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The ability of plants to perceive and react to biotic and abiotic stresses is critical for their health. We recently identified a core set of genes consistently induced by members of the leaf microbiota, termed general non-self response (GNSR) genes. Here we show that GNSR components conversely impact leaf microbiota composition. Specific strains that benefited from this altered assembly triggered strong plant responses, suggesting that the GNSR is a dynamic system that modulates colonization by certain strains. Examination of the GNSR to live and inactivated bacteria revealed that bacterial abundance, cellular composition and exposure time collectively determine the extent of the host response. We link the GNSR to pattern-triggered immunity, as diverse microbe- or danger-associated molecular patterns cause dynamic GNSR gene expression. Our findings suggest that the GNSR is the result of a dose-responsive perception and signalling system that feeds back to the leaf microbiota and contributes to the intricate balance of plant-microbiome interactions., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Endosymbioses have profoundly impacted the evolution of life and continue to shape the ecology of a wide range of species. They give rise to new combinations of biochemical capabilities that promote innovation and diversification 1,2 . Despite the many examples of known endosymbioses across the tree of life, their de novo emergence is rare and challenging to uncover in retrospect 3-5 . Here we implant bacteria into the filamentous fungus Rhizopus microsporus to follow the fate of artificially induced endosymbioses. Whereas Escherichia coli implanted into the cytosol induced septum formation, effectively halting endosymbiogenesis, Mycetohabitans rhizoxinica was transmitted vertically to the progeny at a low frequency. Continuous positive selection on endosymbiosis mitigated initial fitness constraints by several orders of magnitude upon adaptive evolution. Phenotypic changes were underscored by the accumulation of mutations in the host as the system stabilized. The bacterium produced rhizoxin congeners in its new host, demonstrating the transfer of a metabolic function through induced endosymbiosis. Single-cell implantation thus provides a powerful experimental approach to study critical events at the onset of endosymbiogenesis and opens opportunities for synthetic approaches towards designing endosymbioses with desired traits., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

In healthy plants, the innate immune system contributes to maintenance of microbiota homoeostasis, while disease can be associated with microbiome perturbation or dysbiosis, and enrichment of opportunistic plant pathogens like Xanthomonas. It is currently unclear whether the microbiota change occurs independently of the opportunistic pathogens or is caused by the latter. Here we tested if protein export through the type-2 secretion system (T2SS) by Xanthomonas causes microbiome dysbiosis in Arabidopsis thaliana in immunocompromised plants. We found that Xanthomonas strains secrete a cocktail of plant cell wall-degrading enzymes that promote Xanthomonas growth during infection. Disease severity and leaf tissue degradation were increased in A. thaliana mutants lacking the NADPH oxidase RBOHD. Experiments with gnotobiotic plants, synthetic bacterial communities and wild-type or T2SS-mutant Xanthomonas revealed that virulence and leaf microbiome composition are controlled by the T2SS. Overall, a compromised immune system in plants can enrich opportunistic pathogens, which damage leaf tissues and ultimately cause microbiome dysbiosis by facilitating growth of specific commensal bacteria., (© 2024. The Author(s).)

Plant-associated microbiomes contribute to important ecosystem functions such as host resistance to biotic and abiotic stresses. The factors that determine such community outcomes are inherently difficult to identify under complex environmental conditions. In this study, we present an experimental and analytical approach to explore microbiota properties relevant for a microbiota-conferred host phenotype, here plant protection, in a reductionist system. We screened 136 randomly assembled synthetic communities (SynComs) of five bacterial strains each, followed by classification and regression analyses as well as empirical validation to test potential explanatory factors of community structure and composition, including evenness, total commensal colonization, phylogenetic diversity, and strain identity. We find strain identity to be the most important predictor of pathogen reduction, with machine learning algorithms improving performances compared to random classifications (94-100% versus 32% recall) and non-modelled predictions (0.79-1.06 versus 1.5 RMSE). Further experimental validation confirms three strains as the main drivers of pathogen reduction and two additional strains that confer protection in combination. Beyond the specific application presented in our study, we provide a framework that can be adapted to help determine features relevant for microbiota function in other biological systems., (© 2023. The Author(s).)

Resource allocation affects the structure of microbiomes, including those associated with living hosts. Understanding the degree to which this dependency determines interspecies interactions may advance efforts to control host-microbiome relationships. We combined synthetic community experiments with computational models to predict interaction outcomes between plant-associated bacteria. We mapped the metabolic capabilities of 224 leaf isolates from Arabidopsis thaliana by assessing the growth of each strain on 45 environmentally relevant carbon sources in vitro. We used these data to build curated genome-scale metabolic models for all strains, which we combined to simulate >17,500 interactions. The models recapitulated outcomes observed in planta with >89% accuracy, highlighting the role of carbon utilization and the contributions of niche partitioning and cross-feeding in the assembly of leaf microbiomes.

Lanthanides were recently discovered as metals required in the active site of certain methanol dehydrogenases. Since then, the characterization of the lanthanome, that is, proteins involved in sensing, uptake, and utilization of lanthanides, has become an active field of research. Initial exploration of the response to lanthanides in methylotrophs has revealed that the lanthanome is not conserved and that multiple mechanisms for lanthanide utilization must exist. Here, we investigated the lanthanome in the obligate model methylotroph Methylobacillus flagellatus. We used a proteomic approach to analyze differentially regulated proteins in the presence of lanthanum. While multiple known proteins showed induction upon growth in the presence of lanthanum (Xox proteins, TonB-dependent receptor), we also identified several novel proteins not previously associated with lanthanide utilization. Among these was Mfla_0908, a periplasmic 19 kDa protein without functional annotation. The protein comprises two characteristic PepSY domains, which is why we termed the protein lanpepsy (LanP). Based on bioinformatic analysis, we speculated that LanP could be involved in lanthanide binding. Using dye competition assays, quantification of protein-bound lanthanides by inductively coupled plasma mass spectrometry, as well as isothermal titration calorimetry, we demonstrated the presence of multiple lanthanide binding sites that showed selectivity over the chemically similar calcium ion. LanP thus represents the first member of the PepSY family that binds lanthanides. Although the physiological role of LanP is still unclear, its identification is of interest for applications toward the sustainable purification and separation of rare-earth elements., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Auxotrophs are unable to synthesize all the metabolites essential for their metabolism and rely on others to provide them. They have been intensively studied in laboratory-generated and -evolved mutants, but emergent adaptation mechanisms to auxotrophy have not been systematically addressed. Here, we investigated auxotrophies in bacteria isolated from Arabidopsis thaliana leaves and found that up to half of the strains have auxotrophic requirements for biotin, niacin, pantothenate and/or thiamine. We then explored the genetic basis of auxotrophy as well as traits that co-occurred with vitamin auxotrophy. We found that auxotrophic strains generally stored coenzymes with the capacity to grow exponentially for 1-3 doublings without vitamin supplementation; however, the highest observed storage was for biotin, which allowed for 9 doublings in one strain. In co-culture experiments, we demonstrated vitamin supply to auxotrophs, and found that auxotrophic strains maintained higher species richness than prototrophs upon external supplementation with vitamins. Extension of a consumer-resource model predicted that auxotrophs can utilize carbon compounds provided by other organisms, suggesting that auxotrophic strains benefit from metabolic by-products beyond vitamins., (© 2022. The Author(s).)

Host-associated microbiomes harbour hundreds of bacterial species that co-occur, creating the opportunity for manifold bacteria-bacteria interactions, which in turn contribute to the overall community structure. The mechanisms that underlie this self-organization among bacteria remain largely elusive. Here, we studied bacterial interactions in the phyllosphere microbiota. We screened for microbial interactions in planta by adding 200 endogenous strains individually to a 15-member synthetic community and tracking changes in community composition upon colonization of the model plant Arabidopsis. Ninety percent of the identified interactions in planta were negative, and phylogenetically closely related strains elicited consistent effects on the synthetic community, providing support for trait conservation. Community changes could be largely explained by binary interactions; however, we also identified a higher-order interaction of more than two interacting strains. We further focused on a prominent interaction between two members of the Actinobacteria. In the presence of Aeromicrobium Leaf245, the population of Nocardioides Leaf374 was reduced by almost two orders of magnitude. We identified a potent antimicrobial peptidase in Aeromicrobium Leaf245, which resulted in Nocardioides Leaf374 lysis. A respective Leaf245 mutant strain was necessary and sufficient to restore Nocardioides colonization in planta, demonstrating that direct bacteria-bacteria interactions were responsible for the population shift originally observed. Our study highlights the power of synthetic community screening and uncovers a strong microbial interaction that occurs despite a spatially heterogeneous environment., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Differences between species promote stable coexistence in a resource-limited environment. These differences can result from interspecies competition leading to character shifts, a process referred to as character displacement. While character displacement is often interpreted as a consequence of genetically fixed trait differences between species, it can also be mediated by phenotypic plasticity in response to the presence of another species. Here, we test whether phenotypic plasticity leads to a shift in proteome allocation during co-occurrence of two bacterial species from the abundant, leaf-colonizing families Sphingomonadaceae and Rhizobiaceae in their natural habitat. Upon mono-colonizing of the phyllosphere, both species exhibit specific and shared protein functions indicating a niche overlap. During co-colonization, quantitative differences in the protein repertoire of both bacterial populations occur as a result of bacterial coexistence in planta. Specifically, the Sphingomonas strain produces enzymes for the metabolization of xylan, while the Rhizobium strain reprograms its metabolism to beta-oxidation of fatty acids fueled via the glyoxylate cycle and adapts its biotin acquisition. We demonstrate the conditional relevance of cross-species facilitation by mutagenesis leading to loss of fitness in competition in planta. Our results show that dynamic character displacement and niche facilitation mediated by phenotypic plasticity can contribute to species coexistence., (© 2022. The Author(s).)

Leaves and flowers are colonized by diverse bacteria that impact plant fitness and evolution. Although the structure of these microbial communities is becoming well-characterized, various aspects of their environmental origin and selection by plants remain uncertain, such as the relative proportion of soilborne bacteria in phyllosphere communities. Here, to address this issue and to provide experimental support for bacteria being filtered by flowers, we conducted common-garden experiments outside and under gnotobiotic conditions. We grew Arabidopsis thaliana in a soil substitute and added two microbial communities from natural soils. We estimated that at least 25% of the phyllosphere bacteria collected from the plants grown in the open environment were also detected in the controlled conditions, in which bacteria could reach leaves and flowers only from the soil. These taxa represented more than 40% of the communities based on amplicon sequencing. Unsupervised hierarchical clustering approaches supported the convergence of all floral microbiota, and 24 of the 28 bacteria responsible for this pattern belonged to the Burkholderiaceae family, which includes known plant pathogens and plant growth-promoting members. We anticipate that our study will foster future investigations regarding the routes used by soil microbes to reach leaves and flowers, the ubiquity of the environmental filtering of Burkholderiaceae across plant species and environments, and the potential functional effects of the accumulation of these bacteria in the reproductive organs of flowering plants., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The plant microbiota consists of a multitude of microorganisms that can affect plant health and fitness. However, it is currently unclear how the plant shapes its leaf microbiota and what role the plant immune system plays in this process. Here, we evaluated Arabidopsis thaliana mutants with defects in different parts of the immune system for an altered bacterial community assembly using a gnotobiotic system. While higher-order mutants in receptors that recognize microbial features and in defence hormone signalling showed substantial microbial community alterations, the absence of the plant NADPH oxidase RBOHD caused the most pronounced change in the composition of the leaf microbiota. The rbohD knockout resulted in an enrichment of specific bacteria. Among these, we identified Xanthomonas strains as opportunistic pathogens that colonized wild-type plants asymptomatically but caused disease in rbohD knockout plants. Strain dropout experiments revealed that the lack of RBOHD unlocks the pathogenicity of individual microbiota members driving dysbiosis in rbohD knockout plants. For full protection, healthy plants require both a functional immune system and a microbial community. Our results show that the NADPH oxidase RBOHD is essential for microbiota homeostasis and emphasizes the importance of the plant immune system in controlling the leaf microbiota.

Plants, like other multicellular lifeforms, are colonized by microorganisms. How plants respond to their microbiota is currently not well understood. We used a phylogenetically diverse set of 39 endogenous bacterial strains from Arabidopsis thaliana leaves to assess host transcriptional and metabolic adaptations to bacterial encounters. We identified a molecular response, which we termed the general non-self response (GNSR) that involves the expression of a core set of 24 genes. The GNSR genes are not only consistently induced by the presence of most strains, they also comprise the most differentially regulated genes across treatments and are predictive of a hierarchical transcriptional reprogramming beyond the GNSR. Using a complementary untargeted metabolomics approach we link the GNSR to the tryptophan-derived secondary metabolism, highlighting the importance of small molecules in plant-microbe interactions. We demonstrate that several of the GNSR genes are required for resistance against the bacterial pathogen Pseudomonas syringae. Our results suggest that the GNSR constitutes a defence adaptation strategy that is consistently elicited by diverse strains from various phyla, contributes to host protection and involves secondary metabolism.

Methylofuran (MYFR) is a formyl-carrying coenzyme essential for the oxidation of formaldehyde in most methylotrophic bacteria. In Methylorubrum extorquens, MYFR contains a large and branched polyglutamate side chain of up to 24 glutamates. These glutamates play an essential role in interfacing the coenzyme with the formyltransferase/hydrolase complex, an enzyme that generates formate. To date, MYFR has not been identified in other methylotrophs, and it is unknown whether its structural features are conserved. Here, we examined nine bacterial strains for the presence and structure of MYFR using high-resolution liquid chromatography-mass spectrometry (LC-MS). Two of the strains produced MYFR as present in M. extorquens, while a modified MYFR containing tyramine instead of tyrosine in its core structure was detected in six strains. When M. extorquens was grown in the presence of tyramine, the compound was readily incorporated into MYFR, indicating that the biosynthetic enzymes are unable to discriminate tyrosine from tyramine. Using gene deletions in combination with LC-MS analyses, we identified three genes, orf5, orfY, and orf17 that are essential for MYFR biosynthesis. Notably, the orfY and orf5 mutants accumulated short MYFR intermediates with only one and two glutamates, respectively, suggesting that these enzymes catalyze glutamate addition. Upon homologous overexpression of orf5, a drastic increase in the number of glutamates in MYFR was observed (up to 40 glutamates), further corroborating the function of Orf5 as a glutamate ligase. We thus renamed OrfY and Orf5 to MyfA and MyfB to highlight that these enzymes are specifically involved in MYFR biosynthesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacteria colonizing the aerial parts of plants (phyllosphere) are linked to the biology of their host. They impact plant-pathogen interactions and may influence plant reproduction. Past studies have shown differences in composition and structure of the leaf, flower, and host microbiota, but an investigation of the impact of individual taxa on these variations remains to be tested. Such information will help to evaluate disparities and to better understand the biology and evolution of the plant-microbe associations. In the present study, we investigated the community structure, occupancy of host and organ, and the prevalence of phyllosphere bacteria from three host species collected at the same location. Almost all (98%) of bacterial taxa detected in the phyllosphere were not only shared across leaves and flowers, or different plant species but also had a conserved prevalence across sub-environments of the phyllosphere. We also found nonrandom associations of the phylogenetic diversity of phyllosphere bacteria. These results suggest that the phyllosphere microbiota is more conserved than previously acknowledged, and dominated by generalist bacteria adapted to environmental heterogeneity through evolutionary conserved traits.

Multicellular organisms, including plants, are colonized by microorganisms, some of which are beneficial to growth and health. The assembly rules for establishing plant microbiota are not well understood, and neither is the extent to which their members interact. We conducted drop-out and late introduction experiments by inoculating Arabidopsis thaliana with synthetic communities from a resource of 62 native bacterial strains to test how arrival order shapes community structure. As a read-out we tracked the relative abundance of all strains in the phyllosphere of individual plants. Our results showed that community assembly is historically contingent and subject to priority effects. Missing strains could, to various degrees, invade an already established microbiota, which was itself resistant and remained largely unaffected by latecomers. Additionally, our results indicate that individual strains of Proteobacteria (Sphingomonas, Rhizobium) and Actinobacteria (Microbacterium, Rhodococcus) have the greatest potential to affect community structure as keystone species.

The general stress response (GSR) represents an important trait to survive in the environment by leading to multiple stress resistance. In alphaproteobacteria, the GSR is under the transcriptional control of the alternative sigma factor EcfG. Here we performed transcriptome analyses to investigate the genes controlled by EcfG of Sphingomonas melonis Fr1 and the plasticity of this regulation under stress conditions. We found that EcfG regulates genes for proteins that are typically associated with stress responses. Moreover, EcfG controls regulatory proteins, which likely fine-tune the GSR. Among these, we identified a novel negative GSR feedback regulator, termed NepR2, on the basis of gene reporter assays, phenotypic analyses, and biochemical assays. Transcriptional profiling of signaling components upstream of EcfG under complex stress conditions showed an overall congruence with EcfG-regulated genes. Interestingly however, we found that the GSR is transcriptionally linked to the regulation of motility and biofilm formation via the single domain response regulator SdrG and GSR-activating histidine kinases. Altogether, our findings indicate that the GSR in S. melonis Fr1 underlies a complex regulation to optimize resource allocation and resilience in stressful and changing environments.

Until recently, rare-earth elements (REEs) had been thought to be biologically inactive. This view changed with the discovery of the methanol dehydrogenase XoxF that strictly relies on REEs for its activity. Some methylotrophs only contain xoxF, while others, including the model phyllosphere colonizer Methylobacterium extorquens PA1, harbor this gene in addition to mxaFI encoding a Ca 2+ -dependent enzyme. Here we found that REEs induce the expression of xoxF in M. extorquens PA1, while repressing mxaFI, suggesting that XoxF is the preferred methanol dehydrogenase in the presence of sufficient amounts of REE. Using reporter assays and a suppressor screen, we found that lanthanum (La 3+ ) is sensed both in a XoxF-dependent and independent manner. Furthermore, we investigated the role of REEs during Arabidopsis thaliana colonization. Element analysis of the phyllosphere revealed the presence of several REEs at concentrations up to 10 μg per g dry weight. Complementary proteome analyses of M. extorquens PA1 identified XoxF as a top induced protein in planta and a core set of La 3+ -regulated proteins under defined artificial media conditions. Among these was a REE-binding protein that is encoded next to a gene for a TonB-dependent transporter. The latter was essential for REE-dependent growth on methanol indicating chelator-assisted uptake of REEs., (© 2019 The Authors Molecular Microbiology Published by John Wiley & Sons Ltd.)

Two-component systems constitute phosphotransfer signaling pathways and enable adaptation to environmental changes, an essential feature for bacterial survival. The general stress response (GSR) in the plant-protecting alphaproteobacterium Sphingomonas melonis Fr1 involves a two-component system consisting of multiple stress-sensing histidine kinases (Paks) and the response regulator PhyR; PhyR in turn regulates the alternative sigma factor EcfG, which controls expression of the GSR regulon. While Paks had been shown to phosphorylate PhyR in vitro, it remained unclear if and under which conditions direct phosphorylation happens in the cell, as Paks also phosphorylate the single domain response regulator SdrG, an essential yet enigmatic component of the GSR signaling pathway. Here, we analyze the role of SdrG and investigate an alternative function of the membrane-bound PhyP (here re-designated PhyT), previously assumed to act as a PhyR phosphatase. In vitro assays show that PhyT transfers a phosphoryl group from SdrG to PhyR via phosphoryl transfer on a conserved His residue. This finding, as well as complementary GSR reporter assays, indicate the participation of SdrG and PhyT in a Pak-SdrG-PhyT-PhyR phosphorelay. Furthermore, we demonstrate complex formation between PhyT and PhyR. This finding is substantiated by PhyT-dependent membrane association of PhyR in unstressed cells, while the response regulator is released from the membrane upon stress induction. Our data support a model in which PhyT sequesters PhyR, thereby favoring Pak-dependent phosphorylation of SdrG. In addition, PhyT assumes the role of the SdrG-phosphotransferase to activate PhyR. Our results place SdrG into the GSR signaling cascade and uncover a dual role of PhyT in the GSR.

Trans-AT polyketide synthases (PKSs) are a family of biosynthetically versatile modular type I PKSs that generate bioactive polyketides of impressive structural diversity. In this study, we detected, in the genome of several bacteria a cryptic, architecturally unusual trans-AT PKS gene cluster which eluded automated PKS prediction. Genomic mining of one of these strains, the model methylotroph Methylobacterium extorquens AM1, revealed unique epoxide- and cyclopropanol-containing polyketides named toblerols. Relative and absolute stereochemistry were determined by NMR experiments, chemical derivatization, and the comparison of CD data between the derivatized natural product and a synthesized model compound. Biosynthetic data suggest that the cyclopropanol moiety is generated by carbon-carbon shortening of a more extended precursor. Surprisingly, a knock-out strain impaired in polyketide production showed strong inhibitory activity against other methylobacteria in contrast to the wild-type producer. The activity was inhibited by complementation with toblerols, thus suggesting that these compounds modulate an as-yet unknown methylobacterial antibiotic., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Two-component systems are major signal transduction pathways, which consist of histidine kinases and response regulators that communicate through phosphorylation. Here, we highlight a distinct class of single-domain response regulators containing the PFXFATG[G/Y] motif that are activated by a mechanism distinct from the Y-T coupling described for prototypical receiver domains. We first solved the structures of inactive and active SdrG, a representative of the FAT GUY family, and then biochemically and genetically characterized variants in which residues in this motif were mutated. Our results support a model of activation mainly driven by a conserved lysine and reveal that the rotation of the threonine induces the reorganization of several aromatic residues in and around the PFXFATG[G/Y] motif to generate intermediates resembling those occurring during classical Y-T coupling. Overall, this helps define a new subfamily of response regulators that emerge as important players in physiological adaptation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Understanding cells as integrated systems requires that we systematically decipher how single genes affect multiple biological processes and how processes are functionally linked. Here, we used multiprocess phenotypic profiling, combining high-resolution 3D confocal microscopy and multiparametric image analysis, to simultaneously survey the fission yeast genome with respect to three key cellular processes: cell shape, microtubule organization, and cell-cycle progression. We identify, validate, and functionally annotate 262 genes controlling specific aspects of those processes. Of these, 62% had not been linked to these processes before and 35% are implicated in multiple processes. Importantly, we identify a conserved role for DNA-damage responses in controlling microtubule stability. In addition, we investigate how the processes are functionally linked. We show unexpectedly that disruption of cell-cycle progression does not necessarily affect cell size control and that distinct aspects of cell shape regulate microtubules and vice versa, identifying important systems-level links across these processes.

The identity of plant host genetic factors controlling the composition of the plant microbiota and the extent to which plant genes affect associated microbial populations is currently unknown. Here, we use a candidate gene approach to investigate host effects on the phyllosphere community composition and abundance. To reduce the environmental factors that might mask genetic factors, the model plant Arabidopsis thaliana was used in a gnotobiotic system and inoculated with a reduced complexity synthetic bacterial community composed of seven strains representing the most abundant phyla in the phyllosphere. From a panel of 55 plant mutants with alterations in the surface structure, cell wall, defense signaling, secondary metabolism, and pathogen recognition, a small number of single host mutations displayed an altered microbiota composition and/or abundance. Host alleles that resulted in the strongest perturbation of the microbiota relative to the wild-type were lacs2 and pec1. These mutants affect cuticle formation and led to changes in community composition and an increased bacterial abundance relative to the wild-type plants, suggesting that different bacteria can benefit from a modified cuticle to different extents. Moreover, we identified ein2, which is involved in ethylene signaling, as a host factor modulating the community's composition. Finally, we found that different Arabidopsis accessions exhibited different communities, indicating that plant host genetic factors shape the associated microbiota, thus harboring significant potential for the identification of novel plant factors affecting the microbiota of the communities.

Bacillus methanolicus MGA3 is a facultative methylotroph of industrial relevance that is able to grow on methanol as its sole source of carbon and energy. The Gram-positive bacterium possesses a soluble NAD(+) -dependent methanol dehydrogenase and assimilates formaldehyde via the ribulose monophosphate (RuMP) cycle. We used label-free quantitative proteomics to generate reference proteome data for this bacterium and compared the proteome of B. methanolicus MGA3 on two different carbon sources (methanol and mannitol) as well as two different growth temperatures (50°C and 37°C). From a total of approximately 1200 different detected proteins, approximately 1000 of these were used for quantification. While the levels of 213 proteins were significantly different at the two growth temperatures tested, the levels of 109 proteins changed significantly when cells were grown on different carbon sources. The carbon source strongly affected the synthesis of enzymes related to carbon metabolism, and in particular, both dissimilatory and assimilatory RuMP cycle enzyme levels were elevated during growth on methanol compared to mannitol. Our data also indicate that B. methanolicus has a functional tricarboxylic acid cycle, the proteins of which are differentially regulated on mannitol and methanol. Other proteins presumed to be involved in growth on methanol were constitutively expressed under the different growth conditions. All MS data have been deposited in the ProteomeXchange with the identifiers PXD000637 and PXD000638 (http://proteomecentral.proteomexchange.org/dataset/PXD000637, http://proteomecentral.proteomexchange.org/dataset/PXD000638)., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)