Mutation Research, 243 (1990) 267-272 Elsevier
267
MUTLET 0325
Expression of the recA gene is reduced in Escherichia coli topoisomerase I mutants Amparo Urios, Guadalupe Herrera, Vicente Aleixandre and Manuel Blanco lnstituto de Investigaciones Citol6gicas de la Caja de Ahorros de Valencia (Centro asociado del CSIC), 46010 Valencia (Spain) (Accepted 9 November 1989)
Keywords: recA gene; Topoisomerase I; DNA gyrase; SOS mutagenesis
Summary We studied the influence of DNA topological changes on Escherichia coli recA gene expression. This was monitored by measuring ~3-galactosidase activity in cells containing a recA-lacZ fusion. To modulate DNA supercoiling we used mutations in the genes encoding for topoisomerase I and DNA gyrase. After either UV irradiation or treatment with the gyrase inhibitor ciprofloxacin, induction of the recA gene was reduced in topAlO mutants, this reduction being alleviated when gyrA or gyrB mutations causing DNA relaxation were present. A reduced induction of recA was also observed after incubation of cells carrying the recA441 mutation at 42°C in the presence of adenine. Using bacteria deficient in the LexA repressor, we have demonstrated that the topA 10 mutation reduces the constitutive expression of the recA gene. We suggest that the increase in negative supercoiling resulting from topoisomerase I deficiency interferes with transcription from the recA promoter. The reduction in the expression of the recA gene in topAlO bacteria could determine their increased UV sensitivity as well as their partial defectiveness in SOS mutability.
The repair of DNA damage could be influenced by variations in the degree of supercoiling of the DNA molecule (cf. Drlica, 1984; Wang, 1985). This influence might occur either by modifying the access of repair enzymes to the DNA, or by altering the expression of the genes encoding for these enzymes. The latter possibility is particularly interesting given that DNA damage stimulates, in Escherichia coli, the expression of several repair genes as a part of the so-called SOS response. InCorrespondence: Dr. M. Blanco, Instituto de Investigaciones Citol6gicas, Amadeo de Saboya 4, 46010 Valencia (Spain).
duction of the SOS response occurs when an activated form of RecA protein mediates the proteolytic cleavage of the LexA protein, the repressor of recA and the other SOS genes (reviewed by Walker, 1984). An influence of DNA superhelicity on SOSdependent phenomena has been suggested by the absence of UV mutability in Salmonella typhimurium topA mutants, deficient in topoisomerase I (Overbye and Margolin, 1981). In contrast, no significant effect of gyrB mutations on UV mutability was found in E. coli strains (Bridges et al., 1983).
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
268 In this p a p e r we e x a m i n e the influence o f D N A t o p o l o g i c a l c h a n g e s on the expression o f the recA gene in E. coli. D N A s u p e r c o i l i n g was m o d u l a t e d using m u t a t i o n s in the genes e n c o d i n g for either t o p o i s o m e r a s e I or D N A gyrase ( A l e i x a n d r e et al., in press). O u r results i n d i c a t e that a deficiency in t o p o i s o m e r a s e I, due to the t o p A l O m u t a t i o n ( S t e r n g l a n z et al., 1981), reduces the i n d u c e d exp r e s s i o n o f the recA gene, as well as its constitutive e x p r e s s i o n o c c u r r i n g in the a b s e n c e o f the L e x A r e p r e s s o r . This a b n o r m a l e x p r e s s i o n o f recA c o u l d d e t e r m i n e the o t h e r c h a r a c t e r i s t i c s exhibited b y t o p A l O m u t a n t s , such as UV sensitivity a n d low ind u c e d m u t a b i l i t y . W e also show that gyrA a n d g y r B m u t a t i o n s r e d u c i n g the degree o f D N A supercoiling are able to p r o m o t e a significant increase in the e x p r e s s i o n o f the recA gene when t o p o i s o m e r a s e I is deficient.
Materials and methods Bacterial strains, phages and p l a s m i d s T h e E. coli K12 strains used are s h o w n in T a b l e
1. In the t r a n s d u c t i o n s to c o n s t r u c t strains c a r r y i n g t o p A , gyrA o r gyrB m u t a t i o n s , the recipient bacteria contained plasmid plCV90 (Aleixandre a n d B l a n c o , 1987), so t h a t the g e n o t y p e o f the t r a n s d u c t a n t s was r e c o g n i z e d a c c o r d i n g to the level o f a m p i c i l l i n resistance c o n f e r r e d by this p l a s m i d to the host cell: the t o p A l O m u t a t i o n d e c r e a s e d w h e r e a s the gyrA a n d gyrB m u t a t i o n s i n c r e a s e d the cellular level o f a m p i c i l l i n resistance. P l a s m i d l e s s derivatives were o b t a i n e d by s p o n t a n e o u s curing. T h e p h a g e used was k G E 2 7 2 , which carries a ~ ( r e c A ' - l a c Z + ) o p e r o n f u s i o n ( W e i s e m a n n et al., 1984). Media Y M 9 b u f f e r c o n t a i n e d 11 g N a z H P O 4 - 7HzO, 3 g K H z P O 4 , 1 g NH4C1 a n d 5 g N a C I p e r liter. This s o l u t i o n , after it h a d been a u t o c l a v e d , was supp l e m e n t e d with 0.1 m M CaCI2 a n d 1 m M MgSO4. Y M 9 C m e d i u m was Y M 9 b u f f e r s u p p l e m e n t e d with 400 m g c a s a m i n o acids (Difco), 2 g glucose a n d 2 m g t h i a m i n e per liter. Y M 9 C solid m e d i u m
TABLE 1 BACTERIAL STRAINS Strain
Relevant genotype
Source or reference"
GC3217
recA441sfiAll thr leu thi pro argE3 his-4 ilvts topAlO trpC22::TnlO top + uvrA6 thr leu thi pro argE3 his-4 sup-3 7 strA As IC1797 but topAlO topAlO gyrB226 zid::TnlO As IC1797 but (kGE272) As IC1798 but (kGE272) As IC2016 but gyrB226 zid::TnlO As GC3217 but (kGE272) As IC2029 but topAlO trpC22::Tnl0 As GC3217 but topAlO trpC22::TnlO As IC2029 but lexA71::Tn5 uvrA155 As IC2031 but trp + As IC2038 but topAlO trpC22: :TnlO As IC2053 but trp ÷ gyrA944zeg::TnlO As IC2016 but gyrA944 zeg::TnlO
J. George
IC1693 IC1797 IC1798 IC1818 IC2015 IC2016 IC2028 IC2029 IC2030 IC2031 IC2038 IC2049 IC2053 IC2057 IC2083 IC2143
Our collection b Our collection b Our collection b Our collection" This paper This paper PI, IC1818 × IC2016 This paper P1, IC1693xIC2029 P1, IC1693 ×GC3217 This paperd PI, IC1798 × IC2031 P1, IC1693 × IC2038 P1, IC1798 × 1C2053 Our collection e P1, IC2083 x IC2016
P1, PI transduction. b The detailed construction of this strain is described in Aleixandre et al. (in press). cgyrB226 is a gyrB allele causing a decrease in DNA supercoiling (DiNardo et al., 1982). a lexA71::Tn5 and uvrA155 mutations were introduced by P1 transduction from SC30 TFSP (Witkin and Kogoma, 1984). e gyrA944 is a gyrA allele originating both decrease in DNA supercoiling and low level of quinolone resistance (Aleixandre et al., in press).
was Y M 9 C solidified with 1.5°70 Difco agar. L i m i t i n g a r g i n i n e A N B m e d i u m was Y M 9 b u f f e r s u p p l e m e n t e d with 1070 ( v o l / v o l ) Difco n u t r i e n t b r o t h , 2 m g / m l glucose, 60 /~g/ml each o f t h r e o n i n e , leucine, p r o l i n e , histidine, isoleucine a n d valine, 2 tzg/ml o f t h i a m i n e , a n d 1.5070 Difco a g a r . Viable b a c t e r i a were s c o r e d in this m e d i u m s u p p l e m e n t e d with 200 t~g/ml arginine. Soft a g a r c o n t a i n e d 0.607o Difco agar. L B T a n d L A T m e d i a have been d e s c r i b e d p r e v i o u s l y ( A l e i x a n d r e a n d B l a n c o , 1987).
269
UP" irradiation A germicidal lamp with a maximal output at 254 nm was used for UV irradiation. Doses were measured with a Latarjet dosimeter. Ciprofloxacin treatment Bacteria were grown in YM9C at 37°C to about 108 cells/ml. To 1-ml samples, ciprofloxacin (a gift of Bayer) was added to give the desired final concentration and then incubated with shaking at 37°C in the dark. At the indicated times, the samples were assayed for/3-galactosidase activity. Bacterial mutagenesis The reversion of the argE3 ochre mutation was measured as follows. Bacteria grown in YM9C at 37°C to about 10s cells/ml were centrifuged and resuspended at the same concentration in YM9 buffer for UV irradiation. Aliquots of this suspension (0.1 ml) were added to 2.5 ml of soft agar and poured onto ANB plates. Arg ÷ colonies were scored after 3 days at 37°C. For RecA441-dependent mutagenesis 0.1-ml samples of resuspended cells were spread on ANB plates and incubated at the indicated temperature. Measurement of ~-galactosidase /3-Galactosidase activity was assayed in cells grown under appropriate conditions as described by Weisemann et al. (1984). Enzyme assays were performed and units of activity expressed as described by Miller (1972).
Results and discussion
Induced and constitutive expression of the recA gene A low level of/3-galactosidase activity was induced in topAlO mutants containing a recA-lacZ fusion by UV irradiation (Fig. 1A) or by treatment with the gyrase inhibitor ciprofloxacin (Fig. 1B). Induction of recA promoted by activated RecA441 protein was also reduced in the presence of the topAlO mutation (Fig. 1C). The increase in DNA supercoiling occurring in topAlO mutants can be
2O
A
18 ~U
o o~2
"0 "-
/
* 0
o6
10
i[¢ 0
/]
o
/
/
// Y
@
tCF 1
0 lime,
t !
a 2
t 42"c.ad i i 0 1
h
Fig. 1. Effect of topAlO mutation on induction of recA-lacZ fusion. ~-Galactosidase-specific activity was assayed in recA + cells after treatment with either 3 J / m 2 of UV radiation (A) or 0.05 /zg/ml ciprofloxacin (B), and in recA441 mutants after growth at 42°C in the presence of 100/~g/ml adenine (C). Strains (A, B): 0 , top + (IC2015); O, topAlO(IC2016); (C): II, top + (IC2029); E3, topAlO (IC2030).
compensated by means of gyr mutations which reduce supercoiling below that of the wild type (DiNardo et al., 1982; Pruss et al., 1982). We introduced well-defined gyr mutations (Aleixandre et al., in press) into our topAlO strain and observed that the UV-induced level of ~-galactosidase synthesis in the topA gyr double mutants increased significantly, although it was still lower than that in top + (Table 2). A similar result was found after ciprofloxacin treatment (Table 2), which in addition to its inducing ability might cause DNA relaxation. These results indicated that a decrease in supercoiling partially compensated for the topoisomerase I deficiency in order to allow a normal recA gene expression. The low induction of the recA gene in topAlO bacteria could result from either a reduced gene expression or an abnormal inactivation of the LexA repressor by the RecA protease. To differentiate between these 2 possibilities, we introduced the topAlO allele into ceils containing the recA-lacZ fusion and carrying the lexA71::Tn5 null allele, which causes a complete derepression of all SOS genes (Krueger et al., 1983). Fig. 2 shows that the
270
TABLE 2 I N F L U E N C E OF gyrA A N D gyrB M U T A T I O N S ON T H E EXPRESSION OF T H E recA-lacZ GENE FUSION IN topA I0 STRAINS Strain
Relevant genotype
IC2016 IC2143 IC2028 IC2015
¢3-galactosidase acitivity (units/OD60oF
t opA 10 gyr + topAlO gyrA944 topAlO gyrB226 top + gyr +
Uninduced
With UV
With CF
1,770 2,287 1,827 2,390
4,562 6,604 8,507 12,127
5,794 8,063 8,579 12,493
a Early-log-phase cultures, grown at 37°C in YM9C medium, were divided in half. One part was UV-irradiated with 3 J / m 2, and another part was treated with 0.05 ~g/ml ciprofloxacin (CF). After 2 h of additional growth, the samples were assayed for 13-galactosidase activity.
differential rate of/3-galactosidase synthesis from the derepressed recA promoter was lower in the topAlO mutant compared to an isogenic top + strain. In the topAlO gyrB226 double mutant, the rate of 13-galactosidase synthesis was intermediate between those observed in top + and topAlO bacteria (data not shown). Moreover, expression of the recA gene in topAlO cells was increased when they were converted to top ÷ (data not shown). The deficiency in topoisomerase I had no generalized negative effect on the expression of all genes as
1800
1500
E 12oG c 900 o
o
eoo
o
0
300 0
,
0
0.1
i
L
~
i
0.2
0.3
0.4
0.5
O. D . 6 0 0 Fig. 2. Influence of topA 10 mutation on the constitutive expression of the recA-laeZ fusion in lexA71::Tn5 mutants. Samples were periodically taken from log-phase cultures grown at 37°C in YM9C, and assayed for 13-galactosidase activity. Strains: m, top + (IC2038); 0 , topAlO (IC2057).
proved by the fact that B-galactosidase activity after induction of the chromosomal lacZ gene was not reduced in lexA(Def) topAlO bacteria (data not shown). Our results therefore demonstrate that the topoisomerase I deficiency causes a rather specific reduction in the expression of the recA gene. We can speculate on how topAlO mutation might influence recA gene transcription. This influence could be mediated by the change in supercoiling promoted by topoisomerase I deficiency, and therefore the recA gene would be responsive to D N A structure or topology as happens with several prokaryotic genes (cf. Pruss and Drlica, 1989). There are potential cruciform sequences clustered in the first third of the recA gene (Sancar et al., 1980). An increase in negative D N A supercoiling could lead to cruciform extrusion (Panayotatos and Wells, 1981) followed by a disturbance of the transcription from the recA promoter (cf. Horwitz and Loeb, 1988). This possibility is supported by the fact that gyrA and gyrB mutations, which by reducing supercoiling might prevent cruciform extrusion, are able to enhance recA gene expression. The fact that this expression is not restored to the level observed in top + cells could be due to the inability of gyrase mutations to completely compensate for deficiency in topoisomerase I, or to a more direct role of this enzyme in recA expression. A direct involvement of D N A gyrase in the expression of the recA gene has been suggested (Smith, 1983). S O S mutability The diminished expression of the recA gene oc-
271
TABLE 3 UV M U T A B I L I T Y OF t o p A l O DERIVATIVES OF A B 1 8 8 6 : u v r A 6 STRAIN Strain
Genotype
UV dose ( J / m 2)
Survival (%)
IC1797
top +
0
100
9
85 50
392 570
220 600
100 47 15
15 170 180
150 490
1.5 3 IC 1798
0
t o p A 10
1.5
3
curring in topAlO mutants (Figs. 1 and 2) could interfere with their capability to induce the SOS response. A consequence of this induction is the increase in the mutation frequency promoted by a mutagenic repair process (cf. Walker, 1984). Table 3 shows that the topAlO mutation caused a moderate reduction in the frequency of UVinduced Arg ÷ revertants, although it did not completely abolish UV mutability. This result is in contrast with the findings of Overbye and Margolin (1981) indicating a total absence of mutability in S. typhirnurium topA bacteria. It is possible, however, that their results were influenced by the apparent low UV mutability of S. typhimurium strains (Walker, 1984; Herrera et al., 1988). We also examined the RecA441 mutator activity in topAlO mutants. Table 4 shows that this mutator activity was reduced in both lexA ÷ and lexA(Def) ceils carrying the topAlO mutation. The reduction in the induced mutability of the TABLE 4 RecA441 M U T A T O R E F F E C T IN l e x A ÷ A N D l e x A 7 1 : : T n 5 STRAINS CARRYING THE topAlO MUTATION Strain
Genotype
°C
Arg ÷ revertants per plate a - Ad
Arg ÷ revertants/plate
Induced Arg+/107 viable cells
topAlO ceils correlated well with the observed decrease in recA gene expression. We cannot exclude, however, that this mutability is also affected by changes in the expression of other genes such as, for example, the umuDC operon, known to be involved in SOS mutagenesis (Walker, 1984; Blanco et al., 1986). As seen in Table 3, the topAlO mutation increased the sensitivity of excision-deficient cells to UV irradiation. This effect could result from the altered induction of SOS mutagenic repair shown above. Moreover, topoisomerase I deficiency, which is known to negatively influence some recombination pathways (Fishel and Kolodner, 1984; and data not shown), could also affect other repair mechanisms, such as post-replicative recombination repair.
Acknowledgements This work was supported in part by Comisi6n Asesora Cientifica y T6cnica, Grant PB85-0264, and the IIC-K.U. International Molecular Biology Program. A.U. is a recipient of a Carmen y Severo Ochoa Fellowship from the Ayuntamiento de Valencia.
+ Ad
GC3217 IC2049
lexA + top + lexA ÷ topAlO
42 42
58 29
272 86
IC2038 IC2057
lexA71 t o p + lexA71 topAlO
37 37
167 64
482 150
a + Ad: A N B medium with adenine (100 #g/ml).
References
Aleixandre, V., and M. Blanco (1987) Heterogeneity in the level o f ampicillin resistance conferred by pBR322 derivatives with different D N A supercoiling, Mol. Gem Genet., 209, 56-60.
272
Blanco, M., G. Herrera and V. Aleixandre (1986) Different efficiency of UmuDC and MucAB proteins for UV light-induced mutagenesis in Escherichia coli, Mol. Gen. Genet., 205, 234-239. Bridges, B.A., M.W. Southworth and E. Orr (1983) Mutagenic repair in Escherichia coli, VIII. Effect of gyrB mutations on ultraviolet light mutagenesis, Mutation Res., 112, 3-16. DiNardo, S., K.A. Voelkel, R. Sternglanz, A.E. Reynolds and A. Wright (1982) Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes, Cell, 31, 43-51. Drlica, K. (1984) Biology of bacterial deoxyribonucleic acid topoisomerases, Microbiol. Rev., 48,273-289. Fishel, R.A., and R. Kolodner (1984) Escherichia coli strains containing mutations in the structural gene for topoisomerase I are recombination deficient, J. Bacteriol., 160, 1168-1170. Herrera, G., A. Urios, V. Aleixandre and M. Blanco (1988) UV light-induced mutability in Salmonella strains containing the u m u D C or the m u c A B operon: evidence for a umuC function, Mutation Res., 198, 9-13. Horwitz, M.S.Z., and L.A. Loeb (1988) An E. coli promoter that regulates transcription by DNA superhelix-induced cruciform extrusion, Science, 241,703-705. Krueger, J.H., S.J. Elledge and G.C. Walker (1983) Isolation and characterization of Tn5 insertion mutations in the lexA gene of Escherichia coli, J. Bacteriol., 153, 1368-1378. Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Overbye, K.M., and P. Margolin (1981) Role of the s u p X gene in ultraviolet light-induced mutagenesis in Salmonella typhirnurium, J. Bacteriol., 146, 170-178.
Panayotatos, N., and R.D. Wells (1981) Cruciform structures in supercoiled DNA, Nature (London), 289, 466-470. Pruss, G.J., S.H. Manes and K. Drlica (1982) Escherichia coli DNA topoisomerase l mutants: increased supercoiling is corrected by mutations near gyrase genes, Cell, 31, 35-42. Sancar, A., C. Stachelek, W. Konigsberg and W.D. Rupp (1980) Sequences of the recA gene and protein, Proc. Natl. Acad. Sci. (U.S.A.), 77, 2611-2615. Smith, C.L. (1983) recF-dependent induction of recA synthesis by coumermycin, a specific inhibitor of the B subunit of DNA gyrase, Proc. Natl. Acad. Sci. (U.S.A.), 80, 2510-2513. Sternglanz, R., S. DiNardo, K.A. Voelkel, Y. Nishimura, Y. Hirota, K. Becherer, L. Zumstein and J.C. Wang (1981) Mutations in the gene coding for Escherichia coli DNA topoisomerase I affect transcription and transposition, Proc. Natl. Acad. Sci. (U.S.A.), 78, 2747-2751. Walker, G.C. (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli, Microbiol. Rev., 48, 60-93. Wang, J.C. (1985) DNA topoisomerases, Annu. Rev. Biochem., 54, 665-697. Weisemann, J.M., C. Funk and G.M. Weinstock (1984) Measurement of in vivo expression of the recA gene of Escherichia coli by using lacZ gene fusions, J. Bacteriol., 160, 112-121. Witkin, E.M., and T. Kogoma (1984) Involvement of the activated form of RecA protein in SOS mutagenesis and stable DNA replication in Escherichia coli, Proc. Natl. Acad. Sci. (U.S.A.), 81, 7539-7543. Communicated by F.H. Sobels
267
MUTLET 0325
Expression of the recA gene is reduced in Escherichia coli topoisomerase I mutants Amparo Urios, Guadalupe Herrera, Vicente Aleixandre and Manuel Blanco lnstituto de Investigaciones Citol6gicas de la Caja de Ahorros de Valencia (Centro asociado del CSIC), 46010 Valencia (Spain) (Accepted 9 November 1989)
Keywords: recA gene; Topoisomerase I; DNA gyrase; SOS mutagenesis
Summary We studied the influence of DNA topological changes on Escherichia coli recA gene expression. This was monitored by measuring ~3-galactosidase activity in cells containing a recA-lacZ fusion. To modulate DNA supercoiling we used mutations in the genes encoding for topoisomerase I and DNA gyrase. After either UV irradiation or treatment with the gyrase inhibitor ciprofloxacin, induction of the recA gene was reduced in topAlO mutants, this reduction being alleviated when gyrA or gyrB mutations causing DNA relaxation were present. A reduced induction of recA was also observed after incubation of cells carrying the recA441 mutation at 42°C in the presence of adenine. Using bacteria deficient in the LexA repressor, we have demonstrated that the topA 10 mutation reduces the constitutive expression of the recA gene. We suggest that the increase in negative supercoiling resulting from topoisomerase I deficiency interferes with transcription from the recA promoter. The reduction in the expression of the recA gene in topAlO bacteria could determine their increased UV sensitivity as well as their partial defectiveness in SOS mutability.
The repair of DNA damage could be influenced by variations in the degree of supercoiling of the DNA molecule (cf. Drlica, 1984; Wang, 1985). This influence might occur either by modifying the access of repair enzymes to the DNA, or by altering the expression of the genes encoding for these enzymes. The latter possibility is particularly interesting given that DNA damage stimulates, in Escherichia coli, the expression of several repair genes as a part of the so-called SOS response. InCorrespondence: Dr. M. Blanco, Instituto de Investigaciones Citol6gicas, Amadeo de Saboya 4, 46010 Valencia (Spain).
duction of the SOS response occurs when an activated form of RecA protein mediates the proteolytic cleavage of the LexA protein, the repressor of recA and the other SOS genes (reviewed by Walker, 1984). An influence of DNA superhelicity on SOSdependent phenomena has been suggested by the absence of UV mutability in Salmonella typhimurium topA mutants, deficient in topoisomerase I (Overbye and Margolin, 1981). In contrast, no significant effect of gyrB mutations on UV mutability was found in E. coli strains (Bridges et al., 1983).
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
268 In this p a p e r we e x a m i n e the influence o f D N A t o p o l o g i c a l c h a n g e s on the expression o f the recA gene in E. coli. D N A s u p e r c o i l i n g was m o d u l a t e d using m u t a t i o n s in the genes e n c o d i n g for either t o p o i s o m e r a s e I or D N A gyrase ( A l e i x a n d r e et al., in press). O u r results i n d i c a t e that a deficiency in t o p o i s o m e r a s e I, due to the t o p A l O m u t a t i o n ( S t e r n g l a n z et al., 1981), reduces the i n d u c e d exp r e s s i o n o f the recA gene, as well as its constitutive e x p r e s s i o n o c c u r r i n g in the a b s e n c e o f the L e x A r e p r e s s o r . This a b n o r m a l e x p r e s s i o n o f recA c o u l d d e t e r m i n e the o t h e r c h a r a c t e r i s t i c s exhibited b y t o p A l O m u t a n t s , such as UV sensitivity a n d low ind u c e d m u t a b i l i t y . W e also show that gyrA a n d g y r B m u t a t i o n s r e d u c i n g the degree o f D N A supercoiling are able to p r o m o t e a significant increase in the e x p r e s s i o n o f the recA gene when t o p o i s o m e r a s e I is deficient.
Materials and methods Bacterial strains, phages and p l a s m i d s T h e E. coli K12 strains used are s h o w n in T a b l e
1. In the t r a n s d u c t i o n s to c o n s t r u c t strains c a r r y i n g t o p A , gyrA o r gyrB m u t a t i o n s , the recipient bacteria contained plasmid plCV90 (Aleixandre a n d B l a n c o , 1987), so t h a t the g e n o t y p e o f the t r a n s d u c t a n t s was r e c o g n i z e d a c c o r d i n g to the level o f a m p i c i l l i n resistance c o n f e r r e d by this p l a s m i d to the host cell: the t o p A l O m u t a t i o n d e c r e a s e d w h e r e a s the gyrA a n d gyrB m u t a t i o n s i n c r e a s e d the cellular level o f a m p i c i l l i n resistance. P l a s m i d l e s s derivatives were o b t a i n e d by s p o n t a n e o u s curing. T h e p h a g e used was k G E 2 7 2 , which carries a ~ ( r e c A ' - l a c Z + ) o p e r o n f u s i o n ( W e i s e m a n n et al., 1984). Media Y M 9 b u f f e r c o n t a i n e d 11 g N a z H P O 4 - 7HzO, 3 g K H z P O 4 , 1 g NH4C1 a n d 5 g N a C I p e r liter. This s o l u t i o n , after it h a d been a u t o c l a v e d , was supp l e m e n t e d with 0.1 m M CaCI2 a n d 1 m M MgSO4. Y M 9 C m e d i u m was Y M 9 b u f f e r s u p p l e m e n t e d with 400 m g c a s a m i n o acids (Difco), 2 g glucose a n d 2 m g t h i a m i n e per liter. Y M 9 C solid m e d i u m
TABLE 1 BACTERIAL STRAINS Strain
Relevant genotype
Source or reference"
GC3217
recA441sfiAll thr leu thi pro argE3 his-4 ilvts topAlO trpC22::TnlO top + uvrA6 thr leu thi pro argE3 his-4 sup-3 7 strA As IC1797 but topAlO topAlO gyrB226 zid::TnlO As IC1797 but (kGE272) As IC1798 but (kGE272) As IC2016 but gyrB226 zid::TnlO As GC3217 but (kGE272) As IC2029 but topAlO trpC22::Tnl0 As GC3217 but topAlO trpC22::TnlO As IC2029 but lexA71::Tn5 uvrA155 As IC2031 but trp + As IC2038 but topAlO trpC22: :TnlO As IC2053 but trp ÷ gyrA944zeg::TnlO As IC2016 but gyrA944 zeg::TnlO
J. George
IC1693 IC1797 IC1798 IC1818 IC2015 IC2016 IC2028 IC2029 IC2030 IC2031 IC2038 IC2049 IC2053 IC2057 IC2083 IC2143
Our collection b Our collection b Our collection b Our collection" This paper This paper PI, IC1818 × IC2016 This paper P1, IC1693xIC2029 P1, IC1693 ×GC3217 This paperd PI, IC1798 × IC2031 P1, IC1693 × IC2038 P1, IC1798 × 1C2053 Our collection e P1, IC2083 x IC2016
P1, PI transduction. b The detailed construction of this strain is described in Aleixandre et al. (in press). cgyrB226 is a gyrB allele causing a decrease in DNA supercoiling (DiNardo et al., 1982). a lexA71::Tn5 and uvrA155 mutations were introduced by P1 transduction from SC30 TFSP (Witkin and Kogoma, 1984). e gyrA944 is a gyrA allele originating both decrease in DNA supercoiling and low level of quinolone resistance (Aleixandre et al., in press).
was Y M 9 C solidified with 1.5°70 Difco agar. L i m i t i n g a r g i n i n e A N B m e d i u m was Y M 9 b u f f e r s u p p l e m e n t e d with 1070 ( v o l / v o l ) Difco n u t r i e n t b r o t h , 2 m g / m l glucose, 60 /~g/ml each o f t h r e o n i n e , leucine, p r o l i n e , histidine, isoleucine a n d valine, 2 tzg/ml o f t h i a m i n e , a n d 1.5070 Difco a g a r . Viable b a c t e r i a were s c o r e d in this m e d i u m s u p p l e m e n t e d with 200 t~g/ml arginine. Soft a g a r c o n t a i n e d 0.607o Difco agar. L B T a n d L A T m e d i a have been d e s c r i b e d p r e v i o u s l y ( A l e i x a n d r e a n d B l a n c o , 1987).
269
UP" irradiation A germicidal lamp with a maximal output at 254 nm was used for UV irradiation. Doses were measured with a Latarjet dosimeter. Ciprofloxacin treatment Bacteria were grown in YM9C at 37°C to about 108 cells/ml. To 1-ml samples, ciprofloxacin (a gift of Bayer) was added to give the desired final concentration and then incubated with shaking at 37°C in the dark. At the indicated times, the samples were assayed for/3-galactosidase activity. Bacterial mutagenesis The reversion of the argE3 ochre mutation was measured as follows. Bacteria grown in YM9C at 37°C to about 10s cells/ml were centrifuged and resuspended at the same concentration in YM9 buffer for UV irradiation. Aliquots of this suspension (0.1 ml) were added to 2.5 ml of soft agar and poured onto ANB plates. Arg ÷ colonies were scored after 3 days at 37°C. For RecA441-dependent mutagenesis 0.1-ml samples of resuspended cells were spread on ANB plates and incubated at the indicated temperature. Measurement of ~-galactosidase /3-Galactosidase activity was assayed in cells grown under appropriate conditions as described by Weisemann et al. (1984). Enzyme assays were performed and units of activity expressed as described by Miller (1972).
Results and discussion
Induced and constitutive expression of the recA gene A low level of/3-galactosidase activity was induced in topAlO mutants containing a recA-lacZ fusion by UV irradiation (Fig. 1A) or by treatment with the gyrase inhibitor ciprofloxacin (Fig. 1B). Induction of recA promoted by activated RecA441 protein was also reduced in the presence of the topAlO mutation (Fig. 1C). The increase in DNA supercoiling occurring in topAlO mutants can be
2O
A
18 ~U
o o~2
"0 "-
/
* 0
o6
10
i[¢ 0
/]
o
/
/
// Y
@
tCF 1
0 lime,
t !
a 2
t 42"c.ad i i 0 1
h
Fig. 1. Effect of topAlO mutation on induction of recA-lacZ fusion. ~-Galactosidase-specific activity was assayed in recA + cells after treatment with either 3 J / m 2 of UV radiation (A) or 0.05 /zg/ml ciprofloxacin (B), and in recA441 mutants after growth at 42°C in the presence of 100/~g/ml adenine (C). Strains (A, B): 0 , top + (IC2015); O, topAlO(IC2016); (C): II, top + (IC2029); E3, topAlO (IC2030).
compensated by means of gyr mutations which reduce supercoiling below that of the wild type (DiNardo et al., 1982; Pruss et al., 1982). We introduced well-defined gyr mutations (Aleixandre et al., in press) into our topAlO strain and observed that the UV-induced level of ~-galactosidase synthesis in the topA gyr double mutants increased significantly, although it was still lower than that in top + (Table 2). A similar result was found after ciprofloxacin treatment (Table 2), which in addition to its inducing ability might cause DNA relaxation. These results indicated that a decrease in supercoiling partially compensated for the topoisomerase I deficiency in order to allow a normal recA gene expression. The low induction of the recA gene in topAlO bacteria could result from either a reduced gene expression or an abnormal inactivation of the LexA repressor by the RecA protease. To differentiate between these 2 possibilities, we introduced the topAlO allele into ceils containing the recA-lacZ fusion and carrying the lexA71::Tn5 null allele, which causes a complete derepression of all SOS genes (Krueger et al., 1983). Fig. 2 shows that the
270
TABLE 2 I N F L U E N C E OF gyrA A N D gyrB M U T A T I O N S ON T H E EXPRESSION OF T H E recA-lacZ GENE FUSION IN topA I0 STRAINS Strain
Relevant genotype
IC2016 IC2143 IC2028 IC2015
¢3-galactosidase acitivity (units/OD60oF
t opA 10 gyr + topAlO gyrA944 topAlO gyrB226 top + gyr +
Uninduced
With UV
With CF
1,770 2,287 1,827 2,390
4,562 6,604 8,507 12,127
5,794 8,063 8,579 12,493
a Early-log-phase cultures, grown at 37°C in YM9C medium, were divided in half. One part was UV-irradiated with 3 J / m 2, and another part was treated with 0.05 ~g/ml ciprofloxacin (CF). After 2 h of additional growth, the samples were assayed for 13-galactosidase activity.
differential rate of/3-galactosidase synthesis from the derepressed recA promoter was lower in the topAlO mutant compared to an isogenic top + strain. In the topAlO gyrB226 double mutant, the rate of 13-galactosidase synthesis was intermediate between those observed in top + and topAlO bacteria (data not shown). Moreover, expression of the recA gene in topAlO cells was increased when they were converted to top ÷ (data not shown). The deficiency in topoisomerase I had no generalized negative effect on the expression of all genes as
1800
1500
E 12oG c 900 o
o
eoo
o
0
300 0
,
0
0.1
i
L
~
i
0.2
0.3
0.4
0.5
O. D . 6 0 0 Fig. 2. Influence of topA 10 mutation on the constitutive expression of the recA-laeZ fusion in lexA71::Tn5 mutants. Samples were periodically taken from log-phase cultures grown at 37°C in YM9C, and assayed for 13-galactosidase activity. Strains: m, top + (IC2038); 0 , topAlO (IC2057).
proved by the fact that B-galactosidase activity after induction of the chromosomal lacZ gene was not reduced in lexA(Def) topAlO bacteria (data not shown). Our results therefore demonstrate that the topoisomerase I deficiency causes a rather specific reduction in the expression of the recA gene. We can speculate on how topAlO mutation might influence recA gene transcription. This influence could be mediated by the change in supercoiling promoted by topoisomerase I deficiency, and therefore the recA gene would be responsive to D N A structure or topology as happens with several prokaryotic genes (cf. Pruss and Drlica, 1989). There are potential cruciform sequences clustered in the first third of the recA gene (Sancar et al., 1980). An increase in negative D N A supercoiling could lead to cruciform extrusion (Panayotatos and Wells, 1981) followed by a disturbance of the transcription from the recA promoter (cf. Horwitz and Loeb, 1988). This possibility is supported by the fact that gyrA and gyrB mutations, which by reducing supercoiling might prevent cruciform extrusion, are able to enhance recA gene expression. The fact that this expression is not restored to the level observed in top + cells could be due to the inability of gyrase mutations to completely compensate for deficiency in topoisomerase I, or to a more direct role of this enzyme in recA expression. A direct involvement of D N A gyrase in the expression of the recA gene has been suggested (Smith, 1983). S O S mutability The diminished expression of the recA gene oc-
271
TABLE 3 UV M U T A B I L I T Y OF t o p A l O DERIVATIVES OF A B 1 8 8 6 : u v r A 6 STRAIN Strain
Genotype
UV dose ( J / m 2)
Survival (%)
IC1797
top +
0
100
9
85 50
392 570
220 600
100 47 15
15 170 180
150 490
1.5 3 IC 1798
0
t o p A 10
1.5
3
curring in topAlO mutants (Figs. 1 and 2) could interfere with their capability to induce the SOS response. A consequence of this induction is the increase in the mutation frequency promoted by a mutagenic repair process (cf. Walker, 1984). Table 3 shows that the topAlO mutation caused a moderate reduction in the frequency of UVinduced Arg ÷ revertants, although it did not completely abolish UV mutability. This result is in contrast with the findings of Overbye and Margolin (1981) indicating a total absence of mutability in S. typhirnurium topA bacteria. It is possible, however, that their results were influenced by the apparent low UV mutability of S. typhimurium strains (Walker, 1984; Herrera et al., 1988). We also examined the RecA441 mutator activity in topAlO mutants. Table 4 shows that this mutator activity was reduced in both lexA ÷ and lexA(Def) ceils carrying the topAlO mutation. The reduction in the induced mutability of the TABLE 4 RecA441 M U T A T O R E F F E C T IN l e x A ÷ A N D l e x A 7 1 : : T n 5 STRAINS CARRYING THE topAlO MUTATION Strain
Genotype
°C
Arg ÷ revertants per plate a - Ad
Arg ÷ revertants/plate
Induced Arg+/107 viable cells
topAlO ceils correlated well with the observed decrease in recA gene expression. We cannot exclude, however, that this mutability is also affected by changes in the expression of other genes such as, for example, the umuDC operon, known to be involved in SOS mutagenesis (Walker, 1984; Blanco et al., 1986). As seen in Table 3, the topAlO mutation increased the sensitivity of excision-deficient cells to UV irradiation. This effect could result from the altered induction of SOS mutagenic repair shown above. Moreover, topoisomerase I deficiency, which is known to negatively influence some recombination pathways (Fishel and Kolodner, 1984; and data not shown), could also affect other repair mechanisms, such as post-replicative recombination repair.
Acknowledgements This work was supported in part by Comisi6n Asesora Cientifica y T6cnica, Grant PB85-0264, and the IIC-K.U. International Molecular Biology Program. A.U. is a recipient of a Carmen y Severo Ochoa Fellowship from the Ayuntamiento de Valencia.
+ Ad
GC3217 IC2049
lexA + top + lexA ÷ topAlO
42 42
58 29
272 86
IC2038 IC2057
lexA71 t o p + lexA71 topAlO
37 37
167 64
482 150
a + Ad: A N B medium with adenine (100 #g/ml).
References
Aleixandre, V., and M. Blanco (1987) Heterogeneity in the level o f ampicillin resistance conferred by pBR322 derivatives with different D N A supercoiling, Mol. Gem Genet., 209, 56-60.
272
Blanco, M., G. Herrera and V. Aleixandre (1986) Different efficiency of UmuDC and MucAB proteins for UV light-induced mutagenesis in Escherichia coli, Mol. Gen. Genet., 205, 234-239. Bridges, B.A., M.W. Southworth and E. Orr (1983) Mutagenic repair in Escherichia coli, VIII. Effect of gyrB mutations on ultraviolet light mutagenesis, Mutation Res., 112, 3-16. DiNardo, S., K.A. Voelkel, R. Sternglanz, A.E. Reynolds and A. Wright (1982) Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes, Cell, 31, 43-51. Drlica, K. (1984) Biology of bacterial deoxyribonucleic acid topoisomerases, Microbiol. Rev., 48,273-289. Fishel, R.A., and R. Kolodner (1984) Escherichia coli strains containing mutations in the structural gene for topoisomerase I are recombination deficient, J. Bacteriol., 160, 1168-1170. Herrera, G., A. Urios, V. Aleixandre and M. Blanco (1988) UV light-induced mutability in Salmonella strains containing the u m u D C or the m u c A B operon: evidence for a umuC function, Mutation Res., 198, 9-13. Horwitz, M.S.Z., and L.A. Loeb (1988) An E. coli promoter that regulates transcription by DNA superhelix-induced cruciform extrusion, Science, 241,703-705. Krueger, J.H., S.J. Elledge and G.C. Walker (1983) Isolation and characterization of Tn5 insertion mutations in the lexA gene of Escherichia coli, J. Bacteriol., 153, 1368-1378. Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Overbye, K.M., and P. Margolin (1981) Role of the s u p X gene in ultraviolet light-induced mutagenesis in Salmonella typhirnurium, J. Bacteriol., 146, 170-178.
Panayotatos, N., and R.D. Wells (1981) Cruciform structures in supercoiled DNA, Nature (London), 289, 466-470. Pruss, G.J., S.H. Manes and K. Drlica (1982) Escherichia coli DNA topoisomerase l mutants: increased supercoiling is corrected by mutations near gyrase genes, Cell, 31, 35-42. Sancar, A., C. Stachelek, W. Konigsberg and W.D. Rupp (1980) Sequences of the recA gene and protein, Proc. Natl. Acad. Sci. (U.S.A.), 77, 2611-2615. Smith, C.L. (1983) recF-dependent induction of recA synthesis by coumermycin, a specific inhibitor of the B subunit of DNA gyrase, Proc. Natl. Acad. Sci. (U.S.A.), 80, 2510-2513. Sternglanz, R., S. DiNardo, K.A. Voelkel, Y. Nishimura, Y. Hirota, K. Becherer, L. Zumstein and J.C. Wang (1981) Mutations in the gene coding for Escherichia coli DNA topoisomerase I affect transcription and transposition, Proc. Natl. Acad. Sci. (U.S.A.), 78, 2747-2751. Walker, G.C. (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli, Microbiol. Rev., 48, 60-93. Wang, J.C. (1985) DNA topoisomerases, Annu. Rev. Biochem., 54, 665-697. Weisemann, J.M., C. Funk and G.M. Weinstock (1984) Measurement of in vivo expression of the recA gene of Escherichia coli by using lacZ gene fusions, J. Bacteriol., 160, 112-121. Witkin, E.M., and T. Kogoma (1984) Involvement of the activated form of RecA protein in SOS mutagenesis and stable DNA replication in Escherichia coli, Proc. Natl. Acad. Sci. (U.S.A.), 81, 7539-7543. Communicated by F.H. Sobels
