FEMS MicrobiologyLetters 69 (1990) 255-258 Published by Elsevier
255
FEMSLE 04019
The effect of nuclease on transformation efficiency in S e r r a t i a m a r c e s c e n s J u l i o Palomar, J o a n F r a n c e s c G u a s c h , M i q u e l Regu6 a n d M i q u e l Vihas Department of Microbiology' and Parasitology. Health Sciences Division, Faculty of Pharmao'. University o/Barcelona, Barcelona, Spain
Received6 December 1989 Revision received13 February 1990 Accepted 14 February 1990 Key words: Serratia marcescens; Nuclease; Transformation efficiency
1. S U M M A R Y N o differences in the efficiency of transformation were observed from both plasmid and chromosomal D N A in Serratia marcescens 2170 and an extracellular nuclease defective isogenic strain. The efficiency of transformation was the same for Escherichia coil 5K and E. coil containing a recombinant plasmid conferring the ability to synthesize a S. marcescens nuclease. From these resuits we conclude that the extracellular nuclease of S. marcescens 2170 is not the main cause of the low efficiency of transformation observed in this bacterium.
2. I N T R O D U C T I O N The introduction of foreign D N A into bacteria by means of transformation is a basic method in the genetic analysis of bacteria. Only a few bacterial species have the ability to transform by a
Correspondence to: Dr. Miquel Yi/tas, Laboratori de MicrobioIogia, Facultat de Farm/tcia, Universitat de Barcelona, 08028 Barcelona, Sp~n.
natural process ( H a e m o p h i l u s influenzae, Streptococcus p n e u m o n i a e etc.). In other cases, like Escherichia coil, transformation procedures involve some treatments that make the bacteria competent for D N A uptake (i.e. CaC! 2 treatment). Many other species exhibit low transformation rates. Serratia marcescens is an enterobacterium that secretes many proteins like chitinases [1,2], nucleases [3], several proteases [4] and lipascs [5]. This makes S. marcescens a potentially useful microorganism for genetic engineering purposes. However, genetic manipulations in Serratia pose many additional problems when compared with E. coll. First of all, the chromosomal D N A extractions from Serratia need to use special techniques in order to avoid the effect of nuclease on the chromosomal D N A [6]. On the other hand, Serratia exhibits very low efficiencies of transformation. One of the reasons to explain these low efficiencies of transformation is the ability of Serratia to produce and secrete a potent nuclease [7-9]. The nuclease of Serratia was first recognized by Jeffries et al. [10] when they described the ability of S. marcescens to degrade D N A as well as RNA. Further, Eaves and Jeffries [3] described the first purification of the nuclease and several characteristics of the enzyme activity. In
0378-1097/90/$03.50 © 1990 Federation of EuropeanMicrobiologicalSocieties
256 this paper, we present a series of experiments to study the effect of DNase on the transformation efficiencies of Serratia marcescens.
Table 2 Comparative transformation efficiencies(frequency of transformants/viable bacteria) of three different methods using S. marcescens 2170 and 2170c and E. coli 5K Strain
3. M A T E R I A L S A N D M E T H O D S S. marcescens 3.1. Bacterial strains a n d p l a s m i d s
2170
Bacterial strains used in the present study are summarized in Table 1. Plasmids used were pANN202.312 [11] and pBAIK, a plasmid encoding kanamycin resistance. The plasmid pBR328 was used in cloning experiments [12].
S. marcescens
3.2. Transformation procedures
Three alternative methodologies of transformation were assayed in this work. The first one was based on the method from Cohen et al. [13]. An alternative method from Merrick et al. [14] using two steps of freezing and thawing was also used. Finally the method of supercompetent cells described for E. coil by Hanahan [15] was also tested. 3.3. Mutagenesis
To obtain mutants defective in DNase production of S. marcescens we used the T n 5 transposon. A strain derived from 2170c and transformed by the plasmid p T R O Y l l [16] which encodes for the LamB protein (the receptor of lambda phage) is susceptible to infection by lambda particles. Lambda 467 phage ( ~ : : T n S ) was used as mutagenic agent. Mutants were selected on plates con-
Table 1 Bactcrial strains used in this investigation Bacterial strains
Characteristics
S. marcescens 2170 S. marcescens 2170c
wild type 2170 transformed with pANN202.312 and cured 2170c DNase F-, r -k, m -k, rpsL, thr, leu, lacZ 5K carrying pFG891 plasmid DNA
S. marcescens 2170¢JP1 E. coil 5K E. coli 5kdn
Transformation procedure Cohen Merrick Hanahan 2xi0 -6 1.6×10 -6 2.2 x 10-6 3×10 .6
2.5×10 -6
6.9×10 -6
9×10 -2
7.0x 10-2
2.0x 10-2
2170c E. coliSK
raining 100 /tg per ml of kanamycin (lower concentrations allowed the growth of wild type S. m a r c e s c e n s ) and then transferred by replica plating to plates of DNase agar (Difco) with methyl green. Plates were scored after 24 h incubation at 30°C. 3.4. Cloning o f nuclease gene A genetic library from S. marcescens 2170c was
obtained using pBR328 as a vector. Recombinant D N A was used to transform E. coil 5 K and the transformants recovered in plates of TSA containing 3 0 / ~ g / m l of chloroamphenicol, transferred by replica plating onto DNase agar as described above, and the plates scored after 24-48 h incubation. 3.5. Nuclease activity assays
This was done according to the methods described by Bali et al. [17]. The amount of D N A in each well of the microtiter dishes was 10/~g.
4. R E S U L T S A N D D I S C U S S I O N Since the efficiency of transformation of S. marcescens 2170c has proved to be very low using
the standard Cohen method [13], we tried two other alternative methods to make S. marcescens 2170 cells competent for the foreign D N A uptake. As shown in Table 2, no significant differences were observed in the overall transformation efficiencies with the three methods tested. Further experiments were carried out following the method of Merrick [14]. In order to test the effect of the S.
257
Fig. I. Mk'rotiter dish in which the wells were filled with 10/lg of plasmid DNA in 50 mM Tris pH 8. 10 mM MgCI2. 10/~g ethidium bromide and 50 ~1 of supernatant of the following: A: 2170 c from a 24 h culture: B 2170cJPl from a 24 h culture: C: 2170c from a 48 h culture: D: 2170cJPl from a 48 h culture: E: 2170c from a 2 h culture; F: 2170cJPl from a 2 h culture: G sterile broth control: H: E. coli 5K and I: E. coil 5Kdn.
2170 extracellular nuclease on the transformation process, we did transformation experiments using either plasmid and chromosomal D N A into S. m a r c e s c e n s 2170 and an isogenic extracellular nuclease defective mutant. This m u t a n t was obtained by insertional mutagenesis with the transposon Tn5. As can be seen in Fig. 1,
marcescens
Table 3 Transformation efficiancies(Merrick method) in S, marcescens 2170c; S. marcescens 2170cJPl (DNase-); E. coil 5K and E. co/i 5Kdn (pFG891) Strain S. mareescens 2170c S. marcescens 2170cJPl E. co/i 5K E. co/i 5Kdn
Transfom, a~s//tgDNA 31 32 7.2x 10s 7.3 x l0 s
Transformants/pgDNA, average of three experiments.
the levels of extracellular nuclease production in this m u t a n t are negligible. The results of Table 3 show that there was n o increase in the efficiency of transformation in the nuclease defective mutant, as one would expect if the presence of this extracellular nuclease was the main reason for the low efficiency of transformation in S. m a r c e s c e n s . The extracellular nuclease is only one of the possible barriers for incoming foreign DNA. In order to test the effect of the nuclease independently from other S. m a r c e s c e n s factors affecting transformation efficiencies, a gene library of pBR328 was prepared. E. coil 5K transformants were screened for extracellular nuclease production o n D N A agar plates. The recombinant plasmid pFG891, proved to confer tQ E. coil the ability to produce a nuclease a n d allowed to test the effect of the S e r r a t i a nuclease on the transformation process in E. c o i l As shown in Table 3 the efficiencies of transformation into E. coil were not changed by carrying pFG891 plasmid. These resuits suggest that the treatments involved in the development of competence are enough to remove most of the extracellular nuclease. Moreover, in the Merrick et at. [14] method, the time a n d temperature of incubation probably prevents the action of the remaining nuclease on the transforming DNA. Since nuclease seems not to be the actual reason for the low efficiency of transformation in this bacterial species, other causes should be investigated. One of such factors could be the lipopolysaccharide because in another enterobacterium, K l e b s i e l l a p n e u m o n i a e , the O-side chain (antigen O) has been shown to play an important role in transformation efficiencies. In this species, the reduction of LPS O-side chains increases the efficiency of D N A uptake [18]. ACKNOWLEDGEMENT This work was supported by grant PB 86-0034 from Comision lnterministeriai de Ciencia y Tecnologia (C1CYT). REFERENCES [1l MonreaL J. and Reese, E.T. (1969) Can. J. Microbiol. 15, 689-696.
258 [2] Jones, J.D., Grady, K.L., Suslow, T.V. and Bedbrook, J.R. (1986) EMBO J. 5, 467-473. [3] Eaves, G.N. and Jeffries, C.D. (1963) J. Bacteriol. 85, 263-268. [4] Braun, V. and Schmitz, G. (1980) Arch. Microbiol. 124, 55-61. [5] Heller, K. (1979) J. Bacteriol. 154, 413-418. [6] Timmis, K. and Winkler, U. (1973) J. Bacterioi. 113, 508-509. [7] Wirth, R., Friesseneger, A. and Fiedler, S. (1989) Mol. Gen. Genet. 216,175-177. [8] Clegg, S. and Allen, B.L. (1985) FEMS Microbiol. Lett. 27. 257-262. [9] Reid, J.D., Stoufer, S.D. and Ogrydziak, D.M. (1982) Gene 17,107-112. [10] Jeffries, C.D., Holtman, D.F. and Guse, D.G. (19570 J. Bacteriol. 73, 590-591.
[11] Goebel, W. and Hedgpeth, I. (1982) J. Bacteriol. 151, 1290-1298. [12] Soberon, X., Covarrubias, L. and Bolivar, F. (1980) Gene 9, 287-305. [13] Cohen, S.N., Chang, A.C.Y. and Hsu, L. 0972) Proc. Natl. Acad. Sci. U.S.A. 69, 2110-2114. [14] Merrick, M.J., Gibbins, J.R. and Postage. J.R. (1987) J. Gen. Microbioi. 133, 2053-2057. [15] Hanahan, D. (1983) J. Molec. Biol. 166, 557-580. [i6] De Vries, G.E., Raymond, C.K. and Ludwig, R.A. (1984) Proc. Natl. Acad. Sci U.S.A. 81, 0080-6084. [17] Bah, T.K., Saurugger, P.N. and Benedik, M.J. (1987) Gene 57, 183-192. [18] Camprubl, S., Regu~, M. and Tomfis, J. 0989) Can. J. Microbiol. 35, 735-737.
255
FEMSLE 04019
The effect of nuclease on transformation efficiency in S e r r a t i a m a r c e s c e n s J u l i o Palomar, J o a n F r a n c e s c G u a s c h , M i q u e l Regu6 a n d M i q u e l Vihas Department of Microbiology' and Parasitology. Health Sciences Division, Faculty of Pharmao'. University o/Barcelona, Barcelona, Spain
Received6 December 1989 Revision received13 February 1990 Accepted 14 February 1990 Key words: Serratia marcescens; Nuclease; Transformation efficiency
1. S U M M A R Y N o differences in the efficiency of transformation were observed from both plasmid and chromosomal D N A in Serratia marcescens 2170 and an extracellular nuclease defective isogenic strain. The efficiency of transformation was the same for Escherichia coil 5K and E. coil containing a recombinant plasmid conferring the ability to synthesize a S. marcescens nuclease. From these resuits we conclude that the extracellular nuclease of S. marcescens 2170 is not the main cause of the low efficiency of transformation observed in this bacterium.
2. I N T R O D U C T I O N The introduction of foreign D N A into bacteria by means of transformation is a basic method in the genetic analysis of bacteria. Only a few bacterial species have the ability to transform by a
Correspondence to: Dr. Miquel Yi/tas, Laboratori de MicrobioIogia, Facultat de Farm/tcia, Universitat de Barcelona, 08028 Barcelona, Sp~n.
natural process ( H a e m o p h i l u s influenzae, Streptococcus p n e u m o n i a e etc.). In other cases, like Escherichia coil, transformation procedures involve some treatments that make the bacteria competent for D N A uptake (i.e. CaC! 2 treatment). Many other species exhibit low transformation rates. Serratia marcescens is an enterobacterium that secretes many proteins like chitinases [1,2], nucleases [3], several proteases [4] and lipascs [5]. This makes S. marcescens a potentially useful microorganism for genetic engineering purposes. However, genetic manipulations in Serratia pose many additional problems when compared with E. coll. First of all, the chromosomal D N A extractions from Serratia need to use special techniques in order to avoid the effect of nuclease on the chromosomal D N A [6]. On the other hand, Serratia exhibits very low efficiencies of transformation. One of the reasons to explain these low efficiencies of transformation is the ability of Serratia to produce and secrete a potent nuclease [7-9]. The nuclease of Serratia was first recognized by Jeffries et al. [10] when they described the ability of S. marcescens to degrade D N A as well as RNA. Further, Eaves and Jeffries [3] described the first purification of the nuclease and several characteristics of the enzyme activity. In
0378-1097/90/$03.50 © 1990 Federation of EuropeanMicrobiologicalSocieties
256 this paper, we present a series of experiments to study the effect of DNase on the transformation efficiencies of Serratia marcescens.
Table 2 Comparative transformation efficiencies(frequency of transformants/viable bacteria) of three different methods using S. marcescens 2170 and 2170c and E. coli 5K Strain
3. M A T E R I A L S A N D M E T H O D S S. marcescens 3.1. Bacterial strains a n d p l a s m i d s
2170
Bacterial strains used in the present study are summarized in Table 1. Plasmids used were pANN202.312 [11] and pBAIK, a plasmid encoding kanamycin resistance. The plasmid pBR328 was used in cloning experiments [12].
S. marcescens
3.2. Transformation procedures
Three alternative methodologies of transformation were assayed in this work. The first one was based on the method from Cohen et al. [13]. An alternative method from Merrick et al. [14] using two steps of freezing and thawing was also used. Finally the method of supercompetent cells described for E. coil by Hanahan [15] was also tested. 3.3. Mutagenesis
To obtain mutants defective in DNase production of S. marcescens we used the T n 5 transposon. A strain derived from 2170c and transformed by the plasmid p T R O Y l l [16] which encodes for the LamB protein (the receptor of lambda phage) is susceptible to infection by lambda particles. Lambda 467 phage ( ~ : : T n S ) was used as mutagenic agent. Mutants were selected on plates con-
Table 1 Bactcrial strains used in this investigation Bacterial strains
Characteristics
S. marcescens 2170 S. marcescens 2170c
wild type 2170 transformed with pANN202.312 and cured 2170c DNase F-, r -k, m -k, rpsL, thr, leu, lacZ 5K carrying pFG891 plasmid DNA
S. marcescens 2170¢JP1 E. coil 5K E. coli 5kdn
Transformation procedure Cohen Merrick Hanahan 2xi0 -6 1.6×10 -6 2.2 x 10-6 3×10 .6
2.5×10 -6
6.9×10 -6
9×10 -2
7.0x 10-2
2.0x 10-2
2170c E. coliSK
raining 100 /tg per ml of kanamycin (lower concentrations allowed the growth of wild type S. m a r c e s c e n s ) and then transferred by replica plating to plates of DNase agar (Difco) with methyl green. Plates were scored after 24 h incubation at 30°C. 3.4. Cloning o f nuclease gene A genetic library from S. marcescens 2170c was
obtained using pBR328 as a vector. Recombinant D N A was used to transform E. coil 5 K and the transformants recovered in plates of TSA containing 3 0 / ~ g / m l of chloroamphenicol, transferred by replica plating onto DNase agar as described above, and the plates scored after 24-48 h incubation. 3.5. Nuclease activity assays
This was done according to the methods described by Bali et al. [17]. The amount of D N A in each well of the microtiter dishes was 10/~g.
4. R E S U L T S A N D D I S C U S S I O N Since the efficiency of transformation of S. marcescens 2170c has proved to be very low using
the standard Cohen method [13], we tried two other alternative methods to make S. marcescens 2170 cells competent for the foreign D N A uptake. As shown in Table 2, no significant differences were observed in the overall transformation efficiencies with the three methods tested. Further experiments were carried out following the method of Merrick [14]. In order to test the effect of the S.
257
Fig. I. Mk'rotiter dish in which the wells were filled with 10/lg of plasmid DNA in 50 mM Tris pH 8. 10 mM MgCI2. 10/~g ethidium bromide and 50 ~1 of supernatant of the following: A: 2170 c from a 24 h culture: B 2170cJPl from a 24 h culture: C: 2170c from a 48 h culture: D: 2170cJPl from a 48 h culture: E: 2170c from a 2 h culture; F: 2170cJPl from a 2 h culture: G sterile broth control: H: E. coli 5K and I: E. coil 5Kdn.
2170 extracellular nuclease on the transformation process, we did transformation experiments using either plasmid and chromosomal D N A into S. m a r c e s c e n s 2170 and an isogenic extracellular nuclease defective mutant. This m u t a n t was obtained by insertional mutagenesis with the transposon Tn5. As can be seen in Fig. 1,
marcescens
Table 3 Transformation efficiancies(Merrick method) in S, marcescens 2170c; S. marcescens 2170cJPl (DNase-); E. coil 5K and E. co/i 5Kdn (pFG891) Strain S. mareescens 2170c S. marcescens 2170cJPl E. co/i 5K E. co/i 5Kdn
Transfom, a~s//tgDNA 31 32 7.2x 10s 7.3 x l0 s
Transformants/pgDNA, average of three experiments.
the levels of extracellular nuclease production in this m u t a n t are negligible. The results of Table 3 show that there was n o increase in the efficiency of transformation in the nuclease defective mutant, as one would expect if the presence of this extracellular nuclease was the main reason for the low efficiency of transformation in S. m a r c e s c e n s . The extracellular nuclease is only one of the possible barriers for incoming foreign DNA. In order to test the effect of the nuclease independently from other S. m a r c e s c e n s factors affecting transformation efficiencies, a gene library of pBR328 was prepared. E. coil 5K transformants were screened for extracellular nuclease production o n D N A agar plates. The recombinant plasmid pFG891, proved to confer tQ E. coil the ability to produce a nuclease a n d allowed to test the effect of the S e r r a t i a nuclease on the transformation process in E. c o i l As shown in Table 3 the efficiencies of transformation into E. coil were not changed by carrying pFG891 plasmid. These resuits suggest that the treatments involved in the development of competence are enough to remove most of the extracellular nuclease. Moreover, in the Merrick et at. [14] method, the time a n d temperature of incubation probably prevents the action of the remaining nuclease on the transforming DNA. Since nuclease seems not to be the actual reason for the low efficiency of transformation in this bacterial species, other causes should be investigated. One of such factors could be the lipopolysaccharide because in another enterobacterium, K l e b s i e l l a p n e u m o n i a e , the O-side chain (antigen O) has been shown to play an important role in transformation efficiencies. In this species, the reduction of LPS O-side chains increases the efficiency of D N A uptake [18]. ACKNOWLEDGEMENT This work was supported by grant PB 86-0034 from Comision lnterministeriai de Ciencia y Tecnologia (C1CYT). REFERENCES [1l MonreaL J. and Reese, E.T. (1969) Can. J. Microbiol. 15, 689-696.
258 [2] Jones, J.D., Grady, K.L., Suslow, T.V. and Bedbrook, J.R. (1986) EMBO J. 5, 467-473. [3] Eaves, G.N. and Jeffries, C.D. (1963) J. Bacteriol. 85, 263-268. [4] Braun, V. and Schmitz, G. (1980) Arch. Microbiol. 124, 55-61. [5] Heller, K. (1979) J. Bacteriol. 154, 413-418. [6] Timmis, K. and Winkler, U. (1973) J. Bacterioi. 113, 508-509. [7] Wirth, R., Friesseneger, A. and Fiedler, S. (1989) Mol. Gen. Genet. 216,175-177. [8] Clegg, S. and Allen, B.L. (1985) FEMS Microbiol. Lett. 27. 257-262. [9] Reid, J.D., Stoufer, S.D. and Ogrydziak, D.M. (1982) Gene 17,107-112. [10] Jeffries, C.D., Holtman, D.F. and Guse, D.G. (19570 J. Bacteriol. 73, 590-591.
[11] Goebel, W. and Hedgpeth, I. (1982) J. Bacteriol. 151, 1290-1298. [12] Soberon, X., Covarrubias, L. and Bolivar, F. (1980) Gene 9, 287-305. [13] Cohen, S.N., Chang, A.C.Y. and Hsu, L. 0972) Proc. Natl. Acad. Sci. U.S.A. 69, 2110-2114. [14] Merrick, M.J., Gibbins, J.R. and Postage. J.R. (1987) J. Gen. Microbioi. 133, 2053-2057. [15] Hanahan, D. (1983) J. Molec. Biol. 166, 557-580. [i6] De Vries, G.E., Raymond, C.K. and Ludwig, R.A. (1984) Proc. Natl. Acad. Sci U.S.A. 81, 0080-6084. [17] Bah, T.K., Saurugger, P.N. and Benedik, M.J. (1987) Gene 57, 183-192. [18] Camprubl, S., Regu~, M. and Tomfis, J. 0989) Can. J. Microbiol. 35, 735-737.
