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. 2010 Aug 26;285(45):34330–34336. doi: 10.1074/jbc.M110.166314

FIGURE 4.

FIGURE 4.

KdhA is a Kdo hydrolase. A, Kdo hydrolase activity in F. tularensis wild-type and ΔkdhA mutant total membranes. Total membranes of F. tularensis LVS (LVSm) or ΔkdhA (ΔkdhAm) were assayed with Re LPS from S. enterica Re 595 as the substrate. Release of Kdo was measured by HPAEC. B, detection of KdhA protein in LVS total membranes. Total membranes of LVS wild-type and ΔkdhA mutant were analyzed by immunoblotting for the presence of KdhA. Loaded amounts were controlled by probing with mAb 2033 monoclonal antibody, specific to the O-antigen. C, side-chain Kdo trimming in E. coli BL21 expressing the following KdhA orthologs: KdhA from F. tularensis LVS, FTT0399c from F. tularensis Schu S4, HP0580 from H. pylori, and Lpg2939 from L. pneumophila. Total membranes from E. coli BL21 expressing KdhA orthologs were subjected to mild hydrolysis, and released Kdo was measured by HPAEC. Measurements were normalized with the amount of glucose released by TFA treatment and are expressed as -fold induction when compared with BL21/pET45b vector control. The average values ± S.D. of two independent experiments are shown.