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The construction of a whole-cell biosensor for phosphonoacetate, based on the LysR-like transcriptional regulator PhnR from Pseudomonas fluorescens 23F.

Kulakova AN, Kulakov LA, McGrath JW, Quinn JP - Microb Biotechnol (2009)

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).

View Article: PubMed Central - PubMed

Affiliation: The QUESTOR Centre and School of Biological Sciences, The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland.

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Induction of gfp in E. coli DH5α (pPANT3) in the presence of PA (light grey), phosphonoacetaldehyde (white) and arsonoacetate (dark grey). Induction ratio was calculated as SFUx/SFUo, where SFUx refers to the sample containing the inducer and SFUo to the control sample at the same time point: a value of 1.0 corresponds to no fluorescence being detected. Specific fluorescence (SFU) was calculated as described in Experimental procedures. All measurements were made in triplicate. Error bars represent a standard deviation of the mean (n = 3).
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f2: Induction of gfp in E. coli DH5α (pPANT3) in the presence of PA (light grey), phosphonoacetaldehyde (white) and arsonoacetate (dark grey). Induction ratio was calculated as SFUx/SFUo, where SFUx refers to the sample containing the inducer and SFUo to the control sample at the same time point: a value of 1.0 corresponds to no fluorescence being detected. Specific fluorescence (SFU) was calculated as described in Experimental procedures. All measurements were made in triplicate. Error bars represent a standard deviation of the mean (n = 3).

Mentions: Apart from phosphonoacetate, only phosphonoacetaldehyde and arsonoacetate were found to induce fluorescence by the E. coli DH5α (pPANT3) whole‐cell biosensor. Its response to a broad range of concentrations (0.05 µM–50 mM) of these effectors was further tested in liquid LB and compared with that of phosphonoacetate (Fig. 2). These data confirm that phosphonoacetate induces detectable biosensor fluorescence at concentrations of as little as 0.5 µM, and that the response reaches saturation in the presence of approximately 500 µM. By contrast, the biosensor reacts to phosphonoacetaldehyde with some 100‐fold reduced sensitivity, while the maximum response is only some 40% of that obtained using phosphonoacetate (Fig. 2). The decreased level of biosensor response above 500 µM phosphonoacetaldehyde is most likely due to the inhibition of the host's cellular activities by this compound [this was also demonstrated by the almost complete inhibition of growth of E. coli DH5α (pPANT3) on solidified LB medium containing 50 mM phosphonoacetaldehyde]. Arsonoacetate was found to be an even less effective inducer than phosphonoacetaldehyde; it was detected by the DH5α (pPANT3) biosensor at threshold concentrations of between 0.5 and 50 mM – a decrease in sensitivity relative to phosphonoacetate of greater than 1000‐fold – while the maximum level of GFP expression reached only 50% of that produced by phosphonoacetate (Fig. 2).


The construction of a whole-cell biosensor for phosphonoacetate, based on the LysR-like transcriptional regulator PhnR from Pseudomonas fluorescens 23F.

Kulakova AN, Kulakov LA, McGrath JW, Quinn JP - Microb Biotechnol (2009)

Induction of gfp in E. coli DH5α (pPANT3) in the presence of PA (light grey), phosphonoacetaldehyde (white) and arsonoacetate (dark grey). Induction ratio was calculated as SFUx/SFUo, where SFUx refers to the sample containing the inducer and SFUo to the control sample at the same time point: a value of 1.0 corresponds to no fluorescence being detected. Specific fluorescence (SFU) was calculated as described in Experimental procedures. All measurements were made in triplicate. Error bars represent a standard deviation of the mean (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815843&req=5

f2: Induction of gfp in E. coli DH5α (pPANT3) in the presence of PA (light grey), phosphonoacetaldehyde (white) and arsonoacetate (dark grey). Induction ratio was calculated as SFUx/SFUo, where SFUx refers to the sample containing the inducer and SFUo to the control sample at the same time point: a value of 1.0 corresponds to no fluorescence being detected. Specific fluorescence (SFU) was calculated as described in Experimental procedures. All measurements were made in triplicate. Error bars represent a standard deviation of the mean (n = 3).
Mentions: Apart from phosphonoacetate, only phosphonoacetaldehyde and arsonoacetate were found to induce fluorescence by the E. coli DH5α (pPANT3) whole‐cell biosensor. Its response to a broad range of concentrations (0.05 µM–50 mM) of these effectors was further tested in liquid LB and compared with that of phosphonoacetate (Fig. 2). These data confirm that phosphonoacetate induces detectable biosensor fluorescence at concentrations of as little as 0.5 µM, and that the response reaches saturation in the presence of approximately 500 µM. By contrast, the biosensor reacts to phosphonoacetaldehyde with some 100‐fold reduced sensitivity, while the maximum response is only some 40% of that obtained using phosphonoacetate (Fig. 2). The decreased level of biosensor response above 500 µM phosphonoacetaldehyde is most likely due to the inhibition of the host's cellular activities by this compound [this was also demonstrated by the almost complete inhibition of growth of E. coli DH5α (pPANT3) on solidified LB medium containing 50 mM phosphonoacetaldehyde]. Arsonoacetate was found to be an even less effective inducer than phosphonoacetaldehyde; it was detected by the DH5α (pPANT3) biosensor at threshold concentrations of between 0.5 and 50 mM – a decrease in sensitivity relative to phosphonoacetate of greater than 1000‐fold – while the maximum level of GFP expression reached only 50% of that produced by phosphonoacetate (Fig. 2).

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).

View Article: PubMed Central - PubMed

Affiliation: The QUESTOR Centre and School of Biological Sciences, The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland.

Show MeSH
Related in: MedlinePlus