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Viral spread within ageing bacterial populations
Gene 202 (1997) 147–149
Viral spread within ageing bacterial populations E. Ramı´rez, A. Villaverde * Institut de Biologia Fonamental and Departament de Gene`tica i Microbiologia, Universitat Auto`noma de Barcelona, Bellaterra, 08193, Barcelona, Spain Received 4 March 1997; accepted 1 August 1997; Received by M. Salas
Abstract The viral spread within isolated host populations has been studied throughout the growth of P22-infected Salmonella cell colonies. By using an integration mutant of this bacteriophage, horizontal and vertical transmission have been analyzed independently. The data obtained show that both strategies are not simultaneous but consecutive during the colony development. Lytic cycles are tightly repressed during the exponential cell growth but stimulated in independent colonies with remarkable synchrony when the cell division rate decreases. The coincidence of the viral outburst and the decay of bacterial replicative fitness is a new example of the extreme viral competence in exploiting the host cells as dissemination vehicles for viral genomes. © 1997 Elsevier Science B.V. Keywords: P22; Bacteriophage; Lysogeny; Colony growth; SOS response; (Salmonella)
1. Introduction Insights into RNA viruses and retroviruses variability (Domingo et al., 1985; Steinhauer and Holland, 1987; Barre-Sinoussi, 1996) provide further evidence of the extreme adaptability of viral genomes to their continuously changing environment. Viral populations can gain replicative fitness by fixation of naturally occurring mutations that improve their biological properties in a given context. Alternatively, viruses can also stimulate their multiplication in response to conditions that, being adverse for the host, could cause loss of viral genetic information. In this line, reactivation of proviral genomes or expression of lytic genes after damaging host DNA have been described in both bacterial (Lwoff et al., 1950; Roberts and Roberts, 1975) and animal viruses (Blyth et al., 1976; Vogel et al., 1992). These functions are the result of viral mechanisms that have evolved in parallel with cell features to ensure viral maintenance when the host survival itself is compromised. In this work, we have explored the incidence of * Corresponding author. Tel.: +34 3 5812148; Fax: +34 3 5812011; e-mail: [email protected] Abbreviations: cfu, colony-forming unit(s); LB, Luria–Bertani (medium); pfu, plaque-forming unit(s). 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 4 67 - 8
productive horizontal viral transmission and non-productive vertical transference of proviral genomes to daughter cells, within a clonal population of infected bacterial cells. The analysis has been focused on the use of both strategies by the viral genome throughout the life of an isolated host population, and on the adaptative responses of the virus to the ageing, and consequent loss of replicative fitness, of the host cells. By using as a model a mutant bacteriophage unable to integrate its genome, we have detected an asymmetry in the occurrence of these transmission strategies that tends to maintain the viability of infected cells when exponentially growing, but promotes viral production and efficient horizontal spread when entering into the stationary phase.
2. Experimental and discussion We have analyzed the spread of bacteriophage P22 int7 within isolated, lysogenic colonies of Salmonella typhimurium. This virus represents an unique model to study viral dynamics within clonal populations since not all the individuals, even deriving from a single infected cell, carry the proviral genome. The integration-deficient mutant genome remains in an episomal form and does not replicate in lysogen cells (Susskind and Botstein,
148
E. Ramı´rez, A. Villaverde / Gene 202 (1997) 147–149
1978). Consequently, its vertical transmission during cell division is restricted to a mere passage to one of the daughter cells. This unusual situation, in which only a very small fraction of the progeny cells is infected, can result in an extremely high potential for viral propagation among uninfected cells, since conditions promoting reactivation of latent genomes would result in an immediate release of virus particles and the concomitant infection cycles on an increasing number of neighbouring cells. Therefore, the percentage of infected cells throughout the growth of the colony is indicative of the occurrence of lytic cycles, and it allows us to analyze separately both vertical and horizontal transmission strategies. In this scenery, it seemed reasonable to expect that productive lytic episodes in an infected colony would occur randomly and intersected with monotonous, onecell-restricted vertical virus transmission throughout the colony development, resulting in a high and nearly constant fraction of infected individuals and a regular release of virus particles from early cell divisions. Surprisingly, when examining cell populations in P22 int7 lysogenic colonies, the percentage on infected cells was very low, making it undetectable by 23 h ( Fig. 1). At latter times, a rapid increase in the infected cell fraction is detected, becoming in few hours the totality of the population. This suggests an asymmetry in the occurrence of both transmission strategies, in which the vertical, silent passage predominates in the early colony
Fig. 1. Percentage of infected cells within P22 wild type (#) and P22 int7 ($) lysogenic colonies at different times after plating. Drops from phage lysates at approx. 109 pfu/ml were placed on freshly prepared confluent cultures of Salmonella typhimurium LT2 strain, in LB plates that were further incubated at 37°C overnight. Cells from the lysis areas were spread on LB plates (at time 0) to obtain isolated colonies. Once visible, single colonies were carefully sampled at different times and spread on LB plates to study their composition by the analysis of individual cell clones. After incubation for 75 h at 37°C, viral infection in these clones was detected by the lysis areas arising after replica plating on LT2 confluent cultures. Between 50 and 100 clones were analyzed for each sample. Values obtained up to 23 h were lower than 2%.
development, whereas the horizontal propagation takes place later, concomitant to colony ageing. In agreement with this hypothesis, at 15 h, the ratio between free virus particles and host cells is lower than 10−5 in 8 of the 15 analyzed colonies and increases dramatically thereafter to reach about 20 infectious particles per cell at 90 h ( Fig. 2). This increase can only be explained by recurrent infection cycles producing an important viral amplification. Globally, the independent data depicted in Fig. 2 indicate that the outburst of virus particles is notably synchronic in separated colonies. It is worth noting also that it takes place during few host generations and when the bacterial growth rate declines (Fig. 2). By age 15, about 22 cell doublings have occurred with an average generation time of about 40 min. However, a single generation takes place between 15 and 19 h and only six more between 19 and 120 h. In conclusion, viral replication is tightly repressed during the exponential growth but efficiently stimulated during the decay of the growth rate. Then, lysis and recurrent infection cycles result in a several-order increase of infectious free particles. The basis of the proviral reactivation probably lies on the activation of the SOS DNA repair system detected
Fig. 2. Free virus particles relative to viable cells in P22 int7 lysogen colonies ($) and number of cell generations (#). Lysogenic colonies (obtained as in Fig. 1) growing on LB were removed from the plates at different times and resuspended into an sterile 0.9% NaCl solution. After gentle vortexing, an aliquot of the solution was used for viable cell counting, and another treated with chloroform to promote cell lysis, to determine the concentration of infectious particles. Before age 15, no plaque forming units were detected. At 15 h, in 8 of the 16 analyzed colonies, plaques were not observed. The relative concentration of free viruses in these colonies was estimated to be <8.0×10−7, <3.3×10−5, <1.3×10−6, <8.3×10−7, <4.5×10−7, <3.0×10−7, <4.4×10−7 and <5.8×10−7 pfu/cfu, respectively. Cell generations (n) were calculated by colony forming units (cfu) from entire colonies resuspended in 0.9% NaCl at different times, according to the following relationship: cfu=2n. To prevent any underestimation of the number of cell generations, which could be induced by the viral outburst and the consequent cell death, uninfected LT2 colonies were used for this analysis. However, matching data were obtained from P22 int7-infected colonies (not shown).
E. Ramı´rez, A. Villaverde / Gene 202 (1997) 147–149
in grown bacterial colonies ( Taddei et al., 1995). The bacterial SOS response is triggered by DNA damage but also by the arrest of intact DNA replication ( Walker, 1985), and involves the coordinated activity of more than 20 gene products which promote DNA repair and prevent cell division until replication of the cell chromosome is restored (Little and Mount, 1982). The key elements in the regulation of the SOS response are the RecA protein and the LexA repressor of SOS genes. Upon its activation by single-stranded DNA and a nucleoside triphosphate, RecA promotes the autodigestion of LexA ( Kim and Little, 1993) and the lytic repressors of bacteriophages l and P22 (Phizicky and Roberts, 1980; Kim and Little, 1993). In isolated bacterial populations, attenuated P22 virus propagates following two consecutive schemes. During rapid division of host cells, the viral genome is transmitted to the offspring without detectable productive episodes, thus guaranteeing an efficient host spreading. When the growth rate of the host population declines, lytic functions are expressed and the surviving cells become infected in very few generations. This behaviour reflects an extreme proviral sensitivity to the host growth potential, and reveals a programmed adaptability of viral replicative fitness to better maximize the host as a dissemination vehicle for the virus genomes.
Acknowledgement We are indebted to A. Poteete for generously providing wild type P22 bacteriophage and to E. Domingo for helpful discussion. We also thank J. Checa and V. Ferreres for technical assistance. This work has been
149
supported by grants BIO95-0801, CICYT, and 1995SGR 00376, CIRIT, and partially by Ma2 Francesca de Roviralta Foundation. E.R. is recipient of a predoctoral fellowship from MEC.
References Barre-Sinoussi, F., 1996. HIV as the cause of AIDS. Lancet 348, 31–35. Blyth, W.A., Hill, T.J., Field, H.J., Harbour, D.A., 1976. Reactivation of herpes simplex virus infection by ultraviolet light and possible involvement of prostaglandin. J. Gen. Virol. 33, 547–550. Domingo, E., Martı´nez-Salas, E., Sobrino, F. et al., 1985. The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance—a review. Gene 40, 1–8. Kim, B., Little, J.W., 1993. LexA and lambda CI repressors as enzymes: specific cleavage in an intermolecular reaction. Cell 73, 1165–1173. Little, J.W., Mount, D.W., 1982. The SOS regulatory system of Escherichia coli. Cell 29, 11–22. Lwoff, A., Siminovitch, L., Kjeldgaard, N., 1950. Induction de la lyse bacte´riophagique de la totalite´ d’une population microbienne lysoge`ne. CR Acad. Sci. Paris 231, 190–191. Phizicky, E.M., Roberts, J.M., 1980. Kinetics of RecA protein-directed inactivation of repressors of phage lambda and phage P22. J. Mol. Biol. 139, 319–328. Roberts, J.W., Roberts, C.W., 1975. Proteolytic cleavage of bacteriophage lambda repressor in induction. Proc. Natl. Acad. Sci. USA 72, 147–151. Steinhauer, D.A., Holland, J.J., 1987. Rapid evolution of RNA viruses. Annu. Rev. Microbiol. 41, 409–433. Susskind, M., Botstein, D., 1978. Molecular genetics of bacteriophage P22. Microbiol. Rev. 42, 385–413. Taddei, F., Matic, I., Radman, M., 1995. cAMP-Dependent SOS induction and mutagenesis in resting bacterial populations. Proc. Natl. Acad. Sci. USA 92, 11736–11740. Vogel, J., Cepeda, M., Tschachler, E., Napolitano, L.A., Jay, G., 1992. UV activation of human immunodeficiency virus gene expression in transgenic mice. J. Virol. 66, 1–5. Walker, G.C., 1985. Inducible DNA repair systems. Annu. Rev. Biochem. 54, 425–457.
Viral spread within ageing bacterial populations E. Ramı´rez, A. Villaverde * Institut de Biologia Fonamental and Departament de Gene`tica i Microbiologia, Universitat Auto`noma de Barcelona, Bellaterra, 08193, Barcelona, Spain Received 4 March 1997; accepted 1 August 1997; Received by M. Salas
Abstract The viral spread within isolated host populations has been studied throughout the growth of P22-infected Salmonella cell colonies. By using an integration mutant of this bacteriophage, horizontal and vertical transmission have been analyzed independently. The data obtained show that both strategies are not simultaneous but consecutive during the colony development. Lytic cycles are tightly repressed during the exponential cell growth but stimulated in independent colonies with remarkable synchrony when the cell division rate decreases. The coincidence of the viral outburst and the decay of bacterial replicative fitness is a new example of the extreme viral competence in exploiting the host cells as dissemination vehicles for viral genomes. © 1997 Elsevier Science B.V. Keywords: P22; Bacteriophage; Lysogeny; Colony growth; SOS response; (Salmonella)
1. Introduction Insights into RNA viruses and retroviruses variability (Domingo et al., 1985; Steinhauer and Holland, 1987; Barre-Sinoussi, 1996) provide further evidence of the extreme adaptability of viral genomes to their continuously changing environment. Viral populations can gain replicative fitness by fixation of naturally occurring mutations that improve their biological properties in a given context. Alternatively, viruses can also stimulate their multiplication in response to conditions that, being adverse for the host, could cause loss of viral genetic information. In this line, reactivation of proviral genomes or expression of lytic genes after damaging host DNA have been described in both bacterial (Lwoff et al., 1950; Roberts and Roberts, 1975) and animal viruses (Blyth et al., 1976; Vogel et al., 1992). These functions are the result of viral mechanisms that have evolved in parallel with cell features to ensure viral maintenance when the host survival itself is compromised. In this work, we have explored the incidence of * Corresponding author. Tel.: +34 3 5812148; Fax: +34 3 5812011; e-mail: [email protected] Abbreviations: cfu, colony-forming unit(s); LB, Luria–Bertani (medium); pfu, plaque-forming unit(s). 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 4 67 - 8
productive horizontal viral transmission and non-productive vertical transference of proviral genomes to daughter cells, within a clonal population of infected bacterial cells. The analysis has been focused on the use of both strategies by the viral genome throughout the life of an isolated host population, and on the adaptative responses of the virus to the ageing, and consequent loss of replicative fitness, of the host cells. By using as a model a mutant bacteriophage unable to integrate its genome, we have detected an asymmetry in the occurrence of these transmission strategies that tends to maintain the viability of infected cells when exponentially growing, but promotes viral production and efficient horizontal spread when entering into the stationary phase.
2. Experimental and discussion We have analyzed the spread of bacteriophage P22 int7 within isolated, lysogenic colonies of Salmonella typhimurium. This virus represents an unique model to study viral dynamics within clonal populations since not all the individuals, even deriving from a single infected cell, carry the proviral genome. The integration-deficient mutant genome remains in an episomal form and does not replicate in lysogen cells (Susskind and Botstein,
148
E. Ramı´rez, A. Villaverde / Gene 202 (1997) 147–149
1978). Consequently, its vertical transmission during cell division is restricted to a mere passage to one of the daughter cells. This unusual situation, in which only a very small fraction of the progeny cells is infected, can result in an extremely high potential for viral propagation among uninfected cells, since conditions promoting reactivation of latent genomes would result in an immediate release of virus particles and the concomitant infection cycles on an increasing number of neighbouring cells. Therefore, the percentage of infected cells throughout the growth of the colony is indicative of the occurrence of lytic cycles, and it allows us to analyze separately both vertical and horizontal transmission strategies. In this scenery, it seemed reasonable to expect that productive lytic episodes in an infected colony would occur randomly and intersected with monotonous, onecell-restricted vertical virus transmission throughout the colony development, resulting in a high and nearly constant fraction of infected individuals and a regular release of virus particles from early cell divisions. Surprisingly, when examining cell populations in P22 int7 lysogenic colonies, the percentage on infected cells was very low, making it undetectable by 23 h ( Fig. 1). At latter times, a rapid increase in the infected cell fraction is detected, becoming in few hours the totality of the population. This suggests an asymmetry in the occurrence of both transmission strategies, in which the vertical, silent passage predominates in the early colony
Fig. 1. Percentage of infected cells within P22 wild type (#) and P22 int7 ($) lysogenic colonies at different times after plating. Drops from phage lysates at approx. 109 pfu/ml were placed on freshly prepared confluent cultures of Salmonella typhimurium LT2 strain, in LB plates that were further incubated at 37°C overnight. Cells from the lysis areas were spread on LB plates (at time 0) to obtain isolated colonies. Once visible, single colonies were carefully sampled at different times and spread on LB plates to study their composition by the analysis of individual cell clones. After incubation for 75 h at 37°C, viral infection in these clones was detected by the lysis areas arising after replica plating on LT2 confluent cultures. Between 50 and 100 clones were analyzed for each sample. Values obtained up to 23 h were lower than 2%.
development, whereas the horizontal propagation takes place later, concomitant to colony ageing. In agreement with this hypothesis, at 15 h, the ratio between free virus particles and host cells is lower than 10−5 in 8 of the 15 analyzed colonies and increases dramatically thereafter to reach about 20 infectious particles per cell at 90 h ( Fig. 2). This increase can only be explained by recurrent infection cycles producing an important viral amplification. Globally, the independent data depicted in Fig. 2 indicate that the outburst of virus particles is notably synchronic in separated colonies. It is worth noting also that it takes place during few host generations and when the bacterial growth rate declines (Fig. 2). By age 15, about 22 cell doublings have occurred with an average generation time of about 40 min. However, a single generation takes place between 15 and 19 h and only six more between 19 and 120 h. In conclusion, viral replication is tightly repressed during the exponential growth but efficiently stimulated during the decay of the growth rate. Then, lysis and recurrent infection cycles result in a several-order increase of infectious free particles. The basis of the proviral reactivation probably lies on the activation of the SOS DNA repair system detected
Fig. 2. Free virus particles relative to viable cells in P22 int7 lysogen colonies ($) and number of cell generations (#). Lysogenic colonies (obtained as in Fig. 1) growing on LB were removed from the plates at different times and resuspended into an sterile 0.9% NaCl solution. After gentle vortexing, an aliquot of the solution was used for viable cell counting, and another treated with chloroform to promote cell lysis, to determine the concentration of infectious particles. Before age 15, no plaque forming units were detected. At 15 h, in 8 of the 16 analyzed colonies, plaques were not observed. The relative concentration of free viruses in these colonies was estimated to be <8.0×10−7, <3.3×10−5, <1.3×10−6, <8.3×10−7, <4.5×10−7, <3.0×10−7, <4.4×10−7 and <5.8×10−7 pfu/cfu, respectively. Cell generations (n) were calculated by colony forming units (cfu) from entire colonies resuspended in 0.9% NaCl at different times, according to the following relationship: cfu=2n. To prevent any underestimation of the number of cell generations, which could be induced by the viral outburst and the consequent cell death, uninfected LT2 colonies were used for this analysis. However, matching data were obtained from P22 int7-infected colonies (not shown).
E. Ramı´rez, A. Villaverde / Gene 202 (1997) 147–149
in grown bacterial colonies ( Taddei et al., 1995). The bacterial SOS response is triggered by DNA damage but also by the arrest of intact DNA replication ( Walker, 1985), and involves the coordinated activity of more than 20 gene products which promote DNA repair and prevent cell division until replication of the cell chromosome is restored (Little and Mount, 1982). The key elements in the regulation of the SOS response are the RecA protein and the LexA repressor of SOS genes. Upon its activation by single-stranded DNA and a nucleoside triphosphate, RecA promotes the autodigestion of LexA ( Kim and Little, 1993) and the lytic repressors of bacteriophages l and P22 (Phizicky and Roberts, 1980; Kim and Little, 1993). In isolated bacterial populations, attenuated P22 virus propagates following two consecutive schemes. During rapid division of host cells, the viral genome is transmitted to the offspring without detectable productive episodes, thus guaranteeing an efficient host spreading. When the growth rate of the host population declines, lytic functions are expressed and the surviving cells become infected in very few generations. This behaviour reflects an extreme proviral sensitivity to the host growth potential, and reveals a programmed adaptability of viral replicative fitness to better maximize the host as a dissemination vehicle for the virus genomes.
Acknowledgement We are indebted to A. Poteete for generously providing wild type P22 bacteriophage and to E. Domingo for helpful discussion. We also thank J. Checa and V. Ferreres for technical assistance. This work has been
149
supported by grants BIO95-0801, CICYT, and 1995SGR 00376, CIRIT, and partially by Ma2 Francesca de Roviralta Foundation. E.R. is recipient of a predoctoral fellowship from MEC.
References Barre-Sinoussi, F., 1996. HIV as the cause of AIDS. Lancet 348, 31–35. Blyth, W.A., Hill, T.J., Field, H.J., Harbour, D.A., 1976. Reactivation of herpes simplex virus infection by ultraviolet light and possible involvement of prostaglandin. J. Gen. Virol. 33, 547–550. Domingo, E., Martı´nez-Salas, E., Sobrino, F. et al., 1985. The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance—a review. Gene 40, 1–8. Kim, B., Little, J.W., 1993. LexA and lambda CI repressors as enzymes: specific cleavage in an intermolecular reaction. Cell 73, 1165–1173. Little, J.W., Mount, D.W., 1982. The SOS regulatory system of Escherichia coli. Cell 29, 11–22. Lwoff, A., Siminovitch, L., Kjeldgaard, N., 1950. Induction de la lyse bacte´riophagique de la totalite´ d’une population microbienne lysoge`ne. CR Acad. Sci. Paris 231, 190–191. Phizicky, E.M., Roberts, J.M., 1980. Kinetics of RecA protein-directed inactivation of repressors of phage lambda and phage P22. J. Mol. Biol. 139, 319–328. Roberts, J.W., Roberts, C.W., 1975. Proteolytic cleavage of bacteriophage lambda repressor in induction. Proc. Natl. Acad. Sci. USA 72, 147–151. Steinhauer, D.A., Holland, J.J., 1987. Rapid evolution of RNA viruses. Annu. Rev. Microbiol. 41, 409–433. Susskind, M., Botstein, D., 1978. Molecular genetics of bacteriophage P22. Microbiol. Rev. 42, 385–413. Taddei, F., Matic, I., Radman, M., 1995. cAMP-Dependent SOS induction and mutagenesis in resting bacterial populations. Proc. Natl. Acad. Sci. USA 92, 11736–11740. Vogel, J., Cepeda, M., Tschachler, E., Napolitano, L.A., Jay, G., 1992. UV activation of human immunodeficiency virus gene expression in transgenic mice. J. Virol. 66, 1–5. Walker, G.C., 1985. Inducible DNA repair systems. Annu. Rev. Biochem. 54, 425–457.