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Plasmid content evaluation of activated sludge
Pergamon
0043-1354(94)E0088-N
Wat. Res. Vol. 29, No. I, pp. 371-374, 1995 Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0043-1354/95$7.00+ 0.00
P L A S M I D C O N T E N T E V A L U A T I O N OF A C T I V A T E D SLUDGE P. BAUDAl, C. LALLEMENT1 and J. MANEM2 ICentre des Sciences de l'Environnement, Universit6 de Metz, 1 rue des R6collets 57000 Metz, and 2Centre International de Recherches sur l'Eau et l'Environnement (CIRSEE), 38 rue du Pr6sident Wilson, 78230, Le Pecq, France (First received September 1992; accepted in revised form March 1994)
Abstract--Plasmid content of activated sludge was evaluated by two different and complementary approaches. The first one consisted in the isolation of colonies from activated sludge samples, then isolated colonies were grown in nutrient broth and the plasmids were extracted from these cultures. The plasmid extraction procedure used a commercialized kit and the corresponding protocol. By this first approach, 40% of isolated colonies harboring plasmids were found. The second approach was an adaptation of the plasmid extraction kit directly on activated sludge samples. This direct application required several washing steps of sludges samples to avoid plasmid DNA digestion by DNases adsorbed within the sludge. This direct and rapid technique was validated in terms of reproducibility, specificity for plasmid DNA and losses. It allows the isolation of around 1#g of plasmid DNA per 10tl cells in the sludge. The two approaches presented in this paper indicated that the plasmid content of activated sludge is important and its particular role within the wastewater epuration procedure should be investigated. Key words--activated sludge, plasmids, extraction, quantification
INTRODUCTION Past attempts to improve the degradation of organic matter by biological wastewater treatment, have focused on procedures intended to increase the concentration of biomass or increase biomass activity, but, as yet, have not considered the genetic potential of the biomass. However, persistence of or expression of a gene may often be a limiting factor in the purification of water. The fact that particular compounds are not completely biodegraded or that the biodegradation process does not work in particular conditions may be related in some cases to a lack of genes coding for (a) a specific biodegradative pathway, (b) for resistance to toxic compounds present in the influent, or (c) both. Genetic information is classically located on chromosomal DNA (chr DNA) but genes in bacteria are also located on plasmid DNA (pDNA), which is not as big as chr DNA but has the ability to replicate itself. Well identified plasmid genes are often coded for biodegradation pathways (Sayler et al., 1990), antibiotic or metal resistance (Helinski, 1973), and toxine production (Reeves, 1972). Plasmids are not stable structures and they can be lost (Godwin and Slater, 1979) or transferred to other bacteria by several mechanisms: transformation (Jeffrey et al., 1990), conjugation (Trieu-Cuot et al., 1987), mobilization (Geat et al., 1985; Sandt and Herson, 1991), for a review see Reanney et al., (1983). In these conditions, autochtonous plasmids may have a function in xenobiotic biodegradation within the activated
sludge process. Resident plasmids may also play a role in allochtonous plasmid dissemination introduced voluntarily or inadvertently to the activated sludge. Presently very little research has dealt with plasmid DNA extraction from biomass sampled in activated sludge. Most of the publications concerning plasmid extraction procedures concern pure strains grown in the laboratory (most often E. coli). However, some of them concern samples of natural aquatic or soil media (Deflaun et al., 1986; Ogram et al., 1987). It comes from this work that the analysis of DNA content of natural samples is difficult because it needs an extraction step of bacteria from the medium matrix. Particularly, the removal of humic or fulvic acids from the bacterial or DNA fraction is necessary to avoid interferences in DNA fluorimetric quantification. The inactivation of DNases enzymes is also required to avoid DNA digestion during the extraction procedure. The use of denaturants such as the guanidinium thiocyanate or fl mercapthoethanol was recommended in some situations (Boom et al., 1990) but not with activated sludge samples. Moreover, these products must be removed after from the nucleic acid fraction. The enzymatic digestion of DNases by large spectrum proteases such as proteinase K or pronase is another suggested solution (Sommerville et al., 1989) but it needs high protease concentration whose removal from the nucleic acid fraction is responsible from DNA loss. In this study we have evaluated by classical techniques, the proportion of activated sludge colony forming units 371
372
P. BAUDAet al.
( C F U ) harboring plasmids. We have also optimized a second, novel technique for the rapid and direct extraction of plasmic D N A from biomass sampled from an activated sludge process. MATERIAL AND METHODS
Biomass was sampled from an urban activated sludge wastewater treatment process serving the city of Metz (France). Biomass samples were sonicated before use (Vibracell 600 W) at 250 W for 1 min with 80% of active cycles (these sonication conditions allow the recovery of the largest number of colonies), then filtered through a 5/~m porosity gauze filter (UGB) to remove larger particles or persistent aggregates. Resulting biomass was used for both the isolation of pure strains and for direct plasmid DNA (pDNA) extraction. Isolation of strains Serial dilutions of sludge biomass in sterile saline water were plated on nutrient agar (Biomerieux 15139 L) modified with amphotericin 3 mg/1 final concentration to avoid fungi. 100 to 150 single colonies sampled from a single plate were replated once on peptoned broth and isolated 3 successive times on plates incubated at 20°C for 72 h. Plasmid extraction from activated sludge isolated strains In the first part of the study, we used a plasmid extraction method on pure strains isolated from sludges: 150 ml batch suspended cultures were grown on peptoned nutrient broth at room temperature to provide at least 108 cells/ml. Bacteria were harvested by centrifugation at 2500 g for 15 min. Bacterial cells were lysed and plasmids were extracted and purified according to "Qiagen midi prep." kit (Ref. 41021 commercialized by Coger, 79 rue des Morillons 75015 Paris). Protocols and material are detailed in the plasmid extraction kit. The extraction procedure is based on an alkaline lysis of bacterial cell pellet followed by anion exchange chromatography of extracted nucleic acids with specific elution of pDNA. Control of plasmid extraction efficiency in each set of plasmid screening was verified using the strain E. coli UB 1832 (pCE 325) harboring a 6kb plasmid. The efficiency of the plasmid extraction technique was verified with three different plasmid-carrying strains selected to test the procedure: (i) Streptococcus faecalis harboring three plasmids, kindly provided by C. Guimont (University of Nancy, France) this strain was chosen as representative of gram positive strains; (ii) Pseudomonas putida (pWWo) commonly found in activated sludge processes and (iii) E. coil UB 1832 (laCE 328) harboring a 5.5 kb plasmid chosen as a positive standard since the pDNA extraction protocol was optimized for E. coli strains. An 18 h 150 ml shaken culture of each strain was produced in (i) lactose containing broth at 37°C, (ii) in peptoned nutrient broth at 30°C and (iii) in L.B. medium at 37°C for the three strains respectively to provide at least 10s cells/ml. This 150 ml batch of suspended cultures were used following the standard protocol previously presented and recommended by Qiagen. Gel electrophoresis Extracted pDNA was analyzed by 0.50 agarose gel electrophoresis in tris borate EDTA buffer (TBE buffer, Sigma T6400), using a Hind III digest of ). DNA as good migration control (Sigma D4521) according to the Maniatis procedure (Sambrook et al., 1989). Gels were observed at 300nm with an ultraviolet transilluminator and photographed using a Polaroid MP4 camera. Direct plasmid recovery from sludges 100ml volumes of activated sludge were centrifuged at 10000g for 15min and resuspended in 50mlPBS; this
washing procedure was repeated three successive times. These washings decreased the amount of adsorbed DNAses. The final pellet was resuspended in the kit dissolution buffer: Tris EDTA ('rE) pH 7.5. The remaining process was similar to that applied to single bacterial strains. Extracted pDNA was quantified by fluorimetry and visualized by agarose gel electrophoresis. The direct extraction procedure was assayed with respect to procedure reproducibility and specificity for plasmid DNA and plasmid DNA loss during the experiments. Reproducibility test was performed by using direct extraction procedure on 4 x 100ml sludge sampled at different time on the same treatment plant. Purified plasmid DNA was measured by the fluorimetry method and was expressed per bacterial cell estimated by total epifluorescence counts. Specificity of the technique for plasmid DNA was assayed with selective addition of chr DNA to sludge samples, chr DNA standards of 200 and 500 ng were added in 100 ml sludge sample volumes before lysis of cells. Extracted plasmid quantities were compared with that of a control without added chr DNA. Losses of plasmid DNA during the procedure were quantified by adding 500 ng of plasmid DNA (R388, 33 kb plasmid) in the sludge sample before lysis. R388 is a well-known plasmid isolated in an environmental E. coil strain by Datta and Hedges (1972). It was chosen because its size is representative of the size of natural environmental plasmids and it is capable of migration in classical agarose gels (Fujita et aL, 1993). R388 DNA was extracted from an E. coli UB 1832 culture by the Birnboim and Doly technique (Birnboim and Doly, 1979) and quantified by fluorimetry. This strain and the E. coil UB 1832 pCE 325 E. coil UB 1832 pCE 328 were kindly provided by M.C. Lett (University Louis Pasteur, Strasbourg, France). Fluorimetric determination Extracted plasmid was quantified with Hoechst 33258 dye (Sigma B2380) dissolved in saline sodium citrate buffer at a 0.1/zg/ml final concentration using spectrofluorimetry at excitation/emission wavelengths of 342 nm and 443 nm, respectively (Latt, 1973). A calibration curve was obtained from Calf thymus DNA. Total cell counts Results are frequently explained and comparisons made with regard to the initial bacterial cell number in the sample analysed. We determined the total cell number by epifluorescence countings using 4--6 diamidino-2-phenylindole (DAPI) as a fluorochrome (Hoff, 1989). Bacterial suspensions were diluted in Tris-HC1 pH 8.5 and I ml of DAPI (25mg/1) was aded to 9 ml of dilution. After a 15 min contact time, bacterial dilutions were filtered on a black membrane (Millipore) 0.2/~m pore diameter. 30 microscope fields per filter were counted per serial dilution under u.v. light (365 nm).
RESULTS AND DISCUSSION Our study involved two different techniques for plasmid occurrence estimation in activated sludge. First, we estimated sludge plasmid content by extracting plasmids from sludge isolated colonies. Although the extraction procedure on single colonies grown in broth is easily carried out, it is time consuming because of the isolation steps and it includes a deviation due to the selective pressure growth conditions. In fact, only 1-10% of sludge bacterial cells will grow on solid media, moreover the replication step of the cell isolation procedure may induce some modifications of the cell plasmid con-
Plasmid content evaluation tent. For these reasons our plasmid screenings performed on isolated colonies will be an underestimate. During the validity test of the p D N A extraction procedure, we isolated plasmids from 3 known strains: Streptococcus faecalis, Pseudomonas putida and E. coli. We analysed the plasmid content of these strains on agarose gel. Since each strain tested positive, we assumed the plasmid extraction procedure was efficient for most activated sludge bacteria (data not shown). Plasmid content of 125 and 119 isolates from sludge samples taken at two different times was determined and reveals that, respectively, 38 and 42% of the total isolated strains contained at least one plasmid. Figure 1 indicates the number of plasmid D N A bands observed per isolate and the number of isolates providing the p D N A band number. Due to the different conformations of plasmids giving rise to different migration rates in gel electrophoresis, these results cannot be directly related to plasmid number per strain. Nevertheless, we estimated about 40% of isolated cells carrying at least one plasmid (mean of two assays performed at different times) which reflects an important proportion of cells harboring plasmids in the total original sludge sample. In the second technique, we estimated an average base pair number of p D N A per bacteria by extracting plasmids directly from sludges. The main problem in the optimization of this method was directly removing or inactivating the D N A s e s contained in sludges. Several techniques have been tested (including action
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1. Plasmid DNA extracted at three different occasions (I month apart) from biomass sampled from activated sludge Total DNA Total cell Date extract(ng) number pDNA ng/eell Mean bp/cell 3.02 3387 2.31 • 10II 1.45.10-s 13,302 26.02 3760 2.61 • 10tl 1.44.10-s 13,216 2.03 343 2.98.10 I° 1.15.10 ~ 10,552
Table
of SDS, guanidinium thiocyanate, dithiothreitol) and we retained repeated washings of sludges that removed more of contaminants, DNAses, and minimize p D N A losses. It would seem that the DNAses were mostly adsorbed on cells or on the sludge matrix since the extraction of p D N A (Qiagen protocol) on isolated colonies did not promote any D N A digestion. This technique allows a global quantification of the p D N A extract but does not give any information on the real plasmid content of cells in sludges. It would be better to use a plasmid index for sludge expressed in ng of plasmid D N A / g of sludge or per cell. Direct extraction was validated for reproducibility in three experiments performed at different times. Results given in Table 1 show that the plasmid content of activated sludge in the Metz treatment plant does not vary as a function of time during the time scale considered (1 month). Specificity of the technique for direct p D N A recovery from sludge was verified by comparing the efficiency of p D N A extraction with or without addition of foreign chr D N A . Results in Table 2 show quantitative determination of D N A extraction gave no significant variation between test samples with D N A adducts. The D N A added does not interfere with the plasmid D N A extraction technique. On the contrary an addition of p D N A , consisting of 500 ng of a 33 kb plasmid promote a significant consecutive increase of the p D N A extracted from the sludge. Results reported in Table 3 show a 75% recovery for added p D N A during the extraction procedure. Consequently, the p D N A loss related to extraction procedure was estimated at 25%, a range that will allow a useful application in detection of gene without any concentration. Specificity of the technique for plasmid D N A was verified, the standardization and rapidity of the protocol were insured by the use of a kit. The two approaches are complementary and describe differently the activated sludge D N A content. Nevertheless, we must note that neither takes into account loss of bacterial aggregates retained on 5/~m porosity filter during sludge treatment. While the indirect procedure is time consuming, it does allow
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Plasmid bands number Fig. 1. Number of plasmid bands extracted from isolated activated sludge strains. Results are of two separate extraction experiments carried out at different times.
Table 2. Effect of a known chromosomic DNA addition on the plasmid DNA extraction et~ciency. Standard deviations were calculated from triplicate samples Added DNA (ng) 0 200 500 Total extracted pDNA (ng) (o) 3387(39) 3948(110) 3012(350) from 2.31. 10II cells
374
P. BAUDAet al.
Table 3. Effectof a known plasmid DNA addition on plasmid DNA extraction efficiency Added DNA (ng) 0 500 Extracted DNA (ng)(~) from 343(31) 715(12) 2.98.10 I° cells
the identification and further characterization of plasmid D N A . On the other hand, the direct extraction technique is a very interesting one to investigate foreign gene dissemination within activated sludge. Such a technique may be useful for genetically engineered microorganisms, voluntarily or accidentally released. In fact, in case of such a release the probability that the recombinant cells or genes arrive to a water treatment plant is not of no account. This technique would also be useful to follow the dissemination of desired plasmid genes in wastewater treatment which can be introduced in activated sludge to improve treatment (Sayler and Layton, 1990). Plasmids studied should be looked for or detected more easily by hybridization techniques with a specific complementary probe on a direct extract of plasmid D N A from the activated sludge. The detection limit for the recovery of recombinant genes may be lowered by previous amplification of these genes by polymerase chain reaction (PCR) before hybridization (Sayler and Layton, 1990). P C R and hybridization techniques have been used in the same context on D N A extracted from soils contaminated with G E M s (Holben et al., 1988). The conclusion was that the limiting step in the determination of the recombinant genes persistence in soils was the efficiency of the D N A extraction technique. In this respect the second direct technique described in this paper should provide an appreciable help. REFERENCES
Birnboim H. C. and Doly J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmids DNA. Nucl. Acids Res. 7, 1513. Boom R., Sol C. J. A., Salinans M. M. M., Jansen C. L., Wertheim Van Dillen P. M. E. and Van der Nooida J. (1990) Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28, 495-503. Daflaun M. F., Paul J. H. and Davis D. (1986) Simplified method for dissolved DNA determination in aquatic environments. Appl. envir. Microbiol. 52, 654-659.
Datta N. and Hedges W. (1992) Trimetoprim resistance conferred by W plasmids in Enterobacteriaceae. J. gen. Microbiol. 72, 349-355. Fujita M., Ike M. and Susuki H. (1993) Screening of plasmids from wastewater bacteria. War. Res. 27, 949-953. Gear M. A., Chai M. D., Alpert K. B. and Boyer J. C. (1985) Transfer of plasmids pBR322 and pBR325 in wastewater from laboratory strains of Escherichia coli to bacteria indigenous to the waste disposal system. Appl. envir. Microbiol. 49, 836-841. Godwin D. and Slater J. H. (1979) The influence of the growth environment on the stability of a drug resistance plasmid in Escherichia coli K12. J. gen. Microbiol. IIl, 201-210. Helinski D. R. (1973) Plasmid determined resistance to antibiotics: molecular properties of R factors. Ann. Rev. Microbiol. 27, 437-470. Hoff K. A. (1989) Survival of Vibrio anguillarum and Vibrio salmonicida at different salinities. Appl. envir. Microbiol. 55, 1775-1786. Holben W. E., Jansson J. K., Chelm B. K. and Tiedje J. M. (1988) DNA probe method for the detection of specific microorganisms in the soil bacterial community. Appl. envirn. Microbiol. 54, 703-711. Jeffrey W. H., Paul J. H. and Stewart G. J. (1990) Natural transformation of a marine vibrio species by plasmid DNA. MicrobioL Ecol. 19, 259-268. Latt S. A. (1973) Microfluorimetric detection of deoxyribonucleic acid. Replication in human metaphase chromosome. Proc. natn. Acad. Sci. U.S.A. 70, 3396-3399. Ogram A., Sayler G. S. and Barkay T. (1987) The extraction and purification of microbial DNA from sediments. J. Microbiol. Meth. 7, 57-66. Reanney D. C., Gowland P. C. and Slater J. H. (1983) Genetic interactions among microbial communities. In Microbes in their Natural Environments (Edited by Slater J. H., Whittenbury R. and Wimpenny J. W. T.), pp. 379-421. Cambridge Univ. Press. Reeves P. (1972) The Bacteriocins. Springer, Berlin. Sambrook J., Fritsch E. F. and Maniatis T. (1989) Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press, U.S.A. Sandt C. H. and Herson D. S. (1991) Mobilization of the genetically engineered plasmid pHSV 106 from Escherichia coli HB 101 (pHSV 106) to Enterobacter doacae in drinking water. Appl. Envirn. Microbiol. 57, 194-200. Sayler G. S. and Layton A. C. (1990) Environmental application of nucleic acid hybridization. Ann. Rev. Microbiol. 44, 625-648. Sayler G. S., Hoopper, S. W., Layton A. C. and King J. M. H. (1990) Catabolic plasmids of environmental and ecological significance. Microbiol Ecol. 19, I~0. Sommerville C. C., Knight I. T., Straube W. L. and Colwell R. R. (1989) Simple, rapid method for direct isolation of nucleic acids from aquatic environments. Appl. envir. Microbiol. 54, 2185-2191. Trieu-Cuot P., Carlier C., Martin P. and Courvalin P. (1987) Plasmid transfer by conjugation from Escherichia coli to gram positive bacteria. FEMS Microbiol. Lett. 48, 289-294.
0043-1354(94)E0088-N
Wat. Res. Vol. 29, No. I, pp. 371-374, 1995 Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0043-1354/95$7.00+ 0.00
P L A S M I D C O N T E N T E V A L U A T I O N OF A C T I V A T E D SLUDGE P. BAUDAl, C. LALLEMENT1 and J. MANEM2 ICentre des Sciences de l'Environnement, Universit6 de Metz, 1 rue des R6collets 57000 Metz, and 2Centre International de Recherches sur l'Eau et l'Environnement (CIRSEE), 38 rue du Pr6sident Wilson, 78230, Le Pecq, France (First received September 1992; accepted in revised form March 1994)
Abstract--Plasmid content of activated sludge was evaluated by two different and complementary approaches. The first one consisted in the isolation of colonies from activated sludge samples, then isolated colonies were grown in nutrient broth and the plasmids were extracted from these cultures. The plasmid extraction procedure used a commercialized kit and the corresponding protocol. By this first approach, 40% of isolated colonies harboring plasmids were found. The second approach was an adaptation of the plasmid extraction kit directly on activated sludge samples. This direct application required several washing steps of sludges samples to avoid plasmid DNA digestion by DNases adsorbed within the sludge. This direct and rapid technique was validated in terms of reproducibility, specificity for plasmid DNA and losses. It allows the isolation of around 1#g of plasmid DNA per 10tl cells in the sludge. The two approaches presented in this paper indicated that the plasmid content of activated sludge is important and its particular role within the wastewater epuration procedure should be investigated. Key words--activated sludge, plasmids, extraction, quantification
INTRODUCTION Past attempts to improve the degradation of organic matter by biological wastewater treatment, have focused on procedures intended to increase the concentration of biomass or increase biomass activity, but, as yet, have not considered the genetic potential of the biomass. However, persistence of or expression of a gene may often be a limiting factor in the purification of water. The fact that particular compounds are not completely biodegraded or that the biodegradation process does not work in particular conditions may be related in some cases to a lack of genes coding for (a) a specific biodegradative pathway, (b) for resistance to toxic compounds present in the influent, or (c) both. Genetic information is classically located on chromosomal DNA (chr DNA) but genes in bacteria are also located on plasmid DNA (pDNA), which is not as big as chr DNA but has the ability to replicate itself. Well identified plasmid genes are often coded for biodegradation pathways (Sayler et al., 1990), antibiotic or metal resistance (Helinski, 1973), and toxine production (Reeves, 1972). Plasmids are not stable structures and they can be lost (Godwin and Slater, 1979) or transferred to other bacteria by several mechanisms: transformation (Jeffrey et al., 1990), conjugation (Trieu-Cuot et al., 1987), mobilization (Geat et al., 1985; Sandt and Herson, 1991), for a review see Reanney et al., (1983). In these conditions, autochtonous plasmids may have a function in xenobiotic biodegradation within the activated
sludge process. Resident plasmids may also play a role in allochtonous plasmid dissemination introduced voluntarily or inadvertently to the activated sludge. Presently very little research has dealt with plasmid DNA extraction from biomass sampled in activated sludge. Most of the publications concerning plasmid extraction procedures concern pure strains grown in the laboratory (most often E. coli). However, some of them concern samples of natural aquatic or soil media (Deflaun et al., 1986; Ogram et al., 1987). It comes from this work that the analysis of DNA content of natural samples is difficult because it needs an extraction step of bacteria from the medium matrix. Particularly, the removal of humic or fulvic acids from the bacterial or DNA fraction is necessary to avoid interferences in DNA fluorimetric quantification. The inactivation of DNases enzymes is also required to avoid DNA digestion during the extraction procedure. The use of denaturants such as the guanidinium thiocyanate or fl mercapthoethanol was recommended in some situations (Boom et al., 1990) but not with activated sludge samples. Moreover, these products must be removed after from the nucleic acid fraction. The enzymatic digestion of DNases by large spectrum proteases such as proteinase K or pronase is another suggested solution (Sommerville et al., 1989) but it needs high protease concentration whose removal from the nucleic acid fraction is responsible from DNA loss. In this study we have evaluated by classical techniques, the proportion of activated sludge colony forming units 371
372
P. BAUDAet al.
( C F U ) harboring plasmids. We have also optimized a second, novel technique for the rapid and direct extraction of plasmic D N A from biomass sampled from an activated sludge process. MATERIAL AND METHODS
Biomass was sampled from an urban activated sludge wastewater treatment process serving the city of Metz (France). Biomass samples were sonicated before use (Vibracell 600 W) at 250 W for 1 min with 80% of active cycles (these sonication conditions allow the recovery of the largest number of colonies), then filtered through a 5/~m porosity gauze filter (UGB) to remove larger particles or persistent aggregates. Resulting biomass was used for both the isolation of pure strains and for direct plasmid DNA (pDNA) extraction. Isolation of strains Serial dilutions of sludge biomass in sterile saline water were plated on nutrient agar (Biomerieux 15139 L) modified with amphotericin 3 mg/1 final concentration to avoid fungi. 100 to 150 single colonies sampled from a single plate were replated once on peptoned broth and isolated 3 successive times on plates incubated at 20°C for 72 h. Plasmid extraction from activated sludge isolated strains In the first part of the study, we used a plasmid extraction method on pure strains isolated from sludges: 150 ml batch suspended cultures were grown on peptoned nutrient broth at room temperature to provide at least 108 cells/ml. Bacteria were harvested by centrifugation at 2500 g for 15 min. Bacterial cells were lysed and plasmids were extracted and purified according to "Qiagen midi prep." kit (Ref. 41021 commercialized by Coger, 79 rue des Morillons 75015 Paris). Protocols and material are detailed in the plasmid extraction kit. The extraction procedure is based on an alkaline lysis of bacterial cell pellet followed by anion exchange chromatography of extracted nucleic acids with specific elution of pDNA. Control of plasmid extraction efficiency in each set of plasmid screening was verified using the strain E. coli UB 1832 (pCE 325) harboring a 6kb plasmid. The efficiency of the plasmid extraction technique was verified with three different plasmid-carrying strains selected to test the procedure: (i) Streptococcus faecalis harboring three plasmids, kindly provided by C. Guimont (University of Nancy, France) this strain was chosen as representative of gram positive strains; (ii) Pseudomonas putida (pWWo) commonly found in activated sludge processes and (iii) E. coil UB 1832 (laCE 328) harboring a 5.5 kb plasmid chosen as a positive standard since the pDNA extraction protocol was optimized for E. coli strains. An 18 h 150 ml shaken culture of each strain was produced in (i) lactose containing broth at 37°C, (ii) in peptoned nutrient broth at 30°C and (iii) in L.B. medium at 37°C for the three strains respectively to provide at least 10s cells/ml. This 150 ml batch of suspended cultures were used following the standard protocol previously presented and recommended by Qiagen. Gel electrophoresis Extracted pDNA was analyzed by 0.50 agarose gel electrophoresis in tris borate EDTA buffer (TBE buffer, Sigma T6400), using a Hind III digest of ). DNA as good migration control (Sigma D4521) according to the Maniatis procedure (Sambrook et al., 1989). Gels were observed at 300nm with an ultraviolet transilluminator and photographed using a Polaroid MP4 camera. Direct plasmid recovery from sludges 100ml volumes of activated sludge were centrifuged at 10000g for 15min and resuspended in 50mlPBS; this
washing procedure was repeated three successive times. These washings decreased the amount of adsorbed DNAses. The final pellet was resuspended in the kit dissolution buffer: Tris EDTA ('rE) pH 7.5. The remaining process was similar to that applied to single bacterial strains. Extracted pDNA was quantified by fluorimetry and visualized by agarose gel electrophoresis. The direct extraction procedure was assayed with respect to procedure reproducibility and specificity for plasmid DNA and plasmid DNA loss during the experiments. Reproducibility test was performed by using direct extraction procedure on 4 x 100ml sludge sampled at different time on the same treatment plant. Purified plasmid DNA was measured by the fluorimetry method and was expressed per bacterial cell estimated by total epifluorescence counts. Specificity of the technique for plasmid DNA was assayed with selective addition of chr DNA to sludge samples, chr DNA standards of 200 and 500 ng were added in 100 ml sludge sample volumes before lysis of cells. Extracted plasmid quantities were compared with that of a control without added chr DNA. Losses of plasmid DNA during the procedure were quantified by adding 500 ng of plasmid DNA (R388, 33 kb plasmid) in the sludge sample before lysis. R388 is a well-known plasmid isolated in an environmental E. coil strain by Datta and Hedges (1972). It was chosen because its size is representative of the size of natural environmental plasmids and it is capable of migration in classical agarose gels (Fujita et aL, 1993). R388 DNA was extracted from an E. coli UB 1832 culture by the Birnboim and Doly technique (Birnboim and Doly, 1979) and quantified by fluorimetry. This strain and the E. coil UB 1832 pCE 325 E. coil UB 1832 pCE 328 were kindly provided by M.C. Lett (University Louis Pasteur, Strasbourg, France). Fluorimetric determination Extracted plasmid was quantified with Hoechst 33258 dye (Sigma B2380) dissolved in saline sodium citrate buffer at a 0.1/zg/ml final concentration using spectrofluorimetry at excitation/emission wavelengths of 342 nm and 443 nm, respectively (Latt, 1973). A calibration curve was obtained from Calf thymus DNA. Total cell counts Results are frequently explained and comparisons made with regard to the initial bacterial cell number in the sample analysed. We determined the total cell number by epifluorescence countings using 4--6 diamidino-2-phenylindole (DAPI) as a fluorochrome (Hoff, 1989). Bacterial suspensions were diluted in Tris-HC1 pH 8.5 and I ml of DAPI (25mg/1) was aded to 9 ml of dilution. After a 15 min contact time, bacterial dilutions were filtered on a black membrane (Millipore) 0.2/~m pore diameter. 30 microscope fields per filter were counted per serial dilution under u.v. light (365 nm).
RESULTS AND DISCUSSION Our study involved two different techniques for plasmid occurrence estimation in activated sludge. First, we estimated sludge plasmid content by extracting plasmids from sludge isolated colonies. Although the extraction procedure on single colonies grown in broth is easily carried out, it is time consuming because of the isolation steps and it includes a deviation due to the selective pressure growth conditions. In fact, only 1-10% of sludge bacterial cells will grow on solid media, moreover the replication step of the cell isolation procedure may induce some modifications of the cell plasmid con-
Plasmid content evaluation tent. For these reasons our plasmid screenings performed on isolated colonies will be an underestimate. During the validity test of the p D N A extraction procedure, we isolated plasmids from 3 known strains: Streptococcus faecalis, Pseudomonas putida and E. coli. We analysed the plasmid content of these strains on agarose gel. Since each strain tested positive, we assumed the plasmid extraction procedure was efficient for most activated sludge bacteria (data not shown). Plasmid content of 125 and 119 isolates from sludge samples taken at two different times was determined and reveals that, respectively, 38 and 42% of the total isolated strains contained at least one plasmid. Figure 1 indicates the number of plasmid D N A bands observed per isolate and the number of isolates providing the p D N A band number. Due to the different conformations of plasmids giving rise to different migration rates in gel electrophoresis, these results cannot be directly related to plasmid number per strain. Nevertheless, we estimated about 40% of isolated cells carrying at least one plasmid (mean of two assays performed at different times) which reflects an important proportion of cells harboring plasmids in the total original sludge sample. In the second technique, we estimated an average base pair number of p D N A per bacteria by extracting plasmids directly from sludges. The main problem in the optimization of this method was directly removing or inactivating the D N A s e s contained in sludges. Several techniques have been tested (including action
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1. Plasmid DNA extracted at three different occasions (I month apart) from biomass sampled from activated sludge Total DNA Total cell Date extract(ng) number pDNA ng/eell Mean bp/cell 3.02 3387 2.31 • 10II 1.45.10-s 13,302 26.02 3760 2.61 • 10tl 1.44.10-s 13,216 2.03 343 2.98.10 I° 1.15.10 ~ 10,552
Table
of SDS, guanidinium thiocyanate, dithiothreitol) and we retained repeated washings of sludges that removed more of contaminants, DNAses, and minimize p D N A losses. It would seem that the DNAses were mostly adsorbed on cells or on the sludge matrix since the extraction of p D N A (Qiagen protocol) on isolated colonies did not promote any D N A digestion. This technique allows a global quantification of the p D N A extract but does not give any information on the real plasmid content of cells in sludges. It would be better to use a plasmid index for sludge expressed in ng of plasmid D N A / g of sludge or per cell. Direct extraction was validated for reproducibility in three experiments performed at different times. Results given in Table 1 show that the plasmid content of activated sludge in the Metz treatment plant does not vary as a function of time during the time scale considered (1 month). Specificity of the technique for direct p D N A recovery from sludge was verified by comparing the efficiency of p D N A extraction with or without addition of foreign chr D N A . Results in Table 2 show quantitative determination of D N A extraction gave no significant variation between test samples with D N A adducts. The D N A added does not interfere with the plasmid D N A extraction technique. On the contrary an addition of p D N A , consisting of 500 ng of a 33 kb plasmid promote a significant consecutive increase of the p D N A extracted from the sludge. Results reported in Table 3 show a 75% recovery for added p D N A during the extraction procedure. Consequently, the p D N A loss related to extraction procedure was estimated at 25%, a range that will allow a useful application in detection of gene without any concentration. Specificity of the technique for plasmid D N A was verified, the standardization and rapidity of the protocol were insured by the use of a kit. The two approaches are complementary and describe differently the activated sludge D N A content. Nevertheless, we must note that neither takes into account loss of bacterial aggregates retained on 5/~m porosity filter during sludge treatment. While the indirect procedure is time consuming, it does allow
F'fl
vA
I 7
373
i r i i I I I J 9 10 ii 12 13 14 15 16
Plasmid bands number Fig. 1. Number of plasmid bands extracted from isolated activated sludge strains. Results are of two separate extraction experiments carried out at different times.
Table 2. Effect of a known chromosomic DNA addition on the plasmid DNA extraction et~ciency. Standard deviations were calculated from triplicate samples Added DNA (ng) 0 200 500 Total extracted pDNA (ng) (o) 3387(39) 3948(110) 3012(350) from 2.31. 10II cells
374
P. BAUDAet al.
Table 3. Effectof a known plasmid DNA addition on plasmid DNA extraction efficiency Added DNA (ng) 0 500 Extracted DNA (ng)(~) from 343(31) 715(12) 2.98.10 I° cells
the identification and further characterization of plasmid D N A . On the other hand, the direct extraction technique is a very interesting one to investigate foreign gene dissemination within activated sludge. Such a technique may be useful for genetically engineered microorganisms, voluntarily or accidentally released. In fact, in case of such a release the probability that the recombinant cells or genes arrive to a water treatment plant is not of no account. This technique would also be useful to follow the dissemination of desired plasmid genes in wastewater treatment which can be introduced in activated sludge to improve treatment (Sayler and Layton, 1990). Plasmids studied should be looked for or detected more easily by hybridization techniques with a specific complementary probe on a direct extract of plasmid D N A from the activated sludge. The detection limit for the recovery of recombinant genes may be lowered by previous amplification of these genes by polymerase chain reaction (PCR) before hybridization (Sayler and Layton, 1990). P C R and hybridization techniques have been used in the same context on D N A extracted from soils contaminated with G E M s (Holben et al., 1988). The conclusion was that the limiting step in the determination of the recombinant genes persistence in soils was the efficiency of the D N A extraction technique. In this respect the second direct technique described in this paper should provide an appreciable help. REFERENCES
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