JOURNAL OF VIROLOGY, Oct. 1975, p. 854-858 Copyright 01975 American Society for Microbiology

Vol. 16, No. 4 Printed in U.S.A.

Characterization of Transduction by Bacteriophage Ti: Time of Production and Density of Transducing Particles KARIN J. KYLBERG, MARY M. BENDIG, AND HENRY DREXLER* Department of Microbiology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27103 Received for publication 17 April 1975

The transducing activity of two different kinds of premature lysates of Ti-infected cells have been compared to normal lysates. The results show that Ti-transducing particles are made early in the maturation period. The average density of Ti-transducing particles is slightly greater than the density of plaque-forming Ti.

The virulent coliphage Ti destroys the chromosome of its bacterial host and utilizes the DNA breakdown products in the formation of progeny Ti (11; D. Figurski and J. R. Christensen, personal communication). Phage Ti is able to package undegraded bacterial DNA as demonstrated by its ability to transduce a variety of markers (4). Up to the present, Ti has been shown to be capable of transducing 14 distinct bacterial cistrons (4, 7, 8; M. M. Bendig, M.S. thesis, Wake Forest University, WinstonSalem, N. C., 1974). Presumably, Ti is a generalized transducing phage which can transduce any bacterial marker. In addition, Ti is able to transduce PFU of the phages X and Mu from appropriate lysogenic donors to sensitive recipients (H. Drexler and J. R. Christensen, unpublished data; Bendig, M.S. thesis, Wake Forest University, 1974). Experiments were performed to determine whether the destruction of the bacterial chromosome by Ti placed any restrictions on the length of time that bacterial DNA is available for packaging. In addition, the density of transducing particles was studied to determine whether varying amounts of bacterial DNA were present in transducing particles. The results indicate that transducing particles are made early in the maturation period. The density of transducing particles is slightly greater than plaque-forming T1; therefore, the amount of DNA per particle is approximately the same for all markers tested.

Xrex508 (8), and Xrex... (1) contain mutations in the rex gene of phage X. Xrex . and Xrex3J,, were kindly provided by G. Gussin and Xrex.,. by J. R. Christensen. Bacterial stains. All bacterial strains are varieties of Escherichia coli. The donor Su+ (suppresses am mutations) strain is KB-3 and is StrR (streptomycin resistant) and otherwise prototrophic for all markers of interest here. The Su- (does not suppress am mutations) recipient strain is W3350 and is StrR, Gal-, and Lac (sensitive to streptomycin and unable to ferment galactose or lactose, respectively). Varients to W3350 which were utilized include those which were Arg-, Bio-, Pro-, or Trp- (unable to synthesize arginine, biotin, proline, or tryptophan, respectively). Media. Cells for transduction experiments and phage assays were routinely grown in L broth. Cells and phage were assayed on L agar. Saltless nutrient broth was utilized for Ti adsorption; in addition, incubation of Ti-infected cells in saltless nutrient broth results in a prolonged latent period as compared to growth in L broth. Transductants were scored on L agar (for streptomycin resistance), on eosinmethylene blue agar with appropriate sugar added (galactose or lactose), or on a defined synthetic medium containing glucose and agar. Lysates. Artificial, premature lysates (APLs) of Ti-infected cells in saltless nutrient broth were obtained by treating cells with EDTA, lysozyme, and sodium dodecyl sulfate according to the method of Botstein et al. (2). An APL obtained in this fashion was always paired with a normal lysate (NL) obtained from the same batch of infected cells. Natural, premature lysates (NPLs) of Ti-infected cells were obtained by infection of X-lysogenic donors. It has been shown (3) that infection of X-lysogenic hosts by Ti leads to premature lysis of the cells compared to lysis of either nonlysogenic cells or cells lysogenic for Xrex. Therefore, in most instances the effects of natural premature lysis can be evaluated through a comparison of results obtained with lysates obtained from either nonlysogens or Xrex lysogens. Gradients. Three milliliters of stock Tlam was mixed with 9 ml of CsCl solution (0.84 g of CsCl/ml of 1.2% trizma base). The mixture was centrifuged on a

MATERIALS AND METHODS A complete description of the materials, strains, terms, and methods can be found elsewhere (4, 5, 8). Except for new items, only the briefest description is given here. Phage strains. Phage Tlam contains the am (amber) mutations 5 and 11 (4). Phages Xrex., 854

TRANSDUCTION BY Ti

VOL. 16, 1975

Beckman model L centrifuge at 100,000 x g at 4 C for 16 to 18 h in a 50ti head. Three-drop fractions were collected by puncturing the bottom of the centrifuge tube.

RESULTS If transducing and plaque-forming particles are made with equal probability throughout the maturation period, the ratio of transducing to plaque-forming particles (i.e., the efficiency of transduction [EOT ]) should be constant throughout the maturation period. Early production of transducing particles should lead to an EOT which is greater in the early, rather than in the later, part of the maturation period. Finally, late production of transducing particles should lead to an increase in the EOT toward the end of the maturation period. APLs. Su+ cells were infected with Tlam at a multiplicity of about 4. Beginning at 25 min after infection samples were taken at 5-min intervals and lysed artificially (as described in Materials and Methods). Under our conditions (incubation in saltless nutrient broth) the latent period was about 40 min long. The results (not presented) showed that 50 to 90% of the transducing particles were produced by the time 25% of plaque-forming Ti had been made. Therefore, only the earliest APLs (those made at 25 min) have been used to compile the APL data (Table 1). The results for three APL/NL paired lysates showed (Table 1) that the EOT of any APL, for all the markers tested, was always higher than the EOT of the paired NL. This can be seen most easily by observing the ratio of the mean EOT of the APLs to the mean EOT of the NLs (last column, Table 1). A Student's t test for paired samples was performed on the individual experiments; for all markers tested the value of P was less than 0.05. Therefore, the average number of transductants per PFU is significantly greater in the premature lysates. NPLs. The EOT of lysates made on X-lysogenic donors was compared to lysates made on both nonlysogenic and Xrex-lysogenic donors. It was found (Table 2) that the EOT of each of the four markers tested was two to four times higher for Tlam -Su+(A) (i.e., Tlam grown in Su+ cells lysogenic for X) lysates than Tlam -Su+ or Tlam*Su+(Xrex) lysates. The EOTs for Tlam*Su+ and Tlam*Su+(Xrex) lysates were very similar. (The EOTs for all the lysates made on Xrex lysogens were so similar that they have been averaged; however, at least two different types of rex mutants were used to test each marker). It was also found (data not shown) that the

855

TABLE 1. Comparison of Tiam transduction by pairs of APLs and NLsa Marker

Lac+

Pro+

StrR

Trp+

Ga1+

Bio+

Lysate no.

APL-1 NL-1 APL-2 NL-2 APL-3 NL-3 APL-1 NL-1 APL-2 NL-2 APL-3 NL-3 APL-1 NL-1 APL-2 NL-2 APL-3 NL-3 APL-1 NL-1 APL-2 NL-2 APL-3 NL-3 APL-1 NL-1 APL-2 NL-2 APL-3 NL-3 APL-1 NL-1 APL-2 NL-2 APL-3 NL-3

No. exptof 3 2 2 2

3 2 1

1

2 2 2 2 1

3

3 2 2 2

Mean EOT

1.2 x 10-6 5.0x 10-7 1.3 x 10-6 5.1 x 10-7 9.0 x 10-7 4.3 x 10-7 1.2 x 10-6 5.2 x 10-7 1.6 x 10-6 3.9 x 10-7 1.4 x 10-6 6.7 x 10-7 8.2 x 10-1 3.6 x 10-1 8.6 x 10-1 2.5 x 10-6 4.0 x 101.9 x 10-6 4.1x10-x 2.4 x 10-6 5.5 x 10-6 2.9 x 10-6 3.9 x 10-1 2.0 x 10-6 8.4 x 10-7 3.5 x 10-7 1.8 x 10-6 3.9 x 10-7 9.9 X 10-7 3.3 x 10-7 4.7 x 10-1 2.7 x 104.5 x 10-b 3.2 x 10-5 3.9 x 102.0 x 10-

Ratio APL/NL 2.4 2.5 2.1

2.3 4.1

2.1 2.3

3.4 2.1 1.7 1.9

2.0

2.4

4.6 3.0 1.7 1.4

2.0

'A portion of Su+ donors infected with Tlam were

artificially lysed while another portion was allowed to lyse normally. Thus, from each batch of infected cells an APL-NL pair was obtained. In transductions, Surecipients were infected with APL or NL lysates at multiplicities equal to or less than 1. Details of the techniques of transduction can be found in other reports (4-6).

presence of prophage A in recipients had no effect on the results given in Table 2. A Student's t test was used to compare the EOTs of experiments in which Su+ and Su+(X) were the donors. The P value was not greater than 0.05 in any instance. On the contrary, when the EOTs of experiments performed with lysates obtained from Su+ and Su+(Arex) were compared by the t test, the value of P varied from 0.19 (Trp+) to 0.80 (StrR). For the four markers tested the results indi-

856

KYLBERG, BENDIG, AND DREXLER

J. V IROL.

TABLE 2. Comparison of Tlam transduction by lysates made on the donors KB-3, KB-3(X) and

enhanced transduction is equalized or cancelled out in Su- recipients, the EOT of premature lysates was observed to be significantly greater than with normal lysates. For reasons which we are unable to explain, the NLs transduced Su-(X) for Gal+ more efficiently than did lysates from Su+(Xrex) donors. Transduction of Bio+ by NPLs. The EOT of Bio+ is influenced by lysogenization of donors with X (8). The presence of A in donors causes an eightfold decrease in the EOT of Bio+ (Table 4). The EOT is unaffected by X-lysogenization of recipient cells (data not shown). The reason for the peculiar relationship between A lysogeny and Bio+ transduction is not known. It was found that Tlam -Su+(X) lysates transduce Bio+ four times as efficiently as Tlam Su+(Arex) lysates, again demonstrating that premature lysis leads to an increase in the EOT. Parenthetically, the results show that, once the effects of premature lysis have been removed, the presence of a A prophage (i.e., Arex) in donors causes a 34-fold drop in the EOT. Density of Ti-transducing particles. Previously it was reported that Ti-transducing particles have a density which is similar, but not necessarily identical, to Ti PFU. We have examined the transduction of Su - recipients for Arg+, Bio+, Gal+, Lac+, Pro+, and StrR by fractions obtained from density gradient centrifugation of Tlam -Su+ lysates. The distribu-

KB-3(Xrex)a Ratio EOTMarker

Lac+

Pro+

StrR Trp+

Donor

KB-3 KB-3(A) KB-3(Arex) KB-3 KB-3(A) KB-3(Arex) KB-3 KB-3(A) KB-3(Arex) KB-3 KB-3(A) KB-3(Arex)

No. of expt

3 4 4 4 4 5 2 2 2 5 5 6

Mea

EOT

3.0 1.1 3.4 2.6 1.2 3.7 1.7 5.9 1.8 9.0 1.9 4.8

x x x x x x x x x x x x

10' 10'

10' 10-' 10'

10' 10' 10' 106 10-' 106 10-'

lysogenic donor/ EOT-nonlysogenic donor 3.7 1.1 4.6 1.4

3.5 1.1 2.1 0.5

aTlam lysates made on Su+, Su+(A), and Su+(Arex) were used to transduce various Su - recipients for the given markers. See footnote to Table 1 for experimental details.

cate that, when the donor is lysogenic for X, the EOT is two to four times higher than when the donor is either nonlysogenic or lysogenic for Arex.

Transduction of Gal+ by NPLs. The transduction of Gal+ or Bio+ by NPLs was tested separately from other markers because the location of prophage X near to these markers affects the EOT of these markers (5-8). In previous work it was found that Tlam lysates obtained from Su+(X) donors are able to transduce Gal+ to Su- recipients more efficiently than lysates from nonlysogenic donors. Furthermore, the enhanced ability of Tlam Su+(X) lysates to transduce Gal + is abolished by making recipients lysogenic for X. The exact increase in the EOT by Tlam-Su+(X) varies between 35- and 1,000-fold and depends on the donors and recipients employed. The effect of enhanced transduction of Gal+ by X-lysogenic donors obscures the effect of premature lysis caused by the presence of prophage X in donor cells. Since cells lysogenic for Xrex do not lyse prematurely when infected by Ti but do appear to mediate enhanced transduction of Gal+ (Table 3), it is possible to determine the effects of X-mediated, premature lysis by comparing the EOT of Tlam-Su+(Xlysates to Tlam .Su+(Xrex) lysates. Using identical recipients (either nonlysogenic or X-lysogenic) it was observed that the EOT of Gal+ by Tlam -Su+(X) was four to nine times greater than the EOT of Tlam Su+(Xrex) (Table 3). In other words, once the effect of

TABLE 3. Comparison of Tlam transduction of Gal+ by lysates made on the donors KB-3, KB-3(A), and

KB-3(Arex)a Donor

Recipient

No. of expt

Mean

EOT

Ratio EOTKB-3(A)/ EOT-KB-

3(Arex) KB-3 KB-3(A) KB-3(Arex)

W3350 W3350 W3350

KB-3

W3350(A) W3350(A) W3350(A)

KB-3(A) KB-3(Arex)

7 5 2 3 5 4

3.6 x 1.3 x 3.0 x 4.0 x A.9 x 5.2 x

10-' 10-' 1010-' 10-' 10-'

4.3 9.4

aDue to the proximity of prophage A to the galactose operon, Tlam is able to transduce Gal+ more efficiently from Su'(A) donors to Su- recipients than from Su+ donors. Thus the effects of A-mediated natural, premature lysis are obscured. The enhanced transduction of Su - recipients for Gal I by Tlam -Su+(A) lysates is sensitive to A immunity since the enhancement is abolished in Su-(A) recipients. When the ability of Tlam lysates made on Su+(A) and Su+(Xrex) donors to transduce Gal+ are compared to each other (as above), the effects of enhanced transduction due to the proximity of A to the galactose operon are cancelled out and the effect of natural, premature lysis by A can be determined. See footnote to Table 1 for experimental details.

TRANSDUCTION BY Ti

VOL. 16, 1975 TAmBz 4. Comparison of Tlam transduction of Bio+ by lysates made on the donors KB-3, KB-3(X), and

KB-3(Grex)a Ratio Donor

KB-3 KB-3(X)

KB-3(Xrex)

No. of expt

Mean EOT

9 4 4

1.6 x 10-5 2.0 x 10-6 4.7 x 10-7

EOT-KB 3(X)/EOT-

KB-3(Arex)

4.3

a Due to the proximity of prophage A to the biotin operon, Tlam is not able to transduce Bio+ as efficiently from Su+(A) donors to Su- recipients as it is from Su+ donors. Thus, the effect of A-mediated premature lysis of Ti-infected cells is obscured. When the ability of Tlam lysates made on Su+(X) and Su+(Xrex) are compared (as above), the effects of transductional interference caused by the proximity of A to the biotin operon are abolished and the effect of natural, premature lysis by A can be determined. See footnote to Table 1 for experimental details.

(13). In each instance we found that the ratio of transductants to plaque-forming particles is significantly greater in the premature lysates (Tables 1 and 2). Therefore, we conclude that most Ti-transducing particles are formed early in the maturation period. There does not seem to be any relationship between the position of a marker on the E. coli map and the time the marker is picked up by Ti. Ti degrades the bacterial chromosome and utilizes the breakdown products for progeny Ti (11; Figurski and Christensen, personal communication). The results presented here suggest that undegraded bacterial DNA is not available for pickup late in the maturation period. In addition to demonstrating the early production of transducing particles, the experiments in this report permit further characterization of enhanced transduction of Gal+ from DENSITY (G/CM3) 1.515 1.510 1.505

12

tion of plaque-forming Ti and the Pro+ transducing particles are shown in Fig. 1; the other markers give nearly identical results. The results show that the average density of Ti-transducing particles is slightly greater than the density of plaque-forming Ti. The EOT of the various fractions is also plotted in Fig. 1. It is noteworthy that the EOT of Pro+ (and the other markers) displays a definite peak in the more dense part of the gradient. This supports the idea that the transducing particles are, on the average, more dense than plaque-forming Ti.

DISCUSSION The production of transducing particles during a phage's maturation period has been studied for several phages. Zinder (15) found that in P22-infected cells the appearance of transducing particles varied with the marker and even the physiological state of the host cell. Harriman (10) reported that with Pi the production of transducing activity lags behind plaqueforming particles but is fairly steady once it begins. We found with Ti that 50 to 90% of the potential transductants which arise from NLs can already be detected in premature lysates in which only 25% of the total plaque-forming particles have been made. Using six different markers and two different methods of premature lysis we have concentrated on comparing the EOT of the six markers in early, premature lysates to NLs. The six markers are scattered over about two-thirds of the E. coli genetic map

857

1.500

I.

-6

I.-

lz. 11-

;lb: z .13

E-

L4.

IR

.,EI lz-i1

Iz~

j

S 03

.j .1

~~~r

50

.lj

'I~~~~~~~~i- .z

60 70 FRACTION NUMBER

80

FIG. 1. Tlam-Su+ lysates were centrifuged at high speed in CsCI (see Materials and Methods for details) and 3-drop fractions were collected. The figure shows the distribution of plaque-forming particles (a), Pro+ transducing particles (0), and the ratio of transducing particles to plaque formers (i.e., the EOT [0]). For ease of comparison the peaks of the various plots have been aligned. Low values from the high- and low-density regions of the gradient were not plotted because it is not possible to detect transducing particles when their concentration falls much below 103/ml. The concentration of Tlam was less than 1010/ml in all fractions below no. 61 and above no. 76. A straight line resulted when density was plotted against drop number (not shown).

858

KYLBERG, BENDIG, AND DREXLER

Su+ (X) donors to Su recipients (Table 3). If the "enhanced" transduction of Gal+ by Ti grown on X-lysogenic donors is defined as that increase in the EOT that is due to the proximity of prophage X to the galactose operon and exclusive of the X-mediated, premature lysis of Tiinfected cells, it can be deduced that the proximity of X to the galactose operon leads to approximately a 10-fold increase in the transduction of Gal+ (Table 3). In contrast to the transduction of Gal+, it has been found that the EOT of Bio+ drops eightfold when the donor is lysogenic for X. By utilizing donors lysogenic for a phage which does not cause premature lysis of Ti-infected cells (i.e., Xrex lysogens), it has been found that the proximity of X to the biotin operon causes a greater decrease in the EOT of Bio+ than had first been supposed; Table 4 shows that Ti lysates derived from donors lysogenic for Xrex transduce Bio+ 34-fold less efficiently than Tlam-Su+ lysates. Ti-transducing particles carrying a number of different markers have been found to have an average density which is slightly greater than the density of plaque-forming particles (Fig. 1); in this respect Ti resembles phage Pi (14). transducing particles' DNA is approximately broader range of densities than Ti. These observations suggest that, although the length of the transducing particles DNA is approximately uniform, packaging of bacterial DNA is less precise than viral DNA. The observation that the ratio of the transducing to plaque-forming particles (i.e., the EOT; Fig. 1) forms a definite peak in the more dense part of the gradient is curious. If (as observed) the transducing particles are more dense than Ti particles, then the ratio of transductants to plaque formers should increase continuously as density increases. Since plaque formers are more numerous than transducing particles, a point might be reached where too few transducing particles were present to be reproducibly measured or where none were present. We plotted (Fig. 1) the EOT only from fractions which contained abundant transduc-

J. VIROL.

ing particles, giving reproducible EOT values, and found that with increasing density there was a significant drop in the EOT. ACKNOWLEDGMENTS This project was supported by Public Health Service grant AI 07107 from the National Institute of Allergy and Infectious Diseases. We would like to thank Crystal Jarman for her expert technical assistance. L. Kucera made a number of valuable suggestions in the preparation of the manuscript. LITERATURE CITED 1. Astrachan, L., and J. F. Miller. 1972. Regulation of Arex expression after infection of Escherichia coli K by lambda bacteriophage. J. Virol. 9:510-518. 2. Botstein, D., C. H. Waddel, and J. King. 1973. Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. I. Genes, proteins, structures, and DNA maturation. J. Mol. Biol. 80:669-695. 3. Christensen, J. R., and J. M. Geiman. 1973. A new effect of the rex gene of phage X: premature lysis after infection by phage Ti. Virology 56:285-290. 4. Drexler, H. 1970. Transduction by bacteriophage Ti. Proc. Natl. Acad. Sci. U.S.A. 66:1083-1088. 5. Drexler, H. 1972. Transduction of Gal+ by coliphage Ti. I. Role of hybrids of bacterial and prophage A deoxyribonucleic acid. J. Virol. 9:273-279. 6. Drexler, H. 1972. Transduction of Gal+ by coliphage Ti. II. Role of transcription control in efficiency of transduction. J. Virol. 9:280-285. 7. Drexler, H. 1973. Transduction of Gal+ by coliphage Ti. III. Requirement for transcription and translation in recipient cells. J. Virol. 12:1072-1077. 8. Drexler, H., and K. J. Kylberg. 1975. Effect of UV irradiation on transduction by coliphage Ti. J. Virol. 16:263-266. 9. Gussin, G., and V. Peterson. 1972. Isolation and properties of rex- mutants of bacteriophage lambda. J. Virol. 10:760-765. 10. Harriman, P. 1973. Appearance of transducing activity in P1-infected Escherichia coli. Virology 45:324-325. 11. Labaw, L. W. 1952. The origin of phosphorus in the Ti, T5, T6, and T7 bacteriophage of E. coli. J. Bacteriol. 66:429-436. 12. MacHattie, L. A., M. Rhoades, and C. A. Thomas. 1972. Large repetition in the non-permuted nucleotide sequence of bacteriophage Ti DNA. J. Mol. Biol. 72:645-656. 13. Taylor, A. L., and C. D. Trotter. 1972. Linkage map of Escherichia coli strain K-12. Bacteriol. Rev. 36:504524 14. Ting, R. C. 1962. The specific gravity of transducing particles of bacteriophage P1. Virology 16:115-121. 15. Zinder, N. D. 1955. Bacteriol transduction. J. Cell. Comp. Physiol. 45(Suppl. 2):23-49.