Metabolic rewiring compensates for the loss of glutamate and aspartate biosynthesis in<i>Bacillus subtilis</i>

Mardoukhi, Mohammad Saba Yousef; Rapp, Johanna; Irisarri, Iker; Gunka, Katrin; Link, Hannes; Marienhagen, Jan; de Vries, Jan; Stülke, Jörg; Commichau, Fabian M.

doi:10.1101/2023.11.10.566560

Cited by 3 publications

(3 citation statements)

References 111 publications

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“…l -asparagine enters the cell via the importers AimA and BcaP. Within the cell it is degraded to l -aspartate and then subsequently feeds into the tricarboxylic acid cycle (TCC) and production of l -glutamate via a newly discovered AspB-dependent bypass ( 49 ). l -asparagine is exported from the cell due to mutations in the regulators AzlB and YdeC.…”

Section: Discussionmentioning

confidence: 99%

Control of asparagine homeostasis in Bacillus subtilis : identification of promiscuous amino acid importers and exporters

Meißner,

Königshof,

Wrede

et al. 2024

J Bacteriol

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The Gram-positive model bacterium B. subtilis is able to import all proteinogenic amino acids from the environment as well as to synthesize them. However, the players involved in the acquisition of asparagine have not yet been identified for this bacterium. In this work, we used d -asparagine as a toxic analog of l -asparagine to identify asparagine transporters. This revealed that d - but not l -asparagine is taken up by the malate/lactate antiporter MleN. Specific strains that are sensitive to the presence of l -asparagine due to the lack of the second messenger cyclic di-AMP or due to the intracellular accumulation of this amino acid were used to isolate and characterize suppressor mutants that were resistant to the presence of otherwise growth-inhibiting concentrations of l -asparagine. These screens identified the broad-spectrum amino acid importers AimA and BcaP as responsible for the acquisition of l -asparagine. The amino acid exporter AzlCD allows detoxification of l -asparagine in addition to 4-azaleucine and histidine. This work supports the idea that amino acids are often transported by promiscuous importers and exporters. However, our work also shows that even stereo-enantiomeric amino acids do not necessarily use the same transport systems. IMPORTANCE Transport of amino acid is a poorly studied function in many bacteria, including the model organism Bacillus subtilis . The identification of transporters is hampered by the redundancy of transport systems for most amino acids as well as by the poor specificity of the transporters. Here, we apply several strategies to use the growth-inhibitive effect of many amino acids under defined conditions to isolate suppressor mutants that exhibit either reduced uptake or enhanced export of asparagine, resulting in the identification of uptake and export systems for l -asparagine. The approaches used here may be useful for the identification of transporters for other amino acids both in B. subtilis and in other bacteria.

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Section: Discussionmentioning

confidence: 99%

Control of asparagine homeostasis in Bacillus subtilis : identification of promiscuous amino acid importers and exporters

Meißner,

Königshof,

Wrede

et al. 2024

J Bacteriol

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show abstract

“…L-asparagine enters the cell via the importers AimA and BcaP. Within the cell it is degraded to L-aspartate and then subsequently feeds into the tricarboxylic acid cycle (TCC) and production of L-glutamate via a newly discovered AspB-dependent bypass (59). L-asparagine is exported from the cell due to mutations in the regulators AzlB and YdeC.…”

Section: Resultsmentioning

confidence: 99%

Control of asparagine homeostasis inBacillus subtilis: Identification of promiscuous amino acid importers and exporters

Meißner,

Königshof,

Wrede

et al. 2023

Preprint

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Amino acids are the main building block for proteins. The Gram-positive model bacteriumB. subtilisis able to import all proteinogenic amino acids from the environment as well as to synthesize them. However, the players involved in the acquisition of asparagine have not yet been identified for this bacterium. In this work, we used D-asparagine as a toxic analog of L-asparagine to identify asparagine transporters. This revealed that D-but not L-asparagine is taken up by the malate/lactate antiporter MleN. Specific strains that are sensitive to the presence of L-asparagine due to the lack of the second messenger cyclic di-AMP or due to the intracellular accumulation of this amino acid were used to isolate and characterize suppressor mutants that were resistant to the presence of otherwise growth-inhibiting concentrations of L-asparagine. These screens identified the broad-spectrum amino acid importers AimA and BcaP as responsible for the acquisition of L-asparagine. The amino acid exporter AzlCD allows detoxification of L-asparagine in addition to 4-azaleucine and histidine. This work supports the idea that amino acids are often transported by promiscuous importers and exporters. However, our work also shows that even stereo-enantiomeric amino acids do not necessarily use the same transport systems.IMPORTANCETransport of amino acid is a poorly studied function in many bacteria, including the model organismBacillus subtilis. The identification of transporters is hampered by the redundancy of transport systems for most amino acids as well as by the poor specificity of the transporters. Here, we apply several strategies to use the growth-inhibitive effect of many amino acids under defined conditions to isolate suppressor mutants that exhibit either reduced uptake or enhanced export of asparagine, resulting in the identification of uptake and export systems for L-asparagine. The approaches used here may be useful for the identification of transporters for other amino acids both inB. subtilisand other bacteria as well.

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“…The observations presented above suggest that the rfaA mutant might be auxotrophic for glutamate. However, the biosynthetic pathway for glutamate in B. subtilis as well as its regulation have been extensively studied, and there is no indication that proteins in addition to those already identified might play a role in glutamate biosynthesis in B. subtilis 19,20,21 . Therefore, we considered an alternative possibility, i. e. that growth in the absence of glutamate might result in a growth inhibition due to the accumulation of toxic compounds.…”

Section: Glutamate Is Essential For Growth Of the Rfaa Mutantmentioning

confidence: 99%

RfaA (YqhY), a novel adaptor protein, controls metabolite-sensitive protein degradation in Bacillus subtilis

Wicke,

Lentes,

Herrfurth

et al. 2024

Preprint

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The carboxylation of acetyl-CoA is the committed step of fatty acid synthesis catalyzed by the multisubunit acetyl-CoA carboxylase (ACCase). However, the mechanisms that control the activity of this enzyme are poorly understood. Here, we identify the so far unknown protein RfaA (YqhY) of the model bacterium Bacillus subtilis as a regulator of fatty acid acquisition that targets the AccC subunit of ACCase for degradation. RfaA interacts with both AccC and the ClpE unfoldase subunit of the ClpEP protease complex. While the former interaction is not sensitive to the physiological conditions, RfaA interacts with ClpE only in the absence of the global amino group donor glutamate. This results in the degradation of AccC in the absence of glutamate. The inactivation of the rfaA gene results in the accumulation of fatty acids in the cell and in the formation of lipid droplets which are toxic for the bacteria. The reduced fatty acid synthesis in the absence of glutamate as a result of RfaA- and ClpEP-dependent degradation of AccC thus prevents intoxication of the cells by fatty acids. Our findings suggest that the degradation of enzymes that catalyze the committed step of biosynthetic pathways might be an important mechanism to control cellular homeostasis.

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Metabolic rewiring compensates for the loss of glutamate and aspartate biosynthesis inBacillus subtilis

Cited by 3 publications

References 111 publications

Control of asparagine homeostasis in Bacillus subtilis : identification of promiscuous amino acid importers and exporters

Control of asparagine homeostasis in Bacillus subtilis : identification of promiscuous amino acid importers and exporters

Control of asparagine homeostasis inBacillus subtilis: Identification of promiscuous amino acid importers and exporters

RfaA (YqhY), a novel adaptor protein, controls metabolite-sensitive protein degradation in Bacillus subtilis

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