Studies of the Genetics, Function, and Kinetic Mechanism of TagE, the Wall Teichoic Acid Glycosyltransferase in Bacillus subtilis 168

Abstract

The biosynthetic enzymes involved in wall teichoic acid biogenesis in Gram-positive bacteria have been the subject of renewed investigation in recent years with the benefit of modern tools of biochemistry and genetics. Nevertheless, there have been only limited investigations into the enzymes that glycosylate wall teichoic acid. Decades-old experiments in the model Gram-positive bacterium, Bacillus subtilis 168, using phage-resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the wall teichoic acid glycosyltransferase. This study and others have provided only indirect evidence to support a role for TagE in wall teichoic acid glycosylation. In this work, we showed that deletion of tagE resulted in the loss of ␣-glucose at the C-2 position of glycerol in the poly(glycerol phosphate) polymer backbone. We also reported the first kinetic characterization of pure, recombinant wall teichoic acid glycosyltransferase using clean synthetic substrates. We investigated the substrate specificity of TagE using a wide variety of acceptor substrates and found that the enzyme had a strong kinetic preference for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form. Further, we showed that the enzyme recognized its polymeric (and repetitive) substrate with a sequential kinetic mechanism. This work provides direct evidence that TagE is the wall teichoic acid glycosyltransferase in B. subtilis 168 and provides a strong basis for further studies of the mechanism of wall teichoic acid glycosylation, a largely uncharted aspect of wall teichoic acid biogenesis.

Localization and expression of the Bacillus subtilis dl-endopeptidase LytF are influenced by mutations in LTA synthases and glycolipid anchor synthetic enzymes

“…In addition, no significant differences in expression or localization of LytF were observed in the tagE null mutant strain (Fig. 1b, 3f-g), which has a defect in glycosylation of the major WTA (Allison et al, 2011;Lazarevic et al, 2002). A previous report has demonstrated that TagE is only able to use a poly(GroP) polymer as a substrate (Allison et al, 2011).…”

“…1b, 3f-g), which has a defect in glycosylation of the major WTA (Allison et al, 2011;Lazarevic et al, 2002). A previous report has demonstrated that TagE is only able to use a poly(GroP) polymer as a substrate (Allison et al, 2011). Therefore, it is thought that WTA polymer synthesis is initiated by TagF prior to glycosylation.…”

Repeated triggering of sporulation in Bacillus subtilis selects against a protein that affects the timing of cell division

“…The physiological function of this modification is yet unknown (Allison et al, 2011). Together with lipoteichoic acids, WTA accounts for over 60% of the mass of the Gram-positive cell wall.…”

“…This cassette was obtained by PCR on genomic DNA (Supplementary Table S1) of a previously constructed tagE::spec strain (Allison et al, 2011) (gift of professor Eric Brown).…”

Pathways and roles of wall teichoic acid glycosylation in Staphylococcus aureus

“…Decades ago it was shown that glucosylated WTA were required for phage adsorption in B. subtilis 168 (Young, 1967) (Table 1) and the TagE protein (also called GtaA and RodD) of B. subtilis 168 was proposed as the WTA glucosyltransferase based on the indirect genetic evidence (Honeyman and Stewart, 1989;Mauel et al, 1989). Recently, TagE was characterized and demonstrated to be a GroP WTA ␣-glucosyl transferase (Allison et al, 2011). Thus, WTA glycosylation in B. subtilis and S. aureus share similar functions such as phage adsorption and infection.…”