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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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Related in: MedlinePlus

Construction of the PA biosensor plasmid pPANT3. A 1108 bp fragment from the genome of P. fluorescens 23F containing the phnR gene together with the phnA regulatory region was amplified by PCR and cloned into the HindIII‐SacI sites of the promoter probe vector pPROBE‐NT. km, kanamycin‐resistance gene of pPROBE‐NT; T1 and T4, rrnB1 transcriptional terminators of E. coli. ΔphnA, first 29 nt of the phnA gene.
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f1: Construction of the PA biosensor plasmid pPANT3. A 1108 bp fragment from the genome of P. fluorescens 23F containing the phnR gene together with the phnA regulatory region was amplified by PCR and cloned into the HindIII‐SacI sites of the promoter probe vector pPROBE‐NT. km, kanamycin‐resistance gene of pPROBE‐NT; T1 and T4, rrnB1 transcriptional terminators of E. coli. ΔphnA, first 29 nt of the phnA gene.

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.


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)

Construction of the PA biosensor plasmid pPANT3. A 1108 bp fragment from the genome of P. fluorescens 23F containing the phnR gene together with the phnA regulatory region was amplified by PCR and cloned into the HindIII‐SacI sites of the promoter probe vector pPROBE‐NT. km, kanamycin‐resistance gene of pPROBE‐NT; T1 and T4, rrnB1 transcriptional terminators of E. coli. ΔphnA, first 29 nt of the phnA gene.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815843&req=5

f1: Construction of the PA biosensor plasmid pPANT3. A 1108 bp fragment from the genome of P. fluorescens 23F containing the phnR gene together with the phnA regulatory region was amplified by PCR and cloned into the HindIII‐SacI sites of the promoter probe vector pPROBE‐NT. km, kanamycin‐resistance gene of pPROBE‐NT; T1 and T4, rrnB1 transcriptional terminators of E. coli. ΔphnA, first 29 nt of the phnA gene.
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.

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