Genomic Insights into Moderately Thermophilic Methanotrophs of the Genus Methylocaldum.

Considering the increasing interest in understanding the biotic component of methane removal from our atmosphere, it becomes essential to study the physiological characteristics and genomic potential of methanotroph isolates, especially their traits allowing them to adapt to elevated growth temperatures. The genetic signatures of Methylocaldum species have been detected in many terrestrial and aquatic ecosystems. A small set of representatives of this genus has been isolated and maintained in culture. The genus is commonly described as moderately thermophilic, with the growth optimum reaching 50 °C for some strains. Here, we present a comparative analysis of genomes of three Methylocaldum strains-two terrestrial M. szegediense strains (O-12 and Norfolk) and one marine strain, Methylocaldum marinum (S8). The examination of the core genome inventory of this genus uncovers significant redundancy in primary metabolic pathways, including the machinery for methane oxidation (numerous copies of pmo genes) and methanol oxidation (duplications of mxaF, xoxF1-5 genes), three pathways for one-carbon (C1) assimilation, and two methods of carbon storage (glycogen and polyhydroxyalkanoates). We also investigate the genetics of melanin production pathways as a key feature of the genus.

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.

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.

DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms.

Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13C, 18O, or 15N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labelled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labelled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labelling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, or even metagenomes and metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labelled microorganisms. Analysis of labelled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allowed using labelled substrates at environmentally relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies, leading to further breakthroughs in our understanding of novel microbial populations and elucidation of the metabolic function of complex microbial communities. In this chapter, we provide protocols for obtaining labelled DNA, RNA, and proteins that can be used for downstream omics-based analyses.

'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.

Cultivation of ammonia-oxidising archaea on solid medium.

Ammonia-oxidising archaea (AOA) are environmentally important microorganisms involved in the biogeochemical cycling of nitrogen. Routine cultivation of AOA is exclusively performed in liquid cultures and reports on their growth on solid medium are scarce. The ability to grow AOA on solid medium would be beneficial for not only the purification of enrichment cultures but also for developing genetic tools. The aim of this study was to develop a reliable method for growing individual colonies from AOA cultures on solid medium. Three phylogenetically distinct AOA strains were tested: 'Candidatus Nitrosocosmicus franklandus C13', Nitrososphaera viennensis EN76 and 'Candidatus Nitrosotalea sinensis Nd2'. Of the gelling agents tested, agar and Bacto-agar severely inhibited growth of all three strains. In contrast, both 'Ca. N. franklandus C13' and N. viennensis EN76 tolerated Phytagel™ while the acidophilic 'Ca. N. sinensis Nd2' was completely inhibited. Based on these observations, we developed a Liquid-Solid (LS) method that involves immobilising cells in Phytagel™ and overlaying with liquid medium. This approach resulted in the development of visible distinct colonies from 'Ca. N. franklandus C13' and N. viennensis EN76 cultures and lays the groundwork for the genetic manipulation of this group of microorganisms.

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.

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.

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.

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.

Investigating the Role of Root Exudates in Recruiting Streptomyces Bacteria to the Arabidopsis thaliana Microbiome.

Streptomyces species are saprophytic soil bacteria that produce a diverse array of specialized metabolites, including half of all known antibiotics. They are also rhizobacteria and plant endophytes that can promote plant growth and protect against disease. Several studies have shown that streptomycetes are enriched in the rhizosphere and endosphere of the model plant Arabidopsis thaliana. Here, we set out to test the hypothesis that they are attracted to plant roots by root exudates, and specifically by the plant phytohormone salicylate, which they might use as a nutrient source. We confirmed a previously published report that salicylate over-producing cpr5 plants are colonized more readily by streptomycetes but found that salicylate-deficient sid2-2 and pad4 plants had the same levels of root colonization by Streptomyces bacteria as the wild-type plants. We then tested eight genome sequenced Streptomyces endophyte strains in vitro and found that none were attracted to or could grow on salicylate as a sole carbon source. We next used 13CO2 DNA stable isotope probing to test whether Streptomyces species can feed off a wider range of plant metabolites but found that Streptomyces bacteria were outcompeted by faster growing proteobacteria and did not incorporate photosynthetically fixed carbon into their DNA. We conclude that, given their saprotrophic nature and under conditions of high competition, streptomycetes most likely feed on more complex organic material shed by growing plant roots. Understanding the factors that impact the competitiveness of strains in the plant root microbiome could have consequences for the effective application of biocontrol strains.

Soil, senescence and exudate utilisation: characterisation of the Paragon var. spring bread wheat root microbiome.

BACKGROUND: Conventional methods of agricultural pest control and crop fertilisation are unsustainable. To meet growing demand, we must find ecologically responsible means to control disease and promote crop yields. The root-associated microbiome can aid plants with disease suppression, abiotic stress relief, and nutrient bioavailability. The aim of the present work was to profile the community of bacteria, fungi, and archaea associated with the wheat rhizosphere and root endosphere in different conditions. We also aimed to use 13CO2 stable isotope probing (SIP) to identify microbes within the root compartments that were capable of utilising host-derived carbon. RESULTS: Metabarcoding revealed that community composition shifted significantly for bacteria, fungi, and archaea across compartments. This shift was most pronounced for bacteria and fungi, while we observed weaker selection on the ammonia oxidising archaea-dominated archaeal community. Across multiple soil types we found that soil inoculum was a significant driver of endosphere community composition, however, several bacterial families were identified as core enriched taxa in all soil conditions. The most abundant of these were Streptomycetaceae and Burkholderiaceae. Moreover, as the plants senesce, both families were reduced in abundance, indicating that input from the living plant was required to maintain their abundance in the endosphere. Stable isotope probing showed that bacterial taxa within the Burkholderiaceae family, among other core enriched taxa such as Pseudomonadaceae, were able to use root exudates, but Streptomycetaceae were not. CONCLUSIONS: The consistent enrichment of Streptomycetaceae and Burkholderiaceae within the endosphere, and their reduced abundance after developmental senescence, indicated a significant role for these families within the wheat root microbiome. While Streptomycetaceae did not utilise root exudates in the rhizosphere, we provide evidence that Pseudomonadaceae and Burkholderiaceae family taxa are recruited to the wheat root community via root exudates. This deeper understanding crop microbiome formation will enable researchers to characterise these interactions further, and possibly contribute to ecologically responsible methods for yield improvement and biocontrol in the future.