Fig. 8.

NMR and biochemical analysis of purified LTA.

A and B. NMR analysis. Large cultures of S. aureus strains (A) ANG514 (LtaS_SA-expressing) and (B) ANG515 (YfnI-expressing) were grown and LTA purified as described in the Experimental procedures section. One milligram purified LTA was suspended and lyophilized several times in D₂O to exchange ¹H for ²H deuterons and ¹H NMR spectra were recorded at 600 MHz, 300 K. The signals derived from citrate, a buffer component used during LTA purification and retained in the samples are marked in grey. The different signals previously assigned to LTA components (Morath et al., 2002) are colour coded [blue – D-Ala (4 protons per D-Ala group), green – GroP (5 protons per GroP group), orange – CH₂/CH₃ groups of fatty acids (59 protons per lipid anchor)]. The integration values are shown above each signal. Chain length was determined by calculating the ratio of integral values for GroP to CH₂/CH₃ groups in fatty acids and % D-Ala substitution by calculating the ratio of integral values for D-Ala to GroP x 100 and taking into account the number of protons for each signal. NMR analysis was performed on four independently isolated LTA samples for each strain and a representative result is shown.

C and D. Biochemical analysis of LTA. LTA extracted from strains ANG514 (LtaS_SA) and ANG515 (YfnI_BS) was subjected to a biochemical analysis. Phosphate, glucose and D-Ala contents were determined as described in the Experimental procedures section. GroP, D-Ala and glucose solutions of known concentrations were used as standards. The chain length in GroP subunits (C) was determined by calculation of the ratio of phosphate/½ glucose concentration and the % D-Ala substitution (D) by calculating the ratio of D-Ala/phosphate concentration × 100. Biochemical analysis was performed on four independently isolated LTA samples for each strain and the mean and standard deviation is shown. The difference in chain length is statistically significant and indicated with an asterisk (*) (two-tailed P-value of 0.017; unpaired T-test) while the difference in D-Ala modification is not (two-tailed P-value of 0.355, unpaired T-test).