- Email: [email protected]
Location of glucosyl transferase genes on the genetic map of phage T4
SHORT
364
COMMUNICATIONS
and PFEFFERKORN, E. R., 30, 214-223 (1966). 3. WALEN, K. H., Virology 20,230-234 (1963). 4. SIMPSON, R. W., and HAUSER, R. E., Virology 29,654-667 (1966). 5. SIMPSON, R. W., and HAWSER, R. E., Virology in press. 6. KNIGHT, C. A,, Protoplasw~alologia 4, 3-177 (1963). ROBERT W. SIMPSON ROLF E. HAUSER Department of Virology The Public Health Research Institute of the City of New York, Inc. 455 First Avenue New York, New York 10016 Accepted December 4, 1967
2. BURGE, B. W., Virology
location
of Glucosyl
on the Genetic
Transferase
Genes
Map of Phage T4’
coli with phage T4 Infection of Escherichia induces two phage-specific enzymes, an (Yand a p-glucosyl transferase, which glucothe hydroxymethylcytosine all sylate (HMC) residues of the T4 DNA. In the wild-type T4 phage approximately 70% of the HMC groups are a-glucosylated and the remaining 30 % are /3-glucosylated (1). The mechanism responsible for this specific distribution of glucosyl groups onto the HMC residues remains unknown. The isolation of gt mutants of phage T4, unable t,o induce CP and p-glucosyl transferase activities, has been reported (2, 3). Double mutant’s cvgt @gt defective in both enzymes and unable to glucosylate the HMC groups 011 DXA are restricted by some E’. coli strains, but grow normally 011 Xhigella clysenteriae and on the permissive E. coli mutants isolated by Revel (4). Each of the two enzymes alone provides sufficient so that phage possessing glucosylation, either ~1- or p-glucosyl transferase activity can form plaques on hosts that restrict the double mutant agt Pgt. The glucosyl transferase enzymes appear t’o behave in vivo as they do in z&o (6); that is, phage with orlly 1 This work was supported National Institutes of Health Nat,ional Science Foundation S. E. Lrlria.
by grants from the (AI 03038) and the (GB-5304X) to Dr.
TABLE
1
BACTERIAL AND PHAGE STRAINS USED Bacterial strains E. coli B: restrictive for both am and T4 ugt @gt mutants E. coli Brgl; E. coli K12 r6,r24: restrictive for am mutants; permissive for T4 olgt @gt mutants E. coli CR63: permissive for am mutants; restrictive for T4 agt @gt mutants E. coli CR63 rg/8: permissive for both T4 am and T4 olgt pgt mutants; isolated from CRG3 by the technique described by Revel (4) strains’ Bacteriophage T4 agt57 ogt14: lacks both (Y- and fl-glucosyl transferase activities T4 olgt+ pgt14: revertant from T4 ag157 flgtl-lT4 olgt57 Bgt+: revertant from T4 olgt57 ijgtl,4 mutation in gene 56 T4 amE : T4 amE219: mutation in gene 61 T4 amHL6n: mutation in gene .58 mutation in gene 41 T4 amN81: T4 amN122: mutation in gene -t’L T4 amB22: mutation in gene 43 mutation in gene 4-1 T4 amN82: mutation in gene 45 T4 amEl0: T4 amB3: mutation in gene 4(i T4 amA456x5: mutation in gene -17 T4 amBL292: mutation in gene 55 in gene 50 T4 amA : mutation T4 am727: mutation in gene 49
a The symbols oIgt57 and flgtl4 will be abbreviated as (Y and p, respectively. The T4 am mutants were from the collections of C. Levinthal, S. IX. Luria, and J. Wiberg. Each amber mutant will be referred to by the number of the gene in which its mlltation resides; for example. /(n/56 refers to tz?nE51.
a-glucosyl transferase has 70% of its HJIC groups glucosylated, whereas the @-glucosyl transferase alone gives phage with 100% glucosylation of HMC (3). The existence of nonglucosylated double mut’ants T4 agt ,Bgt made it possible to map the t#wo glucosyl t,ransferase genes as here report’ed. All bact,erial and phage strains used are listed in Table 1. The mapping procedure employed was somewhat’ elaborate because of the peculiarity of this system, in which t’he function of either of the two genes is sufficient to produce the unrestricted phenot,ppe. The essential step was to construct a series of
SHORT TABLE TWO-FACTOR
2
TABLE
CROSSES
am42 am43
am44 am45 am46 am47
am55 am49 am50
Parents: amff+p X am+ a/3
Parents: am a @+ X am+ a/3
Cross am 47 a+ am55+ fl X am47+ a am55 9
am+ a+ recombinant@ (%I
am.+ p+ recombinants” (70)
Progeny gtxotype Plaqzles
11.7 12.1 9.6 8.0 7.0 3.8 7.8 16.2 20.0
a Recombinants am+ assayed on Escherichia was assayed by plating were done following the and Lielausis (6).
3
ANALYSIS OF A RANDOM SAMPLE OF THE PROGENY OF THREE-FACTOR CROSSES
Cross
Amber mutant
365
COMMUNICATIONS
0.8 0.3 2.1 1.2 2.7 1.2 3.5 3.7 8.9
(Y+ p and am+ (Y @+ were co& B. The total progeny on CR63 rgZ8. The crosses procedure given by Edgar
phage strains containing one or more amber (=uwz) mutations in various genes and an agt or a /3gt mutation. This was done as follows. A single mutant, T4 Lugt57 /3gtl4, was used as parent strain in order to have the same agt or ,8gt mutation in each recombinant. (The symbols agt57 and /3gtl4 hereafter will be abbreviated as Q: or 0, respectively). First, a series of am mutants in various genes were crossed with T4 CY/3 on the fully permissive host strain CR63 rgZ8. Recombinants am 01 p were selected, and each recombinant was separately crossed with a single T4 Q+ p strain and a single T4 o p+ strain, both of these being one-step spontaneous revertants from T4 cr 0. From each pair of crosses, one T4 am a+ p strain and one T4 am (Y p+ strain were isolated and tested for LY-and p-glucosyl transferase activities (3, 5). Double am strains, when needed, were constructed by crossing single am mutants carrying the appropriate Q or 0 mutations. The various am CY+/3 strains were then crossed with am+ a /3 in order to estimate the genetic distance between the am mutant
am47 af am55+ am47+ (Y am55 am47+ a am55+ am47 ff+ am55 am47+ af am55+ am47 a am55 am47+ (Y+ am55 am47 01 am55+ 47 + Order: + a
Cross am47 a amSS+ p X
am47+ 01+am55 0 Progeny genotype Plaques
80 113 13 13 13 6 1 1 + 55
am47 (Y am55+ am47+ a+ am55 am47+ 01 am55+ am47 Al+ am55 am47+ 01+ am55+ am47 01 am55 am47 a+ am55+ am47+ a am55 47 a Order: + +
Cross
88 120 9 3 18 10 2 1 + 55
Cross am41 j3 am42+ LYX am41+@+ am42 (Y
am41 j3+ am42+ 01 X
am41+ p am42 01 Progeny genotype Plaques
Progeny genotype Plaques
am41 fI+ am42+ am41+ p am42 am4l+ fl am42+ am41 @+ am42 am41+ B+ am42+ am41 @ am42 am41 @ am42+ am41+ fi+ am42 41 + Order: + P
am41 p am42+ am41+ fl+ am42 am41+ fl am42+ am41 p+ am42 am41+ Bf am42+ am41 @ am42 am41 p+ am42+ am41+ fi am42 41 B Order : + +
337 250 13 12 46 19 1 4 +
42
-
99 155 14 11 23 5 4 1 +
42
sites and the a-glucosyl transferase gene. The results of these two-factor crosses, shown in Table 2, suggested that the (Yglucosyl transferase gene was located very close to gene 47, in agreement with a report that the cvgt mutation of phage T2 mapped in this region (7). Similar crosses between the am 1y Of and am+ Q!p strains, also shown in Table 2, did not give consistent recombination frequencies between the p gene and the various am mutants tested. The reason for this behavior may be the low efficiency of plating of all CYpf phage on restrictive strains; as reported before (S), glucosylation by the /I enzyme alone seems to be less valuable in overcoming restriction than glucosylation provided by the a enzyme.
366
SHORT
COMMUNIC.4TIONS
The location of the cu-glucosyl transferase gene between genes 47 and 55 was determined in two ways. First, the following two crosses were performed: am47 am46 CY+fi X am55 a! P and am47 am55 (Y+p X am46 o( p. If t,he cr-glucosyl transferase gene maps between genes 47 and 55, then in the first cross am+ (Y+ fi recombinants can arise by a single crossover event, whereas in the second cross t,hey arise by a triple crossover. The results showed that am+ o(+ p recombinsnt~s were more frequent in the first’ cross (2.3 f 0.2%) than in the second cross (0.31 f O.OS%). The map position of the a-glucosyl transferase gene was verified by analyzing a random sample of the progeny of the cross am47 LY+0 X am55 (Y P and its reciprocal am55 a+ 6 X am47 (Y p. The progeny of these crosses was plated on CR63 rgZ8; single plaques were transferred to buffer and their genotype was analyzed by spotting on the appropriat,e indicator plates. The results are shown in Table 3. The classes of recombinants that occur at the lowest frequency are those which, according to the order 47 - (Y - 55, are expected to arise from double crossovers. 11 location of the p gene near gene 42 was suggested by a series of three-factor crosses with markers in genes 58, 41, 42, and 43. Its position between genes 41 and 42 was verified by testing a random sample of the progeny of two crosses: am41 01p+ X am42 (Y /3, and am41 a p X am42 (Y /3+, for all the relevant markers. The result’s are included in Table 3. In each cross the lowest frequency was that of t’he recombinant classes which, on the basis of the order 41 - /3 - 42, were predicted to require a double crossover. 1n summary, the glucosyl transferase genes have been assigned in the known genetic map of phage T4 (8) the places shown in the following sequence: . . .56 61 - 5s - I1 - ,Bgt- 42 - 43 - 62 - 44 - 45 46 - 47 - agt - 55 - 49. , . . These assignments have been made using only one mmant in each of the gt genes. ACKNOWLEDGMENT I would like to thank Drs. D. H. Hall, S. E. Luria, II. R. Revel, and E. R. Signer for helpful discussions.
REFERENCES 1. JOSSE, J., and KORNBERG, .4.. .I. Hiol. (‘hem. 237, 1968 (1962). 2. HOSODA, J., Biochem. Biophys. ties. (?ornnrun. 27,294 (1967) 5. GEORGOPOULOS, C. P., Biochenl. Biophys. Res. Commun. 28, 179 (1967). 4. REVEL, II. R., Virology 31,688 (1967). 5. KORNBERG, S. Ii., ZIMMEHM.\N, $. H., and KORNBEILG, :k, J. Biol. Chem. 236, 1487 (1961).
6. EDGAR, R. S., and lI~~~.~~~~~~, T., Genetics 49, 635 (1964). 7. RUSSELL, R., personal communication. 8. WOOD, W. B., and EDG.\R, 11. H., Sci. .lm 217, 60 (1967). c.
Department Massachusetts
Cambridge, dccepted
1’. (:Eortc:oroCLos2
of Biology Institute
of Technology
Massachusetts 02159 December 1, 1967
2 Trainee under Microbiology Training GM66602 to the Department of Biology, chusetts Institute of Technology.
Grant Massa-
Bacteriophages Related to 4X174 Showing a Transition between Two Forms with Different Heat Sensitivity and Adsorption Behavior Two forms are known of bacteriophage $X174. The +* form is relatively heat resistant and is not adsorbed by its host at 4”. The 4 form is relat’ively sensitive to heat and attaches it’self readily to Escherichia coli C in t,he coId. In various media at temperat,ures around 37” the 4* form changes into the 4 form. Under certain conditions the opposite transition can occur ( 1). 111 the present communication it will be shown that t,his phenomenon is not characteristic for +X174 but is exhibit’ed also by other small phages. Material and methods. Bacteriophages s13 , p2, and fL originate from Dr. W. Harm (Dallas, Texas), Dr. I~. DeMori (Milan, It,aly), and Dr. K. D. Zinder (l”\‘ew York, ?;ew York), respectively, +R and St,/1 from Dr. D. E. Bradley (Edinburgh, Scot#land). 4X174hlhg is a double mutant, of +X174
364
COMMUNICATIONS
and PFEFFERKORN, E. R., 30, 214-223 (1966). 3. WALEN, K. H., Virology 20,230-234 (1963). 4. SIMPSON, R. W., and HAUSER, R. E., Virology 29,654-667 (1966). 5. SIMPSON, R. W., and HAWSER, R. E., Virology in press. 6. KNIGHT, C. A,, Protoplasw~alologia 4, 3-177 (1963). ROBERT W. SIMPSON ROLF E. HAUSER Department of Virology The Public Health Research Institute of the City of New York, Inc. 455 First Avenue New York, New York 10016 Accepted December 4, 1967
2. BURGE, B. W., Virology
location
of Glucosyl
on the Genetic
Transferase
Genes
Map of Phage T4’
coli with phage T4 Infection of Escherichia induces two phage-specific enzymes, an (Yand a p-glucosyl transferase, which glucothe hydroxymethylcytosine all sylate (HMC) residues of the T4 DNA. In the wild-type T4 phage approximately 70% of the HMC groups are a-glucosylated and the remaining 30 % are /3-glucosylated (1). The mechanism responsible for this specific distribution of glucosyl groups onto the HMC residues remains unknown. The isolation of gt mutants of phage T4, unable t,o induce CP and p-glucosyl transferase activities, has been reported (2, 3). Double mutant’s cvgt @gt defective in both enzymes and unable to glucosylate the HMC groups 011 DXA are restricted by some E’. coli strains, but grow normally 011 Xhigella clysenteriae and on the permissive E. coli mutants isolated by Revel (4). Each of the two enzymes alone provides sufficient so that phage possessing glucosylation, either ~1- or p-glucosyl transferase activity can form plaques on hosts that restrict the double mutant agt Pgt. The glucosyl transferase enzymes appear t’o behave in vivo as they do in z&o (6); that is, phage with orlly 1 This work was supported National Institutes of Health Nat,ional Science Foundation S. E. Lrlria.
by grants from the (AI 03038) and the (GB-5304X) to Dr.
TABLE
1
BACTERIAL AND PHAGE STRAINS USED Bacterial strains E. coli B: restrictive for both am and T4 ugt @gt mutants E. coli Brgl; E. coli K12 r6,r24: restrictive for am mutants; permissive for T4 olgt @gt mutants E. coli CR63: permissive for am mutants; restrictive for T4 agt @gt mutants E. coli CR63 rg/8: permissive for both T4 am and T4 olgt pgt mutants; isolated from CRG3 by the technique described by Revel (4) strains’ Bacteriophage T4 agt57 ogt14: lacks both (Y- and fl-glucosyl transferase activities T4 olgt+ pgt14: revertant from T4 ag157 flgtl-lT4 olgt57 Bgt+: revertant from T4 olgt57 ijgtl,4 mutation in gene 56 T4 amE : T4 amE219: mutation in gene 61 T4 amHL6n: mutation in gene .58 mutation in gene 41 T4 amN81: T4 amN122: mutation in gene -t’L T4 amB22: mutation in gene 43 mutation in gene 4-1 T4 amN82: mutation in gene 45 T4 amEl0: T4 amB3: mutation in gene 4(i T4 amA456x5: mutation in gene -17 T4 amBL292: mutation in gene 55 in gene 50 T4 amA : mutation T4 am727: mutation in gene 49
a The symbols oIgt57 and flgtl4 will be abbreviated as (Y and p, respectively. The T4 am mutants were from the collections of C. Levinthal, S. IX. Luria, and J. Wiberg. Each amber mutant will be referred to by the number of the gene in which its mlltation resides; for example. /(n/56 refers to tz?nE51.
a-glucosyl transferase has 70% of its HJIC groups glucosylated, whereas the @-glucosyl transferase alone gives phage with 100% glucosylation of HMC (3). The existence of nonglucosylated double mut’ants T4 agt ,Bgt made it possible to map the t#wo glucosyl t,ransferase genes as here report’ed. All bact,erial and phage strains used are listed in Table 1. The mapping procedure employed was somewhat’ elaborate because of the peculiarity of this system, in which t’he function of either of the two genes is sufficient to produce the unrestricted phenot,ppe. The essential step was to construct a series of
SHORT TABLE TWO-FACTOR
2
TABLE
CROSSES
am42 am43
am44 am45 am46 am47
am55 am49 am50
Parents: amff+p X am+ a/3
Parents: am a @+ X am+ a/3
Cross am 47 a+ am55+ fl X am47+ a am55 9
am+ a+ recombinant@ (%I
am.+ p+ recombinants” (70)
Progeny gtxotype Plaqzles
11.7 12.1 9.6 8.0 7.0 3.8 7.8 16.2 20.0
a Recombinants am+ assayed on Escherichia was assayed by plating were done following the and Lielausis (6).
3
ANALYSIS OF A RANDOM SAMPLE OF THE PROGENY OF THREE-FACTOR CROSSES
Cross
Amber mutant
365
COMMUNICATIONS
0.8 0.3 2.1 1.2 2.7 1.2 3.5 3.7 8.9
(Y+ p and am+ (Y @+ were co& B. The total progeny on CR63 rgZ8. The crosses procedure given by Edgar
phage strains containing one or more amber (=uwz) mutations in various genes and an agt or a /3gt mutation. This was done as follows. A single mutant, T4 Lugt57 /3gtl4, was used as parent strain in order to have the same agt or ,8gt mutation in each recombinant. (The symbols agt57 and /3gtl4 hereafter will be abbreviated as Q: or 0, respectively). First, a series of am mutants in various genes were crossed with T4 CY/3 on the fully permissive host strain CR63 rgZ8. Recombinants am 01 p were selected, and each recombinant was separately crossed with a single T4 Q+ p strain and a single T4 o p+ strain, both of these being one-step spontaneous revertants from T4 cr 0. From each pair of crosses, one T4 am a+ p strain and one T4 am (Y p+ strain were isolated and tested for LY-and p-glucosyl transferase activities (3, 5). Double am strains, when needed, were constructed by crossing single am mutants carrying the appropriate Q or 0 mutations. The various am CY+/3 strains were then crossed with am+ a /3 in order to estimate the genetic distance between the am mutant
am47 af am55+ am47+ (Y am55 am47+ a am55+ am47 ff+ am55 am47+ af am55+ am47 a am55 am47+ (Y+ am55 am47 01 am55+ 47 + Order: + a
Cross am47 a amSS+ p X
am47+ 01+am55 0 Progeny genotype Plaques
80 113 13 13 13 6 1 1 + 55
am47 (Y am55+ am47+ a+ am55 am47+ 01 am55+ am47 Al+ am55 am47+ 01+ am55+ am47 01 am55 am47 a+ am55+ am47+ a am55 47 a Order: + +
Cross
88 120 9 3 18 10 2 1 + 55
Cross am41 j3 am42+ LYX am41+@+ am42 (Y
am41 j3+ am42+ 01 X
am41+ p am42 01 Progeny genotype Plaques
Progeny genotype Plaques
am41 fI+ am42+ am41+ p am42 am4l+ fl am42+ am41 @+ am42 am41+ B+ am42+ am41 @ am42 am41 @ am42+ am41+ fi+ am42 41 + Order: + P
am41 p am42+ am41+ fl+ am42 am41+ fl am42+ am41 p+ am42 am41+ Bf am42+ am41 @ am42 am41 p+ am42+ am41+ fi am42 41 B Order : + +
337 250 13 12 46 19 1 4 +
42
-
99 155 14 11 23 5 4 1 +
42
sites and the a-glucosyl transferase gene. The results of these two-factor crosses, shown in Table 2, suggested that the (Yglucosyl transferase gene was located very close to gene 47, in agreement with a report that the cvgt mutation of phage T2 mapped in this region (7). Similar crosses between the am 1y Of and am+ Q!p strains, also shown in Table 2, did not give consistent recombination frequencies between the p gene and the various am mutants tested. The reason for this behavior may be the low efficiency of plating of all CYpf phage on restrictive strains; as reported before (S), glucosylation by the /I enzyme alone seems to be less valuable in overcoming restriction than glucosylation provided by the a enzyme.
366
SHORT
COMMUNIC.4TIONS
The location of the cu-glucosyl transferase gene between genes 47 and 55 was determined in two ways. First, the following two crosses were performed: am47 am46 CY+fi X am55 a! P and am47 am55 (Y+p X am46 o( p. If t,he cr-glucosyl transferase gene maps between genes 47 and 55, then in the first cross am+ (Y+ fi recombinants can arise by a single crossover event, whereas in the second cross t,hey arise by a triple crossover. The results showed that am+ o(+ p recombinsnt~s were more frequent in the first’ cross (2.3 f 0.2%) than in the second cross (0.31 f O.OS%). The map position of the a-glucosyl transferase gene was verified by analyzing a random sample of the progeny of the cross am47 LY+0 X am55 (Y P and its reciprocal am55 a+ 6 X am47 (Y p. The progeny of these crosses was plated on CR63 rgZ8; single plaques were transferred to buffer and their genotype was analyzed by spotting on the appropriat,e indicator plates. The results are shown in Table 3. The classes of recombinants that occur at the lowest frequency are those which, according to the order 47 - (Y - 55, are expected to arise from double crossovers. 11 location of the p gene near gene 42 was suggested by a series of three-factor crosses with markers in genes 58, 41, 42, and 43. Its position between genes 41 and 42 was verified by testing a random sample of the progeny of two crosses: am41 01p+ X am42 (Y /3, and am41 a p X am42 (Y /3+, for all the relevant markers. The result’s are included in Table 3. In each cross the lowest frequency was that of t’he recombinant classes which, on the basis of the order 41 - /3 - 42, were predicted to require a double crossover. 1n summary, the glucosyl transferase genes have been assigned in the known genetic map of phage T4 (8) the places shown in the following sequence: . . .56 61 - 5s - I1 - ,Bgt- 42 - 43 - 62 - 44 - 45 46 - 47 - agt - 55 - 49. , . . These assignments have been made using only one mmant in each of the gt genes. ACKNOWLEDGMENT I would like to thank Drs. D. H. Hall, S. E. Luria, II. R. Revel, and E. R. Signer for helpful discussions.
REFERENCES 1. JOSSE, J., and KORNBERG, .4.. .I. Hiol. (‘hem. 237, 1968 (1962). 2. HOSODA, J., Biochem. Biophys. ties. (?ornnrun. 27,294 (1967) 5. GEORGOPOULOS, C. P., Biochenl. Biophys. Res. Commun. 28, 179 (1967). 4. REVEL, II. R., Virology 31,688 (1967). 5. KORNBERG, S. Ii., ZIMMEHM.\N, $. H., and KORNBEILG, :k, J. Biol. Chem. 236, 1487 (1961).
6. EDGAR, R. S., and lI~~~.~~~~~~, T., Genetics 49, 635 (1964). 7. RUSSELL, R., personal communication. 8. WOOD, W. B., and EDG.\R, 11. H., Sci. .lm 217, 60 (1967). c.
Department Massachusetts
Cambridge, dccepted
1’. (:Eortc:oroCLos2
of Biology Institute
of Technology
Massachusetts 02159 December 1, 1967
2 Trainee under Microbiology Training GM66602 to the Department of Biology, chusetts Institute of Technology.
Grant Massa-
Bacteriophages Related to 4X174 Showing a Transition between Two Forms with Different Heat Sensitivity and Adsorption Behavior Two forms are known of bacteriophage $X174. The +* form is relatively heat resistant and is not adsorbed by its host at 4”. The 4 form is relat’ively sensitive to heat and attaches it’self readily to Escherichia coli C in t,he coId. In various media at temperat,ures around 37” the 4* form changes into the 4 form. Under certain conditions the opposite transition can occur ( 1). 111 the present communication it will be shown that t,his phenomenon is not characteristic for +X174 but is exhibit’ed also by other small phages. Material and methods. Bacteriophages s13 , p2, and fL originate from Dr. W. Harm (Dallas, Texas), Dr. I~. DeMori (Milan, It,aly), and Dr. K. D. Zinder (l”\‘ew York, ?;ew York), respectively, +R and St,/1 from Dr. D. E. Bradley (Edinburgh, Scot#land). 4X174hlhg is a double mutant, of +X174