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Vitamin B6 metabolism in the Gram-positive model bacterium Bacillus subtilis

Subject Area Metabolism, Biochemistry and Genetics of Microorganisms
Term from 2020 to 2024
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 449598253
 
Final Report Year 2024

Final Report Abstract

Pyridoxal (PL) and pyridoxal 5’-phosphate (PLP) are “Vitamin B6” vitamers. PLP is a cofactor for many enzymes, and it modulates the activity of DNA-binding transcription factors. Furthermore, vitamin B6 is implicated in oxidative stress responses. Thus, vitamin B6 fulfils a variety of vital functions. Two pathways for de novo PLP synthesis are known. The deoxy 5-xylulose phosphate (DXP)-dependent and the DXP-independent vitamin B6 biosynthetic pathway of which the latter involves only the PdxST complex that consists of the glutaminase PdxT and the PLP synthase PdxS. So far, only few vitamin B6 transporters have been in described. The Gram-positive model bacterium Bacillus subtilis synthesizes PLP via the PdxST complex. B. subtilis must also possess an unknown PL uptake system. Moreover, a survey revealed that B. subtilis contains at least 65 PLP-dependent proteins of which one third are poorly characterized. Thus, even for well-studied model bacteria the complete set of enzymes involved in vitamin B6 metabolism, and the PLP-dependent proteins remain to be identified. Moreover, the PL transporters are unknown in this organism. It also remains to be resolved how the reactive PLP is delivered to the target proteins. Recently, it was shown that evolved B. subtilis pdxST mutants carrying a truncated heterologous DXP-dependent vitamin B6 pathway bypass a block in PLP synthesis. This proposal was aimed at identifying the uptake system for the B6 vitamer PL. While a transport system in B. subtilis that is involved in the uptake of PL could not be identified, a genetic suppressor analysis revealed that PL is highly toxic for B. subtilis and that some of the acquired mutations may reduce PL toxicity. Moreover, the results of the suppressors and the characterization of mutant strains lacking putative transporter genes indicate that B. subtilis does not possess a transport system that is specifically transports PL. It was also aimed at identifying PdxT-independent PdxS variants. A genetic suppressor screen revealed that ammonium limitation due to the lack of PdxT is strictly suppressed by mutations increasing either ammonium assimilation or pdxS gene dosage. This suggests that the catalytic mechanism of PdxS restricts its evolution to become independent of PdxT. Given its reactivity, the PLP synthesized at the PdxST enzyme complex is probably directly transferred to the PLP-dependent proteins. However, in vivocrosslinking and protein purification experiments revealed that an interactome of the PdxST enzyme complex likely does not exist in B. subtilis. Finally, it was confirmed that the native YtoQ protein of unknown function is involved in PLP synthesis in the evolved B. subtilis pdxST mutant strains carrying enzymes of a DXP-dependent vitamin B6 pathway. Structure determination of YtoQ revealed that the protein could be a nucleoside 2-deoxyriboysltransferase. In conclusion this work shows that PLP is required in minute amounts and that the B6 vitamer may enter the cell via unspecific transporters, explaining why only a few PL uptake systems were identified in the past. Moreover, the finding that the overexpression of pdxS is sufficient to cover the cellular need for PLP is important when it comes to the construction of genome reduced bacteria. Finally, further characterization of the B. subtilis mutants synthesizing PLP via a truncated DXP-dependent vitamin B6 will be helpful to elucidate how a non-native pathway implements in an existing metabolic network.

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