Comparison of 16S rRNA gene phylogeny and functional tfdA gene distribution in thirty-one different 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid degraders Jacob Bælum a , Carsten S. Jacobsen a,c,n , William E. Holben b a Geological Survey of Denmark and Greenland (GEUS), Department of Geochemistry, Øster Voldgade 10, DK-1350 Copenhagen K, Denmark b University of Montana, Microbial Ecology Program, Division of Biological Sciences, Missoula, MT-59812, USA c University of Copenhagen, Faculty of Life Sciences, Department of Basic Science and Environment, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark article info Article history: Received 28 September 2009 Keywords: Biodegradation Microbial ecology Phenoxy acid Mineralization Bacterial degraders abstract 31 different bacterial strains isolated using the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, were investigated for their ability to mineralize 2,4-D and the related herbicide 4- chloro-2-methylphenoxyacetic acid (MCPA). Most of the strains mineralize 2,4-D considerably faster than MCPA. Three novel primer sets were developed enabling amplification of full-length coding sequences (CDS) of the three known tfdA gene classes known to be involved in phenoxy acid degradation. 16S rRNA genes were also sequenced; and in order to investigate possible linkage between tfdA gene classes and bacterial species, tfdA and 16S rRNA gene phylogeny was compared. Three distinctly different classes of tfdA genes were observed, with class I tfdA sequences further partitioned into the two sub-classes I-a and I-b based on more subtle differences. Comparison of phylogenies derived from 16S rRNA gene sequences and tfdA gene sequences revealed that most class II tfdA genes were encoded by Burkholderia sp., while class I-a, I-b and III genes were found in a more diverse array of bacteria. & 2010 Elsevier GmbH. All rights reserved. Introduction Bacterial degradation of the phenoxyacetic acid herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) has been intensively studied, and pure cultures of 2,4-D degrading bacteria have been isolated from numerous locations worldwide [1–3]. The similar compound 4-chloro-2-methylphenoxyacetic acid (MCPA) has been less studied, and to our knowledge no bacterial degraders has been isolated based on this compound. Despite structural similarity between MCPA and 2,4-D, recent studies on expression of the functional gene tfdA (involved in the first step of degradation of both compounds) during degradation of the compounds in situ in soil, have revealed that marked differences in functional diversity exist between the degrading populations [4,5]. Most of the organisms that are able to degrade 2,4-D have been isolated from agricultural fields or other locations previously treated with 2,4-D [3], but also few 2,4-D degrading organisms have been isolated from more pristine areas as well [1,6]. Typically, isolates from 2,4-D-naı¨ve locations belong to the group of a-proteobacteria, while organisms isolated from sites with historical exposure typically belong to b- and g-proteobacteria [6]. Even though the first step in the degradation of MCPA and 2,4-D is the same, only few of the strains isolated on 2,4-D have been investigated for their ability to degrade MCPA [23]. Based on uniqueness investigated by repetitive extragenic palindromic PCR (REP–PCR) (data not shown), we chose 31 strains from a collection of four novel and 46 previously described 2,4-D degraders and investigated their abilities towards mineralization of 2,4-D and MCPA (for details on isolation strategy and origin of the novel degraders see S1). This experiment was performed by addition of similar cell numbers of the individual strains to mineral salt medium containing 2.3 mM of the respective phenoxy acid. Mineralization was quantified by radiorespirometry (for further details see S1). Most of the strains mineralized Z40% of the applied 2,4-D relatively rapidly, typically within 5 days, while Z40% of the applied MCPA was mineralized much slower (Table 1). Only strains TFD44, TFD47, TFD48, and TFD49 required more than 5 days to mineralize 2,4-D, while only strains TFD34, TFD36, TFD38, and C. necator JMP134 mineralized MCPA in less than 35 days. Thus, all of the strains except strain TFD38 (which mineralized either compound in 5 days) exhibited a strong preference towards 2,4-D as substrate. ARTICLE IN PRESS Contents lists available at ScienceDirect journal homepage: www.elsevier.de/syapm Systematic and Applied Microbiology 0723-2020/$ - see front matter & 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2010.01.001 Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; MCPA, 4-chloro-2-methyl- phenoxyacetic acid; CDS, coding sequence; a-KG, a-ketoglutarate; bp, base pair n Corresponding author at: The Geological Survey of Denmark and Greenland, Department of Geochemistry, Øster Voldgade 10, DK-1350 Copenhagen K, Denmark. Tel.: + 45 3814 2313. E-mail address: csj@geus.dk (C.S. Jacobsen). Systematic and Applied Microbiology 33 (2010) 67–70