Downloaded from www.microbiologyresearch.org by IP: 23.22.24.125 On: Thu, 25 Feb 2016 12:53:25 Microbiology (2002), 148, 2883–2888 Printed in Great Britain Assimilatory detoxification of herbicides by Delftia acidovorans MC1 : induction of two chlorocatechol 1,2-dioxygenases as a response to chemostress Dirk Benndorf and Wolfgang Babel Author for correspondence : Wolfgang Babel. Tel : 49 341 235 2225. Fax: 49 341 235 2247. e-mail : babelumb.ufz.de UFZ – Centre for Environmental Research Leipzig-Halle, Department of Environmental Microbiology, PF 500135, 04301 Leipzig, Germany Proteome analysis of bacteria that can detoxify harmful organic compounds enables the discovery of enzymes involved in the biodegradation of these substances and proteins that protect the cell against poisoning. Exposure of Delftia acidovorans MC1 to 2,4-dichlorophenoxypropionic acid and its metabolites 2,4-dichlorophenol and 3,5-dichlorocatechol during growth on pyruvate as a source of carbon and energy induced several proteins. Contrary to the general hypothesis that lipophilic or reactive compounds induce heat shock or oxidative stress proteins, no induction of the GroEL, DnaK and AhpC proteins that were used as markers for the induction of heat shock and oxidative stress responses was observed. However, two chlorocatechol 1,2-dioxygenases, identified by amino terminal sequence analysis, were induced. Both enzymes catalyse the conversion of 3,5-dichlorocatechol to 2,4-dichloro-cis,cis-muconate indicating that biodegradation is a major mechanism of resistance in the detoxifying bacterium D. acidovorans MC1. Keywords : resistance mechanism, degradation, chlorophenoxy herbicides, 2D-PAGE INTRODUCTION The herbicide 2-(2,4-dichlorophenoxy)propionic acid is used in crop control. The biodegradation of chloro- phenoxy acid herbicides by microbial communities was first observed several years ago (Duxbury et al., 1970; Evans et al., 1971 ; Pemperton & Fisher, 1977 ; Kilpi, 1980). Since then, various species have been isolated which are able to use at least one of these compounds as the sole source of carbon and energy (Pieper et al., 1988 ; Horvath et al., 1990). The enzymes and the corre- sponding genes involved in the biodegradation pathway have been well described in some species (Fukumori & Hausinger, 1993 ; Kaphammer et al., 1990). The first step of biodegradation is often catalysed by a 2- oxoglutarate dependent dioxygenase, which is encoded by the gene tfdA. The tfdBCDE genes which, like tfdA, ................................................................................................................................................. Abbreviations : 3,5-DCC, 3,5-dichlorocatechol ; 2,4-DCP, 2,4-dichloro- phenol ; 2,4-DCPP, 2,4-dichlorophenoxypropionic acid ; IPG, immobilized pH gradient. The SWISS-PROT accession numbers for the sequences reported in this paper are P83115, P83116 and P83117. belong to the tfd gene cluster, catalyse ortho-cleavage of the aromatic ring of the first metabolite dichlorophenol and the following dechlorination. However, this knowl- edge alone seems to be insufficient to explain the low conversion rates of chlorophenoxy acids observed at polluted sites. Therefore, current research is focused on determining the effects of unfavourable environmental factors on the biodegradation rate and on discovering mechanisms that stabilize the biocatalysts. One un- favourable factor could be growth on 2,4-dichlorophen- oxyacetic acid (2,4-D) itself, which causes the depletion of ATP (Mu  ller et al., 1997). In addition, the presence of 2,4-D induces the synthesis of the heat-shock proteins DnaK and GroEL in Burkholderia sp. YK-2 (Cho et al., 2000), indicating the importance of stress proteins for adaptation. These results are consistent with the estab- lished fact that detoxifying bacteria respond to chemo- stress by inducing stress proteins (Lupi et al., 1995; Uchiyama et al., 1999), as do many other bacteria (Blom et al., 1992 ; van Dyk et al., 1994). Previous studies have shown heat-shock proteins to be induced by a variety of mainly hydrophobic compounds and these chaperones may defend cells against the toxic effects of such compounds (Benndorf et al., 1999). An example of an 0002-5387  2002 SGM 2883