Figure 2.

The CLIBASIA_00460 gene encodes a Sec-dependent presecretory protein. (A) Gene structure of the CLIBASIA_00460 gene. The signal peptide was named 460SP, and the mature form of CLIBASIA_00460 was m460. Two positively charged amino acids, arginine (R) and histine (H), and a central hydrophobic stretch were indicated. (B) Schematic of the prokaryotic expression cassettes for the phoA gene. (C) The 460SP directed the extracellular translocation of the mPhoA lacking its native SP. On indicator LB medium containing 90 μg/ml BCIP, the E. coli cells expressing the fusion protein 460SP-PhoA turned blue after 6 h of incubation at 37°C. IPTG (100 mM) was added to the LB medium to induce lacUV5 promoter and T₇lac promoter, which controls the phoA gene expression, and Na₂HPO₄ (75 mM) was introduced to suppress the endogenous phosphatase activity.