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

Anna Kulakova, Leonid Kulakov, John McGrath, John Quinn

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

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 DH5a 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 DH5a (pPANT3) biosensor, although at much-reduced sensitivities (50 µM phosphonoacetaldehyde and 500 µM arsonoacetate).
Original languageEnglish
Pages (from-to)234-240
Number of pages7
JournalMicrobial Biotechnology
Volume2
Issue number2 SPEC. ISS.
DOIs
Publication statusPublished - Mar 2009

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Biotechnology
  • Biochemistry
  • Bioengineering

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