Figure 7.

Complementation of the chorismate mutase (CM)‐deficient Escherichia coli strain KA12/pKIMP‐UAUC by HsCM1. (A) Schematic representation of the different deletion constructs used for the complementation test. For the nomenclature of the protein domains, see Fig. 5. (B) Western blot analysis of HsCM1 deletion constructs. Proteins were fractionated by sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and blotted on to a poly(vinylidene difluoride) (PVDF) membrane. HsCM1 derivatives were visualized using a polyhistidine (poly‐His) probe conjugated to horseradish peroxidase (HRP). Molecular masses from the size standard are indicated in kilodaltons on the right. (C) CM complementation assay. Different deletion constructs of Hscm1 were transformed into the CM‐deficient E. coli strain KA12/pKIMP‐UAUC (Kast et al., 1996). As a positive control, a construct expressing a CM gene of Bacillus subtilis was used (Sasso et al., 2005). KA12/pKIMP‐UAUC cells transformed with an empty pQE‐30 UA vector were used as negative control. Bacteria were grown for 3 days at 30 °C on minimal medium plates supplemented with or without phenylalanine (M9cY + F and M9cY, respectively).