A methanotrophic bacterium to enable methane removal for climate mitigation.

The rapid increase of the potent greenhouse gas methane in the atmosphere creates great urgency to develop and deploy technologies for methane mitigation. One approach to removing methane is to use bacteria for which methane is their carbon and energy source (methanotrophs). Such bacteria naturally convert methane to CO2 and biomass, a value-added product and a cobenefit of methane removal. Typically, methanotrophs grow best at around 5,000 to 10,000 ppm methane, but methane in the atmosphere is 1.9 ppm. Air above emission sites such as landfills, anaerobic digestor effluents, rice paddy effluents, and oil and gas wells contains elevated methane in the 500 ppm range. If such sites are targeted for methane removal, technology harnessing aerobic methanotroph metabolism has the potential to become economically and environmentally viable. The first step in developing such methane removal technology is to identify methanotrophs with enhanced ability to grow and consume methane at 500 ppm and lower. We report here that some existing methanotrophic strains grow well at 500 ppm methane, and one of them, Methylotuvimicrobium buryatense 5GB1C, consumes such low methane at enhanced rates compared to previously published values. Analyses of bioreactor-based performance and RNAseq-based transcriptomics suggest that this ability to utilize low methane is based at least in part on extremely low non-growth-associated maintenance energy and on high methane specific affinity. This bacterium is a candidate to develop technology for methane removal at emission sites. If appropriately scaled, such technology has the potential to slow global warming by 2050.

A Computational Framework for Identifying Promoter Sequences in Nonmodel Organisms Using RNA-seq Data Sets.

Engineering microorganisms into biological factories that convert renewable feedstocks into valuable materials is a major goal of synthetic biology; however, for many nonmodel organisms, we do not yet have the genetic tools, such as suites of strong promoters, necessary to effectively engineer them. In this work, we developed a computational framework that can leverage standard RNA-seq data sets to identify sets of constitutive, strongly expressed genes and predict strong promoter signals within their upstream regions. The framework was applied to a diverse collection of RNA-seq data measured for the methanotroph Methylotuvimicrobium buryatense 5GB1 and identified 25 genes that were constitutively, strongly expressed across 12 experimental conditions. For each gene, the framework predicted short (27-30 nucleotide) sequences as candidate promoters and derived -35 and -10 consensus promoter motifs (TTGACA and TATAAT, respectively) for strong expression in M. buryatense. This consensus closely matches the canonical E. coli sigma-70 motif and was found to be enriched in promoter regions of the genome. A subset of promoter predictions was experimentally validated in a XylE reporter assay, including the consensus promoter, which showed high expression. The pmoC, pqqA, and ssrA promoter predictions were additionally screened in an experiment that scrambled the -35 and -10 signal sequences, confirming that transcription initiation was disrupted when these specific regions of the predicted sequence were altered. These results indicate that the computational framework can make biologically meaningful promoter predictions and identify key pieces of regulatory systems that can serve as foundational tools for engineering diverse microorganisms for biomolecule production.

Cultivation techniques to study lanthanide metal interactions in the haloalkaliphilic Type I methanotroph "Methylotuvimicrobium buryatense" 5GB1C.

Lanthanide metals are commonly used in technological devices including batteries, computers, catalysts and magnets. Despite their important properties, mining difficulties and pollution concerns limit the number of mines worldwide. Because of these concerns, biometallurgy is an attractive possibility for lanthanide extraction from recycled materials or from contaminated sites. Methylotrophs, bacteria that grow on reduced carbon substrates like methane and methanol, utilize lanthanides for a central reaction in their metabolisms. They must have some mechanism for uptake or trafficking, and are therefore excellent candidates for applying small molecules or proteins for selective lanthanide metal recycling. The haloalkaliphilic methanotroph "Methylotuvimicrobium buryatense" 5GB1C is the fastest growing methanotroph isolated to date, and thus has great industrial potential. The MxaFI enzyme complex uses calcium as a Lewis acid in conjunction with the pyroquinoline quinone cofactor to oxidize methanol, while the alternative enzyme XoxF uses lanthanide metals (e.g. lanthanum and cerium) for the same function. Lanthanide metals, abundant in the earth's crust, strongly repress the transcription of mxaF yet activate the transcription of xoxF, implying that XoxF may be the predominant methanol dehydrogenase in the bacterium's native environment. It may be that lanthanum interaction mechanisms are different from those in other microorganisms. In addition, the facile genetics in this strain and existing background information make it a good study organism for biological lanthanum uptake. The interesting physiology of this organism required empirical work to develop cultivation methods that allow robust assays of gene expression and measurement of lanthanum associated with cell biomass. In this chapter, we show that altering the metal chelator increased the availability of lanthanum to the cell as measured by the specific gene expression response. We also made further alterations to prevent lanthanum precipitation in medium for the growth of haloalkaliphiles.

Expression, purification and properties of the enzymes involved in lanthanide-dependent alcohol oxidation: XoxF4, XoxF5, ExaF/PedH, and XoxG4.

In this chapter we describe logistics, protocols and conditions for expression, purification and characterization of Ln3+-dependent alcohol dehydrogenases representing three distinct phylogenetic clades of these enzymes, classified as XoxF4, XoxF5 and ExaF/PedH. We present data on the biochemical properties of a dozen enzymes, all generated by our group, in a comparative fashion. These enzymes display a range of properties in terms of substrate and metal specificities, pH and ammonium requirement, as well as catalytic constants. In addition, we describe a single novel cytochrome, XoxG4, that likely serves as a natural electron acceptor from XoxF5 in methanotrophs of the Gammaproteobacteria class.

The Entner-Doudoroff Pathway Is an Essential Metabolic Route for Methylotuvimicrobium buryatense 5GB1C.

Methylotuvimicrobium buryatense 5GB1C, a fast-growing gammaproteobacterial methanotroph, is equipped with two glycolytic pathways, the Entner-Doudoroff (ED) pathway and the Embden-Meyerhof-Parnas (EMP) pathway. Metabolic flux analysis and 13C-labeling experiments have shown the EMP pathway is the principal glycolytic route in M. buryatense 5GB1C, while the ED pathway appears to be metabolically and energetically insignificant. However, it has not been possible to obtain a null mutant in the edd-eda genes encoding the two unique enzymatic reactions in the ED pathway, suggesting the ED pathway may be essential for M. buryatense 5GB1C growth. In this study, the inducible P BAD promoter was used to manipulate gene expression of edd-eda, and in addition, the expression of these two genes was separated from that of a downstream gltA gene. The resulting strain shows arabinose-dependent growth that correlates with ED pathway activity, with normal growth achieved in the presence of ∼0.1 g/liter arabinose. Flux balance analysis shows that M. buryatense 5GB1C with a strong ED pathway has a reduced energy budget, thereby limiting the mutant growth at a high concentration of arabinose. Collectively, our study demonstrates that the ED pathway is essential for M. buryatense 5GB1C. However, no known mechanism can directly explain the essentiality of the ED pathway, and thus, it may have a yet unknown regulatory role required for sustaining a healthy and functional metabolism in this bacterium.IMPORTANCE The gammaproteobacterial methanotrophs possess a unique central metabolic architecture where methane and other reduced C1 carbon sources are assimilated through the ribulose monophosphate cycle. Although efforts have been made to better understand methanotrophic metabolism in these bacteria via experimental and computational approaches, many questions remain unanswered. One of these is the essentiality of the ED pathway for M. buryatense 5GB1C, as current results appear contradictory. By creating a construct with edd-eda and gltA genes controlled by P BAD and P J23101 , respectively, we demonstrated the essentiality of the ED pathway for this obligate methanotroph. It is also demonstrated that these genetic tools are applicable to M. buryatense 5GB1C and that expression of the target genes can be tightly controlled across an extensive range. Our study adds to the expanding knowledge of methanotrophic metabolism and practical approaches to genetic manipulation for obligate methanotrophs.

Synthetic Methane-Consuming Communities from a Natural Lake Sediment.

The factors and processes that influence the behavior and functionality of ecosystems inhabited by complex microbiomes are still far from being clearly understood. Synthetic microbial communities provide reduced-complexity models that allow an examination of ecological theories under defined and controlled conditions. In this study, we applied a multiphasic approach to study synthetic methane-oxidizing communities and species interactions as proxies to the natural communities. Our results confirm that, under selective pressures, natural-sediment communities of high complexity simplify rapidly, selecting for several major functional guilds, the major partners in methane oxidation being the Methylococcaceae methanotrophs and the Methylophilaceae methylotrophs, along with minor but persistent partners, members of Burkholderiales and Flavobacteriales As a proof of concept, we established minimalist synthetic communities that were representative of the four functional guilds to demonstrate the dependency of the non-methane-utilizing species on the methanotrophs as the primary carbon-providing species. We observed that in communities consisting of multiple representatives of the key guilds, members of the same guild appeared to compete for resources. For example, when two methanotrophs of the same family were present, the two expressed similar key methanotrophy pathways and responded similarly to changing environmental conditions, suggesting that they perform a similar keystone function in situ Similar observations were made for the Methylophilaceae However, differences were noted in the expression of auxiliary and unique genes among strains of the same functional guild, reflecting differential adaptation and suggesting mechanisms for competition. At the same time, differences were also noted in the performances of partners with specific metabolic schemes. For example, a mutant of Methylotenera mobilis impaired in nitrate utilization behaved as a more efficient cooperator in methane consumption, suggesting that the loss of function may lead to changes in communal behavior. Overall, we demonstrate the robust nature of synthetic communities built of native lake sediment strains and their utility in addressing important ecological questions while using a simplified model.IMPORTANCE The metabolism of methane is an important part of the global carbon cycle. While deciphering the community function and the potential role of the different functional guilds is very difficult when considering native complex communities, synthetic communities, built of species originating from a study site in question, present a simplified model and allow specific questions to be addressed as to carbon, nitrogen, and other nutrient transfer among species in a controlled system. This study applies an ecophysiological approach, as a proof of principle, to an already well-studied model system, contributing to a better understanding of microbial community function and microbial ecosystem processes.

A Mutagenic Screen Identifies a TonB-Dependent Receptor Required for the Lanthanide Metal Switch in the Type I Methanotroph "Methylotuvimicrobium buryatense" 5GB1C.

Several of the metabolic enzymes in methanotrophic bacteria rely on metals for both their expression and their catalysis. The MxaFI methanol dehydrogenase enzyme complex uses calcium as a cofactor to oxidize methanol, while the alternative methanol dehydrogenase XoxF uses lanthanide metals such as lanthanum and cerium for the same function. Lanthanide metals, abundant in the earth's crust, strongly repress the transcription of mxaF yet activate the transcription of xoxF This regulatory program, called the "lanthanide switch," is central to methylotrophic metabolism, but only some of its components are known. To uncover additional components of the lanthanide switch, we developed a chemical mutagenesis system in the type I gammaproteobacterial methanotroph "Methylotuvimicrobium buryatense" 5GB1C and designed a selection system for mutants unable to repress the mxaF promoter in the presence of lanthanum. Whole-genome resequencing for multiple lanthanide switch mutants identified several unique point mutations in a single gene encoding a TonB-dependent receptor, which we have named LanA. The LanA TonB-dependent receptor is absolutely required for the lanthanide switch and controls the expression of a small set of genes. While mutation of the lanA gene does not affect the amount of cell-associated lanthanum, it is essential for growth in the absence of the MxaF methanol dehydrogenase, suggesting that LanA is involved in lanthanum uptake to supply the XoxF methanol dehydrogenase with its critical metal ion cofactor. The discovery of this novel component of the lanthanide regulatory system highlights the complexity of this circuit and suggests that further components are likely involved.IMPORTANCE Lanthanide metals, or rare earth elements, are abundant in nature and used heavily in technological devices. Biological interactions with lanthanides are just beginning to be unraveled. Until very recently, microbial mechanisms of lanthanide metal interaction and uptake were unknown. The TonB-dependent receptor LanA is the first lanthanum receptor identified in a methanotroph. Sequence homology searches with known metal transporters and regulators could not be used to identify LanA or other lanthanide metal switch components, and this method for mutagenesis and selection was required to identify the receptor. This work advances the knowledge of microbe-metal interactions in environmental niches that impact atmospheric methane levels and are thus relevant to climate change.

Rare earth element alcohol dehydrogenases widely occur among globally distributed, numerically abundant and environmentally important microbes.

Lanthanides (Ln3+), known as rare earth elements, have recently emerged as enzyme cofactors, contrary to prior assumption of their biological inertia. Several bacterial alcohol dehydrogenases have been characterized so far that depend on Ln3+ for activity and expression, belonging to the methanol dehydrogenase clade XoxF and the ethanol dehydrogenase clade ExaF/PedH. Here we compile an inventory of genes potentially encoding Ln3+-dependent enzymes, closely related to the previously characterized XoxF and ExaF/PedH enzymes. We demonstrate their wide distribution among some of the most numerically abundant and environmentally important taxa, such as the phylogenetically disparate rhizobial species and metabolically versatile bacteria inhabiting world's oceans, suggesting that reliance on Ln3+-mediated biochemistry is much more widespread in the microbial world than previously assumed. Through protein expression and analysis, we here more than double the extant collection of the biochemically characterized Ln3+-dependent enzymes, demonstrating a range of catalytic properties and substrate and cofactor specificities. Many of these enzymes reveal propensity for oxidation of methanol. This observation, in combination with genome-based reconstruction of methylotrophy pathways for select species suggests a much wider occurrence of this metabolic capability among bacterial species, and thus further suggests the importance of methylated compounds as parts of the global carbon cycling.

Deletion of the Clostridium thermocellum recA gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences.

A limitation to the engineering of cellulolytic thermophiles is the availability of functional, thermostable (≥ 60 °C) replicating plasmid vectors for rapid expression and testing of genes that provide improved or novel fuel molecule production pathways. A series of plasmid vectors for genetic manipulation of the cellulolytic thermophile Caldicellulosiruptor bescii has recently been extended to Clostridium thermocellum, another cellulolytic thermophile that very efficiently solubilizes plant biomass and produces ethanol. While the C. bescii pBAS2 replicon on these plasmids is thermostable, the use of homologous promoters, signal sequences and genes led to undesired integration into the bacterial chromosome, a result also observed with less thermostable replicating vectors. In an attempt to overcome undesired plasmid integration in C. thermocellum, a deletion of recA was constructed. As expected, C. thermocellum ∆recA showed impaired growth in chemically defined medium and an increased susceptibility to UV damage. Interestingly, we also found that recA is required for replication of the C. bescii thermophilic plasmid pBAS2 in C. thermocellum, but it is not required for replication of plasmid pNW33N. In addition, the C. thermocellum recA mutant retained the ability to integrate homologous DNA into the C. thermocellum chromosome. These data indicate that recA can be required for replication of certain plasmids, and that a recA-independent mechanism exists for the integration of homologous DNA into the C. thermocellum chromosome. Understanding thermophilic plasmid replication is not only important for engineering of these cellulolytic thermophiles, but also for developing genetic systems in similar new potentially useful non-model organisms.

Expression of a heat-stable NADPH-dependent alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus 39E in Clostridium thermocellum 1313 results in increased hydroxymethylfurfural resistance.

BACKGROUND: Resistance to deconstruction is a major limitation to the use of lignocellulosic biomass as a substrate for the production of fuels and chemicals. Consolidated bioprocessing (CBP), the use of microbes for the simultaneous hydrolysis of lignocellulose into soluble sugars and fermentation of the resulting sugars to products of interest, is a potential solution to this obstacle. The pretreatment of plant biomass, however, releases compounds that are inhibitory to the growth of microbes used for CBP. RESULTS: Heterologous expression of the Thermoanaerobacter pseudethanolicus 39E bdhA gene, that encodes an alcohol dehydrogenase, in Clostridium thermocellum significantly increased resistance to furan derivatives at concentrations found in acid-pretreated biomass. The mechanism of detoxification of hydroxymethylfurfural was shown to be primarily reduction using NADPH as the cofactor. In addition, we report the construction of new expression vectors for homologous and heterologous expression in C. thermocellum. These vectors use regulatory signals from both C. bescii (the S-layer promoter) and C. thermocellum (the enolase promoter) shown to efficiently drive expression of the BdhA enzyme. CONCLUSIONS: Toxic compounds present in lignocellulose hydrolysates that inhibit cell growth and product formation are obstacles to the commercialization of fuels and chemicals from biomass. Expression of genes that reduce the effect of these inhibitors, such as furan derivatives, will serve to enable commercial processes using plant biomass for the production of fuels and chemicals.

Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature.

Clostridium thermocellum is a leading candidate for the consolidated bioprocessing of lignocellulosic biomass for the production of fuels and chemicals. A limitation to the engineering of this strain is the availability of stable replicating plasmid vectors for homologous and heterologous expression of genes that provide improved and/or novel pathways for fuel production. Current vectors relay on replicons from mesophilic bacteria and are not stable at the optimum growth temperature of C. thermocellum. To develop more thermostable genetic tools for C. thermocellum, we constructed vectors based on the hyperthermophilic Caldicellulosiruptor bescii replicon pBAS2. Autonomously replicating shuttle vectors based on pBAS2 reproducibly transformed C. thermocellum at 60 °C and were maintained in multiple copy. Promoters, selectable markers and plasmid replication proteins from C. bescii were functional in C. thermocellum. Phylogenetic analyses of the proteins contained on pBAS2 revealed that the replication initiation protein RepL is unique among thermophiles. These results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.

Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing.

Restriction-modification (R-M) systems pose a major barrier to DNA transformation and genetic engineering of bacterial species. Systematic identification of DNA methylation in R-M systems, including N(6)-methyladenine (6mA), 5-methylcytosine (5mC) and N(4)-methylcytosine (4mC), will enable strategies to make these species genetically tractable. Although single-molecule, real time (SMRT) sequencing technology is capable of detecting 4mC directly for any bacterial species regardless of whether an assembled genome exists or not, it is not as scalable to profiling hundreds to thousands of samples compared with the commonly used next-generation sequencing technologies. Here, we present 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), a next-generation sequencing method that rapidly and cost efficiently reveals the genome-wide locations of 4mC for bacterial species with an available assembled reference genome. In 4mC-TAB-seq, both cytosines and 5mCs are read out as thymines, whereas only 4mCs are read out as cytosines, revealing their specific positions throughout the genome. We applied 4mC-TAB-seq to study the methylation of a member of the hyperthermophilc genus, Caldicellulosiruptor, in which 4mC-related restriction is a major barrier to DNA transformation from other species. In combination with MethylC-seq, both 4mC- and 5mC-containing motifs are identified which can assist in rapid and efficient genetic engineering of these bacteria in the future.

Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated.

Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. It will also allow the study of glycosylation of CelA itself and its role in the structure and function of this important enzyme in biomass deconstruction.

Heterologous complementation of a pyrF deletion in Caldicellulosiruptor hydrothermalis generates a new host for the analysis of biomass deconstruction.

BACKGROUND: Members of the thermophilic, anaerobic Gram-positive bacterial genus Caldicellulosiruptor grow optimally at 65 to 78°C and degrade lignocellulosic biomass without conventional pretreatment. Decomposition of complex cell wall polysaccharides is a major bottleneck in the conversion of plant biomass to biofuels and chemicals, and conventional biomass pretreatment includes exposure to high temperatures, acids, or bases as well as enzymatic digestion. Members of this genus contain a variety of glycosyl hydrolases, pectinases, and xylanases, but the contribution of these individual enzymes to biomass deconstruction is largely unknown. C. hydrothermalis is of special interest because it is the least cellulolytic of all the Caldicellulosiruptor species so far characterized, making it an ideal naïve system to study key cellulolytic enzymes from these bacteria. RESULTS: To develop methods for genetic manipulation of C. hydrothermalis, we selected a spontaneous deletion of pyrF, a gene in the pyrimidine biosynthetic pathway, resulting in a strain that was a uracil auxotroph resistant to 5-fluoroorotic acid (5-FOA). This strain allowed the selection of prototrophic transformants with either replicating or non-replicating plasmids containing the wild-type pyrF gene. Counter-selection of the pyrF wild-type allele on non-replicating vectors allowed the construction of chromosomal deletions. To eliminate integration of the non-replicating plasmid at the pyrF locus in the C. hydrothermalis chromosome, we used the non-homologous Clostridium thermocellum wild-type pyrF allele to complement the C. hydrothermalis pyrF deletion. The autonomously replicating shuttle vector was maintained at 25 to 115 copies per chromosome. Deletion of the ChyI restriction enzyme in C. hydrothermalis increased the transformation efficiency by an order of magnitude and demonstrated the ability to construct deletions and insertions in the genome of this new host. CONCLUSIONS: The use of C. hydrothermalis as a host for homologous and heterologous expression of enzymes important for biomass deconstruction will enable the identification of enzymes that contribute to the special ability of these bacteria to degrade complex lignocellulosic substrates as well as facilitate the construction of strains to improve and extend their substrate utilization capabilities.

A common structure for the potexviruses.

We have used fiber diffraction, cryo-electron microscopy, and scanning transmission electron microscopy to confirm the symmetry of three potexviruses, potato virus X, papaya mosaic virus, and narcissus mosaic virus, and to determine their low-resolution structures. All three viruses have slightly less than nine subunits per turn of the viral helix. Our data strongly support the view that all potexviruses have approximately the same symmetry. The structures are dominated by a large domain at high radius in the virion, with a smaller domain, which includes the putative RNA-binding site, extending to low radius.