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

Abstract

Phenoxyalkanoic acid (PAA) herbicides are widely used in agriculture. Biotic degradation of such herbicides occurs in soils and is initiated by ␣-ketoglutarate-and Fe 2؉ -dependent dioxygenases encoded by tfdA-like genes (i.e., tfdA and tfdA␣). Novel primers and quantitative kinetic PCR (qPCR) assays were developed to analyze the diversity and abundance of tfdA-like genes in soil. Five primer sets targeting tfdA-like genes were designed and evaluated. Primer sets 3 to 5 specifically amplified tfdA-like genes from soil, and a total of 437 sequences were retrieved. Coverages of gene libraries were 62 to 100%, up to 122 genotypes were detected, and up to 389 genotypes were predicted to occur in the gene libraries as indicated by the richness estimator Chao1. Phylogenetic analysis of in silico-translated tfdA-like genes indicated that soil tfdA-like genes were related to those of group 2 and 3 Bradyrhizobium spp., Sphingomonas spp., and uncultured soil bacteria. Soil-derived tfdA-like genes were assigned to 11 clusters, 4 of which were composed of novel sequences from this study, indicating that soil harbors novel and diverse tfdA-like genes. Correlation analysis of 16S rRNA and tfdA-like gene similarity indicated that any two bacteria with D > 20% of group 2 tfdA-like gene-derived protein sequences belong to different species. Thus, data indicate that the soil analyzed harbors at least 48 novel bacterial species containing group 2 tfdA-like genes. Novel qPCR assays were established to quantify such new tfdA-like genes. Copy numbers of tfdA-like genes were 1.0 ؋ 10 6 to 65 ؋ 10 6 per gram (dry weight) soil in four different soils, indicating that hitherto-unknown, diverse tfdA-like genes are abundant in soils.

Contrasting denitrifier communities relate to contrasting N2O emission patterns from acidic peat soils in arctic tundra

“…The stability of tree topologies was assessed by calculating 10 000 bootstrap replicates (Saitou and Nei, 1987). Diversity measures with 95% confidence intervals were calculated as described previously (Sørensen, 1948;Bray and Curtis, 1957;Hill et al, 2003;Zaprasis et al, 2010). Normalized weighted UniFrac significance was calculated to evaluate differences between the communities of narG, nirK, nirS and nosZ based on phylogenetic information (Lozupone and Knight, 2005;Lozupone et al, 2007).…”

“…The lower limits of quantification were p10 1 gene copy numbers m l À1 of DNA extract. 16S rRNA gene copy numbers were determined concomitantly for all environmental samples to quantify microorganisms that harbor genes associated with denitrification in soils relative to the total bacterial population (Muyzer et al, 1993;Zaprasis et al, 2010). Inhibition of quantitative kinetic realtime PCR was assessed per individual DNA extract according to Zaprasis et al (2010) by spiking soil DNA with pure standard DNA.…”

“…Quantification of narG, nirK, nirS, nosZ and 16S rRNA genes in soil Quantitative kinetic real-time PCRs were performed as described with DNA extracts from three replicate cryoturbated and three unturbated sites in six technical replicates per DNA extract (Zaprasis et al, 2010;Supplementary Materials and methods). Thermal protocols and primers were as described previously (Supplementary Materials and methods, Table 1).…”

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

“…As transcription of cadA is induced by 2,4-D in pure cultures, groups 2 and 3 might employ cadA gene homologs for PAA herbicide degradation (Itoh et al, 2002(Itoh et al, , 2004Kitagawa et al, 2002). 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).…”

“…Group 1 consists of Beta-and Gammaproteobacteria, which harbor tfdA-like genes. Groups 2 and 3 consist of alphaproteobacterial Bradyrhizobiumand Sphingomonas-related organisms, respectively, including potential novel organisms (Fulthorpe et al, 1995;Kamagata et al, 1997;Zaprasis et al, 2010). Group 2 organisms harbor both tfdA-like and cadA genes, whereas only cadA was detected in group 3 organisms (Itoh et al, 2002(Itoh et al, , 2004Kitagawa et al, 2002;Huong et al, 2007Huong et al, , 2008.…”