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 α-ketoglutarate- and Fe ²⁺ -dependent dioxygenases encoded by tfdA -like genes (i.e., tfdA and tfdA α). 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.