Molec. gen. Genet. 155,241
247 (1977) (c~ by Springer-Verlag 1977
Deletion Mutants of Temperate Bacillus subtilis Bacteriophage ~105 Jan-Ingmar Flock Department of Bacteriology, Karolinska Institutet, S-104 01 Stockholm 60, Sweden
Summary. Six deletion mutants of temperate Bacillus subtilis phage d~105 have been isolated on the basis of their increased resistance to chelating agents. The size and position of the deletions was determined by electronmicroscopy of DNA heteroduplexes. All deletions are located in a region about 55-70% from one end of the DNA molecule, in the right half of the known genetic map of the phage. The segment 55-65% does not contain any genes essential for lytic growth or lysogenization. A gene(s) for immunity is located in a segment 65 70% from the left end. By electronmicroscopy of partially denatured d?105 DNA two A-T rich regions have been localized in the right half of the molecule. One of these regions falls within the non-essential 55-65% DNA segment.
Introduction
DNA deletion mutants of several bacteriophages show increased resistance to chelating agents and to heat (Rubenstein, 1968; Parkinson and Huskey, 1971), a fact which makes their isolation quite easy. Part of the genome of temperate phage is not required for lytic growth. In coliphage lambda about 25% and in coliphage P2 about 18% of wild type DNA can be deleted without severely impairing lytic growth of the phage. To identify and orient DNA by electron microscopy heteroduplexes between wild type and deletion mutant and/or partial denaturation maps can be used (Inman, 1967; Davis and Davidson, 1968). Phage qb105 is a temperate phage active on Bacillus subtilis ( Bridsell et al., 1969). The phage attachment site maps outside all known genetic markers (Armentrout and Rutberg, 1970), and might be located at the very end(s) of the mature phage DNA (Armentrout and Rutberg, 1970 ; Chow and Davidson, 1973). Induction of ~105 lysogenic bacteria is not followed
by rapid excision of the prophage as shown for several other phages (Armentrout and Rutberg, 1971). After induction the prophage DNA initially replicates covalently bound to bacterial DNA adjacent to the phage attachment site (Rutberg, 1973). In this property (~105 resembles coliphage Mu-1 (Abelson etal., 1974; Schroeder et al., 1974). However, prophage 00105 integrates at a specific locus (Rutberg, 1969), whereas Mu-1 can integrate anywhere on the bacterial chromosome (Bukhari and Zipser, 1972). We describe here some properties of six deletion mutants of d~105. Heteroduplexes between wild type DNA and DNA from these deletion mutants have been studied in the electron microscope, and the location of the 4)105 immunity gene on the (~105 DNA molecule has been determined. The heteroduplexes seen in the electron microscope have been oriented in relation to the known genetic map of d?105. Finally, we have constructed a partial denaturation map of ~105 mature DNA and oriented this in respect to the position of the deletions.
Materials and Methods Bacteria and Phage. The following strains of Bacillus subtilis were used: W168 (obtained from J. Spizizen), SR135 (su+3, trp-7, spoA9 obtained from J.A. Hoch), BR95 (~plO5tsA15) (phe-A1, ilvC1, trpC2 obtained from J. Spizizen and lysogenized with ~plO5tsA15 in our laboratory), and 3G18 (trp, met, ade) obtained from G. Venema. Wild type ~105 and its mutants, and the media and methods used to grow phage have been described (Armentrout and Rutberg, 1970). ~4C-iabeled T7 D N A was prepared as previously described (Armentrout et al., 1971). Competent cells were prepared from 3G18 as described by Arwert and Venema (1973) and stored in 10% glycerol at 70 ° C. Immediately before use the cells were .% thawed at 37 ° C. PBS1 transduction was performed as prevloulsy described (Rutberg, 1969).
Treatment of ~105 with Chelating Agent. Wild type ~105 was diluted 100-fold into 10 m M Naz-pyrophosphate at 32 ° C. The phage titer dropped about 104-fold in a few minutes. Plate stocks were
J.-I. Flock: Deletion Mutants of Temperate Bacillus subtilis Bacteriophage d~105
242
o= 100~,,~
"O
A
'°/ u 0,1
0.01o
\ 8
2'o
Time(minutes)
JB
J2
Fig. 1. Inactivation of wild type ~b105 with Naz-pyrophosphate. Phage was diluted 100-fold into 10 mM Na2-pyrophosphate at 32° C, and plaque forming particles were assayed at intervals. Stocks of surviving phage were made directly from the plates, and the surviving phage was exposed to a second and then a third cycle of treatment with Na2-pyrophosphate. Symbols : • - - • first cycle, A - - A second cycle, o - - e and third cycle of treatment respectively
made from plates with surviving phage and the phage exposed to a second and third cycle of Na2-pyrophosphate treatment. With successive cycles of treatment phage with increased resistance to Naz-pyrophosphate was selected (Fig. 1). Similar results were obtained using SSC (0.15 NaC1-0.015M Na citrate). Phage from CsCI gradients (see below) was tested for sensitivity to SSC by the "running" temperature method as described by Bertani (1975).
CsCI Density Gradients. Phage stocks were prepared from single plaques and the phage dialyzed against 1/10 nutrient broth (Difco) with 5 x 10-3M MgCI2. The phage stock was then diluted with saturated CsC1 (E. Merck, Darmstadt) to give a density of about 1.48. The phage was centrifuged at 120,000 x g in 6 x 5 ml MSE swing out rotor for 48 h at about 12° C. Two drop fractions were collected from the bottom of the tube, the density of some fractions was determined using an Abbe refractometer. A small sample fi'om each fraction was spotted on W168 indicator. The fractions containing phage were then titrated for plaque-formers and samples from these fractions were tested for sensitivity to SSC as described above. Marker Rescue. Competent 3G18 cells were mixed with phage DNA at 37 ° C. After 10 rain the sample was dduted 10-fold into tubes containing genetically marked phage particles to give a multiplicity of infection of about 10_ After an additional 10min for adsorption of phage the tubes were assayed. The superinfecting phage used was qb105 susC19 and (b105 susJll, respectively, and marker rescue was assayed by plating for wild type recombinants on he non-permissive host W168. Controls for reversions were included in each experiment,
Preparation of Phage DNA .for Electron Microscopy. Phage stocks were prepared and purified by CsC1 density gradient centrifugation as previously described (Rutberg, 1969, Rutberg et al., 1972). DNA was extracted with water saturated phenol.
Preparation of Heteroduplex DNA for Electron Microscopy. Phage DNA at a concentration of about 5 p,g per ml was mixed with 5 gl 0.2 M EDTA, 5 gl 1 M NaOH, to give a final volume of 50 gl. The mixture was left at room temperature for 10 rain and then 5 ~tl 2M Tris buffer pH 7.5 and 50 gl formamide (Merck, recrystallized (Chow et al., 1975)) was added. After one hour at room temperature 10gl was mixed with 101al cytochrome C (Sigma Co) 1 mg per ml, 40 gl formamide, 10 gl 1M Tris buffer pH 8.5 with 0.1M EDTA and 30 gl redistilled water. Fifty p.1 of this mixture was then spread onto a hypophase with 10% formamide in 10 mM Tns buffer pH 8.5. In later experiments heterod uplex DNA was also prepared directly from intact phage particles as described by Davis (Davis and Parkinson, 1971). DNA was collected on 300 mesh parlodion coated copper grids. The preparations were stained for 30 sec in 5 x 10-5 M uranyl acetate, washed in 93% ethanol, rotary shadowed with platinum/ palladium (80/20) and visualized in a Philips EM 200 or EM 301. Length measurements were done in a Scriptographic digitizer connected to a Compucorp 445 Statistician calculator. Single-stranded ~X174 DNA was added to DNA samples for calibration. Grating replicas were also used. Partial Denaturation of 0105 DNA. Phage DNA was added to a spreading solution containing 0.1M Tris buffer pH 8.5, 10 mM EDTA and 0.1 mg cytochrome C per ml in 82% formamide. The DNA was spread on a hypophase containing 50% formamide in 10 mM Tris buffer pH 8.5. Length variation between different molecules was less than 10%. No difference between single and double stranded DNA can be seen with this method.
Experimental Results Isolation o f 9105 Deletion Mutants. Six C l o n e s o f qb105 w i t h i n c r e a s e d r e s i s t a n c e t o N a 2 - p y r o p h o s p h a t e were selected for further studies. All mutants were isolated in different experiments and are of independent origin. M u t a n t s D I : l t a n d D 1 : 2 9 t w e r e p i c k e d as t u r b i d p l a q u e s a n d m u t a n t s D I : l c , 2c a n d 4c w e r e p i c k e d as c l e a r p l a q u e s a f t e r o n e cycle o f t r e a t m e n t w i t h Na2-pyrophosphate. M u t a n t D I I : 6 c w a s p i c k e d as a clear plaque after two cycles of Naz-pyrophosphate treatment.
General Characterization o f the Deletion Mutants. I n CsC1 g r a d i e n t s t h e d e l e t i o n m u t a n t s b a n d a t a l o w e r density than wild phage indicating an increased p r o t e i n t o D N A r a t i o (Fig. 2). It was shown that the four clear plaque mutants are unable to make a functional immunity repressor b e c a u s e t h e y d o n o t c o m p l e m e n t d~105 c t s 2 3 ( T a b l e 1). H o w e v e r , t h e y a r e still s e n s i t i v e t o t h e r e p r e s s o r as t h e y d o n o t g r o w i n 168 (qbl05) b u t a d s o r b t o it. T h e m u t a n t s D I : l t a n d D I : 2 9 t l y s o g e n i z e 168 s t a b l y a n d i n 168 (qbl05 D I : l t ) t h e p r o p h a g e w a s s h o w n b y P B S 1 t r a n s d u c t i o n t o b e l i n k e d t o t h e pheA locus, indicating that the prophage integrates at the
J.-I. Flock: Deletion Mutants of Temperate Bacillus subtilis Bacteriophage 0105
243
Table2. Sizes and positions of deleted regions. The molecular weight of the ¢105 genome is 26.3 x 1 0 6 Daltons (Chow et al., 1972) 10
247 (1977) (c~ by Springer-Verlag 1977
Deletion Mutants of Temperate Bacillus subtilis Bacteriophage ~105 Jan-Ingmar Flock Department of Bacteriology, Karolinska Institutet, S-104 01 Stockholm 60, Sweden
Summary. Six deletion mutants of temperate Bacillus subtilis phage d~105 have been isolated on the basis of their increased resistance to chelating agents. The size and position of the deletions was determined by electronmicroscopy of DNA heteroduplexes. All deletions are located in a region about 55-70% from one end of the DNA molecule, in the right half of the known genetic map of the phage. The segment 55-65% does not contain any genes essential for lytic growth or lysogenization. A gene(s) for immunity is located in a segment 65 70% from the left end. By electronmicroscopy of partially denatured d?105 DNA two A-T rich regions have been localized in the right half of the molecule. One of these regions falls within the non-essential 55-65% DNA segment.
Introduction
DNA deletion mutants of several bacteriophages show increased resistance to chelating agents and to heat (Rubenstein, 1968; Parkinson and Huskey, 1971), a fact which makes their isolation quite easy. Part of the genome of temperate phage is not required for lytic growth. In coliphage lambda about 25% and in coliphage P2 about 18% of wild type DNA can be deleted without severely impairing lytic growth of the phage. To identify and orient DNA by electron microscopy heteroduplexes between wild type and deletion mutant and/or partial denaturation maps can be used (Inman, 1967; Davis and Davidson, 1968). Phage qb105 is a temperate phage active on Bacillus subtilis ( Bridsell et al., 1969). The phage attachment site maps outside all known genetic markers (Armentrout and Rutberg, 1970), and might be located at the very end(s) of the mature phage DNA (Armentrout and Rutberg, 1970 ; Chow and Davidson, 1973). Induction of ~105 lysogenic bacteria is not followed
by rapid excision of the prophage as shown for several other phages (Armentrout and Rutberg, 1971). After induction the prophage DNA initially replicates covalently bound to bacterial DNA adjacent to the phage attachment site (Rutberg, 1973). In this property (~105 resembles coliphage Mu-1 (Abelson etal., 1974; Schroeder et al., 1974). However, prophage 00105 integrates at a specific locus (Rutberg, 1969), whereas Mu-1 can integrate anywhere on the bacterial chromosome (Bukhari and Zipser, 1972). We describe here some properties of six deletion mutants of d~105. Heteroduplexes between wild type DNA and DNA from these deletion mutants have been studied in the electron microscope, and the location of the 4)105 immunity gene on the (~105 DNA molecule has been determined. The heteroduplexes seen in the electron microscope have been oriented in relation to the known genetic map of d?105. Finally, we have constructed a partial denaturation map of ~105 mature DNA and oriented this in respect to the position of the deletions.
Materials and Methods Bacteria and Phage. The following strains of Bacillus subtilis were used: W168 (obtained from J. Spizizen), SR135 (su+3, trp-7, spoA9 obtained from J.A. Hoch), BR95 (~plO5tsA15) (phe-A1, ilvC1, trpC2 obtained from J. Spizizen and lysogenized with ~plO5tsA15 in our laboratory), and 3G18 (trp, met, ade) obtained from G. Venema. Wild type ~105 and its mutants, and the media and methods used to grow phage have been described (Armentrout and Rutberg, 1970). ~4C-iabeled T7 D N A was prepared as previously described (Armentrout et al., 1971). Competent cells were prepared from 3G18 as described by Arwert and Venema (1973) and stored in 10% glycerol at 70 ° C. Immediately before use the cells were .% thawed at 37 ° C. PBS1 transduction was performed as prevloulsy described (Rutberg, 1969).
Treatment of ~105 with Chelating Agent. Wild type ~105 was diluted 100-fold into 10 m M Naz-pyrophosphate at 32 ° C. The phage titer dropped about 104-fold in a few minutes. Plate stocks were
J.-I. Flock: Deletion Mutants of Temperate Bacillus subtilis Bacteriophage d~105
242
o= 100~,,~
"O
A
'°/ u 0,1
0.01o
\ 8
2'o
Time(minutes)
JB
J2
Fig. 1. Inactivation of wild type ~b105 with Naz-pyrophosphate. Phage was diluted 100-fold into 10 mM Na2-pyrophosphate at 32° C, and plaque forming particles were assayed at intervals. Stocks of surviving phage were made directly from the plates, and the surviving phage was exposed to a second and then a third cycle of treatment with Na2-pyrophosphate. Symbols : • - - • first cycle, A - - A second cycle, o - - e and third cycle of treatment respectively
made from plates with surviving phage and the phage exposed to a second and third cycle of Na2-pyrophosphate treatment. With successive cycles of treatment phage with increased resistance to Naz-pyrophosphate was selected (Fig. 1). Similar results were obtained using SSC (0.15 NaC1-0.015M Na citrate). Phage from CsCI gradients (see below) was tested for sensitivity to SSC by the "running" temperature method as described by Bertani (1975).
CsCI Density Gradients. Phage stocks were prepared from single plaques and the phage dialyzed against 1/10 nutrient broth (Difco) with 5 x 10-3M MgCI2. The phage stock was then diluted with saturated CsC1 (E. Merck, Darmstadt) to give a density of about 1.48. The phage was centrifuged at 120,000 x g in 6 x 5 ml MSE swing out rotor for 48 h at about 12° C. Two drop fractions were collected from the bottom of the tube, the density of some fractions was determined using an Abbe refractometer. A small sample fi'om each fraction was spotted on W168 indicator. The fractions containing phage were then titrated for plaque-formers and samples from these fractions were tested for sensitivity to SSC as described above. Marker Rescue. Competent 3G18 cells were mixed with phage DNA at 37 ° C. After 10 rain the sample was dduted 10-fold into tubes containing genetically marked phage particles to give a multiplicity of infection of about 10_ After an additional 10min for adsorption of phage the tubes were assayed. The superinfecting phage used was qb105 susC19 and (b105 susJll, respectively, and marker rescue was assayed by plating for wild type recombinants on he non-permissive host W168. Controls for reversions were included in each experiment,
Preparation of Phage DNA .for Electron Microscopy. Phage stocks were prepared and purified by CsC1 density gradient centrifugation as previously described (Rutberg, 1969, Rutberg et al., 1972). DNA was extracted with water saturated phenol.
Preparation of Heteroduplex DNA for Electron Microscopy. Phage DNA at a concentration of about 5 p,g per ml was mixed with 5 gl 0.2 M EDTA, 5 gl 1 M NaOH, to give a final volume of 50 gl. The mixture was left at room temperature for 10 rain and then 5 ~tl 2M Tris buffer pH 7.5 and 50 gl formamide (Merck, recrystallized (Chow et al., 1975)) was added. After one hour at room temperature 10gl was mixed with 101al cytochrome C (Sigma Co) 1 mg per ml, 40 gl formamide, 10 gl 1M Tris buffer pH 8.5 with 0.1M EDTA and 30 gl redistilled water. Fifty p.1 of this mixture was then spread onto a hypophase with 10% formamide in 10 mM Tns buffer pH 8.5. In later experiments heterod uplex DNA was also prepared directly from intact phage particles as described by Davis (Davis and Parkinson, 1971). DNA was collected on 300 mesh parlodion coated copper grids. The preparations were stained for 30 sec in 5 x 10-5 M uranyl acetate, washed in 93% ethanol, rotary shadowed with platinum/ palladium (80/20) and visualized in a Philips EM 200 or EM 301. Length measurements were done in a Scriptographic digitizer connected to a Compucorp 445 Statistician calculator. Single-stranded ~X174 DNA was added to DNA samples for calibration. Grating replicas were also used. Partial Denaturation of 0105 DNA. Phage DNA was added to a spreading solution containing 0.1M Tris buffer pH 8.5, 10 mM EDTA and 0.1 mg cytochrome C per ml in 82% formamide. The DNA was spread on a hypophase containing 50% formamide in 10 mM Tris buffer pH 8.5. Length variation between different molecules was less than 10%. No difference between single and double stranded DNA can be seen with this method.
Experimental Results Isolation o f 9105 Deletion Mutants. Six C l o n e s o f qb105 w i t h i n c r e a s e d r e s i s t a n c e t o N a 2 - p y r o p h o s p h a t e were selected for further studies. All mutants were isolated in different experiments and are of independent origin. M u t a n t s D I : l t a n d D 1 : 2 9 t w e r e p i c k e d as t u r b i d p l a q u e s a n d m u t a n t s D I : l c , 2c a n d 4c w e r e p i c k e d as c l e a r p l a q u e s a f t e r o n e cycle o f t r e a t m e n t w i t h Na2-pyrophosphate. M u t a n t D I I : 6 c w a s p i c k e d as a clear plaque after two cycles of Naz-pyrophosphate treatment.
General Characterization o f the Deletion Mutants. I n CsC1 g r a d i e n t s t h e d e l e t i o n m u t a n t s b a n d a t a l o w e r density than wild phage indicating an increased p r o t e i n t o D N A r a t i o (Fig. 2). It was shown that the four clear plaque mutants are unable to make a functional immunity repressor b e c a u s e t h e y d o n o t c o m p l e m e n t d~105 c t s 2 3 ( T a b l e 1). H o w e v e r , t h e y a r e still s e n s i t i v e t o t h e r e p r e s s o r as t h e y d o n o t g r o w i n 168 (qbl05) b u t a d s o r b t o it. T h e m u t a n t s D I : l t a n d D I : 2 9 t l y s o g e n i z e 168 s t a b l y a n d i n 168 (qbl05 D I : l t ) t h e p r o p h a g e w a s s h o w n b y P B S 1 t r a n s d u c t i o n t o b e l i n k e d t o t h e pheA locus, indicating that the prophage integrates at the
J.-I. Flock: Deletion Mutants of Temperate Bacillus subtilis Bacteriophage 0105
243
Table2. Sizes and positions of deleted regions. The molecular weight of the ¢105 genome is 26.3 x 1 0 6 Daltons (Chow et al., 1972) 10
