Specialized transduction of tryptophan markers in Escherichia coli K12 by bacteriophage ∅80

VIROLOGY

19,

475482 (1963)

Specialized

Transduction

Escherichia

of Tryptophan

Markers

coli K12 by Bacteriophage

in

480

AIZO MATSUSHIRO Department

of Parasitology,

The Research Institute for Microbial Osaka University, Osaka, Japan Accepted

December

Diseases,

3, 1962

A UV-inducible temperate phagerp80,capable of lysogenizing on E. coli K12, has been isolated. Some of its properties have been investigated. Prophage #SO is closely linked to the tru marker on the chromosome of strain K12. Specialized transducnegative cells tion of try+ genes from t,ryptophan positive (try’) to t~ptoph~ (try-) by #80 has been demonstrated. No markers except those in the try region have been transduced by this phage. The frequency of transduction resembles that of other transduction systems such as X. Some try+ transductants have been found to be heterogenotes. Heterogenotie cultures produce high frequency transducing (HFT) lysates, which contain both active phage rpX0and defective transducing phage $80 dt carrying try genes. Each $60 dt strain carries the set of genes of the tryptophan operon, replacing a part of the phage genome which includes the immunity-related locus.

The relationship between a prophage and the chromosome of its bacterial host is the central problem in lysogcny. One means of studying this relation is to investigate the origin of transducing phage from the prophage of lysogenic bacteria. The best-known system is that of galactose-transduction by phage X. This has been clarified by Morse, Lederberg and Lederberg (1956a, b) , Arber et al. (1957)) Campbell (1957)) and others. The present paper describes a temperate phage #SOwhich has been isolated by author (1961). Prophage &O is located in the general region, where other UV-inducible temperate phages are mapped (Jacob, 1955, 1957), and is closely linked to the tryptophan (try) loci on the K12 chromosome. This phage can specially transduce the try+ genesin E. coli K12. There is a close analogy between the gaZ-transducing system mediated by h and the try-transducing system mediated by phage $30. MATERIALS

AND METHODS

Strains. The bacterial strains used, E. coli K12 and its derivatives, were from the labo-

ratories of Drs. J. Lederberg and C. Yanofsky and were obtained from Drs. Y. Hirota and T. Yura. Strain BSO was one of 564 strains of E. coli that were tested for lysogeny using K12 as indicator strain. Strain B80 is prototrophic, streptomycin sensitive, and insensitive to h and to the T-series phages. Following ultra~7iolet (UV) irradiation, BSO bacteria lyse, liberating phages 480 and (~81 (Nakamura, Matsushiro, and Ozeki, to be published). A nonlysogenic derivative of strain B80, designated as B80 (ly) -, was obtained as a survivor of TJV treatment. Media and Procedures. Bacteria were kept on nutrient agar slants and transferred to liquid media for the experimental cultures, which were grown on a rotary shaker at 37°C. The Davis-Mingioli (1950) minimal medium and L broth (Lennox, 1955) were used. EMB agar and BTB peptone medium were used for sugar fermentation tests. Adsorption of phage and dilutions were done in a medium containing Tris buffer (pH 7.4), 6 x 1O-3 M; CaCl’ , 1O-4 i%f; NaCl, 0.4%; gelatin, 0.005%. For bacterial mating experiments, phage-

475

476

MATSUSHIRO

resistant bacterial cultures were used to avoid reinfection by spontaneously released phage. Equal volumes of growing cultures of F+ (or Hfr) and F- bacteria were mixed, kept without vigorous shaking, and then centrifuged. The cells were resuspended in saline and spread on minimal agar. The recombinants were purified by restreaking and tested. To test for the ability to produce phage, a loopful of bacterial culture was spotted on agar seeded with a sensitive indicator and UV treated. UV irradiations were done with a “Matsuda” germicidal lamp, 15 watt, at SO-cm distance. Immunity was tested by spotting loopfuls of phage on lawns of bacterial cells. Phage lysates were produced by a 40 seconds’ UV treatment of lysogenic bacteria suspended in saline followed by incubation in aerated L-broth until clearing (about 2 hours). The lysates, whether from the lytic growth or induced growth, were assayed by agar layer method. The procedure used for transduction experiments followed that of Lennox (1955). The standard assay for defective +SOdt was similar to that of Kaiser and Hogness (1960) for Adg. A sample of +SOdt to be assayed was diluted and mixed with an equal volume of exponentially growing cells of try- (480) + in suspension medium at 2 x 10” cells per milliliter. After adsorption for 20 minutes at 37”C, 3 ml of soft minimal agar was added and the mixture was poured onto minimal agar plates. After 72 hours of incubation at 37”C, the plates were counted for try+ transductants. RESULTS

Properties

of Phage 480

Phage 480 forms plaques on E. coli strains BSO(ly) -) C, K12 and on Shigella dysenteriae strain SH. Induced or lytic lysates grown on K12 give higher titers on K12 and C than on BSO(ly) - and vice versa (host-induced modification). Lysogenic derivatives of K12 are readily obtained. The K12-480 system was used in all experiments described in this report. All K12 mutants resistant to phage Tl, whether of the Tl-T5’ type or of the Tl’-

try types (Yanofsky and Crawford, 1959), are also resistant to $80. Conversely all mutants resistant to $80 are also resistant to Tl. This finding indicates that the cell receptors for Tl and $80 are either identical or closely overlapping. Preliminary search for a serological relation between h and 480 was negative. Location of the Prophage $80 on the Kl2 Chromosome Crosses between suitably marked strains of F+ or Hfr (Hayes) and F- bacteria were done to establish the location of prophage $80. A cross between F+ Zac+ x F- Zac(@O) + indicated that 480 prophage was located in the region where other inducible prophages are located (Jacob and Wollman, 1957). Hfr (Hayes) x F- crosses are illustrated in Table 1. Crosses 1 and 2, with selection for gal+, show that $80 is only weakly linked to gal, much less so than h (9% vs. 76%). Cross 3, again with gal+ selection, shows that all (28/28) the gal+ recombinant,s that have become sensitive to 480, like the Hfr parent, have also received the try+ marker from the Hfr. From these results, it is concluded that the site of prophage $80 is very close to the try cluster of gene loci. Lennox (1955) and Jacob (1955) have shown that closely linked markers can be co-transduced by phages of the PI group. Acceptor try- bacteria lysogenie for 480 and resistant to $80 were infected with a preparation of phage Plkc grown on nonlysogenic anthbacteria. Try+ transductants were selected on minimal media supplemented by anthranilic acid (25 pg/ml) . The transductant colonies were purified by restreaking and were replicated as follows: (1) on +80-sensitive indicators to test for lysogeny; the agar medium was not supplemented with Ca++ ion, in order to prevent growth of Plkc; (2) on minimal agar to demonstrate the joint transfer of the anth- gene and the try+ gene. The results of these experiments are listed in Table 2. Taken together with Yanofsky and Lennox’s results (1959) they indicate that, the linkage order is as follows: prophage 480, tryptophan synthetase A gene and B gene,. . anth, cys B.

TRANSDUCTION

477

OF TRYPTOPHAN MARKERS

TABLE 1 CROSS 1AND 2”: THE DEGREE OF LINKED TRANSFER

tion; this is of interest because of the finding of Yanofsky and Lennox (1959) that this cys marker can be co-transduced with OF NONLYSOGENY WITH gal+ MARKER IN THE CROSS HAYES Hfr (Ig)-B1- X F-W3102(ly)+gaZ~V try by phage Plkc. , The lysates from induced try+ ($80) + gal+ recomstrains are considered as low frequency binant, 7’ Donor X recipient Cross transducing (LFT) lysates. Table 4 shows data on the frequency of try+ transductants (lY)+ (b-__ obtained with LFT lysates on various recipients. The frequency of the appearance of 24 76 X 3102(x)+ 1 Hfr(ly)(g&+B,-X’) (g&-X’) transductants is of the order of 10V7 per 9 Hfr(ly)- X 3102(480)+ 91 2 plaque-forming phage unit or higher with (gal,+B,-4801) (gaZz-480’) sensitive or lysogenic try- recipients. The frequency is lower for markers on the anth side, as may be expected from the linkage CROSS 3”: THE DESREE OF LINKED TRANSFER OF order: $80, try ,anth. (ly)~m AND try+ WITH gal+ MARKER IN THE CROSS Lysates produced by lytic growth of OF HAYES HFr(ly)-gal+try+SsB1- X F-W4627 phage $80 do not transduce the try markers. (480)+gal-try-SIBI+ This finding is analogous to that of Morse gal+ Sr BI+ recombinants et al. (1956a) for A and the ga2 genes. In Crossing these tests, to avoid any carry-over of transTotal over value ducing phage from previously induced ly(480)(48@+ (%I sates, the phage derived from an induced try+ trytry+ 1 trytry- ($80) f strain is used as an inoculum to ____ prepare a lytic-cycle phage on nonlysogenic 0 0 284 28 try+ cells. E%, 0% 91% (9%) CL Phage 481, originally carried by BBO a The crosses are carried out under usual bacteria together with 480, does not perform conditions and they are plated on the minimal transduction in K12 bacteria, but when galactose agar supplemented with streptomycin. present in a lysate affects the transducing effectiveness of 480 (Nakamura, MatsuSpecialized Transduction of try Markers by shiro, and Ozeki, to be published). Phage $30. The close proximity of the $80 prophage to the try loci suggested the possibility of a relation like that of h to the gal loci. This expectation was borne out by the transduction experiments illustrated in Table 3. The lysates used were produced by induction of +80-lysogenic bacteria. It is clear from the results shown in Table 3 that various auxotrophs with mutations in the try gene cluster, including T24,l T16, and T3 (or other anth- markers), could be made try+ However, no markers by transduction. other than try were transduced by phage $80. Note that a cys- mutant (%Cys B4) could not be made cys+ by $80 transduc1 T24: defect in the step of indole glycerol phosphate synthesis. T16: defect in the step of anthranilic ribulotide synthesis. T3: defect in anthranilic acid synthesis.

TABLE 2 THE CO-TRANSDUCTION OF try+ AND &SO)MARKERS WITH PHAGE Plkc” Genetic constitution of try+ transductants

anth+(480)+ anthf(480)ad-(480)+ ad-(480)-

7 6 42 53

QDonor strain: try+,anth-(480)(adwas T3 from Dr. C. Yanofsky). Acceptor strain: try-, anth+(@O)+ (try- was W3623,A mutant, from Dr. J. Lederberg). Acceptor try-anth+(480)+ bacteria are infected with the transducing phage Plkc grown on donor try+anth-(480)bacteria. Transductants were selected on minimal medium supplemented with anthranilic acid.

MATSUSEIIBO TABLE

3

TABLE

TRANSDUCTXON OF VARIOUS &IARKER~ WITH &AGE r&8@

Marker

try

B4 B8 a B-mutant iad A2 All A23 an A-mutant T24 T16 anth T3 3977 --_I

Strain used

4

FREQUENCY OF LFT TRANSDUCTION AND HETEROGENOTES AMONG TRANSDUCTANTS

Transduction

B4(&30)+S~ B8 (#8o)+sr W4627(#~80)+@

i+ +

AZ(~)+Sr A11(#80)‘Sr A23@8O)+S’

+ + +

W3623($~80)+8~ T24(+80)+Sr TlS(l$8o)+S T3(+80)+Sr 3977(~)+sr

+ + + t +

Recipient 3623(Iy)~23(~~)~ 3623(+81)+ 3623(+80)+(+81)*

Frequenty of transductiona 2.3 3.0 3.5 1.1

X lo-7 x 10-7 x 10-7 x lo-’

Heterogenotes among transductantsb 3140 4/40 4/37 9137

a Transduction uer olauue-forming unit. b Heterogenotes were identified by their ability to give rise t,o HFT-lysates.

Similar findings were made with many of the LFT-try+ transductants produced with a cys-mutant phage 480. All try+ transductant colonies cys-pro-(&o)+s~ Pro from an F- try- gal- recipient were grown Y70(+8O)+S’ thr Y70(#80)+Sr in spots on master plates of minimal glucose leu thiamine Y7o(~o)~sr agar and were replicated onto plates of miniHfrC(#80)+ mal-galactose agar seeded with F- trymeth W3637(480) +Sr gal+ bacteria as indicators. Neither the doarg-methnor bact,eria nor the indicator could grow on arg-methw these plates. After replication the plates W4801 purine were treated with UV to induce lysis. After thyt~~nz~~e 48 hours’ incubation, areas of growth were w3104 gal gal4 observed on t,he replica plates, corresponding Y70(+8O)+S’ gal6 to spots containing heterogenotes on the W4627 (#~80)‘Sr gal6 master plate. Representative results shown W4627(+80)+8’ lac in Table 4 reveal that IO-20% of the try+ w1027 transduetan~ are heterogenotes. Table 5 Y70{~80)+Sf shows that the transducing titer of HFTW4627(#80)-%’ mal W4627(+80)+Sr lysate from these heterogenotes, assayed on XYl W4627(+80)+Sr arab lysogenic try- recipients, is around 109, while the plaque-forming titer is about 10 @Many of these experiments were done using a times higher. The absolute titer of $30~8 lysate of @O which also contained phage#8X. pnrticles is probably higher since the transduction efficiency is probably fess than 1 Transduction Heterogenotes for Ehe try (Arber, 1958j . Locus Most try+ heterogenot.esare unstable and In transduction of gal markers with phage segregate try- derivatives. Sometimes, howh, most transductants are heterogenotes for ever, they give rise to st,able try+ bacteria, the ~a1 loci (Morse et al., 1956a). They in which try+ genes are probably incorpocarry one set of gal genesin a defective prophage hdg (Arber et al., 1957). The LFT- rated into the host genome. Try- segreheterogenotes have two distinctive prop- gants can be detected and counted by reperties : (1) they yield high frequency lica plating. The probability of segregation transducing (HFT) lysates; and (2) they per bacterial division is more frequent than that of the h-gal+ heterogenotes (Morse et are unstable and regularly throw off galaE.:1956a). segregants. cysB

cys4(+8O)+S’ cys-pro-(+80)+Sr

-

TRANSDUCTION

OF TRYPTOPHAN

479

MARKERS

TABLE 5 THE PLAQUE-FORMING TITER (P) AND THE TRANSDUCING TITER (2’) OF VARIOUS HFT Heterogenotic strain

endogenote exogenote

LYSATE~

T per ml

P per mlQ

ratio T/P

Active:

HO

A-Bfanthf A+B+anth+

0.77 x 109

9.84 x 10’0

l/127

Active:

Hl (5-2)

A-B+anth+ A+B+anthde’

1.4 x 109

8.0

x 10’0

l/57

Active: Hl (5-2)

A+B-anth+ A+B+anthd”r

3.0 x 109

8.5 x 10’0

l/28

Defective:

A-B+anth+ A+B+anthde’

2.3 x 103


A-B+anth+ A+B+a.r.de’anthder

1.5 x 109

3.1 x 10’0

Hl

Active: H2

>2.3 X 10s

l/20

a In this case, this value indicates the sum of both 480 and 481 particles, not being assayed separately. As the transducing phages always derive from 480, the true ratio T/P might be higher.

The #?Odt Phage a. Defectiveness of #IO&. The try+ heterogenotes obtained by transduction of lysogenic try- recipients yield, after UV induction, HFT lysates containing two types of particles, normal $30 particles and transducing particles designated as +80dt. When +80dt infects lysogenic recipients carrying an active $30 prophage or when it infects sensitive bacteria with simultaneous infection by active phage 480 as helper, then lysogenic heterogenotes are produced, which are fully immune and again produce HFT-lysate after UV induction. The @Odt from certain heterogenotes can be detected by transduction on lysogenic try- recipients or nonlysogenic recipients simultaneously infected by active helper phage, but not on sensitive try- recipients (see Fig. 1). Apparently this type of @Odt is defective because it cannot lysogenize readily without the existence of active helper $80. Another type of transducing particles can instead transduce both lysogenic and sensitive try- bacteria without helper phage. Many transductants from the sensitive trycells infected with a single particle of this type give rise to defective heterogenotes,

FIG. 1. Linear relationship between the amount of HFT lysate and the number of transductants assayed on try-(~$30)~ (filled circles) and try-(1~)~ (open circles). The lysate 5-2 contained originally 7.7 X 10’ plaque units per milliliter and 1 X 10’ transducing units per milliliter, and was suitably diluted for plating.

which often appear negative in an immunity test and cannot produce any normal phage particles. These defective heterogenotes, however, can produce HFT lysates
480 B

MATSUSIZIRO

(0)

2 3.0Q

? E (D 0 -5.0 $ - 4.0 .$ - 3.0 .p - 2.0 2 -1.0 g

X

83 2.0.c0, .c ii ,o LO2 a 01 H 60

65

70

75

Drop number

Drop number

FIG. 2. Density gradient (preparative) centrifugation of HFT Jysates. IWed circles and open circles indicate transducing and plaque-forming phages, respectively. It is shown that transducing phages are lighter in lysate 5-2 (2a) and heavier in lysate 23-9 (2b) than plaque-forming phages.

used rout~neiy for the assay of @Odt because they are suitable for both types of transducing particles. b. Density gradient centrifugation of @ldt. Weigle et al. (1959) showed that the transducing particles kdg generally differ from those of h in buoyant density as measured in a CsCl density-gradient. Similar experiments were done with LFT- and HFT4~80 lysates. The lysates were purified by differential centrifugation and the phage were resuspended in a solution of C&l with a final density of 1.480 + 0.005 g cm-3 (30°C). Lusteroid tubes were filled with 3 ml of the phage suspension and placed in a swinging bucket rotor (SW 39) of a Spinco preparative centrifuge. After centrifugation for 16 hours at 23,100 rpm, the bottoms of the tubes were pierced with a fine needle. Successive droplets were collected in sepa-

rate tubes containing 1.0 ml of suspension medium. Each tube was assayed for plaqueforining par~ieIes and for transducing particles. LFT lysates obtained by the induction of lysogenic strains carrying (~80 prophage, contain a main band of plaque-forming particles and a broader band of transducing particles extending especially far toward the lower density side. Each HFT lysate prepared from an independently derived heterogenote forms two distinct bands in CsCl gradient,. For example, in HFT-lysate no. 5-2, the transducing particles @Odt are less dense than the active (~80 (Fig. 2a). This is the most frequent case. With another HFT lysate obtained from heterogenote no. 23-9 derived from the transducing phage on the high density portion of an LFT lysate, the transducing particles #80dt are denser than the normal 480 (see Fig. 2b). Each transducing phage +BOdt of independent origin transmits its characteristic density through repeated lysogenization cycles. This situation resembles that observed with hdg (Weigle et al., 1959; Wcigie, 19611. Genetic Markers

Carried

by #8Odt

The density differences among transducing phage +80dt may be due to differences in DNA content per particle, arising from the replacement of a portion of the phage genome by a fragment of the bacterial genome containing the try markers. As shown in Table 6, less than half of the isolates of $&Odt are of the HO type, and contain all the try-anth genes. Another group, the Hl type, contains most of the try genes but Iacks anth. Another type (H2) contains only some of the try genes. The (p80dt phage of lysate 5-2 used in the experiment of Fig. 2a is of type Hl; the phage in lysate 23-9, illustrated in Fig. 2b, is of type HO. This situation, in which ~~erent #80dt isolates carry different portions of a set of bacterial genes, has no analogy in the cases of hdg and of Pldl, in which all transducing phage types appear to contain the entire gal and Zac operon, respectively (Morse et aE., 195612;Cohn et al., 1960; Luria et al’,, 1960).

TRANSDUCTION

OF TRYPTOPHAN

MARKERS

481

TABLE 6 LINKAGE Map OF THE TRYPTOPHAN REGION AND FREQUENCY OF VARIOUS TYPES OF @Ml Shikimic-5-p

3

CYS 4

unth

Anthranilic acid

+

U.T.

Anthranilic ribulotide

3

Swine Indole gly- --+ Tryptophan cerol-phosphate

I.G.P.

B

A

Length of try region carried in each type of @Odt

Prophage 480 Type”

t

t t I

I I -

HO Hl H2 H3 Total examined

Frequency 31 32 14 0 77

a The $30dt designated as HO type carries all the structural genes of the try region. The Hl type lacks the terminal anth locus. The H2 type lacks the anth as well as he or. cistron. DISCUSSION

Zinder and Lederberg (1952) first noted the phenomenon of phage-mediated transduction in Salmonella typhimurium. These workers found that a variety of genetic characters of the host bacteria could be transferred by bacteriophage particles from donor cell to recipient. Such transduction has been referred to as generalized transduction. Later Morse et al. (1956a, b) described another type of transduction, called specialized transduction, involving the E. coli-A phage system. The transduction of try markers by phage 480 described here is apparently a new instance of specialized transduction and has many analogies with the case of gal transduction by phage h. In both cases, transducing lysates can be obtained only from induced lysogenic bacteria, not from lytic cycles of growth. The transductants are at least in part heterogenotes, unstable for the try+ marker, and yield HFT lysates containing a high proportion of transducing particles. In both cases of hdg and @Odt, independently isolated transducing phage differ in their density, and hence presumably in the amount of their DNA contents, some being heavier, others being lighter than normal active phage (Weigle et al., 1959). This situation seems to reflect the heterogeneity of

genetic constitution among independently isolated transducing phage strains. In the case of Xdg, it appears that a segment, of the phage genome has actually been replaced by a segment of the bacterial genome (Arber et al., 1957; Arber, 1958). The segments of the phage genome that are eliminated in the hdg strains always include the, h marker (dg region) but are variable in length. The try-transducing particles, QOdt, have not yet been studied in as great detail as the particles of hdg. The segment of $30 genome presumably replaced by the bacterial try genes has not yet been identified. The bacterial try genes contained in the +80dt always include the A and B genes, but the markers on the anth side are often missing. Lacking an essential part of the phage genome as the result of this incorporation of bacterial genetic materials, the phage becomes defective. Phage xdg can, with low probability, lysogenize a sensitive bacterium and confer on its specific immunity. However, its prophage cannot enter the vegetative cycle. From the fact that the relative frequency of stable transductions by LFT-x was higher than that with HFT-A, Campbell (1958) concluded that LFT-X must contain some kind of gal-transducing particle other than the type found in HFT !ysates.

482

MATSUSHIRO

In another system of transduction by defecaspects of lysogeny. Sump. Chem. Basis Heredity Baltimore 1957, Johns Hopkins Univ. McCollumtive phage particles, that of phage Pldl Pratt Inst. &‘ontrih. 153, 46&498. (Luria et nl., 1960), the transducing particles show various degrees of defectiveness. KAISER, A. D., and HOTNESS,D. (1960). The t.ransformation of Escherichia coli with deosyribonuThe +80-tryptophan transduction system cleic acid isolated from bacteriophage ~dg. J. also shows diversity in defectiveness among Mol. Biol. 2,392-415. the transducing phage strains. Some $80dt LENNOX, E. S. (1955). Transduction of linked strains can fysogenize without helper active genetic characters of the host by bacteriophage phage, others cannot. The defective +8Odt PI. Virology 1,190-206. seemsto lose a portion of the characteristic LURIA, S. E., ADAMS,J. X., and TINa, R. C. (1960). immunity-related loci. Transduction of lactose-utilizing ability among I wish to thank the following for their help during this investigation: Dr. C. Yanofsky, Dr. I. Crawford, and Drs. Esther and Joshua Lederberg for supplying the various genetically marked strains; Drs. T. Yura, H. Ozeki, and Y. Hirota for their manp valuable discussions and for their sound advice throughout this project; Dr. S. E. Luria for enlightened help in the preparation of this paper. The author also wishes to express his grat,itude to Drs. J. Tomizawa and H. Ozeki for the opportunity of working for some weeks in their laboratory. The author is deeplY indebted to Miss S. Kida, Mr. J. Ito, and Mr. K. Sato for their able assistance. This work was supported in part by a subsidy from the Asahi Press. REFERENCES

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