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Improved plasmids containing the Escherichia coli dam gene under the control of the tac promoter
309
Biochimica et Biophysica Acta, 1132 (1992) 309-310 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
BBAEXP 90403
Rapid Report
Improved plasmids containing the Escherichia coil dam gene under the control of the tac promoter Sibajyoti Guha 1 and Wilhelm Guschlbauer Serrice de Bioehimie et G~netique Mol~culaire, B~t. 142, D~partement de Biologie Cellulaire et Mol~eulaire, Centre d'Etudes de Saclay,
Gif-sur-Yrette (France) (Received 19 June 1992) Key words: Plasmid; dam gene; tac promoter; (E. coli) We report the construction of a series of plasmids containing the dam gene under the contol of the tac promoter. Cells containing these plasmids produce about 8 to 10-fold more Dam methyltransferase (Mtase) than the previously used plasmid pTP166 and avoid the use of a high temperature step necessary for the expression of Dam Mtase in the plasmid pDOX1 and thus allow its use for the study of thermosensitive mutants.
T h e d a m g e n e of Escherichia coli c o d e s for D a m m e t h y l t r a n s f e r a s e ( M t a s e ) which m e t h y l a t e s a d e n i n e in t h e s e q u e n c e 5 ' - G A T C - 3 ' . D a m m e t h y l a t i o n is involved in v a r i o u s c e l l u l a r p r o c e s s e s a n d is r e s p o n s i b l e for s t r a n d d i s c r i m i n a t i o n in m u t H L S m e d i a t e d mism a t c h r e p a i r [1]. H i g h D a m m e t h y l a t i o n in E. coli, however, p r o v o k e s h y p e r m u t a t i o n [2,3] as well as S O S r e s p o n s e [4]. T h e s e p r o p e r t i e s have h a m p e r e d t h e construction o f an a p p r o p r i a t e h i g h - c o p y - n u m b e r p l a s m i d o v e r p r o d u c i n g D a m M t a s e . It is t h e r e f o r e n e c e s s a r y to p u t this g e n e u n d e r the c o n t r o l o f a r e g u l a t a b l e p r o m o t e r . A p l a s m i d , pTP166, in which the d a m g e n e is u n d e r t h e c o n t r o l o f t h e i n d u c i b l e tac p r o m o t e r , has b e e n c o n s t r u c t e d [2]. T h e a m o u n t of D a m M t a s e p r o d u c e d from t h e s e p l a s m i d b e a r i n g ceils is, however, not very high, even w h e n the p r o m o t e r is fully i n d u c e d by isopropylthiogalactoside (IPTG). Another plasmid, p D O X 1 , in which the d a m g e n e is p l a c e d u n d e r the c o n t r o l o f a h e a t - i n d u c i b l e p r o m o t e r cI [5], was constructed; from p D O X 1 b e a r i n g cells c o n s i d e r a b l e a m o u n t s of D a m M t a s e can b e isolated. A high temp e r a t u r e for i n d u c t i o n a n d s u b s e q u e n t i n c u b a t i o n excludes its use in t h e r m o s e n s i t i v e E. coli m u t a n t s a n d also w h e n the expression of t h e r m o s e n s i t i v e d a m mutants in the p l a s m i d is studied.
• ~ i ~ d ~ ,. ~X
E
0.25 kb
I tac
X
.,,~"'f~'.,~
co + X b a
/ "
%
p
P
dam
/) P
~ |
Tlad
puc19 I
..~.~Pvull
linearized
04kb',~
Taq fragment
~
E dam
M6
E Ncl
Correspondence to: W. Guschlbauer, Service de Biochimie et G6netique Mol6culaire, B~t. 142, D6partement de Biologie Cellulaire et Mol6culaire, Centre d'Etudes de Saclay, F-91191, Gif-sur-Yvette, France. i Present address: Institut Jacques Monod, Universit6 Paris VII, 2, Place Jussieu, F-75251, Paris Cedex 05, France.
Fig. 1. Schematic drawing of the construction of pDDM6 containing
the dam and /acl genes. Sites: B, BamHI; E, EcoRl; P, PrulI; T, TaqI; X, XbaI. In pDDM3 the dark line around the XbaI site (not to scale) indicates the Bal31 deleted portion. Arrows in pDDM6 indicate polarity of the genes. In pDDM7 the lacl gene is in an orientation opposite to that of plasmid pDDM6.
310 To obtain a high-expression tacpromoter controlled dam gene, the plasmid pTP166 [2] was XbaI linearized and digested by Bal31 to remove nucleotides upstream of the coding sequence of the dam gene (Fig. 1). The pool of the Bal31 digested plasmid was religated in the presence of a XbaI linker (5'-CTCTAGAG-3') and used for transformation of a E. coli dam mutant GM48 [6]. A transformant with one of the plasmids, pDDM3, containing the Xba site had high enzyme yield. But in this plasmid the nucleotides constituting the lac repressor binding site (rb in Fig. 1) had also been removed and it showed only a negligible induction by IPTG. To restore the repressor binding site, the 235 bp EcoRI-XbaI fragment of pDDM3 was removed and replaced by the 0.25 kb EcoR1-XbaI fragment from pTPI66 with the intact repressor binding site. It showed induction by IPTG and synthesized 7-8-fold more enzyme than the pTP166 bearing cells. Cells with this plasmid pDDM4 produced more enzyme even in the absence of IPTG than in the fully induced pTP166. To render repression of the tac promoter more efficient, the lacI gene from plasmid pUC19 [7] in a 0.4 kb TaqI fragment (filled in with Klenow DNA polymerase) was inserted in the unique Pt,uII site of the plasmid pDDM4. Insertions of lacl in both orientations were obtained giving rise to p[asmids pDDM6 and pDDM7, respectively. Uninduced cells containing the plasmid pDDM6 produced considerably lower amount of enzyme as compared to pDDM3 or pDDM4 transformed cells, but a 20-fold induction was obtained by IPTG (Table l). In the induced cells the amount of enzyme was about l 1-fold higher than in the induced cells carrying pTP166. The cells with pDDM7 also produced a very high amount of enzyme but, for unknown reasons, the uninduced level was very high and only 2-fold induction was obtained with IPTG.
TABLE I
Dam-Mtase activity in difJ?rent plasmid bearing E. coli GM48 (dam, dcm) cell extracts Cells were cultured at 37°C in double strength TY medium containing ampicillin (100 m g / m l ) . IPTG (1 mM) was added at A~,0c) = 0.5 to 0.6 in half of the cultures and they were allowed to grow for 3 h before harvesting. Cells were broken in 5 x buffer (w/v) containing 50 mM Tris (pH 8.0), 300 m M NaCI, 5 mM EDTA, 1 mM phenylmethanesulfonyl fluoride and 11)% glycerol. After centrifugation at 121)01) rpm for 10 min, the supernatant was saved and used for assays. Assay mixture (lfl0 ml) contained 1 ml cell-extract (11 + 1 mg protein), 1 mCi [methyl-3H]AdoMet (15 m C i / m M ) , and 10 mg E. coil dam dcm. D N A in 51) m M Tris (pH 8.0), 10 mM E D T A and 2 m M dithiothreitol. Incubation: 31) rain at 37°C. Plasmid in E. coli GM48
Counts/min IPTG
+ IPTG
Induction (-fold)
pTP 166 pDDM 3 pDDM 4 pDDM 6 pDDM 7
2998 58827 20 121 51157 56 638
I 1 022 77716 78002 118 133 131/9(111
3.7 1.5 3.9 23 2.3
We are grateful to Prof. M.G. Marinus for plasmid pTP166. Work supported by European Community grant No. SC1"-CT90-0472 (TSTS). References 1 2 3 4 5
Modrich, P. (1987) Annu. Rev. Biochem. 56, 435-466. Marinus, M.G., Poteete, A. and Arraj, A. (1984) Gene 28, 123-125. Herman, G.E. and Modrich, P. (1981) J. Bacteriol. 145, 644-646. Heitman, J. and Model, P. 11987) J. Bacteriol. 169, 3243 3250. Hiilsmann, K.-H., Quaas, R., Georgalis, Y., Saenger, W. and Hahn, U. (1991) Gene 98, 83-88. 6 Marinus, M.G. (19731 Mol. Gen. Genet. 127, 47-55. 7 Yanisch-Perron, C., Viera, J. and Messing, J. (1985) Gene 33, 103-119.
Biochimica et Biophysica Acta, 1132 (1992) 309-310 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
BBAEXP 90403
Rapid Report
Improved plasmids containing the Escherichia coil dam gene under the control of the tac promoter Sibajyoti Guha 1 and Wilhelm Guschlbauer Serrice de Bioehimie et G~netique Mol~culaire, B~t. 142, D~partement de Biologie Cellulaire et Mol~eulaire, Centre d'Etudes de Saclay,
Gif-sur-Yrette (France) (Received 19 June 1992) Key words: Plasmid; dam gene; tac promoter; (E. coli) We report the construction of a series of plasmids containing the dam gene under the contol of the tac promoter. Cells containing these plasmids produce about 8 to 10-fold more Dam methyltransferase (Mtase) than the previously used plasmid pTP166 and avoid the use of a high temperature step necessary for the expression of Dam Mtase in the plasmid pDOX1 and thus allow its use for the study of thermosensitive mutants.
T h e d a m g e n e of Escherichia coli c o d e s for D a m m e t h y l t r a n s f e r a s e ( M t a s e ) which m e t h y l a t e s a d e n i n e in t h e s e q u e n c e 5 ' - G A T C - 3 ' . D a m m e t h y l a t i o n is involved in v a r i o u s c e l l u l a r p r o c e s s e s a n d is r e s p o n s i b l e for s t r a n d d i s c r i m i n a t i o n in m u t H L S m e d i a t e d mism a t c h r e p a i r [1]. H i g h D a m m e t h y l a t i o n in E. coli, however, p r o v o k e s h y p e r m u t a t i o n [2,3] as well as S O S r e s p o n s e [4]. T h e s e p r o p e r t i e s have h a m p e r e d t h e construction o f an a p p r o p r i a t e h i g h - c o p y - n u m b e r p l a s m i d o v e r p r o d u c i n g D a m M t a s e . It is t h e r e f o r e n e c e s s a r y to p u t this g e n e u n d e r the c o n t r o l o f a r e g u l a t a b l e p r o m o t e r . A p l a s m i d , pTP166, in which the d a m g e n e is u n d e r t h e c o n t r o l o f t h e i n d u c i b l e tac p r o m o t e r , has b e e n c o n s t r u c t e d [2]. T h e a m o u n t of D a m M t a s e p r o d u c e d from t h e s e p l a s m i d b e a r i n g ceils is, however, not very high, even w h e n the p r o m o t e r is fully i n d u c e d by isopropylthiogalactoside (IPTG). Another plasmid, p D O X 1 , in which the d a m g e n e is p l a c e d u n d e r the c o n t r o l o f a h e a t - i n d u c i b l e p r o m o t e r cI [5], was constructed; from p D O X 1 b e a r i n g cells c o n s i d e r a b l e a m o u n t s of D a m M t a s e can b e isolated. A high temp e r a t u r e for i n d u c t i o n a n d s u b s e q u e n t i n c u b a t i o n excludes its use in t h e r m o s e n s i t i v e E. coli m u t a n t s a n d also w h e n the expression of t h e r m o s e n s i t i v e d a m mutants in the p l a s m i d is studied.
• ~ i ~ d ~ ,. ~X
E
0.25 kb
I tac
X
.,,~"'f~'.,~
co + X b a
/ "
%
p
P
dam
/) P
~ |
Tlad
puc19 I
..~.~Pvull
linearized
04kb',~
Taq fragment
~
E dam
M6
E Ncl
Correspondence to: W. Guschlbauer, Service de Biochimie et G6netique Mol6culaire, B~t. 142, D6partement de Biologie Cellulaire et Mol6culaire, Centre d'Etudes de Saclay, F-91191, Gif-sur-Yvette, France. i Present address: Institut Jacques Monod, Universit6 Paris VII, 2, Place Jussieu, F-75251, Paris Cedex 05, France.
Fig. 1. Schematic drawing of the construction of pDDM6 containing
the dam and /acl genes. Sites: B, BamHI; E, EcoRl; P, PrulI; T, TaqI; X, XbaI. In pDDM3 the dark line around the XbaI site (not to scale) indicates the Bal31 deleted portion. Arrows in pDDM6 indicate polarity of the genes. In pDDM7 the lacl gene is in an orientation opposite to that of plasmid pDDM6.
310 To obtain a high-expression tacpromoter controlled dam gene, the plasmid pTP166 [2] was XbaI linearized and digested by Bal31 to remove nucleotides upstream of the coding sequence of the dam gene (Fig. 1). The pool of the Bal31 digested plasmid was religated in the presence of a XbaI linker (5'-CTCTAGAG-3') and used for transformation of a E. coli dam mutant GM48 [6]. A transformant with one of the plasmids, pDDM3, containing the Xba site had high enzyme yield. But in this plasmid the nucleotides constituting the lac repressor binding site (rb in Fig. 1) had also been removed and it showed only a negligible induction by IPTG. To restore the repressor binding site, the 235 bp EcoRI-XbaI fragment of pDDM3 was removed and replaced by the 0.25 kb EcoR1-XbaI fragment from pTPI66 with the intact repressor binding site. It showed induction by IPTG and synthesized 7-8-fold more enzyme than the pTP166 bearing cells. Cells with this plasmid pDDM4 produced more enzyme even in the absence of IPTG than in the fully induced pTP166. To render repression of the tac promoter more efficient, the lacI gene from plasmid pUC19 [7] in a 0.4 kb TaqI fragment (filled in with Klenow DNA polymerase) was inserted in the unique Pt,uII site of the plasmid pDDM4. Insertions of lacl in both orientations were obtained giving rise to p[asmids pDDM6 and pDDM7, respectively. Uninduced cells containing the plasmid pDDM6 produced considerably lower amount of enzyme as compared to pDDM3 or pDDM4 transformed cells, but a 20-fold induction was obtained by IPTG (Table l). In the induced cells the amount of enzyme was about l 1-fold higher than in the induced cells carrying pTP166. The cells with pDDM7 also produced a very high amount of enzyme but, for unknown reasons, the uninduced level was very high and only 2-fold induction was obtained with IPTG.
TABLE I
Dam-Mtase activity in difJ?rent plasmid bearing E. coli GM48 (dam, dcm) cell extracts Cells were cultured at 37°C in double strength TY medium containing ampicillin (100 m g / m l ) . IPTG (1 mM) was added at A~,0c) = 0.5 to 0.6 in half of the cultures and they were allowed to grow for 3 h before harvesting. Cells were broken in 5 x buffer (w/v) containing 50 mM Tris (pH 8.0), 300 m M NaCI, 5 mM EDTA, 1 mM phenylmethanesulfonyl fluoride and 11)% glycerol. After centrifugation at 121)01) rpm for 10 min, the supernatant was saved and used for assays. Assay mixture (lfl0 ml) contained 1 ml cell-extract (11 + 1 mg protein), 1 mCi [methyl-3H]AdoMet (15 m C i / m M ) , and 10 mg E. coil dam dcm. D N A in 51) m M Tris (pH 8.0), 10 mM E D T A and 2 m M dithiothreitol. Incubation: 31) rain at 37°C. Plasmid in E. coli GM48
Counts/min IPTG
+ IPTG
Induction (-fold)
pTP 166 pDDM 3 pDDM 4 pDDM 6 pDDM 7
2998 58827 20 121 51157 56 638
I 1 022 77716 78002 118 133 131/9(111
3.7 1.5 3.9 23 2.3
We are grateful to Prof. M.G. Marinus for plasmid pTP166. Work supported by European Community grant No. SC1"-CT90-0472 (TSTS). References 1 2 3 4 5
Modrich, P. (1987) Annu. Rev. Biochem. 56, 435-466. Marinus, M.G., Poteete, A. and Arraj, A. (1984) Gene 28, 123-125. Herman, G.E. and Modrich, P. (1981) J. Bacteriol. 145, 644-646. Heitman, J. and Model, P. 11987) J. Bacteriol. 169, 3243 3250. Hiilsmann, K.-H., Quaas, R., Georgalis, Y., Saenger, W. and Hahn, U. (1991) Gene 98, 83-88. 6 Marinus, M.G. (19731 Mol. Gen. Genet. 127, 47-55. 7 Yanisch-Perron, C., Viera, J. and Messing, J. (1985) Gene 33, 103-119.