FIGURE 4.

Trafficking of WT Smlt1473 and a C23F mutant. A, sequence of N-terminal signaling peptide of Smlt1473 highlighted in gray and the lipid-modified cysteine 23 residue boxed. A C23F mutant was prepared via site-directed mutagenesis. B, anti-His₆ tag Western blot of E. coli BL21 cultures expressing wild-type Smlt1473 and C23F mutant separated into whole cell lysate and periplasmic fractions. C, E. coli BL21 cultures expressing wild-type Smlt1473 and C23F mutant dotted onto LB plates solidified with 1% agarose, and supplemented with 50 μg/ml of kanamycin and 1 mg/ml of poly-GlcA (top) or poly-ManA (bottom). After a 12-h incubation at 25 °C, the plates were treated with 10% cetylpyridinium chloride. Clearing zone indicates degradation of polysaccharide in surrounding media. D, activity of purified Smlt1473 WT and C23F. Enzyme activity was monitored by absorbance at 235 nm. All reactions were performed in triplicate and error is reported as S.D.