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The role of gene cro in phage development
VIROLOGY
68, 266-269 (1975)
The Role of Gene cro in Phage Development H. EISEN,
M. GEORGIOU, Department
C. P. GEORGOPOULOS,
of Molecular
Biology,
University
GERALD
SELZER
of Geneua. Switzerland
GARY GUSSIN Department
of Zoology,
of Iowa, Iowa City, Iowa 52240
University
IRA HERSKOWITZ Institute
of Molecular
Biologv.
University
of Oregon, Eugene. Oregon 97403
Accepted June 2, 1975 Phage X CTOmutants are unable to grow at 42”. This is due to two effects. One effect, called Tro, prevents the growth of other heteroimmune lamboid phages and is expressed by phage i”cI857cro27bio252 but not by i”cI857cro27bio256. The other effect interferes with phage growth only in cis and is expressed by phage iAc1857cro27bio256.
The product of the regulatory gene cru of phage X is thought to act at two sites on the phage chromosome (Fig. 1). Interaction of the CFO product with the region of the leftward operator and promoter results in turnoff of leftward phage transcription (I, 2). Interaction of the cro product with the rightward operator-promoter region has two effects. First the cro product blocks expression of the c1 gene, presumably by preventing transcription from the repressor maintenance promoter prm (1, 3, 46). In addition the cro product has been shown to interfere with phage transcription initiated at the rightward promoter P,, (2, 4a, 5). Phage mutant in the cro gene cannot grow vegetatively under conditions where synthesis of active repressor can be initiated from the repressor establishment promoter pre. Under these conditions all the infected bacteria become lysogenic. For example, XcI857cro27 is unable to form plaques at 30”. However, introduction of a mutation in either gene ~11 or ~111permits plaque formation at 30” because transcrip266 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
tion of the c1 gene from pre requires the product of these genes (I, 6, 7). We report here another property of phage mutated in the cro gene, namely, their inability to grow at elevated temperatures. Phage i”cI857cro27 is unable to propagate at 42”. Both its efficiency of plating and the phage progeny released per infected bacterium are very low (Table 1). The inability to grow is recessive to the presence of an active cro gene product in the cytoplasm of the infected cell (Table 1). When phage i”cI857cro27cII infects sensitive bacteria at 42”, its ability to subsequently propagate ‘at 30” (permissive conditions because of the presence of the ~112002 mutation) is very rapidly lost (Fig. 2). This suggests that upon cro27 phage change infection at 42”, an irreversible occurs, either in the infecting phage itself or in the capacity of the infected host to propagate the phage. Our experiments (see below) have led us to define at least two phage-mediated effects which are responsi-
SHORT
ble for the inability of i”cI857cro27 phage to propagate at 42”. One effect, called Tro (from the French trop, too much), is due to the overproduction of two phage functions located in the P,-promoted operon. The Tro effect can act in tram to inhibit heteroimmune phage growth (see below) and host protein and RNA synthesis (data not shown). The other effect acts only in cis (see below) to specifically prevent cro 27 phage propagation and does not interfere with the growth of the other phages in the infected cells. Table 2 shows the results of phage production in cells mixedly infected with various phage types at 42”. As expected in bacteria mixedly infected with i”cI857 cro27 and i”cI857cro+, phage growth proceeds normally, presumably because the active cro” product can act in trans. However, in mixed infections between i”cI857cro27 and i4S4cI or iZ1b2cIII phage, no phage progeny of either type is produced, because the croA, cro4s4 and croz’ gene products are not functionally interchangeable and hence the Tro effect of X is expressed. We have localized the region on the X-genome which is necessary for the Tro phenotype by constructing various i”cI857cro27 bio derivatives (Fig. 1) and testing their ability to inhibit i434cI phage “on-ham”~ogy
wlfn
267
COMMUNICATIONS
‘I‘
--
FIG. 1. Genetic map of the early region of phage X. The endpoints of the bio deletions are taken from Signer et al. (8). TABLE
growth at 42”. The results, shown in Table 2, suggest that the Tro effect is due to the presence of the phage region localized between the endpoints of bio252 and bio256, since i”cI857cro27bio252 possesses the full Tro effect, whereas phage i “cI857cro27bio. ‘0 4
.
0
20
10
60
TIME imInI at 42°C
FIG. 2. Effect of time of growth at 42” on subsequent phage production at 33”. E. coli C600 in 0.01 M MgSO, was infected with either i”cI857Nsus7sus53~112002 or its cro27 derivative at a multiplicity of infection of 0.1 phage per bacterium. After 15 min at 23”, the infected cells were centrifuged and resuspended at 10’ cells/ml in tryptone broth. The culture was placed at 42” and at the indicated times aliquots were diluted lOO-fold into tryptone broth and further incubated for 90 min at 33”. Chloroform was then added and the phage titered. (-0-O-1, Infected with iAc1857ik’sus7sus53cI~2002cro27; (-O-O-), infected with i”c1857Nsus7sus53cI12002. 1
EFFICIENCY OF PLATING UNDER VARIOUS CONDITIONS” Infecting
phage
i”~I857~~0+ i^cI857cro27
TC600 su+2
W3350 (i”cI857Nsus7sus53ONQ8)
30”
39”
42”
30”
30”
42”
0.9 5 x10-6
1.0 1.0
1.0 2 x 10-S
1.0 0.9
1.0 1.0
1.0 0.8
“The efficiency of plating is measured as the number of plaques produced by a phage strain on a given bacterial host at various temperatures relative to the number produced on TC600 su’2 at 39”. TC600 is a Toronto-derived C600 strain. The W3550(i”cI857Nsus7sus53ONQ8) strain, before use, was made, cro”-constitutive by growth at 42” for at least ten generations. ONQ8 is a mutation which renders the 0 gene inactive.
268
SHORT
COMMIJNICATIONS
256 shows no Tro effect. Phage i”cI857cro. 276io10, whose endpoint is located between those of bio252 and bio256 shows a much weaker Tro effect (8). Similar rePhage produced Infecting phages sults were obtained using the bio243 deper infected bacterium rivative strain (Fig. 1; data not shown). It thus appears that the Tro phenotype is Type A Type B Type B Type A made up of at least two components. The 8.6 i”cI857cro+ stronger one is located between the end0.01 i”cI857cro27 points of bio252 and biol0 and could in fact 13.5 i’3’cI be the EalO protein reported by Hendrix 21.9 iZ’b2cIII (9) as coming from that region or the ral protein (M. Zabeau, personal communica6.7 8.3 i’34cI i”cI857cro+ tion). The second component is much 7.2 15.4 i2’b2cIII weaker and could be due either to a new gene product located between biol0 and 5.8 6.5 i”cI857cro27 i”cI857cro the N gene or, since it is known that the 0.04 0.2 i’3’cI endpoint of bio256 penetrates the N struci*‘b2cIII 0.02 0.4 tural gene and causes the formation of a 5.9 i”bio256c1857crot more unstable N gene product (ZO), it 5.0 13.6 i’3’cI could be that N itself is the weak Tro component. 0.08 i”bio256cI857cro27 Table 2 reveals that besides the Tro i’3’cI 0.19 8.5 effect which acts in tram, there appears to be also a cis effect which interferes with i”biolOcI857cro+ 5.3 cro27 phage propagation at 42”. This result 4.8 11.2 i’34cI is best seen in mixed infections such as between i”cI857cro27bio256 and i434cI. Ali”biolOcI857cro27 0.07 0.1 2.1 i’3’cI though the bio256 substitution has completely removed the Tro effect allowing a 4.5 i”bio252c1857crot normal production of i434cI phage, the i’3’cI 3.3 10.7 iAc1857cro27bio256 parent itself is not found among the phage progeny. This cis 0.02 i”bio252cI857cro27 effect on cro27 phage development could 0.04 0.35 i’34cI either be due to the overproduction of a “TC600 bacteria were grown in 1% tryptone broth c&acting protein or could be due to the supplemented with 0.2% maltose to 3 x 10’ cells/ml, elevated levels of transcription of the right washed once, resuspended in lo-’ M MgSO, at 1.5 x hand operon. These high levels of P,10’ cells/ml and incubated at 37” for 30 min. Phages mediated transcription could conceivably were added at a multiplicity of infection (m.o.i.) of 5 interfere with either the production or each, except in the case of infection by a single phage function of the small RNA piece (oop, 11) type when a m.o.i. of 10 was used instead. Adsorpwhich has been implicated in the initiation tion was allowed to proceed at room temperature for of X-DNA replication. 20 min, and unadsorbed phages were eliminated by TABLE
2
PHACE GROWTH AT 42” IN CELLS MIXEDLV INFECTED WITH VARIOUS X PHAC.E~
centrifugation. The infected bacteria were diluted lO’-fold in prewarmed tryptone broth and shaken at 42” for 75 min. Chloroform was added to assure lysis and the progeny phage was assayed on the appropriate indicator strains. The i”cI857bio256 and i”cI857bio256cro27 phage strains were constructed by crossing the corresponding i”cI857 phage strain with i’3’bio256 in order to eliminate the sex mutation which was fortuitously associated with the original i”bio256 isolate (Max Gottesman, personal communication).
ACKNOWLEDGMENTS This work was supported by research grants, No. 3.100.73 from the Fonds National Suisse de la Recherche Scientifique and No. GM 18075 from the National Institutes of Health. REFERENCES I. EISEN, H., BRACHET, P., PEREIRA DA SILVA, L., and JACOB, F., Proc. Nat. Acad. Sci. USA 66, 855 (1970).
SHORT
COMMUNICATIONS
2. SLY, U’. S., RABIDEAI., K., and KOI,BER, A., In “The Bacteriophage Lambda” (A.D. Hershey. ed.), p. 575, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1971. 3. CXSTELLAZZI,M., BRACHET, P., and EISEN, H., Mol. Gen. Genet. 117, 211 (1972). 4a. REICHARDT, L,., J. Mol. Biol. 93, 267 (1975). 46. REICHARDT, L., J. Mol. Biol. 93, 289 (1975). 5. ECHOLS, H.. GREEN, L., OPPENHEIM, A. B., OPPENHEIM, A., and HONIGMAN, A., J. Mol. Biol. 80, 203 (1973). 6. SPIEGELMAN, W., REICHARDT, L., YANIF, M., HEINXMANN, S., KAISER, D.. and EISEN, H.,
269
Proc. Nat. Acad. Sci. USA 69, 3156 (1972). 7. REICHARDT, L., and KAISER, A. D., Proc. Nat. Acad. Sci. USA 68, 2185 (1971). 8. SIGNER, E. R., MANI.Y, K. F., and BRUNSTETTEK, M.-A., Virology 39, 137 (1969). 9. HENDHIX, W. R., In “The Bacteriophage Lambda” (A. D. Hershey, ed.), p. 355, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1971. 10. GREENHLATT. J., Proc. Nat. Acad. Sci. USA 69, 3606 (1972). Il. HAYES, S., and SZYHALSKI, W., Fed. Proc. 32, 529 (1973,.
68, 266-269 (1975)
The Role of Gene cro in Phage Development H. EISEN,
M. GEORGIOU, Department
C. P. GEORGOPOULOS,
of Molecular
Biology,
University
GERALD
SELZER
of Geneua. Switzerland
GARY GUSSIN Department
of Zoology,
of Iowa, Iowa City, Iowa 52240
University
IRA HERSKOWITZ Institute
of Molecular
Biologv.
University
of Oregon, Eugene. Oregon 97403
Accepted June 2, 1975 Phage X CTOmutants are unable to grow at 42”. This is due to two effects. One effect, called Tro, prevents the growth of other heteroimmune lamboid phages and is expressed by phage i”cI857cro27bio252 but not by i”cI857cro27bio256. The other effect interferes with phage growth only in cis and is expressed by phage iAc1857cro27bio256.
The product of the regulatory gene cru of phage X is thought to act at two sites on the phage chromosome (Fig. 1). Interaction of the CFO product with the region of the leftward operator and promoter results in turnoff of leftward phage transcription (I, 2). Interaction of the cro product with the rightward operator-promoter region has two effects. First the cro product blocks expression of the c1 gene, presumably by preventing transcription from the repressor maintenance promoter prm (1, 3, 46). In addition the cro product has been shown to interfere with phage transcription initiated at the rightward promoter P,, (2, 4a, 5). Phage mutant in the cro gene cannot grow vegetatively under conditions where synthesis of active repressor can be initiated from the repressor establishment promoter pre. Under these conditions all the infected bacteria become lysogenic. For example, XcI857cro27 is unable to form plaques at 30”. However, introduction of a mutation in either gene ~11 or ~111permits plaque formation at 30” because transcrip266 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
tion of the c1 gene from pre requires the product of these genes (I, 6, 7). We report here another property of phage mutated in the cro gene, namely, their inability to grow at elevated temperatures. Phage i”cI857cro27 is unable to propagate at 42”. Both its efficiency of plating and the phage progeny released per infected bacterium are very low (Table 1). The inability to grow is recessive to the presence of an active cro gene product in the cytoplasm of the infected cell (Table 1). When phage i”cI857cro27cII infects sensitive bacteria at 42”, its ability to subsequently propagate ‘at 30” (permissive conditions because of the presence of the ~112002 mutation) is very rapidly lost (Fig. 2). This suggests that upon cro27 phage change infection at 42”, an irreversible occurs, either in the infecting phage itself or in the capacity of the infected host to propagate the phage. Our experiments (see below) have led us to define at least two phage-mediated effects which are responsi-
SHORT
ble for the inability of i”cI857cro27 phage to propagate at 42”. One effect, called Tro (from the French trop, too much), is due to the overproduction of two phage functions located in the P,-promoted operon. The Tro effect can act in tram to inhibit heteroimmune phage growth (see below) and host protein and RNA synthesis (data not shown). The other effect acts only in cis (see below) to specifically prevent cro 27 phage propagation and does not interfere with the growth of the other phages in the infected cells. Table 2 shows the results of phage production in cells mixedly infected with various phage types at 42”. As expected in bacteria mixedly infected with i”cI857 cro27 and i”cI857cro+, phage growth proceeds normally, presumably because the active cro” product can act in trans. However, in mixed infections between i”cI857cro27 and i4S4cI or iZ1b2cIII phage, no phage progeny of either type is produced, because the croA, cro4s4 and croz’ gene products are not functionally interchangeable and hence the Tro effect of X is expressed. We have localized the region on the X-genome which is necessary for the Tro phenotype by constructing various i”cI857cro27 bio derivatives (Fig. 1) and testing their ability to inhibit i434cI phage “on-ham”~ogy
wlfn
267
COMMUNICATIONS
‘I‘
--
FIG. 1. Genetic map of the early region of phage X. The endpoints of the bio deletions are taken from Signer et al. (8). TABLE
growth at 42”. The results, shown in Table 2, suggest that the Tro effect is due to the presence of the phage region localized between the endpoints of bio252 and bio256, since i”cI857cro27bio252 possesses the full Tro effect, whereas phage i “cI857cro27bio. ‘0 4
.
0
20
10
60
TIME imInI at 42°C
FIG. 2. Effect of time of growth at 42” on subsequent phage production at 33”. E. coli C600 in 0.01 M MgSO, was infected with either i”cI857Nsus7sus53~112002 or its cro27 derivative at a multiplicity of infection of 0.1 phage per bacterium. After 15 min at 23”, the infected cells were centrifuged and resuspended at 10’ cells/ml in tryptone broth. The culture was placed at 42” and at the indicated times aliquots were diluted lOO-fold into tryptone broth and further incubated for 90 min at 33”. Chloroform was then added and the phage titered. (-0-O-1, Infected with iAc1857ik’sus7sus53cI~2002cro27; (-O-O-), infected with i”c1857Nsus7sus53cI12002. 1
EFFICIENCY OF PLATING UNDER VARIOUS CONDITIONS” Infecting
phage
i”~I857~~0+ i^cI857cro27
TC600 su+2
W3350 (i”cI857Nsus7sus53ONQ8)
30”
39”
42”
30”
30”
42”
0.9 5 x10-6
1.0 1.0
1.0 2 x 10-S
1.0 0.9
1.0 1.0
1.0 0.8
“The efficiency of plating is measured as the number of plaques produced by a phage strain on a given bacterial host at various temperatures relative to the number produced on TC600 su’2 at 39”. TC600 is a Toronto-derived C600 strain. The W3550(i”cI857Nsus7sus53ONQ8) strain, before use, was made, cro”-constitutive by growth at 42” for at least ten generations. ONQ8 is a mutation which renders the 0 gene inactive.
268
SHORT
COMMIJNICATIONS
256 shows no Tro effect. Phage i”cI857cro. 276io10, whose endpoint is located between those of bio252 and bio256 shows a much weaker Tro effect (8). Similar rePhage produced Infecting phages sults were obtained using the bio243 deper infected bacterium rivative strain (Fig. 1; data not shown). It thus appears that the Tro phenotype is Type A Type B Type B Type A made up of at least two components. The 8.6 i”cI857cro+ stronger one is located between the end0.01 i”cI857cro27 points of bio252 and biol0 and could in fact 13.5 i’3’cI be the EalO protein reported by Hendrix 21.9 iZ’b2cIII (9) as coming from that region or the ral protein (M. Zabeau, personal communica6.7 8.3 i’34cI i”cI857cro+ tion). The second component is much 7.2 15.4 i2’b2cIII weaker and could be due either to a new gene product located between biol0 and 5.8 6.5 i”cI857cro27 i”cI857cro the N gene or, since it is known that the 0.04 0.2 i’3’cI endpoint of bio256 penetrates the N struci*‘b2cIII 0.02 0.4 tural gene and causes the formation of a 5.9 i”bio256c1857crot more unstable N gene product (ZO), it 5.0 13.6 i’3’cI could be that N itself is the weak Tro component. 0.08 i”bio256cI857cro27 Table 2 reveals that besides the Tro i’3’cI 0.19 8.5 effect which acts in tram, there appears to be also a cis effect which interferes with i”biolOcI857cro+ 5.3 cro27 phage propagation at 42”. This result 4.8 11.2 i’34cI is best seen in mixed infections such as between i”cI857cro27bio256 and i434cI. Ali”biolOcI857cro27 0.07 0.1 2.1 i’3’cI though the bio256 substitution has completely removed the Tro effect allowing a 4.5 i”bio252c1857crot normal production of i434cI phage, the i’3’cI 3.3 10.7 iAc1857cro27bio256 parent itself is not found among the phage progeny. This cis 0.02 i”bio252cI857cro27 effect on cro27 phage development could 0.04 0.35 i’34cI either be due to the overproduction of a “TC600 bacteria were grown in 1% tryptone broth c&acting protein or could be due to the supplemented with 0.2% maltose to 3 x 10’ cells/ml, elevated levels of transcription of the right washed once, resuspended in lo-’ M MgSO, at 1.5 x hand operon. These high levels of P,10’ cells/ml and incubated at 37” for 30 min. Phages mediated transcription could conceivably were added at a multiplicity of infection (m.o.i.) of 5 interfere with either the production or each, except in the case of infection by a single phage function of the small RNA piece (oop, 11) type when a m.o.i. of 10 was used instead. Adsorpwhich has been implicated in the initiation tion was allowed to proceed at room temperature for of X-DNA replication. 20 min, and unadsorbed phages were eliminated by TABLE
2
PHACE GROWTH AT 42” IN CELLS MIXEDLV INFECTED WITH VARIOUS X PHAC.E~
centrifugation. The infected bacteria were diluted lO’-fold in prewarmed tryptone broth and shaken at 42” for 75 min. Chloroform was added to assure lysis and the progeny phage was assayed on the appropriate indicator strains. The i”cI857bio256 and i”cI857bio256cro27 phage strains were constructed by crossing the corresponding i”cI857 phage strain with i’3’bio256 in order to eliminate the sex mutation which was fortuitously associated with the original i”bio256 isolate (Max Gottesman, personal communication).
ACKNOWLEDGMENTS This work was supported by research grants, No. 3.100.73 from the Fonds National Suisse de la Recherche Scientifique and No. GM 18075 from the National Institutes of Health. REFERENCES I. EISEN, H., BRACHET, P., PEREIRA DA SILVA, L., and JACOB, F., Proc. Nat. Acad. Sci. USA 66, 855 (1970).
SHORT
COMMUNICATIONS
2. SLY, U’. S., RABIDEAI., K., and KOI,BER, A., In “The Bacteriophage Lambda” (A.D. Hershey. ed.), p. 575, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1971. 3. CXSTELLAZZI,M., BRACHET, P., and EISEN, H., Mol. Gen. Genet. 117, 211 (1972). 4a. REICHARDT, L,., J. Mol. Biol. 93, 267 (1975). 46. REICHARDT, L., J. Mol. Biol. 93, 289 (1975). 5. ECHOLS, H.. GREEN, L., OPPENHEIM, A. B., OPPENHEIM, A., and HONIGMAN, A., J. Mol. Biol. 80, 203 (1973). 6. SPIEGELMAN, W., REICHARDT, L., YANIF, M., HEINXMANN, S., KAISER, D.. and EISEN, H.,
269
Proc. Nat. Acad. Sci. USA 69, 3156 (1972). 7. REICHARDT, L., and KAISER, A. D., Proc. Nat. Acad. Sci. USA 68, 2185 (1971). 8. SIGNER, E. R., MANI.Y, K. F., and BRUNSTETTEK, M.-A., Virology 39, 137 (1969). 9. HENDHIX, W. R., In “The Bacteriophage Lambda” (A. D. Hershey, ed.), p. 355, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1971. 10. GREENHLATT. J., Proc. Nat. Acad. Sci. USA 69, 3606 (1972). Il. HAYES, S., and SZYHALSKI, W., Fed. Proc. 32, 529 (1973,.