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Structural and Functional Studies on the Interaction of GspC and GspD in the Type II Secretion System

Figure 5

Simultaneous substitution of Ile18 and Asn22 in V. cholerae GspD results in inactivation of protease secretion by V. cholerae.

(A) V. cholerae wild-type or gspD mutant strain (ΔgspD) containing either pMMB or pGspD variants were grown overnight in LB. Culture supernatants were separated from cells by centrifugation and tested for the presence of extracellular protease. The rate of hydrolysis was obtained from three independent experiments, and the results are presented with standard error. Both the pMMB vector control and pGspDI18R/N22Y were below the limits of detection. (B) V. cholerae gspD cells containing either pMMB or pGspD variants were disrupted and subjected to SDS-PAGE and immunoblotting with anti-GspD antibodies to determine the relative level of expression. The positions of molecular mass markers are shown. Arrow indicates monomeric GspD and arrowhead indicates multimeric GspD.

Figure 5