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).