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
Methane, a potent greenhouse gas, requires sustainable mitigation strategies. Here, the microbial upcycling of methane to phytoene, a valuable colorless carotenoid with applications in the cosmeceutical industry was demonstrated. To achieve this goal, a stepwise metabolic engineering approach was employed in Methylocystis sp. MJC1, a methane-oxidizing bacterium. The incorporation of crtE and crtB genes from Deinococcus radiodurans R1 established the phytoene biosynthetic pathway. This pathway was fine-tuned through promoter optimization, resulting in a phytoene production of 450 µg/L from 37 mmol/L methane. Disrupting the ackA gene reduced a by-product, acetate, by 50 % and increased phytoene production by 56 %. Furthermore, overexpressing the dxs gene boosted phytoene titer 3-fold. The optimized strain produced 15 mg/L phytoene from 2 mol/L methane in fed-batch fermentation, a 4-fold increase in phytoene titer and 4-fold in yield. This demonstrates Methylocystis sp. MJC1's potential for efficient phytoene production and presents a novel approach for greenhouse gas reduction.
Assuntos
Engenharia Metabólica , Metano , Methylocystaceae , Metano/metabolismo , Engenharia Metabólica/métodos , Methylocystaceae/metabolismo , Methylocystaceae/genética , Carotenoides/metabolismo , Fermentação , Deinococcus/metabolismo , Deinococcus/genética , Regiões Promotoras GenéticasRESUMO
Methanotrophs are crucial in keeping environmental CH4 emissions in check. However, the contributions of different groups of methanotrophs at terrestrial CH4-oxidation hotspots, such as the oxic-anoxic interface of rice paddies, have shown considerable inconsistency across observations. To address the knowledge gap regarding this inconsistency, methanotrophic microbiomes were enriched from paddy soils in well-mixed CH4-fed batch reactors under six different incubation conditions, prepared as combinations of two CH4 mixing ratios (0.5 and 10%) and three supplemented Cu2+ concentrations (0, 2, and 10 µM). Monitoring of temporal community shifts in these cultures revealed a dominance of Methylocystis spp. in all 0.5%-CH4 cultures, while methanotrophs affiliated to Gammaproteobacteria dominated the 10%-CH4 cultures that were less consistent both temporally and across conditions. The shotgun metagenome analyses of the 0.5%-CH4 cultures corroborated the Methylocystis dominance and, interestingly, showed that copper deficiency did not select for mmoXYZ-possessing methanotrophs. Instead, a mbn cluster, accounting for approximately 5% of the Methylocystis population, was identified, suggesting the ecological significance of methanobactin in Cu-deficient methanotrophy. These findings underscore the important role of Methylocystis spp. in mitigating emissions from terrestrial CH4 hotspots and suggest the feasibility of directed enrichment and/or isolation of Methylocystis spp. for utilization in, for example, methanobactin and polyhydroxybutyrate production.
Assuntos
Metano , Methylococcaceae , Methylocystaceae , Metano/metabolismo , Methylococcaceae/metabolismo , Methylocystaceae/metabolismo , Microbiologia do Solo , MicrobiotaRESUMO
Methane (CH4) and carbon dioxide (CO2) are the dominant greenhouse gases (GHGs) that are increasing at an alarming rate. Methanotrophs have emerged as potential CH4 and CO2 biorefineries. This study demonstrated the synchronous incorporation of CH4 and CO2 into polyhydroxybutyrate (PHB) for the first time using 13C-labeling experiments in methanotrophs. By supplying substantial amounts of CO2, PHB content was enhanced in all investigated type II methanotrophic strains by 140 %, 146 %, and 162 %. The highest content of PHB from CH4 and CO2 in flask-scale cultivation reached 38 % dry cell weight in Methylocystis sp. MJC1, in which carbon percentage in PHB from CO2 was 45 %. Flux balance analysis predicted the critical roles of crotonyl-CoA carboxylase/reductase and phosphoenolpyruvate carboxylase in CO2 recycling. This study provided proof of the conversion of GHGs into a valuable and practical product using methanotrophic bacteria, contributing to addressing GHG emissions.
Assuntos
Dióxido de Carbono , Hidroxibutiratos , Metano , Metano/metabolismo , Dióxido de Carbono/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Methylocystaceae/metabolismo , Isótopos de CarbonoRESUMO
Methanotrophs of the genus Methylocystis are frequently found in rice paddies. Although more than ten facultative methanotrophs have been reported since 2005, none of these strains was isolated from paddy soil. Here, a facultative methane-oxidizing bacterium, Methylocystis iwaonis SD4, was isolated and characterized from rhizosphere samples of rice plants in Nanjing, China. This strain grew well on methane or methanol but was able to grow slowly using acetate or ethanol. Moreover, strain SD4 showed sustained growth at low concentrations of methane (100 and 500 ppmv). M. iwaonis SD4 could utilize diverse nitrogen sources, including nitrate, urea, ammonium as well as dinitrogen. Strain SD4 possessed genes encoding both the particulate methane monooxygenase and the soluble methane monooxygenase. Simple and rapid genetic manipulation methods were established for this strain, enabling vector transformation and unmarked genetic manipulation. Fast growth rate and efficient genetic tools make M. iwaonis SD4 an ideal model to study facultative methanotrophs, and the ability to grow on low concentration of methane implies its potential in methane removal.
Assuntos
Metano , Methylocystaceae , Oryza , Rizosfera , Microbiologia do Solo , Oryza/microbiologia , Methylocystaceae/genética , Methylocystaceae/metabolismo , Methylocystaceae/isolamento & purificação , Metano/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , China , Metanol/metabolismoRESUMO
This study explores the ability of methanotrophs to convert biogas into biopolymers, addressing H2S as a limitation in the utilization of biogas as a carbon source for bioconversion. Transcriptomic analysis was conducted to understand the growth and changes in the expression patterns of Type I and II methanotrophs under varying H2S concentrations. Results suggested that Type II methanotrophs can possess a native H2S utilization pathway. Both Type I and II methanotrophs were evaluated for their growth and polyhydroxybutyrate (PHB) production from biogas. Methylocystis sp. MJC1 and Methylocystis sp. OK1 exhibited a maximum biomass production of 4.0 and 4.5 gDCW/L, respectively, in fed-batch culture, aligning with the transcriptome data. Furthermore, Methylocystis sp. MJC1 produced 2.9 g PHB/L from biogas through gas fermentation. These findings underscore biogas-based biotechnology as an innovative solution for environmental and industrial challenges with further optimization and productivity enhancement research expected to broaden the potential in this field.
Assuntos
Biocombustíveis , Hidroxibutiratos , Hidroxibutiratos/metabolismo , Fermentação , Methylocystaceae/metabolismo , Biomassa , Poliésteres/metabolismo , Metano/metabolismo , Técnicas de Cultura Celular por LotesRESUMO
Climate change and plastic pollution are likely the most relevant challenges for the environment in the 21st century. Developing cost-effective technologies for the bioconversion of methane (CH4) into polyhydroxyalkanoates (PHAs) could simultaneously mitigate CH4 emissions and boost the commercialization of biodegradable polymers. Despite the fact that the role of temperature, nitrogen deprivation, CH4:O2 ratio or micronutrients availability on the PHA accumulation capacity of methanotrophs has been carefully explored, there is still a need for optimization of the CH4-to-PHA bioconversion process prior to becoming a feasible platform in future biorefineries. In this study, the influence of different cultivation broth pH values (5.5, 7, 8.5 and 10) on bacterial biomass growth, CH4 bioconversion rate, PHA accumulation capacity and bacterial community structure was investigated in a stirred tank bioreactor under nitrogen deprivation conditions. Higher CH4 elimination rates were obtained at increasing pH, with a maximum value of 50.4 ± 2.7 g CH4·m-3·h-1 observed at pH 8.5. This was likely mediated by an increased ionic strength in the mineral medium, which enhanced the gas-liquid mass transfer. Interestingly, higher PHB accumulations were observed at decreasing pH, with the highest PHB contents recorded at a pH 5.5 (43.7 ± 3.4 %w·w-1). The strong selective pressure of low pH towards the growth of Type II methanotrophic bacteria could explain this finding. The genus Methylocystis increased its abundance from 34 % up to 85 and 90 % at pH 5.5 and 7, respectively. On the contrary, Methylocystis was less abundant in the community enriched at pH 8.5 (14 %). The accumulation of intracellular PHB as energy and carbon storage material allowed the maintenance of high CH4 biodegradation rates during 48 h after complete nitrogen deprivation. The results here obtained demonstrated for the first time a crucial and multifactorial role of pH on the bioconversion performance of CH4 into PHA.
Assuntos
Methylocystaceae , Poli-Hidroxialcanoatos , Poli-Hidroxibutiratos , Carbono/metabolismo , Metano/metabolismo , Methylocystaceae/metabolismo , Nitrogênio/metabolismo , Concentração de Íons de HidrogênioRESUMO
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
Methylocystis spp. are known to have a low salt tolerance (≤1.0% NaCl). Therefore, we tested various amino acids and other well-known osmolytes for their potential to act as an osmoprotectant under otherwise growth-inhibiting NaCl conditions. Adjustment of the medium to 10 mM asparagine had the greatest osmoprotective effect under severe salinity (1.50% NaCl), leading to partial growth recovery of strain SC2. The intracellular concentration of asparagine increased to 264 ± 57 mM, with a certain portion hydrolyzed to aspartate (4.20 ± 1.41 mM). In addition to general and oxidative stress responses, the uptake of asparagine specifically induced major proteome rearrangements related to the KEGG level 3 categories of "methane metabolism," "pyruvate metabolism," "amino acid turnover," and "cell division." In particular, various proteins involved in cell division (e.g., ChpT, CtrA, PleC, FtsA, FtsH1) and peptidoglycan synthesis showed a positive expression response. Asparagine-derived 13C-carbon was incorporated into nearly all amino acids. Both the exometabolome and the 13C-labeling pattern suggest that in addition to aspartate, the amino acids glutamate, glycine, serine, and alanine, but also pyruvate and malate, were most crucially involved in the osmoprotective effect of asparagine, with glutamate being a major hub between the central carbon and amino acid pathways. In summary, asparagine induced significant proteome rearrangements, leading to major changes in central metabolic pathway activity and the sizes of free amino acid pools. In consequence, asparagine acted, in part, as a carbon source for the growth recovery of strain SC2 under severe salinity. IMPORTANCE Methylocystis spp. play a major role in reducing methane emissions into the atmosphere from methanogenic wetlands. In addition, they contribute to atmospheric methane oxidation in upland soils. Although these bacteria are typical soil inhabitants, Methylocystis spp. are thought to have limited capacity to acclimate to salt stress. This called for a thorough study into potential osmoprotectants, which revealed asparagine as the most promising candidate. Intriguingly, asparagine was taken up quantitatively and acted, at least in part, as an intracellular carbon source under severe salt stress. The effect of asparagine as an osmoprotectant for Methylocystis spp. is an unexpected finding. It may provide Methylocystis spp. with an ecological advantage in wetlands, where these methanotrophs colonize the roots of submerged vascular plants. Collectively, our study offers a new avenue into research on compounds that may increase the resilience of Methylocystis spp. to environmental change.
Assuntos
Asparagina , Methylocystaceae , Asparagina/metabolismo , Methylocystaceae/metabolismo , Ácido Aspártico , Proteoma/metabolismo , Cloreto de Sódio/metabolismo , Carbono/metabolismo , Aminoácidos/metabolismo , Metano/metabolismo , Estresse Salino , Piruvatos/metabolismoRESUMO
Biodegradable polyhydroxybutyrate (PHB) can be produced from methane by some type II methanotroph such as the genus Methylocystis. This study presents the comparative genomic analysis of a newly isolated methanotroph, Methylocystis sp. MJC1 as a biodegradable PHB-producing platform strain. Methylocystis sp. MJC1 accumulates up to 44.5% of PHB based on dry cell weight under nitrogen-limiting conditions. To facilitate its development as a PHB-producing platform strain, the complete genome sequence of Methylocystis sp. MJC1 was assembled, functionally annotated, and compared with genomes of other Methylocystis species. Phylogenetic analysis has shown that Methylocystis parvus to be the closest species to Methylocystis sp. MJC1. Genome functional annotation revealed that Methylocystis sp. MJC1 contains all major type II methanotroph biochemical pathways such as the serine cycle, EMC pathway, and Krebs cycle. Interestingly, Methylocystis sp. MJC1 has both particulate and soluble methane monooxygenases, which are not commonly found among Methylocystis species. In addition, this species also possesses most of the RuMP pathway reactions, a characteristic of type I methanotrophs, and all PHB biosynthetic genes. These comparative analysis would open the possibility of future practical applications such as the development of organism-specific genome-scale models and application of metabolic engineering strategies to Methylocystis sp. MJC1.
Assuntos
Metano , Methylocystaceae , Filogenia , Metano/metabolismo , Genômica , Methylocystaceae/genética , Methylocystaceae/metabolismoRESUMO
Chlorimuron-ethyl is a widely used herbicide in agriculture. However, uncontrolled chlorimuron-ethyl application causes serious environmental problems. Chlorimuron-ethyl can be effectively degraded by microbes, but the underlying molecular mechanisms are not fully understood. In this study, we identified the possible pathways and key genes involved in chlorimuron-ethyl degradation by the Chenggangzhangella methanolivorans strain CHL1, a Methylocystaceae strain with the ability to degrade sulfonylurea herbicides. Using a metabolomics method, eight intermediate degradation products were identified, and three pathways, including a novel pyrimidine-ring-opening pathway, were found to be involved in chlorimuron-ethyl degradation by strain CHL1. Transcriptome sequencing indicated that three genes (atzF, atzD, and cysJ) are involved in chlorimuron-ethyl degradation by strain CHL1. The gene knock-out and complementation techniques allowed for the functions of the three genes to be identified, and the enzymes involved in the different steps of chlorimuron-ethyl degradation pathways were preliminary predicted. The results reveal a previously unreported pathway and the key genes of chlorimuron-ethyl degradation by strain CHL1, which have implications for attempts to enrich the biodegradation mechanism of sulfonylurea herbicides and to construct engineered bacteria in order to remove sulfonylurea herbicide residues from environmental media.
Assuntos
Herbicidas , Methylocystaceae , Poluentes do Solo , Biodegradação Ambiental , Herbicidas/metabolismo , Methylocystaceae/metabolismo , Pirimidinas/metabolismo , Poluentes do Solo/metabolismo , Compostos de Sulfonilureia/metabolismoRESUMO
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
The design of efficient cultivation strategies to produce bioplastics from biogas is crucial for the implementation of this biorefinery process. In this work, biogas-based polyhydroxybutyrate (PHB) production and CH4 biodegradation performance was investigated for the first time in a stirred tank bioreactor inoculated with Methylocystis parvus str. OBBP. Decreasing nitrogen loading rates in continuous mode and alternating feast:famine regimes of 24 h-cycles, and alternating feast:famine regimes of 24 h:24 h and 24 h:48 h were tested. Continuous N feeding did not support an effective PHB production despite the occurrence of nitrogen limiting conditions. Feast-famine cycles of 24 h:24 h (with 50% stoichiometric nitrogen supply) supported the maximum PHB production (20 g-PHB m-3 d-1) without compromising the CH4-elimination capacity (25 g m-3 h-1) of the system. Feast:famine ratios ≤1:2 entailed the deterioration of process performance at stoichiometric nitrogen inputs ≤60%.
Assuntos
Biocombustíveis , Methylocystaceae , Reatores Biológicos , Metano/metabolismo , Methylocystaceae/metabolismo , Nitrogênio/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
In nature, methane is oxidized to methanol by two enzymes, the iron-dependent soluble methane monooxygenase (sMMO) and the copper-dependent particulate MMO (pMMO). While sMMO's diiron metal active site is spectroscopically and structurally well-characterized, pMMO's copper sites are not. Recent EPR and ENDOR studies have established the presence of two monocopper sites, but the coordination environment of only one has been determined, that within the PmoB subunit and denoted CuB. Moreover, this recent work only focused on a type I methanotrophic pMMO, while previous observations of the type II enzyme were interpreted in terms of the presence of a dicopper site. First, this report shows that the type II Methylocystis species strain Rockwell pMMO, like the type I pMMOs, contains two monocopper sites and that its CuB site has a coordination environment identical to that of type I enzymes. As such, for the full range of pMMOs this report completes the refutation of prior and ongoing suggestions of multicopper sites. Second, and of primary importance, EPR/ENDOR measurements (a) for the first time establish the coordination environment of the spectroscopically observed site, provisionally denoted CuC, in both types of pMMO, thereby (b) establishing the assignment of this site observed by EPR to the crystallographically observed metal-binding site in the PmoC subunit. Finally, these results further indicate that CuC is the likely site of biological methane oxidation by pMMO, a conclusion that will serve as a foundation for proposals regarding the mechanism of this reaction.
Assuntos
Cobre/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Methylocystaceae/enzimologia , Oxigenases/química , Oxigenases/metabolismo , Methylocystaceae/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
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
In coastal aquatic ecosystems, prokaryotic communities play an important role in regulating the cycling of nutrients and greenhouse gases. In the coastal zone, estuaries are complex and delicately balanced systems containing a multitude of specific ecological niches for resident microbes. Anthropogenic influences (i.e. urban, industrial and agricultural land uses) along the estuarine continuum can invoke physical and biochemical changes that impact these niches. In this study, we investigate the relative abundance of methanogenic archaea and other prokaryotic communities, distributed along a land use gradient in the subtropical Burnett River Estuary, situated within the Great Barrier Reef catchment, Australia. Microbiological assemblages were compared to physicochemical, nutrient and greenhouse gas distributions in both pore and surface water. Pore water samples from within the most urbanised site showed a high relative abundance of methanogenic Euryarchaeota (7.8% of all detected prokaryotes), which coincided with elevated methane concentrations in the water column, ranging from 0.51 to 0.68 µM at the urban and sewage treatment plant (STP) sites, respectively. These sites also featured elevated dissolved organic carbon (DOC) concentrations (0.66 to 1.16 mM), potentially fuelling methanogenesis. At the upstream freshwater site, both methane and DOC concentrations were considerably higher (2.68 µM and 1.8 mM respectively) than at the estuarine sites (0.02 to 0.66 µM and 0.39 to 1.16 mM respectively) and corresponded to the highest relative abundance of methanotrophic bacteria. The proportion of sulfate reducing bacteria in the prokaryotic community was elevated within the urban and STP sites (relative abundances of 8.0%- 10.5%), consistent with electron acceptors with higher redox potentials (e.g. O2, NO3-) being scarce. Overall, this study showed that ecological niches in anthropogenically altered environments appear to give an advantage to specialized prokaryotes invoking a potential change in the thermodynamic landscape of the ecosystem and in turn facilitating the generation of methane-a potent greenhouse gas.
Assuntos
Archaea/isolamento & purificação , Estuários , Metano/metabolismo , Methanococcales/isolamento & purificação , Methylocystaceae/isolamento & purificação , Microbiota , Águas Salinas , Microbiologia da Água , Agricultura , Compostos de Amônio/metabolismo , Criação de Animais Domésticos , Archaea/metabolismo , Carbono/metabolismo , Ecossistema , Água Doce/análise , Água Doce/microbiologia , Gases de Efeito Estufa/análise , Habitação , Indústrias , Methanococcales/metabolismo , Methylocystaceae/metabolismo , Nitratos/metabolismo , Oxirredução , Queensland , Águas Salinas/análise , Salinidade , Sulfatos/metabolismo , Temperatura , Termodinâmica , Purificação da ÁguaRESUMO
Upland soil clusters alpha and gamma (USCα and USCγ) are considered a major biological sink of atmospheric methane and are often detected in forest and grassland soils. These clusters are phylogenetically classified using the particulate methane monooxygenase gene pmoA because of the difficulty of cultivation. Recent studies have established a direct link of pmoA genes to 16S rRNA genes based on their isolated strain or draft genomes. However, whether the results of pmoA-based assays could be largely represented by 16S rRNA gene sequencing in upland soils remains unclear. In this study, we collected 20 forest soils across China and compared methane-oxidizing bacterial (MOB) communities by high-throughput sequencing of 16S rRNA and pmoA genes using different primer sets. The results showed that 16S rRNA gene sequencing and the semi-nested polymerase chain reaction (PCR) of the pmoA gene (A189/A682r nested with a mixture of mb661 and A650) consistently revealed the dominance of USCα (accounting for more than 50% of the total MOB) in 12 forest soils. A189f/A682r successfully amplified pmoA genes (mainly RA14 of USCα) in only three forest soils. A189f/mb661 could amplify USCα (mainly JR1) in several forest soils but showed a strong preferential amplification of Methylocystis and many other type I MOB groups. A189f/A650 almost exclusively amplified USCα (mainly JR1) and largely discriminated against Methylocystis and most of the other MOB groups. The semi-nested PCR approach weakened the bias of A189f/mb661 and A189f/A650 for JR1 and balanced the coverage of all USCα members. The canonical correspondence analysis indicated that soil NH4+-N and pH were the main environmental factors affecting the MOB community of Chinese forest soils. The RA14 of the USCα group prefers to live in soils with low pH, low temperature, low elevation, high precipitation, and rich in nitrogen. JR1's preferences for temperature and elevation were opposite to RA14. Our study suggests that combining the deep sequencing of 16S rRNA and pmoA genes to characterize MOB in forest soils is the best choice.
Assuntos
Bactérias/metabolismo , Metano/metabolismo , Microbiota , Microbiologia do Solo , Bactérias/isolamento & purificação , China , Florestas , Genes Bacterianos , Methylocystaceae/isolamento & purificação , Methylocystaceae/metabolismo , Oxirredução , Reação em Cadeia da Polimerase , RNA Bacteriano/análise , RNA Ribossômico 16S/análiseRESUMO
Methane, a non-expensive natural substrate, is used by Methylocystis spp. as a sole source of carbon and energy. Here, we assessed whether Methylocystis sp. strain SC2 is able to also utilize hydrogen as an energy source. The addition of 2% H2 to the culture headspace had the most significant positive effect on the growth yield under CH4 (6%) and O2 (3%) limited conditions. The SC2 biomass yield doubled from 6.41 (±0.52) to 13.82 (±0.69) mg cell dry weight per mmol CH4, while CH4 consumption was significantly reduced. Regardless of H2 addition, CH4 utilization was increasingly redirected from respiration to fermentation-based pathways with decreasing O2/CH4 mixing ratios. Theoretical thermodynamic calculations confirmed that hydrogen utilization under oxygen-limited conditions doubles the maximum biomass yield compared to fully aerobic conditions without H2 addition. Hydrogen utilization was linked to significant changes in the SC2 proteome. In addition to hydrogenase accessory proteins, the production of Group 1d and Group 2b hydrogenases was significantly increased in both short- and long-term incubations. Both long-term incubation with H2 (37 d) and treatments with chemical inhibitors revealed that SC2 growth under hydrogen-utilizing conditions does not require the activity of complex I. Apparently, strain SC2 has the metabolic capacity to channel hydrogen-derived electrons into the quinone pool, which provides a link between hydrogen oxidation and energy production. In summary, H2 may be a promising alternative energy source in biotechnologically oriented methanotroph projects that aim to maximize biomass yield from CH4, such as the production of high-quality feed protein.
Assuntos
Hidrogênio/metabolismo , Metano/metabolismo , Methylocystaceae , Methylocystaceae/genética , Methylocystaceae/metabolismoRESUMO
Bensulfuron-methyl is an herbicide widely used for weed control although its residues cause damage to other crops during crop rotations. In this study, the biodegrading activity of bensulfuron-methyl by a plant growth-promoting bacterial strain was carried out. Methylopila sp. DKT isolated from soil was determined for bensulfuron-methyl degradation and phosphate solubilization in the liquid media and soil. Moreover, the effects of the herbicide on peanut development and the role of Methylopila sp. DKT on the growth promotion of peanut were investigated. The results showed that the isolate effectively utilized the compound as a sole carbon source and solubilized low soluble inorganic phosphates. Methylopila sp. DKT also utilized 2-amino-4,6-dimethoxypyrimidine, a metabolite of bensulfuron-methyl degradation, as a sole carbon and energy source, and released ammonium and nitrate. The supplementation with Methylopila sp. DKT in soil increased the peanut biomass and the phosphorus content in the plant. In addition, the inoculation with Methylopila sp. DKT in soil and peanut cultivation increased the bensulfuron-methyl degradation by 57.7% for 1 month, which suggests that both plants and the bacterial isolate play a key role in herbicide degradation. These results indicate that the studied strain has a high potential for soil remediation and peanut growth promotion.