Transcriptional regulation is insufficient to explain substrate‐induced flux changes in <i>Bacillus subtilis</i>

Chubukov, Victor; Uhr, M.; Chat, Ludovic Le; Kleijn, Roelco J.; Jules, Matthieu; Link, Hannes; Aymerich, Stéphane; Stelling, Jörg; Sauer, Uwe

doi:10.1038/msb.2013.66

Cited by 161 publications

(211 citation statements)

References 71 publications

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“…The transport activities of GltP and YveA might thus be physiologically relevant under growth conditions different from those tested by us. Indeed, the level of the gltP transcript varies in response to the type of carbon source available (67). Furthermore, transcriptional data obtained in a tiling array study examining more than 100 growth conditions (44) demonstrate that gltP expression is upregulated in cells that swarm, grow on solid media, enter stationary phase, or proceed to form spores.…”

Section: Discussionmentioning

confidence: 99%

Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis

Zaprasis

Bleisteiner

Kerres

et al. 2015

Appl Environ Microbiol

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bThe data presented here reveal a new facet of the physiological adjustment processes through which Bacillus subtilis can derive osmostress protection. We found that the import of proteogenic (Glu, Gln, Asp, Asn, and Arg) and of nonproteogenic (Orn and Cit) amino acids and their metabolic conversion into proline enhances growth under otherwise osmotically unfavorable conditions. Osmoprotection by amino acids depends on the functioning of the ProJ-ProA-ProH enzymes, but different entry points into this biosynthetic route are used by different amino acids to finally yield the compatible solute proline. Glu, Gln, Asp, and Asn are used to replenish the cellular pool of glutamate, the precursor for proline production, whereas Arg, Orn, and Cit are converted into ␥-glutamic semialdehyde/⌬ 1 -pyrroline-5-carboxylate, an intermediate in proline biosynthesis. The import of Glu, Gln, Asp, Asn, Arg, Orn, and Cit did not lead to a further increase in the size of the proline pool that is already present in osmotically stressed cells. Hence, our data suggest that osmoprotection of B. subtilis by this group of amino acids rests on the savings in biosynthetic building blocks and energy that would otherwise have to be devoted either to the synthesis of the proline precursor glutamate or of proline itself. Since glutamate is the direct biosynthetic precursor for proline, we studied its uptake and found that GltT, an Na ؉ -coupled symporter, is the main uptake system for both glutamate and aspartate in B. subtilis. Collectively, our data show how effectively B. subtilis can exploit environmental resources to derive osmotic-stress protection through physiological means. Bacillus subtilis is a resident of the upper layers of the soil and of the rhizosphere, and it can also efficiently colonize root surfaces (1-3). The blueprint of its genome (4) bears the hallmarks of a bacterium that can exploit many plant-produced compounds for its growth. Accordingly, a considerable portion of the coding capacity of the B. subtilis chromosome (5) is devoted to highaffinity import systems (6) that allow the scavenging of a wide spectrum of nutrients. Reoccurring and persisting high osmolarity in the soil ecosystem (7) is a situation in which B. subtilis can take advantage of the import of plant-produced compounds (8-10) for its physiological adjustment to these unfavorable environmental conditions (7, 11).As in many bacterial species (11-13), cellular adaptation of B. subtilis to both sudden and sustained increases in the external osmolarity involves a two-stage process (7,14). It initially encompasses the uptake of large quantities of potassium as an emergency stress reaction to curb water efflux (14, 15) and, subsequently, the replacement of part of this ion by organic osmolytes, such as proline (Pro) and glycine betaine (GB), to decrease the ionic strength of the cytoplasm and to optimize its solvent properties (14,(16)(17)(18)(19). These organic osmolytes, commonly referred to as compatible solutes, are highly compliant with cellular physiolog...

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Section: Discussionmentioning

confidence: 99%

Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis

Zaprasis

Bleisteiner

Kerres

et al. 2015

Appl Environ Microbiol

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“…This immediate response leads to a major redistribution of carbon flux between assimilation and energy production (23), and the results presented here suggest that this response is at least partially responsible for preventing the accumulation of formaldehyde during the transition. Nontranscriptional regulatory mechanisms have been suggested to play an important role in controlling metabolic fluxes (39), and this is another example in which such a mechanism contributes to an effective acclimation after a metabolic perturbation (27).…”

Section: Discussionmentioning

confidence: 99%

Methenyl-Dephosphotetrahydromethanopterin Is a Regulatory Signal for Acclimation to Changes in Substrate Availability in Methylobacterium extorquens AM1

2015

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During an environmental perturbation, the survival of a cell and its response to the perturbation depend on both the robustness and functionality of the metabolic network. The regulatory mechanisms that allow the facultative methylotrophic bacterium Methylobacterium extorquens AM1 to effect the metabolic transition from succinate to methanol growth are not well understood. Methenyl-dephosphotetrahydromethanopterin (methenyl-dH 4 MPT), an early intermediate during methanol metabolism, transiently accumulated 7-to 11-fold after addition of methanol to a succinate-limited culture. This accumulation partially inhibited the activity of the methylene-H 4 MPT dehydrogenase, MtdA, restricting carbon flux to the assimilation cycles. A strain overexpressing the gene (mch) encoding the enzyme that consumes methenyl-dH 4 MPT did not accumulate methenyl-dH 4 MPT and had a growth rate that was 2.7-fold lower than that of the wild type. This growth defect demonstrates the physiological relevance of this enzymatic regulatory mechanism during the acclimation period. Changes in metabolites and enzymatic activities were analyzed in the strain overexpressing mch. Under these conditions, the activity of the enzyme coupling formaldehyde with dH 4 MPT (Fae) remained constant, with concomitant formaldehyde accumulation. Release of methenyl-dH 4 MPT regulation did not affect the induction of the serine cycle enzyme activities immediately after methanol addition, but after 1 h, the activity of these enzymes decreased, likely due to the toxicity of formaldehyde accumulation. Our results support the hypothesis that in a changing environment, the transient accumulation of methenyl-dH 4 MPT and inhibition of MtdA activity are strategies that permit flexibility and acclimation of the metabolic network while preventing the accumulation of the toxic compound formaldehyde. IMPORTANCEThe identification and characterization of regulatory mechanisms for methylotrophy are in the early stages. We report a nontranscriptional regulatory mechanism that was found to operate as an immediate response for acclimation during changes in substrate availability. Methenyl-dH 4 MPT, an early intermediate during methanol oxidation, reversibly inhibits the methylene-H 4 MPT dehydrogenase, MtdA, when Methylobacterium extorquens is challenged to switch from succinate to methanol growth. Bypassing this regulatory mechanism causes formaldehyde to accumulate. Fae, the enzyme catalyzing the conversion of formaldehyde to methylene-dH 4 MPT, was also identified as another potential regulatory target using this strategy. The results herein further our understanding of the complex regulatory network in methylotrophy and will allow us to improve metabolic engineering strategies of methylotrophs for the production of value-added products. Methylobacterium extorquens AM1 is a facultative methylotroph that is capable of growth on both one-carbon (C 1 ) compounds, such as methanol and methylamine, and multicarbon compounds, such as succinate. Methanol metabolism is predic...

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“…Similar conclusions on flux control in central metabolism were recently drawn for microorganisms. For Bacillus subtilis grown under a variety of physiological conditions, changes in enzyme concentrations were insufficient to explain the observed changes in flux in central metabolism (Chubukov et al, 2013).…”

Section: Metabolic Versus Transcriptional Controlmentioning

confidence: 98%

Quantitative Multilevel Analysis of Central Metabolism in Developing Oilseeds of Oilseed Rape during in Vitro Culture

et al. 2015

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Seeds provide the basis for many food, feed, and fuel products. Continued increases in seed yield, composition, and quality require an improved understanding of how the developing seed converts carbon and nitrogen supplies into storage. Current knowledge of this process is often based on the premise that transcriptional regulation directly translates via enzyme concentration into flux. In an attempt to highlight metabolic control, we explore genotypic differences in carbon partitioning for in vitro cultured developing embryos of oilseed rape (Brassica napus). We determined biomass composition as well as 79 net fluxes, the levels of 77 metabolites, and 26 enzyme activities with specific focus on central metabolism in nine selected germplasm accessions. Overall, we observed a tradeoff between the biomass component fractions of lipid and starch. With increasing lipid content over the spectrum of genotypes, plastidic fatty acid synthesis and glycolytic flux increased concomitantly, while glycolytic intermediates decreased. The lipid/starch tradeoff was not reflected at the proteome level, pointing to the significance of (posttranslational) metabolic control. Enzyme activity/flux and metabolite/flux correlations suggest that plastidic pyruvate kinase exerts flux control and that the lipid/starch tradeoff is most likely mediated by allosteric feedback regulation of phosphofructokinase and ADP-glucose pyrophosphorylase. Quantitative data were also used to calculate in vivo mass action ratios, reaction equilibria, and metabolite turnover times. Compounds like cyclic 39,59-AMP and sucrose-6-phosphate were identified to potentially be involved in so far unknown mechanisms of metabolic control. This study provides a rich source of quantitative data for those studying central metabolism.Seeds develop by absorbing nutrients from their mother plant and using these to synthesize a combination of starch, protein, and lipid. The size and number of seeds that finally develop determine the crop's yield, while their composition determines the end-use quality of the crop. The conversion of nutrients into storage products involves a complex network of metabolic reactions, many of which are subject to transcriptional, translational, and posttranslational regulation. Attempting to engineer seed composition clearly requires a firm understanding of these regulatory networks.The seed's central metabolism differs markedly from those of both a photosynthesizing leaf and a root. In most species, the immature seed is green for a period during its development, so during this phase it is regarded as being photoheterotrophic. A further level of complexity arises as a result of spatial heterogeneity within the seed (Rolletschek et al., 2011; Borisjuk et al.,

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Transcriptional regulation is insufficient to explain substrate‐induced flux changes in Bacillus subtilis

Cited by 161 publications

References 71 publications

Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis

Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis

Methenyl-Dephosphotetrahydromethanopterin Is a Regulatory Signal for Acclimation to Changes in Substrate Availability in Methylobacterium extorquens AM1

Quantitative Multilevel Analysis of Central Metabolism in Developing Oilseeds of Oilseed Rape during in Vitro Culture

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