The effect of methane and methanol on the terrestrial ammonia-oxidizing archaeon 'Candidatus Nitrosocosmicus franklandus C13'.

The ammonia monooxygenase (AMO) is a key enzyme in ammonia-oxidizing archaea, which are abundant and ubiquitous in soil environments. The AMO belongs to the copper-containing membrane monooxygenase (CuMMO) enzyme superfamily, which also contains particulate methane monooxygenase (pMMO). Enzymes in the CuMMO superfamily are promiscuous, which results in co-oxidation of alternative substrates. The phylogenetic and structural similarity between the pMMO and the archaeal AMO is well-established, but there is surprisingly little information on the influence of methane and methanol on the archaeal AMO and terrestrial nitrification. The aim of this study was to examine the effects of methane and methanol on the soil ammonia-oxidizing archaeon 'Candidatus Nitrosocosmicus franklandus C13'. We demonstrate that both methane and methanol are competitive inhibitors of the archaeal AMO. The inhibition constants (Ki ) for methane and methanol were 2.2 and 20 µM, respectively, concentrations which are environmentally relevant and orders of magnitude lower than those previously reported for ammonia-oxidizing bacteria. Furthermore, we demonstrate that a specific suite of proteins is upregulated and downregulated in 'Ca. Nitrosocosmicus franklandus C13' in the presence of methane or methanol, which provides a foundation for future studies into metabolism of one-carbon (C1) compounds in ammonia-oxidizing archaea.

Analysis of Essential Isoprene Metabolic Pathway Proteins in Variovorax sp. Strain WS11.

Isoprene monooxygenase (IsoMO, encoded by isoABCDEF) initiates the oxidation of the climate-active gas isoprene, with the genes isoGHIJ and aldH nearly always found adjacent to isoABCDEF in extant and metagenome-derived isoprene degraders. The roles of isoGHIJ and aldH are uncertain, although each is essential to isoprene degradation. We report here the characterization of these proteins from two model isoprene degraders, Rhodococcus sp. strain AD45 and Variovorax sp. strain WS11. The genes isoHIJ and aldH from Variovorax and aldH from Rhodococcus were expressed individually in Escherichia coli as maltose binding protein fusions to overcome issues of insolubility. The activity of two glutathione S-transferases from Variovorax, IsoI and IsoJ was assessed with model substrates, and the conversion of epoxyisoprene to the intermediate 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB) was demonstrated. The next step of the isoprene metabolic pathway of Variovorax is catalyzed by the dehydrogenase IsoH, resulting in the conversion of HGMB to 2-glutathionyl-2-methyl-3-butenoic acid (GMBA). The aldehyde dehydrogenases (AldH) from Variovorax and Rhodococcus were examined with a variety of aldehydes, with both exhibiting maximum activity with butanal. AldH significantly increased the rate of production of NADH when added to the IsoH-catalyzed conversion of HGMB to GMBA (via GMB), suggesting a synergistic role for AldH in the isoprene metabolic pathway. An in silico analysis of IsoG revealed that this protein, which is essential for isoprene metabolism in Variovorax, is an enzyme of the formyl CoA-transferase family and is predicted to catalyze the formation of a GMBA-CoA thioester as an intermediate in the isoprene oxidation pathway. IMPORTANCE Isoprene is a climate-active gas, largely produced by trees, which is released from the biosphere in amounts equivalent to those of methane and all other volatile organic compounds combined. Bacteria found in many environments, including soils and on the surface of leaves of isoprene-producing trees, can grow on isoprene and thus may represent a significant biological sink for this globally significant volatile compound and remove isoprene before it escapes to the atmosphere, thus reducing its potency as a climate-active gas. The initial oxidation of isoprene by bacteria is mediated by isoprene monooxygenase encoded by the genes isoABCDEF. In isoprene-degrading bacteria, a second gene cluster, isoGHIJ, is also present, although the exact role in isoprene degradation by the proteins encoded by these genes is uncertain. This investigation sheds new light on the roles of these proteins in the isoprene oxidation pathway in two model isoprene-degrading bacteria of the genera Rhodococcus and Variovorax.

'Omics-guided prediction of the pathway for metabolism of isoprene by Variovorax sp. WS11.

Bacteria that inhabit soils and the leaves of trees partially mitigate the release of the abundant volatile organic compound, isoprene (2-methyl-1,3-butadiene). While the initial steps of isoprene metabolism were identified in Rhodococcus sp. AD45 two decades ago, the isoprene metabolic pathway still remains largely undefined. Limited understanding of the functions of isoG, isoJ and aldH and uncertainty in the route of isoprene-derived carbon into central metabolism have hindered our understanding of isoprene metabolism. These previously uncharacterised iso genes are essential in Variovorax sp. WS11, determined by targeted mutagenesis. Using combined 'omics-based approaches, we propose the complete isoprene metabolic pathway. Isoprene is converted to propionyl-CoA, which is assimilated by the chromosomally encoded methylmalonyl-CoA pathway, requiring biotin and vitamin B12, with the plasmid-encoded methylcitrate pathway potentially providing robustness against limitations in these vitamins. Key components of this pathway were induced by both isoprene and its initial oxidation product, epoxyisoprene, the principal inducer of isoprene metabolism in both Variovorax sp. WS11 and Rhodococcus sp. AD45. Analysis of the genomes of distinct isoprene-degrading bacteria indicated that all of the genetic components of the methylcitrate and methylmalonyl-CoA pathways are not always present in isoprene degraders, although incorporation of isoprene-derived carbon via propionyl-CoA and acetyl-CoA is universally indicated.

Hydrazines as Substrates and Inhibitors of the Archaeal Ammonia Oxidation Pathway.

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) perform key steps in the global nitrogen cycle, the oxidation of ammonia to nitrite. While the ammonia oxidation pathway is well characterized in AOB, many knowledge gaps remain about the metabolism of AOA. Hydroxylamine is an intermediate in both AOB and AOA, but homologues of hydroxylamine dehydrogenase (HAO), catalyzing bacterial hydroxylamine oxidation, are absent in AOA. Hydrazine is a substrate for bacterial HAO, while phenylhydrazine is a suicide inhibitor of HAO. Here, we examine the effect of hydrazines in AOA to gain insights into the archaeal ammonia oxidation pathway. We show that hydrazine is both a substrate and an inhibitor for AOA and that phenylhydrazine irreversibly inhibits archaeal hydroxylamine oxidation. Both hydrazine and phenylhydrazine interfered with ammonia and hydroxylamine oxidation in AOA. Furthermore, the AOA "Candidatus Nitrosocosmicus franklandus" C13 oxidized hydrazine into dinitrogen (N2), coupling this reaction to ATP production and O2 uptake. This study expands the known substrates of AOA and suggests that despite differences in enzymology, the ammonia oxidation pathways of AOB and AOA are functionally surprisingly similar. These results demonstrate that hydrazines are valuable tools for studying the archaeal ammonia oxidation pathway. IMPORTANCE Ammonia-oxidizing archaea (AOA) are among the most numerous living organisms on Earth, and they play a pivotal role in the global biogeochemical nitrogen cycle. Despite this, little is known about the physiology and metabolism of AOA. We demonstrate in this study that hydrazines are inhibitors of AOA. Furthermore, we demonstrate that the model soil AOA "Ca. Nitrosocosmicus franklandus" C13 oxidizes hydrazine to dinitrogen gas, and this reaction yields ATP. This provides an important advance in our understanding of the metabolism of AOA and expands the short list of energy-yielding compounds that AOA can use. This study also provides evidence that hydrazines can be useful tools for studying the metabolism of AOA, as they have been for the bacterial ammonia oxidizers.

Purification and Characterization of the Isoprene Monooxygenase from Rhodococcus sp. Strain AD45.

Isoprene (2-methyl-1,3-butadiene) is a climate-active gas released to the atmosphere in large quantities, comparable to methane in magnitude. Several bacteria have been isolated which can grow on isoprene as a sole carbon and energy source, but very little information is available about the degradation of isoprene by these bacteria at the biochemical level. Isoprene utilization is dependent on a multistep pathway, with the first step being the oxidation of isoprene to epoxy-isoprene. This is catalyzed by a four-component soluble diiron monooxygenase, isoprene monooxygenase (IsoMO). IsoMO is a six-protein complex comprising an oxygenase (IsoABE), containing the di-iron active site, a Rieske-type ferredoxin (IsoC), a NADH reductase (IsoF), and a coupling/effector protein (IsoD), homologous to the soluble methane monooxygenase and alkene/aromatic monooxygenases. Here, we describe the purification of the IsoMO components from Rhodococcus sp. AD45 and reconstitution of isoprene-oxidation activity in vitro. Some IsoMO components were expressed and purified from the homologous host Rhodococcus sp. AD45-ID, a Rhodococcus sp. AD45 strain lacking the megaplasmid which contains the isoprene metabolic gene cluster. Others were expressed in Escherichia coli and purified as fusion proteins. We describe the characterization of these purified components and demonstrate their activity when combined with Rhodococcus sp. AD45 cell lysate. Demonstration of IsoMO activity in vitro provides a platform for further biochemical and biophysical characterization of this novel soluble diiron center monooxygenase, facilitating new insights into the enzymatic basis for the bacterial degradation of isoprene. IMPORTANCE Isoprene is a highly abundant climate-active gas and a carbon source for some bacteria. Analyses of the genes encoding isoprene monooxygenase (IsoMO) indicate this enzyme is a soluble diiron center monooxygenase in the same family of oxygenases as soluble methane monooxygenase, alkene monooxygenase, and toluene monooxygenase. We report the initial biochemical characterization of IsoMO from Rhodococcus, the first from any bacterium, describing the challenging purification and reconstitution of in vitro activity of its four components. This study lays the foundation for future detailed mechanistic studies of IsoMO, a key enzyme in the global isoprene cycle.

A marine plasmid hitchhiking vast phylogenetic and geographic distances.

Horizontal gene transfer (HGT) plays an important role in bacterial evolution and serves as a driving force for bacterial diversity and versatility. HGT events often involve mobile genetic elements like plasmids, which can promote their own dissemination by associating with adaptive traits in the gene pool of the so-called mobilome. Novel traits that evolve through HGT can therefore lead to the exploitation of new ecological niches, prompting an adaptive radiation of bacterial species. In this study, we present phylogenetic, biogeographic, and functional analyses of a previously unrecognized RepL-type plasmid found in diverse members of the marine Roseobacter group across the globe. Noteworthy, 100% identical plasmids were detected in phylogenetically and geographically distant bacteria, revealing a so-far overlooked, but environmentally highly relevant vector for HGT. The genomic and functional characterization of this plasmid showed a completely conserved backbone dedicated to replication, stability, and mobilization as well as an interchangeable gene cassette with highly diverse, but recurring motifs. The majority of the latter appear to be involved in mechanisms coping with toxins and/or pollutants in the marine environment. Furthermore, we provide experimental evidence that the plasmid has the potential to be transmitted across bacterial orders, thereby increasing our understanding of evolution and microbial niche adaptation in the environment.

Competition-based screening helps to secure the evolutionary stability of a defensive microbiome.

BACKGROUND: The cuticular microbiomes of Acromyrmex leaf-cutting ants pose a conundrum in microbiome biology because they are freely colonisable, and yet the prevalence of the vertically transmitted bacteria Pseudonocardia, which contributes to the control of Escovopsis fungus garden disease, is never compromised by the secondary acquisition of other bacterial strains. Game theory suggests that competition-based screening can allow the selective recruitment of antibiotic-producing bacteria from the environment, by providing abundant resources to foment interference competition between bacterial species and by using Pseudonocardia to bias the outcome of competition in favour of antibiotic producers. RESULTS: Here, we use RNA-stable isotope probing (RNA-SIP) to confirm that Acromyrmex ants can maintain a range of microbial symbionts on their cuticle by supplying public resources. We then used RNA sequencing, bioassays, and competition experiments to show that vertically transmitted Pseudonocardia strains produce antibacterials that differentially reduce the growth rates of other microbes, ultimately biassing the bacterial competition to allow the selective establishment of secondary antibiotic-producing strains while excluding non-antibiotic-producing strains that would parasitise the symbiosis. CONCLUSIONS: Our findings are consistent with the hypothesis that competition-based screening is a plausible mechanism for maintaining the integrity of the co-adapted mutualism between the leaf-cutting ant farming symbiosis and its defensive microbiome. Our results have broader implications for explaining the stability of other complex symbioses involving horizontal acquisition.

Revealing the community and metabolic potential of active methanotrophs by targeted metagenomics in the Zoige wetland of the Tibetan Plateau.

The Zoige wetland of the Tibetan Plateau is one of the largest alpine wetlands in the world and a major emission source of methane. Methane oxidation by methanotrophs can counteract the global warming effect of methane released in the wetlands. Understanding methanotroph activity, diversity and metabolism at the molecular level can guide the isolation of the uncultured microorganisms and inform strategy-making decisions and policies to counteract global warming in this unique ecosystem. Here we applied DNA stable isotope probing using 13 C-labelled methane to label the genomes of active methanotrophs, examine the methane oxidation potential and recover metagenome-assembled genomes (MAGs) of active methanotrophs. We found that gammaproteobacteria of type I methanotrophs are responsible for methane oxidation in the wetland. We recovered two phylogenetically novel methanotroph MAGs distantly related to extant Methylobacter and Methylovulum. They belong to type I methanotrophs of gammaproteobacteria, contain both mxaF and xoxF types of methanol dehydrogenase coding genes, and participate in methane oxidation via H4 MPT and RuMP pathways. Overall, the community structure of active methanotrophs and their methanotrophic pathways revealed by DNA-SIP metagenomics and retrieved methanotroph MAGs highlight the importance of methanotrophs in suppressing methane emission in the wetland under the scenario of global warming.

Assessing the Toxicity and Mitigating the Impact of Harmful Prymnesium Blooms in Eutrophic Waters of the Norfolk Broads.

Prymnesium parvum is a toxin-producing microalga, which causes harmful algal blooms globally, frequently leading to massive fish kills that have adverse ecological and economic implications for natural waterways and aquaculture alike. The dramatic effects observed on fish are thought to be due to algal polyether toxins, known as the prymnesins, but their lack of environmental detection has resulted in an uncertainty about the true ichthyotoxic agents. Using qPCR, we found elevated levels of P. parvum and its lytic virus, PpDNAV-BW1, in a fish-killing bloom on the Norfolk Broads, United Kingdom, in March 2015. We also detected, for the first time, the B-type prymnesin toxins in Broads waterway samples and gill tissue isolated from a dead fish taken from the study site. Furthermore, Norfolk Broads P. parvum isolates unambiguously produced B-type toxins in laboratory-grown cultures. A 2 year longitudinal study of the Broads study site showed P. parvum blooms to be correlated with increased temperature and that PpDNAV plays a significant role in P. parvum bloom demise. Finally, we used a field trial to show that treatment with low doses of hydrogen peroxide represents an effective strategy to mitigate blooms of P. parvum in enclosed water bodies.

A four-helix bundle stores copper for methane oxidation.

Methane-oxidizing bacteria (methanotrophs) require large quantities of copper for the membrane-bound (particulate) methane monooxygenase. Certain methanotrophs are also able to switch to using the iron-containing soluble methane monooxygenase to catalyse methane oxidation, with this switchover regulated by copper. Methane monooxygenases are nature's primary biological mechanism for suppressing atmospheric levels of methane, a potent greenhouse gas. Furthermore, methanotrophs and methane monooxygenases have enormous potential in bioremediation and for biotransformations producing bulk and fine chemicals, and in bioenergy, particularly considering increased methane availability from renewable sources and hydraulic fracturing of shale rock. Here we discover and characterize a novel copper storage protein (Csp1) from the methanotroph Methylosinus trichosporium OB3b that is exported from the cytosol, and stores copper for particulate methane monooxygenase. Csp1 is a tetramer of four-helix bundles with each monomer binding up to 13 Cu(I) ions in a previously unseen manner via mainly Cys residues that point into the core of the bundle. Csp1 is the first example of a protein that stores a metal within an established protein-folding motif. This work provides a detailed insight into how methanotrophs accumulate copper for the oxidation of methane. Understanding this process is essential if the wide-ranging biotechnological applications of methanotrophs are to be realized. Cytosolic homologues of Csp1 are present in diverse bacteria, thus challenging the dogma that such organisms do not use copper in this location.

Poplar phyllosphere harbors disparate isoprene-degrading bacteria.

The climate-active gas isoprene (2-methyl-1,3-butadiene) is released to the atmosphere in huge quantities, almost equaling that of methane, yet we know little about the biological cycling of isoprene in the environment. Although bacteria capable of growth on isoprene as the sole source of carbon and energy have previously been isolated from soils and sediments, no microbiological studies have targeted the major source of isoprene and examined the phyllosphere of isoprene-emitting trees for the presence of degraders of this abundant carbon source. Here, we identified isoprene-degrading bacteria in poplar tree-derived microcosms by DNA stable isotope probing. The genomes of isoprene-degrading taxa were reconstructed, putative isoprene metabolic genes were identified, and isoprene-related gene transcription was analyzed by shotgun metagenomics and metatranscriptomics. Gram-positive bacteria of the genus Rhodococcus proved to be the dominant isoprene degraders, as previously found in soil. However, a wider diversity of isoprene utilizers was also revealed, notably Variovorax, a genus not previously associated with this trait. This finding was confirmed by expression of the isoprene monooxygenase from Variovorax in a heterologous host. A Variovorax strain that could grow on isoprene as the sole carbon and energy source was isolated. Analysis of its genome confirmed that it contained isoprene metabolic genes with an identical layout and high similarity to those identified by DNA-stable isotope probing and metagenomics. This study provides evidence of a wide diversity of isoprene-degrading bacteria in the isoprene-emitting tree phyllosphere and greatly enhances our understanding of the biodegradation of this important metabolite and climate-active gas.

Facultative methanotrophs - diversity, genetics, molecular ecology and biotechnological potential: a mini-review.

Methane-oxidizing bacteria (methanotrophs) play a vital role in reducing atmospheric methane emissions, and hence mitigating their potent global warming effects. A significant proportion of the methane released is thermogenic natural gas, containing associated short-chain alkanes as well as methane. It was one hundred years following the description of methanotrophs that facultative strains were discovered and validly described. These can use some multi-carbon compounds in addition to methane, often small organic acids, such as acetate, or ethanol, although Methylocella strains can also use short-chain alkanes, presumably deriving a competitive advantage from this metabolic versatility. Here, we review the diversity and molecular ecology of facultative methanotrophs. We discuss the genetic potential of the known strains and outline the consequent benefits they may obtain. Finally, we review the biotechnological promise of these fascinating microbes.

Microbial metabolism of isoprene: a much-neglected climate-active gas.

The climate-active gas isoprene is the major volatile produced by a variety of trees and is released into the atmosphere in enormous quantities, on a par with global emissions of methane. While isoprene production in plants and its effect on atmospheric chemistry have received considerable attention, research into the biological isoprene sink has been neglected until recently. Here, we review current knowledge on the sources and sinks of isoprene and outline its environmental effects. Focusing on degradation by microbes, many of which are able to use isoprene as the sole source of carbon and energy, we review recent studies characterizing novel isoprene degraders isolated from soils, marine sediments and in association with plants. We describe the development and use of molecular methods to identify, quantify and genetically characterize isoprene-degrading strains in environmental samples. Finally, this review identifies research imperatives for the further study of the environmental impact, ecology, regulation and biochemistry of this interesting group of microbes.

Inhibition of Ammonia Monooxygenase from Ammonia-Oxidizing Archaea by Linear and Aromatic Alkynes.

Ammonia monooxygenase (AMO) is a key nitrogen-transforming enzyme belonging to the same copper-dependent membrane monooxygenase family (CuMMO) as the particulate methane monooxygenase (pMMO). The AMO from ammonia-oxidizing archaea (AOA) is very divergent from both the AMO of ammonia-oxidizing bacteria (AOB) and the pMMO from methanotrophs, and little is known about the structure or substrate range of the archaeal AMO. This study compares inhibition by C2 to C8 linear 1-alkynes of AMO from two phylogenetically distinct strains of AOA, "Candidatus Nitrosocosmicus franklandus" C13 and "Candidatus Nitrosotalea sinensis" Nd2, with AMO from Nitrosomonas europaea and pMMO from Methylococcus capsulatus (Bath). An increased sensitivity of the archaeal AMO to short-chain-length alkynes (≤C5) appeared to be conserved across AOA lineages. Similarities in C2 to C8 alkyne inhibition profiles between AMO from AOA and pMMO from M. capsulatus suggested that the archaeal AMO has a narrower substrate range than N. europaea AMO. Inhibition of AMO from "Ca Nitrosocosmicus franklandus" and N. europaea by the aromatic alkyne phenylacetylene was also investigated. Kinetic data revealed that the mechanisms by which phenylacetylene inhibits "Ca Nitrosocosmicus franklandus" and N. europaea are different, indicating differences in the AMO active site between AOA and AOB. Phenylacetylene was found to be a specific and irreversible inhibitor of AMO from "Ca Nitrosocosmicus franklandus," and it does not compete with NH3 for binding at the active site.IMPORTANCE Archaeal and bacterial ammonia oxidizers (AOA and AOB, respectively) initiate nitrification by oxidizing ammonia to hydroxylamine, a reaction catalyzed by ammonia monooxygenase (AMO). AMO enzyme is difficult to purify in its active form, and its structure and biochemistry remain largely unexplored. The bacterial AMO and the closely related particulate methane monooxygenase (pMMO) have a broad range of hydrocarbon cooxidation substrates. This study provides insights into the AMO of previously unstudied archaeal genera, by comparing the response of the archaeal AMO, a bacterial AMO, and pMMO to inhibition by linear 1-alkynes and the aromatic alkyne, phenylacetylene. Reduced sensitivity to inhibition by larger alkynes suggests that the archaeal AMO has a narrower hydrocarbon substrate range than the bacterial AMO, as previously reported for other genera of AOA. Phenylacetylene inhibited the archaeal and bacterial AMOs at different thresholds and by different mechanisms of inhibition, highlighting structural differences between the two forms of monooxygenase.

Streptomyces Endophytes Promote Host Health and Enhance Growth across Plant Species.

Streptomyces bacteria are ubiquitous in soils and are well known for producing secondary metabolites, including antimicrobials. Increasingly, they are being isolated from plant roots, and several studies have shown they are specifically recruited to the rhizosphere and the endosphere of the model plant Arabidopsis thaliana Here, we test the hypothesis that Streptomyces bacteria have a beneficial effect on A. thaliana growth and could potentially be used as plant probiotics. To do this, we selectively isolated streptomycetes from surface-washed A. thaliana roots and generated high-quality genome sequences for five strains, which we named L2, M2, M3, N1, and N2. Reinfection of A. thaliana plants with L2, M2, and M3 significantly increased plant biomass individually and in combination, whereas N1 and N2 had a negative effect on plant growth, likely due to their production of polyene natural products which can bind to phytosterols and reduce plant growth. N2 exhibits broad-spectrum antimicrobial activity and makes filipin-like polyenes, including 14-hydroxyisochainin which inhibits the take-all fungus, Gaeumannomyces graminis var. tritici N2 antifungal activity as a whole was upregulated ∼2-fold in response to indole-3-acetic acid (IAA), suggesting a possible role during competition in the rhizosphere. Furthermore, coating wheat seeds with N2 spores protected wheat seedlings against take-all disease. We conclude that at least some soil-dwelling streptomycetes confer growth-promoting benefits on A. thaliana, while others might be exploited to protect crops against disease.IMPORTANCE We must reduce reliance on agrochemicals, and there is increasing interest in using bacterial strains to promote plant growth and protect against disease. Our study follows up reports that Arabidopsis thaliana specifically recruits Streptomyces bacteria to its roots. We test the hypotheses that they offer benefits to their A. thaliana hosts and that strains isolated from these plants might be used as probiotics. We isolated Streptomyces strains from A. thaliana roots and genome sequenced five phylogenetically distinct strains. Genome mining and bioassays indicated that all five have plant growth-promoting properties, including production of indole-3-acetic acid (IAA), siderophores, and aminocyclopropane-1-carboxylate (ACC) deaminase. Three strains significantly increased A. thaliana growth in vitro and in combination in soil. Another produces potent filipin-like antifungals and protected germinating wheat seeds against the fungal pathogen Gaeumannomyces graminis var. tritici (wheat take-all fungus). We conclude that introducing Streptomyces strains into the root microbiome provides significant benefits to plants.

Genome Scale Metabolic Model of the versatile methanotroph Methylocella silvestris.

BACKGROUND: Methylocella silvestris is a facultative aerobic methanotrophic bacterium which uses not only methane, but also other alkanes such as ethane and propane, as carbon and energy sources. Its high metabolic versatility, together with the availability of tools for its genetic engineering, make it a very promising platform for metabolic engineering and industrial biotechnology using natural gas as substrate. RESULTS: The first Genome Scale Metabolic Model for M. silvestris is presented. The model has been used to predict the ability of M. silvestris to grow on 12 different substrates, the growth phenotype of two deletion mutants (ΔICL and ΔMS), and biomass yield on methane and ethanol. The model, together with phenotypic characterization of the deletion mutants, revealed that M. silvestris uses the glyoxylate shuttle for the assimilation of C1 and C2 substrates, which is unique in contrast to published reports of other methanotrophs. Two alternative pathways for propane metabolism have been identified and validated experimentally using enzyme activity tests and constructing a deletion mutant (Δ1641), which enabled the identification of acetol as one of the intermediates of propane assimilation via 2-propanol. The model was also used to integrate proteomic data and to identify key enzymes responsible for the adaptation of M. silvestris to different substrates. CONCLUSIONS: The model has been used to elucidate key metabolic features of M. silvestris, such as its use of the glyoxylate shuttle for the assimilation of one and two carbon compounds and the existence of two parallel metabolic pathways for propane assimilation. This model, together with the fact that tools for its genetic engineering already exist, paves the way for the use of M. silvestris as a platform for metabolic engineering and industrial exploitation of methanotrophs.

Trace-gas metabolic versatility of the facultative methanotroph Methylocella silvestris.

The climate-active gas methane is generated both by biological processes and by thermogenic decomposition of fossil organic material, which forms methane and short-chain alkanes, principally ethane, propane and butane. In addition to natural sources, environments are exposed to anthropogenic inputs of all these gases from oil and gas extraction and distribution. The gases provide carbon and/or energy for a diverse range of microorganisms that can metabolize them in both anoxic and oxic zones. Aerobic methanotrophs, which can assimilate methane, have been considered to be entirely distinct from utilizers of short-chain alkanes, and studies of environments exposed to mixtures of methane and multi-carbon alkanes have assumed that disparate groups of microorganisms are responsible for the metabolism of these gases. Here we describe the mechanism by which a single bacterial strain, Methylocella silvestris, can use methane or propane as a carbon and energy source, documenting a methanotroph that can utilize a short-chain alkane as an alternative to methane. Furthermore, during growth on a mixture of these gases, efficient consumption of both gases occurred at the same time. Two soluble di-iron centre monooxygenase (SDIMO) gene clusters were identified and were found to be differentially expressed during bacterial growth on these gases, although both were required for efficient propane utilization. This report of a methanotroph expressing an additional SDIMO that seems to be uniquely involved in short-chain alkane metabolism suggests that such metabolic flexibility may be important in many environments where methane and short-chain alkanes co-occur.

Insights into toxic Prymnesium parvum blooms: the role of sugars and algal viruses.

Prymnesium parvum is a toxin-producing microalga that causes harmful algal blooms globally, which often result in large-scale fish kills that have severe ecological and economic implications. Although many toxins have previously been isolated from P. parvum, ambiguity still surrounds the responsible ichthyotoxins in P. parvum blooms and the biotic and abiotic factors that promote bloom toxicity. A major fish kill attributed to P. parvum occurred in Spring 2015 on the Norfolk Broads, a low-lying set of channels and lakes (Broads) found on the East of England. Here, we discuss how water samples taken during this bloom have led to diverse scientific advances ranging from toxin analysis to discovery of a new lytic virus of P. parvum, P. parvum DNA virus (PpDNAV-BW1). Taking recent literature into account, we propose key roles for sialic acids in this type of viral infection. Finally, we discuss recent practical detection and management strategies for controlling these devastating blooms.

Methylamine as a nitrogen source for microorganisms from a coastal marine environment.

Nitrogen is a key limiting resource for biomass production in the marine environment. Methylated amines, released from the degradation of osmolytes, could provide a nitrogen source for marine microbes. Thus far, studies in aquatic habitats on the utilization of methylamine, the simplest methylated amine, have mainly focussed on the fate of the carbon from this compound. Various groups of methylotrophs, microorganisms that can grow on one-carbon compounds, use methylamine as a carbon source. Non-methylotrophic microorganisms may also utilize methylamine as a nitrogen source, but little is known about their diversity, especially in the marine environment. In this proof-of-concept study, stable isotope probing (SIP) was used to identify microorganisms from a coastal environment that assimilate nitrogen from methylamine. SIP experiments using 15 N methylamine combined with metagenomics and metaproteomics facilitated identification of active methylamine-utilizing Alpha- and Gammaproteobacteria. The draft genomes of two methylamine utilizers were obtained and their metabolism with respect to methylamine was examined. Both bacteria identified in these SIP experiments used the γ-glutamyl-methylamide pathway, found in both methylotrophs and non-methylotrophs, to metabolize methylamine. The utilization of 15 N methylamine also led to the release of 15 N ammonium that was used as nitrogen source by other microorganisms not directly using methylamine.

Trimethylamine N-oxide metabolism by abundant marine heterotrophic bacteria.

Trimethylamine N-oxide (TMAO) is a common osmolyte found in a variety of marine biota and has been detected at nanomolar concentrations in oceanic surface waters. TMAO can serve as an important nutrient for ecologically important marine heterotrophic bacteria, particularly the SAR11 clade and marine Roseobacter clade (MRC). However, the enzymes responsible for TMAO catabolism and the membrane transporter required for TMAO uptake into microbial cells have yet to be identified. We show here that the enzyme TMAO demethylase (Tdm) catalyzes the first step in TMAO degradation. This enzyme represents a large group of proteins with an uncharacterized domain (DUF1989). The function of TMAO demethylase in a representative from the SAR11 clade (strain HIMB59) and in a representative of the MRC (Ruegeria pomeroyi DSS-3) was confirmed by heterologous expression of tdm (the gene encoding Tdm) in Escherichia coli. In R. pomeroyi, mutagenesis experiments confirmed that tdm is essential for growth on TMAO. We also identified a unique ATP-binding cassette transporter (TmoXWV) found in a variety of marine bacteria and experimentally confirmed its specificity for TMAO through marker exchange mutagenesis and lacZ reporter assays of the promoter for genes encoding this transporter. Both Tdm and TmoXWV are particularly abundant in natural seawater assemblages and actively expressed, as indicated by a number of recent metatranscriptomic and metaproteomic studies. These data suggest that TMAO represents a significant, yet overlooked, nutrient for marine bacteria.