Surface display of monkey metallothionein α tandem repeats and EGFP fusion protein on Pseudomonas putida X4 for biosorption and detection of cadmium.
Monkey metallothionein α domain tandem repeats (4mMTα), which exhibit high cadmium affinity, have been displayed for the first time on the surface of a bacterium using ice nucleation protein N-domain (inaXN) protein from the Xanthomonas campestris pv (ACCC-10049) as an anchoring motif. The shuttle vector pIME, which codes for INAXN-4mMTα-EGFP fusion, was constructed and used to target 4mMTα and EGFP on the surface of Pseudomonas putida X4 (CCTCC-209319). The surface location of the INAXN-4mMTα-EGFP fusion was further verified by western blot analysis and immunofluorescence microscopy. The growth of X4 showed resistance to cadmium presence. The presence of surface-exposed 4mMTα on the engineered strains was four times higher than that of the wild-type X4. The Cd²⁺ accumulation by X4/pIME was not only four times greater than that of the original host bacterial cells but was also remarkably unaffected by the presence of Cu²⁺ and Zn²⁺. Moreover, the surface-engineered strains could effectively bind Cd²⁺ under a wide range of pH levels, from 4 to 7. P. putida X4/pIME with surface-expressed 4mMTα-EGFP had twice the cadmium binding capacity as well as 1.4 times the fluorescence as the cytoplasmic 4mMTa-EGFP. These results suggest that P. putida X4 expressing 4mMTα-EGFP with the INAXN anchor motif on the surface would be a useful tool for the remediation and biodetection of environmental cadmium contaminants.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
Xiaochuan
X
He
Wenli
W
Chen
Qiaoyun
Q
Huang
D020816:Amino Acid Motifs; D000818:Animals; D001673:Biodegradation, Environmental; D002104:Cadmium / Q000032:analysis / Q000378:metabolism*; D052918:Environmental Restoration and Remediation / Q000379:methods*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D000882:Haplorhini; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020080:Tandem Repeat Sequences
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D002104:Cadmium; D049452:Green Fluorescent Proteins; D008668:Metallothionein