Project description:Until recently, rare-earth elements (REEs) had been thought to be biologically inactive. This view changed with the discovery of the methanol dehydrogenase (Mdh) 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 Mdh. Using reporter assays and a suppressor screen, we found that 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 revealed XoxF as a top induced protein in planta and resulted in the identification of a core set of La3+-regulated proteins under defined artificial media conditions. Among these, we identified a potential 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.
Project description:Efforts towards microbial conversion of lignin to value-added products face many challenges because lignin’s methoxylated aromatic monomers release toxic C1 byproducts such as formaldehyde. The ability to grow on methoxylated aromatic acids (e.g., vanillic acid) has recently been identified in certain clades of methylotrophs, bacteria characterized by their unique ability to tolerate and metabolize high concentrations of formaldehyde. Here, we use a phyllosphere methylotroph isolate, Methylobacterium extorquens SLI 505, as a model to identify the fate of formaldehyde during methylotrophic growth on vanillic acids. M. extorquens SLI 505 displays concentration-dependent growth phenotypes on vanillic acid without concomitant formaldehyde accumulation. We conclude that M. extorquens SLI 505 overcomes potential metabolic bottlenecks from simultaneous assimilation of multicarbon and C1 intermediates by allocating formaldehyde towards dissimilation and assimilating the ring carbons of vanillic acid heterotrophically. We correlate this strategy with maximization of bioenergetic yields and demonstrate that formaldehyde dissimilation for energy generation rather than formaldehyde detoxification is advantageous for growth on aromatic acids. M. extorquens SLI 505 also exhibits catabolite repression during growth on methanol and low concentrations of vanillic acid, but no diauxie during growth on methanol and high concentrations of vanillic acid. Results from this study outline metabolic strategies employed by M. extorquens SLI 505 for growth on a complex single substrate that generates both C1 and multicarbon intermediates and emphasizes the robustness of M. extorquens for biotechnological applications for lignin valorization.
Project description:Plants are colonized by a variety of microorganisms, the plant microbiota. In the phyllosphere, the above-ground parts of plants, bacteria are the most abundant inhabitants. Most of these microorganisms are not pathogenic and the plant responses to commensals or to pathogen infection in the presence of commensals are not well understood. We report the Arabidopsis leaf transcriptome after 3 to 4 weeks of colonization by Methylobacterium extorquens PA1 and Sphingomonas melonis Fr1, representatives of two abundant genera in the phyllosphere, compared to axenic plants. In addition, we also sequenced the transcriptome of Arabidopsis 2 and 7 days after spray-infection with a low dose of P. syringae DC3000 and in combination with the commensals.
Project description:Differential analysis of Methylobacterium extorquens DM4 in methanol versus dichloromethane condition using shotgun label free MS1 quantification approach
Project description:Organisms cope with physiological stressors through acclimatizing mechanisms in the short-term, and through adaptive mechanisms over evolutionary timescales. Whereas the former offer a consistent and largely predictable buffer against stressors, myriad paths of adaptation are often possible. Our work examined whether knowledge of acclimatizing responses could be informative ofM-BM- aspects of future adaptation, using as a model system a strain of Methylobacterium extorquens AM1 that was experimentally engineered and then evolved with a novel central metabolism. The engineered strain is markedly slower and less fit than wild-type, which is reflected in microarray analyses by hundreds of genes with differential expression, and also altered levels NAD(P)(H) metabolites. Yet after 600 generations of evolution in the lab, eight replicate populations founded from an engineered ancestor showed substantial but variable improvements in growth using the engineered metabolic pathway. Using information from wild-type, engineered, and adapted physiological states, we determined the extent to which physiological processes were restored, unrestored, reinforced, or novel after experimental evolution. Overall, we found that the vast majority of gene expression perturbations from the engineered strain are restored to wild-type like conditions after experimental evolution but were accompanied by a modest number of unrestored processes, varying instances of novel expression, and a few rare instances where expression changes from acclimation were reinforced through adaptive evolution. One such example was in the reinforced up-regulation of pntAB transhydrogenase, whose increased expression and activity correlated with the restoration of NAD(P)(H) metabolism towards wild-type levels and with increased growth rate in the evolved lineages. Thus, while trajectories of physiological adaptation may still be difficult to predict a priori, our results demonstrate that information from acclimatizing responses can provide a M-bM-^@M-^\directionM-bM-^@M-^] to hypothesize which changes in physiology arose as a consequence of adaptation versus those that may have caused itM-BM- . Single-color microarray hydridization of total RNA isolated from mid-exponentially grown cultures of wildtype, engineered, and eight experimentally evolved populations of Methylobacterium extorquens AM1, each represented by three biological replicates