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Vitamin B6 metabolism and underground metabolic routes in the Gram-positive bacterium Bacillus subtilis

dc.contributor.advisorCommichau, Fabian Prof. Dr.
dc.contributor.authorRichts, Björn
dc.titleVitamin B6 metabolism and underground metabolic routes in the Gram-positive bacterium Bacillus subtilisde
dc.contributor.refereeCommichau, Fabian Prof. Dr.
dc.description.abstractengGenome-reduced bacteria give the opportunity to study essential gene functions but also give the starting point for biotechnological production pathways. The MiniBacillus project aims to produce a genome reduced Bacillus subtilis strain with only a defined set of necessary genes. To identify new deletable genes, we studied the vitamin B6 metabolic pathway since the active form of vitamin B6 pyridoxal-5’-phosphate (PLP) is involved in about 4% of all known enzymatic reactions. In the Gram-positive soil bacterium B. subtilis at least 65 vitamin B6-dependent proteins were identified. In this study, mainly three aspects of vitamin B6 metabolism were studied: The PdxST vitamin B6 synthesis complex, transport and detoxication of vitamin B6 and, the role of the low molecular weight (LMW) thiol bacillithiol (BSH) together with the ytoQ gene in a heterologous vitamin B6 synthesis pathway. In B. subtilis vitamin B6 is produced by a complex of PdxS and PdxT. The glutaminase domain PdxT cleaves off ammonia from glutamine and transfers it to the synthase domain PdxS. Vitamin B6 even can be produced by PdxS in absence of PdxT, when high concentrations of ammonium are present. A suppressor screen was performed with the pdxT mutant on medium containing low amounts of ammonium and an amplification of a 15 kb region including the pdxS gene was identified as the main suppression mechanism. Moreover, in some suppressors the ammonium channel nrgA was upregulated, leading to a growth advantage especially when pdxS was not overexpressed. Besides that, the pdxS gene and its promotor region did not acquire beneficial mutations even if the amplification relevant recombinase gene recA was deleted. Furthermore, the nucleobase:cation symporter family proteins and the ECF transporter were excluded as vitamin B6 transporters and an upregulation of the Ars operon was identified as a detoxification mechanism for toxic PL levels. A PL-auxotroph B. subtilis mutant harboring only the last two genes of the DXP-dependent vitamin B6 synthesis pathway from E. coli, formed suppressor mutants, which deleted the bacillithiol synthesis gene bshC and upregulated the ytoQ gene. It was shown that cultivation on medium containing cysteine repealed the beneficial effect. Cysteine can also act as a LMW thiol indicating a role of YtoQ and BSH in oxidative stress response. Furthermore, a proposed underground metabolism pathway for the synthesis of PLP could be excluded, involving the genes cpgA, serA, serS and thrB. To increase general fitness and get insights about deleterious effects of the genome reduction in the MiniBacillus strains, the two genome-reduced strains PG10 (36% reduction) and PG39 (40% reduction) were evolved in complex medium. For PG39 a deregulation of genes mainly involved in oxidative stress response was identified as suppression mechanism. In PG10 over 25 genes harbored mutations. Interestingly, a downregulation of the mhqNOP operon was found, which was identified as highly upregulated in the
dc.contributor.coRefereeStülke, Jörg Prof. Dr.
dc.contributor.thirdRefereeDaniel, Rolf Prof. Dr.
dc.contributor.thirdRefereePöggeler, Stefanie Prof. Dr.
dc.contributor.thirdRefereeHeimel, Kai Prof. Dr.
dc.contributor.thirdRefereeIschebeck, Till Dr.
dc.subject.enggenome-reduced bacteriade
dc.subject.engvitamin B6 metabolismde
dc.subject.engBacillus subtilisde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

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