| By the bacteria cell-surface display techniques, an inducible bacterial cell-surface display vector pCIM has been constructed. The ice nucleation activity protein identified from Xanthomonas campestris ATCC33913 (inaX) was used as an anchoring motif. A cluster of 4 direct repeats of monkey metallothioneinαdomain (mMT4α) was used as the target protein. By the way of C-terminal fusion to the N-domain of inaX, mMT4αwas successfully displayed on the surface of the heavy metal-resistant bacterial strain Pseudomonas putida X4 (hereinafter short for X4). The cadmium adsorption capability of the engineered strain X4/pCIM and its symbiosis system with plants were significantly higher than those of X4. Consequently, the engineered strain X4/pCIM is promising to be used in the bioremediation of cadmium-contaminated environment.Based on the broad-host-range vector pTR102, fusion expression of the gene inaX and mMT4αwere feasible. The ice nucleation protein N terminal gene (inaX-N) from X. campestris and mMT4αwere cloned, inserted into pTR102 and renamed as pCIM. The expression was regulated by an upstream cadmium-specific responsible promoter CadR. By electroporation, pCIM was introduced into P. putida X4. The fusion expression protein was induced by cadmium ions at 100μmol/L. The results of cell grading assay and SDS-PAGE indicated that the fusion protein was localized on the bacterial outer membrane.The engineered strain X4/pCIM showed higher resistance to cadmium than X4. Bacteriostatic test of cadmium indicated that minimal inhibition concentration (MIC) of X4/pCIM was 2 mmol/L higher than X4. Under static status, the adsorption process of cadmium finished in 5 minutes. The adsorption capacity of X4/pCIM for Cd was 2 times larger than that of X4 in growing culture.Hydroponic culture experiment was carried out with wheat, cowpea, maize and rhizosphere bacteria. The results indicated that the association of X4/pCIM and X4 with plants stabilized heavy metals. |
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