VIROLOGY
72,
Isolation
33-44
(1976)
and Characterization of a A Specialized Transducing for the Escherichia co/i DNA Ligase Gene MICHAEL
National
Institutes
of Health,
National
Institute
Phage
M. GO’M’ESMAN’ of Arthritis, Metabolism Maryland 20014
Accepted
February
and Digestive
Diseases,
Bethesda,
16,1976’
The isolation and characterization of two plaque-forming specialized transducing derivatives of phage A, each carrying the entire Escherichia coli DNA ligase gene flig) as well as most of two closely linked genes involved in membrane transport of phosphosugars (all of p&-H and part of ptsl) are described. These phages were isolated from a lysate produced after induction of a A phage inserted at an abnormal attachment site within the p&Z gene. Their structure is analogous to that of the hpbio transducing phages. Extracts of lig- E. coli strains lysogenized by Aplig contain wild-type levels of DNA ligase activity, while during vegetative growth of one of these phages, DNA ligase activity in crude extracts increases up to 30-fold.
for the DNA ligase genes (kg) of E. coli is described in this paper. By genetic and Lambdoid specialized transducing electron microscopic heteroduplex mapphages have been isolated for a large numping studies, the structure of these phages ber of E. coli genes, and their value in studying regulation of transcription and is shown to be analogous to that of the translation of specific genetic material has hpbio’s (Signer, 1969; Davidson and Szybeen well demonstrated. The recent devel- balski, 1971). Genetic material adjacent to opment of a technique for isolating inser- the Zig locus (Gottesman et al., 1973), coding for the entire ptsH gene and most of tions of phage A at abnormal attachment sites scattered throughout the E. coli ge- the pt.sZ gene, is also carried by these nome (Shimada et al., 1972, 1973) has facil- phages. Cultures infected by one hplig contain large amounts of DNA ligase. A reitated the construction of such specialized transducing phage. In particular, this cent report has described the isolation of a transducing phage for technique has allowed the isolation of lambda-specialized transducing phage for a few of the genes the E. coli DNA ligase gene using an in technique (Cameron et concerned with the essential functions of vitro hybridization al., 1975). DNA such as replication (hdpolC; Shizuya et al., 1974) and transcription (Xdrib MATERIALS AND METHODS Kirschbaum, 1973). Phages carrying esBacterial and phage strains. Bacterial sential bacterial genes are useful both as a strains were all derivatives of E. coli K12 vehicle for increasing gene dosage and and are listed in Table 1. therefore the amount of gene product Phage strains, unless noted, were derivwithin the cell and as a genetic tool for atives of hcI857. AredS, bio69, bioll, studying the poorly understood control of biolOimm2lc.t.Sam7, and imm434c.t. essential functions. were from the collection of M. E. GottesThe isolation of two independent plaqueman. Ximm2lhySAam Barn was from J. forming X specialized transducing phages W. Little (Liedke-Kulke and Kaiser, ’ Current address: Department of Anatomy, Har1967), and As.d. was from J. Weil (Weil et vard Medical School, Boston, Massachusetts. al., 1975). T4 strains T4amE605 (from R. S. INTRODUCTION
33 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
34
MICHAEL
M. GO’M’ESMAN TABLE
BACTERIAL
Strain
1 STRAINS
Genotype
N2134
Hfr (O-thr-leu-lac) Afgal- Aattbio) KS302 Ared3 locptsl
N2625
N2134 gal+ Aatt+ bio+
FF7059 N1624 N1626 N2668 N2641 N2235 N2234 YMC N2217 N2299 FF8011 FF8020 FF8029 CHE-11 2570
F- strA ptsZ105 FF7059 pts+lig+ FF7059 pts+lig4 FF7059 pts+lig ts-7 FF7059 pts +recA56 FF7059 pts+poZA 1 FF7059 pts+ (P2 lysogen) F+ me1 su& YMC lig ts-7 trvi N2217 (Ared3Sam7) F-ptsH11 proC strA F-ptsH20 proC strA FptsH29 proC strA Hfr (O-thyA-argA-pts) p&Z211 Hfr (O-lysA-pheA-purF)thi ptsz Hfr (O-str-mtl-met) ilul88 argl xyl4 purl supN23 ptsZ F- ilv his strA ptsl N2625 (AptsZ) strA proC recA1 ptsZ40/F’98 F’98 was derived from KL98 and carries markers from supN to tyrA
KS302
2547A 911 IVA N2631 FF7040/F’98
Source and/or reference Shimada et al. (19731 M. E. Got&man. Ared lysogen of KS302 This work. Pl transductant of N2134 W. Epstein Gottesman et al. (19731 Got&man et al. (1973) Gottesman et al. (1973) Gottesman et al. (1973) Gottesman et al. (1973) Gottesman et al. (1973) M. Yarmolinsky Gottesman et al. (1974) This work W. Epstein W. Epstein W. Epstein W. Epstein Wang et al. (1969) Wang et al. (1969) Wang et al. (1969) This work; see Table II W. Epstein
Edgar) and T4am H39Xr59 (from H. Ber- cose-6-phosphate were from Sigma Chemiger) carry amber mutations in their ligase cal Company. structural gene and T7 LG3, sent by F. W. Isolation of A lysogens. Lysogens carryStudier, has a deletion of the T7 ligase ing immh were selected by plating on EMBO plates seeded with lo9 h&I and 10’ gene. et Media and plating techniques. Phage hh80cI (Shimada et al., 1972;Gottesman were assayed either on TB or BBL tryptial., 1974). Lysogens of A and Ared inserted into case plates (Gottesman et al., 1974). Bacterial lawns for plating or lysogenization by the pts genes were selected as follows A phage were grown to saturation in (modified from the general technique deTBMM (tryptone broth supplemented with scribed by Shimada et al., 1973). Bacterial strain KS302A(gal-Aatt-bio) was infected 0.01 M MgS04 and 0.2% maltose), centrifuged, resuspended in an equal volume of with A or Ared at a multiplicity of 10 and TMG (0.01 M Tris, pH 8, 0.01 M MgS04, appropriate dilutions of the infected cells 0.01% gelatin), and aerated for 30 min. were spread on sorbitol-MacConkey plates The following media were also used: LB seeded with lo9 Ab2cI and lo9 AhSOcI. agar (Bosner, 1973), EMBO agar (Gottes- White surviving colonies were streaked plates to conman and Yarmolinsky, 1968a), Mac- out on mannitol-MacConkey The notation Conkey indicator plates, and minimal me- firm the pts- phenotype. Ared lot ptsI has been used to indicate dia supplemented with either sorbitol (0.2%) or glucose-6-phosphate (0.2%) that the strain is a lysogen in which Ared (Gottesman et al., 1973). Sorbitol and glu- is inserted in the gene ptsl.
ISOLATION
PI transduction procedure and scoring of lig genotype. The generalized transduction ofpts and Zig markers by phage Pl has been previously described (Gottesman et al., 1973). pts+ bacteria were selected by
their growth on sorbitol-minimal agar and the Zig markers were scored by their ability or inability to support the growth of phage T4am30 and T4am3OrII mutants (gene 30 is the T4 ligase gene). In most cases, growth of the phage T7 strain, LG-3, which is deleted for the T7 ligase genes, was used to substantiate the bacterial ligase phenotype (Gottesman et al., 1973). If more than five bacterial clones were to be scored for lig, testing was done by replica plating from microwell plates with a metal multipronged applicator onto EMBO plates spread with appropriate tester phage (Gottesman et al., 1973). Determination of presence of Znt function. The presence or absence of int on
the various hplig’s described in this work was determined as previously described (Gottesman et al., 1974). Construction
of derivatives of Aplig. Xplig3limm2lc.t.Sam7 and hpZig9AOlimm-
35
OF hplig
al., 1974); or (2) by preparation of high titer plate lysates followed directly by banding in CsCl. The latter technique allowed simple and rapid preparation of purified phage stocks with titers of approx lO”/ml which were suitable as a source of DNA for electron microscopy. CsCl gradients were centrifuged for 24 hr in a Beckman Spinco 65 rotor at 34,000 rpm. Phage bands were extracted from the sides of the tubes to avoid contamination by bacteral DNA. Prior to extraction of DNA and formation of heteroduplexes, phage were dialyzed against TMG. Formation of heteroduplexes, electron microscopy, and measurement and calibration of photographed heteroduplexes were done as originally described by Davis et al. (1971) and modified by Gottesman et al. (1974). RESULTS
Construction and Mapping of the A Insertions in pts A Phages which carry mutations
in their to in DNA
red (exo or bet) or gam genes are unable
plate on bacterial strains deficient ligase (lig) or DNA polymerase I (polA) (Zissler et aZ., 1971a; Gottesman et al., 1973). The general scheme for isolation of ApZig’s involved the insertion of a Ared (exe- and bet-) into the gene pts (Epstein et al., 1970), which is known to be 90% cotransduced by phage Pl with Zig (Gottesman et al., 1973). These lysogens were induced and phages in these lyPreparation of crude extracts and assay thermally of DNA ligase. Crude extracts were pre- sates which were able to plate on Zigpared by sonicaton and assayed using the strains (while still retaining the Bed- phe3H-dAT joining assay as previously de- notype as shown by their failure to plate scribed (Gottesman et al., 1973). A unit of on poZA hosts) were selected. The pts locus appears to be one of the DNA ligase activity is defined as the joining of 100 nmole of 3H-(dAT), in 30’ (Mod- preferred locations for insertion of phage A when the normal bacterial Aatt site has rich and Lehman, 1970). Weisberg, and Preparation of phage for electron micro- been deleted (Shimada, scopic heteroduplex mapping. In order to Gottesman, 1973). In the work reported here, three independent insertions of perform electron microscopic heteroduplex mapping of the phage described in this Ared in pts in strain KS302 were selected work it was first necessary to prepare puri- as described in Materials and Methods. fied, relatively high titer stocks. These These insertions arose at an overall frestocks were prepared in one of two ways: quency of 1 per lo3 A lysogens or 1 per lo5 (1) by induction of a lysogen of the phage infected cells. strain in question, followed by polyethylSingle lysogens of A phage at abnormal ene glycol precipitation and banding in an bacterial attachment sites yield low titer equilibrium CsCl gradient (Gottesman et lysates of plaque-forming phage after in2lc.t.Sam7 were constructed by crossing the appropriate Xplig with AbiolOimm21c.t.Sam7 in YMC(su&) and selecting phage carrying inn21 and lig on N2299 (su& Zigts = 7 Ared3Sam7) at 32”. These phage were scored for Sam7 by their ability to plate on the su& host YMC and their inability to plate on the su host N1624.
36
MICHAEL
M.
GOTTESMAN
duction because of their abnormal hybrid gion. A series of these has been mapped attachment sites (P * A’ and A. P’) (Shi- against known ptsl and ptsH mutants (D. mada et al., 1972). Tandem double lyso- J. Gruol and W. Epstein, personal commugens are able to excise by the ter system nication). All of the deletions begin within with relatively high efficiency and give the ptsl gene and several extend clockwise much higher titer lysates (Got&man and on the E. coli genetic map (Taylor and Yarmolinsky, 1968b). In this work, single Trotter, 1967) out of theptsl gene and into lysogens, which give small phage bursts a locus which is required for S042- utilizawhen induced, were chosen since these tion (W. Epstein, personal communicawould be expected to give a relatively high tion). This locus appears to be analogous to frequency of abnormal excision events the cysA locus mapped in Salmonella tyafter induction, and hence a high yield of phimurium and linked by transduction to specialized transducing phage per plaque- pts in this organism (Cordaro and Roseforming phage. One strain carrying a man, 1972). None of the deletions tested by hred3 insertion inpts, strain N2134, gave Gruol and Epstein extend into the ptsH a phage burst of 5 x lop4 PFU/induced gene, which is closely linked to ptsI and cell, as compared to a known tandem dou- has been mapped by three-factor crosses ble lysogen of A in pts which gave a burst counterclockwise to ptsl. These data place of ca. lo/induced cell. Strain N2134 was the Ared insertion in strain N2625 inptsl. selected for further study. Data presented below will show that the Strain N2134 was unable to grow on Aplig phages generated from strain N2134 minimal media containing sorbitol, mancarry most, but not all of ptsl, and the nitol, glucose, lactose, or maltose as car- entire ptsH and Zig genes which are counbon source. The multiple sugar defect and terclockwise to ptsl. These data confirm immh were 100% cotransduced in strain the conclusion of Gruol and Epstein from N2134. These genotypes were 90% cotrans- bacterial deletion mapping that Ared is duced with Zig, confirming that hred3 was inserted into geneptsl in strain N2134 and probably inserted in pts. its derivative N2625. Utilizing 42” survivors of N2625, a proBy selecting high temperature (42”) surphage deletion map of Ared inserted in vivors of strain N2625 (a bio+ att+ gal+ Pl transductant of N2134), it is possible to p&I can be constructed. Table 2 demonobtain bacterial deletions in the pts re- strates that the order of genes in the proTABLE ORIENTATION Number of independent isolates”
Strain designation
1 1 1 2
N2684 N2680 N2681 N2679; N2682 N2685; LE-10 N2631
2 1
OF
hred3 INSERTION
Growth on SO,b
IN
2
p&Z BY PROPHAGE
Complementation
DELETION
by prophage
MAPPING
deletions
of A amber
mutants’
NOPQRAFLIJ + + +
-
+ -
-
-
-
+
-
-
+ -
+ -
+ +
-
-
-
-
-
+ + + -
+ + + +
+ + + +
+ + + +
+ + + +
-
-
-
-
(1 These deletion strains were selected from isolated clones of strain N2625 (Ared locptsll for survival at 42”. All plated A and hence were nonimmune, but carried other phage genes as indicated by the complementation tests in this table. b Some of the deletions selected as described above were unable to utilize SOd2- as sole sulfur source, but could use SOz2-. This was determined by testing for growth on minimal agar plates with either 0.01 M (NH&SO, alone or 0.01 M (NH,l,SO, and 0.01 M Na$O, as sulfur source. c Lawns of the deletion strains were prepared as described in Materials and Methods and spotted with 2 ~1 of the indicated A amber mutants ka., 10’ PFU/mll. A positive complementation test was complete lysis and a negative test showed no lysis. A lawn of YMC (su&) was included as a control.
ISOLATION
phage serted normal other sites These
is the same as that when A is inthrough the phage att site at its bacterial attachment site or at well-studied abnormal attachment (Shimada et al., 1972 and 1973). data also establish the orientation of Ared in N2625 which is with int clockwise from the b2 region (see line 3, Fig. 11, the opposite of the orientation of A at its normal bacterial attachment site. Isolation of Aplig
Low titer lysates of strain N2134 (Ared lot ptsl) were prepared by thermal induction and the phages were concentrated and banded in CsCl gradients as described in Materials and Methods. The purified phages were initially screened for growth on strain N1626 (Zig4) at 42” and one of several minute plaques (frequency of appearance, 5 x lO+VPFU) was purified and chosen for further study (ApZig9). This phage was eventually propagated through a number of hosts, including a strain carrying a lop mutation (lop is a cis-dominant control element of Zig which increases levels of DNA ligase in cell extracts by a factor of 5; Gottesman et al., 1973). No evidence could be obtained that the lop mutation had been acquired by the phage; however, a large plaque variant was recovered which appeared to be identical with the parent phage except for the acquisition of a deletion in the b2 region (see heteroduplex mapping data below). This phage has been designated ApZig9AOl. In a second selection scheme, the lysates from N2134 were selected for growth on
Phage
red
mm
37
OF hplig
strain N2234 (P2 lysogen) since such a selection identifies phage which are the product of an abnormal excision (Zissler et al., 1971b) and screened for growth on strain N2668 (Zig ts-7). A large plaqueformer was obtained (frequency of appearance, 3 x lo-“IPFU) which has been designated hpZig31. Table 3 summarizes the efficiency of plating and plaque morphology of these Aplig’s, hred3, Abio69, and Abioll. These hbio transducing phage have deletions of A DNA similar to the deletions
AD*9
.xPllW
FIG. 1. Formation of the lysogen Ared lot ptsl and the isolation of ApZig9 and Aplig31 from this insertion. The figure and notation is in the style of Shimada et al. (1973).
TABLE 3 EFFICIENCY OF PLATING OF hdids Efficiency of plating” ligts-7
1 recA56 P2 lysogen tYP@ Aplig9 + 1.1
72,
Isolation
33-44
(1976)
and Characterization of a A Specialized Transducing for the Escherichia co/i DNA Ligase Gene MICHAEL
National
Institutes
of Health,
National
Institute
Phage
M. GO’M’ESMAN’ of Arthritis, Metabolism Maryland 20014
Accepted
February
and Digestive
Diseases,
Bethesda,
16,1976’
The isolation and characterization of two plaque-forming specialized transducing derivatives of phage A, each carrying the entire Escherichia coli DNA ligase gene flig) as well as most of two closely linked genes involved in membrane transport of phosphosugars (all of p&-H and part of ptsl) are described. These phages were isolated from a lysate produced after induction of a A phage inserted at an abnormal attachment site within the p&Z gene. Their structure is analogous to that of the hpbio transducing phages. Extracts of lig- E. coli strains lysogenized by Aplig contain wild-type levels of DNA ligase activity, while during vegetative growth of one of these phages, DNA ligase activity in crude extracts increases up to 30-fold.
for the DNA ligase genes (kg) of E. coli is described in this paper. By genetic and Lambdoid specialized transducing electron microscopic heteroduplex mapphages have been isolated for a large numping studies, the structure of these phages ber of E. coli genes, and their value in studying regulation of transcription and is shown to be analogous to that of the translation of specific genetic material has hpbio’s (Signer, 1969; Davidson and Szybeen well demonstrated. The recent devel- balski, 1971). Genetic material adjacent to opment of a technique for isolating inser- the Zig locus (Gottesman et al., 1973), coding for the entire ptsH gene and most of tions of phage A at abnormal attachment sites scattered throughout the E. coli ge- the pt.sZ gene, is also carried by these nome (Shimada et al., 1972, 1973) has facil- phages. Cultures infected by one hplig contain large amounts of DNA ligase. A reitated the construction of such specialized transducing phage. In particular, this cent report has described the isolation of a transducing phage for technique has allowed the isolation of lambda-specialized transducing phage for a few of the genes the E. coli DNA ligase gene using an in technique (Cameron et concerned with the essential functions of vitro hybridization al., 1975). DNA such as replication (hdpolC; Shizuya et al., 1974) and transcription (Xdrib MATERIALS AND METHODS Kirschbaum, 1973). Phages carrying esBacterial and phage strains. Bacterial sential bacterial genes are useful both as a strains were all derivatives of E. coli K12 vehicle for increasing gene dosage and and are listed in Table 1. therefore the amount of gene product Phage strains, unless noted, were derivwithin the cell and as a genetic tool for atives of hcI857. AredS, bio69, bioll, studying the poorly understood control of biolOimm2lc.t.Sam7, and imm434c.t. essential functions. were from the collection of M. E. GottesThe isolation of two independent plaqueman. Ximm2lhySAam Barn was from J. forming X specialized transducing phages W. Little (Liedke-Kulke and Kaiser, ’ Current address: Department of Anatomy, Har1967), and As.d. was from J. Weil (Weil et vard Medical School, Boston, Massachusetts. al., 1975). T4 strains T4amE605 (from R. S. INTRODUCTION
33 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
34
MICHAEL
M. GO’M’ESMAN TABLE
BACTERIAL
Strain
1 STRAINS
Genotype
N2134
Hfr (O-thr-leu-lac) Afgal- Aattbio) KS302 Ared3 locptsl
N2625
N2134 gal+ Aatt+ bio+
FF7059 N1624 N1626 N2668 N2641 N2235 N2234 YMC N2217 N2299 FF8011 FF8020 FF8029 CHE-11 2570
F- strA ptsZ105 FF7059 pts+lig+ FF7059 pts+lig4 FF7059 pts+lig ts-7 FF7059 pts +recA56 FF7059 pts+poZA 1 FF7059 pts+ (P2 lysogen) F+ me1 su& YMC lig ts-7 trvi N2217 (Ared3Sam7) F-ptsH11 proC strA F-ptsH20 proC strA FptsH29 proC strA Hfr (O-thyA-argA-pts) p&Z211 Hfr (O-lysA-pheA-purF)thi ptsz Hfr (O-str-mtl-met) ilul88 argl xyl4 purl supN23 ptsZ F- ilv his strA ptsl N2625 (AptsZ) strA proC recA1 ptsZ40/F’98 F’98 was derived from KL98 and carries markers from supN to tyrA
KS302
2547A 911 IVA N2631 FF7040/F’98
Source and/or reference Shimada et al. (19731 M. E. Got&man. Ared lysogen of KS302 This work. Pl transductant of N2134 W. Epstein Gottesman et al. (19731 Got&man et al. (1973) Gottesman et al. (1973) Gottesman et al. (1973) Gottesman et al. (1973) Gottesman et al. (1973) M. Yarmolinsky Gottesman et al. (1974) This work W. Epstein W. Epstein W. Epstein W. Epstein Wang et al. (1969) Wang et al. (1969) Wang et al. (1969) This work; see Table II W. Epstein
Edgar) and T4am H39Xr59 (from H. Ber- cose-6-phosphate were from Sigma Chemiger) carry amber mutations in their ligase cal Company. structural gene and T7 LG3, sent by F. W. Isolation of A lysogens. Lysogens carryStudier, has a deletion of the T7 ligase ing immh were selected by plating on EMBO plates seeded with lo9 h&I and 10’ gene. et Media and plating techniques. Phage hh80cI (Shimada et al., 1972;Gottesman were assayed either on TB or BBL tryptial., 1974). Lysogens of A and Ared inserted into case plates (Gottesman et al., 1974). Bacterial lawns for plating or lysogenization by the pts genes were selected as follows A phage were grown to saturation in (modified from the general technique deTBMM (tryptone broth supplemented with scribed by Shimada et al., 1973). Bacterial strain KS302A(gal-Aatt-bio) was infected 0.01 M MgS04 and 0.2% maltose), centrifuged, resuspended in an equal volume of with A or Ared at a multiplicity of 10 and TMG (0.01 M Tris, pH 8, 0.01 M MgS04, appropriate dilutions of the infected cells 0.01% gelatin), and aerated for 30 min. were spread on sorbitol-MacConkey plates The following media were also used: LB seeded with lo9 Ab2cI and lo9 AhSOcI. agar (Bosner, 1973), EMBO agar (Gottes- White surviving colonies were streaked plates to conman and Yarmolinsky, 1968a), Mac- out on mannitol-MacConkey The notation Conkey indicator plates, and minimal me- firm the pts- phenotype. Ared lot ptsI has been used to indicate dia supplemented with either sorbitol (0.2%) or glucose-6-phosphate (0.2%) that the strain is a lysogen in which Ared (Gottesman et al., 1973). Sorbitol and glu- is inserted in the gene ptsl.
ISOLATION
PI transduction procedure and scoring of lig genotype. The generalized transduction ofpts and Zig markers by phage Pl has been previously described (Gottesman et al., 1973). pts+ bacteria were selected by
their growth on sorbitol-minimal agar and the Zig markers were scored by their ability or inability to support the growth of phage T4am30 and T4am3OrII mutants (gene 30 is the T4 ligase gene). In most cases, growth of the phage T7 strain, LG-3, which is deleted for the T7 ligase genes, was used to substantiate the bacterial ligase phenotype (Gottesman et al., 1973). If more than five bacterial clones were to be scored for lig, testing was done by replica plating from microwell plates with a metal multipronged applicator onto EMBO plates spread with appropriate tester phage (Gottesman et al., 1973). Determination of presence of Znt function. The presence or absence of int on
the various hplig’s described in this work was determined as previously described (Gottesman et al., 1974). Construction
of derivatives of Aplig. Xplig3limm2lc.t.Sam7 and hpZig9AOlimm-
35
OF hplig
al., 1974); or (2) by preparation of high titer plate lysates followed directly by banding in CsCl. The latter technique allowed simple and rapid preparation of purified phage stocks with titers of approx lO”/ml which were suitable as a source of DNA for electron microscopy. CsCl gradients were centrifuged for 24 hr in a Beckman Spinco 65 rotor at 34,000 rpm. Phage bands were extracted from the sides of the tubes to avoid contamination by bacteral DNA. Prior to extraction of DNA and formation of heteroduplexes, phage were dialyzed against TMG. Formation of heteroduplexes, electron microscopy, and measurement and calibration of photographed heteroduplexes were done as originally described by Davis et al. (1971) and modified by Gottesman et al. (1974). RESULTS
Construction and Mapping of the A Insertions in pts A Phages which carry mutations
in their to in DNA
red (exo or bet) or gam genes are unable
plate on bacterial strains deficient ligase (lig) or DNA polymerase I (polA) (Zissler et aZ., 1971a; Gottesman et al., 1973). The general scheme for isolation of ApZig’s involved the insertion of a Ared (exe- and bet-) into the gene pts (Epstein et al., 1970), which is known to be 90% cotransduced by phage Pl with Zig (Gottesman et al., 1973). These lysogens were induced and phages in these lyPreparation of crude extracts and assay thermally of DNA ligase. Crude extracts were pre- sates which were able to plate on Zigpared by sonicaton and assayed using the strains (while still retaining the Bed- phe3H-dAT joining assay as previously de- notype as shown by their failure to plate scribed (Gottesman et al., 1973). A unit of on poZA hosts) were selected. The pts locus appears to be one of the DNA ligase activity is defined as the joining of 100 nmole of 3H-(dAT), in 30’ (Mod- preferred locations for insertion of phage A when the normal bacterial Aatt site has rich and Lehman, 1970). Weisberg, and Preparation of phage for electron micro- been deleted (Shimada, scopic heteroduplex mapping. In order to Gottesman, 1973). In the work reported here, three independent insertions of perform electron microscopic heteroduplex mapping of the phage described in this Ared in pts in strain KS302 were selected work it was first necessary to prepare puri- as described in Materials and Methods. fied, relatively high titer stocks. These These insertions arose at an overall frestocks were prepared in one of two ways: quency of 1 per lo3 A lysogens or 1 per lo5 (1) by induction of a lysogen of the phage infected cells. strain in question, followed by polyethylSingle lysogens of A phage at abnormal ene glycol precipitation and banding in an bacterial attachment sites yield low titer equilibrium CsCl gradient (Gottesman et lysates of plaque-forming phage after in2lc.t.Sam7 were constructed by crossing the appropriate Xplig with AbiolOimm21c.t.Sam7 in YMC(su&) and selecting phage carrying inn21 and lig on N2299 (su& Zigts = 7 Ared3Sam7) at 32”. These phage were scored for Sam7 by their ability to plate on the su& host YMC and their inability to plate on the su host N1624.
36
MICHAEL
M.
GOTTESMAN
duction because of their abnormal hybrid gion. A series of these has been mapped attachment sites (P * A’ and A. P’) (Shi- against known ptsl and ptsH mutants (D. mada et al., 1972). Tandem double lyso- J. Gruol and W. Epstein, personal commugens are able to excise by the ter system nication). All of the deletions begin within with relatively high efficiency and give the ptsl gene and several extend clockwise much higher titer lysates (Got&man and on the E. coli genetic map (Taylor and Yarmolinsky, 1968b). In this work, single Trotter, 1967) out of theptsl gene and into lysogens, which give small phage bursts a locus which is required for S042- utilizawhen induced, were chosen since these tion (W. Epstein, personal communicawould be expected to give a relatively high tion). This locus appears to be analogous to frequency of abnormal excision events the cysA locus mapped in Salmonella tyafter induction, and hence a high yield of phimurium and linked by transduction to specialized transducing phage per plaque- pts in this organism (Cordaro and Roseforming phage. One strain carrying a man, 1972). None of the deletions tested by hred3 insertion inpts, strain N2134, gave Gruol and Epstein extend into the ptsH a phage burst of 5 x lop4 PFU/induced gene, which is closely linked to ptsI and cell, as compared to a known tandem dou- has been mapped by three-factor crosses ble lysogen of A in pts which gave a burst counterclockwise to ptsl. These data place of ca. lo/induced cell. Strain N2134 was the Ared insertion in strain N2625 inptsl. selected for further study. Data presented below will show that the Strain N2134 was unable to grow on Aplig phages generated from strain N2134 minimal media containing sorbitol, mancarry most, but not all of ptsl, and the nitol, glucose, lactose, or maltose as car- entire ptsH and Zig genes which are counbon source. The multiple sugar defect and terclockwise to ptsl. These data confirm immh were 100% cotransduced in strain the conclusion of Gruol and Epstein from N2134. These genotypes were 90% cotrans- bacterial deletion mapping that Ared is duced with Zig, confirming that hred3 was inserted into geneptsl in strain N2134 and probably inserted in pts. its derivative N2625. Utilizing 42” survivors of N2625, a proBy selecting high temperature (42”) surphage deletion map of Ared inserted in vivors of strain N2625 (a bio+ att+ gal+ Pl transductant of N2134), it is possible to p&I can be constructed. Table 2 demonobtain bacterial deletions in the pts re- strates that the order of genes in the proTABLE ORIENTATION Number of independent isolates”
Strain designation
1 1 1 2
N2684 N2680 N2681 N2679; N2682 N2685; LE-10 N2631
2 1
OF
hred3 INSERTION
Growth on SO,b
IN
2
p&Z BY PROPHAGE
Complementation
DELETION
by prophage
MAPPING
deletions
of A amber
mutants’
NOPQRAFLIJ + + +
-
+ -
-
-
-
+
-
-
+ -
+ -
+ +
-
-
-
-
-
+ + + -
+ + + +
+ + + +
+ + + +
+ + + +
-
-
-
-
(1 These deletion strains were selected from isolated clones of strain N2625 (Ared locptsll for survival at 42”. All plated A and hence were nonimmune, but carried other phage genes as indicated by the complementation tests in this table. b Some of the deletions selected as described above were unable to utilize SOd2- as sole sulfur source, but could use SOz2-. This was determined by testing for growth on minimal agar plates with either 0.01 M (NH&SO, alone or 0.01 M (NH,l,SO, and 0.01 M Na$O, as sulfur source. c Lawns of the deletion strains were prepared as described in Materials and Methods and spotted with 2 ~1 of the indicated A amber mutants ka., 10’ PFU/mll. A positive complementation test was complete lysis and a negative test showed no lysis. A lawn of YMC (su&) was included as a control.
ISOLATION
phage serted normal other sites These
is the same as that when A is inthrough the phage att site at its bacterial attachment site or at well-studied abnormal attachment (Shimada et al., 1972 and 1973). data also establish the orientation of Ared in N2625 which is with int clockwise from the b2 region (see line 3, Fig. 11, the opposite of the orientation of A at its normal bacterial attachment site. Isolation of Aplig
Low titer lysates of strain N2134 (Ared lot ptsl) were prepared by thermal induction and the phages were concentrated and banded in CsCl gradients as described in Materials and Methods. The purified phages were initially screened for growth on strain N1626 (Zig4) at 42” and one of several minute plaques (frequency of appearance, 5 x lO+VPFU) was purified and chosen for further study (ApZig9). This phage was eventually propagated through a number of hosts, including a strain carrying a lop mutation (lop is a cis-dominant control element of Zig which increases levels of DNA ligase in cell extracts by a factor of 5; Gottesman et al., 1973). No evidence could be obtained that the lop mutation had been acquired by the phage; however, a large plaque variant was recovered which appeared to be identical with the parent phage except for the acquisition of a deletion in the b2 region (see heteroduplex mapping data below). This phage has been designated ApZig9AOl. In a second selection scheme, the lysates from N2134 were selected for growth on
Phage
red
mm
37
OF hplig
strain N2234 (P2 lysogen) since such a selection identifies phage which are the product of an abnormal excision (Zissler et al., 1971b) and screened for growth on strain N2668 (Zig ts-7). A large plaqueformer was obtained (frequency of appearance, 3 x lo-“IPFU) which has been designated hpZig31. Table 3 summarizes the efficiency of plating and plaque morphology of these Aplig’s, hred3, Abio69, and Abioll. These hbio transducing phage have deletions of A DNA similar to the deletions
AD*9
.xPllW
FIG. 1. Formation of the lysogen Ared lot ptsl and the isolation of ApZig9 and Aplig31 from this insertion. The figure and notation is in the style of Shimada et al. (1973).
TABLE 3 EFFICIENCY OF PLATING OF hdids Efficiency of plating” ligts-7
1 recA56 P2 lysogen tYP@ Aplig9 + 1.1
