Distinct Pathways Carry Out α and β Galactosylation of Secondary Cell Wall Polysaccharide in Bacillus anthracis.

Bacillus anthracis, the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is required for the retention of surface layer (S-layer) and S-layer homology (SLH) domain proteins. Genetic disruption of the SCWP biosynthetic pathway impairs growth and cell division. B. anthracis SCWP is comprised of trisaccharide repeats composed of one ManNAc and two GlcNAc residues with O-3-α-Gal and O-4-β-Gal substitutions. UDP-Gal, synthesized by GalE1, is the substrate of galactosyltransferases that modify the SCWP repeat. Here, we show that the gtsE gene, which encodes a predicted glycosyltransferase with a GT-A fold, is required for O-4-β-Gal modification of trisaccharide repeats. We identify a DXD motif critical for GtsE activity. Three distinct genes, gtsA, gtsB, and gtsC, are required for O-3-α-Gal modification of trisaccharide repeats. Based on the similarity with other three-component glycosyltransferase systems, we propose that GtsA transfers Gal from cytosolic UDP-Gal to undecaprenyl phosphate (C55-P), GtsB flips the C55-P-Gal intermediate to the trans side of the membrane, and GtsC transfers Gal onto trisaccharide repeats. The deletion of galE1 does not affect growth in vitro, suggesting that galactosyl modifications are dispensable for the function of SCWP. The deletion of gtsA, gtsB, or gtsC leads to a loss of viability, yet gtsA and gtsC can be deleted in strains lacking galE1 or gtsE We propose that the loss of viability is caused by the accumulation of undecaprenol-bound precursors and present an updated model for SCWP assembly in B. anthracis to account for the galactosylation of repeat units.IMPORTANCE Peptidoglycan is a conserved extracellular macromolecule that protects bacterial cells from turgor pressure. Peptidoglycan of Gram-positive bacteria serves as a scaffold for the attachment of polymers that provide defined bacterial interactions with their environment. One such polymer, B. anthracis SCWP, is pyruvylated at its distal end to serve as a receptor for secreted proteins bearing the S-layer homology domain. Repeat units of SCWP carry three galactoses in B. anthracis Glycosylation is a recurring theme in nature and often represents a means to mask or alter conserved molecular signatures from intruders such as bacteriophages. Several glycosyltransferase families have been described based on bioinformatics prediction, but few have been studied. Here, we describe the glycosyltransferases that mediate the galactosylation of B. anthracis SCWP.