Leaf microbiome dysbiosis triggered by T2SS-dependent enzyme secretion from opportunistic Xanthomonas pathogens.

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.

Lanpepsy is a novel lanthanide-binding protein involved in the lanthanide response of the obligate methylotroph Methylobacillus flagellatus.

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.

Generation of an Escherichia coli strain growing on methanol via the ribulose monophosphate cycle.

Methanol is a liquid with high energy storage capacity that holds promise as an alternative substrate to replace sugars in the biotechnology industry. It can be produced from CO2 or methane and its use does not compete with food and animal feed production. However, there are currently only limited biotechnological options for the valorization of methanol, which hinders its widespread adoption. Here, we report the conversion of the industrial platform organism Escherichia coli into a synthetic methylotroph that assimilates methanol via the energy efficient ribulose monophosphate cycle. Methylotrophy is achieved after evolution of a methanol-dependent E. coli strain over 250 generations in continuous chemostat culture. We demonstrate growth on methanol and biomass formation exclusively from the one-carbon source by 13C isotopic tracer analysis. In line with computational modeling, the methylotrophic E. coli strain optimizes methanol oxidation by upregulation of an improved methanol dehydrogenase, increasing ribulose monophosphate cycle activity, channeling carbon flux through the Entner-Doudoroff pathway and downregulating tricarboxylic acid cycle enzymes. En route towards sustainable bioproduction processes, our work lays the foundation for the efficient utilization of methanol as the dominant carbon and energy resource.

Dynamic character displacement among a pair of bacterial phyllosphere commensals in situ.

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.

A general non-self response as part of plant immunity.

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.

Mass spectrometry-based approaches to study lanthanides and lanthanide-dependent proteins in the phyllosphere.

Rare-earth elements (REEs) were recently discovered to be biologically significant. The finding was originally made with the methanol dehydrogenase XoxF, which depends on REEs for its activity, and reports of lanthanide-utilizing bacteria have since expanded. Environmental proteomics allows the identification of proteins specifically induced by the presence of lanthanides or can provide insights into the preferred use of lanthanide-dependent and -independent isoenzymes, for example. Here we describe protocols for the growth and subsequent mass spectrometry-based proteome analysis of bacteria obtained from controlled artificial media and from the phyllosphere of the model plant Arabidopsis thaliana. In addition, the use of inductively coupled plasma mass spectrometry (ICP-MS) is described for the quantification of REEs in biological samples.

Use of rare-earth elements in the phyllosphere colonizer Methylobacterium extorquens PA1.

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 Ca2+ -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 (La3+ ) 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 La3+ -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.

Transposon Sequencing Uncovers an Essential Regulatory Function of Phosphoribulokinase for Methylotrophy.

Methylotrophy is the ability of organisms to grow at the expense of reduced one-carbon compounds, such as methanol or methane. Here, we used transposon sequencing combining hyper-saturated transposon mutagenesis with high-throughput sequencing to define the essential methylotrophy genome of Methylobacterium extorquens PA1, a model methylotroph. To distinguish genomic regions required for growth only on methanol from general required genes, we contrasted growth on methanol with growth on succinate, a non-methylotrophic reference substrate. About 500,000 insertions were mapped for each condition, resulting in a median insertion distance of five base pairs. We identified 147 genes and 76 genes as specific for growth on methanol and succinate, respectively, and a set of 590 genes as required under both growth conditions. For the integration of metabolic functions, we reconstructed a genome-scale metabolic model and performed in silico essentiality analysis. In total, the approach uncovered 95 genes not previously described as crucial for methylotrophy, including genes involved in respiration, carbon metabolism, transport, and regulation. Strikingly, regardless of the absence of the Calvin cycle in the methylotroph, the screen led to the identification of the gene for phosphoribulokinase as essential during growth on methanol, but not during growth on succinate. Genetic experiments in addition to metabolomics and proteomics revealed that phosphoribulokinase serves a key regulatory function. Our data support a model according to which ribulose-1,5-bisphosphate is an essential metabolite that induces a transcriptional regulator driving one-carbon assimilation.