Arch Microbiol (1992) 158:302-308
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-uponTyne, 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 carbamate 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 degradation of carbofuran phenol. Key words: Pesticide catabolism - Soil bacterium Catabolic plasmid - Carbofuran - Carbamate insecticide
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 probelms 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; C F P - , 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 Bactopeptone. YPCF as above but with the addition of 1 mM carbofuran. EPTC, S-ethyl-N,N-dipropylthiocarbamate.2,4-D, 2,4-dichlorophenoxyacetlc acid. NAG, N-acetylglucosamine. 3-HB, 3-hydroxybutyrate
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 degradation 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 recognized (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, W M l l l which was isolated using plasmid-assisted molecular breeding (Karns et al. 1986b), has been particularly well characterized. 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 hydrolase activity (Karns et al. 1986a). Subsequent cloning of the hydrolase gene and Southern hybridization of the cloned gene with the original plasmid DNA demonstrated however, that the hydrolase gene was present on the plasmid. A yellow pigmented Gram-negative organism originally 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
CH3NH2+CO2 - ! OH
t CO2 + H20 Fig. 1. Degradation of carbofuran by bacterial isolates. Reaction carried out by Achromobacter sp. WM111 (1). Reactions carried out by strain MS2d (2)
subculture in minimal mineral salts medium containing KH2PO4, lg; CaSO4, 0.05 g; MgC12 96 H20, 0.05 g; (NH4)2SO4, 0.1 g; trace elements (Barnett and Ingrams 1955), 1 ml; dissolved in 800 ml of glass double distilled water, adjusted to pH 6.5 with 5M K O H and made up to 1 1 with double distilled water. Carbofuran was added to sterile minimal medium as the sole source of carbon and energy
Table 1. Bacterial strains and plasmids
to a final concentration of 0.25 mM. For detection and isolation of plasmids strain MS2d was grown in MS containing 5 g yeast extract, 5 g Bactopeptone and 1 mM carbofuran dissolved in 1 litre of distilled water (YPCF medium). All media were steam sterilised (121 ~ 103 kPa), and carbofuran was added to warm (45 ~ medium from a concentrated filter-sterilised stock solution containing 10 mg carbofuran dissolved in 1 ml acetone. All cultures were incubated at 25 ~ All chemicals used were purchased from BDH (Lutterworth, UK) unless otherwise stated and were of analytical grade. Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl N-methylcarbamate) (99% pure) was a gift from Bayer, UK. Carbofuran phenol (2,3-dihydro-2,2-dimethylbenzofuran-7-ol) was prepared by alkaline hydrolysis of carbofuran in an aqueous solution, followed by acidification and extraction into dichloromethane which was evaporated to dryness under vacuum and the residue weighed, redissolved in acetone to a final concentration of 10 mg carbofuran phenol/ml and analysed by H P L C to ensure complete conversion to carbofuran phenol. Carbofuran and carbofuran phenol concentrations in culture supernatants were determined by H P L C (Chapman et al. 1985) with detection by measurement of absorption at 200 nm. When a large number of strains were to be tested for carbofuran hydrolase activity a colorimetric procedure for the detection of carbofuran phenol was used (Chapman et al. 1985). Where appropriate, the results were confirmed by H P L C analysis. The carbofuran degrading isolate was characterized microscopically, biochemically using an API 20NE strip and by analysis of cellular fatty acids, respiratory quinone content and the nature of the pigments produced by strain MS2d. Quinone analysis was done according to Minnikin et al. (1984) and cellular fatty acids were extracted and analysed by capillary gas chromatography (Gillan 1983) using the methods of Embley et al. (1987) and Minnikin et al. (t980). Pigments were extracted and analysed using the procedures recommended by Reichenbach et al. (1980). Plasmids in wild-type, cured and transconjugant strains of strain MS2d were visualised using the miniprep method of Wheatcroft and Williams (1981). Details of electrophoresis conditions are given in the figure legends. In an attempt to induce loss of the carbofuran phenol degrading phenotype or carbofuran hydrolase activity, strain MS2d or its derivatives cured of the ability to catabolise carbofuran phenol, were grown in YP medium containing either 10 to 100mg of ethidium bromide/l, 1 to 5 mg mitomycin c/l or 10 to 100 mg rifampicin/1. Serial dilutions of the cultures containing the highest concentration of curing agent which permitted growth were plated out on Y P C F agar. Colonies developing after several days incubation were replica-plated on to minimal carbofuran agar (containing 2 m M carbofuran) and colonies from the master plate which failed to grow on the minimal medium were streaked out on fresh Y P C F agar and their ability to hydrolyse carbofuran or degrade carbofuran phenol was tested in liquid minimal medium and liquid YP medium to ensure that inability to grow at the expense of the chemicals was not due to auxotrophy. In addition, growth of strain MS2d in YP medium with no added carbofuran was used as a curing procedure. Filter matings were carried out using the procedure of Simon et al. (1986).
Plasmids and comments
pIH1 to pIH7 Cured derivative of MS2d lacking pIH3 Transconjugant strains obtained by mating MS2d and MS2dC
CFH +, C F P +, RiP CFH +, C F P - , Rig
Head et al. 1988 This study
C F H § CFP § Rig
TC1 TC14 TC28
Abbreviations: Rift, Rifampicin resistant; RiP, Rifampicin sensitive; CFH +, carbofuran hydrolase activity; C F H - , no carbofuran hydrolase activity; C F P +, ability to catabolise carbofuran phenol; C F P - , inability to catabolise carbofuran phenol
cellular pigments were not flexirubins since they did not undergo bathochromic shifts in absorbance maxima and ring-labelled 14C tyrosine was not incorporated into the pigment molecules. Analysis of the cellular fatty acids of strain MS2d revealed predominantly even-numbered unbranched fatty acids (Table 2), unlike bacteria of the FlavobacteriumCytophaga complex which contain largely C15 and C17 fatty acids with significant amounts of branched and hydroxyfatty acids (Oyaizu and Komagata 1981; Holmes et al. 1984). These observations indicate that strain MS2d cannot be reasonably accommodated in the genus Flavobacterium or indeed any described genus of yellowpigmented non-fruiting gliding bacteria.
Characterization of strain MS2d Strain MS2d was originally isolated from a field soil exhibiting enhanced degradation of the insecticide carbofuran (Head et al. 1988). The non-motile, yellow-pigmented, gram-negative rod (5 to 15 pro) was catalase- and oxidase-positive and gave negative results for all the tests included on an API 20NE strip (Table 2). Additional tests showed that the strain could grow at the expense of 3-hydroxybutyrate but could not hydrolyse cellulose, cellobiose, chitin or pectin, and the organism did not show gliding motility in chamber cultures as described by Reichenbach and Dworkin (1981). These results prompted its original identification as a Flavobacterium sp. (Head 1990; Holmes et al. 1984; Weeks 1974). Further investigation revealed that strain MS2d contained only ubiquinones with no menaquinones being detected, a feature inconsistent with inclusion in the genus Flavobacterium (Table 2). It was also found that the Table 2. Characteristics of strain M S 2 d
Degradation of carbofuran by strain MS2d The rate of carbofuran degradation by strain MS2d was comparable to that observed in other carbofuran degrading bacteria Karns et al. 1986b; Chaudhry and All
A. Morphology and biochemical characteristics G r a m stain Oxidase Urease Arginine Dehydrolase
Motiliy" NO3 reduction /~-Galactosidase Aesculin Hydrolysis
C a r b o n source assimilation Glucose Mannitol Gluconate Cellobiose Caprate Citrate Phenylacetate
Arabinose NAG Rhamnose Cellulose Adipate Succinate Methylamine
Mannose Maltose Pectin Chitin Malate 3-HB
O t h e r characteristics Ubiquinones
B. Major cellular fatty acids present in strain MS2d R e t e n t i o n time (rain) b
% of total
14.99 • c 26.22• 26,54 • 0.023 29.06 • 0.026 29.21 • 0.021
11.982 15.744 15.857 15.972 16.685
12.000 15.758 15.856 16.000 16.686
12:0 16:1A9 16:lAll 16:0 18:1A9
27,4 23s 4.2 6,2 32,7
a G l i d i n g motility as d e t e r m i n e d in c h a m b e r cultures a n d motility in h a n g i n g d r o p preparations b F a t t y acid m e t h y l esters ( F A M E s ) extracted from strain M S 2 d were analysed u s i n g a P e r k i n - E l m e r 8240 Series gas c h r o m a t o g r a p h fitted with a n OV-1 fused silica capillary c o l u m n a n d flame ionization detector. T h e following t e m p e r a t u r e p r o g r a m m e was used for analyses: injection at 100 ~ h e a t e d to 240 ~ at 5 ~ a n d held at 240 ~ for 10 min. Eluted p e a k s a n d their relative areas were quantified u s i n g a Trio c o m p u t i n g integrator (Trivector Systems) C a l c u l a t e d equivalent c h a i n length (ECLc,~c) values were d e t e r m i n e d by the m e t h o d of GiUan (1983) ECLIit a n d FAMEI~t values were o b t a i n e d f r o m the s a m e source a n d used to identify t h e fatty acids present in strain MS2d. D a t a are the m e a n of three separate analyses a n d are p r e s e n t e d as the m e a n _+ s t a n d a r d deviation. Abbreviations: N A G , N - a c e t y l g l u c o s a m i n e . 3-HB, 3 - h y d r o x y b u t y r a t e
Fig. 2. Metabolism of carbofuran by wild-type, cured and transconjugant strains of strain MS2d. MS medium containing carbofuran (0.25 mM) was inoculated with wild-type strain MS2d (11); a cured derivative, MS2dC (A) or a transeonjugant strain, TC1, (e) obtained by mating MS2d with MS2dC; carbofuran phenol accumulated in flasks moculated with strain MS2dC (A); carbofuran an unmoculated flasks (). Carbofuran and carbofuran phenol concentrations were determined by HPLC. Data are the mean of three replicate samples. Deviations from the mean of less than 5% were omitted
at the expense of carbofuran from wild-type MS2d to rifampicin resistant derivatives of cured strains. Transfer 'frequencies were low (1.84 _+ 0.12 x 10 -7 transconjugants per recipient) but greater than the frequency of spontaneous rifampicin resistant donor cells on mineral salts agar with carbofuran provided as the sole source of carbon and energy and containing 100 mg rifampicin/1, or the reversion of cured strains to wild-type phenotype. Neither of these events were ever detected but rifampicin resistant mutants of MS2d could be obtained on YP agar, with added rifampicin, at a frequency of 1.70 +_ 0.32 x 10 -8. The ability to grow on carbofuran phenol could not be transferred to plasmid-free Escherichia coli JM83 or Pseudomonas putida PAW130 (data not shown). Degradation of carbofuran by wild-type, cured and transconjugant derivatives of strain MS2d, which regained the ability to degrade the insecticide without accumulation of carbofuran phenol, is shown in Fig. 2. Carbofuran hydrolase ( C F H +) activity was never lost as a result of any of the curing procedures used. The patterns of loss and transfer of carbofuran phenol degrading phenotype (CFP +) suggested plasmid involvement in metabolism of the insecticide. When lysates of wild-type, cured and transconjugant strains were examined for the presence of extrachromosomal DNA, complex plasmid patterns were revealed. One plasmid,
1988). Strain MS2d was capable of completely degrading carbofuran provided at a concentration of 0.25 m M within 48 h, without accumulation of carbofuran phenol or any other metabolites detectable by ultraviolet absorption between 200 and 300 nm (Fig. 2). When higher concentrations of carbofuran (2 raM) were provided in the culture medium, red-pigmented metabolites were released into the culture supernatant. This has been noted in other cultures of carbofuran-degrading bacteria (Venkateswarlu and Sethunathan 1984).
Association of plasmid plH3 with metabolism of carbofuran phenol When examined for the presence of plasmid D N A strain MS2d was found to harbour a large number of plasmid species (Fig. 3A). Derivatives of strain MS2d incapable of growth on carbofuran as sole carbon and energy source were readily obtained. Simply growing the bacterium in YP medium, in the absence of carbofuran, until the culture reached stationary growth phase (48 to 60 h) resulted in approximately 70% (67 + 7.3%) of the cells losing the ability to completely degrade the carbofuran molecule. These cured strains did, however, retain carbofuran hydrolase activity, It proved possible to transfer the ability to grow
Fig. 3A, B. Plasmid content of wild-type flavobacterium MS2d and derivative strains. A Plasmid content of wild-type strain MS2d. The plasmid DNA was prepared by the method of Wheatcroft and Williams (1981) and purified on a caesium chloride gradient. Electrophoresis was at 250 rnA for 3 h in a 0.6% agarose gel in TAE. B Plasmid content of: lane 1, wild-type strain MS2d (CFH +, CFP+); lane 2, strain MS2dC (CFH +, CFP-) a cured derivative of strain MS2d; lanes 3-5, transconjugant stratus (CFH +, CFP +) obtained by mating MS2d and MS2dC; lane 3, TC1; lane 4, TC14; lane 5, TC28. Electrophoresis was carried out at 40 mA for 16 h in a 0.6% agarose gel in TAE (Tris-acetate, 40 raM, EDTA, 2 mM, pH 8.0) Plasmids pIH4 and pIH5 were not resolved under these electrophoresis conditions and appear as a diffuse band below plH3 ([). Contaminating chromosomal DNA is indicated (I-)
306 designated pIH3, was, however,aiways present in strains capable of totally catabolizing carbofuran and always absent in strains unable to degrade carbofuran phenol (Fig. 3). Plasmid pIH3 was larger than plasmid pAtc58 (199 kb) from Agrobacterium tumefaciens C58 (Head 1990) and an accurate size determination was not possible.
Strain MS2d, resembled members of the genus Flavobacterium on the basis of biochemical tests. However, more detailed investigations revealed that the bacterium did not belong in that genus and was also not a yellow-pigmented gliding bacterium. Flexirubin-type pigments and menaquinones characteristic of the genus Flavobacterium and several genera of gliding bacteria (Reichenbach, et al. 1981) were not detected in strain MS2d. The recently amended genus Flavobacterium (Holmes et al. 1984) contains only organisms with flexirubins and menaquinones but none with ubiquinones. In addition, the composition of the cellular fatty acids of strain MS2d precluded its identification as a Flavobacterium sp. On the basis of these data, strain MS2d could not be assigned to any of the commonly encountered taxa of aerobic, yellow-pigmented bacteria. Bacteria with fatty acid profiles and quinone content similar to strain MS2d were previously assigned to the genus Flavobacterium (Reichenbach and Weeks 1981; Weeks 1974). These strains had high % G + C and have been shown by D N A : rRNA hybridization experiments to be unrelated to genuine Flavobacterium spp. or Cytophaga spp. (Bauwens and DeLey 1981; Fautz et al. 1981). One of these strains, Flavobacterium devorans, has now been reclassified as Pseudomonas paucimobilis and others too have been shown to have affiliations with the genus Pseudomonas. Using the ultra-violet light sensitivity method of McMeekin (1977) strain MS2d was estimated to have a low % G + C compared to Pseudomonas spp. This coupled with the limited range of substrates utilized by strain MS2d and its lack of motility suggested, however, that the carbofuran-catabolizing isolate was not a pseudomonad. Similar limited substrate utilization has been noted previously with a pyrazon-degrading bacterium, which in common with strain MS2d, had eluded classification (Eberspficher and Lingens 1981) until 16S rRNA based analyses indicated the organism was a member of the alpha-subdivision of the proteobacteria, but not closely related to other species belonging to the alphasubdivision and has been designated Phenylobacterium immobile (Ludwig et aI. 1984). Though strain MS2d is unlikely to be related to this organism it does illustrate the point made by several investigators that isolates obtained from terrestrial and aquatic environments may be difficult to classify using standard methodologies and often do not fall into well-defined taxa (Holmes et al. 1984; Reichenbach and Weeks 1981). Until more sophisticated taxonomic analyses such as 16S rRNA cataloguing or sequencing can be carried out it was considered
reasonable that strain MS2d should not correctly be placed in any genus for reason of convenience alone. A number of isolates capable of transforming carbofuran have been isolated, and several taxa including Streptomyces (Venkateswarlu and Sethunathan 1984), Arthrobacter (Ramanand etal. 1988), Achromobacter (Karns et al. 1986b), Pseudomonas and Flavobacterium (Chaudhry and Ali 1988) are represented. Not all of these strains degraded the insecticide rapidly; some required up to 40 days to mineralize the compound completely. The extent to which they transformed the molecule also varied and on this basis three groups could be defined: isolates which hydrolyzed the carbamate ester and used the methylamine produced as a nitrogen source; those which catalyzed the same transformation but utilised methylamine as a carbon source; strains which hydrolyzed the carbamate side chain but catabolized the carbofuran phenol produced. Strain MS2d fell into the last of these groups. Experiments with ~4C furan ring-labelled carbofuran demonstrated that carbofuran was converted to ~4CO2, cellular material and soluble products. Ortho- and meta-cleavage activities could not be detected in strain MS2d and the mechanisms of carbofuran phenol catabolism remain unclear (Head 1990). In addition to the observation that carbofuran phenol degradation is mediated by a plasmid, initial evidence that a carbofuran hydrolase gene is present on a second 79 kb plasmid, pIH4, present in strain MS2d has also been obtained (Head 1990). Transposon mutagenesis of strain MS2d using Tn5-mob (Simon, 1984) resulted in the isolation of mutants lacking carbofuran hydrolase activity (CFH-). Southern hybridisation studies have shown the transposon insert to be present on plasmid pIH4. The evidence, however, is not conclusive. The C F H + phenotype could not be cured even by prolonged growth in the presence of rifampicin, ethidium bromide or mitomycin c. It was also not possible to transfer carbofuran hydrolase activity to a number of Escheriehia coli or Pseudomonas strains, though lack of expression of the gene in an alternative host could conceivably account for this observation. Tomasek and Karns (1989) have previously noted that the carbofuran hydrolase gene from Achromobacter sp. WM111 is only poorly expressed in host strains other than that from which the gene was isolated. Further the possibility that a transposon insertion in a chromosomally located gene has caused loss of the phenotype with a second copy of the transposon being present on the plasmid has not been resolved. The transposon also caused substantial rearrangements in the plasmid genome of strain MS2d, though plasmid pIH4 was never obviously affected by this, making it difficult to associate the carbofuran hydrolase phenotype unequivocally with a specific plasmid. Plasmid involvement in the catabolism of xenobiotic chemicals, including pesticides, has been extensively documented. Several of the pesticides known to be susceptible to enhanced degradation in soil are degraded via plasmid-encoded metabolic routes. Catabolism of EPTC (Tam et al. 1987), 2,4-D (Pemberton and Fisher 1977) and organophosphortus compounds (Serdar et al.
307 1982), all of which have proved subject to enhanced degradation under field conditions (Felsot 1989; Suett and Walker 1988; Roeth 1986; Walker and Suett 1986) are now known to be mediated by plasmid-encoded catabolic sequences. With the advent o f practical problems, in which spread o f catabolic plasmids may be o f prime importance, it is important that the mechanisms controlling plasmid spread in the environment are elucidated. This study reveals the role of catabolic plasmids in the degradation of the insecticide carbofuran, one o f the c o m p o u n d s causing considerable concern as a result of its enhanced degradation. Another report has also demonstrated plasmid involvement in the degradation of carbofuran, though in this case it was carbofuran hydrolysis by an Achromobacter sp. which was mediated by the plasmidencoded gene (Tomasek and Karns 1989). With evidence that genes required for carbofuran catabolism in strain MS2d are plasmid-borne, cloning and sequencing of the carbofuran catabolic genes may allow oligonucleotide probes specific for the gene to be developped. This could be used to plot the development and spread of catabolic activities resulting in enhanced carbofuran degradation in the field, and the methodologies required to make these studies practical have already been developed (Holben et al. 1988; Steffan and Atlas 1988). The ultimate aim of this approach would be the development of means to prevent the development or spread of enhanced degradation before its effects are manifested in significant reductions in crop yields and commercial profits in the farming community. Acknowledgements. We are grateful to Dr. H.J. Gilbert for invaluable advice and assistance and many critical discussions. We are also indebted to Dr. A.J. McCarthy and Dr. J. R. Saunders for helpful comments and revision of the original manuscript. This work was funded by a SERC-CASE studentship awarded to RBC and DLS.
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