JOURNAL OF BACTERIOLOGY, Sept., 1965 Vol. 90, No. 3Copyright © 1965 American Society for Microbiology Printed in U.S.A.

Multiple-Tailed T4 BacteriophageKENDALL 0. SMITH AND MELVIN TROUSDALE

Department of Virology and Epidemiology, Baylor University College of Medicine, Houston, 7'exas

Received for publication 10 May 1965

ABSTRACT

SMITH, KENDALL 0. (Baylor University College of Medicine, Houstoni, Tex.), andMELVIN TROUSDALE. Multiple-tailed T4 bacteriophage. J. Bacteriol. 90:796-802. 1965.-T4 phage particles which appeared to have multiple-tails were observed. Experimentswere designed to minimize the possibility that superimposed particles might accountfor this appearance. Double-tailed particles occurred at a frequellcy as high as 10%.Triple- and quadruple-tailed particles were extremely rare. All attempts to isolatepure lines of multiple-tailed phage have failed. Multiple-tailed phage particles wereproduced in highest frequency by Escherichia coli cells in the logarithmic growthphase which had been inoculated at a multiplicity of abouit 2.

The morphology of T bacteriophages has beenstudied extensively (Williams and Fraser, 1953,1956; Brenner et al., 1959; Bradley, 1963). Todate, however, we are aware of no reports de-scribing the occurrence of more than one tail perphage head. The present report describes themorphology of what appear to be multiple-tailedT4 phage particles and the conditions which favortheir production.

MATERIALS AND METHODS

Stocks of T4 bacteriophage and Escherichiacoli B were obtained from M. Mandel, M. D. An-derson Hospital and Tumor Institute, Houston,Tex. Growth of E. coli and phage was at 37 C inTryptose Phosphate Broth (Difco). After visibleclearing of infected E. coli suspensions, clarifica-tion was accomplished by low-speed centrifuga-tion (500 X g for 5 to 10 min). Phage was preparedfor electron microscopy by methods previouslydescribed (Sharp, 1960; Smith and Melnick, 1962;Penterik and Taylor, 1962). The preparativemethod of Smith and Melnick (1962) was used inmost cases. Staining was accomplished by use ofexcess amounts of uranyl acetate, which tends tomask positive staining and gives a negative stain-ing effect (Smith, Gehle, and Trousdale, 1965).Plaque assays of phage were done by mixing ap-propriate phage dilutions with E. coli and warmsoft agar (50 C), spreading the mixtures onto tryp-tose-phosphate-agar plates (Difco), and incubat-ing the plates at 37 C. E. coli colony counts weredone by the common pour-plate technique. Growthin E. coli was followed turbidimetrically by remov-ing small samples at regular intervals and readingper cent transmittance at a wavelength of 660 miAof a spectrophotometer. The calculated multiplic-ity of infection referred to in the text is defined asthe ratio of the plaque-forming phage concentra-tion to the number of E. coli colony-producing

units. Density-gradient experiments were done asdescribed previously, with the use of gradientspreformed with solutions of cesium chloride(Smith, in press).

RESULTS

Frequency of occurrence and morphology of mul-ple-tailed phage particles. Over a period of severalyears, we have noticed occasional particles in ourT4 phage stocks which appeared to have multipletails. The fractions of particles displaying twotails varied greatly in different passages, butwere usually between 0.5 and 8%. Triple- andquadruple-tailed particles were extremely rare.(Only one quadruple-tailed particle has been seenin over 10,000 individual particles we have ex-amined). Figure 1 illustrates the appearance ofparticles that seem to have single, double, triple,and quadruple tails. Figure 2 shows two double-tailed particles at greater magnification so thatthe fine structures can be studied. Both tails ofdouble-tailed particles often displayed a highdegree of structural organization. The periodicbands of tail subunits, the tail plates, the tailfibrils, and the tail sheaths were often visible onboth tails. The most common angle formed be-tween the tails was 700 (see Fig. 1 to 5). Smaller orlarger angles were seen very rarely.

Possibility of artifact production. We consideredthe possibility that such "double-tailed" formswereirmpe suposed heads, since two-dimensionalelectron microscopy might give this impression.For this reason, we employed metal shadowingto compare the heights of the particles. Figure 3shows the results of chromium metal shadowing.It can be seen that shadow lengths of double- andsingle-tailed particles are almost identical. Figure

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MULTIPLE-TAILED BACTERIOPHAGE

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FIG. 1. Phage particles appearing to possess one, two, three, and four tails. (The bar on this and the fol-lowing micrographs equals 100 m,u.)

4 shows two fields containing both single- anddouble-tailed particles which were stained andphotographed at high magnification in an electronmicroscope. The stained preparations were thenremoved from the microscope and metal-shad-owed, the same fields were relocated in the micro-scope, and the particles were again photographed.This procedure permitted the examination of aparticle's fine structure and provided informationabout its height. Each micrograph shows that

single- and double-tailed particles cast shadowsof about equal lengths. This offers strongf evidencethat these are not superimposed particles, whichwould give the illusion of double tails on a singlehead. It seems extremely unlikely, however, thattwo, three, or four heads could be so perfectlyaligned in one plane as to give the impression ofbeing only one structure. Moreover, structuralcontinuity between head and tails can be seen inseveral of the particles (Fig. 1, 2, 4).

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SMITH ANI) TROUSI)ALE.

Fi]u'1. 2. Phage particles 'which appe(ll to have two t(lils.

FIG. 3. Phage particles shadowed with chromium metal, showing that shadows cast bly single- and doUble-tailed particles are approxitmiately the samtie.

The number of unattached tails in most phagesuspensions was very small (note their absence inthe low magnification micrograph, Fig. 3). There-fore, chance superimp)osition of single-tailedp)hage particles upon unattached tails could cer-tainly not account for the high fr equency ofmutltiple-tailed l)articles which we have observed.Multiple-tailed phage particles can be signifi-

cantly concentrated by equilibrium density-

gradient centrifugation. AMost single-tailed par-ticles have a density of 1.49, whereas many mul-tiple-tailed particles are somewhat more buovant(density = 1.44). N-early all confusing extraneousdebris and fragmented palticles were eliminatedfrom these preparations. Figure 5 shows a fieldof lparticles (density = 1.44) taken from such adensity gradient. Complete separation of multi-ple-tailed forms would be difficult by this method

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MULTIPLE-TAILED BACTERIOPHAGE

FIG. 4. Phage particles stained, photographed, removed from the microscope, shadowed with chromiummetal, then photographed again. (Shadows cast by single- and double-tailed particles are approximatelythe same.)

because of the heterogeneity of single-tailedparticle density (range = 1.40 to 1.51).To rule out the possibility that our usual

pseuodoreplication l)rocedure from an agar sur-

face (Smith and Melnick, 1962) in some wayproduced artifacts resembling multiple-tailedparticles, we applied the preparative method ofPenterik and Ta3lor (1962). This method in-

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SMITH AND TROUSDALE

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FIG. 5 Low-magnification micrograph of cesium chloride-puirified phage particles (density = 1.44)showing several multiple-tailed particles (see arrows).

volves the dialysis of salts and fluids through asemipermeable membrane, followed by metalshadowing of the deposited virus particles. Theresults obtained by this method were the same;i.e., multiple-tailed phage were easily recognizedand were present in about the same relativenumbers as observed in other preparations.

Attempts to isolate pure lines of multiple-tailedphage. In the course of this study, we attemptedto determine whether pure populations of double-tailed phage could be isolated by growth fromsingle plaques. Sixty individual plaques werepicked for inoculation into log-phage E. coli, andthe progeny from each plaque was examinedmicroscopically. None of the passages yielded asignificantly higher proportion of multiple-tailedparticles than was present in the original inocu-lum, namely, about 5%. Another suspension,which contained about 10% multiple-tailedphage, was similarly plaqued, and 30 individualplaques were subcultured in log-phase E. coli.Again, no significantly higher numbers of multi-

pie-tailed phage were seen in these suspensions.If we assume that they are both viable and breedtrue, the probability of our having isolated a puremultiple-tailed line by this procedure is well over95%.

Conditions influencing the production of multiple-tailed phage particles. Experiments were designedto determine whether the bacterial growth phaseat the time of inoculation or the size of the inocu-lum could influence the production of multiple-tailed phage. A 1.0-ml volume of stationary-phaseE. coli was inoculated into 100 ml of warm broth,and the resulting bacterial growth at 37 C wasmeasured by frequent readings on samples in aspectrophotometer. The bacterial growth patternis shown in Fig. 6. Samples (15 ml each) of bac-terial suspension were removed at intervals of1.5, 3.0, and 5.0 hr after inoculation of the E. coli,and each sample was inoculated with 6.0 X 108plaque-forming units of phage. After inoculation,each suspension was incubated at 37 C for 4 hr toallow maximal phage growth. Spectrophotometric

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readings were then made at the end of the 4-hrperiod on each of the three phage-infected speci-mens. The "clearing" effect was apparent only inthe logarithmic-phase E. coli suspension (seeFig. 6). The final yield of infectious phage permilliliter was greatest from the logarithmic-phaseE. coli, as is shown in Fig. 7. Figure 7 shows, inaddition, the results of a similar experiment inwhich samples of E. coli were removed and inocu-lated with phage (as above) at 0.5-hr intervalsduring the lag phase. It was concluded from theseand other experiments, as might be expected,that the maximal yields of infectious phage wereobtainable from bacterial cells in the logarithmic-growth phase.The frequency with which multiple-tailed par-

ticles were lproduced in the above exl)eriments is

shown in Table 1. At a glance, these data suggestthat multiple-tailed particles are produced withhigher frequency by cells in the lag phase (5.5%)than by cells in the logarithmic phase (0.9%). It isimportant to note, however, that each bacterialsuspension was inoculated with the same amountof phage, and that the concentration of bacteriaincreased rapidly after 1.5 hr. The infection mul-tiplicity for each suspension (shown in Table 1)thus decreased sharply after the lag phase be-cause of the rapid increase in the concentrationof bacteria. Therefore, two variables were opera-tional during these experiments: (i) changes inthe bacterial growth l)hase and (ii) changes in theinfection multiplicitv.

Experiments were next designed to determinewhether varying only the infection multiplicitywould influence the l)roduction of multiple-tailedparticles. Samples of E. coli cells in the logarithmicphase of growth (3 hr after broth inoculation,showing 54% transmittance and having a colonycount of 4.0 X 108 per milliliter) were inoculated

TABLE 1. Effects of varying the Escherichia coligrowth phase at the time of inoculation and themultiplicity of infection upon the production

of multiple-tailed phage particlesAge of E. Per cent mul-coli at tie Growth Multiplicity tiple-tailedof phage in' Gowl phase of infection* tple-agledoculation particles

hr

0.5 Lag 11 2.61.0 Lag 5.5 2.01.5, 1.5t Lag 1.9, 1.9 5.5, 4.62.0 Early loga- 1.1 2.1

rithmic3.0t Logarithmic 0.10 0.95.0t Stationiary 0.04 Too few to

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t Experiment 2.

E. coli colony count

TABLE 2. Effect of varying the infection multi-plicity upon the production of multiple-tailed

phage particles in logarithmic-phaseEscherichia coli

Per cent multiple-tailed phage particlesMultiplicity of ______________________

infection* -Expt 1 Eaxpt 2

0.10 1.3 1.00.50 2.2 2.82.0 6.0 4.9

*Calculationis based ons E. coli colony count attime of inioculation.

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SMITH ANI) TROUSDALE

so as to give calculated multiplicities of 0.1, 0.5,and 2.0. Each inoculated bacterial suspensioncleared visibly within about 1 hi, but was allowedto incubate for a total of 4 hr after inoculation,as before. Table 2 summarizes the results of twosuch experiments. These data indicate that vari-ation in infection multiplicity has a marked effectupon the production of multiple-tailed phageparticles. Infection multiplicities much higherthan 2 resulted in a marked suppression of totalphage yields (as much as 99%) without obviouslyincreasing multiple-tailed particle production. Weconcluded, therefore, that the maximal produc-tion of multiple-tailed phage particles occurs inE. coli cells which have been inoculated at a mul-tiplicity of about 2, either during the late lagphase or during the early logarithmic phase.Multiple-cycle growth of phage consistently pro-

duced low yields of multiple-tailed particles(fewerthan 1%).

DISCUSSION

One explanation for the failure of previousinvestigators to report the occurrence of multi-ple-tailed phage particles is that the p)articles are

not produced in large numbers by the usualmethods of passage. A common and efficientprocedure for growing large phage stocks con-

sists of inoculating bacterial cells in the log-arithmic-growth phase with small concentrationsof phage, thus allowing multiple cycles of phagegrowth. Our experiments indicate that these con-

ditions are probably the lpoorest for producingmultiple-tailed lparticles. If the incidence of mul-tiple-tailed particles were low, they could easilybe overlooked, or discounted as superimp)osedparticles. The highest prol)ortions of these parti-cles are l)roduced by single growth cycles of phagein actively growing bacterial cells. These condi-tions require quantitatively exact combinationsof E. coli and phage, and such conditions are

seldom used for sim)le phage passage.We have examined another T4 phage straiin

which had been obtained from William D. AMc-Bride when he was a member of this department(the original source is unknown). This strain alsocontained occasional double-tailed particles.Therefore, production of such p)articles is notlimited to only one strain of T4 phage. We havenot examined other phage ty)es sufficiently toconclude whether or not they contain multiple-tailed heads.The biological significance of multiple-tailed

phage l)articles is not clear. Our attemlts to iso-

late pure lines containing multiple-tailed particleshave failed. This suggests that multiple-tailedphage production is the result of structural errorsin assembly rather than of genetic variation.There is nothing obvious in the morphology ofthese p)articles to suggest that they are deficientor unusual, other than their possession of super-numerarv tails. At this time, we can only saythat some T4 phage heads possess multiple sitesfavorable for the attachment of tail structures.No information is at hand to allow us to saywhether more than one tail is functional.

ACKNOWLEDGMENTS

This investigation was supported by PublicHealth Service grant Al 05382 from the NationalInstitute of Allergy and Infectious Diseases,anid by Research Career Development Award1-K3-CA-13,120 from the National Institutes ofHealth.

ADDENDUM

Since this work was completed, E. Kellenberger,at the 1965 Aninual Meetinig of the Society ofAmerican Microbiologists (Office of Naval Re-search Lecture), reported that he had observeddouble-tailed T coliphages.

LITER.ATUIRE CITED

BRADLEY, D. E. 1963. The structure of coliphages.J. Gen Microbiol. 31:435-445.

BRENNEiR, S., G. STREISINGER, R. W. HORNE,S. P. CHAMPE, L. BARNETT, S. BENZER, ANDM. W. REES. 1959. Structural components ofbacteriophage. J. Mol. Biol. 1:281-292.

PENTERIK, L., AND J. TAYLOR. 1962. The lowereddrop method for the preparation of specimensof partially purified virus lysates for quantita-tive electroni micrographic anialyses. Virology18:359-371.

SHARP, I). G. 1960. SedimentatioIn counting ofparticles via electron microscopy. Intern.Kongr. Elektronenimikroskopie, 4, Berlin,1958, Verhanidl., p. 542-548.

SMITH, K. O., W. D. GEHLE, AND M\1. D. TRous-DALE. 1965. Architecture of the adenovirus cap-sid. J. Bacteriol. 90:254-261.

SMITH, K. O., AND J. L. MELNICK. 1962. A methodfor staininig virus particles anid identifying theirniucleic acid type in the electron microscope.Virology 17:480-490.

WILLIAMS, R. C., AND I). FIIASElt. 1953. Morphol-ogy of the seven T-bacteriophages. J. Bacteriol.66:458-469.

WILLIAMS, R. C., AND 1). FRAsER. 1956. Structuralarid funictionial differentiation in T2 bacterio-phage. Virology 2:289-307.

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