The construction of a whole-cell biosensor for phosphonoacetate, based on the LysR-like transcriptional regulator PhnR from Pseudomonas fluorescens 23F.
The phnA gene that encodes the carbon-phosphorus bond cleavage enzyme phosphonoacetate hydrolase is widely distributed in the environment, suggesting that its phosphonate substrate may play a significant role in biogeochemical phosphorus cycling. Surprisingly, however, no biogenic origin for phosphonoacetate has yet been established. To facilitate the search for its natural source we have constructed a whole-cell phosphonoacetate biosensor. The gene encoding the LysR-type transcriptional activator PhnR, which controls expression of the phosphonoacetate degradative operon in Pseudomonas fluorescens 23F, was inserted in the broad-host-range promoter probe vector pPROBE-NT, together with the promoter region of the structural genes. Cells of Escherichia coli DH5α that contained the resultant construct, pPANT3, exhibited phosphonoacetate-dependent green fluorescent protein fluorescence in response to threshold concentrations of as little as 0.5 µM phosphonoacetate, some 100 times lower than the detection limit of currently available non-biological analytical methods; the pPANT3 biosensor construct in Pseudomonas putida KT2440 was less sensitive, although with shorter response times. From a range of other phosphonates and phosphonoacetate analogues tested, only phosphonoacetaldehyde and arsonoacetate induced green fluorescent protein fluorescence in the E. coli DH5α (pPANT3) biosensor, although at much-reduced sensitivities (50 µM phosphonoacetaldehyde and 500 µM arsonoacetate).
The QUESTOR Centre and School of Biological Sciences, The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland.
Anna N
AN
Kulakova
Leonid A
LA
Kulakov
John W
JW
McGrath
John P
JP
Quinn
D000469:Alkaline Phosphatase; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D009876:Operon; D010746:Phosphonoacetic Acid / Q000032:analysis / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D000469:Alkaline Phosphatase; D010744:Phosphoric Monoester Hydrolases; C097254:phosphonoacetate hydrolase; D010746:Phosphonoacetic Acid
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