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A recombinant foot-and-mouth disease virus antigen inhibits DNA replication and triggers the SOS response in Escherichia coli
ELSEVIER
FEMS Microbiology
Letters 129 (1995) 157-162
A recombinant foot-and-mouth disease virus antigen inhibits DNA replication and triggers the SOS response in Escherichia coli A. Benito, E. Viaplana, J.L. Corchero, X. Carbonell, A. Villaverde
*
Institut de Biologia Fonamental and Departament de Gen&ica i Microbiologia, Universitat Authnoma de Barcelona, Bellaterra, 08193 Barcelona, Spain Received 28 March 1995; accepted 13 April 1995
Abstract The 30 gene of foot-and-mouth disease virus encodes the viral RNA dependent RNA polymerase, also called virus infection associated (VIA) antigen, which is the most important serological marker of virus infection. This 30 gene from a serotype Cl virus has been cloned and overexpressed in Escherichiu coli under the control of the strong lambda lytic promoters. The resulting 51 kDa recombinant protein has been shown to be immunoreactive with sera from infected animals. After induction of gene expression, an immediate and dramatic arrest of cell DNA synthesis occurs, similar to that produced by genotoxic doses of the drug mitomycin C. This effect does not occur during the production of either a truncated VIA antigen or other related and non-related viral proteins. The inhibition of DNA replication results in a subsequent induction of the host SOS DNA-repair response and in an increase of the mutation frequency in the surviving cells. Keywords: Escherichia coli; Recombinant protein; Virus infection associated antigen (VIA); Foot-and-mouth disease virus
(FMDV); SOS response
1. Introduction Overexpression of plasmid-encoded foreign genes in Escherichia coli allows to obtain eukaryotic polypeptides for both basic research and industrial purposes. Many recombinant proteins from mammalian cells or viruses are toxic for E. coli and consequently promote the selection of plasmid-free cells, even when produced at low levels. Several
* Corresponding author. Tel: (343) 5812011; E-mail: [email protected]
037%1097/95/$09.50 SD1
5812148;
Fax:
(343)
0 1995 Federation of European Microbioiogical
0378-1097(95)00151-4
independent strategies such as obtaining fusion proteins, the deletion of toxic domains, or the promotion of inclusion bodies formation can contribute to reducing these undesirable effects [l]. Although a general basis for the cytotoxicity of heterologous proteins on bacteria has not been found, some mechanisms have been proposed for specific proteins, mainly enzymes, whose activity might interfere with normal cell functions. Moreover, the overproduction of many heterologous proteins causes expression of lon [2] and other heat-shock genes 131, whose activity is usually associated with the recovery from cellular stress. Societies. All rights reserved
158
A. Benito et al. /FEMS Microbiology Letters 129 (1995) 157-162
In this report, we describe the molecular cloning and the expression in E. coli of the 30 gene of foot-and-mouth disease virus (FMDV) under the control of the bacteriophage lambda thermosensitive mutant repressor CI857. This gene encodes the viral replicase, also known as the virus infection associated (VIA) antigen [4], which is a powerful antigenic marker of virus replication in infected animals. The synthesis of VIA antigen immediately blocks cell DNA replication, induces transcription of SOS genes and significantly increases the mutation frequency in the surviving cells. This particular toxic effect, promoted by the unmodified VIA antigen, offers a new and interesting model to study the genetics and regulation of stress responses in recombinant E. coli cells.
2. Materials and methods
2.1. Plasmids, strains and cloning procedures Plasmid pBR-VFACl-18.5 carries a long region of cDNA from FMDV genome (isolate CS&l), including the complete 30 gene [5]. Plasmid DNA was obtained by alkaline lysis and digested to completion with EcoRI. Amplification of the 30 gene segment was done by using the high fidelity Vent R polymerase (Biolabs) in the presence of 250 PM of each dNTP and 3.5 mM MgSO,. Upstream 3Dl (5’-GGGTTGATCGTTGAT-3’) and downstream 3D2 (5’-GGGAA’ITCTGCGCGTCCGCACAC-3’) primers were designed to amplify the complete 30 gene. The 1410-bp long DNA fragment was recovered from agarose gel and introduced into plasmid pJLA602 [6] previously digested with Ncol and treated with PolKI. The resulting recombinant vector p3D12 encodes 30 gene under the control of pL and pR promoters placed in tandem and also carries the cZ8.57 repressor gene. p3D12 was further introduced in both E. coli strains MC1061 araD139 mcrB A(araABC-leu)7697 Ala&74 galU galK h&h! rpsL thi strA, and HBlOl hsdS20 (r; rn,) h&M supE44 arall galK2 lacy1 proA rpsL20 ~$15 mtll recAl3 mcrB. The recombinant 3D protein is expected to carry an extra methionine at the N-terminus and a short peptide of 13 aa at the C-terminus. p3DCla is a p3D12 derivative in which a stop codon
has been introduced by cutting, filling-in and ligating by the unique Clal site within the 30 gene. The product encoded by p3DCla is a truncated VIA antigen carrying only the 132 N-terminal amino acids plus a short missense C-terminal peptide. 2.2. Growth, conditions for gene expression analysis of the recombinant antigens
and
Recombinant cultures were grown in LB medium with 100 pg/ml ampicillin at 28” C until an OD,,, of about 0.3. Then, shaker flasks were transferred to a water bath prewarmed at 42” C and the induction was extended for 4 h. Periodically, samples were taken and quantitative analysis of recombinant proteins was done by densitometry of Coomassie bluestained gels. Western blot was also performed as described elsewhere [7] using sera from FMDV infected pigs and from rabbits immunized against a recombinant, FMDV serotype 0 VIA antigen. Total protein was determined by standard procedures [8]. 2.3. Monitoring SOS gene expression levels and analysis of mutation frequency GE864 is a MC4100 derivative containing a chromosomal cea::lacZ gene fusion controlled by LexA repressor [9] and was used to monitor SOS response after inducing 30 gene expression from p3D12. Several pJLA602 derivatives, and also a different CI857-based expression vector (pMV8), all of them encoding other viral genes, were introduced into GE864 as controls. pM619VPl encodes an inactive /3-galactosidase enzyme carrying an inserted segment of VP1 FMDV protein [lo], pMV8 encodes a tnmcated VP1 and the non-structural 2A and a truncated 2B FMDV proteins [ll], and pJVP1, pJXC1 and pJRV26 encode the complete FMDV VPl, bacteriophage P22 p9 and rabbit hemorragic disease virus p60 proteins. These constructs are detailed elsewhere. The presence of all these proteins in induced cultures was checked by Western blot using appropriate sera. PGalactosidase enzymatic units are presented as corrected by the optical density of the cultures. AB1157 is Escherichia coli F; thrl, let&, hisG4, proA2, argE3, thil, lacY1, galk2, aral4, xy11.5, mtll, tsxK33, rpo1122, rpsL, and Al32463 is its recAl3 derivative. Cultures of these strains, once
A. Ben&oet al. / FEMS Microbiology Letters 129 (1995) 157-162
159
transformed with p3D12, were grown in CAM9 plus 0.2% glucose and 100 fig/ml ampicillin and 30 gene expression was induced as described above. After 2 h, cells were concentrated by centrifugation and plated on M9 plus glucose and all the required amino acids (at 25 pg/ml) but without histidine. Colonies were counted after incubation at 28” C during 48 h. pJLacZ is a pJLA602 derivative producing the complete E. coli P-galactosidase [7] and was used as a control. 2.4. Pulse-labelling
of cell DNA
GE864/p3D12 and GE864/p3DCla cells were growing in CAM9 medium plus ampicillin and glucose (at standard concentrations) at 28” C until exponential phase. Cultures were then submitted to temperature shift or treated with 1.6 pg/ml mitomycin C. The rate of DNA synthesis was measured by 2-min pulse labelling of 0.5 ml samples as described [12], with [3H]-thymidine (1 &i; specific activity 85 Ci/mM).
3. Results and discussion Cells carrying p3D12 were growing at 28” C until the exponential phase. After induction by shifting to 42” C, a protein of about 51 kDa was observed in cell extracts. This product was specifically recognixed by polyclonal sera from FMDV-infected pigs and by a rabbit polyclonal serum raised against FMDV serotype 0 VIA antigen [13] (Fig. 1). At 3 h post-induction, the recombinant protein present in cell extracts was estimated to be 5% (in MC10611 and 18% (in HBlOl) of the total cell protein. In MC1061 (RecA+) cells expressing the recombinant 30 gene, a clear inhibition of cell division was repeatedly observed in different experiments (not shown) but not in MC1061 cells harbouring pJLA602 after shifting to 42” C. This symptom of cellular stress can be evoked in E. coli by several stimuli. One of the best known is the induction of the SOS DNA repair system which is controlled by RecA and LexA proteins [14]. This inducible mechanism is triggered in response to the DNA damage produced by W-irradiation, by treatment with chemical genotoxic compounds and by inhibition of DNA
Fig. 1. Coomassie brilliant blue stained PAGE of HESlOl/p3DCla (1) and HBlOl/p3D12 (2) cells after 3 h of induction. The recombinant VIAA is immunodetected by a polyclonal sera obtained against a recombinant, fused VIAA from isolated 0, Kb (3) and by serum of a FMDV-infected pig (4). An arrow indicates the position of the Sl-kDa product.
replication in conditional mutants. During SOS response, RecA protein is activated and about 20 LexA-repressed SOS genes are coordinately transcribed [15] by RecA-mediated LexA self-cleavage. The product of one of them, &A, is responsible for inhibition of cell division. To investigate whether filamentation promoted by the VIA antigen could be related with DNA repair activities, SOS induction was explored in p3D12-carrying cells by studying transcription of a tightly repressed SOS gene called tea [16]. Results presented in Fig. 2 confirm that the synthesis of the recombinant 3D protein promotes expression of tea. As a control, other recombinant viral proteins were produced in the same thermo-inducible system but none of them revealed SOS-inducing activity. Moreover, a truncated 3D protein (3DCla) only carrying the 132 N-terminal amino acids plus a short missense C-terminal peptide was also inefficient as an inducer of tea expression (Fig. 2B). These results indicate a specific ability of 30 gene product in promoting SOS response for which a N-terminal segment of the protein is not sufficient. The levels of tea transcription achieved during synthesis of VIA antigen were comparable to those
A. Benito et al. /FEMS
160
Microbiology
Letters 129 (1995) 157-162
B
Time
Fig. 2. tea gene expression levels (in /?-galactosidase enzymatic units). (A) GF%4/p3D12 cells incubated at 28 and 42” C in absence ( - MC) or presence (+ MC) of 1.6 /.&g/ml mitomycin C; (B) GE864 cells carrying different pJLA602 derivatives (with the exception of pMV8) incubated at 42” C in absence ( -) and in presence of mitomycin C ( + 1.
obtained after treating the cells with 1.6 pg/ml of mitomycin C (Fig. 2A). This DNA crosslinking drug is a powerful inhibitor of DNA replication and has a good SOS-inducing activity at the dose used in this work. Since interruption of DNA replication is the most common mechanism for activation of RecA, DNA synthesis was monitored by [3H]-thymidine pulse labelling in induced p3D12 carrying cells. As shown in Fig. 3, a dramatic arrest of DNA synthesis was observed immediately after induction of the full-length 30 gene expression but not of the mutant, truncated 3DClu gene. This fact was detected immediately after thermal induction and even before the recombinant protein was evident by Western blot analysis (not shown). The inhibition of DNA synthesis explains by itself the induction of SOS system by
Table 1 His- reversion Plasmid p3D12 pJLacZ
frequency
in E. coli cells 2 h after expression
AH1157 28” C
42” C
2.3 f 0.9 X 1O-9 1.2 f 0.4 x 10-s
7.8 f 1.1 1.0 f 0.6
X x
Fig. 3. DNA synthesis was studied in heat-induced GE864/p3D12 (0) and GE864/p3DCla (V) cells. A GE864/p3DlZ culture was kept at 28” C and treated with 1.6 pg/ml mitomycin C (0). The arrow indicates the moment of induction at 42” C or the addition of the drug.
activation of RecA protein and the subsequent RecA-assisted self-cleavage of LexA repressor. However, other alternative mechanisms for 3Dmediated expression of SOS genes (such as putative interactions between VIA antigen and proteins RecA and LexA) were investigated with negative results (not shown). Therefore, the 3D promoted inhibition of DNA replication seems to be the only cause of the SOS response. As a consequence of the SOS-dependent errorprone DNA replication, a significant enhancement of the mutation frequency was also observed (Table 1). Surprisingly, even in RecA- cells that are deficient in the SOS response and mutagenesis, a slight increase in the mutation frequency was also detected. This fact suggests that, irrespective of the indirect SOS-dependent mutagenesis caused by VIA antigen, the viral protein itself might have a direct, although low, mutagenic activity.
of recombinant
10-s lo-’
(min)
FMDV 30 and E. coli 1acZ genes AH2463 28” C
42’ C
8.0 f 0.8 X lo-’ 1.8 + 0.4 X 1O-9
4.1 f 1.3 x 1o-8 1.9 f 0.5 x lo- 9
A. Ben&o et al. / FEMS Microbiology Letters 129 (1995) 157-162
The results presented in this work demonstrate that the FMDV VIA antigen is able to block DNA synthesis in vivo, resulting in the induction of DNA repair mechanisms. As far as we know, the only recombinant viral protein that has been shown to induce the SOS response when overproduced in bacterial cells is the A’ protein of the bacteriophage @X174 [17], because this polypeptide is involved in the shut-down of host DNA synthesis during virus multiplication. The features of 3D protein described above represent the first observation of a protein involved in RNA replication which interferre with DNA replication. However, it is feasible that other recombinant mammalian or viral proteins could also produce toxicity on E. coli by stimulating DNA repair responses. On the other hand, and since the genetics of E. coli SOS system are well characterized, the controlled production of VIA antigen could offer a good model to study the performance of recombinant cells during an extremely complex stress situation. On the other hand, data from FMDV persistent infections in cell cultures have revealed a co-evolution of both cell and resident virus [18] and a rapid phenotypic and genotypic variation of the host cells during the establishment of persistance [19]. Therefore, it has been suggested that a yet unidentified mechanism associated with virus replication could be responsible for this cellular genetic instability [19]. Although the mechanics by which the 30 gene product can trigger DNA repair system in recombinant E. coli cells could be restricted to this non-natural system or favoured by the lack of compartimentation in bacteria, the immediate arrest of DNA replication after induction of gene expression might be the consequence of a natural, intrinsic activity of the FMDV 3D protein that could also stimulate analogous DNA repair systems in FMDV-infected mammalian cells. Further experiments with recombinant 3D protein expressed in cell culture would help to check this possibility.
Acknowledgements We thank J. Checa and V. Farreres for technical assistance, J.E.G. McCarthy and M. Schnarr for generously providing plasmid pJLA602 and purified
161
I.exA protein respectively, and E. Domingo, J. Martin, S. de la Luna and M. Defais for critical comments and discussion. We are also indebted to F. Sobrino and J. Plana for the generous gift of polyclonal sera against 3D. This work has been supported by CICYT, Spain (grant BI092-05031, and partially by CIRIT of Catalonia. A.B. and X.C. are recipients of predoctoral fellowships from MEC and DGU respectively.
References [I] Marston, F.A.O. (1986) The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem. J. 240, 1-12. [2] Goff, S. and Goldberg, A.L. (1985) Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes. Cell 41, 587-595. [3] Allen, S.P., Polaxxi, J.O., Gierse, J.K. and Easton, A.M. (1992) Two novel heat shock genes encoding proteins produced in response to heterologous protein expression in Escherichia coli. J. Bacterial. 174, 6938-6947. [4] Newman, J.F.E., Carhvright, B., Doel, T.R. and Brown, F. (1979) Purification and identification of the RNA-dependent RNA polymerase of foot-and-mouth disease virus. J. Gen. Virol. 45, 497-507. [5] Villanueva, N., Dkila, M., Ortin, J. and Domingo, E. (1983) Molecular cloning of cDNA from foot-and-mouth disease virus Cl-Santa Pau (C-S8). Sequence of protein-VPl-coding segment. Gene 23, 185-194. 161 Schauder, B., Blocker, H., Frank, B. and McCarthy, J.E.G. (19871 Inducible expression vectors incorporating the Escherichia coli apE translational initiation region. Gene 52, 279-283. [7] Benito, A., Vidal, M. and Villaverde, A. (1993) Enhanced production of p,-controlled recombinant proteins and plasmid stability in Escherichia coli RecA+ strains. J. Biotechnol. 29, 299-306. [8] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248254. [9] Larminat, F. and Defais, M. (1989) Modulation of the SOS response by truncated RecA proteins. Mol. Gen. Genet. 216, 106-112. [lo] Benito, A. and Villaverde, A. (1994) Insertion of a 27 amino acid viral peptide in different zones of Escherichiu coli /3-galactosidase: Effects on the enzyme activity. FEMS Microbiol. Lett. 123, 107-112. [ll] Vidal, M., Cair6, J., Mateu, M.G. and Villaverde, A. (1991) Molecular cloning and expression of the VP1 gene of footand-mouth disease virus C, in E. coli: effect on bacterial cell viability. Appl. Microbial. Biotechnol. 35, 788-792.
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[12] Khidhir, A., Casaregola, S. and Holland, LB. (1985) Mechanism of transient inhibition of DNA synthesis in ultravioletirradiated E. coli: Inhibition is independent of recA whilst recovery requires RecA protein itself and an additional, inducible SOS function. Mol. Gen. Genet. 199, 133-140. [13] Strebel, K., Beck, E., Strohmaier, K. and Schaller, H. (1986) Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins. J. Virol. 57, 983-991. [14] Walker, G.C. (1984) Mutagenesis and inducibles responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol. Rev. 48, 60-92. [15] Peterson, K.R., Ossana, N., Thliveris, A.T., Ennis, D.G. and Mount, D.W. (1988) Derepression of specific genes promotes DNA repair and mutagenesis in Escherichia coli. J. Bacteriol. 170, 1-4.
Letters 129 (1995) 157-162 [16] Ebina, Y., Takahara, Y., Kishi, F., Nakazawa, A. and Brent, R. (1983) LexA protein is a repressor of the colicin El gene. J. Biol. Chem. 258, 13258-13261. 1171 Colasanti, J. and Denhardt, D.T. (1985) Expression of the cloned bacteriophage @X174 A* gene in Escherichia co& inhibits DNA replication and cell division. J. Viral. 53, 807-813. [18] de La Terre, J.C., Martinez-Salas, E., Diez, J., Villaverde, A., Gebauer, F., Rocha, E., Davila, M. and Domingo, E. (1988) Coevolution of cells and viruses in a persistent infection of foot-and-mouth disease virus in cell culture. J. Virol. 62, 2050-2058. [19] Martin, A.M., Carrillo, E.C., Sevilla, N. and Domingo, E. (1994) Rapid cell variation can determine the stablishment of a persistent viral infection. Proc. Natl. Acad. Sci. USA 91, 3705-3709.
FEMS Microbiology
Letters 129 (1995) 157-162
A recombinant foot-and-mouth disease virus antigen inhibits DNA replication and triggers the SOS response in Escherichia coli A. Benito, E. Viaplana, J.L. Corchero, X. Carbonell, A. Villaverde
*
Institut de Biologia Fonamental and Departament de Gen&ica i Microbiologia, Universitat Authnoma de Barcelona, Bellaterra, 08193 Barcelona, Spain Received 28 March 1995; accepted 13 April 1995
Abstract The 30 gene of foot-and-mouth disease virus encodes the viral RNA dependent RNA polymerase, also called virus infection associated (VIA) antigen, which is the most important serological marker of virus infection. This 30 gene from a serotype Cl virus has been cloned and overexpressed in Escherichiu coli under the control of the strong lambda lytic promoters. The resulting 51 kDa recombinant protein has been shown to be immunoreactive with sera from infected animals. After induction of gene expression, an immediate and dramatic arrest of cell DNA synthesis occurs, similar to that produced by genotoxic doses of the drug mitomycin C. This effect does not occur during the production of either a truncated VIA antigen or other related and non-related viral proteins. The inhibition of DNA replication results in a subsequent induction of the host SOS DNA-repair response and in an increase of the mutation frequency in the surviving cells. Keywords: Escherichia coli; Recombinant protein; Virus infection associated antigen (VIA); Foot-and-mouth disease virus
(FMDV); SOS response
1. Introduction Overexpression of plasmid-encoded foreign genes in Escherichia coli allows to obtain eukaryotic polypeptides for both basic research and industrial purposes. Many recombinant proteins from mammalian cells or viruses are toxic for E. coli and consequently promote the selection of plasmid-free cells, even when produced at low levels. Several
* Corresponding author. Tel: (343) 5812011; E-mail: [email protected]
037%1097/95/$09.50 SD1
5812148;
Fax:
(343)
0 1995 Federation of European Microbioiogical
0378-1097(95)00151-4
independent strategies such as obtaining fusion proteins, the deletion of toxic domains, or the promotion of inclusion bodies formation can contribute to reducing these undesirable effects [l]. Although a general basis for the cytotoxicity of heterologous proteins on bacteria has not been found, some mechanisms have been proposed for specific proteins, mainly enzymes, whose activity might interfere with normal cell functions. Moreover, the overproduction of many heterologous proteins causes expression of lon [2] and other heat-shock genes 131, whose activity is usually associated with the recovery from cellular stress. Societies. All rights reserved
158
A. Benito et al. /FEMS Microbiology Letters 129 (1995) 157-162
In this report, we describe the molecular cloning and the expression in E. coli of the 30 gene of foot-and-mouth disease virus (FMDV) under the control of the bacteriophage lambda thermosensitive mutant repressor CI857. This gene encodes the viral replicase, also known as the virus infection associated (VIA) antigen [4], which is a powerful antigenic marker of virus replication in infected animals. The synthesis of VIA antigen immediately blocks cell DNA replication, induces transcription of SOS genes and significantly increases the mutation frequency in the surviving cells. This particular toxic effect, promoted by the unmodified VIA antigen, offers a new and interesting model to study the genetics and regulation of stress responses in recombinant E. coli cells.
2. Materials and methods
2.1. Plasmids, strains and cloning procedures Plasmid pBR-VFACl-18.5 carries a long region of cDNA from FMDV genome (isolate CS&l), including the complete 30 gene [5]. Plasmid DNA was obtained by alkaline lysis and digested to completion with EcoRI. Amplification of the 30 gene segment was done by using the high fidelity Vent R polymerase (Biolabs) in the presence of 250 PM of each dNTP and 3.5 mM MgSO,. Upstream 3Dl (5’-GGGTTGATCGTTGAT-3’) and downstream 3D2 (5’-GGGAA’ITCTGCGCGTCCGCACAC-3’) primers were designed to amplify the complete 30 gene. The 1410-bp long DNA fragment was recovered from agarose gel and introduced into plasmid pJLA602 [6] previously digested with Ncol and treated with PolKI. The resulting recombinant vector p3D12 encodes 30 gene under the control of pL and pR promoters placed in tandem and also carries the cZ8.57 repressor gene. p3D12 was further introduced in both E. coli strains MC1061 araD139 mcrB A(araABC-leu)7697 Ala&74 galU galK h&h! rpsL thi strA, and HBlOl hsdS20 (r; rn,) h&M supE44 arall galK2 lacy1 proA rpsL20 ~$15 mtll recAl3 mcrB. The recombinant 3D protein is expected to carry an extra methionine at the N-terminus and a short peptide of 13 aa at the C-terminus. p3DCla is a p3D12 derivative in which a stop codon
has been introduced by cutting, filling-in and ligating by the unique Clal site within the 30 gene. The product encoded by p3DCla is a truncated VIA antigen carrying only the 132 N-terminal amino acids plus a short missense C-terminal peptide. 2.2. Growth, conditions for gene expression analysis of the recombinant antigens
and
Recombinant cultures were grown in LB medium with 100 pg/ml ampicillin at 28” C until an OD,,, of about 0.3. Then, shaker flasks were transferred to a water bath prewarmed at 42” C and the induction was extended for 4 h. Periodically, samples were taken and quantitative analysis of recombinant proteins was done by densitometry of Coomassie bluestained gels. Western blot was also performed as described elsewhere [7] using sera from FMDV infected pigs and from rabbits immunized against a recombinant, FMDV serotype 0 VIA antigen. Total protein was determined by standard procedures [8]. 2.3. Monitoring SOS gene expression levels and analysis of mutation frequency GE864 is a MC4100 derivative containing a chromosomal cea::lacZ gene fusion controlled by LexA repressor [9] and was used to monitor SOS response after inducing 30 gene expression from p3D12. Several pJLA602 derivatives, and also a different CI857-based expression vector (pMV8), all of them encoding other viral genes, were introduced into GE864 as controls. pM619VPl encodes an inactive /3-galactosidase enzyme carrying an inserted segment of VP1 FMDV protein [lo], pMV8 encodes a tnmcated VP1 and the non-structural 2A and a truncated 2B FMDV proteins [ll], and pJVP1, pJXC1 and pJRV26 encode the complete FMDV VPl, bacteriophage P22 p9 and rabbit hemorragic disease virus p60 proteins. These constructs are detailed elsewhere. The presence of all these proteins in induced cultures was checked by Western blot using appropriate sera. PGalactosidase enzymatic units are presented as corrected by the optical density of the cultures. AB1157 is Escherichia coli F; thrl, let&, hisG4, proA2, argE3, thil, lacY1, galk2, aral4, xy11.5, mtll, tsxK33, rpo1122, rpsL, and Al32463 is its recAl3 derivative. Cultures of these strains, once
A. Ben&oet al. / FEMS Microbiology Letters 129 (1995) 157-162
159
transformed with p3D12, were grown in CAM9 plus 0.2% glucose and 100 fig/ml ampicillin and 30 gene expression was induced as described above. After 2 h, cells were concentrated by centrifugation and plated on M9 plus glucose and all the required amino acids (at 25 pg/ml) but without histidine. Colonies were counted after incubation at 28” C during 48 h. pJLacZ is a pJLA602 derivative producing the complete E. coli P-galactosidase [7] and was used as a control. 2.4. Pulse-labelling
of cell DNA
GE864/p3D12 and GE864/p3DCla cells were growing in CAM9 medium plus ampicillin and glucose (at standard concentrations) at 28” C until exponential phase. Cultures were then submitted to temperature shift or treated with 1.6 pg/ml mitomycin C. The rate of DNA synthesis was measured by 2-min pulse labelling of 0.5 ml samples as described [12], with [3H]-thymidine (1 &i; specific activity 85 Ci/mM).
3. Results and discussion Cells carrying p3D12 were growing at 28” C until the exponential phase. After induction by shifting to 42” C, a protein of about 51 kDa was observed in cell extracts. This product was specifically recognixed by polyclonal sera from FMDV-infected pigs and by a rabbit polyclonal serum raised against FMDV serotype 0 VIA antigen [13] (Fig. 1). At 3 h post-induction, the recombinant protein present in cell extracts was estimated to be 5% (in MC10611 and 18% (in HBlOl) of the total cell protein. In MC1061 (RecA+) cells expressing the recombinant 30 gene, a clear inhibition of cell division was repeatedly observed in different experiments (not shown) but not in MC1061 cells harbouring pJLA602 after shifting to 42” C. This symptom of cellular stress can be evoked in E. coli by several stimuli. One of the best known is the induction of the SOS DNA repair system which is controlled by RecA and LexA proteins [14]. This inducible mechanism is triggered in response to the DNA damage produced by W-irradiation, by treatment with chemical genotoxic compounds and by inhibition of DNA
Fig. 1. Coomassie brilliant blue stained PAGE of HESlOl/p3DCla (1) and HBlOl/p3D12 (2) cells after 3 h of induction. The recombinant VIAA is immunodetected by a polyclonal sera obtained against a recombinant, fused VIAA from isolated 0, Kb (3) and by serum of a FMDV-infected pig (4). An arrow indicates the position of the Sl-kDa product.
replication in conditional mutants. During SOS response, RecA protein is activated and about 20 LexA-repressed SOS genes are coordinately transcribed [15] by RecA-mediated LexA self-cleavage. The product of one of them, &A, is responsible for inhibition of cell division. To investigate whether filamentation promoted by the VIA antigen could be related with DNA repair activities, SOS induction was explored in p3D12-carrying cells by studying transcription of a tightly repressed SOS gene called tea [16]. Results presented in Fig. 2 confirm that the synthesis of the recombinant 3D protein promotes expression of tea. As a control, other recombinant viral proteins were produced in the same thermo-inducible system but none of them revealed SOS-inducing activity. Moreover, a truncated 3D protein (3DCla) only carrying the 132 N-terminal amino acids plus a short missense C-terminal peptide was also inefficient as an inducer of tea expression (Fig. 2B). These results indicate a specific ability of 30 gene product in promoting SOS response for which a N-terminal segment of the protein is not sufficient. The levels of tea transcription achieved during synthesis of VIA antigen were comparable to those
A. Benito et al. /FEMS
160
Microbiology
Letters 129 (1995) 157-162
B
Time
Fig. 2. tea gene expression levels (in /?-galactosidase enzymatic units). (A) GF%4/p3D12 cells incubated at 28 and 42” C in absence ( - MC) or presence (+ MC) of 1.6 /.&g/ml mitomycin C; (B) GE864 cells carrying different pJLA602 derivatives (with the exception of pMV8) incubated at 42” C in absence ( -) and in presence of mitomycin C ( + 1.
obtained after treating the cells with 1.6 pg/ml of mitomycin C (Fig. 2A). This DNA crosslinking drug is a powerful inhibitor of DNA replication and has a good SOS-inducing activity at the dose used in this work. Since interruption of DNA replication is the most common mechanism for activation of RecA, DNA synthesis was monitored by [3H]-thymidine pulse labelling in induced p3D12 carrying cells. As shown in Fig. 3, a dramatic arrest of DNA synthesis was observed immediately after induction of the full-length 30 gene expression but not of the mutant, truncated 3DClu gene. This fact was detected immediately after thermal induction and even before the recombinant protein was evident by Western blot analysis (not shown). The inhibition of DNA synthesis explains by itself the induction of SOS system by
Table 1 His- reversion Plasmid p3D12 pJLacZ
frequency
in E. coli cells 2 h after expression
AH1157 28” C
42” C
2.3 f 0.9 X 1O-9 1.2 f 0.4 x 10-s
7.8 f 1.1 1.0 f 0.6
X x
Fig. 3. DNA synthesis was studied in heat-induced GE864/p3D12 (0) and GE864/p3DCla (V) cells. A GE864/p3DlZ culture was kept at 28” C and treated with 1.6 pg/ml mitomycin C (0). The arrow indicates the moment of induction at 42” C or the addition of the drug.
activation of RecA protein and the subsequent RecA-assisted self-cleavage of LexA repressor. However, other alternative mechanisms for 3Dmediated expression of SOS genes (such as putative interactions between VIA antigen and proteins RecA and LexA) were investigated with negative results (not shown). Therefore, the 3D promoted inhibition of DNA replication seems to be the only cause of the SOS response. As a consequence of the SOS-dependent errorprone DNA replication, a significant enhancement of the mutation frequency was also observed (Table 1). Surprisingly, even in RecA- cells that are deficient in the SOS response and mutagenesis, a slight increase in the mutation frequency was also detected. This fact suggests that, irrespective of the indirect SOS-dependent mutagenesis caused by VIA antigen, the viral protein itself might have a direct, although low, mutagenic activity.
of recombinant
10-s lo-’
(min)
FMDV 30 and E. coli 1acZ genes AH2463 28” C
42’ C
8.0 f 0.8 X lo-’ 1.8 + 0.4 X 1O-9
4.1 f 1.3 x 1o-8 1.9 f 0.5 x lo- 9
A. Ben&o et al. / FEMS Microbiology Letters 129 (1995) 157-162
The results presented in this work demonstrate that the FMDV VIA antigen is able to block DNA synthesis in vivo, resulting in the induction of DNA repair mechanisms. As far as we know, the only recombinant viral protein that has been shown to induce the SOS response when overproduced in bacterial cells is the A’ protein of the bacteriophage @X174 [17], because this polypeptide is involved in the shut-down of host DNA synthesis during virus multiplication. The features of 3D protein described above represent the first observation of a protein involved in RNA replication which interferre with DNA replication. However, it is feasible that other recombinant mammalian or viral proteins could also produce toxicity on E. coli by stimulating DNA repair responses. On the other hand, and since the genetics of E. coli SOS system are well characterized, the controlled production of VIA antigen could offer a good model to study the performance of recombinant cells during an extremely complex stress situation. On the other hand, data from FMDV persistent infections in cell cultures have revealed a co-evolution of both cell and resident virus [18] and a rapid phenotypic and genotypic variation of the host cells during the establishment of persistance [19]. Therefore, it has been suggested that a yet unidentified mechanism associated with virus replication could be responsible for this cellular genetic instability [19]. Although the mechanics by which the 30 gene product can trigger DNA repair system in recombinant E. coli cells could be restricted to this non-natural system or favoured by the lack of compartimentation in bacteria, the immediate arrest of DNA replication after induction of gene expression might be the consequence of a natural, intrinsic activity of the FMDV 3D protein that could also stimulate analogous DNA repair systems in FMDV-infected mammalian cells. Further experiments with recombinant 3D protein expressed in cell culture would help to check this possibility.
Acknowledgements We thank J. Checa and V. Farreres for technical assistance, J.E.G. McCarthy and M. Schnarr for generously providing plasmid pJLA602 and purified
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I.exA protein respectively, and E. Domingo, J. Martin, S. de la Luna and M. Defais for critical comments and discussion. We are also indebted to F. Sobrino and J. Plana for the generous gift of polyclonal sera against 3D. This work has been supported by CICYT, Spain (grant BI092-05031, and partially by CIRIT of Catalonia. A.B. and X.C. are recipients of predoctoral fellowships from MEC and DGU respectively.
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