis new model is correct, but it is already clear that the exactdetails of the copper switch mechanism are more complex thanpreviously thought.

PHYSIOLOGICAL FUNCTIONS

Copper Acquisition

Although mb has a high affinity for copper, its importance incopper acquisition by methanotrophs is still unclear. NonnativeCu1�-mbs have been shown to be taken up by methanotrophs

FIG 7 Model for the regulation of gene expression in the mmo, pmo, and mbn gene clusters by copper, mb, and MmoD. (A) Low copper/biomass ratio; (B) highcopper/biomass ratio. (Reproduced from reference 187 with permission of the publisher.)




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and facilitate switchover to pMMO (133). Copper uptake andswitchover are faster if the native mb is used but still take morethan 24 h. However, the role of mb in meeting the overall copperrequirements of the cell is still in question. It has been found thatM. trichosporium OB3b possesses at least two mechanisms for cop-per uptake, one clearly based on mb and involving active transportof copper-mb complexes and another, nonspecific passive trans-port pathway (192). It was also found that the amount of copperassociated with biomass was the same regardless of whether cop-per was added as CuSO4 or as the Cu

1�-mb complex at the sameconcentration. Subsequently, it was found for M. trichosporiumOB3b that when the gene for the precursor polypeptide of mb(mbnA) is knocked out, copper is still associated with biomass,and it increases with increasing amounts of copper in the growthmedium, as does copper found associated with wild-type cultures.The role of mb in copper uptake thus remains unclear. It should bekept in mind that these studies were performed using a well-de-fined growth medium with limited diversity of copper speciationand little if any copper-containing precipitate. It is tempting tospeculate that mb may have a more significant role in copperuptake in situ, where copper speciation and distribution will bemuch more complex and will include copper associated with awide range of organic materials (e.g., humic and fulvic acids), aswell as found either sorbed onto or part of various mineralphrases. Indeed, it has been shown that expression of pmoA isstrongly dependent on the form of copper present, with copperassociated with metal oxides found to induce smaller changes inpmoA expression, and that concomitant addition of mb increasedthe magnitude of the copper switch in M. trichosporium OB3b(176, 193).

Do Methanobactins Have a Role as Signaling Molecules?

Given the small size of mbs and their secretion into the environ-ment when copper availability is low, a role for mbs as signalingmolecules may be envisaged. Indeed, addition of mb-OB3b en-hances expression of mmoX in in cultures of both wild-type M.trichosporium OB3b and its mbnA::Gmr mutant (184, 187), sug-gesting that mb may control gene expression by acting as a signal-ing molecule. Further, given that all known forms of mb havesignificant structural similarity, mbs may allow for cross-species

communication. Recent studies show that this is indeed the case.mb-SB2 increased both mmoX expression and sMMO activity inM. trichosporium OB3b in the presence of copper. However, it hadno significant effect (P 0.05) on expression of either pmoA ormbnA, nor did it increase the amount of cell-associated copper(228). The addition of mb-SB2 preloaded with copper, however,reduced both mmoX and mbnA expression when M. trichosporiumOB3b was grown in the absence of any added copper, and nosMMO activity was detected. The latter results were likely due tothe increased amount of cell-associated copper and may reflect therole of mb in copper uptake.

Membrane Development

Several studies have demonstrated a direct correlation betweenintracytoplasmic membrane development and expression ofpMMO in methanotrophs (33, 35, 66, 109, 114, 194). As statedabove, mbnA plays a secondary role in the regulation of both themembrane-bound and soluble MMO (see Regulation of Gene Ex-pression above). However, deletion of mmoD along with the restof the sMMO operon had little effect on intracytoplasmic mem-brane development in M. trichosporium OB3b (Fig. 9C and D). Incontrast, a deletion mutation in the mbnA structural gene for mbappears to affect intracytoplasmic membrane development wellbeyond its effects on pMMO expression (187) (Fig. 9E and F).Currently, knowledge of the effect(s) of mb on intracytoplasmicmembrane development is observational, and additional researchis required to determine whether mb is directly involved in thedevelopment of these membranes in methanotrophs.

Detoxification of Reactive Oxygen Species

Copper is one of the essential metals required by microorgan-isms for growth (179). Like iron, however, copper in oxygen-ated solutions can generate a variety of toxic reactive oxygenspecies via Fenton and Haber-Weiss reactions (195–197). Thecoordination of copper by mb is likely to reduce this toxicity, asobserved with the iron- and copper-binding siderophoreschizokinen (146). Nevertheless, both copper-bound and cop-per-free forms of mb-OB3b have been shown to reduce dioxy-gen to superoxide in the presence of a reductant (198). In gen-eral, this would represent a daunting problem for bacteria that

FIG 8 Revised regulatory scheme for expression of the mmo, pmo, and mbn gene clusters as a function of copper, mb, and MbnI. (A) Low copper/biomass ratio;(B) high copper/biomass ratio.

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accumulate high concentrations of Cu-mb. Perhaps not unex-pectedly, the superoxide dismutase (SOD) activity of Cu-mb isapproximately 30,000 times higher than the oxidase activityassociated with either mb-OB3b or Cu-mb-OB3b. In addition,in the presence of a reductant, Cu-mb-OB3b also has a hydro-gen peroxide reductase (HPR) activity which is approximately50 times the oxidase activity (198). The overall result of thesethree activities is the reduction of O2 to H2O with concomitantloss of reductant. In addition to their protective roles in thecontext of mb-catalyzed oxidations, high SOD and HPR activ-ities of mb are also likely to provide protection against oxygenradicals that are potentially formed during methane oxidationand respiration of dioxygen (196, 199, 200) and may thus ex-plain the stabilizing effects of mb-OB3b on pMMO activity incell extracts (66, 73, 198). Similar activities have also been ob-served for mb-SB2 (N. L. Bandow et al., unpublished results),and we predict that this is a property of all mbs.

MEDICAL, INDUSTRIAL, AND ENVIRONMENTALIMPLICATIONS

Metal Mobilization or Immobilization for Remediation ofPolluted Subsurface Environments

Based on the published yields of mb in laboratory cultures (136,138), methanotrophs are predicted to export 3 to 50 copies of mb

per cell per second depending on the copper concentration duringgrowth. Given this high export rate and the fact that mb can bindmost transition and near-transition metals, it is likely that mb isable to influence metal mobilization and thus bioavailability ofdifferent metals in soil systems and that this affects the structureand functioning of microbial communities. Recent studies haveshown that mb will bind both inorganic and organic forms ofmercury (154) and thereby protect the host bacterium as well asother bacteria from mercury toxicity (184). However, additionalstudies are required to determine which metals are affected intheir mobility by mbs and the effects of mbs on the soil microflora.

Formation of Gold Nanoparticles

The potential application of gold nanoparticles as anti-inflamma-tory agents, in the targeting of cancer cells, in the detection ofmelamine, as biosensors, and in glucose oxidation has motivatedthe development of various production methods for such materi-als (201–206). Most approaches require either high (100°C) orlow (18°C) temperatures in the presence of reducing and stabi-lizing agents. Alternatively, microbially mediated production ofgold nanoparticles, both intracellularly and extracellularly, hasalso been extensively investigated (207). However, the size distri-bution of gold nanoparticles produced by microbial processes isusually quite broad, ranging from 10 to 6,000 nm (208). Further,most reported processes yield particles with a wide variety ofshapes (spherical, octahedral, triangular, irregular, etc.), and ratesof formation vary over a large range, from minutes to hours (209).Crucially, both mb-OB3b and mb-SB2 have been shown to effi-ciently reduce Au3� ions to metallic gold (typically 5 ions/mb)and in so doing to form well-defined size distributions of sphericalnanoparticles (137, 148). In a further study with mb-SB2, goldspherical nanoparticles of well-defined sizes (2.0 � 0.7 nm) wereformed, with mb-SB2 suggested to act both as a reductant and as astabilizing agent, thereby demonstrating that the use of mb-SB2represents a viable approach to produce well-defined gold nano-particles in this size range (137).

Possible Therapeutic Treatment of Human Ailments

Copper has been implicated as a key factor in the development ofa variety of human ailments, perhaps most notably Wilson’s dis-ease (210, 211), in which affected individuals have mutations thatinactivate the Cu-transporting P-type ATPase ATP7B (212), im-peding copper export to the bile and causing copper accumulationin the liver. Based on in vivo and in vitro studies, copper accumu-lation specifically affects liver mitochondria, via copper-inducedmodification of membrane protein thiols leading to multiproteincross-linking (213). In the final stages of Wilson’s disease, oxida-tive damage, primarily in the liver, is observed. Other symptomsof Wilson’s disease include serious neurological issues that canlead to additional psychiatric disorders (210, 214, 215).

There is as yet no cure for Wilson’s disease. Currently, treat-ment consists of one or more of the following options: (i) the useof chelating agents to remove copper via enhanced urine excre-tion, (ii) implementation of a low-copper diet, and (iii) zinc sup-plements added to the diet to stimulate production of the copper-binding protein metallothionein (210, 214). Of these options, theuse of chelating agents is the most commonly prescribed treat-ment, with two drugs, penicillamine and trientine, currently ap-proved by the U.S. Food and Drug Administration (FDA). Peni-cillamine, a breakdown product of penicillin, binds copper (216)

FIG 9 Transmission electron micrographs of wild-type M. trichosporiumOB3b (A and B), an M. trichosporium OB3b sMMO deletion mutant (C andD), and an M. trichosporium OB3b mb deletion mutant (E and F) cultured innitrate mineral salts (NMS) medium (A, C, and E) or NMS medium amendedwith 5 �M CuSO4 (B, D, and F).




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and also induces the production of metallothionein. Although theclinical benefit of penicillamine is widely reported, it has seriousside effects, including bone marrow suppression, degeneration ofelastic tissue, and proteinuria. In a large fraction of cases (20 to50%), neurological deterioration is observed (217). In such cases,the polyamine trientine, which has fewer reported side effects thanpenicillamine (217) and a comparable if not higher affinity forcopper, is substituted. In both penicillamine and trientine treat-ments, copper is excreted through the urine and not the bile. An-other chelating agent, tetrathiomolybdate, allows for copper to beexcreted from liver primarily into the blood, but some evidencesuggests that part of the copper may also be transferred to the bile(219) and that insoluble copper is found in both liver and kidneysat tetrathiomolybdate/copper ratios greater than two (219–221).The physiological impact of such an increased burden of copper inthe blood, as well as the formation of insoluble copper, is unclear.

mbs may represent a superior alternative as a copper-chelatingagent to treat Wilson’s disease, as indicated by preliminary studies.In the animal model for Wilson’s disease, the Long Evans Cinna-mon (LEC) rat, intravenous application of mb-OB3b extractedcopper from the liver into the bile as Cu-mb-OB3b, without mea-surable side effects (213, 222). It was also found that mb-OB3breversed both the buildup of copper in liver mitochondria andprotein cross-linking, allowing the liver to function normally(213), and also stripped copper from metallothionein (222). In-soluble forms of copper were not observed. Lastly, and in contrastto other chelators used for the treatment of Wilson’s disease, mb-OB3b was shown to work in circumstances of advanced liver fail-ure, further suggesting that mb may be a viable alternative in thetreatment of Wilson’s disease in the fut

methanobactin and the link between copper and bacterial ...a second methanotroph, methylococcus...

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