title: A powerful molecular engineering tool provided efficient Chlamydomonas mutants as bio-sensing elements for herbicides detection.
abstract: This study was prompted by increasing concerns about ecological damage and human health threats derived by persistent contamination of water and soil with herbicides, and emerging of bio-sensing technology as powerful, fast and efficient tool for the identification of such hazards. This work is aimed at overcoming principal limitations negatively affecting the whole-cell-based biosensors performance due to inadequate stability and sensitivity of the bio-recognition element. The novel bio-sensing elements for the detection of herbicides were generated exploiting the power of molecular engineering in order to improve the performance of photosynthetic complexes. The new phenotypes were produced by an in vitro directed evolution strategy targeted at the photosystem II (PSII) D1 protein of Chlamydomonas reinhardtii, using exposures to radical-generating ionizing radiation as selection pressure. These tools proved successful to identify D1 mutations conferring enhanced stability, tolerance to free-radical-associated stress and competence for herbicide perception. Long-term stability tests of PSII performance revealed the mutants capability to deal with oxidative stress-related conditions. Furthermore, dose-response experiments indicated the strains having increased sensitivity or resistance to triazine and urea type herbicides with I(50) values ranging from 6 × 10(-8) M to 2 × 10(-6) M. Besides stressing the relevance of several amino acids for PSII photochemistry and herbicide sensing, the possibility to improve the specificity of whole-cell-based biosensors, via coupling herbicide-sensitive with herbicide-resistant strains, was verified.
authors.affiliation: Institute of Crystallography, National Research Council, Monterotondo Scalo, Rome, Italy. maya.lambreva@ic.cnr.it
authors.forename: Maya D
authors.initials: MD
authors.lastname: Lambreva
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Giardi
authors.forename: Irene
authors.initials: I
authors.lastname: Rambaldi
authors.forename: Amina
authors.initials: A
authors.lastname: Antonacci
authors.forename: Sandro
authors.initials: S
authors.lastname: Pastorelli
authors.forename: Ivo
authors.initials: I
authors.lastname: Bertalan
authors.forename: Ivan
authors.initials: I
authors.lastname: Husu
authors.forename: Udo
authors.initials: U
authors.lastname: Johanningmeier
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Rea
mesh_terms: D000222:Adaptation, Physiological / Q000187:drug effects; D019943:Amino Acid Substitution; D001280:Atrazine / Q000633:toxicity; D015374:Biosensing Techniques; D016825:Chlamydomonas reinhardtii / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development / Q000502:physiology; D002734:Chlorophyll / Q000378:metabolism; D004579:Electron Transport / Q000187:drug effects; D005453:Fluorescence; D005609:Free Radicals / Q000633:toxicity; D005818:Genetic Engineering / Q000379:methods*; D006540:Herbicides / Q000633:toxicity*; D006801:Humans; D057230:Limit of Detection; D009154:Mutation / Q000235:genetics*; D009502:Neutrons; D018384:Oxidative Stress / Q000187:drug effects; D045332:Photosystem II Protein Complex / Q000378:metabolism; D011522:Protons
chemical_list: D005609:Free Radicals; D006540:Herbicides; D045332:Photosystem II Protein Complex; D011522:Protons; D002734:Chlorophyll; D001280:Atrazine