The construction of a whole-cell biosensor for phosphonoacetate, based on the LysR-like transcriptional regulator PhnR from Pseudomonas fluorescens 23F.
Bottom Line: 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.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).
Affiliation: The QUESTOR Centre and School of Biological Sciences, The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland.Show MeSH
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Mentions: A 1108 bp fragment of the phosphonoacetate degradative gene cluster from P. fluorescens 23F (Kulakova et al., 2001), consisting of the entire LTTR regulatory gene phnR, the promoter regions for phnR and its associated structural genes (phnA and phnB), and the first 29 5′‐end nucleotides of the phosphonoacetate hydrolase gene (phnA), was amplified and cloned into pPROBE vectors as described in Experimental procedures. This led to the creation of a phnR–ΔphnA–gfp transcriptional fusion (Fig. 1). Transformation of cells of Escherichia coli DH5α‐T1R (further designated as DH5α) with pPROBE::phnR–ΔphnA constructs allowed for the selection of clones showing an elevated, statistically significant, fluorescence response to the presence of phosphonoacetate in the medium when compared with phosphonoacetate‐free controls. Analysis of these strains demonstrated that the fluorescence values produced by pPROBE‐NT‐based constructs were approximately two times higher than those based on pPROBE‐TT under similar conditions. In the light of this finding, a pPROBE‐NT::phnR–ΔphnA plasmid designated pPANT3 was used in subsequent biosensor optimization studies.
Affiliation: The QUESTOR Centre and School of Biological Sciences, The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland.