Development of highly-sensitive microbial biosensors by mutation of the nahR regulatory gene.
NahR, a transcriptional regulator for naphthalene degradation in response to salicylate, is a central element in the microbial biosensor for detection of naphthalene and salicylate. To maximize the sensitivity of the biosensor, we have chosen a rational design of highly-sensitive microbial biosensors by introducing site directed mutagenesis to nahR gene. Eight single mutants (N169A, N169C, N169K, N169S, R248H, R248M, R248Q, and R248Y) were made at residues 169 and 248 known as the central inducer-recognition and the C-terminal multimerization domain. The effects of these mutations were examined by monitoring expression of a firefly luciferase (luc) reporter gene under the control of NahR. We found that all mutants at residues 248 and N169C show increased sensitivity (maximum ∼50-fold) compared to wild type, respectively. R248M shows response even at toxic concentration, 5mM. The results show the feasibility and potential versatility of mutational approach for the development of the highly-sensitive microbial biosensors.
Shin
Hae Ja
HJ
Division of Energy Bioengineering, Dongseo University, Busan 617-716, Republic of Korea. hjshin@dongseo.ac.kr
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D003001:Cloning, Molecular; D049409:Luciferases, Firefly / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009281:Naphthalenes / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012459:Salicylates / Q000032:analysis*; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; C063173:NahR protein, Bacteria; D009281:Naphthalenes; D011994:Recombinant Proteins; D012459:Salicylates; D014157:Transcription Factors; C031721:naphthalene; D049409:Luciferases, Firefly