VIROLOGY 42,621-632 (1970)

ES18, a General Transducing Phage for Smooth and

Nonsmooth Salmonella typhimurium’

TSENG-TONG KU02 AND B. A. D. STOCKER

Department of Medical Microbiology, Stanford University School of Medicine, Stanford, California 94805

Accepted July 14, 1970

Bacteriophage ES18 derived from typing phage 18 of the Salmonella typhimurium phage-typing scheme of Callow, is hear-labile and serologically and morphologically unlike phage P22 (a typical phage of Boyd’s group A). Unlike phage P22 it attacks all classes of nonsmooth, as well as smooth, derivatives of LT2 lines “cured” of prophage Fels 2. A host-range variant, ES18hf , overcomes the (partial) resistance conferred by lysogeny for Fels 2. Phage ES18 does not lysogeniae pro deletion mutants lacking the preferred P22 prophage integration site. Lysogeny for P22 confers im- munity to ES18 but ES18 lysogens are still sensitive to P22. Phage ES18 is a general transducing phage, effecting both complete and abortive transduction. Experiments to demonstrate transduction must take account of the very slow adsorption rate of this phage. The observed frequency of (complete) transduction of various loci by ES18 was lower, by a factor of 10-106, than the corresponding rates of transduction by phage P22. Lysogeny of the recipient for P22 increased the yield of transductants evoked by ES18 (though it lowered the yield of P22 transductants). For several pairs of linked loci the rates of cotransduction by ES18 and by P22 were about the same; this suggests that the chromosome fragments present in transducing particles of the two phages are of similar lengths. The activity of ES18 and ES18.hl on nonsmooth mutants makes them useful for the transductional mapping of rfa genes in S. typhimurium.

INTRODUCTION

Rough mutants of Salmonella typhimurium are resistant to the general transducing phage P22, probably because somatic lipo- polysaccharide side chains made up of more than one 0 repeating unit constitute the adsorption site for this phage. Nonsmooth mutants, except some that are leaky (Gemski and Stocker, 1967), are therefore unsus- ceptible to transduction by phage P22. Several other general transducing phages active on this species, including those of groups Al-A2, A3, and A4 of Boyd and Bidwell (1957), phage L (Bezdek and Amati,

1 This investigation was supported by Public Health Service Research Grant No. AI 07168 and Training Grant No. AI-82 both from the National Institute of Allergy and Infectious Diseases.

2 Present address: Barnes Hospital, St. Louis, Missouri 63110.

1967), and MG40 (Grabnar and Hartman, 1968), are probably all related to P22 and, like it, “smooth-specific”. For an investiga- tion of the genetics of rough mutants, we sought a general transducing phage active on both smooth and rough forms of S. typhi- murium. Phage ES& derived from typing phage 18 in the S. typhimurium phage-typing scheme of Callow (1959), is known to be active on both smooth and rough forms of a particular subline of S. typhimurium strain LT2 (R. G. Wilkinson, University of London Ph.D. Thesis, 1966). We here report experi- ments showing that this phage can effect general transduction, and described certain of its properties.

MATERIALS AND METHODS

Bacterial strains and phages. Bacterial strains differing from their wild-type parents

621

622 KU0 AND STOCKER

by only a single character are indicated by Media. The nutrient agar and broth used the name of the parent strain and the new were “Oxoid” blood agar base medium character, e.g., LT2 pyrEIW5. Multiply (CR255) and nutrient extract broth No. 2 marked strains, assigned strain numbers, (CM67). Davis defined medium with gly- are listed in Table 1. The phage termed ES18 cerol (0.2 %, v/v) in place of glucose was derives from a single-plaque isolate made by used in t,ransduction experiments. For selec- R. G. Wilkinson (University of London t,ion of hut+ t’ransductants, i.e., those able to Ph.D. Thesis, 1966) from a sample of t’yping use histidine as nitrogen source, the am- phage 18 (Callow, 1959) received from Dr. monium sulfate of the defined medium was E. S. Anderson of the Ent,eric Reference replaced by sodium sulfate and m-hi&dine, Laborat’ory, Central Public Health Labora- 0.05 %, was added as sole nitrogen source. tory, Colindale, London, N.W. 9. Phage P22 For experiments on transduction of motility wild-type was used for lysogenization, its a semisolid nutrient medium containing Clear-Plaque variant P22.C2 for testing phage sensitivity, and either P22 wild-type or it,s

0.3 % agar and 8 % gelatin in nutrient broth

integration-deficient variant P22.L4 (= was used.

d-4) (Smith and Levine, 1967; Rao, 1968) Phage methods. Phage ES18 was propa-

for transduction. Other phages used were gated by t,he soft-agar-layer method, with a

9NA, a smooth-specific phage active on LT2 phage inoculum of about 10’ PFU per 14 cm

strains even if lysogenic for P22 (P. Gemski, diameter nutrient agar plate. The super-

cited by Stocker, 1969), and P221.cS, active natant fraction of the material harvested on rough mutants of LT2 except those after overnight incubation at 36” was lysogenic for P22 (Subbaiah and Stocker, sterilized by Seitz or membrane filtration or 1964). by exposure to chloroform. Phage stock-

Strain No.”

SA624 SD14

SL :428

SL1025 SL1026

SL1027b SL1102

SL1547 SL2229 SL3501 SL3622 SR120 SU687

TABLE 1

MULTIPLY MARKED BACTERIAL STRAINS

Description

LT2 pyrEi cysEl705 LT2 metA trpB2 Hl -b H2-e,n,x

“cured of Fels 2” SR120 rjc-9%

= SR120 (Fels 2) = SD14 (Fels 2)

SD14 jlaA66 strAl20 x$-404 metE551 SL1027 rfaE543

LT7 cysES0 gal-851 LTS pyrE126 xyl-41Y rha-4Y9 rja-9% = SL1027 (ES18) = SL1027 (P22) LT2 “cured of Fels 2” LT2 trpA52 cysB12 pyrF1.46

Reference

Spicer and Datta (1959)

N. D. Zinder (pers. comm.), Naide et al. (1965)

R. G. Wilkinson and B. A. D. Stocker (unpublished)

Wilkinson and Stocker (1968) R. G. Wilkinson and B. A. D.

Stocker (unpublished) K. E. Sanderson (pers. comm.) P. Gemski (pers. comm.)

Zinder (1958) K. E. Sanderson (pers. comm.)

a Strains with prefixes SA or SU are from collection of K. E. Sanderson; with prefix SL or SD from collection of B. A. D. Stocker; with prefix SR from collection of N. D. Zinder.

b The previously undetected metE mutation in strain SL1027 (R. G. Wilkinson, University of London, Ph.D. Thesis, 1966; Gemski and Stocker, 1967) was discovered in the course of transduction experiments. It apparently arose in consequence of the mutagen exposure used to evoke the xyl mutation. The strain also seems to carry a modifier of strA, causing rapid, instead of slow, growth in the presence of high con- centrations of streptomycin.

'l'JLANS:UUC;'I‘lWN - - .__I -__l-l-ml BY PHAGE ES18 623

were usually titrated by the “drop-on-lawn” method (Gemski and Stocker, 1967).

Isolation and testing of lysogenic derivatives. Strains lysogenic for P22.cf or for ES18 were obtained by streaking out growth from areas of lysis produced by these phages and then testing repurified clones. Overnight broth cultures of the presumed lysogens were treated with chloroform and tested for pres- ence of phage active on appropriate indicator strains: the smooth strain SL1027, sensitive to both phages; a rough (heptose-negative) derivative, SL1102, unable to absorb P22 but sensitive to ES18; and a smooth, ES& lysogenic derivative, SL3501, sensitive to P22 but resistant to ESl8. A mixture of P22 and ES18 would have att’acked all three indicator strains. The clones were also tested for sensitivity to phages P22.&, ES18, 9NA and P221.&. The pattern of resistance con- ferred by lysogeny is discussed below, under Results.

the recipient strain and 0.1 ml of phage diluted so as to give the desired input ratio, then poured over a plate of selective medium.

Nonexacting transductants of cysB and cysE recipients were selected on nutrient agar, where they gave rise to papillae in the thin film of growth of the cystine-requiring parent strain (cf. Clowes, 1959). Colonies were counted after 1,2, or 3 days’ incubation at 36”.

RESULTS

Properties of Phage ES18

Transduction methods. Three methods were used. In the “broth” met.hod phage was added to an unshaken overnight 36” broth culture of the recipient strain, to a phage: bacterium ratio of about 12:l. After 15 min at room temperature, for phage P22, or 30 min for phage ES& the cells were collected by centrifugation, resuspended in saline and samples spread on selective medium. It was later found that in such experiments most of the phage ES18 remained unadsorbed, and was discarded in the supernatant. The other two methods were intended to avoid this loss.

Phage ES18 when tested on a fully sensi- tive LT2 derivative, such as strain SL1027, produced almost clear plaques, about 1 mm diameter, on surface-inoculated nutrient agar plates, and somewhat larger plaques in soft agar layers. The plaque counts obtained by the latter method were somewhat higher, usually by a factor of about 1.1. Only scanty growt’h was seen in areas of confluent lysis. Lysates of fully sensitive strains made by the soft-agar-layer method commonly had titers of lOlo to 1O’l PFU/ml, bhose made in broth had titers of about log.

In the “drop-on-lawn” method plates of selective medium were inoculated by flood- ing with a broth culture of the recipient strain and pipetting off excess culture. After the surface of the agar had dried, drops (volume 0.01 ml) of ES18 lysates, diluted m broth to titers between 1 and 5 X log PFU per milliliter, were deposited on the agar; drops of the required volume are delivered by 23-gauge needles (external diameter 0.64 mm)-we used “Luer Stub Adapters, 23 gauge,” from Clay-Adams, Inc., New York, attached to Luer-tip needle pipettes, from Bellco Glass, Inc., Vineland, New Jersey.

Phage ES18 was much more heat-labile than A phages, such as P22. Exposure in broth at 60” for 30 min reduced the titer by about half, and 5 min exposure at 70” caused more than 99 % loss of activity. Exposure to chloroform did not inact’ivate. Filtration through membranes of average pore diame- ter 0.45 nm resulted in a loss of no more than 20 % of PFU titer. There was no appreciable drop in titer on storage at 4”. Serum from a rabbit immunized with ES18 had a K value of 159/min for ES& but did not neutralize phage P22 even during 60 min contact at a final serum concentration of $$. Under the same conditions it likewise effected no de- tectable neutralization of phage P221&, which is morphologically similar to ES18 (R. G. Wilkinson, University of London Ph.D. Thesis, 1966).

In the third, “soft-agar-layer,” method, 3 ml of soft minimal agar at 45” was seeded with 0.1 ml of an overnight broth culture of

Phage ES18 was very poorly adsorbed when added to broth cultures of LT2 deriva- tives. In one experiment phage ES18 was added (to give a phage: bacterium ratio of 1: ZOO) to a broth culture (viable count 5.5 X lox/ml) of a smooth LT2 strain, LT2 pyrEIdS, in the presence of chloramphenicol (0.1 mg/ml) to prevent phage multiplication.

624 KU0 AND STOCKER

At intervals samples were diluted into chloroform-saturated broth, t’o destroy ir- reversibly adsorbed phage, then titrated on the indicator strain SL1027. After 50 min the phage titer had decreased by only about a half, corresponding to an adsorption rate value, K, of 2.8 X 10-ll/bacterium/min. In another experiment phage ES18 was added, at a low phage: bacterium ratio, to a young broth culture of the fully sensitive strain SL1027, with chloramphenicol, 0.1 mg/ml; at intervals samples were mixed with rabbit anti-ESlS serum, to neutralize unadsorbed phage, then titrated for content of infectious centers. After 40 min only 12 % of the input phage was recoverable as nonneutralizable infectious centers. The K value calculated from this experiment was 6 X 10~‘l/bat- terium/min. The nutrient broth used in these experiments contains sodium chloride, 0.5%. We did not try the effect of varying the cation content of the medium. However, the presence of sodium citrate, 1 %, in mini- mal agar caused only a slight reduction, less than 50%, in the efficiency of plating of phage ES18 and it is therefore unlikely that this phage requires divalent cations for ad- sorption or penetration.

UV-irradiation of phage ES18 caused ex- ponential inactivat’ion, down to 10-d survi-

val, at a rate about the same as for phage P22. Irradiated phage ES18 plated with very low efficiency on a UV-sensitive LT2 mutant known to be unable to effect host-cell re- activation of UV-irradiated phage P22. The results indicate that phage ES18 is about as susceptible to host-cell reactivation as phage P22.

Host Range of ES18 and Its Variant ES18.hl

Phage ES18 was originally grown on a smooth LT2 line, SD14 (Spicer and Datta, 1959) and tested on many sublines of SD14, including the smooth strain used as indica- tor, SL1027, and many nonsmooth mutants; the latter included mutants of classes rfb, rfc, and various subclasses of $a (see M5ike18; and Stocker, 1969, for different classes of non- smooth mutants). Phage ES18 plated with equal efficiency on all these strains (Table 2). It also plated with the same efficiency on the (supposedly) nonlysogenic S. typhimurium strain Ql of Boyd (Boyd and Bidwell, 1957). On many LT2 lines other than those derived from SD14 phage ES18 was almost inactive (efficiency of plating less than lo+). Strain SD14 is LT2 metA trpB2 given genes Hl-b and H2-e,n,x from S. abony by P22 transduction but nonlysogenic for P22

TABLE 2

EFFICIENCY OF PLATING OF ES18 AND ES18.hl ON Salmonella typhimurium LINES

E.o.~.~ Strain Description

ES18 ES18.hl

LT2 wild-typeb (i.e., lysogenic for Fels 2) <10-E 1 SR120 LT2 “cured” of Fels 2 1 1 SL1025 SR120 (Fels 2) lo-6 1 SD14 LT2 line “cured” of Fels 2 1 1 SL1026 SD14 (Fels 2) <lV6 1 SL1027 LT2 line “cured” of Fels 2 1 1 SL1102c SL1027 rfaE (heptose-negative rough) 1 1

S. typhimurium Ql 1 1 Ql (Fels 1) 1 1 Ql (Fels 2) <10-e 1

SL1547 LT7d cysE gal <10-G <lo-”

a Determined by “drop-on-lawn” method. In tests where e.o.p. was <1V6 or 1V6 phage of titer >108 PFU/ml produced “thinning” of lawn of bacterial growth.

b Many LT2 lines, smooth or rough, presumably not cured for Fels 2, behaved similarly. c Several nonsmooth mutants of other classes, derived from SL1027, behaved similarly. d Several other LT7 lines behaved similarly.

TRANSDUCTION BY PHAGE ES18 625

(Spicer and Datta, 1959). Strain SD14 was found by Wilkinson and Stocker (unpub- lished data) to be “cured” of a B phage (Boyd, 1950) carried by LT2 wild-type, apparently identical with the B phage miss- ing in Zinder’s strain SR120, described as “LT2 cured of a B phage” (Zinder, 1958). Wilkinson and Stocker (unpublished data) found this phage to be indist,inguishable by the criterion of lysogenic immunity from one termed B8 by Boyd (personal communica- tion). Yamamoto (1967) has shown that strain LT2 wild-type is lysogenic for t’wo phages, which he terms Fels 1 and Fels 2. Strains SD14 and SR120 carry Fels 1 but not Fels 2 (Yamamoto, personal communica- tion). We shall hereinafter use Yamamoto’s names for the temperate phages cerried by strain LT2. To test whether their lysogeny for Fels 2 accounted for the resistance of most LT2 lines to phage ES18 bhe latter phage was titrated by the drop method on a subline of SD14 and on SR120 (both being LT2 lines cured of Fels 2), on their deriva- t,ives relysogenized with Fels 2 (Wilkinson and Stocker, unpublished data) and on S. typhimurium strain Ql (Boyd and Bidwell, 1957) and its derivatives made lysogenic for Fels 1 or Fels 2, received from Dr. Yama- moto. The efficiency of plating of ES18 was about the same on the t,wo cured LT2 lines, on strain Ql and on Ql carrying Fels 1; but was less than lo+ on the relysogenized LT2 lines and on Ql carrying Fels 2 (Table 2). We conclude that the almost complete re- sistance of most LT2 lines to phage ES18 results from their lysogeny for Fels 2.

Drops of phage ES18 of titer 10s/ml or greater, applied to various LT2 lines pro- duced slight “thinning” of growth on nu- trient agar-presumably because the cells adsorbed and were killed by phage ES18 even though they did not support its mul- tiplication. Often a few small clear plaques were visible in the thinned growth. A phage line obtained from one such plaque, pro- duced by phage ES18 applied to SL3770 (a pyrf transductant of LT2 pyrEl.25) yielded an extended-host-range variant which we termed ESlS.hl. Phage ESlS.hl, even after propagation on a ‘(cured” LT2 line, pro- duced the same number of plaques on

“cured” LT2 or Ql as on the related lines carrying Fels 2; no other differences from ES18 were noticed. Presumably the ESlS.hl phage arose by a mut’ation in ES18 enabling it to overcome the resistance conferred by Fels 2-but the possibility that the change arose by recombinat’ion, rather than muta- tion, is not excluded.

A few other strains of S. typhimurium were tested for sensitivity to ES18 A deriva- tive of strain R27471 (MacPhee and Stocker, 1969) was fully sensitive bo ES18 and ESlS.hl. Strain LT7 was resistant to both ES18 and ES18.hl (efficiency of plating < lo- “), and an attempt to isolate a host-range variant active on LT7 was unsuccessful. Test’s on strains of a few other Salmonella species showed that some group B and some group D strains were sensitive to ESlS and ES18.hl. r\‘one of four E’. coli strains tested (strains K12, B and C and a strain of anti- genie character 0111: B4) were sensitive to either phage.

Lysogeny, Lysogenic Immunity, and Prophage Attachment Site

Streaking from the scanty growth in areas of lysis caused by ES18 or ES18.h1 yielded colonies most of which were lysogenic for the corresponding phage. To test for resistance conferred by lysogeny, the fully sensitive “LT2 cured” smooth indicator strain, SL1027, its derivatives lysogenic for ESlS or for P22, and its rough (heptose-negative) mutant SL1102, made lysogenic for ES18 or nonlysogenic, were tested for sensitivity to phages ES18 and ES18.h1, to the smooth- specific phages 1’22.~2 and 9NA, and to a rough-specific phage which does not attack rough strains lysogenic for P22, i.e., phage P221.c2 (Yamamoto, 1967) (Table 3). As expected, hosts carrying prophage ES18 were unaffected by phages ES18 and ESlS.hl. In the case of the smooth host lysogeny for ES18 also conferred (incomplete) resis- tance to the smooth-specific phage 9NA, but not t,o phage P22.cd. In the rough host carriage of ES18 conferred resistance not not only to ES18 but also to the rough- specific phage P221.c2. The smooth strain carrying P22 was resistant to ES18 and ESlS.h1, as well as to P22, but still sensitive

626 KU0 AND STOCKER

Description

TABLE 3

EFFECT OF LYSOGENY ON PHAGE SENSITIVITY -

Strain Action of phages”

S-specific R-specific SR NO.

P22 ’ cz 9NA P221 .cz ES18 ES18.1~1

LT2 smooth, “cured” of Feis 2

SR120, SD14, and + + - + + SL1027

“Cured” LT2 smooth re- SL1026 and SL1025 f + - -

lysogenieed’with Fe& 2 +

“Cured” LT2, smooth, SL3501 + - - - -

made ES18-lysogenic “Cured” LT2, smooth, SL3622 - + - - -

made P22-lysogenic “Cured” LT2, rough SL1102 - - + + + “Cured” LT2, rough, made SL3503 - - - - -

ESl%lysogenic

a Tested by “drop-on-lawn” method, with phages of titer lo* PFU/ml; + = clearing; f = thinning; - = ,no effect.

to 9NA. Thus, lysogeny for ES18 confers resistance to the smooth-specific phage 9NA and to the rough-specific phage P221; and lysogeny for P22 confers resistance to ES18 (and its variant ES18.U) and, as previously reported, to the rough-specific phage P221. As noted above (under Host Range), lyso- geny for Fels 2 confers incomplete resistance to ES18, but not to ESlS.hl; it did not con- fer immunity to any of the other phages t’ested. Presumably the resistances to certain heterologous phages of hosts made lysogenic for P22, ES18, or Fels 2 result from lysogenic immunity-but we have not excluded the possibility of lysogenic conversion, with an alteration of cell surface such that phage- adsorbing capacity is lost.

We attempted to make LT2 lysogenic for both P22 and ES18. Two lysogenic deriva- tives of SL1027 were used. Strain SL3622, already lysogenic for P22, was exposed to ES18, and strain SL3501, lysogenic for ES18, was exposed to P22 (wild-type). Reisolated clones were tested to determine what phage they released and for phage sensitivity (see Methods). No double lysogens were ob- tained. All of 12 clones from the P22-lyso- genie strain exposed to ES18 remained P22- lysogenic and nonlysogenic for ES18. In the reciprocal test, i.e., the ESlS-lysogenic strain exposed to P22, of 15 clones tested two re-

mained ES18-lysogenic only, 6 were cured of ES18 but nonlysogenic for P22 and 7 were P22-lysogenic but no longer released ES18. Thus, exposure to P22 usually caused loss of ES18 lysogeny, with or without lysogeniza- tion by P22.

The similarity of phage ES18 to phage P22 in certain properties suggested that phage ES18 might be integrated at the (preferred) prophage integration site for P22, near proA (Smith and Levine, 1965; Smith and Stocker, 1966; Young and Hartman, 1966). Mutants with deletions in t’he proAB region were therefore tested for susceptibility to lyso- genization by ES18, and, as a control, by P22. Three LT7 mutants in which the P22 attachment site is deleted, proAlO7, proAB47, and proABl26 (Smith and Levine, 1965), were as expected unsusceptible to lysogenization by P22; and none of them could be lysogenized by ES18. By contrast three LT2 delet’ion mutants in which the P22 site is unaffected, viz. proA15, proBS1, and proAB21 (Smith and Levine, 1965), were easily lysogenized by either P22 or ES18. These experiments were inconclusive in that the three deletion mutants unsusceptible to lysogenization by ES18 were all derivatives of strain LT7, the three susceptible to lyso- genization of strain LT2. We therefore tested derivatives of strain SL3613, which is our

TRANSDUCTION BY PHAGE ES18 627

designation for the purE66 mutant of LT7 proAB47, whose deletion includes the P22 attachment site. Strain SL3613, like its parent LT7 proAB47, was unsusceptible to lysogenization by P22 or ES18 So also was a (P22-sensitive) pur+ derivative of SL3613 obtained by transduction with P22.LJ. By contrast P22-lysogenic and ES18lysogenic forms were easily isolated from a similarly obtained pro+ derivative of SL3613. It thus appeared that mutants lacking the preferred P22 integration site were unsusceptible to lysogenization by ES18 Perhaps ES18 is integrated as prophage at the same site.

Phage P22, like other phages of group Al-A2, converts, determining production of 0 antigenic factor 1 (Stocker et al., 1960). The smooth indicator strain SL1027 and its derivatives lysogenic for P22 or ES18 were tested by slide-agglutination with an anti- 1,3,19 serum. Only the P22-lysogenic strain was agglutinated.

Transduction by ES18 and ES18.hl

The transducing ability of phage ES18 was originally tested by the “broth” method, using as recipients various stable or nearly stable auxotrophic mutants of strains LT2 and LT7, all more or less resistant to phage ES18 The ES18 stocks used had been grown on fully sensitive LT2 lines wild-type in respect of the nutritional character con- cerned. Transductants were obtained, at fre- quencies between 1 and 5 X lOus per plaque- forming unit, from LT2 derivatives carrying proC4, pyrEi and argEll6, and from LT7 derivatives carrying purE66, proAB47, proC90, proC91, proC110, and cysES0. The discovery that phage ES18 was very poorly adsorbed, at least under the conditions used in the “broth” transduction method, sug- gested that the observed low yield of trans- ductants might result from most of the transducing particles remaining unadsorbed and therefore being discarded during the washing procedure. The “drop-on-lawn” met,hod was therefore devised. In tests with SL1547 (= LT7 cy.sE gal) as recipient cys+ transductants were evoked by application of drops (volume 0.01 ml) of several ES18 lysates of cys+ donors, adjusted to titers between 1 and 5 X log PFU/ml; the average

yield of transductants was 5 X lo-’ per PFU compared with an average of 4.6 X lo+ in three experiments by the broth method, with input ratios of c. 15. Similar results were obtained with phage ES18.h1. With SU687 (= LT2 trpA cysB pyrF) as recipient the rates of transduction by phage ES18.h1 for trpA were 1.2 X 10~*/PFU by the broth method and 7.8 X lo-’ by the drop-on-lawn method; and for pyrF 4.5 X 10M8/PFU by the broth method, 1.7 X lo+ by the drop-on-lawn method. Thus for all comparisons the number of transductants obtained per input phage particle was be- tween X 30 and X 100 greater in the drop- on-lawn method than in the broth method.

Less than 5% of transductants obtained by the broth method were lysogenic for ES18, even when the phage: bacterium input ratio was 20: 1 or greater. By contrast about 30 % of transductants obtained by t’he drop- on-lawn method were lysogenic. We assume that the transducing particle is defective and unable to lysogenize, but that some trans- ductants become lysogenic by secondary infection. This would presumably be more likely in the drop-on-lawn procedure, where all the input phage remains available.

Nonsmooth strains, with various defects in lipopolysaccharide synthesis, were used both as donors and as recipients in transduc- tion with ES18 or ESlS.hl. Rates of trans- duction were not obviously different from experiments with smooth strains. In tests in which the donor strain carried the same mutation as the recipient (instead of its wild-type allele), either no or very few non- exacting colonies were obtained-as ex- pected. To test for abortive transduct’ion a mixture of a &A recipient, strain SL3622, with phage ES18 grown on a jZa+ donor strain was streaked on semisolid medium. Both swarms and trails were obtained. This indicates that phage ES18 effects both com- plete and abortive transduction of motility.

For several pairs of linked loci the rates of cotransduction by phage ES18, ranging from about 0.01 for pyrE with cysE t’o about 0.3 for rfaG with pyrE, were about the same as for cotransduction by phage P22, observed by us or recorded by others (Table 4). We observed about 0.3 cotransduction of galE

628 KU0 AND STOCKER

TABLE 4

COTRANSDUCTION OF PAIRS OF MARKERS BY PHAGES ES18 AND P22

Donor Allele Recipient

Selected Unselected Strain No. Genotype

Rate of cotransductiona by phage

ES18 or ES18.hl P22 or P22. L4

CySEf PYTE+ SA624 LT2 pyrE185 cysElYO9 0.01 (l/117) 0.016 hut 4

- LT2 rjb 0.2@ (17/60) 0.6d

pyrE+ rfa@ - LT2 pyrEi 0.31 (34/119) 0.35 @O/57)

PYLE+ rfaF/ - LT2 pyrEI 0.10 (16/156) 0.08 (7/92)

wrE+ rja+ SL22290 LT2 pyrE125 rja-9% 0.18 (11/60) 0.20 (5/24)

a Figures in parentheses are number of transductants with unselected donor allele over number of transductants tested.

b Data of Dr. K. E. Sanderson (personal communication). c In the ES18 experiments the donor was LT2 gaZEbO3 (strain LTZ-Ml of Fukasawa and Nikaido,

1961) given the ability to utilize histidine as nitrogen source by P22.LCq transduction of (part of) the hut region (Meiss et al., 1969) from strain 49-3H-7, an LT2 derivative received from Dr. W. J. Brill. The transduction data are pooled from experiments with several LT2 rfb strains as recipients, all gal+ and unable to utilize histidine (Meiss et al., 1969). Phage ES18.hl was used for transduction.

d Data from Meiss et al. (1969) and Brill and Magasanik (1969). B ES18 data pooled from experiments involving three pyr+ rjaG donors, SL1032 (rjaG@l), SL1062

(rfaG489) and SL1005 (rfaG572). The P22 data concern a “leaky” rja donor, SL3603 (rjaG6’56). For account of rfa alleles see Wilkinson and Stocker (1968), M%kel% and Stocker (1969), and T. Kuo (Ph.D. thesis, Stanford University, 1969).

f ES18 data pooled from experiments involving three pyr+ rfaF donors, SL1181 (rfaF611), SL1182 (rjaF537) and SL1183 (rjaF546). The P22 data concern a “leaky” rja donor, SL3600 (rjaF667).

0 This strain, received from Dr. P. Gemski, is a part-rough mutant, of phage-resistance pattern D-l (Stocker, 1969) and probably has a leaky defect at rfaJ or rfaK (Gemski and Stocker, 1967, P. Gemski, $ersonal communication).

with hut by phage ESlS, whereas a rate of about 0.6 is reported for phage P22 (Brill and Magasanik, 1969; Meiss et al., 1969)- but this difference may reflect differences in the alleles and met’hods used rather than a true difference in rates of cotransduction by the two phages. Phage ESlS, like phage P22 (Sanderson, 1967), could simultaneously transduce trpA+, cysB+, and pyrF+ to strain SU687. No persistent segregation of either selected or unselected transduced character was observed. The expression of mutant (and therefore presumably recessive) alleles co- transduced from t)he donor establishes that the usual final product of transduction in this system, as in other general transduction systems, results from gene substitution, not addition.

To test whether the transducing particles in the ES18 lysates had the external charac- ters of ESlS phage particles, a lysate of titer c. 1Ol2 PFU/ml was incubat>ed at 36” for 60 min with an equal volume of anti-ESlS serum diluted MO ; t’he mixture, and a con-

trol sample incubated without serum, were then titrated for content of plaque-forming units and for transducing activity (by the drop-on-lawn method) on a pyrEI recip- ient. The serum treatment reduced the plaque titer from 6.5 X 1011 to 5.9 X log/ ml, i.e., by about 99 %; the number of pyr+ transductants evoked by t.he serum-treated sample was 10, compared with 549 for the control sample-a reduction of about 98%.

E$ect of Lysogeny, Input Ratio, and CV Irradiation on Yield of Transcluctants

Ko or very few trp+ transductants were obtained when phage ES18 or ES18.M was applied to the fully sensitive LT2 derivative SL1027, by the drop-on-lawn method or by the soft-agar-layer method. The appearance of the drop areas suggested that this might be because transductants were killed by secondary infection m-ith normal phage par- ticles. LT2 lines cured of Fels 2 become resistant to ESlS and ESlS.hl if they are made lysogenic for either ES18 or P22, and

partly resistant to ES18 if made lysogenic for Fels 2. Such lysogenic derivatives were compared with their fully sensitive parents as transductional recipients. The fully sensi- tive strains used were SD14 (LT2 trpB metA, etc., cured of Fels 2) and its$a str xyl metE derivative, SL1027. The lysogenic derivatives tested were SD14 relysogenized with Fels 2 and derivatives of SL1027 lyso- genie for ES18 or P22; trp+ transductants were selected by the soft-agar-layer method using phages ES18 and ESlSM and, for comparison, P22, all at phage:bacterium input ratios of about 4: 1 (Table 5). The number of trpf transductants evoked by P22 was not, much affected by lysogeny of the recipient for Fels 2 or ESlS, but was con- siderably reduced when the recipient carried P22, as previously reported (Zinder, 1955). Making the recipient lysogenic for Fels 2 or ES18 had little effect on the very low yield of trp+ transductants evoked by ES18 or ES1S.M. By contrast making it lysogenic for P22 resulted in a great increase in the number of trp+ transductants obtained by exposure to either ES18 or ES1S.M. How- ever, even with the P22-lysogenic recipient the yields of ES18 and ES1S.M transduc- tant,s were considerably less than in the combination of nonlysogenic recipient and transduction by phage P22. In other experi- ments using the drop-on-lawn method lyso- geny of the recipient for P22 increased the yield of cys+ transductants evoked from SL1547 (= LT7 cysES0 gaZK) by ES18 or ESlSN, and of pyrf transductlant’s evoked

TRANSDUCTION BY PHAGE ES18 629

TABLE 5

NUMBERV OF trpB+ TRANSDUCTANTS EVOKED FROM “CURED” LT2 trpB metA RECIPIENTS AND THEIR DERIVATIVES LYSOGENIC FOR FELS 2, ES18, OR P22

from LT2 pyrEld5 by phage ESlS, even though in each of these combinations the recipient strain, not lysogenic for P22, ap- peared completely or almost completely resistant to the phage(s) used for transduc- tion. In the experiment summarized in Table 6 the effect of lysogeny for ES18 or P22 on frequency of trp+ transductants evoked from the fully sensitive strain SL1027 by ES18 at different input ratios was tested by the soft-agar-layer method. For all three recip- ients the yield increased with increased input ratio, though not proportionately. In this experiment lysogeny for P22 greatly in- creased, and lysogeny for ES18 moderately increased, the number of transductants ob- tained. The effect of phage: bact,erium input ratio was also tested by the drop-on-lawn method, by applying drops of ESlS, ESlSM and P22.L4 lysates, adjusted to contain between 1.5 X lOa and 2 X lOlo PFU/ml, to plates surface-inoculated with SL1547 (= LT7 cysE gal) or its derivat’ives made lyso- genie for ES18 or P22. For all three recip- ients, the numbers of cys+ colonies evoked by P22 increased consistently with increasing phage concentration, t,hough not! linearly; and was much less for the P22-lysogenic recipient t’han for the other two. In the case of the P22-lysogenic recipient increased con- centrations of ES18 and ESlSM evoked larger numbers of cys+ colonies. When the recipient was nonlysogenic or carried pro- phage ESlS, the numbers of transductant colonies evoked by ES18 or ES1S.M were less, and there was less effect of increased

Recipient DescriptiorG Input ratio

Transducing phage

ES18 ES18~hl P22

SD14 LT2 trpB, etc., “cured of Fels 2” 5 0 0 453 SL1026 SD14 lysogenized with Fels 2 3 3 1 484 SL1027 SD14 $a, etc., “cured of Fels 2” 4 2 0 346 SL3501 SL1027 lysogenixed with ES18 4 3 1 255 SL3622 SL1027 lysogenized with P22 4 36 27 85

a trp+ transductants were selected by the soft-agar-layer method. The phage:bacterium input ratio was about 4:l.

b For details of these strains see Table 1. They all carry the same trpB allele.

630 KU0 AND STOCKER

phage concentration. In this experiment, as in several others, the yield of cys+ transduc- tants evoked from the PM-lysogenic recip- ient by ES18h1, calculated per plaque- forming unit, was about the same as the yield of transductants evoked by P22 from the nonlysogenic and ES18lysogenic recipients, and the corresponding frequencies of ES18 transduction were lower by a factor of about one quarter.

In the experiment recorded in Table 7, the effect of UV irradiation on transducing efficacy of phage ES18 was tested by the drop-on-lawn method, with LT2 pyrEl25

TABLE 6

EFFECT OF IMPUT RATIO AND OF LYS~GENY OF RECIPIENT ON RATE OF TRANSDUCTION

OF trpB BY PHAGE ES18

Recipient No.” of transduc-

tants at input ratio

- Strain No. Description 0.5

SL1027 LT2 trpB, etc., “cured 0 of Fe!s 2”

SL3501 SL1027 (ES@ 2 SL3622 SL1027 (P22) 35

5 30 ~~

3 17

18 75 112 222

a Number of lrp+ transductant colonies obtained by soft-agar-layer method from inoculum of about 2 x 108 bacteria and ES18 to stated phage: bac- terium input ratio.

TABLE 7

EFFECT OF ULTRAVIOLET IRRADIATION OF ES18 ON RATE OF TR.4NSDUCTlON OF pyrE

Recipient

- I

Strain

LT2 pyrEi SL3826 SL3827

Description

(carries Fels 2) pyrEl25 (P22) pyrEi (ES18)

-

No. of trans- ductants”

NOD irradi-

ated

P2t%

102 263 287 419

54 63

-

a Number of pyr+ transductant colonies result- ing from application of four drops (vol. 0.01 ml each) of ES18 lysate on plates of defined medium surface-inoculated with stated recipient strain.

* Plaque-forming titer before and after irradia- tion 3.8 X 109/ml and 3.0 X lO*/ml, respectively.

and its ES18-lysogenic and P224ysogenic derivatives as recipients. The irradiation re- duced the plaque-forming titer by 92 %, but resulted in small increases (X 1.2 to X 2.6) in the number of transductant colonies ob- tained.

DISCUSSION

We investigated bacteriophage ES18 be- cause it was known to attack various non- smooth LT2 lines, so that if able to transduce it could be used for transductional mapping of rfa loci. It proved to be a general trans- ducing phage; the transduct’ional mapping of rfa loci by phage ES18 and its variant ESl8.M will be reported elsewhere. Phage ES18 was derived from typing phage 18 of the S. typhimurium phage-typing scheme of Callow (1959), itself believed to derive from a temperate phage carried by a lysogenic X. typhimurium strain. Temperate phages iso- lated from S. typhimurium fall into an A group, of heat-stable phages, and a B group, of heat-labile phages (Boyd, 1950; Boyd et al., 1951; Boyd and Bidwell, 1957). The type phages of all twelve subclasses of group A effect general transduction (B. A. D. Stocker and H. Ozeki, unpublished data) about as efficiently as phage P22, which itself falls in class Alb of Boyd and Bidwell (1957). All the A phages are serologically related and so may be presumed t,o be closely related phylogenetically. Attempts to de- monstrate transduction by B phages were not successful (Stocker, unpublished). Phage ES18 differs from the A phages by (a) its heat-lability; (b) lack of serological relat’ion- ship, as shown by failure of anti-ES18 serum to neutralize P22 (t’his paper) and of anti- P22 serum to neutralize ES18 (R. G. Wilkin- son, University of London Ph.D. Thesis, 1966) ; (c) morphology, phage ES18 having a long tail whereas phage P22, a typical A phage, has only a very short tail; (d) adsorp- t,ion specificity, phage ES18 being indifferent to the lipopolysaccharide character whereas P22 (and so far as is known the other A phages) is O-specific.

ES18 shows some surprising similarities to phage P22, however, which suggests that it may be somehow related to the A phages. Thus: (a) it did not lysogenize mutants de- leted for the preferred attachment site for phage P22; (b) lysogeny for ES18, as for

TRANSDUCTION BY PHAGE ES18 631

P22, conferred immunity to the rough- specific phage P221; (c) lysogeny for P22 conferred immunity to ES18 though ES18 did not protect against P22; (d) exposure to P22 often resulted in curing of lysogeny for ESlS; (e) both phages are general transduc- ing phages, effecting abortive and complete transduction and the cotransduction of various pairs of linked loci at similar rates, which suggest’s that the bacterial chromo- some fragments present in transducing par- ticles of the two phages are of about the same length. Dr. N. Yamamoto (personal communication) has observed that phage ES18 recombines with phage P22 at rather high frequency (10M3 to 10-d) despite their serological unrelatedness and he suggests that phage ES18 may have arisen in the same way as phage P221, that is by recom- bination of the genome of a group A phage with a group B prophage present in the pro- pagating strain (Yamamoto, 1967). Such an origin might account for the above-listed differences and similarities of P22 and ES18 if the group B parent were the source of genes determining the non-A characters (long tail, adsorption character, heat lability, and some features of the complex pattern of lysogenic immunity) and the group A parent the source of genes determining preferred integration site, some features of the lyso- genie immunity pattern and the ability to effect general transduction.

Transduction by phage ES18, so far as was investigated, seemed more or less similar to transduction by phage P22. However, the very slow adsorption of phage ES18 necessi- tated precautions, such as the “drop-on- lawn” or soft-agar-layer methods which we used, t’o permit continued adsorption of phage after plating. When the recipient strain was fully sensitive to phage ES18 (specifically an LT2 line cured of Fels 2) the yield of transductants was extremely low. We suspect, but have not proved, that this is because transductants are killed by secondary infection with normal ES18 par- ticles. Though we made no measurements the appearance of plaques and confluent lysis areas suggests that the lytic cycle, or at least killing, rather than lysogenization, is the usual outcome of single or multiple in- fection of fully sensitive strains with normal

ES18 phage particles. Lysogenization of such fully sensitive strains by phage P22 made them into good recipients for ES18 trans- duction-perhaps by making them immune to killing by ES18. Whatever the explanation the observation suggests that the superinfec- tion exclusion mechanism of P22-lysogenic cells (Rao, 1968) does not act on bacterial DNA injected by ES18 transducing parti- cles. Lysogenization of fully sensitive recip- ients by ES18 or Fels 2 was much less effec- tive in raising the rate of ES18 transduction, even though strains lysogenic for ES18 or Fels 2 are resistant to ES18 as judged by spot t,ests. Strains lysogenic for P22 (wild- type) are poor recipients in P22 trans- duction, it is believed because the bacterial DNA injected by P22 transducing particles is affected by the P22-prophage-determined superinfection exclusion mechanism (Rao, 1968). If prophage ES18 similarly deter- mines an ESlS-specific superinfection exclu- sion mechanism it might account, for the low transduction rates observed when the recip- ient was ESlS-lysogenic instead of P22- lysogenic. We do not know the explanation for the failure of lysogeny with Fels 2 to increase the yield of transductants from fully sensitive recipient st’rains. Even when the recipient was P22-lysogenic the yield of transductants obtained by use of ES18 was generally less (by a factor of 10-100) t’han the yield of transductants using P22 and a nonlysogenic recipient. We do not know whether the lower rate results from a lower proportion of transducing part,icles in ES18 lysates, from incomplete adsorption or from other causes. Phage ES18 effect’ed abortive as well as complete transduction, and the yield of complete transductants was some- what increased by UV-irradiation of the transducing lysate-presumably, as in t’he case of P22 transduction, because the irra- diation increased the probability of integra- tion of the transduced gene.

Phage ES18 and its extended-host-range variant ES18.M have proved useful in trans- duction using nonsmooth donor or non- smoot’h recipient strains, derived from strain LT2. Of a few other strains tested, all non- lysogenic for A phages, strain M7471 was sensitive to ES18 whereas strain LT7 was resistant to both ES18 and ES18.M. How-

632 KU0 AND STOCKER

ever, even strains resistant to ES18 (either because of lysogeny for an A or B phage or for unknown reasons) could be used success- fully as transductional recipients. The nature of the bacterial surface component to which phage ESlS is adsorbed remains unknown. The very slow rate of adsorption suggests that it may be only a minor component, at least in smooth bacteria.

ACKNOWLEDGMENTS

Tseng-tong Kuo held a Postdoctoral Fellow- ship from the C. F. Aaron Fellowship Fund, Stanford University School of Medicine.

We thank Drs. R. G. Wilkinson, W. J. Brill, N. Yamamoto, K. Sanderson, and P. Gemski for provision of strains and permission to cite un- published results.

This work is based on the Ph.D. dissertation (Stanford University, 1969) of Tseng-tong Kuo.

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