Figure 1.

Galactosylation of LTA and WTA in a serovar 4b strain is controlled by two separate genes. (a) Agarose gel electrophoresis of PCR‐amplified regions flanking genes gtlB and gttB from WT 1042 and the 1042ΔgtlB and 1042ΔgttB mutant strains. Primers used for the PCR amplification recognize sequences flanking the genes of interest by ~ 600 bp. (b) LTA analysis by western blot. Whole cell extracts were separated on SDS‐PAGE gels and LTA detected using an LTA‐specific antibody, which recognizes the glycerol phosphate backbone of LTA. Positive signal represents undecorated LTA. Lower panel: SDS‐PAGE gel visualization of total extracts to demonstrate equal loading. (c) NMR spectra of the LTA isolated from the indicated strains. Labeled peaks represent the major protons in the sample, while missing galactosylated protons are indicated with arrows. The LTA structure for each strain is indicated to the right. The unlabeled major peaks are derived from the residual citrate buffer used during the extraction process. (d) Liquid chromatographic separation and MS‐based identification of WTA monomer residues derived from WT 1042 and the indicated mutants. Major peaks are labeled with their assigned structures based on the m/z. The chromatograms are aligned on the same time axis to allow for comparison. Peaks appearing at 1–2 min represent ionized or undigested species that elute without separation