Phage P1 temperature-sensitive mutants with defects in the lytic pathway

VIROLOGY

105,

52-59

(1980)

Phage Pl Temperature-Sensitive JOHN B. RAZZA, Department

Mutants

CHARLES

of Microbiology,

Emory

with Defects in the Lytic Pathway

A. WATKINS, University

Accepted April

AND

JUNE

School of Medicine,

ROTHMAN

Atlanta,

SCOTT

Georgia $0%‘8

15, 1980

We have partially characterized the first 10 mutants of bacteriophage Pl that are temperature sensitive in functions required for lytic growth. The 10 mutants fall into six different complementation groups, one of which includes a previously described amber mutant. The approximate position of these five groups was established by deletion mapping. Based on temperature shift experiments with the ts mutants, each of the six affected genes is required for lytic growth only later than 30 mln after infection. Two of these genes, 11 and 12, probably code for structural proteins of the Pl virion since temperature-sensitive mutants in these genes show greater thermolability than wild type Pl phage. One of the ts mutations is suppressed by the presence of an amber suppressor in the host.

have been recently identified for most Pl amber mutants studied (Walker and Walker, unpublished). Recent correlation of the physical and genetic map of Pl (Mural et al., 1979; Sternberg, 1979) makes it clear that there remain regions of DNA in which mutants have not yet been isolated. Thus, isolation of new mutants in the lytic pathway is expected to lead to identification of new genes, possibly including essential replication functions. In many other phages, genes essential for growth have been identified and mapped by taking advantage of conditional lethal mutants, both nonsense and temperature sensitive. Many nonsense mutants of Pl have been isolated and mapped (Scott, 1968; Walker and Walker, 1975,1976), but no temperature-sensitive mutants in essential lytic growth functions have yet been described. In contrast to control of lysogeny in Pl (reviewed by Scott, 1980), very little is known about the lytic growth and morphogenesis of this phage. By hybridization of mRNA produced at different times after thermal induction of a Pl prophage with specific fragments of Pl DNA generated by digestion with restriction endonucleases, Mural (1978) found that the right 40% of the Pl genetic map is transcribed before the left. Beyond this, no resolution of PI genes into clusters expressed at different times in

INTRODUCTION

Phage Pl is a large complex temperate bacteriophage with an icosahedral head, an intricate tail composed of a tube and contractile sheath, and a base plate with tail fibers attached (Anderson and Walker, 1960). In other viral systems, temperaturesensitive (ts) mutants have made possible the identification of the protein product of a specific gene, since this product is often temperature sensitive in vitro. Furthermore, the presence in the virion of the product of a particular gene may sometimes be detected by increased thermal sensitivity of the mutant virus particles compared to the particles of the wild type. Therefore, characterization of temperature-sensitive mutants of Pl is expected to aid in our understanding of viral functions. Pl differs from other well-studied temperate phages because its prophage DNA replicates separately from the host chromosome, as a plasmid (Ikeda and Tomizawa, 1968). Thus, Pl must have two different systems for replication control: one to maintain a single prophage per host genome and one for vegetative replication during lytic growth. None of the Pl mutants so far described is defective in vegetative replication (Scott, unpublished; Walker and Walker, unpublished); in fact morphological defects 0042-6322/80/110052-03$02.00/O Copyright All rights

Q 1980 by Academic Press, Inc. of reproduction in any form reserved

52

TABLE

Pl TEMPERATURE-SENSITIVE

MUTANTS

1

MATERIALS

BACTERIAL STRAINS

53 AND METHODS

Media. Saline-calcium diluent, LB broth, LBC broth, LB agar, and top agar are as deReference Strain scribed by Scott (1974). Bacterial strains. Bacterial strains used Shigella dysenteriae in this work are listed in Table 1. The extent Luriaet al. (1960) Sh-16 of the Pl genome present in defective prophages and cloned fragments as determined Escherichia coli Su-1 K175 Garen et al. (1965) by marker rescue analysis is presented in SW1 (see Fig. 1) K175(Plcry) Scott (1970, 1973) Fig. 1. The plasmids pBR322 and ColElAp’ Su” K140 Horiuehi and were employed as vectors in the construcZinder (1967) tion of pRM206 and pRM116, respectively SW1 K141 Horiuchi and Zinder (1967) (Mural et al., 1979). K142 Horiuchi and SU-2 Phage strains. Pl+ and Plvir” are deZinder (1967) scribed by Scott (1968). The temperaturesu-3 Horiuchi and K143 sensitive Pl mutants were isolated following Zinder (1967) mutagenesis with hydroxylamine (Scott, SlY Shimada et al. 594 (1972) 1968). N99 SU” Gottesman and Complementation tests. Cross-streak Yarmolinsky complementation tests were performed as w=-9 described by Scott (1968). Stocks of temN99(pRM206) Su” (Pl&mHI-5) Muralet al. (1979) (see Fig. 1) perature-sensitive mutants at 10s/ml were K204 SUO Scott (1970) employed. The mutants tsl, ts4, ts5, ts7, K204(pIH1972) Sue (Pldl) Shafferman et al. tslCh@, tsllvir”, tsl2vir”, ts21, ts22, and (see Fig. 1) (1978) ts27 were cross-streaked with each other K336 sue Scott (1974) K336(pRM116) SuO(PLEcoRI-10) Mural et al. (1979) and with 24 Plvir”am mutants (vir”aml.4, (see Fig. 1) vir”am62, vir”am23, vir”am73, vir”am20, RHO(P7dZ) Su+ (see Fig. 1) Chesney and virSam2.31, vir”am33, vir”am72, vir”am66, Scott (1978) am74, wir”am3.21, vir”am75, wir”aml80, vir”RHO(P7d4) Su+ (see Fig. 1) Chesney and am115, vir”am5.19, virsum61, vir”um’7.14, Scott (1978) vir”am30, viVam29, virSam139, virSam8. 13, vir%mlO. 11, viFam9.16, vir”um43). the lytic cycle has been accomplished, and Deletion mapping. Deletion mapping was little is known about when or how Pl genes accomplished as described by Scott (1975) are turned on and off. The timing of essential with the following modification: after infunctions in viral growth can often be eluci- fection and removal of unadsorbed phage, dated from studies of viral mutants with the infected culture was resuspended in 3 ml altered temperature sensitivity because of 0.01 M Tris buffer, pH 7.4, and UV irramutant gene products can frequently be diated at 68 cm from a GE germicidal bulb rapidly inactivated or activated by temper- for 40 set in a glass petri plate. Cells were ature shifts during the lytic growth cycle. then diluted lOOO-fold into prewarmed 34 For these reasons, we have characterized LB broth containing 5 x lop2 M CaCl, and some mutants of phage Pl that are temper- aliquots removed and assayed for viable ature sensitive for functions required in the cells (post UV) and extracellular phage. The lytic cycle. Temperature shift experiments culture was then aerated for 90 min at 34”. indicate that the mutations described here Following sterilization with chloroform, the are in functions required late during phage phage were assayed on Sh-16 at 34 and 42”. growth. Some of these mutations produce The burst size was calculated as plaquedefects in structural virion proteins, as in- forming units released after chloroform dicated by the decreased thermal stability treatment, per viable cell (assayed just prior of the particles. to infection). Relevant genotype

54

RAZZA, WATKINS, AND SCOTT

VtRIl

C8134

FIG. 1. Genetic map of phage Pl. Numbers above the top line refer to amber mutants and the location of tsll. Below the line are vir for virulent mutant and e for clear plaque former% Dotted vertical lines indicate deletion endpoints. Shaded bars are the regions deleted in the defective prophages and absent in pRM116 and pRMZO6. This figure is based on data from this paper and from Scott and Kropf (1977), Scott (1968, 19’70,1972), Chesney and Scott (1975), Baumstark and Scott (unpublished), Mural et al. (1979), and Walker and Walker (1976). This map is not drawn to scale.

Heat inactivation. A suspension of lo5 phage per ml of LBC was heated to 60” for 60 min then chilled quickly in an ice bath. The titer on Sh-16 at 34” was compared before and after heating. In each sample, Pl+ (turbid plaque) or Plvir” (clear plaque) was included as an internal control. Temperature-shift experiments. E. coli strain 594/h was grown to 1 X lo8 cells/ml in LB broth at 37”, concentrated threefold by centrifugation, and infected in Tris buffer (pH 7.4; 0.01 M) at a m.o.i. of 5 with the appropriate ts mutant. Adsorption was allowed to proceed at 0” for 15 min and unadsorbed phage were removed by centrifugation at 4”. Infected cells were diluted to 103/ml and divided into two flasks of LBC, one prewarmed to 42” (negative control) and one at 34” (positive control) and incubated with aeration. At 30 min postinfection, a sample from the 34” flask was diluted lofold to LBC at 42” to accomplish the temperature shift-up and incubation was continued. In temperatures shift-down experiments, samples were removed from the 42” flask at 30 min after infection, diluted lo-fold to LBC at 34”, and incubation was continued. For phage assays, 1 ml samples were removed, treated with chloroform, and plated on Sh-16 at 34”.

RESULTS

Complementation Analysis Cross-streak complementation tests indicate that all the Pl temperature-sensitive mutants tested complement each other with the following exceptions: ts 1 fails to complement ts12; and ts5, ts7 and ts27 do not complement each other; nor do ts21 and ts22. Numbers 11 through 16 were assigned to the complementation groups thus defined (Table 2). In cross-streak tests, 24 amber mutants (see Materials and Methods for list of the mutants used) complement all of the ts mutants except for tell, which does not complement am’75 and may therefore be in the same operon as this amber mutant. No further mapping studies were performed to determine the map location of tsll. One of the ts mutants, ts15.10, plates efficiently at both 34 and 42” on any Su+ host used (see Table l), but on an Suehost plates with an efficiency of only 2 x 10e5 at 42 (data not shown). It is possible, therefore, that ts15.10 is an amber mutant which, when unsuppressed, produces a protein fragment that is functional at 34“ but not at 42”. Alternatively, in an Su+ host, another protein (possibly host-encoded) may make the Pl gene 15 protein nonessential.

Pl TEMPERATURE-SENSITIVE

mutants used, however, cross streaks between am33 and either ts12, ts5, or ts7 produce single isolated plaques, as do cross streaks between ts4 and am62 and between tsl0 and am180. This leads to the tentative conclusion that these ts mutants are located nearam23, am62, andam180, respectively. On the basis of this tentative map assignment, prophages with appropriate deletions were selected for mapping studies.

TABLE 2 COMPLEMENTATION

Complementation group

Mutant ts1, ts12

11 12 13 14 15 16

55

MUTANTS

ts5, ts7, ts27 ts21, ts22 ts4

ts10 tsll, am75

In addition to complementation information, the cross streak test provides some insight into the genetic distance between mutants. Plaques growing where the indicator bacteria are doubly infected (where the two streaks cross) result both from complementation of the mutants and from the production of wild type recombinants. If the two mutants are closely linked genetically, the recombination frequency will be low and single isolated plaques will be visible at the intersection of the streaks. In most cases, the ts mutants give completely confluent phage growth at the cross point with the am

Deletion Mapping The approximate location of eight ts mutants was determined by deletion mapping (see Fig. 1 for extent of deletions). Each defective lysogen was infected with a ts mutant, irradiated with UV light, diluted, and grown for 90 min at 34”. The phage released were assayed under permissive and nonpermissive conditions. The frequency of ts+ phage rescued varies between 0.5 and 5% of the total phage yield (Table 3). tsl and ts12 (complementation group 11) and ts5, ts7, and ts27 (complementation group 12) were all rescued by the BamHI-5 fragment present in pRM.206. ts22 (group 13) and ts4 (group 14) are rescued by the EcoRI-10 fragment of pRM116, and tsl0 (group 15) is

TABLE 3 DELETION MAPPING OF ts MUTANTS”

Complementation group 11

Infecting @we

Lysogens being infected

Burst size

Recombination frequency (wild type/total progeny)

t.91

N99(pRM206) K175(Plcry) N99(pRM206)

5.9

5.4 x 10-Z <10-s 4.6 x 1O-2

2.6 0.4 7.9

ts1 ts 12vir” 12

9.1

ts27

N99(pRM206) N99(pRM206) N99(pRM206) K175(Plcry)

13

ts22

K336(pRM116)

4.3

9.0 x 10-S

14

ts4

K336(pRM116)

6.1

2.3 x 1O-2

tsl@vir”

RHO(P7d2) RHO(P7d4) K204(pIH1972)

3.4 3.7 4.1

4.5 x 10-a 4.8 x 1O-3 2.8 x 1O-2

ts5

ts7 ts27

15

ts ltiir” ts lOuiF

3.4 x 10-Z 5.5 x 10-Z 4.8 x 10-Z 110-s

a The infected cells are UV irradiated and grown 90 min at 34”. Burst size is plaque-forming units released after chloroform treatment, per viable cell (assayed just prior to infection). Wild type phage grow on Sh-16 at 42”; total progeny are assayed on Sh-16 at 34”. Revertants to temperature resistance are less than 10mJfor all phage stocks.

56

RAZZA, WATKINS, AND SCOTT

rescued by the defective prophages pIH1972, P7d2 and P7d4. The complementation group designations of the ts mutants have been placed on the map in Fig. 1 at the locations indicated by these marker rescue results. After the map locations of the ts mutants had been determined, additional cross streak tests were performed with the nearest available amber mutant (Walker and Walker, 1976; Scott and Kropf, unpublished) on both sides of each ts mutant and complementation was observed. (The one exception to this is that we are unable for technical reasons to work with am4.7 which is just left of am62, so group 13 or 14 may include this amber mutant.) Thus, within the reliability of cross streak complementation tests (see Discussion), groups 11-15 define new genes. Heat Sensitivity of the Phage If the product of a ts mutant is a structural protein of the virion, the mutant phage particles may show greater thermal sensitivity than wild type. To test this, a mixture of wild type and mutant phage was heated at 60“ for 1 hr and the relative inactivation (mutant/wild type) was determined (Table 4). The members of complementation group 11 (ts12 and tsl) show thermal sensitivity in this test, although the effect on ts12 is much more pronounced than that on tsl. This implies that gene 11 codes for a protein present in the phage particle and that tsl is a different allele from ts 12. One of the members of cistron 12 also exhibits thermal sensitivity. ts27 shows pronounced thermal inactivation, while ts5 and ts7 may be slightly more thermosensitive than the wild type. This presumably means that gene 12 also codes for a product present in the phage particle (see Discussion). Virions defective in gene 13 (ts22), 14 (ts4), 15 (tslO), and 16 (tsll) do not appear to be significantly more heat labile than wild type. Shi&Up Experiments To determine whether the product of the mutated gene is required either early (before 30 min postinfection), late (after 30 min postinfection), or continuously in the phage

TABLE 4 HEAT INACTIVATIONS

Complementation group

Relative survivaP

Phage

Percentage survivors

Pl+ control

7.9

11 11

ts1 ts12viF

3.9 0.04

.72 .004

12 12 12

ts5 ts27

7.4 6.0 1.1

.67 .72 .20

13

ts22

a.7

.82

14

ts4

6.2

.86

15

ts louir”

12.4

.85

16

ts11

11.6

.88

ts7

a Phage were heated to 60” for 60 min in LBC (see Materials and Methods). To provide an internal control, Pl+ or Plvir” was mixed with each mutant tested and differentiated by plaque morphology. The data represent the average of three separate heat inactivation tests. b Relative survival is the ratio of mutant to wild type at 60 min divided by the same ratio at 0 min.

lytic cycle, one step growth experiments including temperature shifts were performed. For a shift-up experiment, an aliquot of the ts mutant-infected culture was transferred from 34 to 42” at 30 min after infection, and the extracellular phage were assayed at 30-min intervals after the shift (see Methods). If the burst size in the upshifted culture is similar to that obtained for the 34” control, it can be concluded that the altered gene product is required in the lytic cycle before 30 min. Conversely, if the burst is greatly reduced following the shiftup in temperature, the mutant affects a product required either late (after 30 min) or continuously for lytic growth. For the one mutant tested in each complementation group (tsll.1, 12.27, 13.22, 14.4, 15.10, and 16.ll), the burst size at 90 min after shifting the temperature up to 42” was the same as the burst size of the 42” negative control culture (data not shown). At 34”, the lowest burst size for any mutant was 4.5. Therefore, it appears that all of the mutants tested are

Pl TEMPERATURE-SENSITIVE B. IS II.12

A. IS I I. I

i IO

5 .

MUTANTS

4

f ;:I c.lyArTR

0

30

60

90

120

150

180

30

60

90

120

150

180

210

TIME (minutes)

I

E. Is 14.4

24

E ts 15.10

20 IO E 0

30

60

90

I20

150

TIME

I60

(minutes)

FIG. 2. Temperature shift-down. E. coli 594/h was infected with each ts mutant at an m.o.i. of 5. Control cultures were incubated at both 34 and 42”. At 30 min after infection, l-ml aliquots were transferred from 42 to 34” and incubated with aeration. Phage were assayed at 30-min intervals thereafter on Sh-16 at 34”. 0 0, 34” control; A A, 42” control; 0 0, shifted from 42 to 34” at 30 min after infection.

defective in functions required late or continuously for lytic growth. Shifl-Down

Experiments

To distinguish late from continuous functions, shift-down experiments were performed. If the function is required throughout the lytic cycle, then no growth will proceed at the nonpermissive high temperature and when the culture is shifted-down the latent period may be extended by the time spent under nonpermissive conditions. Alternatively, if the thermal inactivation of the protein is irreversible and the protein is

required continuously, presence of the inactive protein or absence of the active one may lead to formation of a complex that kills the infected cells or binds other essential phage proteins. In this case, it is possible that no phage will be produced even after prolonged incubation at the permissive temperature. On the other hand, if the function is needed only late in the cycle and the inactivation is reversible, a shift-down before the time of action of the gene product will produce a normal burst after a normal latent period; if the inactivation is irreversible and the protein is required late following an extended latent period there may

58

RAZZA,

WATKINS,

be a reduced or normal burst produced (because the protein synthesized at low temperature is active). An aliquot of each ts mutant-infected culture was transferred from 42 to 34” at 30 min after infection and the extracellular phage was assayed at 30min intervals after the shift (Fig. 2). All of the mutants tested except ts12 and ts27 have a normal latent period after the shift to the permissive temperature. Although the final burst size for ts11.12 and ts12.27 is the same as that of the control, the latent period for these two mutants is extended by the time of incubation at the nonpermissive temperature (30 min).

AND SCOTT

dicate that the wild type alleles of the ts mutants of groups 11 and 12 are located in the Pl fragment present in pRM206 (BamHI-5), groups 13 and 14 are located in the fragment present in pRM116 (EcoRI-lo), and group 15 is in the Pl DNA present in pIH1972. The virions of representatives of two complementation groups, 11 and 12, exhibit an enhanced thermolability relative to wild type Pl. It thus seems likely that the products of mutants in groups 11 and 12 are structural proteins of the Pl particle. Both of these genes are located in the BamHI-5 fragment of Pl suggesting a possible clustering of genes for major Pl structural proteins in this region of the genome. This DISCUSSION region of the Pl map, which is homologous The 10 temperature-sensitive mutants of to the G region of phage Mu, presumably phage Pl characterized above fall into six codes for proteins involved in synthesis and different complementation groups named assembly of the tail fibers since mutations ll- 16, only one of which (16) contains a in this region affect the phage host range mutant that has been previously described. specificity (Toussaint et al., 1978). It is Complementation tests utilized 22 amber therefore probable that genes 11 and 12 code mutants that are well distributed across the for tail fiber structural proteins. The finding Pl genetic map. From a correlation of the that not all of the mutants in the complephysical and genetic maps of Pl (Mural et mentation groups 11 and 12 exhibit enal., 1979; Sternberg, 1979), it is obvious that hanced thermal sensitivity relative to wild long stretches of DNA remain in which no type Pl may mean that the location of the mutation within the gene affects the degree mutants have yet been identified. Although, it would not be surprising for these 10 tem- of thermosensitivity of the resulting properature-sensitive mutations to define five tein. This is expected since some parts of a new genes, we cannot be certain of this be- protein are more important functionally cause a positive result in a cross streak com- than others and some parts may be less plementation test may be caused by recom- sensitive to thermal denaturation because of bination in addition to complementation (see secondary and tertiary configuration of the Walker and Walker, 1976, for discussion of protein. It is also possible that thermal inactivation of the products of most of the mucomplementation spot tests). However, since two complementation groups of ts mu- tants with lesions in genes 11 and 12 is retants are within each of two “linkage versible, even when the proteins are part of clusters” (Walker and Walker, 1976) (13 and the virion structure, and that renaturation 14 are in II and 11 and 12 are in IV), it seems occurs during the assay. Thermal inactivalikely that complementation plays a more tion of the products of tall. 12 and ts12.27, important role than recombination in pro- on the other hand, may be irreversible. Since a shift of the infected cells from perducing phage growth in these cross streak tests. tsll does not complement with am75, missive to nonpermissive temperature at 30 and is therefore likely to be in the same min postinfection does not lead to phage production, none of the six genes in which operon with this mutation. The locations of the ts mutants on the Pl these mutants are located appears to code genetic map are consistent with the results for a function exclusively required early in of the complement&ion analyses; all of the the infectious cycle. In shift-down experiments, mutants repmembers of a given complementation group are close to each other, at least by the rough resenting each of the six genes give a nordeletion mapping employed. Our results in- mal burst after a normal latent period,

Pl TEMPERATURE-SENSITIVE

implying that all of these genes function late in infection, after the shift has occurred, and that the thermally denatured protein products of these genes renature before they are required for growth. However, unlike the other mutants in the same complementation groups, mutants ts11.12 and ts12.27 (which have by far the greatest virion thermal lability of all the mutants tested) exhibit an increase in the latent period approximately equal to the time of incubation at the nonpermissive temperature. This behavior is expected for mutants in a “lateacting” gene whose defective proteins are irreversibly damaged by heat (see above paragraph) and are required only late in the vegetative growth cycle. After the shift down, the latent period is extended while the mutant gene products are synthesized at the permissive temperature to replace the proteins that have been heat inactivated. Different lesions in the same proteins produced by other mutants in 11 and 12 may result in protein products in which thermal damage is rapidly reversible, so for these mutants no extension of the latent period occurs following return to permissive temperature. ACKNOWLEDGMENTS This work was supported by NIH Grant CA11673 to J.R.S. We are grateful to Drs. J. and D. Walker for helpful criticisms of the manuscript. REFERENCES ANDERSON,T. F., and WALKER, D. H. (1960). Morphological variants of the bacteriophage Pl. Science 132, 1488. CHESNEY, R. H., and SCOTT,J. R. (1975). Superinfection immunity and prophage repression in phage Pl. II. Mapping of the immunity difference and ampicillin resistance loci of Pl and 4amp. Virology 67, 375-384.

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MUTANTS

59

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SCOTT,J. R. (1974). A turbid plaque-forming mutant of phage Pl that cannot lysogenize Escherichia coli. Virology

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SCOTT,J. R. (1980). Immunity and repression in bacteriophages Pl and P7. Current Topics Micro. Immunol. l&49-65. SHAFFERMAN,A., GELLER,J., and HERTMAN,I. (1978). Genetic and physical characterization of Pldlw prophage and its derivatives. Virology 86, 115- 126. SHIMADA, K., WEISBERG, R., and GOTTESMAN, M. E. (1972). Prophage A at unusual chromosomal locations. I. Location of the secondary attachment sites and the properties of the lysogens. J. Mol. Biol. 63, 483-503.

STERNBERG,N. (1979). A characterization of bacteriophage PI DNA fragments cloned in a A vector. Virology 96, 129- 142. TOUSSAINT,A., LEFEBVRE, N., SCOTT,J. R., COWAN, J. A., DEBRUIJN, F., and BUKHARI, A. E. (19’78). Relationships between temperate phages Mu and Pl. Virology 89, 146- 161. WALKER, D. H., and WALKER, J. T. (1975). Genetic studies of coliphage Pl. I. Mapping by use of prophage deletions. J. Virol. 16, 525-534. WALKER, D. H., and WALKER, J. T. (1976). Genetic studies of caliphage Pl. III. Extended genetic map. J. Viral. 20, 177-187.