VIROLOGY
67,
136-143
(1975)
Defective ANN Department
of Biology
Prophage
in Escherichia
STRATHERN
and Institute
of
IRA
AND
Molecular
Biology,
Accepted
April
co/i K12 Strains HERSKOWITZ
University
of
Oregon,
Eugene,
Oregon
97403
21, 1975
Many E. coli K12 strains carry genes analogous to the late gene positive regulator (gene Q) and lysis genes (genes S and R) of phage X. This block of genes (called qsr’) has been identified by isolating X strains in which these genes have replaced X genes Q. S, and R. Late gene control by at least some of these phages is similar to that of previously recognized substitution mutants (Xp4 and Xqin). Xqsr’ phages cannot be recovered from a strain (AB1157) which lacks other genes (rat, recE, reu) attributed to a defective prophage. Qsr’ and these other genes may, therefore, be derived from a common prophage. INTRODUCTION
The X substitution mutations, p4 and independently isolated 9in, although (Jacob and Wollman, 1954; Sato and Campbell, 1970), are strikingly similar in physiological behavior and genetic structure. Genetic and electron microscopic mapping show that X DNA corresponding to genes Q, S, and R and the late gene promoter has been deleted and is replaced by DNA which does not hybridize to X (Fig. 1) (Sato and Campbell, 1970; Fiandt et al., 1971; Herskowitz and Signer, 1974). The inserted DNA’s in p4 and qin mutations are homologous, as seen by electron microscopy of p4lqin DNA heteroduplexes (Fiandt et al., 1971). The substituted DNA of a p4 phage codes for a Q-like gene with a specificity of late gene expression distinct from X: the “Q” of p4 can activate late genes of a p4 or a qin prophage but not the late genes of a X prophage (Herskowitz and Signer, 1974). This substituted DNA presumably also codes for analogs to genes S and R since lysis is required for plaque formation (Campbell and de1 CampilloCampbell, 1963; Goldberg and Howe, 1969). The similarities between p4 and qin substitutions suggest that they have a common origin. We show here that under appropriate conditions p4- and qin- like substitutions can be recovered from many E. coli K12 strains. Henderson and Weil 136
Copyright
@ 1975 by Academic
Press,
Inc.
utilizing a different technique have made similar observations described in the preceding paper (Henderson and Weil, 1975). MATERIALS
AND
METHODS
1. Media. X broth is 1% tryptone and 0.25% NaCl; h top agar contains, in addition, 0.65% agar. YM broth is X broth supplemented with 0.2% maltose and 0.1”) yeast extract. TC plates contain 1% trypticase (BBL), 0.5%’ NaCl, and 1.2”; agar. TCMg plates contain in addition 0.01 M MgSO,. MacConkey plates contain 4% MacConkey agar base (Difco) supplemented with 1% sugar. Citrate plates contain 1.05% K2HPOs, 0.45%) KH2P04, 0.005% MgSOa.7 HzO, 0.1% (NHh)$Od, 0.05% sodium citrate, 1.5% agar, 0.2%’ glucose and 1 .O pg/ml of thiamine-HCl. Phage dilutions were made in SM (Weigle et al., 1969), sometimes supplemented with 0.01 M MgSO,. 2. Phage and bacterial strains. See Table 1 and Fig. 2. The R594 isolate from this lab (R594A), D. Botstein (R594B), D. Henderson (R594H), and F. Stahl (R594S) are all su- (see Table 6), Gal- and Lac(tested on MacConkey plates), stP (able to grow in presence of 200 I*g/ml of streptomycin), and able to grow on citrate plates unsupplemented with amino acids. Deletion mutations bso,, bso2, and b,,, were obtained by plating stocks of XQ5$,, XQ5,,1S7, and XS7R5, respectively, on citrate
DEFECTIVE
PROPHAGE
plates with a lawn of bacterial strain QD5003. Large plaques were picked, purified, and retested for ability to form plaques on citrate plates. Under the conditions used, these phages form plaques as well as Xb2. 3. Construction of lysogens. Lysogens carrying X62, hb802, or Xb803 were formed efficiently in strain M1742, which carries the right prophage hybrid attachment site (Herskowitz and Signer, 1974). 4. Trans-induction infection experiments. These were one-step growth ex-
periments performed at low multiplicity of infection as described in Herskowitz and Signer (1974). Phage yield was calculated as phage produced per infected cell. 5. Phage stocks. High-titer phage stocks
IN
Phage
AND
6. Plaque
strains’
Ml07
$1 QD5003 R594A,
B, H, S
MI742 KRO KR3a KR6 KR6003 QR.5220 QR5366 TO1 E. coli B OP834
formation
Reference
and relevant
plates.
STRAINS”
strains”
Genotype
on citrate
1
BACTERIAL
Xc1857 in.6 red3 Xb2 X b2p4 XQam73, XQam57, XQam501, XSam7, XRam5 Ximm434, Ximm21 XbSOl Qam57 Sam7, Xb802 Qam501 Sam7, Xb803 Sam7 Ram5 Bacterial
137
K12
were made by picking a single plaque with a capillary tube and adding it to 1.0 ml of a fresh saturated bacterial culture diluted three-fold into X broth. After adsorption for 20 min at room temperature, 7.5 ml of X top agar was added and the mixture distributed to freshly poured TC plates (in some cases TCMg plates were used). After 4-5 hr at 37”, CHCl, was added to the plates, the layer of top agar collected, and the supernatant fluid collected after low speed centrifugation. Phage stocks produced in QR5366 were grown in the presence of 200 pg/ml of streptomycin to increase the efficiency of nonsense suppression in this strain. Phage stocks were assayed on TC or TCMg plates.
TABLE PHAGE
E. coli
Herskowitz Kellenberger Herskowitz Herskowitz Herskowitz See Materials
properties
and Signer (1970a) et al. (1960) and Signer (1974) and Signer (1970a) and Signer (1970b) and Methods Reference
suI+, permissive for amber mutations suII+, permissive for amber mutations, (hi+ revertant of C600) suIII+, permissive for amber mutations str’, gal-, lac-, su-; nonpermissive for amber mutations. su-recA-, carries right hybrid prophage attachment recA-, su-, lac-, trp.,suIII+ derivative of KRO ret+ derivative of KRO suIII+ derivative of KR6 AB1157 su- (= JC5220) AB1157 suII+ thyA-,malB(ho), suI+ derivative of R594A r-m(nonrestricting), gal-, met-, su-
or source
site
E. Signer Signer and Weil
or source
(1968)
Goldberg and Howe (1969) Campbell (1965), see Materials and Methods Herskowitz and Signer (1970a) E. Signer F. Ausubel (see Fig. 2) E. Signer (see Fig. 2) A. Strathern (see Fig. 2) J. Gillen E. Signer T. Ogawa (see Fig. 2) U. Kuhnlein (Wood, 1966)
a Abbreviations used: c, clear plaque; b, buoyant density alteration (DNA deletion); imm, immunity; am, amber; int, integration deficiency; red, recombination deficiency; pin, Q-independent. Two different classes of X mutants, both called Xgin, have been isolated as pseudorevertants of XQ- (Herskowitz and Signer, 1970b; Sato and Campbell, 1970). This paper refers only to those of Sato and Campbell. Italics are used for genetic loci (e.g., Q gene) and Roman type for functional products (e.g., Q+ phenotype). b Recombinants were formed according to the procedure of Signer and Weil (1968). c All strains except OP834 are E. coli K12. The genealogies of the strains are described in Fig. 2, Bachmann (1972), and Henderson and Weil (1975). Lysogens of MI742 were constructed as described in Materials and Methods.
138
STRATHERN
AND
Ability to form plaques in the presence of chelating agents (citrate) was tested by streaking a phage suspension onto a bacterial lawn (QD5003 or R$94A) on citrate plates at 34”. Under our plating conditions, A+, himm434 and hb2p4 produce no growth or a limited swath at high phage density; Ximm21 produces small plaques, and Xb2 produces a large plaque. The test works best if X top agar is used. RESULTS
I. Pseudorevertants
of X Mutants
Previous work indicated that the p4 and qin substitutions carry genes analogous to Q, S, and R of X (Herskowitz and Signer, 1974). If such analogs exist in E. coli strains, it should be possible to isolate pseudorevertants of XQ-, XS-, and ARmutants which carry these genes. In practice, we have looked for revertants of double or triple mutants (AQ-S-, XQ-R-, AS-R-, and AQ-Q-R-) so that rare pseudoreversion will not be obscured by true reversion. The p4 substitution mutation results in a net increase of approximately 9% DNA. Under ordinary growth conditions (see Materials and Methods) particles with this much extra DNA are unstable or cannot be formed unless the phage carries a deletion elsewhere in the phage DNA (Herskowitz, 1971; Weil et al., 1972). Consequently, we have used phages carrying deletions in the nonessential b2 region. Table 2 shows that revertants (shown below to be pseudorevertants) are found at a frequency of approximately lo- 5 in stocks of X mutants defective in genes Q and S or genes S and R. Revertants are observed only if the parent carries a deletion. A
10
F
20 ’
70
clw 80,’ r’
a. SR “xY
FIG. 1. Physical map of X DNA from phage particles, based on electron microscopy of DNA heteroduplexes (adapted from Davidson and Szybalski, 19711. Numbers indicate percent of X DNA length. Filled circle (0) between Q and S indicates position of the late gene promoter. The p4 mutation removes X DNA between positions 84 and 95, replacing it with DNA of approximately 20% the size of X DNA (Fiandt et al., 1971; Henderson and Weil, 1975).
HERSKOWITZ TABLE
REVERSIONOF Q-SPhage
AQ67S, AQmS, AS,& Aho, Qd, &m Qm‘3, Aboa S,R,
2
AND S-R-
X MUTANTS
Frequency
of revertantsa
67,
136-143
(1975)
Defective ANN Department
of Biology
Prophage
in Escherichia
STRATHERN
and Institute
of
IRA
AND
Molecular
Biology,
Accepted
April
co/i K12 Strains HERSKOWITZ
University
of
Oregon,
Eugene,
Oregon
97403
21, 1975
Many E. coli K12 strains carry genes analogous to the late gene positive regulator (gene Q) and lysis genes (genes S and R) of phage X. This block of genes (called qsr’) has been identified by isolating X strains in which these genes have replaced X genes Q. S, and R. Late gene control by at least some of these phages is similar to that of previously recognized substitution mutants (Xp4 and Xqin). Xqsr’ phages cannot be recovered from a strain (AB1157) which lacks other genes (rat, recE, reu) attributed to a defective prophage. Qsr’ and these other genes may, therefore, be derived from a common prophage. INTRODUCTION
The X substitution mutations, p4 and independently isolated 9in, although (Jacob and Wollman, 1954; Sato and Campbell, 1970), are strikingly similar in physiological behavior and genetic structure. Genetic and electron microscopic mapping show that X DNA corresponding to genes Q, S, and R and the late gene promoter has been deleted and is replaced by DNA which does not hybridize to X (Fig. 1) (Sato and Campbell, 1970; Fiandt et al., 1971; Herskowitz and Signer, 1974). The inserted DNA’s in p4 and qin mutations are homologous, as seen by electron microscopy of p4lqin DNA heteroduplexes (Fiandt et al., 1971). The substituted DNA of a p4 phage codes for a Q-like gene with a specificity of late gene expression distinct from X: the “Q” of p4 can activate late genes of a p4 or a qin prophage but not the late genes of a X prophage (Herskowitz and Signer, 1974). This substituted DNA presumably also codes for analogs to genes S and R since lysis is required for plaque formation (Campbell and de1 CampilloCampbell, 1963; Goldberg and Howe, 1969). The similarities between p4 and qin substitutions suggest that they have a common origin. We show here that under appropriate conditions p4- and qin- like substitutions can be recovered from many E. coli K12 strains. Henderson and Weil 136
Copyright
@ 1975 by Academic
Press,
Inc.
utilizing a different technique have made similar observations described in the preceding paper (Henderson and Weil, 1975). MATERIALS
AND
METHODS
1. Media. X broth is 1% tryptone and 0.25% NaCl; h top agar contains, in addition, 0.65% agar. YM broth is X broth supplemented with 0.2% maltose and 0.1”) yeast extract. TC plates contain 1% trypticase (BBL), 0.5%’ NaCl, and 1.2”; agar. TCMg plates contain in addition 0.01 M MgSO,. MacConkey plates contain 4% MacConkey agar base (Difco) supplemented with 1% sugar. Citrate plates contain 1.05% K2HPOs, 0.45%) KH2P04, 0.005% MgSOa.7 HzO, 0.1% (NHh)$Od, 0.05% sodium citrate, 1.5% agar, 0.2%’ glucose and 1 .O pg/ml of thiamine-HCl. Phage dilutions were made in SM (Weigle et al., 1969), sometimes supplemented with 0.01 M MgSO,. 2. Phage and bacterial strains. See Table 1 and Fig. 2. The R594 isolate from this lab (R594A), D. Botstein (R594B), D. Henderson (R594H), and F. Stahl (R594S) are all su- (see Table 6), Gal- and Lac(tested on MacConkey plates), stP (able to grow in presence of 200 I*g/ml of streptomycin), and able to grow on citrate plates unsupplemented with amino acids. Deletion mutations bso,, bso2, and b,,, were obtained by plating stocks of XQ5$,, XQ5,,1S7, and XS7R5, respectively, on citrate
DEFECTIVE
PROPHAGE
plates with a lawn of bacterial strain QD5003. Large plaques were picked, purified, and retested for ability to form plaques on citrate plates. Under the conditions used, these phages form plaques as well as Xb2. 3. Construction of lysogens. Lysogens carrying X62, hb802, or Xb803 were formed efficiently in strain M1742, which carries the right prophage hybrid attachment site (Herskowitz and Signer, 1974). 4. Trans-induction infection experiments. These were one-step growth ex-
periments performed at low multiplicity of infection as described in Herskowitz and Signer (1974). Phage yield was calculated as phage produced per infected cell. 5. Phage stocks. High-titer phage stocks
IN
Phage
AND
6. Plaque
strains’
Ml07
$1 QD5003 R594A,
B, H, S
MI742 KRO KR3a KR6 KR6003 QR.5220 QR5366 TO1 E. coli B OP834
formation
Reference
and relevant
plates.
STRAINS”
strains”
Genotype
on citrate
1
BACTERIAL
Xc1857 in.6 red3 Xb2 X b2p4 XQam73, XQam57, XQam501, XSam7, XRam5 Ximm434, Ximm21 XbSOl Qam57 Sam7, Xb802 Qam501 Sam7, Xb803 Sam7 Ram5 Bacterial
137
K12
were made by picking a single plaque with a capillary tube and adding it to 1.0 ml of a fresh saturated bacterial culture diluted three-fold into X broth. After adsorption for 20 min at room temperature, 7.5 ml of X top agar was added and the mixture distributed to freshly poured TC plates (in some cases TCMg plates were used). After 4-5 hr at 37”, CHCl, was added to the plates, the layer of top agar collected, and the supernatant fluid collected after low speed centrifugation. Phage stocks produced in QR5366 were grown in the presence of 200 pg/ml of streptomycin to increase the efficiency of nonsense suppression in this strain. Phage stocks were assayed on TC or TCMg plates.
TABLE PHAGE
E. coli
Herskowitz Kellenberger Herskowitz Herskowitz Herskowitz See Materials
properties
and Signer (1970a) et al. (1960) and Signer (1974) and Signer (1970a) and Signer (1970b) and Methods Reference
suI+, permissive for amber mutations suII+, permissive for amber mutations, (hi+ revertant of C600) suIII+, permissive for amber mutations str’, gal-, lac-, su-; nonpermissive for amber mutations. su-recA-, carries right hybrid prophage attachment recA-, su-, lac-, trp.,suIII+ derivative of KRO ret+ derivative of KRO suIII+ derivative of KR6 AB1157 su- (= JC5220) AB1157 suII+ thyA-,malB(ho), suI+ derivative of R594A r-m(nonrestricting), gal-, met-, su-
or source
site
E. Signer Signer and Weil
or source
(1968)
Goldberg and Howe (1969) Campbell (1965), see Materials and Methods Herskowitz and Signer (1970a) E. Signer F. Ausubel (see Fig. 2) E. Signer (see Fig. 2) A. Strathern (see Fig. 2) J. Gillen E. Signer T. Ogawa (see Fig. 2) U. Kuhnlein (Wood, 1966)
a Abbreviations used: c, clear plaque; b, buoyant density alteration (DNA deletion); imm, immunity; am, amber; int, integration deficiency; red, recombination deficiency; pin, Q-independent. Two different classes of X mutants, both called Xgin, have been isolated as pseudorevertants of XQ- (Herskowitz and Signer, 1970b; Sato and Campbell, 1970). This paper refers only to those of Sato and Campbell. Italics are used for genetic loci (e.g., Q gene) and Roman type for functional products (e.g., Q+ phenotype). b Recombinants were formed according to the procedure of Signer and Weil (1968). c All strains except OP834 are E. coli K12. The genealogies of the strains are described in Fig. 2, Bachmann (1972), and Henderson and Weil (1975). Lysogens of MI742 were constructed as described in Materials and Methods.
138
STRATHERN
AND
Ability to form plaques in the presence of chelating agents (citrate) was tested by streaking a phage suspension onto a bacterial lawn (QD5003 or R$94A) on citrate plates at 34”. Under our plating conditions, A+, himm434 and hb2p4 produce no growth or a limited swath at high phage density; Ximm21 produces small plaques, and Xb2 produces a large plaque. The test works best if X top agar is used. RESULTS
I. Pseudorevertants
of X Mutants
Previous work indicated that the p4 and qin substitutions carry genes analogous to Q, S, and R of X (Herskowitz and Signer, 1974). If such analogs exist in E. coli strains, it should be possible to isolate pseudorevertants of XQ-, XS-, and ARmutants which carry these genes. In practice, we have looked for revertants of double or triple mutants (AQ-S-, XQ-R-, AS-R-, and AQ-Q-R-) so that rare pseudoreversion will not be obscured by true reversion. The p4 substitution mutation results in a net increase of approximately 9% DNA. Under ordinary growth conditions (see Materials and Methods) particles with this much extra DNA are unstable or cannot be formed unless the phage carries a deletion elsewhere in the phage DNA (Herskowitz, 1971; Weil et al., 1972). Consequently, we have used phages carrying deletions in the nonessential b2 region. Table 2 shows that revertants (shown below to be pseudorevertants) are found at a frequency of approximately lo- 5 in stocks of X mutants defective in genes Q and S or genes S and R. Revertants are observed only if the parent carries a deletion. A
10
F
20 ’
70
clw 80,’ r’
a. SR “xY
FIG. 1. Physical map of X DNA from phage particles, based on electron microscopy of DNA heteroduplexes (adapted from Davidson and Szybalski, 19711. Numbers indicate percent of X DNA length. Filled circle (0) between Q and S indicates position of the late gene promoter. The p4 mutation removes X DNA between positions 84 and 95, replacing it with DNA of approximately 20% the size of X DNA (Fiandt et al., 1971; Henderson and Weil, 1975).
HERSKOWITZ TABLE
REVERSIONOF Q-SPhage
AQ67S, AQmS, AS,& Aho, Qd, &m Qm‘3, Aboa S,R,
2
AND S-R-
X MUTANTS
Frequency
of revertantsa
