Suppressor-sensitive mutants of coliphage ∅80

VIROLOGY

34, 637-649

(1968)

Suppressor-Sensitive KOKI

Department

SATO, YOSHITAKE

of Microbial

Genetics,

Department

NISHIMUNE, AND AIZO Research

HACHIRO

AND

Mutants

of Chemistry,

of Coliphage

MITSUNOBU MATSUSHIRO

Institute Osaka,

for Microbial

$80’

SAT02, REFIK

Diseases,

Osaka

University,

NUMICH3,

Suita City,

Japan

INOKUCH14

AND

HARUO

National

Institute

of Health

Accepted

January

9,1968

OZEK14

of Japan,

Tokyo,

Japan

About fifty suppressor sensitive (sus) mutants of phage $80 were isolated after hydroxylamine treatment, and these were classified into fourteen cistrons. In vitro and in vivo complementation experiments revealed that at least five cistrons were concerned with head formation and that at least six cistrons were concerned with tail formation in 480. Some presumed “early” mutants were also found. Defective lysogens were isolated from a &O-lysogenic nonpermissive strain using the colicin plate method (Gratia, 1966). In these strains deletions which affected the colicin B receptor gene extended for varying distances into prophage $80. Marker rescue experiments were carried out with these deletion lysogens by infecting various sus mutants, and the gene order in prophage &30 was determined. Clustering of all head genes and also of tail genes, as in phage X, were demonstrated by the results of the prophage deletion mapping as well as the two-factor crosses performed among the sus mutants. Moreover, the gross gene arrangement of $80 was also similar to that of phage h: namely, the cluster of head genes was found to be located at one end of the vegetative map of 480, being followed by that of tail genes, and presumed “early” genes are located at the other end of the map.

of the genetics, physiology, and morphogeneConditional lethal mutations in bacterio- sis of the phages. Among various temperate coliphages phage T4 (Epstein et al., 1963), in X (Campbell, 1961), or in P22 (Gough and Levine, known, phage X is the only phage which has personal communication) were found to be been studied extensively by utilizing condidistributed widely over the phage genome tional lethal mutations. Recently the study and therefore to be useful in investigations of Weigle (1966) revealed that genes controlling the formation of the head of phage particles form a cluster and similarly the tail 1 Supported in part by a grant from The Jane Coffin Memorial Fund for Medical Research genestogether form another cluster. Since in (Fund’s Project No. 188). vitro assembly tests have made possible the 2 Present address: First Department of Oral classification of head or tail mutations, it and Maxillofacial Surgery, Osaka University has become less laborious to characterize Dental School, Osaka, Japan. suppressor-sensitive mutants. It would be 3 Present address: Faculty of Agriculture, interesting to determine the generality of Laboratory of Plant Pathology, Sarajevo Univerthe in vitro assembly phenomenon with other sity, Zagrebacka 18, Yugoslavia. phages. To examine this problem we have 4 Present address: Department of Genetics, chosen 480, as we have been working with Faculty of Science, Osaka University, Toyonaka City, Osaka, Japan. this phage, which is known to transduce INTRODUCTION

637

63X

SAT0

ET AL.

tryptophan genes specifically (Matsushiro, 1963). In addition, it would be useful to obtain knowledge about t,he genetic map of phage 480, since it has recently been used for a variety of purposes (Franklin et al., 1965; Signer, 1966; Radding et al., 1967). Two types of conditional lethal mutants have been reported, namely amber or suppressor-sensitive (sus) and temperature-sensitive types. Unfortunately, however, our preliminary test indicated that 480 was naturally temperature-sensitive, not forming plaques at high temperature (seealso Signer and Beckwith, 1966). Later, one of us (H. I.) found that the infection process was temperature-sensitive, alt.hough the particles were not, so. Hence we decided to select szlsmutan&. About fift,y sus mutants have been accumulated in our laboratories. This paper is concerned with the classification of these mutants into cist,rons, as well as into head and tail groups. The genetic map of 480 was constructed mainly by deletion mapping, a met,hod which was described by Franklin et al. (1965). Fortunately, prophage $80 is known to locate near a region of the Eseherichia coli chromosomeat which various delet,ion mutat,ions occur frequently (Tl*try deletion), and deletion often extends into prophage $SO. These deletion mutant,s can easily be selected .by isolating resistant mutants against cohcm B (Gratia, 1966). By analyzing these deletion mutants of prophage $180,Franklin et al. (1965) found that all the deletions started at one definite end of prophage, namely t’he h (host range) end. Their results, however, were obtained by using the hybrid phage of 480 and X, and the markers involved were essent.ially those of X, not of 480. Since 480 susmutants as well as defective lysogens became available, we carried out deletion mapping of phage $80 and could determine the linear order of genes on prophage +SO.The genetic dist,ancesbetween these geneswere roughly estimated from the data of t,wo-factor crosses by infecting two different sus mutants in various combina,tions. These results together have suggested that t,he permutation of the genetic map of $80 from “vegetative” to “prophage” took place in t.he same manner as with X (seeCampbell, 1962).

MATERIALS

AND

METHODS

Bacterial strains. The nonpermissive (pm-) strains used were Escherichia coli W3350 (Campbell, 1961) and its strepromycin-resistant mutant 594 (Weigle, 1966), which were kindly supplied by Drs. A Campbell and J. Weigle. A tryptophan-requiring (try-) derivative was selected from 594 and was lysogenized with $80. The strain 594 (480) try- was used for the isolation of defective lysogens. The permissive (pm+) strain used was E. coli CBOOS F-thr+Zeu+Tl, ~~ZUC-SUII~, which was prepared from C600 F-thrleu-Tl, 5TZa~-~~~~f(Campbell, 1961) by crossing with Hfr H and selecting for 480 sensitivity among thr+ leu+ recombinants (th?,, threonine; Zeu, leucine; Zac, lactose; Tl, 5”rr, sensitive or resistant t’o the infection of phage Tl and T5; $SO shares the same receptor as Tl). Strain E. coli CA5013 (kindly supplied by Dr. E. R. Signer) was also used as a permissive strain, which carried sul+ (Signer et al., 1965). The colicin B producer CA18 was provided by Dr. P. Fredericq through Dr. J. I’. Gratia. Ph.age strains. Phage $80 has been described previously (Matsushiro, 1963). The hybrid phage imsnhsx (from Dr. N. Franklin), being able to infect, colicin B-resistant strains, was employed for testing immunity against +SO (i@88o) in defective lysogens isolated. Isolation of sus mutants. Suppressor-sensit’ive (sus) mutants of 480 have been isolated from hydroxylamine-treated phage populations (Freese et al., 1961). Phage was incubated at 37” in 1 M NH,OH*HCl plus 0.5 M KaCl (pH 7.3) to a survival of about lo-“. Plaques developed on C6OOS or CA5013 lawns were randomly picked and tested on the pm- strain. L%S mutant’s obtained were designated in the order of their isolation as susl, sus2, . . . Lysogenic strains for these sus mutants were prepared with pm+ and pm- strains; these were indicated as C6OOS(susl), 594 (susl), etc. Phage lysates were prepared by UV-induction or mitomycin C induction of lysogens. Media. Ilambda broth, lambda agar, and Tl medium have been described elsewhere (Matsushiro el aZ., 1964) Classijicat~ionof xsusmutants into head and

SUS

MUTANTS

OF

tail groups (in vitro complementation test). In general the methods used in phage X (Weigle, 1966) were followed. Two given lysates from pm- lysogens of .YUS phages were mixed together and then tested for the formation of active phage in vitro. This is referred to as the in vitro complementat’ion test. Complementation test (in vivo). Spot tests used for phage X (Campbell, 1961) were generally followed. Certain combinations of sue mutants, which gave ambiguous results by the spot tests, were further investigated by measuring the burst sizes. Nonpermissive bacteria 594, lysogenic for a given sus mutant, were irradiated with an inducing dose of ultraviolet light (UV) and superinfected with the other sus mutant at a multiplicity of 2. The infected mixture was incubated at 37” for 150 minutes. The details of the procedure are given in the legend of Table 3. The burst size of the when cells were incontrol experiment, fected with the wild-type 480 was taken as 100, and the result with the sus mutant was expressed as a percentage of the control. Selection of defective lysogens. From +80lysogenic 594 try-, many colicin B-resistant mutants were isolated by the colicin plate method (Gratia, 1966). Among the resistant strains, those which produced no infective phage and yet retained i4*O (tested by the hybrid phage PoPA) were presumed to be defective lysogens carrying at least a part of the prophage genome. These strains were used for deletion mapping of prophage 480. Deletion mapping of prophage 480 (Marker rescueexperiments). Defective lysogens were UV-induced and superinfected with 480 sus mutants at a multiplicity of 0.5. The mixtures were incubated at 37” for 150 minutes, and the titers of each lysate were assayed with pm- indicator to count the number of wild-type recombinants. Cross procedure. Two-factor crosseswere performed according to Campbell (1959). Pm+ bacteria were infected with two sus mutants at a multiplicity of 4 each. After 20 minutes of adsorption, anti-480 serum was added, and 10 minutes later the culture was diluted 1:5 X lo3 in broth and incubated at 37” for 120 minutes. The

COLIPHAGE

639

480

lysates were assayed with pm+ indicators.

and pm-

RESULTS

Isolation of 480 susMutants Hydroxylamine-treated phage were plated with pm+ indicators and sus mutants were selected as described in Methods. Approximately one mutant was isolated for every 500 plaques tested, and 53 sus mutants have been accumulated so far. 480 susl through sus7were isolated using the C6OOS indicator and the remainder using CA5013. Mutant sus6 grew well on C6OOSbut not so well on CA5013; the efficiency of plating of the latter for the sus6mutation was about three times lower than the former. The other mutants responded equally well to both suppressors. Mutants susl2 and 34 were so leaky that their characterization has been postponed. Mutants ~2~~16,17, 18, 19, 20, and 53 were also rather leaky and were used only in the deletion mapping. Excluding these leaky mutants, lysates prepared by UV induction of pm+ lysogens of sus phages ordinarily gave titers of log to lOlo plaque-forming units per milliliter on pm+ and lo3 to lo* per milliliter on pmindicator bacteria. In Vitro Complementation First we tested the defective lysates of 594 (r#~SOsusl)through 594 (~80~~~10) in all possible combinations for the formation of active phage particles in vitro. When two lysates were mixed, infectious particles were formed in the test tube with certain combinations (Table 1). Thus it appeared that assembly of head and tail, resulting in the formation of active phage particles, took place not only in phage X (Weigle, 1966) but also in 480. This test divided sus mutants into two groups: head donors (tail sus mutants) and tail donors (head sus mutants), which were determined by analyzing the genotype of reconstituted phage. The classification of head and tail donors was also confirmed by sucrose gradient centrifugation analysis of the active components in the defective lysates, which agreed with the comparable values that had been reported for tail (47s) and head (650s) components in phage X (Weigle, 1966). More-

SAT0

640

over, we found that X heads can be activated with 480 tails as efficiently as with h tails but that 480 heads cannot be activated with x tails. The details of these experiments will be reported elsewhere (Inokuchi and Ozeki, in preparation). From Table 1, susl, 6,9, and 10 are considered tail donors and sus2, 5, and 7 are considered head donors. With these head or tail donors, the other sus mutants were also classified by the in vitro complementation test. The results are shown in Table 2. Thirteen mutants are shown to have a defect in head formation, and twentyfive mutants a defect in tail formation. In vitro complementation tests with mutants sus21, 24, 39, and 46 were unsuccessful, but, these mutations have been assigned to “gene 6, ” “gene 11,” “gene 12,” and “gene 13,” respectively, from the results of in vivo complementation and mapping (see Figs. 1 and 2). Three other mutants, AU&, 37, and 50, showed no complementation with either donor, but as described later the comparison of the vegetative and prophage maps of @SO indicated that these mutations are concerned with early functions. TABLE

Defective lysate susl sus6 sus9 s11s10 sus2 sus5 sus7

Tail

1.1 0.14

Head

donors

sus 1 sus 6 sus9 0.95

I

1.2 1.1 1.4

donors

sgs 10

~21s 2

sus 5 sus 7

1.5 0.9 1.6 1.1

100 84 84 50 1.3

31 38 45 31 1.5 1.6

170 130 150 140 26 20 19

a Two defective lysates of 480 sus mutants were mixed in equal volume and incubated for 1 hour at room temperature. The titer of active phage was assayed. The numbers given in the table are titers divided by 103. Tail and head donors are determined by analyzing the genotype of assembled particles. Plaques of the reconstituted phage were examined for their genotype by complementation spot tests using relevant sus mutants. The results were confirmed by separat,ing the head and tail components in sucrose gradient rentrifugation.

ET AL. TABLE CLASSIFICATION AND

2

OF ~$80 sus

MUTANTS

Incubated Defective lysate

None susl susa sus6 sus9 SUSlO susl5 sus30 sus32 sus36 sus43 SUS4.i

sus-29 sus31 su42 sus4 suw s&F7 SliSll sllsl3 .su.s14 sus22 sus23 sus26 sus26 sus27 ms28 slLs29 sus31 S&S33 sus3.i s us38 sus40

.sus41 .Pus42 sus44 sus47 sus48 .sus52

INTO

TAIL GROUPS BY in COMPLEMENTATION~

Self

0.002 2.6 0.03 0.08 0.18 0.19 2.1 0.11 0.93 0.01 1.1 1.1 0.002 0.14 0.74 1.0 0.17 0.05 0.05 0.1 0.89 I.4 0.07 0.34 0.18 3.7 0.23 0.08 0.01 0.06 0.003 0.86 0.07 0.26 0.49 0.44 0.08 0.05

Fith

HUD

Vitro

lvsate

of

sus 2 (head donor)

sus 10 (tail donor)

0.14 92 lli 113 114 9.5 116 129 I.3 96 108 99 132 122 0.3 1.3 1 ..i 0 x 0.25 0.21 0.34 1.9 1.0 0.25 0.48 0.28 3.8 0.37 0.29 0.17 0.17 0.l.i 1.0 0.21 0.4,5 0.84 0.8.5 0.24 0.24

0.18 0.25 3 3 0.22 0.4 0.44 0.4 2 .5 0.48 2.1 0.23 2.0 2.0 0.28 7-f 138 105 91 72 111 42 85 97 97 60 94 79 190 92 76 5X 48 133 146 122 115 173 127 77

____~

a Defective lysates of sus mutants in the first column were mixed with head or tail donor and complemented in vitro as described in the legend of Table 1. The numbers given in the table are titers divided by 103.

In Vivo Complementalion Following the classification described above. comnlementation tests in vivo were

SUS

MUTANTS

OF

COLIPHAGE

performed among members of the head or tail group. These spot tests led to positive, negative, or equivocal results. Pairs which showed equivocal results in spot tests were investigated further with burst-size tests (Edgar et al., 1964). Nonpermissive host bacteria lysogenic for a sus mutant were UV-induced and superinfected with another sus mutant phage. The mixture was incubated and assayed for its titer. As a control, the same bacteria were induced and superinfected with the wild-type phage. The results of complementation experiments among head mutants are presented in Table 3. Thirteen head sus mutants were thus classed into five genes, “gene 1” through “gene 5,” indicating that at least five cistrons were concerned with the formation of the head in $80. Sus21 was placed in “gene

6,” complementing all the other sus mutants, but was not grouped into either head or tail mutants. “Gene 6” was included in the table because deletion mapping data showed that the gene was located between head and tail clusters on the map (see Figs. 1 and 2). Table 4 presents the complementation data among tail mutants. Here “gene 6” was again included in the table. Sus24 and su.s46 did not complement sus22 (“gene 11”) and sus28 (“gene 13’7, respectively, indicating mutations in the same cistron. Sus39 complemented all other sus mutants and hence was assigned to “gene 12,” Although classification of sus39 into head or tail donor was unsuccessful, deletion mapping data indicated that mutation sus39 was located in the tail region. From Table 4,

TABLE

3

In Vivo COMPLEMENTATION BETWEEN HUD Superinfecting sus mutant lysogenized in strain 594

Gene 1

sus

Gene

Gene

Gene

1 sus3 sus30

0.01 0.72

2 susl susl5 sus32

33 39 +

3 sus6 sus51

+ 00

4 sus36 sus43 sus45

+ + i-

Gene 2

szls mutant

Gene 3

MUTANTS" phage

Gene 4

Gene 5

Gene 6

sus

sus

SUS

sus

sus

SUS

30

1

15

32

9

10

49

sus 21

1.2 0.08

$-b +

19 +

+

84 +

+ +

0.001 0.63 3.9

3.2 0.3( 3.1

71

+ + +

3 __~Gene

1

641

$80

26 + + + 52

59 +

+ + +

+ + +

$1 ‘4 j9 + $6

33 +

1

1.0 17 1.9 1 1 0.0: 2 + 52 100 + + +

0.82 0.3 + + +

18 + +

57

16 +

0.22’ 0.9846 14 28 21

15

66 37

+

$

-k 16

0.0 2.1 4.3

+ +

1 1.5 0.48 1.4

+ 33

+ +

00

+ + +

61

+ +

2.4 + 2.6 + O.O! 3 +

+ + +

sus

+ + +

I 00

61 + + +

1.oo 93 I.oo

100 + + + +

0.41 0.00: 2 1.3 22 1 0.93 0.0: 2 + - a Pm- strain 594 lysogenic for a sus mutant, described in the first column, was suspended in Tl medium at 3 X 108/ml, UV-induced, and superinfected with various sus phages at a multiplicity of 2. After 20 minutes adsorption at 37”, antiphage serum was added to give 99% inactivation of unadsorbed phage in 10 minutes. The mixture was then diluted 1: lo4 in lambda broth and aerated at 37” for 150 minutes. The resulting phage titers were determined with pm+ indicator. The control infecting the wild-type 480 instead of sus mutant was also performed, and the numbers in the table are burst sizes expressed as percent of the control. Data obtained are arranged according to the gene order determined by the mapping results described later. b We did not measure the burst sizes for the combinations which gave clearly positive or negative results in spot complementation tests, and these are indicated as + or -, respectively, in the table. Gene

5 susl0 sus49

+38 +

+

+,+

LO +

58 +

+

1s

+I+/+

33

13 +

36

+

sus

mutants

96 + +

sus7 11 SW22 sus47

gene

73 +

lo

gene

84

suslh sus42

9

gene

sus2

90 33 +b + + +

89

8

gene

susll susl3 sus25 SW31 sus33 sus35

594

51

18

38

18

-I+

+

OS8 -

0.001

gene 6 sus21 susll

sus4

7

gene

Gogenized in strain

20

30

+ + +

+

+

-

0.001 0.013 -

SW13

h

42

+ + +

+

+

+

0.67 0.69 0.22

sus25

yiu;p

14

+ + +

+

+

+

0.51 0.53 0.5 0.044

sus31

gene

30

+ + +

+

+

f

0.63 0.33 0.24

7 sus33

Complementation

4

22

+ f +

+

+

+

.0.34 0.69 0.5 0.09 0.18

.sus35

Tail

23

+ + +

+

+

+

0.61 0.76 0.06 0.01 0.63 -

sus41

Superinfecting

between

TABLE

22

+ + +

+

+

+

0.77 0.36

+ + 28

16 18

20

0.006

61 + + + + +

4; 40

39 35

1.8

28

61 + + + t +

mutant phage gene 8 sus48 sus2 SW4

e

Mutantsa

+ + +

25 +

2.1

40

100 + + + + +

gene 9 sus5

19 + +

48 +

0.53

29

78 73 + + + +

sus40

44 27 +

0.17

22

45

66 + + + + +

susl4

4:

+

1.3 3.5

+

41

74 + + + + +

+ 57 47

1.5 0.2

18

55

63 38 + + + +

gene 10 sus23 sus42

9

z= r

;3 m

67

+ 69

sus4

9

10 susl4 sus42

gene

l1

12 sus39

13 sus.28

gene

gene

gene

sus22 sus26 sus27 sus29 sus38 sus47 sus52

SUS7

+

39

0.01 1.2 1.9 3.4 1.4 3.9 2.1

55

gene

sus2

8

+

gene

susll

sus7

7

594

gene

sus mutant Gogenized in strain

+

40

1.7 0.4 2.6 2.8 2.3 4.5 -

80 86

+

+

+

0.71 -

+ +

+

+

+

42 +

sus24

sus22

viva

88

88

38

54

41

5.7 1.6

3.4 0.28

6.8 1.5 66

0.24 2.4 1.9 2.1 0.01

+ 54

+ +

0.95 1.2 0.63 0.02 1.5

+

+

+

+

+

+

gene 11 sus29 sus27

Superinfecting

Complementation

2.4 2.1 0.03 0.47 3.3

+ +

+

+

+

sus26

--In sus

between

20

+

0.58 0.59 1.2 2.5 1.5 0.01 2.8 3.0

+ +

+

52

+

Tail

+ +

+

0.21 6.8

2.7 4.9

77 57

43

+

100

sus47

phage

Mutants

+

0.12 1.5 5.6 3.9 2.4 1.6 3.9 3.5

+ +

+

+

+

sus44

mutant

4 (cont'd)

sus38

TABLE

31

+

0.78 1.2 1.4 1.0 0.42 2.3 3.4 0.02

+ +

?-

+

+

sus52

60

0.94

29 39 67 73 72 + 40 39

58 +

75

84

68

gene12 sus39

0.17

52

34 28 20 20 + + 46 32

-I+

27

24

40

sus28

Eerie

0.33

+

19 + + + + + + +

+ +

+

+

+

13 sus46

644

SAT0 TABLE DELETION

-

~701950 1120 1160 1830 96( L821740 1020 630 1070 65( !282 770 1040 640 890 lOl(

780 504 640

-l-l-l-l-l0 336 0 143 ---

5601 331 610’ 29(

280 60

Gene

1 ~14.~3 s us30

0 0 -

Gene

2 susl sus32 SW15

-t

Gene

4 sus36 sus43 sus45 susl6

-

-. -

Gene

Gene

.? sus9 ,sualO sus49 6

sus21

G6 _-

G7

G8

350 201 -__-

0 132 438 0 42 593 0 168

166 660 338

330 990 981

24~ + ’ +

270 239 262

64 94

287 228

+

190 68

+ +

410 139 267

0’ 0

0 183 0 49

0

0 0 0

----

---

0

--

G5

921 299

0

SUSl7

suxl8 ausl9 sus20 -

‘24

lysogens

G2 --__

0 0 0 -

G3

~$30”

;1 14 sus8 sus37 sus50

3 sus6 sus51

OF PROPHAGE Defective

Gene

Gene

5

MAPPING

Superinfecting szls phage

ET

0 125 978 0 142 425 0 180 1180 -_-

0 0 0

0 0 0

0

0

0 0 0 0 0

239 132 262 139 42

12: + lO( +

260 215 195 295 40

-_0 00

0 0

0 0

0

0

0 0

0 8. 50 0 21 96 0 101 156

0

0

1:

52

-I--.

- i Oi / O/ Oi (

303 - I (L Defective lysogens, 3 X 108/ml, were UVinduced and superinfected with sus mutant phage at a multiplicity of 0.5. The mixtures were incubated at 37” for 20 minutes, unadsorbed phage was removed by centrifugation, and the cells were diluted 1:102 in lambda broth. After inctlbation at ?7” for 150 minutes, the titers of the wild-type recombinants in each mixture were assayed with pm- indicator. The numbers given in the table are phage titers divided by 4 X 10”. Only the combinations with Gl through G8 were presented in the table, since all sus mutants in “gene 11” and “gene 1” through “gene 6” gave SUSf rerombinants when infe:ted the defective lysogens of G9 through G16. b The combinations the phage titers of which were not measured, but were tested by spotting

AL.

it is concluded that at least. six cistrons, “gene 7” through “gene 13” except “gene 12,” are concerned with tail format’ion in ~$80. As for the presumed early mut’ants, s&3, 37, and 50, very little complementation (by spot tests) and a close linkage (t’he recombination frequency between su.sS and sus37 was 0.06, that between sus8 and sus50 was 0.1, and that between sus37 and sus50 was 0.01) were observed among t,hem, and they may belong to the same ciat’ron. Deletion Mapping

of Prophage 480

Among several hundreds of colicin B-resistant mutants, 42 mutants produced no active phage, although thev still maintained ;bsO. These strains are coniidered to contain deletions in the prophage $80 genome. Since the st,rains isolated were minus the receptor for Tl and colicin B, which was common to $80 adsorption, we transferred to these defective lysogens an episome I”‘try (E’redericq, 1965) which carried the Tl and colicin B receptor gene, in order to allow infection with $180 sus particles. With these defective lysogens, the rescue of sus+ alleles into .sus phage was investigated. This may be referred to as deletion mapping of prophage $80, by which the gene order of the prophage can be determined in all-or-nothing fashion with respect to the yields of the wild-type recombinants. The results are presented in Tables 5 and 6. In the control experiments, neither defective lysogens alone (UV-induced) nor nonlysogenic p/j,- strains infected with sus phage, produced act’ive phage. When a UV-induced defective lysogen was superinfected with various s=us mut,antnts, with cert,ain sus mut’ants, sus+ phage were formed, while with the other not, indicating the presence or absence of S.LIS+allele in t)he defectjive lysogen. Forty-two defective lysogens isolated were classed into 16 groups, Gl through G16, according to the extent of their deletions. The numbers of defect#ive lysogens belong________the mixture of a defective lysogen (UV irradiated) and a SW phage on pmindicator lawn, are indicated ill this table by + (positive) or - (lregstive) .

SUS TABLE DELETION

MAPPING

-

OF PROPHAGE

Gene

8

Gene

9 sus4 sus5 sus40

_.-

10 susl4 sus23 szrs42

-

11 sus47 sus7 sus22 sus24 s-us38 sus44 SUS29 sus26 sus27 sus52

-

Gene

12 sus3:

-

Gene

13 sus2f sus46

-

Gene

Gene

sus2

lysogens

>! ?

Gt 7 susll susl3 sus25 sus31 sus33 sus35 sus41 sus48

&30a

-

-. Gene

OF

6

Defective Superinfecting SW phage

MUTANTS

--

-_ -

---__-_

0 0 0 0 0 0 0 0

f $7 106341 92243 : SO 68215620880 f 58 139259 84145 : !I 93215 72 121 I14 59130 92 90 : 16 46323223 Sb c17 89135 + 0 23 52165 + _.- -_ - -- ---__.--_ - b 0/ 0 47150 ---_--__

_.-

-_ -

0 0 0

0202260 0 157 326 0353367 ---______

0 0 36 55 0 0 148 91 0 0155236 - -- _--__-~_

180 310 7701320 137 187 149 174 123 188 102 236 157 204 120 400

+

77

+ +

+

-t 280 330

+

69 50 210 463

162 492 965

500 142 377 39 105 256 564 149 119 131

880 440 370 523 187 447 294 186 140 224

0 0 0

-- ---

0 70162 0 0 30 89 0 0137 269 0 0 93 213 0 0 0 32 62 0 0 36 150 0 0 0 15 0 0 0 0 0 j 0 0 0 0 0 0 0 ---__-~~ 000 _--_-__~

l-

-

0 0

535

526 ea.5 5881310

0 242 0 0

679 239 725 560 443 266 380 970

0 0

0 0

411 14 6

a The procedures are the same as described in the legend of Table 5. The numbers in the table are titers of the wild-type recombinants divided the combinations with G8 by 4 X 104. Only through G16 were shown in the table, since all sus mutants in “gene 7” through “gene 13” gave no susf recombinants when infected the defective lysogens of Gl through G7. 6 + or - indicates positive or negative results, respectively, in spot test, the mixture of a UVirradiated defective lysogen and a sus phage being spotted on pm- indicator lawn.

COLIPHAGE

+8o

645

ing to each group are: Cl (l), G2 (6), G3 (11, G4 (11, G5 (2), G6 Cl), G7 (l), G8 (2), G9 (l), GlO (l), Gil (2>, G12 (2), G13 (3), G14 (3), G15 (6), and Cl6 (9). The results shown in Table 5 are best interpreted by arranging genes in the order presented in Fig. 1, since a deletion missing “gene 12” is also lacking “gene 13,” deletions missing “gene 11” lack “gene 12” and “gene 13” simultaneously, and so forth. Thus the order of genes can be arranged in a linear map, with “gene 13” at the righthand end. Since all the defective lysogens examined retained immunity against 480 (P”), it would be reasonable to place i+SO at the leftmost part on the prophage map. These results are schematically represented in Fig. 1, which shows the deletion end points as well as the order of sus mutations on prophage 480. As seen in the figure, “gene 14” is located next to PO, followed by the head and tail genes on the prophage map. The sequence is permuted in the vegetative map as described in the following section. Two-Factor

Crosses

The distances between two mutational sites were determined by two-factor crosses, which also gave the gross vegetative map. First, the leftmost gene in the head region and that in the tail region were determined. Deletion mapping data suggested that “gene 1” and “gene 7” were located at the leftmost part in the head and tail regions, respectively. Consequently, crosses were carried out between mutant sus3 (“gene 1”) or susll (“gene 7”) and the other .YUSmutants. Approximate recombination frequencies were estimated. Figure 2 is a summary of the results. The vegetative map of 480 was constructed by two-factor cross data as well as deletion mapping data, since the results of two-factor crosses are not necessarily conclusive. Complementation classes are numbered from the left to the right, and referred to as gene number. On the vegetative map, genes are arranged as “head,” “tail ” “i,” and ‘(early,” from the left to the hght. DISCUSSION

Fifty-three sus mutants of coliphage $80 were isolated and characterized. Most of

FIG.

are not

tatIces

present,

UI)PG,

deletiotls

shown;

under

map

-I

8

37 50

T

8 I

to scale.

t

2

the prophage

of ~$80 and

Zigzag

line

6 51

I

3

the

synthesis;

lysogcns.

of t hr prophagr

itldicates

! 1

16 17 18 19 20

T I

5 Y

boundary

Tlr,

’

I t

between

defective

, , , I I I

prophage

2

4 5 40

I

9

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I

10

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-r

and

TI ba:terisl

to phage

t

I 26 27 52

deletion

28 46

-r

13

I

at the

39

I

12

and

gene

biosynthesis.

line)

are

I&s-

also

of prophage

points

represent end types

top

(broken

are dif’ferelrt chromosome

(;16

indicat,e

--\I

B; f,~u, tryptophan chromosomes.

coli

29

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nl1meral.s

11

lines throw@

The

7 22 24 38 44

t -T-

a11d coliciu

011 the F:scherichiu

(:I

Vertical

lysogens. lysogens.

sus m\ltations.

in

markers resistance

genetic

retained

: I

I <

48

T + I

a

-c

in tlefeztivr

13 25 31 33 35 41

l11

carried

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represent

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i

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I

6

21

deletions

9 10 49

(solid

#

, I I

t

Neighborillg

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36 43 45

-I-

4

chromosome

variolls

15

7

defective

diphosphoglucose

ill itldividual

uridine

I

1 32

the portions

:

I

1

3 30

!

indicate

those

lilies

alid

1. Prophage

horizontal

numbers

L

G9 -

G8

G7

G6

G5

G4

G3

G2

Gl

I

I

14 r--x---

SUS MUTANTS

Head Formation

OF COLIPHAGE

Tail

.I 11 13 25 31 33 35 41

&30

Formation 13

I 48 5 40

t

l28 46 42 1

24 38 44

52

t

0.52 2.2

< (

c f 4 4 4 l < <

5.2 _

\ I Lot -

647 Early 1?UI1ct I- i14 l8 37 50

i

>

5.8 6.2

> + 6.6 7 ,,

.

>

10.0

/

10.2

>

10.4

> 10.8

l

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>

14.8 18.6

t

P >

FIG. 2. Vegetative map of $230 made from the results of two-factor crosses and deletion mapping. The numerals over the phage chromosome represent gene numbers, and those under the chromosome represent sus mutations. Zoc stands for the region where the integration into host chromosome may take place (Radding et al., 1967), and i stands for the immunity locus. Vertical lines indicate the boundary between neighboring genes. The numbers over the arrows are recombination frequencies between two sus markers, which were calculated from the results of two-factor crosses.

them are concerned with the formation of structural proteins of phage particles, namely, 13 mutants for head formation and 27 mutants for tail formation; the remaining mutants could not be classified as either head or t,ail, and three of them might be concerned with “early” functions. There are at least five cistrons for head formation and six cistrons (seven cistrons if “gene 12” is added) for tail formation. These numbers of cistrons for each function are somewhat comparable to the numbers of cistrons found in phage A, i.e., six head genes and seven tail genes (Weigle, 1966). Our data from deletion mapping and recombination experiments revealed that all the head genes were linked together forming a cluster of genes, and similarly all tail genes formed

another cluster. This situation is quite similar to that found in phage A. Moreover, the genetic map of vegetative phage r#80 is more or less parallel to that of phage h in respect to the location of head, tail, and early groups. The prophage map of 1#80 has been suggested by Franklin et ab. (1965) by using the hybrid phage i@"h+h, which is also parallel to that of phage X. Deletion mapping of phage 480 described here is in agreement with their result. This type of gene arrangement is not specific for phage X, but might have some generality among temperate phages. Figures 1 and 2 present prophage and vegetative maps of ~$80, respectively. Permuted sequence was also observed in 480 between vegetative and prophage maps with respect to Dhe early,

64S

SAT0

head, and tail genes. The positions of lot at which integration takes place and the immunity locus i have been suggested as it is shown in the figure, and lot should be located between h and i, as in phage X (Franklin el al., 1965; Nakamura and Ozeki, unpublished results; Signer and Beckwith, 1966; Radding et al., 1967). Thus, although not complete, t)he genetic map of $180 turns out to be quite similar to that of phage X, yet, it, may differ in detail. This has led to further investigat’ions of the detailed comparison of each gene between 480 and X. Our preliminary results of complementation tests between head sus mutants of 480 and X have indicated so far that the function of “gene 5” of $80 corresponds to that of gene ,4 of X, that of ‘
ET

AL.

(Smith 1968).

et

al.,

1966;

Andoh

and

Ozeki,

REFERENCES ANDOH, A., and OZEKI, H. (1968). Suppressor gene su 3t of E. coli, a structure gene for tyrosine tRNA. Proc. Natl. iicad. Sci. U.S., in press. C.IMPBELL, A. (1959). Ordering of genetic sites in bacteriophage X by the use of galactose-transducing defectjive phages. Virology 9, 293-305. CAMPBFZLL, A. (1961). Sensitive mutants of bacteriophage X. Virology 14, 22-32. C.IMPBIGLL, A. (1962). Episomes. Advan. Genet. 11, 101-14.5. EDG.IR, R. S., ~NNH.IRDT, G. H., and EPSTHN, R. H. (1964). A comparative genetic study of conditional lethal mutations of bacteriophage T4D. Genetics 49, 635-648. EPSTEIN, R. H., BOLLS, A., SIWNBERG, C. M., KELLENBERGER, E., BOY DE L.\ TOUR, E., and CHEVSLLF,Y, R.; EDGAR, R. S., SUSMAN, M., I)~:NHARDT, G. H., and LIELAUSIS, A. (1963). Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Bid. 28, 375-394. FRANKLIN: 5. C., DOVN, W. F., and Y.INOFSKY, C. (1965). The linear insertion of a prophage into the chromosome of E. coli shown by deletion mapping. Biochem. Biophjya. Res. Commm. 18, 91&923. FRIADE:RICQ, P. (1965). Genetics of colicinogenic factors. Zentr. Bakteriol. Parasitenk., Abt. I. Orig. 196, 142-151. FREEZSE, E., Bsu~z, E., and BAI.JTZ-FREI*:S~?:, E. (1961). The chemical and mutagenic specificity of hydroxylamine. Proc. NatE. Acad. Sci. U.S. 47, 84.5St%. MATMUSHIRO, A. (1963). Specialized transduction of tryptophan markers in Escherichia coEi K12 by bact,eriorJhage @SO. Virology 19, 47.5482. MATSLJSHIHO, A., SATO, Ii., and K1u.4, S. (1964). Characteristics of the transducing elements of bacteriophage $80. Virology 23, 299-306. RADDING, C. M., S~IRER, J., and THOMAS, R. (1967). The st’ructural gene for X exonuclease. Proc. Satl. Acad. Sci. U.S. .57, 277-283. SHAPIRO, J. A. (1966). Chromosomal location of the gene determining uridine diphosphoglucose format’ion in Escherichia coli K12. ,I. Bncteriol. 92, 518-520. SIGNI~R, E. R. (1966). Interaction of prophages at the attao sit’e with the chromosome of Escherichia coli. J. Mol. Biol. 15, 243-255. SIGNER, E. R., and BIXKWWH, J. R. (1966). Transposition of the lac region of Escherichia coli. III. The me:hanism of attachment of bacterio-

SUS MUTANTS phage

480 to the bacterial chromosome. J. 22, 33-51. R., BECKWITH, J. R., and BRENNER, S. (1965). Mapping of suppressor loci in Escherichia coli. J. Mol. Biol. 14, 153-166. SMITH, J. D., ABELSON, J. N., CLARK, B. F. C., Mol. Biol. SIGNER, E.

OF COLIPHAGE

#30

649

H. M. and BRENNER, S. (1966). Studies on amber suppressor tRNA. Cold Spring Harbor Symp. Quant. Biol. 31, 479-485. WEIGLE, J. (1966). Assembly of phage lambda in vitro. Proc. Natl. Acad. Sci. U.S. 55, 14621466. GOODMAN,