Growth and transformation of phage T4 in Escherichia coli B4 , Salmonella, Aerobacter, Proteus, and Serratia

VIROLOGY

39, 153-161 (1969)

Growth

and

Transformation

B / 4, Salmonella, ALLEN Department

of Phage

Aerobacter,

C. WAIS

AND

Proteus,

EDWARD

of Molecular Biology

and Microbiology, Boston, Massachusetts

T4 in Escher-i&a and

co/i

Serratia

B. GOLDBERG Tufts University 02111

School of Medicine,

Accepted May 12, 1969

Spheroplasts of the genera Escherichia, Salmonella, Aerobacter, Proteus, and Serratia can be infected by T4 phage which have been exposed to 6 M urea. The interaction between the phage and the bacteria does not require either phage tail fibers or the specific tail fiber receptors of the bacteria. These findings imply two modes of infection, one of which may precede the other, in the normal phage infection process. Transformation of T4 phage by denatured DNA fragments has been demonstrated with Aerobacter as the host organism. In Aerobacter, transformation is more efficient than in Escherichia, and at low DNA concentrations Aerobacter yields lOO-fold more transformed phage than Escherichia. INTRODUCTION

Veldhuisen et al. (1968) have studied growth of phage T4 in spheroplasts of Escherichia coli infected with urea-treated phage. Similar particles prepared from phage T2, although unable to infect whole cells of E. coli strain B, can infect spheroplasts prepared either from strain B or strain B/2, a mutant lacking the specific phage-receptor sites (Fraser et al., 1957). Weidel (1958) showed that receptor sites for phage T2 probably reside in the lipoprotein of the cell envelope, and those for phage T4 in lipopolysaccharide. Work presented here indicates that infection of spheroplasts by ureatreated T4 phage is mediated neither by specific tail fiber receptor sites on the cell wall nor by tail fibers of the phage. Thus, part of the natural mechanism of host recognition and host-range limitation of T4 is bypassed during this mode of infection. Urea-treated T4 phage can infect and grow in spheroplasts of Aerobacter aerogenes, Salmonella an&urn, Proteus vulgaris, and Serratia marcescens. This shows that only the initial adsorption step, and not problems of genome injection or replication or protein synthesis, prevents T4 phage replication in these bacteria.

Phage T4, growing in spheroplasts of E. coli B can incorporate genetic markers derived from native or denatured DNA in the medium (Veldhuisen and Goldberg, 1968). Use of urea-treated T4 infected spheroplasts of A. aerogenes rather than E. coli B/4 enhances the sensitivity of the marker assay over loo-fold, at low concentrations of denatured DNA. MATERIALS

Bacteria and bacteriophages. E. coli B and K12.112.12 (hh) (called E. coli K(X)) were obtained from Dr. A. D. Hershey; E. coli BB from Dr. S. Champe; E. coli B/4 from Dr. S. E. Luria; E. coli Boll’ (Bsu+) from Dr. F. Stahl. Bsu+ carries an amber suppressor mutation. S. anatum was obtained from Dr. A. Wright, and A. aerogenes (ATCC 8724), P. vulgaris (ATCC 13315), and X. marcescens (SM-Sr-11) from Dr. L. S. Baron. Bacillus megaterium KM was obtained from Dr. M. Schaechter, and Pseudomonas aeruginosa was from the departmental collection. T4D bacteriophages with the markers ~~41,rIIT3, and rIIel, were obtained from Dr. A. Doermann and ama5 from Dr. R. Edgar. Stocks of arn4c5 were grown on Bsu+ and fiberless phage am455.B

153 Copyright

@ 1969 by Academic

PESJ,

Inc.

AND METHODS

were obtained by a single cycle of gro\vt,h of ur~~.Bsu+ on E. coli B. ~11 stocks were grown on E. coli BB. Media. Synthetic medium (LXTG): The Tris-buffered medium of Hershey (1955) was modified to contain the following millimolar concentrations of components; NaCl, 9.2; KCl, 4.0; NH&I, 21; CaClz 0.10; MgC12, 10; Tris (pH 7.4), 100; KHZ04, 0.64; Na&304, 0.16; glucose, 2.8. v broth: 3 g of meat extract, 3 g of yeast extract, 10 g of peptone (all Difco products), 2 g of glucose, 4 g of MgS04.7Hz0, and 200 ml of distilled water. The broth was poured SW cessively through a glass fiber filter and a 0.45 p membrane filter (Millipore, Bedford, Massachusetts) to sterilize it. P broth: 10 g of peptone, 5 g of NaCl, 3 g of beef extract, 1 g of glucose, and 1 liter of distilled water. The broth was autoclaved at 121” for 20 min. Washing fluid: 0.01 M Tris (pH 7.4), 0.001 M MgCl*, 0.1% NaCl, 0.01% gelatin. Urea-treated phage. Phage particles (10” to 1.5 X 1012) in 0.1 ml washing fluid were added to 0.2 ml of a 9 M urea solution containing 2% bovine serum albumin (Pentex) and 0.1 M sodium phosphate, pH 7.0. After 10 min at 37”, 0.2 ml of this mixture was added to 1.8 ml of 2 % albumin in 0.1 M phosphate, pH 7.0. This treatment reduced the titer on bacteria by a factor of lo6 or more. Spheroplasts. One milliliter of an overnight culture of E. co&i B or B/4 in LSTG was subcultured in 19 ml of the same medium and aerated at 37” (for about 4 hours) until the increase in optical density stopped (about OD550 = 0.6). A portion (16.6 ml) of the subculture was added to 2.8 g of sterile sucrose in a 250-ml Erlenmeyer flask. Then 2 ml of V broth and 10 mg (1640 units/mg) of sodium penicillin G were added. The mixture was incubated at 32” for 2 hours. This procedure yielded about 2 X lo8 spheroplasts and lo4 colony-forming bacteria per milliliter. To prepare spheroplasts of A. aerogenes, 19 ml of LSTG was inoculated with 0.4 ml of broth culture aerated overnight at 37”. The subculture was aerated at 37” until it reached an ODsso = 0.6 and then combined with sucrose, V broth, and 30 mg of penicillin, as described for E. coli B and B/4.

Further modifications for other species are listed in the text. DNA. Phage DNA was extracted with phenol and denatured by boiling 3 min and chilling on ice (Goldberg, 1966). Sphewplast infection an.clphage transformatiolz assay. A standard infection mixture consisted of 0.5 ml of spheroplasts diluted with an equal volume of 0.01 M sodium phosnhate, pH 7.0, and 0.1 ml of ureatreated phage. In a standard transformation assay, DNA was dissolved in the phosphate buffer and the mixture was incubated 15 min before the addition of urea-treated phage. All incubations were at 37”. After the required incubation, phage were diluted and titered on E. coli B and E. coli K(X) plating bacteria to assay total phage and r+ phage, respectively. The transformed (rf) phage are those which have rescued rf markers from the input DNA. The transformation frequency is the ratio, transformed phage/ total phage. Xpheroplast dilution medium (SDM) and titration of infective centers. The dilution medium consisted of 166 ml of LSTG, 20 ml of V broth, 28 g of sucrose, 90 ml of 0.01 M sodium phosphate, pH 7.0, and 5 ml of 30 % bovine serum albumin (Pentex). Top agar for plating infective centers was of similar composition with 0.8% agar. To improve plating efficiency of infective centers, a 3-ml aliquot of SDM top agar was spread over the usual bottom agar 1 hour before plating infective centers. RESULTS AND DISCUSSION

The Physiology of Infection of E. coli B Spheroplasts by Urea-Treated T4 Phage The time course of phage production in the standard spheroplast infection mixture is shown in Fig. 1. Four phases are discernible in the curve for the undilut.ed infection mixture. An eclipse period of about 15 min is followed by a rapid rise, representing the progeny of the initial infection by ureatreated phage. This is followed by a period, between 30 and 40 min after infection, in which there is no net phage production. A second cycle of net phage production starts at 40 min. Since this course of phage production is unaltered by the presence of 100

T4 TRANSFORMATION

TIME

IN

AEROBACTER

155

IN MINUTES

FIG. 1. Time course of phage production in the standard infection mixture. At zero time, 3 X IO9ureatreated T4Drr3 phage per milliliter were added in a standard infection mixture containing spheroplasts of Escherichia coli B. After a lo-min adsorption period, an aliquot of the mixture was diluted lOO-fold into SDM. The diluted and undiluted mixtures were titered at intervals for phage production. Titers obtained for the 1:lOO dilution were multiplied by 100 before plotting. Undiluted infection mixture, @--a; diluted infection mixture, O---O.

fig of pancreatic DNase per milliliter, added before the urea-treated phage (unpublished experiments), the phage must deliver its chromosome to the spheroplast by some process which protects the DNA from DNase. The simplest explanation is that the phage adsorbs to the spheroplast before releasing its DNA. Figure 1 also shows that if the infected spheroplasts are diluted in SDM 10 min after infection to prevent further urea-treated phage or progeny phage interactions, the first growth cycle rises higher and the second cycle disappears. The plateau period in the undiluted culture is probably due to adsorption and eclipse of progeny (see below) at a rate equal to that of phage production. The second growth cycle in the undiluted incubation mixture may be the result of growth of the eclipsed progeny phage as well as late infections by urea-treated phage. Infections initiated by urea-treated phage during in initial adsorption period can thus be studied after dilution of the infection mixture. The ability of untreated T4 phage to infect spheroplasts is shown in Fig. 2. The growth pattern obtained varied with differ-

ent batches of spheroplasts. With any given batch of spheroplasts the yield of progeny was proportional to phage input. These data indicate that the second growth cycle, referred to in Fig. 1, is caused in part, and to a variable extent, by progeny phage infections. The development of infective centers in the infections initiated by urea-treated phage is shown in Fig. 3. The result is similar to that obtained when whole cells are infected with untreated T4 phage. Bursts start between 25 and 30 min after the addition of phage, and most are completed by 50 min. The apparent burst size (phage yield per infective center) is 280. The burst size was also measured by isolating single bursts (Adams, 1959). The results are listed in Table 1; 95 % of the fertile tubes yielded more than 60 plaques. The mean burst size for three experiments is 185. From this it appears that about two-thirds of infected spheroplasts that would yield phage in liquid culture produce plaques when plated in the infective center assay. The number of urea-treated particles required to initiate phage growth in a single

156

WAIS

AND

FIG. 2. Infection of spheroplasts with untreated phage. 1 X lo9 untreated T4Dacal phage were used in place of urea-treated phage in a standard infection mixture containing spheroplasts of Eschetichia cola’ B. Input phage were inactivated by antiserum added 10 min after infection. The mixture was diluted X 104 into Spheroplast dilution medium (SDM) at 13 min after infection and titered at intervals. Titers were multiplied by lo4 before plotting.

spheroplast can be determined by measuring phage production as a function of the concentration of input particles. Figure 4 is a graph on logarithmic axes of the relation between input of urea-treated phage particles and yield of phage progeny. The slope is 1.0, indicating that a single urea-treated particle can initiate a productive infection. Since the input is 10 times the yield and the mean burst size is about 200, only 1 in 2 X lo3 urea-treated particles gives a productive infection in a spheroplast. The fraction of spheroplasts capable of being infected by urea-treated phage was determined by infecting spheroplasts at different concentrations with an excess of urea-treated phage and titering infective centers. The infected cultures from which infective centers were plated were incubated until a maximum phage titer was reached. A mean burst size was then calculated from these experiments. In Table 2, the burst size data are compared to the mean burst size of 185 obtained in single-burst experiments, and a suitable correction for the efficiency of plating of infective centers is

GOLDBERG

I

LI 15 TIME

I

I

I

30

45

60

J

IN MINUTES

FIG. 3. The development

of infective centers in urea-treated phage infection. A standard infection mixture containing spheroplasts of Escherichia coli B was prepared with 3 X lo8 T4Drrs urea-treated phage per milliliter and diluted lOOfold 10 min after infection. Infective centers were titered by dilution in spheroplast dilution medium (SDM); total phage were titered by dilution into hypotonic medium. Infective centers, O--O ; total phage, a-----@.

made. Table 2 shows that 14-22% of the spheroplasts visible in the phase contrast microscope can be infected under these conditions. In summary, a subpopulation of ureatreated phage can infect a subpopulation of E. coli spheroplasts in a 1: 1 interaction. For the parameters we have tested (eclipse and latent periods and burst size), phage growth within spheroplasts is similar to that within whole cells. Mode of Adsorption of Urea-Treated Phage: Lack of Requirement for Specific Cell Wall Receptor Sites and Phage Tail Fibers E. coli B/4 is a T4-resistant coli B that does not adsorb lacks the specific cell wall Urea-treated T4 phage will plasts of E. coli B/4 and yield

mutant of E. T4 because it receptor sites. infect spheroapproximately

T4 TRANSFORMATION TABLE SINGLE-BURST

1

TABLE

EXPERIMENT”

Number of tubes with no phage

Total plaques on 50 plates

Mean burst size

1 2 3

17 15 35

10,038 10,427 3,185

200 174 182

5 A standard infection mixture containing spheroplasts of Escherichia coli B with 3.0 X log urea-treated T4DrG1 phage per milliliter was diluted 3 X 104 in spheroplast dilution medium (SDM) 10 min after infection. Single drops of this diluted mixture were distributed by means of a Pasteur pipet,te into 50 tubes containing 0.3 ml SDM. The mean drop size was 0.04 ml. One hour later each tube was titered after the addition of 2 ml of top agar. A Poisson distribution of infected spheroplasts in the tubes was assumed.

,

I07 / I08

I

I

I09

10’0

INPUT

2

FRACTION OF SPHEROPLASTS INFECTIRLER

Expt. No.

b

1<57

IN AEROBACTER

PARTlCLES

FIG. 4. Phage yield as a function of ureatreated phage input. Standard infection mixtures containing spheroplasts of Escherichia coli B with various concentrations of urea-treated T4Da~41 phage were diluted loo-fold 10 min after infection and titered 90 min after infection.

the same number of phage as with E. coli B prepared under the same conditions. Figure 5 shows the time course of phage production in B/4 spheroplasts. Untreated phage T4 will not infect B/4 spheroplasts, indicating that, contrary to the infection of spheroplasts by urea-treated phage, specific cell wall phage receptor sites are required for untreated phage infection of spheroplasts.

Spheroplast dilution Visible spheroplasts/ml Infective tenters/ml Spheroplasts infectible (uncorrected) Phage yield/ml Calculat,ed burst, size Spheroplasts infectible (corret ted)

~

1:l

/

1:5

1:50

1 .O X lo8 2.0 X 107 z.0 x 106 18.7 X lo6 1.8 X lo6 ’ i 8.7% 9.0%

t.0 x 105 10.0%

4.0 X IO9 5.2 X 10’ 6.9 X 107 460 290 350 220/,

147;

17%

a Standard infection mixtures were prepared with dilutions of Escherichia coli B spheroplasts in spheroplast dilution medium (SDM) and 5 X lOlo urea-treated T4Dacrl phage per milliliter. At 10 min after infection these mixtures were further diluted into SDM, titered for infective centers immediately and for phage 60 min later. Correction for fraction of spheroplasts infectible was made by assuming a real burst size of 185.

T4 phage particles without tail fibers can be produced by infecting E. coli B with an amber phage (cMz~~Jwhich is mut.ant in gene 34 (Edgar and Lielausis, 1965). These particles will not infect either whole cells or spheroplasts of E. coZi B or E. coli Bsu+ as do normal fibered phage treated in this manner, as shown in Table 3. From these data we conclude that tail fibers are not required for infection of spheroplasts by urea-treated phage, although they are required for infection of spheroplasts by normal phage. Thus, neither the specific tail fiber receptor sites of the host cell wall nor the phage tail fibers are involved in the infection of spheroplasts by urea-treated T4 phage. Host Range of Urea-Treated Phage Infection Since B/4 spheroplasts are infectible by urea-treated T4 phage, we tested spheroplasts of other bacterial genera to determine whether the host range of T4 could be extended by this technique. Table 4 lists strains that will, when converted to spheroplasts,

15,s

WAIS AND GOLDBElZG TABLE Hosr

ItANGE

Strain

Escherichia

coli B

n I07

E. coli B/4

E

Salmonella anatum Aerobacter aerogenes Proteus vulgaris Serratia marcescens

ki 4 a

106

30

60 TIME

90

120

(50

180

IN MINUTES

FIG. 5. Time course of phage production in Escherichia coli B/4. A standard infection mixture containing spheroplasts of E. coli B/4 and 3 X lo9

urea-treated T4DrI1~1 phage per milliliter prepared and titered at intervals. TABLE

3

GROWTH OF UREA-TREATED

Treatment Urea-treated phage (3 X log/ml input) Untreated phage (1 X log/ml input)

was

FIBERLESS

PHAGE~

Phage am.m. B

rs,.BB

1.5 x 10’

4.0 x 107

<106

1.3 x 10’

0 Numbers in this table are titers obtained after 120 min growth of the input phage on Escherichia coli Bsu+ spheroplasts. Growth of untreated phage was determined as described in Fig. 2. The titer of the am,ae.B stock was estimated by comparing the optical density at 260 rnb with that of the rsi stock. Spheroplasts were prepared as described for E. coli B, except a log phase broth culture at ODjso = 0.6 in place of an LSTG culture was added to penicillin, sucrose, and V broth. The only visible cellular structures after penicillin treatment were 1 X 10’ spheroplasts per milliliter. support the growth of urea-treated T4 phage. Cultures that do not readily produce spheroplasts (as determined by microscopic examination) when exposed to penicillin,

OF

4 T1”

UREA-TREATED

Penicillin (units/ ml)

Iftt$ time (hours)

820 820 8200 2460 8200 8200

5 5 3 6 5 6

1.ieltl

3 x 109 x 109

3 6 7 3 7

X 10’ x 10'9

x 106 x 109

a Spheroplasts of Salmonella, Aerobacter, Proleus, and Serratia were prepared according to the procedure described for E. coli B in the standard infection mixture with the following changes. Log phase broth cultures at ODj50 = 0.6 instead of LSTG cultures were used. The penicillin concentrations and times of incubation of infection mixtures are listed above. Each spheroplast preparation was infected with 5 X lOlo ureaControl treated T4Drsl phage per milliliter. mixtures of urea-treated phage and lysed spheroplasts yielded about 100 phage, and the titers did not increase on further incttbation. give lower yields. Thus, the variation in yield among these species may be due to the amount of unremoved cell nature and/or wall material. Alternatively, the differences in phage yields may reflect differences in phage growth rates or burst sizes. We have

tried, unsuccessfully, to grow T4 phage in this manner on Pseudomonas aeruginosa and Racillus megaterium. The Phage Transformation Assay Using A. aerogenes Xpheroplasts The superior yield of phage in the A. aerogenesinfection suggested that this species might be particularly useful in the phage transformation assay. All strains listed were tested for phage transformation activity with denatured DNA. Transformed phage could be found only in E. coli B and B/4 and in A. aerogenes infections. In all cases the maximal transformation frequency was about KY. Phage T4rI1, growing in E. coli B, B/4, or A. aerogenes spheroplasts after infection by urea-treated phage can incorporate r+ markers added to the medium as denatured DNA. E. coli B and B/4 give similar trans-

T4 TRANSFORMATION

formation frequencies. Use of B/4 is preferred because the progeny of the ureatreated phage infection cannot be inactivated by adsorption and cannot initiate further infections as in E. coli B spheroplast preparations. A. aerogenes spheroplasts have the same advantages since they do not adsorb intact phage T4, as shown in Table 5. While urea-treated T4 phage do infect and grow in Aerobacter spheroplasts, untreated T4 phage do not even adsorb to them. Infection and growth of T4 phage in Aerobacter are unaffected by 100 pg of pancreatic DNase per milliliter (E.G., unpublished experiments). Thus, it seems likely that Aerobacter has no receptors for T4 phage tail fibers, but an adsorption site for urea-treated phage can be uncovered by spheroplasting the bacteria. In addition, the Aerobacter host provides a more sensitive transformation assay. Figure 6A compares the relation between transformation frequency and donor DNA concentration for B/4 and Aerobacter. Less DKA is needed to saturate Aerobacter than Escherichia. The relation between phage-transformed and DXA concentration for Aerobacter and Escherichia is compared in Fig. 6B. It shows that at all DiSA concentrations more phage are transformed when Aerobacter is used than when E. coli B/4 is used as host. Transformation is easily detected in Aerobacter at low DNA conTABLE

5

GROWTH AND ADSORPTION OFPHAGE T~ON SPHEROPLASTS OF Escherichia coli B, E. coli B/4, AND Aerobacter aerogenes”

Adsorption after 12 min Growth in 2 hours

E. coli B

E. coli B/4

A. aerogenes

42%

2%

0%

<106

<105

1.4 x

109

a Growth of untreated phage on spheroplasts was measured as described in Fig. 2. Growth titers in this table were taken 2 hours after infection. Phage inactivation by adsorption was measured by adding 1 X lo9 T4Drel phage per milliliter to each spheroplast preparation and bursting the infected spheroplasts by dilution after a 12-min incubation. The unadsorbed phage were then titered.

IN AEROBACII’ER

159

centrations (0.01 pg/ml) which yield no transformed phage in Escherichia. The relatively greater efficiency of the Aerobacter transformation system diminishes as the DNA concentration increases. The Aerobatter transformation system is therefore more useful than the Escherichia system for establishing the relation between transformation and size of donor DNA as well as for assaying DNA fragments for genetic content where the transformation efficiency or the amount of DNA available is small. CONCLUDING

REMARKS

Investigation of the mechanism by which urea-treated phage infect spheroplasts has led to a technique by which T4 phage can be grown in many genera of bacteria which are usually resistant to T4. With A. aerogenes it was shown not only that transformation can occur, but that the transformation system is more sensitive and efficient than in E. coli. It is possible that under suitable conditions T4 transformation can be shown in other genera as well. Bacteria will not support phage growth if the phage do not adsorb and eject their DKA. In addition, the phage genome must enter the host cell intact and the protein synthetic and nucleic acid replicating apparatus of the bacteria and phage must be compatible. We have shown that there are two modes of infection for T4 phage, one for intact phage and intact bacteria and one for a.ltered phage and altered bacteria. Given the assumption that urea treatment alters the phage only in its adsorptive and/or DNA ejection properties and that penicillin treatment alters the various host, species only in their adsorptive properties, we have shown that diverse types of gram-negative bacteria lacking specific T4 receptors allow the T4 phage genome to enter. The fact that T4 phage can grow on these cells when the first attachment stage is bypassed shows that these hosts do not restrict T4 genomes and that, the protein synthetic and nucleic acid replicating apparatus of these genera are compatible with growth of T4. We are now investigating the growth of nonglucosylated T-even phage and amber mutants

160

EDNA3 yg/ml

+, 5,

w4 t-i

05

I 2.0

7 1.0 CCNAI

gg/ml

FIG. 6. Aerobacter aerogenes and Escher&a coli B/4 phage transformation: frequency of transformation and transformed phage as functions of DNA concentration. Standard transformation mixtures were prepared containing spheroplasts of E. coli B/4 or A. aerogenes and various concentrations of heat-denatured T4Dac41 DNA. These mixtures were infected with 3 X 10”’ urea-treated T4DrI101 phage/ml and titered on E. coli K(x), 180 min after infection. Transformation frequency (r+/rII) and r+ phage per milliliter were plotted as a function of DNA concentration in 6A and 6B, respectively. B/4, O-0; Aerobatter, 0-0.

to survey the distribution of restriction and suppression systems. The unknown mechanism of urea-treated phage adsorption and ejection may be related to the second stage in the normal adsorption process. According to this view,

we would postulate that during the normal infection cycle when the tail fibers of the infecting phage attach to the specific receptor sites on the bacterial surface, the phage is modified to activate a second attachment apparatus, which then finds a more internal

T4 TRANSFORMATION

receptor site. Only then can the DNA be ejected. According to this model the penicillin (or lysozyme)’ treatment uncovers the internal receptor site located in the various genera of bacteria and the urea treatment activates the second attachment apparatus of the phage. If one accepts the hypothesis, then it would have to be assumed that under normal conditions the second attachment apparatus is activated by the attachment of the tail fibers. The combined treatments of both bacteria and phage would thus obviate the first stage of adsorption. Though alternative models can be constructed, this one fits the proposals of Simon and Anderson (1967). Their work implicates the short fibers or the needle, both of which extend from the base plate to the inner cell surface, as the second attachment apparatus. We are now investigating this point. We suggest that the internal receptor site corresponding to the second stage of phage attachment is similar in the various gramnegative genera. This would contrast with the extreme specificity of the T4 phage tail fiber receptor sites. The broad occurrence of a common internal receptor site would further suggest that a primitive phage, perhaps without species-specific tail fibers, once existed and that its host was a primitive bacterium from which evolved the gramnegative genera we have discussed. This primitive bacterium may not have had as differentiated a cell wall as our present genera with their specific receptor sites in the lipopolysaccharide and lipoprotein layers. The location and chemical nature of the postulated receptor site for the second attachment apparatus is now being investigated. ACKNOWLEDGMENTS

This work was supported by awards GM-13511 of the National Institutes of Health and GB-5923 of the National Science Foundation. One of us 1 Urea-treated phage infection of lysozyme spheroplasts has been demonstrated by Fraser et al. (1957) and Goldfarb et al. (1966) as well as in our laboratory (unpublished results).

IN

AEROBACTER

161

(A. W.) is a predoctoral fellow of the N. S. F. and the other (E. G.) is a career development awardee of the U. S. Public Health Service, GM-7567. We thank Dr. D. M. Trilling for suggesting that we try infection of bacterial genera other than Escherichia with phage T4. We also thank Linda Baumann for first demonstrating that urea-treated fiberless phage can infect E. coli spheroplasts, as shown in this article. We acknowledge the help of Patsy Arscott for her technical assistance and in preparing the manuscript and Dr. S. E. Luria for valuable criticism. REFERENCES ADAMS, M. H. (1959). “Bacteriophages.” Wiley (Interscience), New York. EDGAR, R. S., and LIELAUSIS, I. (1965). Serological studies with mutants of phage T4D defective in genes determining tail fiber structure. Genetics 52, 1187-1200. FRASER, D., MAHLER, H. R., SHUG, A. L., and THOMAS, C. A., JR. (1957). The injection of subcellular Escherichia c&i, strain B, with a DNA preparation from T2 bacteriophage. Proc. N&Z. Acad. Sci. U. S. 43, 939-947.

GOLDBERG,E. B. (1966). The amount of DNA between genetic markers in phage T4. Proc. NatZ. Ad. Sci. U. S. 56, 1457-1463. GOLDFARB, D. M., ARDEEVA, A. V., BLINOVA, S. V., SERGEEVA, S. N., and LEVINA, G. A. (1966). Phage T4rIIB-638 transformation by DNA of phage T4~11+. Genetika 7, 143-156. HERSHEY, A. D. (1955). An upper limit to the protein content of the germinal substances of bacteriophage T2. Virology 1, 108-127. SIMON, L. D., and ANDERSON, T. F. (1967). The infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope. I. Attachment and penetration. Virology 32, 279-297.

VELDHUISEN, G., and GOLDBERG, E. B. (1968). Genetic transformation of bacteriophage T4. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, Eds.), Vol. 12 B, pp. 858-863. Academic Press, New York. VELDHUISEN, G., POELMAN, M. C., and COHEN, J. A. (1968). Genetic transformation of the bacteriophage T4. I. An outline and some properties of the phage transformation system. Riochim. Biophys. Actu 161, 94-108. WEIDEL, W. (1953). Bacterial viruses (with particular reference to adsorption/penetration). Ann. Rev. Microbial. 12, 27-48.