Transcription dynamics of the functional tfdA gene during MCPA herbicide degradation by Cupriavidus necator AEO106 (pRO101) in agricultural soil

Mette Haubjerg Nicolaisen, Jacob Bælum, Carsten Suhr Jacobsen, Jan Sørensen

Research output: Contribution to journalArticleResearchpeer-review

74 Citations (Scopus)

Abstract

A modified protocol for simultaneous extraction of RNA and DNA, followed by real-time polymerase chain reaction quantification, was used to investigate tfdA gene expression during in situ degradation of the herbicide MCPA (4-chloro-2-methylphenoxy-acetic acid) in soil. tfdA encodes an α-ketoglutarate-dependent dioxygenase catalysing the first step in the degradation pathway of MCPA and 2,4-D (2,4-dichlorophenoxy-acetic acid). A linear recovery of tfdA mRNA over three orders of magnitude was shown, and the tfdA mRNA level was normalized using the tfdA mRNA/DNA ratio. The density of active cells required for tfdA mRNA detection was 10 5 cells g -1 soil. Natural soil microcosms inoculated with Cupriavidus necator (formerly Ralstonia eutropha) AEO106 (pRO101) cells were amended with four different MCPA concentrations (2, 20, 50 and 150 mg kg -1). Mineralization rates were estimated by quantification of 14CO 2 emission from degradation of 14C-MCPA. tfdA mRNA was detected 1 h after amendment at all four concentrations. In soils amended with 2 and 20 mg kg -1, the mRNA/DNA ratio for tfdA demonstrated a sharp transient maximum of tfdA expression from no to full expression within 3 and 6 h respectively, followed by a decline and complete loss of expression after 19 and 43 h. A more complex pattern of tfdA expression was observed for the higher 50 and 150 mg kg -1 amendments; this coincided with growth of C. necator AEO106 (pRO101) in the system. Repeated amendment with MCPA after 2 weeks in the 20 mg kg -1 scenario revealed a sharp increase of tfdA mRNA, and absence of a mineralization lag phase. For all amendments, tfdA mRNA was detectable only during active mineralization, and thus revealed a direct correlation between tfdA mRNA presence and microbial degrader activity. The present study demonstrates that direct analysis of functional gene expression dynamics by quantification of mRNA can indeed be made in natural soil.

Original languageEnglish
Pages (from-to)571-579
Number of pages9
JournalEnvironmental Microbiology
Volume10
Issue number3
DOIs
Publication statusPublished - Mar 2008

Programme Area

  • Programme Area 2: Water Resources

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