Rémi Peyraud

Methylobacterium Genome Sequences: A Reference Blueprint to Investigate Microbial Metabolism of C1 Compounds from Natural and Industrial Sources

Background Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared. Methodology/Principal Findings The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name “island integration determinant” (iid). Conclusion/Significance These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.

Demonstration of the ethylmalonyl-CoA pathway by using 13C metabolomics

The assimilation of one-carbon (C1) compounds, such as methanol, by serine cycle methylotrophs requires the continuous regeneration of glyoxylate. Instead of the glyoxylate cycle, this process is achieved by a not yet established pathway where CoA thioesters are known to play a key role. We applied state-of-the-art metabolomics and 13C metabolomics strategies to demonstrate how glyoxylate is generated during methylotrophic growth in the isocitrate lyase-negative methylotroph Methylobacterium extorquens AM1. High-resolution mass spectrometry showed the presence of CoA thioesters specific to the recently proposed ethylmalonyl-CoA pathway. The operation of this pathway was demonstrated by short-term 13C-labeling experiments, which allowed determination of the sequence of reactions from the order of label incorporation into the different CoA derivatives. Analysis of 13C positional enrichment in glycine by NMR was consistent with the predicted labeling pattern as a result of the operation of the ethylmalonyl-CoA pathway and the unique operation of the latter for glyoxylate generation during growth on methanol. The results also revealed that 2 molecules of glyoxylate were regenerated in this process. This work provides a complete pathway for methanol assimilation in the model methylotroph M. extorquens AM1 and represents an important step toward the determination of the overall topology of its metabolic network. The operation of the ethylmalonyl-CoA pathway in M. extorquens AM1 has major implications for the physiology of these methylotrophs and their role in nature, and it also provides a common ground for C1 and C2 compound assimilation in isocitrate lyase-negative bacteria.

Genome-scale reconstruction and system level investigation of the metabolic network of Methylobacterium extorquens AM1

BackgroundMethylotrophic microorganisms are playing a key role in biogeochemical processes - especially the global carbon cycle - and have gained interest for biotechnological purposes. Significant progress was made in the recent years in the biochemistry, genetics, genomics, and physiology of methylotrophic bacteria, showing that methylotrophy is much more widespread and versatile than initially assumed. Despite such progress, system-level description of the methylotrophic metabolism is currently lacking, and much remains to understand regarding the network-scale organization and properties of methylotrophy, and how the methylotrophic capacity emerges from this organization, especially in facultative organisms.ResultsIn this work, we report on the integrated, system-level investigation of the metabolic network of the facultative methylotroph Methylobacterium extorquens AM1, a valuable model of methylotrophic bacteria. The genome-scale metabolic network of the bacterium was reconstructed and contains 1139 reactions and 977 metabolites. The sub-network operating upon methylotrophic growth was identified from both in silico and experimental investigations, and 13C-fluxomics was applied to measure the distribution of metabolic fluxes under such conditions. The core metabolism has a highly unusual topology, in which the unique enzymes that catalyse the key steps of C1 assimilation are tightly connected by several, large metabolic cycles (serine cycle, ethylmalonyl-CoA pathway, TCA cycle, anaplerotic processes). The entire set of reactions must operate as a unique process to achieve C1 assimilation, but was shown to be structurally fragile based on network analysis. This observation suggests that in nature a strong pressure of selection must exist to maintain the methylotrophic capability. Nevertheless, substantial substrate cycling could be measured within C2/C3/C4 inter-conversions, indicating that the metabolic network is highly versatile around a flexible backbone of central reactions that allows rapid switching to multi-carbon sources.ConclusionsThis work emphasizes that the metabolism of M. extorquens AM1 is adapted to its lifestyle not only in terms of enzymatic equipment, but also in terms of network-level structure and regulation. It suggests that the metabolism of the bacterium has evolved both structurally and functionally to an efficient but transitory utilization of methanol. Besides, this work provides a basis for metabolic engineering to convert methanol into value-added products.

Complete Genome Sequences of Six Strains of the Genus Methylobacterium

The complete and assembled genome sequences were determined for six strains of the alphaproteobacterial genus Methylobacterium, chosen for their key adaptations to different plant-associated niches and environmental constraints.

Engineering Methylobacterium extorquens for de novo synthesis of the sesquiterpenoid α-humulene from methanol

Over the last 10 to 15 years, metabolic engineering of microbes has become a versatile tool for high-level de novo synthesis of terpenoids, with the sesquiterpenoids armopha-1,4-diene, farnesene and artemisinic acid as prime examples. However, almost all cell factory approaches towards terpenoids to date have been based on sugar as the raw material, which is mainly used as a food resource and subject to high price volatilities. In this study we present de novo synthesis of the sesquiterpenoid α-humulene from the abundantly available non-food carbon source methanol by metabolically engineered Methylobacterium extorquens AM1. Expression of α-humulene synthase from Zingiber zerumbet in combination with farnesyl pyrophosphate (FPP) synthase from Saccharomyces cerevisiae led to concentrations of up to 18 mg/L α-humulene. Introduction of a prokaryotic mevalonate pathway from Myxococcus xanthus in combination with ribosome binding site optimization of α-humulene and FPP synthases increased product concentration 3-fold. This value was additionally raised by 30% using a carotenoid synthesis deficient mutant strain. Final product concentrations of up to 1.65 g/L were obtained in methanol limited fed-batch cultivations, which is the highest titer of de novo synthesized α-humulene reported to date. This study demonstrates the potential of M. extorquens as a future platform strain for the production of high-value terpenoids from the alternative carbon source methanol.

A Resource Allocation Trade-Off between Virulence and Proliferation Drives Metabolic Versatility in the Plant Pathogen Ralstonia solanacearum

Bacterial pathogenicity relies on a proficient metabolism and there is increasing evidence that metabolic adaptation to exploit host resources is a key property of infectious organisms. In many cases, colonization by the pathogen also implies an intensive multiplication and the necessity to produce a large array of virulence factors, which may represent a significant cost for the pathogen. We describe here the existence of a resource allocation trade-off mechanism in the plant pathogen R. solanacearum. We generated a genome-scale reconstruction of the metabolic network of R. solanacearum, together with a macromolecule network module accounting for the production and secretion of hundreds of virulence determinants. By using a combination of constraint-based modeling and metabolic flux analyses, we quantified the metabolic cost for production of exopolysaccharides, which are critical for disease symptom production, and other virulence factors. We demonstrated that this trade-off between virulence factor production and bacterial proliferation is controlled by the quorum-sensing-dependent regulatory protein PhcA. A phcA mutant is avirulent but has a better growth rate than the wild-type strain. Moreover, a phcA mutant has an expanded metabolic versatility, being able to metabolize 17 substrates more than the wild-type. Model predictions indicate that metabolic pathways are optimally oriented towards proliferation in a phcA mutant and we show that this enhanced metabolic versatility in phcA mutants is to a large extent a consequence of not paying the cost for virulence. This analysis allowed identifying candidate metabolic substrates having a substantial impact on bacterial growth during infection. Interestingly, the substrates supporting well both production of virulence factors and growth are those found in higher amount within the plant host. These findings also provide an explanatory basis to the well-known emergence of avirulent variants in R. solanacearum populations in planta or in stressful environments.

Co-Consumption of Methanol and Succinate by Methylobacterium extorquens AM1

Methylobacterium extorquens AM1 is a facultative methylotrophic Alphaproteobacterium and has been subject to intense study under pure methylotrophic as well as pure heterotrophic growth conditions in the past. Here, we investigated the metabolism of M. extorquens AM1 under mixed substrate conditions, i.e., in the presence of methanol plus succinate. We found that both substrates were co-consumed, and the carbon conversion was two-thirds from succinate and one-third from methanol relative to mol carbon. 13C-methanol labeling and liquid chromatography mass spectrometry analyses revealed the different fates of the carbon from the two substrates. Methanol was primarily oxidized to CO2 for energy generation. However, a portion of the methanol entered biosynthetic reactions via reactions specific to the one-carbon carrier tetrahydrofolate. In contrast, succinate was primarily used to provide precursor metabolites for bulk biomass production. This work opens new perspectives on the role of methylotrophy when substrates are simultaneously available, a situation prevailing under environmental conditions.

Emerging Trends in Molecular Interactions between Plants and the Broad Host Range Fungal Pathogens Botrytis cinerea and Sclerotinia sclerotiorum

Fungal plant pathogens are major threats to food security worldwide. Sclerotinia sclerotiorum and Botrytis cinerea are closely related Ascomycete plant pathogens causing mold diseases on hundreds of plant species. There is no genetic source of complete plant resistance to these broad host range pathogens known to date. Instead, natural plant populations show a continuum of resistance levels controlled by multiple genes, a phenotype designated as quantitative disease resistance. Little is known about the molecular mechanisms controlling the interaction between plants and S. sclerotiorum and B. cinerea but significant advances were made on this topic in the last years. This minireview highlights a selection of nine themes that emerged in recent research reports on the molecular bases of plant-S. sclerotiorum and plant-B. cinerea interactions. On the fungal side, this includes progress on understanding the role of oxalic acid, on the study of fungal small secreted proteins. Next, we discuss the exchanges of small RNA between organisms and the control of cell death in plant and fungi during pathogenic interactions. Finally on the plant side, we highlight defense priming by mechanical signals, the characterization of plant Receptor-like proteins and the hormone abscisic acid in the response to B. cinerea and S. sclerotiorum, the role of plant general transcription machinery and plant small bioactive peptides. These represent nine trends we selected as remarkable in our understanding of fungal molecules causing disease and plant mechanisms associated with disease resistance to two devastating broad host range fungi.

Enhanced in planta Fitness through Adaptive Mutations in EfpR, a Dual Regulator of Virulence and Metabolic Functions in the Plant Pathogen Ralstonia solanacearum

Experimental evolution of the plant pathogen Ralstonia solanacearum, where bacteria were maintained on plant lineages for more than 300 generations, revealed that several independent single mutations in the efpR gene from populations propagated on beans were associated with fitness gain on bean. In the present work, novel allelic efpR variants were isolated from populations propagated on other plant species, thus suggesting that mutations in efpR were not solely associated to a fitness gain on bean, but also on additional hosts. A transcriptomic profiling and phenotypic characterization of the efpR deleted mutant showed that EfpR acts as a global catabolic repressor, directly or indirectly down-regulating the expression of multiple metabolic pathways. EfpR also controls virulence traits such as exopolysaccharide production, swimming and twitching motilities and deletion of efpR leads to reduced virulence on tomato plants after soil drenching inoculation. We studied the impact of the single mutations that occurred in efpR during experimental evolution and found that these allelic mutants displayed phenotypic characteristics similar to the deletion mutant, although not behaving as complete loss-of-function mutants. These adaptive mutations therefore strongly affected the function of efpR, leading to an expanded metabolic versatility that should benefit to the evolved clones. Altogether, these results indicated that EfpR is a novel central player of the R. solanacearum virulence regulatory network. Independent mutations therefore appeared during experimental evolution in the evolved clones, on a crucial node of this network, to favor adaptation to host vascular tissues through regulatory and metabolic rewiring.

Advances on plant-pathogen interactions from molecular toward systems biology perspectives

Summary In the past 2 decades, progress in molecular analyses of the plant immune system has revealed key elements of a complex response network. Current paradigms depict the interaction of pathogen‐secreted molecules with host target molecules leading to the activation of multiple plant response pathways. Further research will be required to fully understand how these responses are integrated in space and time, and exploit this knowledge in agriculture. In this review, we highlight systems biology as a promising approach to reveal properties of molecular plant–pathogen interactions and predict the outcome of such interactions. We first illustrate a few key concepts in plant immunity with a network and systems biology perspective. Next, we present some basic principles of systems biology and show how they allow integrating multiomics data and predict cell phenotypes. We identify challenges for systems biology of plant–pathogen interactions, including the reconstruction of multiscale mechanistic models and the connection of host and pathogen models. Finally, we outline studies on resistance durability through the robustness of immune system networks, the identification of trade‐offs between immunity and growth and in silico plant–pathogen co‐evolution as exciting perspectives in the field. We conclude that the development of sophisticated models of plant diseases incorporating plant, pathogen and climate properties represent a major challenge for agriculture in the future.