AEM Accepts, published online ahead of print on 30 October 2009

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Appl. Environ. Microbiol. doi:10.1128/AEM.01727-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Novel and Diverse tfdA-like Genes Encoding Putative Phenoxyalkanoic Acid Herbicide Degrading Dioxygenases are Abundant in Soil

Adrienne Zaprasis, Ya-Jun Liu, Shuang-Jiang Liu, Harold L. Drake, and Marcus A. Horn^*

Department of Ecological Microbiology, University of Bayreuth, Dr.-Hans-Frisch-Straße 1-3, 95440 Bayreuth, Germany; State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, P. R. China

^* To whom correspondence should be addressed. Email: marcus.horn{at}unibay-reuth.de.

Phenoxyalkanoic acid (PAA) herbicides are widely used in agriculture. Biotic degradation of such herbicides occurs in soils and is initiated by -ketoglutarate- and Fe²⁺-dependent dioxygenases encoded by tfdA-like genes (i.e., tfdA and tfdA). Novel primers and 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 3-5 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 sp., Sphingomonas sp., 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 - 65 x 10⁶ per gram soil dry weight in four different soils, indicating that hitherto unknown, diverse tfdA-like genes are abundant in soils.