Direct analysis of tfdA gene expression by indigenous bacteria in phenoxy acid amended agricultural soil

Abstract

Expression of the functional gene tfdA involved in degradation of phenoxyacetic acids such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA) was investigated during degradation scenarios in natural unseeded soil samples. The results illustrate how messenger RNA (mRNA)-based analysis is well suited to quantitatively study the activity of specific microbial populations in soil using phenoxyacetic acid biodegradation as a model system. Via quantitative real-time PCR, a clear response to the presence of phenoxy acids was shown during degradation in soil amended with 20 mg 2,4-D or MCPA per kg soil. Further, we found a relatively high degree of correlation between expression of the functional gene and the rates of mineralization. Melting curve analyses of real-time PCR products, supported by tfdA-denaturing gradient gel electrophoresis analysis showed that, although only class I tfdA genes were apparent in the indigenous microbial population, class III tfdA genes became predominant during incubation, and were the only genes expressed during degradation of MCPA in soil. In contrast, both classes were expressed during degradation of the structurally similar compound 2,4-D. The ability to quantify microbial transcripts directly in environmental samples will have a profound impact on our understanding of microbial processes in the environment in future studies.

Abundance of Novel and Diverse tfdA -Like Genes, Encoding Putative Phenoxyalkanoic Acid Herbicide-Degrading Dioxygenases, in Soil

“…S2). Other studies of tfdA-like gene sequence diversity in soil without prior enrichment and/or isolation are still rather scarce and detect primarily group 1 tfdA-like genes indicative of Beta-and Gammaproteobacteria (3)(4)(5)20). However, enrichments and analysis of isolates from soils revealed the presence of alphaproteobacterial PAA degraders in soils (29,34) and are consistent with the detection of alphaproteobacterial tfdA-like genes in this study.…”

“…tfdA-like genes accounted for up to 3% of bacterial 16S rRNA genes. Copy numbers obtained in other studies with group 1 tfdA-like-gene-specific qPCR assays vary from 10 2 to 10 5 per gram (dry weight) soil prior to PAA incubation (3,5,20), indicating that group 2 and 3 genes outnumber group 1 genes in certain soils and highlighting a hitherto unknown PAA degradation potential in soils.…”

The earthworm Aporrectodea caliginosa stimulates abundance and activity of phenoxyalkanoic acid herbicide degraders

“…Structural genes associated with MCPA degradation in soil MCPA degradation in soil is generally associated with class I and III tfdA genes of the betaproteobacterial group 1 organisms in soil (Baelum et al, 2006(Baelum et al, , 2008 and organisms not capable of degrading the PAA 2,4-D harbor tfdA-like genes (Hogan et al, 1997). However, gene products of tfdA-like genes are capable of PAA conversion (Itoh et al, 2002), indicating a potential role of tfdA-like genes for a (co-metabolic) conversion of MCPA.…”

“…Diverse and new genes encoding putative PAA oxygenases were recently identified in an agricultural soil (Zaprasis et al, 2010). However, to date only group 1 tfdA-like genes are associated with PAA degradation in soil (Baelum et al, 2006(Baelum et al, , 2008Baelum and Jacobsen, 2009;Rodriguez-Cruz et al, 2010).…”

Influence of Heterogeneous Ammonium Availability on Bacterial Community Structure and the Expression of Nitrogen Fixation and Ammonium Transporter Genes during in Situ Bioremediation of Uranium-Contaminated Groundwater

“…One method for elucidating environmental factors that may influence the growth and activity of microorganisms is by quantifying mRNA transcripts for key metabolic genes. Transcript abundance has previously been used to quantify genes involved in remediation processes (9,10), and to diagnose the physiological status of bacteria during in situ bioremediation (11)(12)(13). Recently, this approach has been effective for understanding the central metabolism physiology, oxidative, and heavy metal stress response of Geobacteraceae involved in acetate-stimulated U(VI) Mouser,4 bioremediation (12,14,15).…”