JOURNAL OF VIROLOGY, Aug. 1976, p. 313-317 Copyright © 1976 American Society for Microbiology
Vol. 19, No. 2 Printed in U.S.A.
Genetic Characterization of a 080 Transducing Bacteriophage Carrying the Histidine Operon of Salmonella typhimurium LESLIE S. ISAKI
AND
MARY J. VOLL*
Department of Microbiology, University of Maryland, College Park, Maryland 20742
Received for publication 19 February 1976
A 480 transducing phage, 480immXdhis, carrying the Salmonella his-gnd region, was characterized by immunity studies, tonB deletion analysis, and marker rescue analysis. 480immXdhis retains the phage immunity region ofthe +80-X hybrid phage from which it was derived. Bacterial genes replace most late phage genes. Deletion analysis shows the prophage gene order to be immX-hisgnd and indicates the orientation of the his operon to be hisOGDCBHAFIEgnd. The structure of 480immXdhis is remarkably similar to two independently isolated 0480 phages that carry the his-gnd region of Escherichia coli and that, like q580immXdhis, were derived by directed gene transposition to the tonB locus. A derivative of 480immXdhis that is 480 immune is also reported. A 480 phage, carrying the his operon and gnd gene of Salmonella typhimurium, was isolated previously (22) by the method of directed gene transposition (11). In this communication the phage will be referred to as 4)80immXdhis. Smith and Tong (18) showed that the his operon carried by the phage is under normal regulatory control by histidine. These authors introduced onto the phage a hisO constitutive mutation (his01242), and, to facilitate recovery of large phage yields, the XSam7 mutation. 480immXdhis and derivatives of it are being employed as sources of DNA for in vitro studies of his operon regulation (2, 13, 19). This paper describes the genetic structure of 80immXdhis as determined by deletion and marker rescue analysis. Its genetic structure is found to be remarkably similar to that reported for two 480 phages (3, 4, 23) that were also isolated by the directed gene transposition technique and that carry the his-gnd region of Escherichia coli. 480immXdhis retains the X immunity region of the +80-X hybrid phage from which it was derived. Isolation and characterization of a 480immune derivative of the phage are also described. MATERIALS AND METHODS Media. Minimal medium and growth supplements are as described previously (22). Gluconate minimal medium is E mineral salts medium (21) supplemented with 0.5% glucono-lactone (Fisher Scientific Co.). Rich medium is antibiotic medium 3 (AM3 broth) (Difco) and antibiotic medium 2 (AM2 agar) (Difco). Soft AM2 agar is one-half concentrated AM2 agar. Bacterial strains, bacteriophage, and colicins. E. coli strains are listed in Table 1. Strain SB2339 was
obtained by transduction of SB2334 to His+ with a
,080immXdhis, XcI857h80h lysate. Coliphages are
listed in Table 2. UV-induced lysates of 480 amber mutants were prepared from 480 amber E. coli lysogens obtained from L. Soll. Colicin V and B lysates were prepared as described by Gottesman and Beckwith (11). Bacteria were grown at 350C except for
AcI857h80h lysogens, which were grown at 32°C. 480 lysogens were induced by brief exposure of AM3 broth cultures to UV as described previously (22). XcI857h80h lysogens were induced by heating AM3 broth cultures at 42°C for 15 to 20 min. Good efficiency of plating of 480 phages was obtained with fresh host cultures prepared by inoculating a loopful of broth culture into 3.0 ml of AM3 broth and incubating overnight without aeration. Specialized transduction. A sample (0.1 ml) of an overnight AM3 broth culture and 0.1 ml of an appropriately diluted phage lysate were mixed directly on the surface of selective agar plates. Plates were scored for transductants after 4 to 5 days of incubation. When isolating single lysogens by transduction, cells and phage were added to the selection plate in a soft-agar overlay (E medium containing 0.7% agar). Immunity determinations. Samples (ca. 0.01 ml) of diluted phage suspensions were spotted on AM2 soft-agar lawns seeded with 0.1 ml of host culture, and the plates were incubated overnight. XcI857h80h was used to test for X immunity, with 480h phage as a control for phage adsorption. q580 was used to test for 480 immunity, with colicin V and B lysate serving as the phage adsorption controls. tonB deletion analysis. Single, defective lysogens were grown in minimal medium lacking histidine, subcultured in nutrient broth (Difco), and treated with Ti phage. Survivors were tested for histidine requirement (His-), for tryptophan requirement (Trp-), and for inability to use gluconate as the sole carbon source (Gnd-) by replica plating to appropri313
314
ISAKI AND VOLL
J. VIROL.
TABLE 1. Escherichia coli strains Strains
Genotype and comments Source or reference
SB2201 his3153, (his-gnd) dele22 tion; thi-, (lacpro)x,.. deletion; contains a UAG suppressor SB2331 SB2201 (080immXdhis, 22 XcI857h8Oh) SB2334 SB2201 (480) 22 SB2335 SB2201 (XcI857h8Oh) 22 SB2339 SB2201 (k80immXdhis, This paper XX30 XX33 XX24 RW84
AcI857h80h, 080)
SB2201 This paper (480immSOdhis, ¢80) SB2201 (k80immXdhis) This paper SB2201 This paper
(080immSOdhis)
eda- edd- strr (hisgnd) deletion XX47 RW84 (480immXdhis) XX27 RW84 (q80imm&kdhis) W3110 Wild type hisC463 hisC463 X178 F'trp+ ColV, Bllac-1 74 tonB trp his strr (080)
23
This paper This paper R. L. Sinsheimer 10 W. S. Reznikoff
TABLE 2. Bacteriophages Name
480
Ti k80h
Comments and source
Single plaque isolate from E. coli X178
P. E. Hartman Host range mutant of k80 able to lyse tonB mutants; H. Berger XcI857h80h 480-X hybrid having A immunity region, 080 host range mutation, and cI857 temperaturesensitive mutation; W. S. Reznikoff '80immXdhis" A-immune defective transducing phage carrying his-gnd region of S. typhimurium; derived from AcI857h80h (22) 480-immune derivative of 080immS0dhis -080immAdhis; this paper a Called 480h dhis+ immA cI857 by Smith and Tong (18) and 080 ximm dhis+ by Kasai (13).
ately supplemented minimal plates. Minimal plates lacking histidine but containing histidinol were used to test for the presence of a functional hisD gene (Hol+) (1). Marker rescue. AM3 broth cultures of single, defective lysogens were induced, and a sample (0.05 ml) of the induced culture and 0.1 ml of an AM3 broth culture of a nonpermissive strain (hisC463 or W3110) were plated together in a soft AM2 agar lawn (mixed lawn). As controls, the nonpermissive strains were plated alone. Samples (ca. 0.01 ml) of graded dilutions of 080 amber mutant lysates were
spotted on the mixed and control lawns. Marker rescue was scored as positive if the phage titer on the mixed lawns was 100-fold or greater than the titer on the control lawns. RESULTS
Isolation of (80immXdhis single lysogens. To characterize 080immXdhis, it was necessary to obtain strains singly lysogenic for the transducing phage. Attempts to do so by transducing a nonlysogenic host with induced lysates of the doubly lysogenic strain SB2331 at low multiplicity of infection were unsuccessful. His+ single lysogens were obtained from a permanent stock culture of SB2331 maintained as a stab in a paraffin-sealed vial containing soft minimal agar. Fifty-seven colony isolates from the culture were tested for spontaneous release of phage by replication to lawns of sensitive host bacteria. Three of six isolates that failed to release phage were still His+, indicating that they contained the defective transducing phage but had lost the active phage. These three isolates also yielded no active phage in supernatants of heat-induced cultures. One of the isolates, XX33, was used as the source of prophage for a second His+ single lysogen, XX47. A heatinduced culture of XX33 was superinfected with 080, and the resulting lysate was used to transduce the his-gnd deletion strain RW84 to His+ at a low multiplicity of infection. XX47 is a transductant that does not release active phage. The presence of a chromosomal edd mutation in XX47 allows it to be assayed for phage-encoded gnd gene activity by nutritional assay (8). The buoyant density of the transducing phage of XX33 and of XX47 was measured to determine whether it conformed to that of the original his phage. Phage released from XX33 and XX47 by superinfection of heat-induced cultures with Ac1857h80h was centrifuged in cesium chloride gradients and found to have the same relative buoyant density as that reported for the his prophage of SB2331 (T. Kasai; C. B. Bruni and R. G. Martin, cited in reference 22). Transducing activity (assayed on strain SB2335) banded at a somewhat lighter position in the gradients than did AcI857h80h lytic activity. Presence of phage immunity region in 480immAdhis. XX33 and XX47 were assayed for A immunity. The strains were sensitive to infection by 480h but resistant to infection by Xc1857h80h (see Table 4), indicating that the A immunity region was retained on the phage. tonB deletion analysis. To determine the relative positions of the phage immunity region, the his operon and the gnd gene on
GENETIC STRUCTURE OF 480dhis SALMONELLA
VOL. 19, 1,976
315
080immXdhis strains XX33 and XX47 were sis with a gene 4 amber mutant phage was subjected to tonB deletion analysis (9), the rele- performed, but we were unable to obtain disvant gene order in the tonB region being tinct results with this mutant and our strains. Figure 1 shows the prophage structure of att480-tonB-trp. XX33 and XX47 cultures were infected with Ti, and surviving clones were 080immXdhis as determined in these studies. assayed for the presence of the entire his operon TABLE 3. Phenotypes of Tl-resistant isolates of (His+), the hisD gene (Hol+), the trp operon XX33 and XX47 (Trp+), and the gnd gene (Gnd+; XX47 only). The phenotypes of the Ti-resistant isolates that XX33 isolates XX47 isolates were missing one or more of the gene functions Phenotype No. Phenotypea No. assayed, and hence could be assumed to have 7 His+ Gnd+ Trp2 extended tonB deletions, are listed in Table 3. His+ TIp1 The isolation of His- Trp- classes indicates that Hol+ His- Trp- 1 His+ Gnd- Trp7 HoI+ His- Gnd- Trp5 the prophage is located at the att480 site. The His- Trp8 His- Gnd-Trp50 isolation from XX47 of His+ Gnd- and of Hol+ His- Trp+ His- Gnd- Trp+ 9 His- Gnd- classes indicates that the order of His+ GndTrp+ 1 the his operon and gnd gene relative to trp is a The Gnd phenotype was confirmed by enzymatic his-gnd-trp. The isolation from both strains of a Hol+ His- class indicates that the orientation of assay (15) for one isolate each of the first five classes the his operon with respect to gnd is his- listed. OGDCBHAFIE-gnd. All other phenotypes are TABLE 4. Assay for prophage immunity region in consistent with the above-mentioned gene orXX33 and XX47 Ti-resistant isolates der. No. of Lysisa by: To determine the position of the prophage immunity region, a number of isolates of the lates Strain or phenotype AcI857 His- classes of each strain were tested for the tested 48 h80h presence of X immunity (Table 4). All strains + + RW84 SB2201, tested were immune to infection by XcI857h80h. SB2334 + The presence of the phage immunity region in SB2335, XX33, XX47 + these strains indicates the order immX-his- XX33 isolates: gnd-trp. + 2 His- TrpMarker rescue analysis. XX33 and XX47 + 2 His- Trp+ + 1 Hol+ His- Trpwere heat induced and superinfected with a series of 080 amber mutants, and wild-type XX47 isolates: + 3 His- Gnd- TIpphage production was assayed on nonpermis+ His- Gnd- Trp+ 3 sive hosts. Rescue of a wild-type allele was + 2 Hol+ HisGndTrpin with mutant observed phage genes 1, 2, 3, 14, a 15, 17, 18, and 19 but not with phage mutant in Approximately 104 PFU were spotted on host genes 5, 6, 7, 8, 9, 10, 11, and 13. Rescue analy- cell lawns. +, Lysis observed; -, no lysis observed. a.
genes 080 genes X
F
int B cM N clcIl P 15
Function b. 080dhis
14
Q R
16 1718 19) (I 2 3 4 56 78910111213
Early Functions 8
ci
Vol. 19, No. 2 Printed in U.S.A.
Genetic Characterization of a 080 Transducing Bacteriophage Carrying the Histidine Operon of Salmonella typhimurium LESLIE S. ISAKI
AND
MARY J. VOLL*
Department of Microbiology, University of Maryland, College Park, Maryland 20742
Received for publication 19 February 1976
A 480 transducing phage, 480immXdhis, carrying the Salmonella his-gnd region, was characterized by immunity studies, tonB deletion analysis, and marker rescue analysis. 480immXdhis retains the phage immunity region ofthe +80-X hybrid phage from which it was derived. Bacterial genes replace most late phage genes. Deletion analysis shows the prophage gene order to be immX-hisgnd and indicates the orientation of the his operon to be hisOGDCBHAFIEgnd. The structure of 480immXdhis is remarkably similar to two independently isolated 0480 phages that carry the his-gnd region of Escherichia coli and that, like q580immXdhis, were derived by directed gene transposition to the tonB locus. A derivative of 480immXdhis that is 480 immune is also reported. A 480 phage, carrying the his operon and gnd gene of Salmonella typhimurium, was isolated previously (22) by the method of directed gene transposition (11). In this communication the phage will be referred to as 4)80immXdhis. Smith and Tong (18) showed that the his operon carried by the phage is under normal regulatory control by histidine. These authors introduced onto the phage a hisO constitutive mutation (his01242), and, to facilitate recovery of large phage yields, the XSam7 mutation. 480immXdhis and derivatives of it are being employed as sources of DNA for in vitro studies of his operon regulation (2, 13, 19). This paper describes the genetic structure of 80immXdhis as determined by deletion and marker rescue analysis. Its genetic structure is found to be remarkably similar to that reported for two 480 phages (3, 4, 23) that were also isolated by the directed gene transposition technique and that carry the his-gnd region of Escherichia coli. 480immXdhis retains the X immunity region of the +80-X hybrid phage from which it was derived. Isolation and characterization of a 480immune derivative of the phage are also described. MATERIALS AND METHODS Media. Minimal medium and growth supplements are as described previously (22). Gluconate minimal medium is E mineral salts medium (21) supplemented with 0.5% glucono-lactone (Fisher Scientific Co.). Rich medium is antibiotic medium 3 (AM3 broth) (Difco) and antibiotic medium 2 (AM2 agar) (Difco). Soft AM2 agar is one-half concentrated AM2 agar. Bacterial strains, bacteriophage, and colicins. E. coli strains are listed in Table 1. Strain SB2339 was
obtained by transduction of SB2334 to His+ with a
,080immXdhis, XcI857h80h lysate. Coliphages are
listed in Table 2. UV-induced lysates of 480 amber mutants were prepared from 480 amber E. coli lysogens obtained from L. Soll. Colicin V and B lysates were prepared as described by Gottesman and Beckwith (11). Bacteria were grown at 350C except for
AcI857h80h lysogens, which were grown at 32°C. 480 lysogens were induced by brief exposure of AM3 broth cultures to UV as described previously (22). XcI857h80h lysogens were induced by heating AM3 broth cultures at 42°C for 15 to 20 min. Good efficiency of plating of 480 phages was obtained with fresh host cultures prepared by inoculating a loopful of broth culture into 3.0 ml of AM3 broth and incubating overnight without aeration. Specialized transduction. A sample (0.1 ml) of an overnight AM3 broth culture and 0.1 ml of an appropriately diluted phage lysate were mixed directly on the surface of selective agar plates. Plates were scored for transductants after 4 to 5 days of incubation. When isolating single lysogens by transduction, cells and phage were added to the selection plate in a soft-agar overlay (E medium containing 0.7% agar). Immunity determinations. Samples (ca. 0.01 ml) of diluted phage suspensions were spotted on AM2 soft-agar lawns seeded with 0.1 ml of host culture, and the plates were incubated overnight. XcI857h80h was used to test for X immunity, with 480h phage as a control for phage adsorption. q580 was used to test for 480 immunity, with colicin V and B lysate serving as the phage adsorption controls. tonB deletion analysis. Single, defective lysogens were grown in minimal medium lacking histidine, subcultured in nutrient broth (Difco), and treated with Ti phage. Survivors were tested for histidine requirement (His-), for tryptophan requirement (Trp-), and for inability to use gluconate as the sole carbon source (Gnd-) by replica plating to appropri313
314
ISAKI AND VOLL
J. VIROL.
TABLE 1. Escherichia coli strains Strains
Genotype and comments Source or reference
SB2201 his3153, (his-gnd) dele22 tion; thi-, (lacpro)x,.. deletion; contains a UAG suppressor SB2331 SB2201 (080immXdhis, 22 XcI857h8Oh) SB2334 SB2201 (480) 22 SB2335 SB2201 (XcI857h8Oh) 22 SB2339 SB2201 (k80immXdhis, This paper XX30 XX33 XX24 RW84
AcI857h80h, 080)
SB2201 This paper (480immSOdhis, ¢80) SB2201 (k80immXdhis) This paper SB2201 This paper
(080immSOdhis)
eda- edd- strr (hisgnd) deletion XX47 RW84 (480immXdhis) XX27 RW84 (q80imm&kdhis) W3110 Wild type hisC463 hisC463 X178 F'trp+ ColV, Bllac-1 74 tonB trp his strr (080)
23
This paper This paper R. L. Sinsheimer 10 W. S. Reznikoff
TABLE 2. Bacteriophages Name
480
Ti k80h
Comments and source
Single plaque isolate from E. coli X178
P. E. Hartman Host range mutant of k80 able to lyse tonB mutants; H. Berger XcI857h80h 480-X hybrid having A immunity region, 080 host range mutation, and cI857 temperaturesensitive mutation; W. S. Reznikoff '80immXdhis" A-immune defective transducing phage carrying his-gnd region of S. typhimurium; derived from AcI857h80h (22) 480-immune derivative of 080immS0dhis -080immAdhis; this paper a Called 480h dhis+ immA cI857 by Smith and Tong (18) and 080 ximm dhis+ by Kasai (13).
ately supplemented minimal plates. Minimal plates lacking histidine but containing histidinol were used to test for the presence of a functional hisD gene (Hol+) (1). Marker rescue. AM3 broth cultures of single, defective lysogens were induced, and a sample (0.05 ml) of the induced culture and 0.1 ml of an AM3 broth culture of a nonpermissive strain (hisC463 or W3110) were plated together in a soft AM2 agar lawn (mixed lawn). As controls, the nonpermissive strains were plated alone. Samples (ca. 0.01 ml) of graded dilutions of 080 amber mutant lysates were
spotted on the mixed and control lawns. Marker rescue was scored as positive if the phage titer on the mixed lawns was 100-fold or greater than the titer on the control lawns. RESULTS
Isolation of (80immXdhis single lysogens. To characterize 080immXdhis, it was necessary to obtain strains singly lysogenic for the transducing phage. Attempts to do so by transducing a nonlysogenic host with induced lysates of the doubly lysogenic strain SB2331 at low multiplicity of infection were unsuccessful. His+ single lysogens were obtained from a permanent stock culture of SB2331 maintained as a stab in a paraffin-sealed vial containing soft minimal agar. Fifty-seven colony isolates from the culture were tested for spontaneous release of phage by replication to lawns of sensitive host bacteria. Three of six isolates that failed to release phage were still His+, indicating that they contained the defective transducing phage but had lost the active phage. These three isolates also yielded no active phage in supernatants of heat-induced cultures. One of the isolates, XX33, was used as the source of prophage for a second His+ single lysogen, XX47. A heatinduced culture of XX33 was superinfected with 080, and the resulting lysate was used to transduce the his-gnd deletion strain RW84 to His+ at a low multiplicity of infection. XX47 is a transductant that does not release active phage. The presence of a chromosomal edd mutation in XX47 allows it to be assayed for phage-encoded gnd gene activity by nutritional assay (8). The buoyant density of the transducing phage of XX33 and of XX47 was measured to determine whether it conformed to that of the original his phage. Phage released from XX33 and XX47 by superinfection of heat-induced cultures with Ac1857h80h was centrifuged in cesium chloride gradients and found to have the same relative buoyant density as that reported for the his prophage of SB2331 (T. Kasai; C. B. Bruni and R. G. Martin, cited in reference 22). Transducing activity (assayed on strain SB2335) banded at a somewhat lighter position in the gradients than did AcI857h80h lytic activity. Presence of phage immunity region in 480immAdhis. XX33 and XX47 were assayed for A immunity. The strains were sensitive to infection by 480h but resistant to infection by Xc1857h80h (see Table 4), indicating that the A immunity region was retained on the phage. tonB deletion analysis. To determine the relative positions of the phage immunity region, the his operon and the gnd gene on
GENETIC STRUCTURE OF 480dhis SALMONELLA
VOL. 19, 1,976
315
080immXdhis strains XX33 and XX47 were sis with a gene 4 amber mutant phage was subjected to tonB deletion analysis (9), the rele- performed, but we were unable to obtain disvant gene order in the tonB region being tinct results with this mutant and our strains. Figure 1 shows the prophage structure of att480-tonB-trp. XX33 and XX47 cultures were infected with Ti, and surviving clones were 080immXdhis as determined in these studies. assayed for the presence of the entire his operon TABLE 3. Phenotypes of Tl-resistant isolates of (His+), the hisD gene (Hol+), the trp operon XX33 and XX47 (Trp+), and the gnd gene (Gnd+; XX47 only). The phenotypes of the Ti-resistant isolates that XX33 isolates XX47 isolates were missing one or more of the gene functions Phenotype No. Phenotypea No. assayed, and hence could be assumed to have 7 His+ Gnd+ Trp2 extended tonB deletions, are listed in Table 3. His+ TIp1 The isolation of His- Trp- classes indicates that Hol+ His- Trp- 1 His+ Gnd- Trp7 HoI+ His- Gnd- Trp5 the prophage is located at the att480 site. The His- Trp8 His- Gnd-Trp50 isolation from XX47 of His+ Gnd- and of Hol+ His- Trp+ His- Gnd- Trp+ 9 His- Gnd- classes indicates that the order of His+ GndTrp+ 1 the his operon and gnd gene relative to trp is a The Gnd phenotype was confirmed by enzymatic his-gnd-trp. The isolation from both strains of a Hol+ His- class indicates that the orientation of assay (15) for one isolate each of the first five classes the his operon with respect to gnd is his- listed. OGDCBHAFIE-gnd. All other phenotypes are TABLE 4. Assay for prophage immunity region in consistent with the above-mentioned gene orXX33 and XX47 Ti-resistant isolates der. No. of Lysisa by: To determine the position of the prophage immunity region, a number of isolates of the lates Strain or phenotype AcI857 His- classes of each strain were tested for the tested 48 h80h presence of X immunity (Table 4). All strains + + RW84 SB2201, tested were immune to infection by XcI857h80h. SB2334 + The presence of the phage immunity region in SB2335, XX33, XX47 + these strains indicates the order immX-his- XX33 isolates: gnd-trp. + 2 His- TrpMarker rescue analysis. XX33 and XX47 + 2 His- Trp+ + 1 Hol+ His- Trpwere heat induced and superinfected with a series of 080 amber mutants, and wild-type XX47 isolates: + 3 His- Gnd- TIpphage production was assayed on nonpermis+ His- Gnd- Trp+ 3 sive hosts. Rescue of a wild-type allele was + 2 Hol+ HisGndTrpin with mutant observed phage genes 1, 2, 3, 14, a 15, 17, 18, and 19 but not with phage mutant in Approximately 104 PFU were spotted on host genes 5, 6, 7, 8, 9, 10, 11, and 13. Rescue analy- cell lawns. +, Lysis observed; -, no lysis observed. a.
genes 080 genes X
F
int B cM N clcIl P 15
Function b. 080dhis
14
Q R
16 1718 19) (I 2 3 4 56 78910111213
Early Functions 8
ci
