Novel 2,4-Dichlorophenoxyacetic Acid Degradation Genes from Oligotrophic Bradyrhizobium sp. Strain HW13 Isolated from a Pristine Environment

Abstract

The tfd genes of Ralstonia eutropha JMP134 are the only well-characterized set of genes responsible for 2,4-dichlorophenoxyacetic acid (2,4-D) degradation among 2,4-D-degrading bacteria. A new family of 2,4-D degradation genes, cadRABKC, was cloned and characterized from Bradyrhizobium sp. strain HW13, a strain that was isolated from a buried Hawaiian soil that has never experienced anthropogenic chemicals. The cadR gene was inferred to encode an AraC/XylS type of transcriptional regulator from its deduced amino acid sequence. The cadABC genes were predicted to encode 2,4-D oxygenase subunits from their deduced amino acid sequences that showed 46, 44, and 37% identities with the TftA and TftB subunits of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) oxygenase of Burkholderia cepacia AC1100 and with a putative ferredoxin, ThcC, of 2,4-Dichlorophenoxyacetic acid (2,4-D) is a manufactured herbicide that has been widely used for the control of broadleaf weeds since its introduction in the 1940s. Many 2,4-Ddegrading microorganisms have been isolated from agricultural, urban, and industrial soils and sediments (2,3,9,22,30,50), and the catabolic pathway of 2,4-D mineralization in Ralstonia eutropha JMP134 has been extensively characterized (8-10, 14, 19, 25, 26, 32-35, 38, 41-43, 48). In JMP134, 2,4-D is transformed to 2,4-dichlorophenol (2,4-DCP) by ␣-ketoglutarate-dependent 2,4-D dioxygenase encoded by tfdA, and 2,4-DCP is subsequently hydroxylated by 2,4-DCP hydroxylase encoded by tfdB to form 3,5-dichlorocatechol (3,5-DCC). 3,5-DCC is further metabolized through an intradiol ring cleavage pathway encoded by tfdCDEF (Fig. 1). These genes are located on plasmid pJP4.Most 2,4-D-degrading bacteria isolated from human-disturbed sites contain tfdA gene homologs. They include various copiotrophic, fast-growing genera in the ␤ and ␥ subdivisions of the Proteobacteria and have been classified as class I 2,4-D degraders (24). Ka et al. reported another group of 2,4-D degraders (class II) that were also isolated from disturbed sites but have neither tfdA gene homologs nor ␣-ketoglutarate-dependent 2,4-D dioxygenase activity (21-23). This group is composed of copiotrophic, fast-growing strains in the ␣ subdivision of the Proteobacteria, mostly belonging to the genus Sphingomonas. Fulthorpe et al. (17) and Kamagata et al. (24) isolated 2,4-D degraders from noncontaminated, pristine soils, degraders which have neither tfdA gene homologs nor ␣-ketoglutarate-dependent 2,4-D dioxygenase activity and, in contrast to those of the other two classes, grow slowly. This group of 2,4-D degraders (class III) is affiliated with the Bradyrhizobium-Agromyces-Nitrobacter-Afipia cluster (A. Saitou, H. Mitsui, and T. Hattori, Abstr. 11th Meet. Jpn. Soc. Microb. Ecol., p. 26, 1995) of oligotrophic bacteria in the ␣ subdivision of the Proteobacteria. The existence of three distinct ecological and genetic classes of 2,4-D degraders indicates a diversity of 2,4-D degradation genes and perhaps of pathways among 2,4-D degraders. However, the 2,4-D ...

Direct analysis of tfdA gene expression by indigenous bacteria in phenoxy acid amended agricultural soil

“…Functional diversity within 2,4-D-degrading isolates has been studied in great detail (Fulthorpe et al, 1995;Vallaeys et al, 1996;Kamagata et al, 1998;Kitagawa et al, 2002), but information on dynamics and diversity among the potential degraders (that is, organisms encoding tfdA and tfdA-like genes) and the active degraders (that is, organisms actually expressing tfdA or tfdA-like genes) in situ in soil is still rather scarce. Baelum et al (2006) studied growth and functional diversity among potential degraders in one soil.…”

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

“…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. 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).…”

“…MCPA degradation in soil is mainly biotic, and MCPAdegrading bacteria are ubiquitous in soil (Oh et al, 1995). Microorganisms that utilize and degrade MCPA have been isolated from different environmental samples and are aerobes mainly of the Alpha-, Beta-and Gammaproteobacteria (Bollag et al, 1967;Mü ller and Babel, 1999;Marriott et al, 2000;Kleinsteuber et al, 2001;Kitagawa et al, 2002;Hoffmann et al, 2003;Macur et al, 2007).…”

“…The first step in the degradation of MCPA is initiated by oxygenases encoded by cadAB or tfdA-like genes (that is, tfdA and tfdAa; Streber et al, 1987;Itoh et al, 2002Itoh et al, , 2004Kitagawa et al, 2002). Such oxygenases cleave the ether-bonded acetate side chain of MCPA to produce 2-methyl-4-chlorophenol (MCP) (Fukumori and Hausinger, 1993a, b;McGowan et al, 1998).…”

A Quinoline Antibiotic from Rhodococcus erythropolis JCM 6824

“…erythropolis JCM 6824 was cultured in W-minimal medium [2] supplemented with succinate (0.2%, w/v), sucrose (0.2%, w/v), and casamino acids (0.2%, w/v). Stock seed cultures (10 6 cfu/ml) were maintained at Ϫ80°C in 10% glycerol.…”