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Free keywords:
Bacillus subtilis, YumC, Fer, YkuN, YkuP, Bacterial Cytological Profiling (BCP),
peptidoglycan synthesis
Abstract:
YumC is an essential protein of Bacillus subtilis. Biochemical studies showed that YumC is a ferredoxin/flavodoxin-NAD(P)+ reductase (FNR). Ferredoxins (Fd) and flavodoxins (Fld) are small electron transport proteins, and FNRs catalyze electron transfer between NAD(P)+/NAD(P)H and Fd/Fld. YumC can use NADPH as an electron donor to reduce Fd/Fld, or Fd/Fld as an electron donor to reduce NAD(P)+. Although the enzymology of YumC has been detailed and the crystal structure determined, its biological function in Bacillus subtilis is unknown. In this thesis, I investigated the in vivo role of YumC by combining inducible protein degradation and fluorescence microscopy. We used a system that enables us to quickly degrade a specific protein in the cell. We first showed that degradation of YumC resulted in complete inhibition of cellular growth, thereby confirming that YumC is essential in B. subtilis. We then used fluorescence microscopy to observe the effects of YumC degradation on cell morphology. We observed elongated cells with multiple chromosomes. The cytological profile of YumC degradation matched those of the degradation of different essential proteins involved in peptidoglycan (PG) biosynthesis, and also of treatment with antibiotics that inhibit PG biosynthesis, strongly suggesting that YumC is necessary for PG synthesis. We next studied whether YumC participates in PG synthesis through Fd, Fer, and Flds, YkuN and YkuP. Strains lacking Fer, YkuN or YkuP individually are viable. However, strains lacking the three proteins simultaneously are not viable. We, therefore, constructed a strain with deletions of ykuN and ykuP, in which Fer is can be subjected to inducible degradation. Fer degradation in this background completely arrested cell growth, and produced a cytological profile compatible with PG synthesis inhibition, indicating that YumC participates in PG synthesis via Fer, YkuP and YkuN. Finally, we performed a genetic screen for mutations that rescue cell viability upon YumC degradation. Using genetic analysis and whole-genome sequencing, we found that all isolated clones contained mutations that prevented YumC degradation, and therefore none of them actually compensated for loss of YumC. Overall, the findings of this thesis suggest that YumC is involved in PG synthesis.
