title: 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 alpha-ketoglutarate- and Fe2+-dependent dioxygenases encoded by tfdA-like genes (i.e., tfdA and tfdAalpha). 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 1.0x10(6) to 65x10(6) per gram (dry weight) soil in four different soils, indicating that hitherto-unknown, diverse tfdA-like genes are abundant in soils.
authors.affiliation: Department of Ecological Microbiology, Dr.-Hans-Frisch-Str. 1-3, University of Bayreuth, 95440 Bayreuth, Germany.
authors.forename: Adrienne
authors.initials: A
authors.lastname: Zaprasis
authors.forename: Ya-Jun
authors.initials: YJ
authors.lastname: Liu
authors.forename: Shuang-Jiang
authors.initials: SJ
authors.lastname: Liu
authors.forename: Harold L
authors.initials: HL
authors.lastname: Drake
authors.forename: Marcus A
authors.initials: MA
authors.lastname: Horn
mesh_terms: D020369:Bradyrhizobium / Q000235:genetics; D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D049308:Dioxygenases / Q000235:genetics* / Q000378:metabolism*; D014644:Genetic Variation; D006540:Herbicides / Q000378:metabolism*; D056186:Metagenomics; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D020578:Sphingomonas / Q000235:genetics
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D006540:Herbicides; D012336:RNA, Ribosomal, 16S; D049308:Dioxygenases