- Email: [email protected]
Horizontal transfer of bacterial heavy metal resistance genes and its applications in activated sludge systems
~ Pergamon
Wal. Sci. T~ch. Vol. 37, No. 4-5. pp. 465-468, 1998. iC> 1998 IAWQ. Published by Elsevier Science lid Prinled in Oreal Ontain.
PH: S0273-1223(98)OO147-4
0273-1223198 $19'00 + 0-00
HORIZONTAL TRANSFER OF BACTERIAL HEAVY METAL RESISTANCE GENES AND ITS APPLICAnONS IN ACTIVATED SLUDGE SYSTEMS Qinghan Dong, Dirk Springeal, Jef Schoeters, Gust Nuyts, Max Mergeay I and Ludo Diels V1aamse Inste/ling voor Technologisch Ondenoek (VITO), Environmental Technology, Boeretang 200, B-24oo Mol. Belgium
ABSTRACf The bacterial nickel (Ni) resistance determinant ncc-nre of Alcaligenes 31A strain cloned to an IncQ broad• hosl-range plasmid pKT240 gave nse to pMOL222. The plasmid was subsequently mobilized into various Eubacteria and found to confer an increased Ni resistance on these recipients. An increase of Ni resistance was also observed after the transfer of pMOL222 into activated sludge bacteria by plate mating. The dissemination of pMOL222 into an activated sludge pilot stabilized the system during a heavy metal shock loading wilh 0.25 mM Ni. © 1998IAWQ. Published by ElseVier Science Ltd
KEYWORDS Activated sludge; gene transfer; heavy metal resistance. INTRODUCfION Many bioremediation systems, such as activated sludge systems in wastewater treatment, are based on the use of natural associations of microorganisms. When shock loads of toxic components such as heavy metals or xenobiotics occur. the activity of the system may be adversely affected if it is devoid of resistance or degradation mechanisms. Introduction of genetic information for resistance or degradation into bacterial popUlations of these systems would provide a tool for resistance to toxic compounds in bioremediation systems. The bacterial consortia could then react adaptively against the polluting environment by activating gene dissemination. A prototype of adaptation by horizontal gene transfer in bacterial populations is the Iransfer of antibiotic resistance genes among pathogenic bacteria (Courvalin, 1994). We report here on the horizontal transfer and expression of the Ni resistance determinant ncc-nre in various phyla of Eubacteria, including members of a bacterial population from activated sludge. The application of this heterologous transfer 10 a laboratory-scale activated sludge system is described.
I
Corresponding author.
JlI!>T J7:./5-'
465
466
Q. DONG et al.
METHODS Bacterial strains. plasmjds. and construction of pMOL222 All of the strains and plasmids used in this study are listed in Table I. The 14.5 kb Bamffl fragment containing the ncc-nre loci was isolated from a recombinant plasmid pVDZ'2::TBA9 (Schmidt and Schlegel, 1994). The fragment was subsequently cloned into the IncQ plasmid pKT240, derived from RSFIOIO. The resulting hybrid plasmid was named pMOL222. The strain AE2399 wa~ constructed by transferring the broad-host-range mobilizing plasmid pMOL96 into AEI970 containing pMOL222. Mobilization of pMOL222 into the
slud~e
bacterial community on a&ar plate
For the mobilization on agar plates, CM 2034 (E. coli S 17-I/pMOL222) was used as the donor strain. Portions of donors and recipients (sludge samples) at 10 I0 cfu/ml were mixed at a ratio of I: I. The mixture (5m!) was filtered through a 0.2 ~m Millipore paper filter. placed on the surface of 869 agar plates, and incubated for 40 h at 30·C. The transconjugants were selected on Tris minimal media (Mergeay et at., 1985) supplemented either with glucose, gluconate, glycerol, cellobiose, starch. phenol. or benzoate as a carbon source, and with different concentrations between 0.5 and 30 mM of Ni. Table I. Bacterial strains and plasmids BAC'rnRIAL STRAIN E. coli DHiOB CM404 CM43S (SI7-1) CMI962 (011108) CM2034 (817.1) P. pntido PUT7g (P. pI/lido UWC 6) PUT79 A. cutrophus AEgiS AEI970 AE2399 Plosmids pMOL221 pMOl.222 pMOL96 (pES1)
PLASMID
RELEVANTMARI;ERS
REFERENCES
pMOL222 pMOL222
Leu' KIlII Mob' Ira' Mob' Ira' Pro' Nil,Kml Nil,KllI1
OIDCQ..BRL Figurski ., 01. (1979) Simon., 01. (1983) Ulis study Ulis study
pMOL22I
Up' Up'Tc l
Fry J. pcrsonnl comJII. Ulis study
KIlI I Tcl Api Nil
Ri~
Springocl ., 01. (1993) this study
Km l Tc l Api Nil
Uli. study
pVDZ'2::nl»nrc pKT240::ncc-nrc lndigcnons mobilising plasmid of a ncw incollll,nlihilitv ~roup
Schimils.' 01. (1994) Ulis study Top ct oJ. (1994)
pRI<2013
RP4 pMOL222 pMOL222 pMOL96
Actiyated slud~e and synthetjc wastewater samples The sludge samples originated from a municipal wastewater treatment plant at Dessel, Belgium and from a laboratory-scale activated sludge pilot treating synthetic wa~tewater. The synthetic wastewater contained 1% milk powder (Nestl~) and urea (30 mg/I). The COD of the synthetic sewage was about 1100 mg/1. Laboratory-scale activated slud~e system lBjobox) Two flexible laboratory-scale pilot systems named Biobox were set up. A diagram of the pilot system is shown in Fig. I. The aeration vessel, settling vessel and the supports were stainless steel. The active volume of the system was 8 I. The intermittent feeding and continuous sludge recycling were achieved by 2 peristaltic pumps Masterj7.ex (Cole-Parmer Instrument Co., Chicago, IL60648) which were controlled by 2 digital timers according to the desired mode of operation. Aeration was supplied by an aerator module that
Horizontal transfer of bacterial heavy metal resistance genes
467
sparged compressed air from the bottom of the vessel and served to mix the vessel contents. The flow rate was fixed at II I per day which corresponded to a hydraulic retention time (HRT) of 17.5 h. RESULTS AND DISCUSSION Transfer of pMOL222 to Eschericba coli. Pseudomonas puMa. Alcalifenes eutroM us . Sphinfobacterium he,par;num individually The introduction of Ni resistance plasmid pMOL222 to E. Coli DH lOB by electroporation gave rise to the strain CM1962. The parental strain DHIOB has a maximum tolerable concentration (MTC) for Ni < 0.5 mM. while CMI962 has a MTC of 4 mM. The mobilization of the same plasmid from CMI962 to P. putida PUT78. A. eutrophus AE815. or S. heparinum (ATCC 13125) was carried out with the help of CM404. After the plasmid transfer. the maximum tolerable concentration MTC of these host strains on Tris mineral medium increased from < 0.5 mM to I mM in P. putida. from 0.3 mM to 30 mM in A. eutrophus and from < 0.2 mM to 0.8 mM in S. heparinum. The last case also indicated that the ncc-nre plasmid could be mobilized and expressed in phyla that are quite distant from Proteobacteria. such as CytophagalF1avobacteria and Bacteroides.
r;====(IJ:J===:::::;--;========(Vlt)=:==;--, VII
Figure I. Schematic representallon of the BlObox system.
Transfer of pMOL222 into the activated slud&e bacterial community on a&ar plates The transfer and the expression of pMOL222 to an indigenous bacterial population was studied by growing mixed CM2034 and activated sludge bacteria on a filter as described above. The transconjugants were selected by the Ni resistance of the cfu forming population on mineral media with various carbon sources. In total. 45 colonies were further purified on selection media containing glucose. gluconate. phenol. benzoate. cellobiose or starch as carbon source. 19 bacteria were Gram-positive. 20 were Gram-negative and 6 were Gram variable. By using a set of primers corresponding to an ncc sequence. PCR demonstrated that all of these 45 colonies may contain nec-like DNA sequences. Hybridization by using pMOL222 as probe showed that 38 out of 45 isolated strains hosted the unaltered plasmid. Further identification using the Biolog test showed that some of isolated Gram-positive bacteria were related to Arthrobacter. while some of isolated Gram-negative bacteria were identified as Comamonas, Pseudomonas, or Alcaligenes/Ralstonia. Our study demonstrated for the first time that the heavy metal resistance marker could be transferred and expressed not only within Proteobacteria but also in Flavobacteria and in Gram-positive bacteria. Enhancement of the adaptive capability of activated slud&e facin& heaVY metal influx by &ene dissemjnal!on IblOau&mentationl in a pjlot system Two laboratory-scale activated sludge pilots (pilot L and pilot R) fed with the synthetic wastewater were set up a~ de~cribed above. The addition of 100 ml of AE2399 at 00 = 1.0 to the pilot L occurred on Day 0, (the final den~ity i~ about 107/ml in reactor). before the introduction of synthetic wastewater containing 0.25 mM
468
Q.
DONG et al.
Ni which occurred on Day I for both pilots. The COD of effluent was monitored and is displayed in Fig. 2. Before introduction of Ni in the influent, the CODeff from the pilot L (bioaugmented) and the control pilot R (non-bioaugmented) was mea~ured at respectively 40.5 mgll and 19.3 mgll. After Ni introduction, the CODeft· from both pilots increased. For the bioaugmented reactor the CODeff increased slightly and returned to its initial values (40-50 mgll) by Day 22. For the control reactor the CODeff was increased gradually and stabilized around 100 mgll by Day 22, leading to a 2-fold increase in CODeff' On Day 22, an incident most likely high loading rate or high Ni concentration occurred. This event resulted in a sudden increase of CODeff to 524 mgll for non-bioaugmented pilot and an increase of CODeff to 131 mgll for the bioaugmented pilot. This unintentional incident indicated once more that bioaugmented activated sludge could provide much stronger resistance to what was probably an abrupt increase in metal concentration. Bloaugmentatlon expo pilots treating synthetic wastewater
600 500
E'
r . 'C :::l
• '0 E
0 0 U
f
-+:,:elOaugm."R1Ot [". __ Non Bioaug. R .-
400
--~-
-
---
I
-- - --
300
,efoaugmenfation]
200
/
100
/
.Niaddition
I
0 0
5
10
15
tlmeiS,YI'
25
30
35
40
Figute 2. CODeff in bioaugmented and non-bioaugmented reactors. The timing for the strain addition and Ni introductions 24 h later is indicated. CONCLUSION This study is the first report of a horizontal transfer of heavy metal resistance genes within a large range of bacterial species. especially between Gram-positive and Gram-negative bacteria. To accomplish this, two characteristics of a horizontal gene transfer have to be joined in a heavy metal resistance locus with a wide expression range and a versatile conjugative system. This "universal" transfer ability based on the of RFS 10 10 was fully displayed not only under laboratory conditions (agar plate mating) as described by Gormley and Davies (1991), but also in the much more harsh conditions of the activated sludge ecosystem where the interaction between microbes is often hostile. In conclusion, the successful dissemination of these genetic materials for bioremediation, such as heavy metal resistance genes. in indigenous microbial populations reveals the potential for horizontal gene transfer in environmental engineering. REFERENCES Courvalin. P. (1994). Transfer of antibiotic resistance genes between gram-positive and gram-negative bacteria. Alltimicrob. AIII'II/s Chemorher.• 38. 1447-1451. Gormley E. P. and Davies J. (1991). Transfer of plasmid RSFIOIO by conjugation from Escherichic coli to Streptomyces lividalls and Ml'cobacterium smegmatis. J. Bacteriol.• 173.6705-6708. Figurski, D. H. and Helinski. D. R. (1979). Replication of an origin-containing derivative of plasmid RK2 dependent on ta plasmid function provided in trans. Proc. Natl. Acad. Sci. USA. 76.1648-1652. Mergeay. M.. Nies, D., Schlegel. H. G.. Gerits, J., Charles. P. and Van Gijsegem. F. (1985). Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J. Bacteriol., 162. 328-334. Schmidt. T. and Schlegel. H. G. (1994). Combined nickel-cobalt-cadmium resistance encoded by the nee locus of Alcaligenes xyloslJxidCllIS 31A. J. Bacteriol.• 176. 7015-7054.
Springael. D.. Diels, L.. Hooyberghs. L .• Kreps. S. and Mergeay, M. (1993). Construction and characterization of heavy metal resistant haloaromatic degrading Alcaligenes eutrophus strains. Appl. Environ. Microbiol.• 59, 334-339. Top. E.. Dc Smet, I.. Verstraete. W.. Dijkmans. R. and Mergeay. M. (1994). Exogenous isolation of mobilizing plasmids from polluted soils and sludges. Appl. Environ. Microbiol., 60, 831-839.
Wal. Sci. T~ch. Vol. 37, No. 4-5. pp. 465-468, 1998. iC> 1998 IAWQ. Published by Elsevier Science lid Prinled in Oreal Ontain.
PH: S0273-1223(98)OO147-4
0273-1223198 $19'00 + 0-00
HORIZONTAL TRANSFER OF BACTERIAL HEAVY METAL RESISTANCE GENES AND ITS APPLICAnONS IN ACTIVATED SLUDGE SYSTEMS Qinghan Dong, Dirk Springeal, Jef Schoeters, Gust Nuyts, Max Mergeay I and Ludo Diels V1aamse Inste/ling voor Technologisch Ondenoek (VITO), Environmental Technology, Boeretang 200, B-24oo Mol. Belgium
ABSTRACf The bacterial nickel (Ni) resistance determinant ncc-nre of Alcaligenes 31A strain cloned to an IncQ broad• hosl-range plasmid pKT240 gave nse to pMOL222. The plasmid was subsequently mobilized into various Eubacteria and found to confer an increased Ni resistance on these recipients. An increase of Ni resistance was also observed after the transfer of pMOL222 into activated sludge bacteria by plate mating. The dissemination of pMOL222 into an activated sludge pilot stabilized the system during a heavy metal shock loading wilh 0.25 mM Ni. © 1998IAWQ. Published by ElseVier Science Ltd
KEYWORDS Activated sludge; gene transfer; heavy metal resistance. INTRODUCfION Many bioremediation systems, such as activated sludge systems in wastewater treatment, are based on the use of natural associations of microorganisms. When shock loads of toxic components such as heavy metals or xenobiotics occur. the activity of the system may be adversely affected if it is devoid of resistance or degradation mechanisms. Introduction of genetic information for resistance or degradation into bacterial popUlations of these systems would provide a tool for resistance to toxic compounds in bioremediation systems. The bacterial consortia could then react adaptively against the polluting environment by activating gene dissemination. A prototype of adaptation by horizontal gene transfer in bacterial populations is the Iransfer of antibiotic resistance genes among pathogenic bacteria (Courvalin, 1994). We report here on the horizontal transfer and expression of the Ni resistance determinant ncc-nre in various phyla of Eubacteria, including members of a bacterial population from activated sludge. The application of this heterologous transfer 10 a laboratory-scale activated sludge system is described.
I
Corresponding author.
JlI!>T J7:./5-'
465
466
Q. DONG et al.
METHODS Bacterial strains. plasmjds. and construction of pMOL222 All of the strains and plasmids used in this study are listed in Table I. The 14.5 kb Bamffl fragment containing the ncc-nre loci was isolated from a recombinant plasmid pVDZ'2::TBA9 (Schmidt and Schlegel, 1994). The fragment was subsequently cloned into the IncQ plasmid pKT240, derived from RSFIOIO. The resulting hybrid plasmid was named pMOL222. The strain AE2399 wa~ constructed by transferring the broad-host-range mobilizing plasmid pMOL96 into AEI970 containing pMOL222. Mobilization of pMOL222 into the
slud~e
bacterial community on a&ar plate
For the mobilization on agar plates, CM 2034 (E. coli S 17-I/pMOL222) was used as the donor strain. Portions of donors and recipients (sludge samples) at 10 I0 cfu/ml were mixed at a ratio of I: I. The mixture (5m!) was filtered through a 0.2 ~m Millipore paper filter. placed on the surface of 869 agar plates, and incubated for 40 h at 30·C. The transconjugants were selected on Tris minimal media (Mergeay et at., 1985) supplemented either with glucose, gluconate, glycerol, cellobiose, starch. phenol. or benzoate as a carbon source, and with different concentrations between 0.5 and 30 mM of Ni. Table I. Bacterial strains and plasmids BAC'rnRIAL STRAIN E. coli DHiOB CM404 CM43S (SI7-1) CMI962 (011108) CM2034 (817.1) P. pntido PUT7g (P. pI/lido UWC 6) PUT79 A. cutrophus AEgiS AEI970 AE2399 Plosmids pMOL221 pMOl.222 pMOL96 (pES1)
PLASMID
RELEVANTMARI;ERS
REFERENCES
pMOL222 pMOL222
Leu' KIlII Mob' Ira' Mob' Ira' Pro' Nil,Kml Nil,KllI1
OIDCQ..BRL Figurski ., 01. (1979) Simon., 01. (1983) Ulis study Ulis study
pMOL22I
Up' Up'Tc l
Fry J. pcrsonnl comJII. Ulis study
KIlI I Tcl Api Nil
Ri~
Springocl ., 01. (1993) this study
Km l Tc l Api Nil
Uli. study
pVDZ'2::nl»nrc pKT240::ncc-nrc lndigcnons mobilising plasmid of a ncw incollll,nlihilitv ~roup
Schimils.' 01. (1994) Ulis study Top ct oJ. (1994)
pRI<2013
RP4 pMOL222 pMOL222 pMOL96
Actiyated slud~e and synthetjc wastewater samples The sludge samples originated from a municipal wastewater treatment plant at Dessel, Belgium and from a laboratory-scale activated sludge pilot treating synthetic wa~tewater. The synthetic wastewater contained 1% milk powder (Nestl~) and urea (30 mg/I). The COD of the synthetic sewage was about 1100 mg/1. Laboratory-scale activated slud~e system lBjobox) Two flexible laboratory-scale pilot systems named Biobox were set up. A diagram of the pilot system is shown in Fig. I. The aeration vessel, settling vessel and the supports were stainless steel. The active volume of the system was 8 I. The intermittent feeding and continuous sludge recycling were achieved by 2 peristaltic pumps Masterj7.ex (Cole-Parmer Instrument Co., Chicago, IL60648) which were controlled by 2 digital timers according to the desired mode of operation. Aeration was supplied by an aerator module that
Horizontal transfer of bacterial heavy metal resistance genes
467
sparged compressed air from the bottom of the vessel and served to mix the vessel contents. The flow rate was fixed at II I per day which corresponded to a hydraulic retention time (HRT) of 17.5 h. RESULTS AND DISCUSSION Transfer of pMOL222 to Eschericba coli. Pseudomonas puMa. Alcalifenes eutroM us . Sphinfobacterium he,par;num individually The introduction of Ni resistance plasmid pMOL222 to E. Coli DH lOB by electroporation gave rise to the strain CM1962. The parental strain DHIOB has a maximum tolerable concentration (MTC) for Ni < 0.5 mM. while CMI962 has a MTC of 4 mM. The mobilization of the same plasmid from CMI962 to P. putida PUT78. A. eutrophus AE815. or S. heparinum (ATCC 13125) was carried out with the help of CM404. After the plasmid transfer. the maximum tolerable concentration MTC of these host strains on Tris mineral medium increased from < 0.5 mM to I mM in P. putida. from 0.3 mM to 30 mM in A. eutrophus and from < 0.2 mM to 0.8 mM in S. heparinum. The last case also indicated that the ncc-nre plasmid could be mobilized and expressed in phyla that are quite distant from Proteobacteria. such as CytophagalF1avobacteria and Bacteroides.
r;====(IJ:J===:::::;--;========(Vlt)=:==;--, VII
Figure I. Schematic representallon of the BlObox system.
Transfer of pMOL222 into the activated slud&e bacterial community on a&ar plates The transfer and the expression of pMOL222 to an indigenous bacterial population was studied by growing mixed CM2034 and activated sludge bacteria on a filter as described above. The transconjugants were selected by the Ni resistance of the cfu forming population on mineral media with various carbon sources. In total. 45 colonies were further purified on selection media containing glucose. gluconate. phenol. benzoate. cellobiose or starch as carbon source. 19 bacteria were Gram-positive. 20 were Gram-negative and 6 were Gram variable. By using a set of primers corresponding to an ncc sequence. PCR demonstrated that all of these 45 colonies may contain nec-like DNA sequences. Hybridization by using pMOL222 as probe showed that 38 out of 45 isolated strains hosted the unaltered plasmid. Further identification using the Biolog test showed that some of isolated Gram-positive bacteria were related to Arthrobacter. while some of isolated Gram-negative bacteria were identified as Comamonas, Pseudomonas, or Alcaligenes/Ralstonia. Our study demonstrated for the first time that the heavy metal resistance marker could be transferred and expressed not only within Proteobacteria but also in Flavobacteria and in Gram-positive bacteria. Enhancement of the adaptive capability of activated slud&e facin& heaVY metal influx by &ene dissemjnal!on IblOau&mentationl in a pjlot system Two laboratory-scale activated sludge pilots (pilot L and pilot R) fed with the synthetic wastewater were set up a~ de~cribed above. The addition of 100 ml of AE2399 at 00 = 1.0 to the pilot L occurred on Day 0, (the final den~ity i~ about 107/ml in reactor). before the introduction of synthetic wastewater containing 0.25 mM
468
Q.
DONG et al.
Ni which occurred on Day I for both pilots. The COD of effluent was monitored and is displayed in Fig. 2. Before introduction of Ni in the influent, the CODeff from the pilot L (bioaugmented) and the control pilot R (non-bioaugmented) was mea~ured at respectively 40.5 mgll and 19.3 mgll. After Ni introduction, the CODeft· from both pilots increased. For the bioaugmented reactor the CODeff increased slightly and returned to its initial values (40-50 mgll) by Day 22. For the control reactor the CODeff was increased gradually and stabilized around 100 mgll by Day 22, leading to a 2-fold increase in CODeff' On Day 22, an incident most likely high loading rate or high Ni concentration occurred. This event resulted in a sudden increase of CODeff to 524 mgll for non-bioaugmented pilot and an increase of CODeff to 131 mgll for the bioaugmented pilot. This unintentional incident indicated once more that bioaugmented activated sludge could provide much stronger resistance to what was probably an abrupt increase in metal concentration. Bloaugmentatlon expo pilots treating synthetic wastewater
600 500
E'
r . 'C :::l
• '0 E
0 0 U
f
-+:,:elOaugm."R1Ot [". __ Non Bioaug. R .-
400
--~-
-
---
I
-- - --
300
,efoaugmenfation]
200
/
100
/
.Niaddition
I
0 0
5
10
15
tlmeiS,YI'
25
30
35
40
Figute 2. CODeff in bioaugmented and non-bioaugmented reactors. The timing for the strain addition and Ni introductions 24 h later is indicated. CONCLUSION This study is the first report of a horizontal transfer of heavy metal resistance genes within a large range of bacterial species. especially between Gram-positive and Gram-negative bacteria. To accomplish this, two characteristics of a horizontal gene transfer have to be joined in a heavy metal resistance locus with a wide expression range and a versatile conjugative system. This "universal" transfer ability based on the of RFS 10 10 was fully displayed not only under laboratory conditions (agar plate mating) as described by Gormley and Davies (1991), but also in the much more harsh conditions of the activated sludge ecosystem where the interaction between microbes is often hostile. In conclusion, the successful dissemination of these genetic materials for bioremediation, such as heavy metal resistance genes. in indigenous microbial populations reveals the potential for horizontal gene transfer in environmental engineering. REFERENCES Courvalin. P. (1994). Transfer of antibiotic resistance genes between gram-positive and gram-negative bacteria. Alltimicrob. AIII'II/s Chemorher.• 38. 1447-1451. Gormley E. P. and Davies J. (1991). Transfer of plasmid RSFIOIO by conjugation from Escherichic coli to Streptomyces lividalls and Ml'cobacterium smegmatis. J. Bacteriol.• 173.6705-6708. Figurski, D. H. and Helinski. D. R. (1979). Replication of an origin-containing derivative of plasmid RK2 dependent on ta plasmid function provided in trans. Proc. Natl. Acad. Sci. USA. 76.1648-1652. Mergeay. M.. Nies, D., Schlegel. H. G.. Gerits, J., Charles. P. and Van Gijsegem. F. (1985). Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J. Bacteriol., 162. 328-334. Schmidt. T. and Schlegel. H. G. (1994). Combined nickel-cobalt-cadmium resistance encoded by the nee locus of Alcaligenes xyloslJxidCllIS 31A. J. Bacteriol.• 176. 7015-7054.
Springael. D.. Diels, L.. Hooyberghs. L .• Kreps. S. and Mergeay, M. (1993). Construction and characterization of heavy metal resistant haloaromatic degrading Alcaligenes eutrophus strains. Appl. Environ. Microbiol.• 59, 334-339. Top. E.. Dc Smet, I.. Verstraete. W.. Dijkmans. R. and Mergeay. M. (1994). Exogenous isolation of mobilizing plasmids from polluted soils and sludges. Appl. Environ. Microbiol., 60, 831-839.