MbnC is not required for the formation of the N-terminal oxazolone in the methanobactin from Methylosinus trichosporium OB3b.

Methanobactins (MBs) are ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The post-translational modification that define 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, 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 Δ mbn C mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry and solution NMR spectroscopy. MB-OB3b from Δ mbn C was missing the C-terminal Met and also found to contain a Pro and a Cys in place of the pyrrolidiny-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, nanoparticle formation, as well as for 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 post-translational modification(s) of the two oxazolone groups are not identical.

Switching Between Methanol Accumulation and Cell Growth by Expression Control of Methanol Dehydrogenase in Methylosinus trichosporium OB3b Mutant

Methanotrophs have been used to convert methane to methanol at ambient temperature and pressure. In order to accumulate methanol using methanotrophs, methanol dehydrogenase (MDH) must be downregulated as it consumes methanol. Here, we describe a methanol production system wherein MDH expression is controlled by using methanotroph mutants. We used the MxaF knockout mutant of Methylosinus trichosporium OB3b. It could only grow with MDH (XoxF) which has a cerium ion in its active site and is only expressed by bacteria in media containing cerium ions. In the presence of 0 μM copper ion and 25 μM cerium ion, the mutant grew normally. Under conditions conducive to methanol production (10 μM copper ion and 0 μM cerium ion), cell growth was inhibited and methanol accumulated (2.6 μmol·mg−1 dry cell weight·h−1). The conversion efficiency of the accumulated methanol to the total amount of methane added to the reaction system was ~0.3%. The aforementioned conditions were repeatedly alternated by modulating the metal ion composition of the bacterial growth medium.

The Ethylmalonyl-CoA Pathway for Methane-based Biorefineries: A Case Study of Using Methylosinus trichosporium OB3b, an Alpha-proteobacterial Methanotroph, for Producing 2-Hydroxyisobutyric Acid and 1,3-Butanediol from Methane

The ethylmalonyl-CoA pathway is one of three known anaplerotic pathways that replenish tricarboxylic acid cycle intermediates and plays a major role in the carbon metabolism of many alpha-proteobacteria including Methylosinus...

Enhancement of nitrous oxide emissions in soil microbial consortia via copper competition between proteobacterial methanotrophs and denitrifiers

Unique means of copper scavenging have been identified in proteobacterial methanotrophs, particularly the use of methanobactin, a novel ribosomally synthesized post-translationally modified polypeptide that binds copper with very high affinity. The possibility that copper sequestration strategies of methanotrophs may interfere with copper uptake of denitrifiers in situ and thereby enhance N2O emissions was examined using a suite of laboratory experiments performed with rice paddy microbial consortia. Addition of purified methanobactin from Methylosinus trichosporium OB3b to denitrifying rice paddy soil microbial consortia resulted in substantially increased N2O production, with more pronounced responses observed for soils with lower copper content. The N2O emission-enhancing effect of the soil’s native mbnA-expressing Methylocystaceae methanotrophs on the native denitrifiers was then experimentally verified with a Methylocystaceae-dominant chemostat culture prepared from a rice paddy microbial consortium as the inoculum. Lastly, with microcosms amended with varying cell numbers of methanobactin-producing Methylosinus trichosporium OB3b before CH4 enrichment, microbiomes with different ratios of methanobactin-producing Methylocystaceae to gammaproteobacterial methanotrophs incapable of methanobactin production were simulated. Significant enhancement of N2O production from denitrification was evident in both Methylocystaceae-dominant and Methylococcaceae-dominant enrichments, albeit to a greater extent in the former, signifying the comparative potency of methanobactin-mediated copper sequestration while implying the presence of alternative copper abstraction mechanisms for Methylococcaceae. These observations support that copper-mediated methanotrophic enhancement of N2O production from denitrification is plausible where methanotrophs and denitrifiers cohabit.IMPORTANCE Proteobacterial methanotrophs, groups of microorganisms that utilize methane as source of energy and carbon, have been known to utilize unique mechanisms to scavenge copper, namely utilization of methanobactin, a polypeptide that binds copper with high affinity and specificity. Previously the possibility that copper sequestration by methanotrophs may lead to alteration of cuproenzyme-mediated reactions in denitrifiers and consequently increase emission of potent greenhouse gas N2O has been suggested in axenic and co-culture experiments. Here, a suite of experiments with rice paddy soil slurry cultures with complex microbial compositions were performed to corroborate that such copper-mediated interplay may actually take place in environments co-habited by diverse methanotrophs and denitrifiers. As spatial and temporal heterogeneity allow for spatial coexistence of methanotrophy (aerobic) and denitrification (anaerobic) in soils, the results from this study suggest that this previously unidentified mechanism of N2O production may account for significant proportion of N2O efflux from agricultural soils.

Enhancement of nitrous oxide emissions in soil microbial consortia via copper competition between proteobacterial methanotrophs and denitrifiers

Unique means of copper scavenging have been identified in proteobacterial methanotrophs, particularly the use of methanobactin, a novel ribosomally synthesized post-translationally modified polypeptide that binds copper with very high affinity. The possibility that copper sequestration strategies of methanotrophs may interfere with copper uptake of denitrifiers in situ and thereby enhance N2O emissions was examined using a suite of laboratory experiments performed with rice paddy microbial consortia. Addition of purified methanobactin from Methylosinus trichosporium OB3b to denitrifying rice paddy soil microbial consortia resulted in substantially increased N2O production, with more pronounced responses observed for soils with lower copper content. The N2O emission-enhancing effect of the soil’s native mbnA-expressing Methylocystaceae methanotrophs on the native denitrifiers was then experimentally verified with a Methylocystaceae-dominant chemostat culture prepared from a rice paddy microbial consortium as the inoculum. Lastly, with microcosms amended with varying cell numbers of methanobactin-producing Methylosinus trichosporium OB3b before CH4 enrichment, microbiomes with different ratios of methanobactin-producing Methylocystaceae to gammaproteobacterial methanotrophs incapable of methanobactin production were simulated. Significant enhancement of N2O production from denitrification was evident in both Methylocystaceae-dominant and Methylococcaceae-dominant enrichments, albeit to a greater extent in the former, signifying the comparative potency of methanobactin-mediated copper sequestration while implying the presence of alternative copper abstraction mechanisms for Methylococcaceae. These observations support that copper-mediated methanotrophic enhancement of N2O production from denitrification is plausible where methanotrophs and denitrifiers cohabit.ImportanceProteobacterial methanotrophs, groups of microorganisms that utilize methane as source of energy and carbon, have been known to utilize unique mechanisms to scavenge copper, namely utilization of methanobactin, a polypeptide that binds copper with high affinity and specificity. Previously the possibility that copper sequestration by methanotrophs may lead to alteration of cuproenzyme-mediated reactions in denitrifiers and consequently increase emission of potent greenhouse gas N2O has been suggested in axenic and co-culture experiments. Here, a suite of experiments with rice paddy soil slurry cultures with complex microbial compositions were performed to corroborate that such copper-mediated interplay may actually take place in environments co-habited by diverse methanotrophs and denitrifiers. As spatial and temporal heterogeneity allow for spatial coexistence of methanotrophy (aerobic) and denitrification (anaerobic) in soils, the results from this study suggest that this previously unidentified mechanism of N2O production may account for significant proportion of N2O efflux from agricultural soils.