FIG. 2.

Western blot of cultures of strains carrying the different NagB-expressing plasmids. (A) LAA20 (ΔnagB::cm) carrying a pXE1-derived plasmid expressing NagB-L1, -L2, -L3, or -L4 or MC4100 carrying the vector plasmid pXE1 was grown in glucose, GlcN, or GlcNAc medium, as indicated, to late exponential phase (A₆₅₀ = 0.8). Bacteria were chilled, harvested by centrifugation, and lysed by sonication and boiling in sodium dodecyl sulfate sample buffer. Aliquots (0.25 A₆₅₀ units) were analyzed on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to Hybond-C membrane, and treated with anti-NagA and anti-NagB as described previously (1). S, standards (100 ng NagA and NagB); C, control LAA20 without any plasmid grown in glucose. Note that NagB is poorly retained on the membranes, resulting in a low signal. The band running just faster than NagB is due to contaminant antibodies reacting with a protein of molecular weight similar to that of NagB and which is equally present in all lanes. (B) NagB protein levels due to each construct and growing on the different media were quantified by using the ImageQuant program of PhosphorImager. For each blot, the levels of NagB were normalized to the level of the NagB protein from the NagB-L4 construct growing in glucose. The numbers are the relative levels ± standard deviations of three or four experiments.