Structural and functional characterisation of the bacillus subtilis uridylyltransferase YngB

Abstract

Nucleotide-activated sugars are key sugar donors for glycosylation processes in bacteria. In Bacillus subtilis strain 168, UDP-glucose is used for the decoration of the cell wall polymer wall teichoic acid (WTA) with glucose residues and for the production of membrane glucolipids. UDP-glucose is synthesised by UTP:glucose-1-phosphate uridylyltransferases (UGPases) from UTP and glucose-1-phosphate. In B. subtilis, the GtaB enzyme is essential for UDP-glucose production under standard aerobic growth conditions as WTA lacks glucose decoration and glucolipids are not produced in gtaB mutant strains. Interestingly, bioinformatic predictions indicate that additional UGPases are encoded in the B. subtilis genome including YngB, but the function of YngB has not been characterised to date. In this study, the crystal structure of YngB was solved and unveiled that YngB has the typical fold and all required features of a functional UGPase. Furthermore, using an in vitro enzyme assay it was shown that YngB has UGPase activity and uses UTP and glucose-1-phosphate as substrates. As for the function of YngB in B. subtilis, expression of YngB from an inducible synthetic promoter in a gtaB mutant resulted in glucose decoration on WTA and production of glucolipids, showing that YngB has UGPase activity in bacteria. Furthermore, as part of this study, the hypothesis that YngB is only expressed from its native promoter in B. subtilis under anaerobic conditions was investigated. This was done by growing wild-type and mutant strains of B. subtilis anaerobically. Indeed, YngB-dependent glucose decoration on WTA and glucolipids production could be detected in B. subtilis propagated under anaerobic growth conditions, showing that yngB is expressed from its native promoter under these growth conditions. Furthermore, based on the operon structure and regulation of yngB expression, it can be proposed that YngB potentially forms part of a multi-component glycosylation system functioning during anaerobic growth of B. subtilis. Lastly, in the final part of this study, preliminary data for the structural and functional characterisations of five different membrane-linked GT-A-fold glycosyltransferases involved in multi-component systems forming part of glycosylation pathways in bacteria are presented.