Discovery and heterologous biosynthesis of glycosylated polyketide luteodienoside A reveals unprecedented glucinol-mediated product offloading by a fungal carnitine O-acyltransferase domain.

Luteodienoside A is a novel glycosylated polyketide produced by the Australian fungus Aspergillus luteorubrus MST-FP2246, consisting of an unusual 1-O-ß-d-glucopyranosyl-myo-inositol (glucinol) ester of 3-hydroxy-2,2,4-trimethylocta-4,6-dienoic acid. Mining the genome of A. luteorubrus identified a putative gene cluster for luteodienoside A biosynthesis (ltb), harbouring a highly reducing polyketide synthase (HR-PKS, LtbA) fused at its C-terminus to a carnitine O-acyltransferase (cAT) domain. Heterologous pathway reconstitution in Aspergillus nidulans, substrate feeding assays and gene truncation confirmed the identity of the ltb cluster and demonstrated that the cAT domain is essential for offloading luteodienoside A from the upstream HR-PKS. Unlike previously characterised cAT domains, the LtbA cAT domain uses glucinol as an offloading substrate to release the product from the HR-PKS. Furthermore, the PKS methyltransferase (MT) domain is capable of catalysing gem-dimethylation of the 3-hydroxy-2,2,4-trimethylocta-4,6-dienoic acid intermediate, without requiring reversible product release and recapture by the cAT domain. This study expands the repertoire of polyketide modifications known to be catalysed by cAT domains and highlights the potential of mining fungal genomes for this subclass of fungal PKSs to discover new structurally diverse secondary metabolites.

Complete genome sequences of Methylococcus capsulatus (Norfolk) and Methylocaldum szegediense (Norfolk) isolated from a landfill methane biofilter.

Here we report the complete genome sequence of two moderately thermophilic methanotrophs isolated from a landfill methane biofilter, Methylococcus capsulatus (Norfolk) and Methylocaldum szegediense (Norfolk).

Whole-cell studies of substrate and inhibitor specificity of isoprene monooxygenase and related enzymes.

Co-oxidation of a range of alkenes, dienes, and aromatic compounds by whole cells of the isoprene-degrading bacterium Rhodococcus sp. AD45 expressing isoprene monooxygenase was investigated, revealing a relatively broad substrate specificity for this soluble diiron centre monooxygenase. A range of 1-alkynes (C2 -C8 ) were tested as potential inhibitors. Acetylene, a potent inhibitor of the related enzyme soluble methane monooxygenase, had little inhibitory effect, whereas 1-octyne was a potent inhibitor of isoprene monooxygenase, indicating that 1-octyne could potentially be used as a specific inhibitor to differentiate between isoprene consumption by bona fide isoprene degraders and co-oxidation of isoprene by other oxygenase-containing bacteria, such as methanotrophs, in environmental samples. The isoprene oxidation kinetics of a variety of monooxygenase-expressing bacteria were also investigated, revealing that alkene monooxygenase from Xanthobacter and soluble methane monooxygenases from Methylococcus and Methylocella, but not particulate methane monooxygenases from Methylococcus or Methylomicrobium, could co-oxidise isoprene at appreciable rates. Interestingly the ammonia monooxygenase from the nitrifier Nitrosomonas europaea could also co-oxidise isoprene at relatively high rates, suggesting that co-oxidation of isoprene by additional groups of bacteria, under the right conditions, might occur in the environment.

Fungal Duel between Penicillium brasilianum and Aspergillus nomius Results in Dual Induction of Miktospiromide A and Kitrinomycin A.

Cocultivation of the fungi Penicillium brasilianum MST-FP1927 and Aspergillus nomius MST-FP2004 resulted in the reciprocal induction of two new compounds, miktospiromide A (1) from A. nomius and kitrinomycin A (2) from P. brasilianum. A third new compound, kitrinomycin B (3), was also identified from an axenic culture of P. brasilianum, along with the previously reported compounds austalide K (4), 17S-dihydroaustalide K (5), verruculogen (6), and fumitremorgin B (7). The structures of 1-3 were elucidated by detailed spectroscopic analysis and DFT calculations, while 4-7 were identified by comparison to authentic standards. The genome of A. nomius MST-FP2004 was sequenced, and a putative biosynthetic gene cluster for 1 was identified. Compound 2 showed activity against murine melanoma NS-1 cells (LD99 7.8 µM) and the bovine parasite Tritrichomonas foetus (LD99 4.8 µM).

Alcohols as inhibitors of ammonia oxidizing archaea and bacteria.

Ammonia oxidizers are key players in the global nitrogen cycle and are responsible for the oxidation of ammonia to nitrite, which is further oxidized to nitrate by other microorganisms. Their activity can lead to adverse effects on some human-impacted environments, including water pollution through leaching of nitrate and emissions of the greenhouse gas nitrous oxide (N2O). Ammonia monooxygenase (AMO) is the key enzyme in microbial ammonia oxidation and shared by all groups of aerobic ammonia oxidizers. The AMO has not been purified in an active form, and much of what is known about its potential structure and function comes from studies on its interactions with inhibitors. The archaeal AMO is less well studied as ammonia oxidizing archaea were discovered much more recently than their bacterial counterparts. The inhibition of ammonia oxidation by aliphatic alcohols (C1-C8) using the model terrestrial ammonia oxidizing archaeon 'Candidatus Nitrosocosmicus franklandus' C13 and the ammonia oxidizing bacterium Nitrosomonas europaea was examined in order to expand knowledge about the range of inhibitors of ammonia oxidizers. Methanol was the most potent specific inhibitor of the AMO in both ammonia oxidizers, with half-maximal inhibitory concentrations (IC50) of 0.19 and 0.31 mM, respectively. The inhibition was AMO-specific in 'Ca. N. franklandus' C13 in the presence of C1-C2 alcohols, and in N. europaea in the presence of C1-C3 alcohols. Higher chain-length alcohols caused non-specific inhibition and also inhibited hydroxylamine oxidation. Ethanol was tolerated by 'Ca. N. franklandus' C13 at a higher threshold concentration than other chain-length alcohols, with 80 mM ethanol being required for complete inhibition of ammonia oxidation.

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.

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.

Peering down the sink: A review of isoprene metabolism by bacteria.

Isoprene (2-methyl-1,3-butadiene) is emitted to the atmosphere each year in sufficient quantities to rival methane (>500 Tg C yr-1 ), primarily due to emission by trees and other plants. Chemical reactions of isoprene with other atmospheric compounds, such as hydroxyl radicals and inorganic nitrogen species (NOx ), have implications for global warming and local air quality, respectively. For many years, it has been estimated that soil-dwelling bacteria consume a significant amount of isoprene (~20 Tg C yr-1 ), but the mechanisms underlying the biological sink for isoprene have been poorly understood. Studies have indicated or confirmed the ability of diverse bacterial genera to degrade isoprene, whether by the canonical iso-type isoprene degradation pathway or through other less well-characterized mechanisms. Here, we review current knowledge of isoprene metabolism and highlight key areas for further research. In particular, examples of isoprene-degraders that do not utilize the isoprene monooxygenase have been identified in recent years. This has fascinating implications both for the mechanism of isoprene uptake by bacteria, and also for the ecology of isoprene-degraders in the environments.

'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.

Oceanospirillales containing the DMSP lyase DddD are key utilisers of carbon from DMSP in coastal seawater.

BACKGROUND: Ubiquitous and diverse marine microorganisms utilise the abundant organosulfur molecule dimethylsulfoniopropionate (DMSP), the main precursor of the climate-active gas dimethylsulfide (DMS), as a source of carbon, sulfur and/or signalling molecules. However, it is currently difficult to discern which microbes actively catabolise DMSP in the environment, why they do so and the pathways used. RESULTS: Here, a novel DNA-stable isotope probing (SIP) approach, where only the propionate and not the DMS moiety of DMSP was 13C-labelled, was strategically applied to identify key microorganisms actively using DMSP and also likely DMS as a carbon source, and their catabolic enzymes, in North Sea water. Metagenomic analysis of natural seawater suggested that Rhodobacterales (Roseobacter group) and SAR11 bacteria were the major microorganisms degrading DMSP via demethylation and, to a lesser extent, DddP-driven DMSP lysis pathways. However, neither Rhodobacterales and SAR11 bacteria nor their DMSP catabolic genes were prominently labelled in DNA-SIP experiments, suggesting they use DMSP as a sulfur source and/or in signalling pathways, and not primarily for carbon requirements. Instead, DNA-SIP identified gammaproteobacterial Oceanospirillales, e.g. Amphritea, and their DMSP lyase DddD as the dominant microorganisms/enzymes using DMSP as a carbon source. Supporting this, most gammaproteobacterial (with DddD) but few alphaproteobacterial seawater isolates grew on DMSP as sole carbon source and produced DMS. Furthermore, our DNA-SIP strategy also identified Methylophaga and other Piscirickettsiaceae as key bacteria likely using the DMS, generated from DMSP lysis, as a carbon source. CONCLUSIONS: This is the first study to use DNA-SIP with 13C-labelled DMSP and, in a novel way, it identifies the dominant microbes utilising DMSP and DMS as carbon sources. It highlights that whilst metagenomic analyses of marine environments can predict microorganisms/genes that degrade DMSP and DMS based on their abundance, it cannot disentangle those using these important organosulfur compounds for their carbon requirements. Note, the most abundant DMSP degraders, e.g. Rhodobacterales with DmdA, are not always the key microorganisms using DMSP for carbon and releasing DMS, which in this coastal system were Oceanospirillales containing DddD. Video abstract.

Genome Characterisation of an Isoprene-Degrading Alcaligenes sp. Isolated from a Tropical Restored Forest.

Isoprene is a climate-active biogenic volatile organic compound (BVOC), emitted into the atmosphere in abundance, mainly from terrestrial plants. Soil is an important sink for isoprene due to its consumption by microbes. In this study, we report the ability of a soil bacterium to degrade isoprene. Strain 13f was isolated from soil beneath wild Himalayan cherry trees in a tropical restored forest. Based on phylogenomic analysis and an Average Nucleotide Identity score of >95%, it most probably belongs to the species Alcaligenes faecalis. Isoprene degradation by Alcaligenes sp. strain 13f was measured by using gas chromatography. When isoprene was supplied as the sole carbon and energy source at the concentration of 7.2 × 105 ppbv and 7.2 × 106 ppbv, 32.6% and 19.6% of isoprene was consumed after 18 days, respectively. Genome analysis of Alcaligenes sp. strain 13f revealed that the genes that are typically found as part of the isoprene monooxygenase gene cluster in other isoprene-degrading bacteria were absent. This discovery suggests that there may be alternative pathways for isoprene metabolism.

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.

Identification of active gaseous-alkane degraders at natural gas seeps.

Natural gas seeps release significant amounts of methane and other gases including ethane and propane contributing to global climate change. In this study, bacterial actively consuming short-chain alkanes were identified by cultivation, whole-genome sequencing, and stable-isotope probing (SIP)-metagenomics using 13C-propane and 13C-ethane from two different natural gas seeps, Pipe Creek and Andreiasu Everlasting Fire. Nearly 100 metagenome-assembled genomes (MAGs) (completeness 70-99%) were recovered from both sites. Among these, 16 MAGs had genes encoding the soluble di-iron monooxygenase (SDIMO). The MAGs were affiliated to Actinobacteria (two MAGs), Alphaproteobacteria (ten MAGs), and Gammaproteobacteria (four MAGs). Additionally, three gaseous-alkane degraders were isolated in pure culture, all of which could grow on ethane, propane, and butane and possessed SDIMO-related genes. Two Rhodoblastus strains (PC2 and PC3) were from Pipe Creek and a Mycolicibacterium strain (ANDR5) from Andreiasu. Strains PC2 and PC3 encoded putative butane monooxygenases (MOs) and strain ANDR5 contained a propane MO. Mycolicibacterium strain ANDR5 and MAG19a, highly abundant in incubations with 13C-ethane, share an amino acid identity (AAI) of 99.3%. We show using a combination of enrichment and isolation, and cultivation-independent techniques, that these natural gas seeps contain a diverse community of active bacteria oxidising gaseous-alkanes, which play an important role in biogeochemical cycling of natural gas.

The effect of lanthanum on growth and gene expression in a facultative methanotroph.

The biological importance of lanthanides has only recently been identified, initially as the active site metal of the alternative methanol dehydrogenase (MDH) Xox-MDH. So far, the effect of lanthanide (Ln) has only been studied in relatively few organisms. This work investigated the effects of Ln on gene transcription and protein expression in the facultative methanotroph Methylocella silvestris BL2, a widely distributed methane-oxidizing bacterium with the unique ability to grow not just on methane but also on other typical components of natural gas, ethane and propane. Expression of calcium- or Ln-dependent MDH was controlled by Ln (the lanthanide switch) during growth on one-, two- or three-carbon substrates, and Ln imparted a considerable advantage during growth on propane, a novel result extending the importance of Ln to consumers of this component of natural gas. Two Xox-MDHs were expressed and regulated by Ln in M. silvestris, but interestingly Ln repressed rather than induced expression of the second Xox-MDH. Despite the metabolic versatility of M. silvestris, no other alcohol dehydrogenases were expressed, and in double-mutant strains lacking genes encoding both Ca- and Ln-dependent MDHs (mxaF and xoxF5 or xoxF1), growth on methanol and ethanol appeared to be enabled by expression of the soluble methane monooxygenase.

Genome Mining of Aspergillus hancockii Unearths Cryptic Polyketide Hancockinone A Featuring a Prenylated 6/6/6/5 Carbocyclic Skeleton.

Activation of a cryptic polyketide synthase gene cluster hkn from Aspergillus hancockii via overexpression of the gene-cluster-specific transcription factor HknR led to the discovery of a novel polycyclic metabolite, which we named hancockinone A. The compound features an unprecedented prenylated 6/6/6/5 tetracarbocyclic skeleton and shows moderate antibacterial activity. Heterologous expression, substrate feeding, and in vitro assays confirmed the role of cytochrome P450 HknE in constructing the five-membered ring in hancockinone A from the precursor neosartoricin B.

Isoprene-degrading bacteria associated with the phyllosphere of Salix fragilis, a high isoprene-emitting willow of the Northern Hemisphere.

BACKGROUND: Isoprene accounts for about half of total biogenic volatile organic compound emissions globally, and as a climate active gas it plays a significant and varied role in atmospheric chemistry. Terrestrial plants are the largest source of isoprene, with willow (Salix) making up one of the most active groups of isoprene producing trees. Bacteria act as a biological sink for isoprene and those bacteria associated with high isoprene-emitting trees may provide further insight into its biodegradation. RESULTS: A DNA-SIP experiment incubating willow (Salix fragilis) leaves with 13C-labelled isoprene revealed an abundance of Comamonadaceae, Methylobacterium, Mycobacterium and Polaromonas in the isoprene degrading community when analysed by 16S rRNA gene amplicon sequencing. Metagenomic analysis of 13C-enriched samples confirmed the abundance of Comamonadaceae, Acidovorax, Polaromonas, Variovorax and Ramlibacter. Mycobacterium and Methylobacterium were also identified after metagenomic analysis and a Mycobacterium metagenome-assembled genome (MAG) was recovered. This contained two complete isoprene degradation metabolic gene clusters, along with a propane monooxygenase gene cluster. Analysis of the abundance of the alpha subunit of the isoprene monooxygenase, isoA, in unenriched DNA samples revealed that isoprene degraders associated with willow leaves are abundant, making up nearly 0.2% of the natural bacterial community. CONCLUSIONS: Analysis of the isoprene degrading community associated with willow leaves using DNA-SIP and focused metagenomics techniques enabled recovery of the genome of an active isoprene-degrading Mycobacterium species and provided valuable insight into bacteria involved in degradation of isoprene on the leaves of a key species of isoprene-emitting tree in the northern hemisphere.

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.

Evaluation of Benzguinols as Next-Generation Antibiotics for the Treatment of Multidrug-Resistant Bacterial Infections.

Our recent focus on the "lost antibiotic" unguinol and related nidulin-family fungal natural products identified two semisynthetic derivatives, benzguinols A and B, with unexpected in vitro activity against Staphylococcus aureus isolates either susceptible or resistant to methicillin. Here, we show further activity of the benzguinols against methicillin-resistant isolates of the animal pathogen Staphylococcus pseudintermedius, with minimum inhibitory concentration (MIC) ranging 0.5-1 µg/mL. When combined with sub-inhibitory concentrations of colistin, the benzguinols demonstrated synergy against Gram-negative reference strains of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa (MICs of 1-2 µg/mL in the presence of colistin), whereas the benzguinols alone had no activity. Administration of three intraperitoneal (IP) doses of 20 mg/kg benzguinol A or B to mice did not result in any obvious adverse clinical or pathological evidence of acute toxicity. Importantly, mice that received three 20 mg/kg IP doses of benzguinol A or B at 4 h intervals exhibited significantly reduced bacterial loads and longer survival times than vehicle-only treated mice in a bioluminescent S. aureus murine sepsis challenge model. We conclude that the benzguinols are potential candidates for further development for specific treatment of serious bacterial infections as both stand-alone antibiotics and in combination with existing antibiotic classes.

Semisynthesis and biological evaluation of a focused library of unguinol derivatives as next-generation antibiotics.

In this study, we report the semisynthesis and in vitro biological evaluation of thirty-four derivatives of the fungal depsidone antibiotic, unguinol. Initially, the semisynthetic modifications were focused on the two free hydroxy groups (3-OH and 8-OH), the three free aromatic positions (C-2, C-4 and C-7), the butenyl side chain and the depsidone ester linkage. Fifteen first-generation unguinol analogues were synthesised and screened against a panel of bacteria, fungi and mammalian cells to formulate a basic structure activity relationship (SAR) for the unguinol pharmacophore. Based on the SAR studies, we synthesised a further nineteen second-generation analogues, specifically aimed at improving the antibacterial potency of the pharmacophore. In vitro antibacterial activity testing of these compounds revealed that 3-O-(2-fluorobenzyl)unguinol and 3-O-(2,4-difluorobenzyl)unguinol showed potent activity against both methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MIC 0.25-1 µg mL-1) and are promising candidates for further development in vivo.