RESUMO
Aerobic methanotrophs, or methane-consuming microbes, are strongly dependent on copper for their activity. To satisfy this requirement, some methanotrophs produce a copper-binding compound, or chalkophore, called methanobactin (MB). In addition to playing a critical role in methanotrophy, MB has also been shown to have great promise in treating copper-related human diseases, perhaps most significantly Wilson's disease. In this congenital disorder, copper builds up in the liver, leading to irreversible damage and, in severe cases, complete organ failure. Remarkably, MB has been shown to reverse such damage in animal models, and there is a great deal of interest in upscaling MB production for expanded clinical trials. Such efforts, however, are currently hampered as (1) the natural rate of MB production rate by methanotrophs is low, (2) the use of methane as a substrate for MB production is problematic as it is explosive in air, (3) there is limited understanding of the entire pathway of MB biosynthesis, and (4) the most attractive form of MB is produced by Methylocystis sp. strain SB2, a methanotroph that is genetically intractable. Herein, we report heterologous biosynthesis of MB from Methylocystis sp. strain SB2 in an alternative methanotroph, Methylosinus trichosporium OB3b, not only on methane but also on methanol. As a result, the strategy described herein not only facilitates enhanced MB production but also provides opportunities to construct various mutants to delineate the entire pathway of MB biosynthesis, as well as the creation of modified forms of MB that may have enhanced therapeutic value.
Assuntos
Imidazóis , Methylocystaceae , Methylosinus trichosporium , Oligopeptídeos , Methylosinus trichosporium/metabolismo , Methylosinus trichosporium/genética , Imidazóis/metabolismo , Oligopeptídeos/metabolismo , Methylocystaceae/metabolismo , Methylocystaceae/genética , Metano/metabolismo , Engenharia Metabólica/métodosRESUMO
In Wilson disease (WD), liver copper (Cu) excess, caused by mutations in the ATPase Cu transporting beta (ATP7B), has been extensively studied. In contrast, in the gastrointestinal tract, responsible for dietary Cu uptake, ATP7B malfunction is poorly explored. We therefore investigated gut biopsies from WD patients and compared intestines from two rodent WD models and from human ATP7B knock-out intestinal cells to their respective wild-type controls. We observed gastrointestinal (GI) inflammation in patients, rats and mice lacking ATP7B. Mitochondrial alterations and increased intestinal leakage were observed in WD rats, Atp7b-/- mice and human ATP7B KO Caco-2 cells. Proteome analyses of intestinal WD homogenates revealed profound alterations of energy and lipid metabolism. The intestinal damage in WD animals and human ATP7B KO cells did not correlate with absolute Cu elevations, but likely reflects intracellular Cu mislocalization. Importantly, Cu depletion by the high-affinity Cu chelator methanobactin (MB) restored enterocyte mitochondria, epithelial integrity, and resolved gut inflammation in WD rats and human WD enterocytes, plausibly via autophagy-related mechanisms. Thus, we report here before largely unrecognized intestinal damage in WD, occurring early on and comprising metabolic and structural tissue damage, mitochondrial dysfunction, and compromised intestinal barrier integrity and inflammation, that can be resolved by high-affinity Cu chelation treatment.
Assuntos
ATPases Transportadoras de Cobre , Cobre , Degeneração Hepatolenticular , Mucosa Intestinal , Camundongos Knockout , Degeneração Hepatolenticular/metabolismo , Degeneração Hepatolenticular/patologia , Degeneração Hepatolenticular/tratamento farmacológico , Animais , Humanos , ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , Cobre/metabolismo , Ratos , Camundongos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Mucosa Intestinal/efeitos dos fármacos , Masculino , Células CACO-2 , Feminino , Adulto , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Intestinos/patologia , Intestinos/efeitos dos fármacos , Adulto JovemRESUMO
IMPORTANCE: Aerobic methanotrophs play a critical role in the global carbon cycle, particularly in controlling net emissions of methane to the atmosphere. As methane is a much more potent greenhouse gas than carbon dioxide, there is increasing interest in utilizing these microbes to mitigate future climate change by increasing their ability to consume methane. Any such efforts, however, require a detailed understanding of how to manipulate methanotrophic activity. Herein, we show that methanotrophic activity is strongly controlled by MmoD, i.e., MmoD regulates methanotrophy through the post-transcriptional regulation of the soluble methane monooxygenase and controls the ability of methanotrophs to collect copper. Such data are likely to prove quite useful in future strategies to enhance the use of methanotrophs to not only reduce methane emissions but also remove methane from the atmosphere.
Assuntos
Methylosinus trichosporium , Methylosinus trichosporium/genética , Oxigenases/genética , Metano , CobreRESUMO
Copper plays a critical role in controlling greenhouse gas emissions as it is a key component of the particulate methane monooxygenase and nitrous oxide reductase. Some methanotrophs excrete methanobactin (MB) that has an extremely high copper affinity. As a result, MB may limit the ability of other microbes to gather copper, thereby decreasing their activity as well as impacting microbial community composition. Here, we show using forest soil microcosms that multiple forms of MB; MB from Methylosinus trichosporium OB3b (MB-OB3b) and MB from Methylocystis sp. strain SB2 (MB-SB2) increased nitrous oxide (N2 O) production as well caused significant shifts in microbial community composition. Such effects, however, were mediated by the amount of copper in the soils, with low-copper soil microcosms showing the strongest response to MB. Furthermore, MB-SB2 had a stronger effect, likely due to its higher affinity for copper. The presence of either form of MB also inhibited nitrite reduction and generally increased the presence of genes encoding for the iron-containing nitrite reductase (nirS) over the copper-dependent nitrite reductase (nirK). These data indicate the methanotrophic-mediated production of MB can significantly impact multiple steps of denitrification, as well as have broad effects on microbial community composition of forest soils.
Assuntos
Methylocystaceae , Methylosinus trichosporium , Cobre , Óxido Nitroso , Methylocystaceae/genética , Methylosinus trichosporium/genética , Nitrito Redutases , SoloRESUMO
Methanobactins (MBs) are ribosomally produced and post-translationally modified peptides (RiPPs) that are used by methanotrophs for copper acquisition. The signature post-translational modification of MBs is the formation of two heterocyclic groups, either an oxazolone, pyrazinedione or imidazolone group, with an associated thioamide from an X-Cys dipeptide. The precursor peptide (MbnA) for MB formation is found in a gene cluster of MB-associated genes. The exact biosynthetic pathway of MB formation is not yet fully understood, and there are still uncharacterized proteins in some MB gene clusters, particularly those that produce pyrazinedione or imidazolone rings. One such protein is MbnF, which is proposed to be a flavin monooxygenase (FMO) based on homology. To help to elucidate its possible function, MbnF from Methylocystis sp. strain SB2 was recombinantly produced in Escherichia coli and its X-ray crystal structure was resolved to 2.6â Å resolution. Based on its structural features, MbnF appears to be a type A FMO, most of which catalyze hydroxylation reactions. Preliminary functional characterization shows that MbnF preferentially oxidizes NADPH over NADH, supporting NAD(P)H-mediated flavin reduction, which is the initial step in the reaction cycle of several type A FMO enzymes. It is also shown that MbnF binds the precursor peptide for MB, with subsequent loss of the leader peptide sequence as well as the last three C-terminal amino acids, suggesting that MbnF might be needed for this process to occur. Finally, molecular-dynamics simulations revealed a channel in MbnF that is capable of accommodating the core MbnA fragment minus the three C-terminal amino acids.
Assuntos
Methylocystaceae , Oxigenases de Função Mista , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , NADP/metabolismo , Methylocystaceae/química , Methylocystaceae/metabolismo , Cristalografia por Raios X , AminoácidosRESUMO
BACKGROUND & AIMS: Excess copper causes hepatocyte death in hereditary Wilson's disease (WD). Current WD treatments by copper-binding chelators may gradually reduce copper overload; they fail, however, to bring hepatic copper close to normal physiological levels. Consequently, lifelong daily dose regimens are required to hinder disease progression. This may result in severe issues due to nonadherence or unwanted adverse drug reactions and also due to drug switching and ultimate treatment failures. This study comparatively tested bacteria-derived copper binding agents-methanobactins (MBs)-for efficient liver copper depletion in WD rats as well as their safety and effect duration. METHODS: Copper chelators were tested in vitro and in vivo in WD rats. Metabolic cage housing allowed the accurate assessment of animal copper balances and long-term experiments related to the determination of minimal treatment phases. RESULTS: We found that copper-binding ARBM101 (previously known as MB-SB2) depletes WD rat liver copper dose dependently via fecal excretion down to normal physiological levels within 8 days, superseding the need for continuous treatment. Consequently, we developed a new treatment consisting of repetitive cycles, each of â¼1 week of ARBM101 applications, followed by months of in-between treatment pauses to ensure a healthy long-term survival in WD rats. CONCLUSIONS: ARBM101 safely and efficiently depletes excess liver copper from WD rats, thus allowing for short treatment periods as well as prolonged in-between rest periods.
Assuntos
Degeneração Hepatolenticular , Ratos , Animais , Degeneração Hepatolenticular/tratamento farmacológico , Degeneração Hepatolenticular/metabolismo , Cobre , Eliminação Hepatobiliar , Fígado/metabolismo , Quelantes/farmacologia , Quelantes/uso terapêuticoRESUMO
Methanotrophs require copper for their activity as it plays a critical role in the oxidation of methane to methanol. To sequester copper, some methanotrophs secrete a copper-binding compound termed methanobactin (MB). MB, after binding copper, is reinternalized via a specific outer membrane TonB-dependent transporter (TBDT). Methylosinus trichosporium OB3b has two such TBDTs (MbnT1 and MbnT2) that enable M. trichosporium OB3b to take up not only its own MB (MB-OB3b) but also heterologous MB produced from other methanotrophs, e.g., MB of Methylocystis sp. strain SB2 (MB-SB2). Here, we show that uptake of copper in the presence of heterologous MB-SB2 can either be achieved by initiating transcription of mbnT2 or by using its own MB-OB3b to extract copper from MB-SB2. Transcription of mbnT2 is mediated by the N-terminal signaling domain of MbnT2 together with an extracytoplasmic function sigma factor and an anti-sigma factor encoded by mbnI2 and mbnR2, respectively. Deletion of mbnI2R2 or excision of the N-terminal region of MbnT2 abolished induction of mbnT2. However, copper uptake from MB-SB2 was still observed in M. trichosporium OB3b mutants that were defective in MbnT2 induction/function, suggesting another mechanism for uptake copper-loaded MB-SB2. Additional deletion of MB-OB3b synthesis genes in the M. trichosporium OB3b mutants defective in MbnT2 induction/function disrupted their ability to take up copper in the presence of MB-SB2, indicating a role of MB-OB3b in copper extraction from MB-SB2. IMPORTANCE Methanotrophs play a critical role in the global carbon cycle, as well as in future strategies for mitigating climate change through their consumption of methane, a trace atmospheric gas much more potent than carbon dioxide in global warming potential. Copper uptake is critical for methanotrophic activity, and here, we show different approaches for copper uptake. This study expands our knowledge and understanding of how methanotrophs collect and compete for copper, and such information may be useful in future manipulation of methanotrophs for a variety of environmental and industrial applications.
Assuntos
Methylocystaceae , Methylosinus trichosporium , Methylosinus trichosporium/genética , Methylosinus trichosporium/metabolismo , Cobre/metabolismo , Metanol/metabolismo , Dióxido de Carbono/metabolismo , Methylocystaceae/genética , Methylocystaceae/química , Methylocystaceae/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Metano/metabolismoRESUMO
Methylmercury is a potent neurotoxin that biomagnifies through food webs and which production depends on anaerobic microbial uptake of inorganic mercury (Hg) species. One outstanding knowledge gap in understanding Hg methylation is the nature of bioavailable Hg species. It has become increasingly obvious that Hg bioavailability is spatially diverse and temporally dynamic but current models are mostly built on single thiolated ligand systems, omitting ligand exchanges and interactions, or the inclusion of dissolved gaseous phases. In this study, we used a whole-cell anaerobic biosensor to determine the role of a mixture of thiolated ligands on Hg bioavailability. Serendipitously, we discovered how the diffusion of trace amounts of exogenous biogenic H2 S, originating from anaerobic microbial ligand degradation, can alter Hg speciation - away from H2 S production site - to form bioavailable species. Regardless of its origins, H2 S stands as a mobile mediator of microbial Hg metabolism, connecting spatially separated microbial communities. At a larger scale, global planetary changes are expected to accelerate the production and mobilization of H2 S and Hg, possibly leading to increased production of the potent neurotoxin; this work provides mechanistic insights into the importance of co-managing biogeochemical cycle disruptions.
Assuntos
Mercúrio , Compostos de Metilmercúrio , Poluentes Químicos da Água , Anaerobiose , Ligantes , Mercúrio/metabolismo , Compostos de Metilmercúrio/metabolismo , Neurotoxinas , Poluentes Químicos da Água/metabolismoRESUMO
Aerobic methanotrophic activity is highly dependent on copper availability, and methanotrophs have developed multiple strategies to collect copper. Specifically, when copper is limiting (ambient concentrations less than 1 µM), some methanotrophs produce and secret a small modified peptide (less than 1,300 Da) termed methanobactin (MB) that binds copper with high affinity. As MB is secreted into the environment, other microbes that require copper for their metabolism may be inhibited as MB may make copper unavailable; e.g., inhibition of denitrifiers as complete conversion nitrate to dinitrogen involves multiple enzymes, some of which are copper-dependent. Of key concern is inhibition of the copper-dependent nitrous oxide reductase (NosZ), the only known enzyme capable of converting nitrous oxide (N2O) to dinitrogen. Herein, we show that different forms of MB differentially affect copper uptake and N2O reduction by Pseudomonas stutzeri strain DCP-Ps1 (that expresses clade I NosZ) and Dechloromonas aromatica strain RCB (that expresses clade II NosZ). Specifically, in the presence of MB from Methylocystis sp. strain SB2 (SB2-MB), copper uptake and nosZ expression were more significantly reduced than in the presence of MB from Methylosinus trichosporium OB3b (OB3b-MB). Further, N2O accumulation increased more significantly for both P. stutzeri strain DCP-Ps1 and D. aromatica strain RCB in the presence of SB2-MB versus OB3b-MB. These data illustrate that copper competition between methanotrophs and denitrifying bacteria can be significant and that the extent of such competition is dependent on the form of MB that methanotrophs produce. IMPORTANCE Herein, it was demonstrated that the different forms of methanobactin differentially enhance N2O emissions from Pseudomonas stutzeri strain DCP-Ps1 (harboring clade I nitrous oxide reductase) and Dechloromonas aromatica strain RCB (harboring clade II nitrous oxide reductase). This work contributes to our understanding of how aerobic methanotrophs compete with denitrifiers for the copper uptake and also suggests how MBs prevent copper collection by denitrifiers, thus downregulating expression of nitrous oxide reductase. This study provides critical information for enhanced understanding of microbe-microbe interactions that are important for the development of better predictive models of net greenhouse gas emissions (i.e., methane and nitrous oxide) that are significantly controlled by microbial activity.
Assuntos
Methylocystaceae , Methylosinus trichosporium , Pseudomonas stutzeri , Betaproteobacteria , Cobre/metabolismo , Imidazóis , Methylocystaceae/metabolismo , Óxido Nitroso/metabolismo , Oligopeptídeos , Pseudomonas stutzeri/metabolismoRESUMO
Copper is an important component of methanotrophic physiology, as it controls the expression and activity of alternative forms of methane monooxygenase (MMO). To collect copper, some methanotrophs secrete a chalkophore- or copper-binding compound called methanobactin (MB). MB is a ribosomally synthesized posttranslationally modified polypeptide (RiPP) that, after binding copper, is collected by MbnT, a TonB-dependent transporter (TBDT). Structurally different forms of MB have been characterized, and here, we show that different forms of MB are collected by specific TBDTs. Further, we report that in the model methanotroph, Methylosinus trichosporium OB3b, expression of the TBDT required for uptake of a different MB made by Methylocystis sp. strain SB2 (MB-SB2) is induced in the presence of MB-SB2, suggesting that methanotrophs have developed specific machinery and regulatory systems to actively take up MB from other methanotrophs for copper collection. Moreover, the canonical "copper switch" in M. trichosporium OB3b that controls expression of alternative MMOs is apparent if one of the two TBDTs required for MB-OB3b and MB-SB2 uptake is knocked out, but is disrupted if both TBDTs are knocked out. These data indicate that MB uptake, including the uptake of exogenous MB, plays an important role in the copper switch in M. trichosporium OB3b and, thus, overall activity. Based on these data, we propose a revised model for the copper switch in this methanotroph that involves MB uptake. IMPORTANCE In this study, we demonstrate that different TBDTs in the model methanotroph Methylosinus trichosporium OB3b are responsible for uptake of either endogenous MB or exogenous MB. Interestingly, the presence of exogenous MB induces expression of its specific TBDT in M. trichosporium OB3b, suggesting that this methanotroph is able to actively take up MB produced by others. This work contributes to our understanding of how microbes collect and compete for copper and also helps inform how such uptake coordinates the expression of different forms of methane monooxygenase. Such studies are likely to be very important to develop a better understanding of methanotrophic interactions via synthesis and secretion of secondary metabolites such as methanobactin and thus provide additional means whereby these microbes can be manipulated for a variety of environmental and industrial purposes.
Assuntos
Methylosinus trichosporium , Cobre , Imidazóis , Methylosinus trichosporium/genética , Oligopeptídeos , Oxigenases/genéticaRESUMO
Methanobactins (MBs) are ribosomally synthesized and posttranslationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The posttranslational modification that defines MBs is the formation of two heterocyclic groups with associated thioamines from X-Cys dipeptide sequences. Both heterocyclic groups in the MB from Methylosinus trichosporium OB3b (MB-OB3b) are oxazolone groups. The precursor gene for MB-OB3b is mbnA, which is part of a gene cluster that contains both annotated and unannotated genes. One of those unannotated genes, mbnC, is found in all MB operons and, in conjunction with mbnB, is reported to be involved in the formation of both heterocyclic groups in all MBs. To determine the function of mbnC, a deletion mutation was constructed in M. trichosporium OB3b, and the MB produced from the ΔmbnC mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry, and solution nuclear magnetic resonance (NMR) spectroscopy. MB-OB3b from the ΔmbnC mutant was missing the C-terminal Met and was also found to contain a Pro and a Cys in place of the pyrrolidinyl-oxazolone-thioamide group. These results demonstrate MbnC is required for the formation of the C-terminal pyrrolidinyl-oxazolone-thioamide group from the Pro-Cys dipeptide, but not for the formation of the N-terminal 3-methylbutanol-oxazolone-thioamide group from the N-terminal dipeptide Leu-Cys. IMPORTANCE A number of environmental and medical applications have been proposed for MBs, including bioremediation of toxic metals and nanoparticle formation, as well as the treatment of copper- and iron-related diseases. However, before MBs can be modified and optimized for any specific application, the biosynthetic pathway for MB production must be defined. The discovery that mbnC is involved in the formation of the C-terminal oxazolone group with associated thioamide but not for the formation of the N-terminal oxazolone group with associated thioamide in M. trichosporium OB3b suggests the enzymes responsible for posttranslational modification(s) of the two oxazolone groups are not identical.
Assuntos
Methylosinus trichosporium , Cobre/metabolismo , Imidazóis/metabolismo , Oligopeptídeos/metabolismo , Oxazolona/metabolismo , Oxigenases/metabolismoRESUMO
Aerobic methanotrophy is strongly controlled by copper, and methanotrophs are known to use different mechanisms for copper uptake. Some methanotrophs secrete a modified polypeptide-methanobactin-while others utilize a surface-bound protein (MopE) and a secreted form of it (MopE*) for copper collection. As different methanotrophs have different means of sequestering copper, competition for copper significantly impacts methanotrophic activity. Herein, we show that Methylomicrobium album BG8, Methylocystis sp. strain Rockwell, and Methylococcus capsulatus Bath, all lacking genes for methanobactin biosynthesis, are not limited for copper by multiple forms of methanobactin. Interestingly, Mm. album BG8 and Methylocystis sp. strain Rockwell were found to have genes similar to mbnT that encodes for a TonB-dependent transporter required for methanobactin uptake. Data indicate that these methanotrophs "steal" methanobactin and such "theft" enhances the ability of these strains to degrade methylmercury, a potent neurotoxin. Further, when mbnT was deleted in Mm. album BG8, methylmercury degradation in the presence of methanobactin was indistinguishable from when MB was not added. Mc. capsulatus Bath lacks anything similar to mbnT and was unable to degrade methylmercury either in the presence or absence of methanobactin. Rather, Mc. capsulatus Bath appears to rely on MopE/MopE* for copper collection. Finally, not only does Mm. album BG8 steal methanobactin, it synthesizes a novel chalkophore, suggesting that some methanotrophs utilize both competition and cheating strategies for copper collection. Through a better understanding of these strategies, methanotrophic communities may be more effectively manipulated to reduce methane emissions and also enhance mercury detoxification in situ.
Assuntos
Compostos de Metilmercúrio , Methylosinus trichosporium , Cobre/metabolismo , Imidazóis/metabolismo , Compostos de Metilmercúrio/metabolismo , Methylosinus trichosporium/genética , Methylosinus trichosporium/metabolismo , Oligopeptídeos/metabolismoRESUMO
Methanotrophic bacteria catalyze the aerobic oxidation of methane to methanol using Cu-containing enzymes, thereby exerting a modulating influence on the global methane cycle. To facilitate the acquisition of Cu ions, some methanotrophic bacteria secrete small modified peptides known as "methanobactins," which strongly bind Cu and function as an extracellular Cu recruitment relay, analogous to siderophores and Fe. In addition to Cu, methanobactins form complexes with other late transition metals, including the Group 12 transition metals Zn, Cd, and Hg, although the interplay among solution-phase configurations, metal interactions, and the spectroscopic signatures of methanobactin-metal complexes remains ambiguous. In this study, the complexation of Zn, Cd, and Hg by methanobactin from Methylocystis sp. strain SB2 was studied using a combination of absorbance, fluorescence, extended x-ray absorption fine structure (EXAFS) spectroscopy, and time-dependent density functional theory (TD-DFT) calculations. We report changes in sample absorbance and fluorescence spectral dynamics, which occur on a wide range of experimental timescales and characterize a clear stoichiometric complexation dependence. Mercury L3-edge EXAFS and TD-DFT calculations suggest a linear model for HgS coordination, and TD-DFT suggests a tetrahedral model for Zn2+ and Cd2+. We observed an enhancement in the fluorescence of methanobactin upon interaction with transition metals and propose a mechanism of complexation-hindered isomerization drawing inspiration from the wild-type Green Fluorescent Protein active site. Collectively, our results represent the first combined computational and experimental spectroscopy study of methanobactins and shed new light on molecular interactions and dynamics that characterize complexes of methanobactins with Group 12 transition metals.
Assuntos
Quelantes/química , Complexos de Coordenação/química , Imidazóis/química , Methylocystaceae/química , Oligopeptídeos/química , Elementos de Transição/química , Quelantes/efeitos da radiação , Complexos de Coordenação/efeitos da radiação , Fluorescência , Imidazóis/efeitos da radiação , Luz , Metais Pesados/química , Metais Pesados/efeitos da radiação , Estrutura Molecular , Oligopeptídeos/efeitos da radiação , Espectrometria de Fluorescência , Elementos de Transição/efeitos da radiaçãoRESUMO
Methanobactins (MBs) are small (<1,300-Da) posttranslationally modified copper-binding peptides and represent the extracellular component of a copper acquisition system in some methanotrophs. Interestingly, MBs can bind a range of metal ions, with some being reduced after binding, e.g., Cu2+ reduced to Cu+. Other metal ions, however, are bound but not reduced, e.g., K+. The source of electrons for selective metal ion reduction has been speculated to be water but never empirically shown. Here, using H218O, we show that when MBs from Methylocystis sp. strain SB2 (MB-SB2) and Methylosinus trichosporium OB3b (MB-OB3) were incubated in the presence of either Au3+, Cu2, or Ag+, 18,18O2 and free protons were released. No 18,18O2 production was observed in the presence of either MB-SB2 or MB-OB3b alone, gold alone, copper alone, or silver alone or when K+ or Mo2+ was incubated with MB-SB2. In contrast to MB-OB3b, MB-SB2 binds Fe3+ with an N2S2 coordination and will also reduce Fe3+ to Fe2+. Iron reduction was also found to be coupled to the oxidation of 2H2O and the generation of O2. MB-SB2 will also couple Hg2+, Ni2+, and Co2+ reduction to the oxidation of 2H2O and the generation of O2, but MB-OB3b will not, ostensibly as MB-OB3b binds but does not reduce these metal ions. To determine if the O2 generated during metal ion reduction by MB could be coupled to methane oxidation, 13CH4 oxidation by Methylosinus trichosporium OB3b was monitored under anoxic conditions. The results demonstrate that O2 generation from metal ion reduction by MB-OB3b can support methane oxidation. IMPORTANCE The discovery that MB will couple the oxidation of H2O to metal ion reduction and the release of O2 suggests that methanotrophs expressing MB may be able to maintain their activity under hypoxic/anoxic conditions through the "self-generation" of dioxygen required for the initial oxidation of methane to methanol. Such an ability may be an important factor in enabling methanotrophs to not only colonize the oxic-anoxic interface where methane concentrations are highest but also tolerate significant temporal fluctuations of this interface. Given that genomic surveys often show evidence of aerobic methanotrophs within anoxic zones, the ability to express MB (and thereby generate dioxygen) may be an important parameter in facilitating their ability to remove methane, a potent greenhouse gas, before it enters the atmosphere.
Assuntos
Imidazóis/metabolismo , Metais Pesados/metabolismo , Metano/metabolismo , Methylocystaceae/metabolismo , Oligopeptídeos/metabolismo , Oxigênio/química , Água/química , Metais Pesados/química , OxirreduçãoRESUMO
Aerobic methane-oxidizing bacteria of the Alphaproteobacteria have been found to express a novel ribosomally synthesized post-translationally modified polypeptide (RiPP) termed methanobactin (MB). The primary function of MB in these microbes appears to be for copper uptake, but MB has been shown to have multiple capabilities, including oxidase, superoxide dismutase and hydrogen peroxide reductase activities, the ability to detoxify mercury species, as well as acting as an antimicrobial agent. Herein, we describe the diversity of known MBs as well as the genetics underlying MB biosynthesis. We further propose based on bioinformatics analyses that some methanotrophs may produce novel forms of MB that have yet to be characterized. We also discuss recent findings documenting that MBs play an important role in controlling copper availability to the broader microbial community, and as a result can strongly affect the activity of microbes that require copper for important enzymatic transformations, e.g. conversion of nitrous oxide to dinitrogen. Finally, we describe procedures for the detection/purification of MB, as well as potential medical and industrial applications of this intriguing RiPP.
Assuntos
Alphaproteobacteria/genética , Alphaproteobacteria/metabolismo , Imidazóis/química , Metano/metabolismo , Oligopeptídeos/química , Oligopeptídeos/genética , Cobre/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Oxirredução , Processamento de Proteína Pós-TraducionalRESUMO
Microbial production of the neurotoxin methylmercury (MeHg) is a significant health and environmental concern, as it can bioaccumulate and biomagnify in the food web. A chalkophore or a copper-binding compound, termed methanobactin (MB), has been shown to form strong complexes with mercury [as Hg(II)] and also enables some methanotrophs to degrade MeHg. It is unknown, however, if Hg(II) binding with MB can also impede Hg(II) methylation by other microbes. Contrary to expectations, MB produced by the methanotroph Methylosinus trichosporium OB3b (OB3b-MB) enhanced the rate and efficiency of Hg(II) methylation more than that observed with thiol compounds (such as cysteine) by the mercury-methylating bacteria Desulfovibrio desulfuricans ND132 and Geobacter sulfurreducens PCA. Compared to no-MB controls, OB3b-MB decreased the rates of Hg(II) sorption and internalization, but increased methylation by 5- to 7-fold, suggesting that Hg(II) complexation with OB3b-MB facilitated exchange and internal transfer of Hg(II) to the HgcAB proteins required for methylation. Conversely, addition of excess amounts of OB3b-MB or a different form of MB from Methylocystis strain SB2 (SB2-MB) inhibited Hg(II) methylation, likely due to greater binding of Hg(II). Collectively, our results underscore the complex roles of microbial exogenous metal-scavenging compounds in controlling net production and bioaccumulation of MeHg in the environment.IMPORTANCE Some anaerobic microorganisms convert inorganic mercury (Hg) into the neurotoxin methylmercury, which can bioaccumulate and biomagnify in the food web. While the genetic basis of microbial mercury methylation is known, factors that control net methylmercury production in the environment are still poorly understood. Here, it is shown that mercury methylation can be substantially enhanced by one form of an exogenous copper-binding compound (methanobactin) produced by some methanotrophs, but not by another. This novel finding illustrates that complex interactions exist between microbes and that these interactions can potentially affect the net production of methylmercury in situ.
Assuntos
Desulfovibrio desulfuricans/metabolismo , Poluentes Ambientais/metabolismo , Geobacter/metabolismo , Imidazóis/metabolismo , Mercúrio/metabolismo , Methylosinus trichosporium/metabolismo , Oligopeptídeos/metabolismo , MetilaçãoRESUMO
Aerobic methanotrophs are an intriguing group of microbes with the singular ability to consume methane as their sole source of carbon and energy. As such, methanotrophs are receiving increased attention to control methane emissions to limit future climate change. Methanotrophs have a wide range of other applications, including pollutant remediation and methane valorization (e.g. conversion of methane to protein, bioplastics, and biodiesel amongst other products). Methanotrophs also produce a novel copper-binding compound, methanobactin, that has significant potential for the treatment of copper-related human pathologies. Here we provide an overview of aerobic methanotrophy, describe current and future applications of these unique microbes, as well as discuss various strategies one can consider to better realize the opportunities these microbes present.
Assuntos
Tecnologia Biomédica/métodos , Metabolismo Energético/fisiologia , Recuperação e Remediação Ambiental/métodos , Indústrias/métodos , Metano/metabolismo , Methanobacteriales/metabolismo , Bactérias/metabolismo , Fungos/metabolismo , Regulação Bacteriana da Expressão Gênica , Imidazóis/metabolismo , Methanobacteriales/classificação , Methanobacteriales/fisiologia , Oligopeptídeos/metabolismoRESUMO
Nitrate and nitrite reduction are of paramount importance for nitrogen assimilation and anaerobic metabolism, and understanding the specific roles of each participating reductase is necessary to describe the biochemical balance that dictates cellular responses to their environments. The soluble, cytoplasmic siroheme NADH-nitrite reductase (Nir) in Escherichia coli is necessary for nitrate/nitrite assimilation but has also been reported to either "detoxify" nitrite, or to carry out fermentative ammonification in support of anaerobic catabolism. Theoretically, nitrite detoxification would be important for anaerobic growth on nitrate, during which excess nitrite would be reduced to ammonium. Fermentative ammonification by Nir would be important for maximization of non-respiratory ATP production during anaerobic growth in the presence of nitrite. Experiments reported here were designed to test the potential role of Nir in fermentative ammonification directly by growing E. coli along with mutant strains lacking Nir or the respiratory nitrite reductase (Nrf) under anaerobic conditions in defined media while monitoring nitrogen utilization and fermentation metabolites. To focus on the role of Nir in fermentative ammonification, pH control was used in most experiments to eliminate nitrite toxicity due to nitric acid formation. Our results demonstrate that Nir confers a significant benefit during fermentative growth that reflects fermentative ammonification rather than detoxification. We conclude that fermentative ammonification by Nir allows for the energetically favorable fermentation of glucose to formate and acetate. These results and conclusions are discussed in light of the roles of Nir in other bacteria and in plants.
Assuntos
Escherichia coli/enzimologia , Fermentação , Nitrito Redutase (NAD(P)H)/metabolismo , Compostos de Amônio/metabolismo , Anaerobiose , Escherichia coli/genética , Nitrito Redutase (NAD(P)H)/genética , Nitrito Redutases/genética , Nitrito Redutases/metabolismo , Nitritos/metabolismoRESUMO
BACKGROUND & AIMS: In Wilson disease, ATP7B mutations impair copper excretion into bile. Hepatic copper accumulation may induce mild to moderate chronic liver damage or even acute liver failure. Etiologic factors for this heterogeneous phenotype remain enigmatic. Liver steatosis is a frequent finding in Wilson disease patients, suggesting that impaired copper homeostasis is linked with liver steatosis. Hepatic mitochondrial function is affected negatively both by copper overload and steatosis. Therefore, we addressed the question of whether a steatosis-promoting high-calorie diet aggravates liver damage in Wilson disease via amplified mitochondrial damage. METHODS: Control Atp7b+/- and Wilson disease Atp7b-/- rats were fed either a high-calorie diet (HCD) or a normal diet. Copper chelation using the high-affinity peptide methanobactin was used in HCD-fed Atp7b-/- rats to test for therapeutic reversal of mitochondrial copper damage. RESULTS: In comparison with a normal diet, HCD feeding of Atp7b-/- rats resulted in a markedly earlier onset of clinically apparent hepatic injury. Strongly increased mitochondrial copper accumulation was observed in HCD-fed Atp7b-/- rats, correlating with severe liver injury. Mitochondria presented with massive structural damage, increased H2O2 emergence, and dysfunctional adenosine triphosphate production. Hepatocellular injury presumably was augmented as a result of oxidative stress. Reduction of mitochondrial copper by methanobactin significantly reduced mitochondrial impairment and ameliorated liver damage. CONCLUSIONS: A high-calorie diet severely aggravates hepatic mitochondrial and hepatocellular damage in Wilson disease rats, causing an earlier onset of the disease and enhanced disease progression.
Assuntos
Dieta , Degeneração Hepatolenticular/patologia , Fígado/patologia , Mitocôndrias/patologia , Animais , Ácidos e Sais Biliares/biossíntese , Cobre/sangue , ATPases Transportadoras de Cobre/metabolismo , Progressão da Doença , Fígado Gorduroso/patologia , Feminino , Hepatócitos/patologia , Hepatócitos/ultraestrutura , Degeneração Hepatolenticular/sangue , Inflamação/patologia , Lipídeos/biossíntese , Fígado/metabolismo , Fígado/ultraestrutura , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Peptídeos/farmacologia , Proteoma/metabolismo , RatosRESUMO
Nonendospore-forming pathogenic bacteria in the long-term survival (LTS) phase can remain viable for months or years and may show reduced susceptibility to various antimicrobial interventions. In the present study, we investigated the response of LTS phase Salmonella enterica serovar Typhimurium (ATCC 14028) to ultraviolet (UV) radiation in 0.85% (w/v) saline and apple juice and the extent of sublethal injury in LTS phase survivors. The LTS-phase Salmonella Typhimurium cells were cultured at 35°C for 14 days in tryptic soy broth with 0.6% (w/v) yeast extract (TSBYE). Exponential- and stationary-phase cells, cultured in TSBYE (35°C) for 2.5 and 18 h, respectively, served as control samples. Cells (107 CFU [colony-forming unit]/mL) from each physiological state were exposed to UV light in saline (80 µW/cm2) and apple juice (1500 µW/cm2). The Salmonella Typhimurium survivors were plated for enumeration on either tryptic soy agar with 0.6% yeast extract or xylose-lysine-tergitol 4 (XLT4) agar and colonies counted after incubation (35°C, 24 h). Of all the growth phases tested, LTS phase cells were consistently impacted the least by UV treatment (p < 0.05). In saline, D-values of exponential, stationary, and LTS Salmonella Typhimurium were 0.35, 0.38, and 0.49 min, respectively. D-values in apple juice at pH 3.63 and pH 5.65 were 2.52, 3.19, and 3.57 min and 3.24, 3.50, and 4.18 min, respectively. UV radiation (80 µW/cm2) of Salmonella Typhimurium in saline for 2.5 min reduced the number of exponential- and stationary-phase cells by â¼7.19 and 6.30 log10 CFU/mL, respectively. In contrast, LTS cells were only reduced by 5.08 log10 CFU/mL. Among the three physiological states, LTS phase cells had the least sublethal injury in the surviving population (p < 0.05). These results indicate that the LTS state cross-protects Salmonella Typhimurium against UV radiation and should be considered in determination of the UV radiation D-value for this pathogen.