Arch Microbiol (1992) 158:302-308 Archives of Hicrnldology 9 Springer-Verlag1992 Characterization of a carbofuran-degrading bacterium and investigation of the role of plasmids in catabolism of the insecticide carbofuran Ian M. Head 1, Ronald B. Cain 1, and David L. Suett 2 1 Environmental Microbiology Laboratory, Department of Agricultural and Environmental Sciences, The University, Newcastle-upon- Tyne, NE1 7RU, UK z Horticultural Research International, Wellesbourne, Warwick, CV35 9EF, UK Received December 23, 1991/Accepted May 14, 1992 Abstract. A bacterium capable of using the carbamate insecticide carbofuran as a sole source of carbon and energy, was isolated from soil. The ability to catabolise carbofuran phenol, produced by cleavage of the carb- amate ester linkage of the insecticide, was lost at very high frequency when the bacterium was grown in the absence of carbofuran. Plasmid analyses together with curing and mating experiments indicated that the presence of a large plasmid (pIH3, > 199 kb) was required for the degrada- tion of carbofuran phenol. Key words: Pesticide catabolism - Soil bacterium - Catabolic plasmid - Carbofuran - Carbamate in- secticide In recent years, the performance of several soil-acting pesticides has been compromised by their enhanced degradation in soil. The list of compounds affected by this phenomenon includes insecticides, herbicides and fungicides (Roeth 1986; Walker and Suett 1986; Felsot 1989). With each class of compounds pest control pro- belms have arisen (Rhaman et al. 1979; Felsot et al. 1982; Suett 1987) and bacteria capable of rapidly degrading the problem compound can be isolated from soils where reduced pest control has occurred or rapid dissapearance Correspondence to current address: I.M. Head, Department of Genetics and Microbiology, Life Sciences Building, University of Liverpool, P.O. Box 147, Liverpool, L69 3BX, UK Abbrewations: Rif~, Rifampicin resistant; RiP, Rifampicin sensitive; CFH +, Carbofuran hydrolase activity present; CFH-, Carbofuran hydrolase activity absent; CFP +, ability to degrade carbofuran phenol present; CFP-, ability to degrade carbofnran phenol absent. MS, mineral salts medium. MSCF minimal mineral salts medium containing0.25 mM carbofuran as sole source of carbon and energy. YP, MS medium containing 5 g/1 yeast extract and 5 g/1 Bacto- peptone. YPCF as above but with the addition of 1 mM carbofuran. EPTC, S-ethyl-N,N-dipropylthiocarbamate. 2,4-D, 2,4-dichlorophe- noxyacetlc acid. NAG, N-acetylglucosamine. 3-HB, 3-hydroxybuty- rate of the chemical can be demonstrated (Felsot et al. 1981; Head et al. 1988; Mueller et al. 1988). The involvement of plasmid-encoded catabolic sequences in pesticide de- gradation has been widely decumented (Pemberton and Fisher 1977; Serdar et al. 1982; Mulbry et al. 1986; Tam et al. 1987; Vega et al. 1988), and the potential for spread of plasmid-encoded catabolic genes has long been rec- ognized (Waid 1972). The N-methylcarbamate insecticide carbofuran is used worldwide to control soil-borne insect pests of corn, rice and brassicas. Continued use of the insecticide at single field sites has led to the problem of enhanced degradation and consequent reduced efficacy, and several bacterial isolates capable of transforming the carbofuran molecule to varying degrees have been obtained (Karns et al. 1986b; Chaudhry and Ali 1988; Ramanand et al. 1988; Head 1990). Achromobacter sp, WMlll which was isolated using plasmid-assisted molecular breeding (Karns et al. 1986b), has been particularly well charac- terized. This strain hydrolysed the carbamate side chain of carbofuran, liberating methylamine which served as a nitrogen source for the bacterium (Fig. 1). This strain harboured a large plasmid (> 100 kb) but initially, the plasmid could not be associated with carbofuran hy- drolase activity (Karns et al. 1986a). Subsequent cloning of the hydrolase gene and Southern hybridization of the cloned gene with the original plasmid DNA demon- strated however, that the hydrolase gene was present on the plasmid. A yellow pigmented Gram-negative organism origi- nally assigned to the genus Flavobacterium, was also isolated for its ability to degrade carbofuran (Head et al. 1988) but was obtained by enrichment from a field soil where enhanced degradation was occurring (Suett 1986). Evidence for plasmid involvement in the catabolism of carbofuran phenol, the product of carbofuran hydrolysis is presented for this strain and revision of its taxonomic status is reported. Materials and methods The carbofuran degrading bacterium strain MS2d and its derivatives are listed in Table 1. Strain MS2d was maintained by weekly