Primary Adsorption Site of Phage PBS1: the

JOURNAL OF VIROLOGY, Mar. 1968, p. 256-264Copyright © 1968 American Society for Microbiology

Vol. 2, No. 3Printed in U.S.A.

Primary Adsorption Site of Phage PBS1: theFlagellum of Bacillus subtilis

LINDA M. RAIMONDO, NANCY P. LUNDH, AND RAFAEL J. MARTINEZ

Department of Bacteriology, University of California, Los An1geles, California 90024

Received for publication 1 December 1967

The adsorption of Bacillus subtiiis phage PBS1 was studied, and it was demon-strated that the primary adsorption site for this phage is the flagellum of B. subtilis.The capacity of flagella to function for motility may be lost without the loss of theircapacity to adsorb the phage and permit infection. Deoxyribonucleic acid injectionby the phage is inhibited by cyanide, suggesting the requirement for cellular energy

in the infection process.

Joys (9) and Frankel and Joys (7) recentlysuggested that the receptor site for phage PBS1(16) is the flagellum of Bacillus subtilis. Theycorrelated susceptibility to phage adsorption andinfection with motility of the organism, anddemonstrated that four flagella-less (fa-) mutantsand one flagellated but nonmotile mutant (mot-)of B. subtilis do not adsorb PBS1. They con-cluded that functional flagella were requiredfor phage adsorption. The extensive investiga-tions of Meynell (11) and of Schade, Adler, andRis (14) have shown that the chi phage of Sal-monella also exhibits flagellotropic properties.

Interest in this laboratory for the past severalyears has focused on the bacterial flagellum.For this reason, and because of the possiblebiological significance that phage adsorptionon the bacterial flagellum might have, a moreextensive investigation on the general propertiesof phage PBS1 was undertaken.

MATERIALS AND METHODS

Organisms and growth medium. B. subtilis strainsSB19, SBI71, 168, and SC3, 4, 6, and 23 were used inthese studies. SB19 is a streptomycin-resistant proto-trophic derivative of 168. SB171 is afla- derivative of168 (trp) obtained from E. Lederberg. Strains SC3,4, 6, and 23 are paralyzed mutants of 168 whoseflagella lack the long period helix (Martinez et al.,submittedfor publication). The other fla- mutants usedwere from our collection. The medium used forpropagation and dilution of cells was TY broth (12).Bottom layers and soft agars for plating contained1.5% and 0.6% agar in TY broth, respectively. Themineral salts solution from the minimal medium de-scribed by Spizizen (15) was used for dilution of thephages. Cultures of SB19 were grown in TY broth at37 C to a cell density of 108 to 5 X 108 per ml anddiluted as required. Under these conditions, morethan 90% of the cell population showed translational

motility. Bacteria were grown at 37 C with vigorousaeration. A modification of the assay for free phagedeveloped by W. B. Pritkin (Ph.D. Thesis, Univ. ofCalifornia, Los Angeles, 1967) was found to be themost reproducible of all those tried. One milliliter ofdiluted phage suspension (or infected bacteria) wasmixed with 0.05 ml of indicator cells (actively motileB. subtilis SB 19, at a density of 5 X 108 per ml);10 min was allowed for adsorption. Then, 0.1 ml ofthe mixture was added to 2.5 ml of soft agar held at50 C; 0.15 ml of the SB19 suspension was added andthis mixture was poured over the 1.5% TY agarplate as a uniform layer. Fresh bottom-layer plateswere used with an adsorbent pad on the lid. The plateswere incubated right side up in a humidified incu-bator at 26 C for 12 to 14 hr. Because of inherentdifficulties in the enumeration of PBS1 particles, afree phage control was included in all experiments;this control is labeled "phage control" in the tables.

Preparationi of protoplasts. Exponentially growingcultures of SB19 were harvested and washed bycentrifugation with 10-3 M tris(hydroxymethyl)-aminomethane (Tris) buffer (pH 7.6). The cells wereresuspended in a solution containing lysozyme (100jug/ml), sucrose (200 mg/ml), and Tris (100 Ag/ml,pH 8.0). The suspension was incubated at 37 C forapproximately 30 min, during which time the con-version of vegetative cells to protoplasts was followedby phase-contrast microscopy. The protoplasts weresedimented by centrifugation at 5,900 X g for 10 minand resuspended gently in TY broth containing 20%sucrose.

Deflagellation and flagella regeneration. Activelymotile cultures of SB19 or 168 were deflagellated inthe cold in a Sorvall Omnimixer for 1 min with arheostat setting of 100 v. The cells were sedimented bycentrifugation, washed once with TY broth, andresuspended in TY broth. The cell suspension wasincubated at 37 C on a reciprocal shaker to allowflagella regeneration.

Phage adsorption was measured either by thechloroform method or by centrifuging the bacteria-

256

ADSORPTION SITE OF PHAGEPBS2

phage complexes at 3,600 X g for 15 min and assayingthe supernatant solution for free phage (2).

Anti-PBS1 antiserum with a K value of 2,450 was agift of W. Pritikin. Anti-flagellar antiserum was pre-pared by the method of Ada et al. (1) and was puri-fled by the method of Grant and Simon (8).

Electron microscopy was performed with a HitachiHU 11A microscope. Specimens were stained with2% uranyl acetate.

RESULTS

Growth characteristics of PBSJ. One-stepgrowth curves of PBS1 on SB19 grown on TYbroth at 37 C showed a latent period of 35 to40 min and an average burst size of 23. Thesedata agree well with those reported by Taka-hashi (16).

Figure 1 shows the kinetics of adsorption ofPBS1 by SB19 at various phage to bacteriaratios (P/B). In these experiments, air wasvigorously bubbled through the culture, a con-dition required for optimal efficiency of adsorp-tion and infection. The adsorption rate constantcalculated from the experiments with P/B ofless than 1 was 1.44 x 10-8 ml/min. This rateis about 10-fold faster than that reported for thechi phage (11, 13) and for most B. subtilis phagesexcept SP1 3 (4). At P/B greater than 1, the ad-

1000

0

C,)

WA

O_

0

z

TIME (min)FIG. 1. Kinetics of adsorption of PBSJ by Bacillus

subtilis SBI9. To actively motile SBJ9 cells in TYbroth at 37 C phage was added at various P/B ratiosand the mixture was bubbled vigorously with air. Attime intervals, samples were removed and assayed forfree phage by the chloroform method. (A) P/B =

2.5; (A) P/B = 10; (0) P/B = 0.02; (0) P/B =

0.05.

sorption kinetics show a biphasic response. Theslope of the curves after the initial 2 to 3 min ofadsorption, however, depended on the P/B used.From consideration of the dimensions of the

head of this phage (6), one would expect thedeoxyribonucleic acid (DNA) content to begreater than that of Escherichia coli phage T5,and indeed a value of 2 X 108 daltons has beenrecently reported (5). Lanni (10) has shown that20 to 25 min is required for formation of phageT5-bacteria complexes which are insensitive toshear. The kinetics of infection of SB19 by PBS1were measured by deflagellating cells after phageadsorption. At time zero, actively motile bacteriawere mixed with phage at P/B of 0.4 in TYbroth and incubated at 37C with vigorousaeration. At 5-min intervals, 0.5-ml sampleswere removed and diluted with 4.5 ml of coldsalts solution. The cells were immediately de-flagellated; 0.05 ml of anti-PBS1 antiserum wasadded and, after dilution, the number of infectedcenters was determined. Infection was relativelyrapid, being complete within 10 min (Table 1).Similar experiments in which samples were takenat 1-min intervals showed that infected centerswere not formed prior to 4 min after mixing.

Effect of cyanide on adsorption and infection.KCN at a final concentration of 10-3 M causesimmediate cessation of motility of B. subtilis.Table 2 shows the results of adsorption of PBS1by SB19 rendered nonmotile by 10-3M KCN.Adsorption was measured by the chloroformmethod. It appears that cells rendered non-motile by KCN do not adsorb phage. If adsorp-tion was measured by centrifuging the cyanide-treated cell-phage complex, however, only 10%of the input phage remained in the supernatantfluid (Table 3). No reduction in phage titer wasfound if E. coli or killed SB19 were substitutedfor the motile SB19 cells. These data suggest

TABLE 1. Kinetics of in,ection of SBI9 by PBSJPTime Infected bacterial centers/ml

min

0 <1X1055 1.2X 108

10 2.3 X 10815 2.1 X 10820 2.1 X 108

a To 5 X 108 cells (per ml) of SB19 in TY brothat 37 C was added PBSl to give a final concentra-tion of 2 X 108/ml. At 5-min intervals, 0.5-mlsamples were diluted into 4.5 ml of salts solution,and the bacteria were deflagellated. Anti-PBSIantiserum was added to the samples, the mixtureswere then diluted, and the number of infectedcenters were determined.

257VOL. 2, 1968

RAIMONDO, LUNDH, AND MARTINEZ

TABLE 2. Effect ofKCN on the adsorption of PBSJby SBI9 measured by the CHCl3 methoda

Sample Unadsorbed phage

Phage control 8.4 X 107Cell control (no CN-) 2.0 X 105Cells + CN- 8.1 X 107

a To 1.0 X 108 cells (per ml) of SB19 in TYbroth at 37 C was added KCN to 10-3 M; the cul-ture was shaken for 3 min in a stoppered tube.PBS1 was added to give a P/B of 1, and the mix-ture was shaken for 15 min more at 37 C. Thenumbers of unadsorbed phages were then deter-mined.

TABLE 3. Effect ofKCN on the adsorption of PBSJby SBI9 measured by the centrifugation

methoda

Sample Unadsorbed phages


a See Table 2 for details. Adsorption was meas-ured by centrifuging the bacteria-phage mixtureat 3,600 X g for 15 min and assaying the super-natant fluid for free phages.

that chloroform separates any phage-bacteriacomplexes formed under KCN. To test thispossibility, both the centrifugation and chloro-form methods of measuring adsorption wereused in the same experiment. Actively motilebacteria were incubated for 15 min with phages(P/B = 1); chloroform was added to one sampleand free phage was measured. The other samplewas centrifuged; the supernatant fraction wasassayed for free phage, and the sediment wasresuspended in the initial volume of salts solu-tion containing 10-3 M cyanide and anti-PBS1antiserum. One-half of the volume of the re-suspended sediment was deflagellated and bothparts of this sample were diluted and plated forinfected centers. The data (Table 4) show thatsome adsorption takes place in the presence ofcyanide, as evidenced by the facts that there wasa reduction in the number of free phage in thesupernatant fraction and that infected centerswere formed by the sedimented cells. The dataalso suggest that infection does not occur in thepresence of cyanide, since the sedimented cellsdid not form infected centers after deflagellation.

Adsorption site for PBSJ. The structure ofphage PBS1 was described in detail by Eiserling(6). A unique feature of this phage is the threehelical tail fibers with a pitch of 350 A. Afterphage adsorption, these helical tail fibers are

found wrapped around the bacterial flagellum(Fig. 2 and 3). There does not appear to be asingle specific site on the flagellum requisite forattachment, since at high P/B the entire lengthof the flagellum can be covered with phages (Fig.3 and 4).To examine further the requirement for flagella

in the adsorption process, actively motile cells,deflagellated cells, and deflagellated cells whoseflagella had been permitted to regenerate weretested for their ability to adsorb PBS1. Freshlydeflagellated cells did not adsorb the phage to anyextent, whereas bacteria whose flagella had re-generated did adsorb the phage (Table 5). Iso-lated flagella, either intact or prepared byshearing, did not reduce phage titer, nor didthey compete with motile cells in the adsorptionprocess. Occasionally, we observed phagesattached to isolated flagella, but never a reduc-tion in phage titer, suggesting that, if adsorptionto isolated flagella does occur, it is reversible.

It was of interest to determine whether therelative length of the flagella was a critical factorin the adsorption and infection processes.Samples of a deflagellated culture of SB19 weredispensed into tubes and incubated with aerationat 37 C in TY broth. Under these conditions,B. subtilis will regenerate flagella maximallywithin 20 to 25 min. At timed intervals, phagewas added to duplicate tubes (P/B = 1) andincubation was continued for 5 min more topermit adsorption. One of the duplicate tubeswas treated with chloroform, and free phage wasdetermined; the other sample was again de-flagellated, anti-PBS1 antiserum was added toremove unadsorbed phage, and the number ofinfected centers in this sample was determined(Table 6). It appears that the adsorption process

TABLE 4. Effect ofKCN on the adsorption of PBSIby SBI9 measured by a combination of the CHCl3

and centrifugation methodsa

Sample (supernatant) phages


Sample (sediment) centers

Cell control (no CN-) >1.0 X 108Cells + CN- 3.7 X 107Deflagellated cells + CN- <1.0 X 106

aSee Table 2 for details. Centrifugation ofphages in the presence of E. coli or killed SB19did not reduce the titer of phages from the super-natant fluid.

258 J. VIROL.

Fio. 2. PBS) adsorbed to the flagella of Bacillus subtilis SBI9. Negatively stained with 2% uranyl acetate.X 120,000.

FIG. 3. PBS) adsorbed to Bacillus subtilis SBI9. Negatively stained with 2% uranyl acetate. X 61,000.259

-n.


FIG. 4. PBSJ adsorbed to Bacillus subtilis SC6. Negatively stained with 2% uranyl acetate. X 22,000.

TABLE 5. Adsorption of PBSI to deflagellated cellsand cells with regenerated flagellaa

Sample UnadsorbedSample ~~~~phagesControl cells ...................... 2.0 X 104Deflagellated cells ................. 6.8 X 106Cells with regenerated flagella ... 2.0 X 104Isolated flagella.................... 8.0 X 106Phage control ..................... 8.4 X 106

a To 107 cells (per ml) of SB19 which had beenfreshly deflagellated, cells whose flagella hadregenerated, control cells, and isolated flagellawas added PBSl to a P/B of 1. The mixtures wereincubated for 15 min at 37 C in TY broth, andunadsorbed phages were measured by the CHC13method.

is more rapid as the relative length of the flagellumincreases, i.e., the longer the time permitted forflagella regeneration, the greater the reductionin free phage.The complexing of specific antiflagellar anti-

body to the surface of flagella results in immobili-zation of the bacteria. We determined whetherflagella coated with antibody could act as re-ceptors for PBS1. A culture of SB19 was diluted

TABLE 6. Effect of relative flagella length onadsorption and infection of SBI9 by PBSJa

Time for flagella Unadsorbed Infected bacterialregeneration phages centers

min

0 4.2 X 107 <1.0 x 10510 4.4 X 107 7.7X 10615 2.0X 107 4.8X 10620 9.6X 106 1.2X 10730 8.0X 106 1.5X 10750 3.0X 106 1.5X 107

a SB19 cells were deflagellated, washed oncewith TY broth, suspended in TY broth in separatetubes, and incubated at 37 C with aeration. Attime intervals, phage was added to duplicatetubes (P/B = 1) and incubation was continuedfor 5 min more. One of the tubes was assayed foradsorption by the CHCl3 method, and the othertube was deflagellated again, anti-PBSI antiserumwas added, and the number of infected bacterialcenters was determined.

to contain approximately 107 cells/ml so that,upon addition of antiflagellar antibody, the cellswould not agglutinate. After mixing bacteriawith purified antiflagellar antibody for 15 min at

260 J. VIROL.

ADSORPTION SITE OF PHAGE PBS1

37 C, phage was added at a P/B ratio of 0.6 to1.0 and incubation was continued. Samples weretaken promptly at the time of addition of thephage and after 15 min of incubation. Phageadsorption was measured by the chloroformmethod. No adsorption of phage was detectedwhen flagella were first complexed with theirspecific antibody (Table 7). The purified antibodypreparation did not exhibit any neutralizingeffect on the phage, since no reduction in phagetiter was observed when free phage were treatedwith antibody under the same conditions.

It has been demonstrated by Weilbull (17) thatprotoplasts of flagellated bacilli retain theirflagella yet are nonmotile. We measured the ad-sorption of PBS1 by protoplasts of SB19 todetermine whether the cell wall was involved inthe adsorption process. The protoplast prepara-tions in 20% sucrose were incubated with phageat P/B of 0.2 on a reciprocal shaker, and ad-sorption was measured by the chloroform tech-nique. To determine whether the protoplastpreparations retained significant amounts of cellwall, the adsorption of phage SP8 was also meas-ured. Phage SP8 is known to adsorb to the cellwall of B. subtilis (W. R. Romig, personal com-munication).It is evident (Table 8) that PBS1was adsorbed by the protoplasts, whereas therewas no reduction in the titer of SP8. Althoughincubation was carried out for 60 min, no burstswere detected in any of the experiments. However,one cannot conclude that infection and phagematuration did not occur. Bursts obtained fromprotoplasts may be as low as 5% of the normal(3), so that a similar reduction of the burst forPBS1 might not be detectable. Alternatively,infection and phage maturation may not havetaken place in the protoplast preparations. Fur-ther, under the conditions employed there was areduction in the optical density of the protoplastpreparations, suggesting that some lysis was oc-curring.

TABLE 7. Effect of specific antiflagellar antibodyon PBSJ adsorptions

Sample Time Unadsorbed

~~~phages

min

Control (no antibody) 0 5.0 X 10615 1.5 X 106

Cells treated with antiflagel- 0 6.7 X 106lar antibody 15 6.9 X 106

a SB19 cells (5 X 106/ml) were treated withspecific antiflagellar antibody for 15 min at 37 C;phage was then added (P/B = 1.2) and incubationwas continued for 15 min more. Unadsorbedphages were measured by the CHCl3 method.

TABLE 8. Adsorption of PBSJ by protoplastpreparations of SB19a

Unadsorbed phagesTime

PBS1 SP8

min

0 1.7 X 108 3.8 X 1071 1.4X 1085 6.9 X 107 3.6 X 107

15 2.9 X 107 4.4 X 10730 9.8 X 106 4.0 X 10745 5.0 X 103

a Protoplast preparations of SB19 in TY brothcontaining 20% sucrose were mixed with PBS1(P/B = 0.2) at 37 C in a reciprocal shaker. At timeintervals, samples were assayed by the CHC13method for free phage. Phage SP8 was used ascontrol. Phage SP8 was found to adsorb normallyto vegetative cells in 20% sucrose.

The possibility was considered that interactionbetween the helical tail fibers of the phage and theflagellum might alter the attachment mechanismof the phage. Attachment to the cell wall of bac-teria could conceivably then follow. To test thispossibility, 5 X 107 SB19 cells were incubatedfor 5 min with phage at P/B of 40, at which time5 x 108 cells of SB171 (fla-) were added andincubation was continued for 10 min more.A control tube did not receive the SB171 cells.Anti-PBS1 antiserum was then added, and thenumber of infected centers was determined. Ifthe attachment mechanism of PBS1 were modi-fied by reaction with the flagellum, we wouldanticipate that the number of infected centerswould be greater in the tubes having SB171cells, owing to transfer of modified phages fromSB19 flagella. The numbers of infected centers ob-tained in the tubes having SB171 (30.3 X 106)were essentially the same as those in the controltubes (36.9 x 106).

Frankel and Joys (7) reported that four non-flagellated mutants of B. subtilis would notadsorb PBS1. We extended this observation with>50 fla- mutants of 168, and confirmed thatnone adsorbed the phage. Frankel and Joys (7)also reported that a paralyzed mutant of B.subtilis did not adsorb PBS1, and concluded fromthis observation that functional flagella were re-quired for PBS1 adsorption. However, we foundthat PBS1 adsorption does occur with four mu-tants of 168 which are nonmotile because theflagella formed by these cells lack the long periodhelix (Fig. 4). Table 9 gives the data obtained foradsorption of PBS1 by mutant SC4. The mutantslacking the long period helix not only adsorb thephage but are also susceptible to phage infection

261VOL. 2, 1968


(Table 10). Thus, the capacity of flagella tofunction for motility may be lost without lossof their capacity to adsorb phage and permitinfection.We tested the effect of adsorption of PBS1 by

SB19 on the motility of the cells. Meynell, usingchi phage (11), and Frankel and Joys, usingPBS1 (7), reported the cessation of motility of thebacteria when mixed with phage at high P/B.Both of the phage preparations employed bythese workers were crude lysates. Phages purifiedby banding in CsC1 were used in our experi-ments at various P/B ratios. The cultures ofSB19 used showed more than 95% motile cells.Cells were mixed with phage, and the fraction ofthe cells remaining motile was estimated by exami-nation in a phase microscope. The time intervalbetween mixing bacteria and phage and scoringfor motility was about 15 to 30 sec. At P/B of 1,there were approximately 50% motile cells; atP/B of 2.5, there were 10 to 25% motile cells;at P/B of 5, no motile cells were observed.These numbers agree fairly well with the Poissondistribution for phage adsorption, suggesting

TABLE 9. Adsorption of PBSI by Bacillus subtilisSC4a

Sample Unadsorbed phages

SB19 <1.0x105SC 4 6.0 X 106Phage control 1.2 X 108

a Strains SB19 and SC4 were mixed with phage(P/B = 0.1) for 20 min at 37 C with vigorousaeration, Phage adsorption was measured by theCHCl3 method.

TABLE 10. One-step growth of PBSJ on Bacillussubtilis SC6a

Time Infected bacterial centers

min

15 1.9 X 10620 2.2 X 10625 2.1 X 10630 2.3 X 10635 2.0 X 10640 2.3X10645 3.2 X 10650 4.5 X 10655 1.2 X 10760 1.6x10770 3.0 X 107

a Strain SC6 was mixed with phage (P/B 1)in TY broth at 37 C with vigorous aeration. Attime intervals, samples of the culture were re-moved and one-step growth characteristics weremeasured by the method of Adams (2).

that one plaque-forming unit when attached toone flagellum on the bacterial cell induces arapid cessation of motility.

DISCUSSIONThe data presented in this communication

clearly demonstrate that phage PBS1 attaches tothe flagella of B. subtilis. These data may be sum-marized as follows.

In electron micrographs, the helical tail fibersof the phage are clearly seen, wrapped aroundthe flagella. Rarely, if ever, are the phages seenattached to the cell body, especially if P/B ratiosof less than 10 are used.The fla- mutants do not adsorb the phage.

This in itself would not be compelling evidencethat flagella are involved in phage adsorption.It is possible that fla- mutants may have alteredcell envelopes, perhaps due to a secondary muta-tion or because the absence of flagella modifiesthe conformation of the envelope.

Deflagellated cells do not adsorb PBS1, norare they susceptible to phage infection. This ismore direct evidence for the involvement offlagella in the adsorption process, especially whencoupled with the observation that the same cul-ture, when permitted to regenerate flagella, be-comes susceptible to phage infection.When the surface of flagella is coated with

specific antibody, phage adsorption is completelyinhibited. This evidence might suggest that func-tional flagella are required for phage adsorption.This interpretation appears highly unlikely whenone considers that paralyzed mutants, as well ascells rendered nonmotile by treatment withcyanide, adsorb phage with essentially the samekinetics as do normal cells. Alternative explana-tions which appear more tenable are that coatingthe flagellum with antibody increases the func-tional diameter of the flagellum, thus preventingthe helical tail fibers of the phage from attaching,or that attachment sites on the flagellum arecoated with antibody, thus hindering the adsorp-tion mechanism of the phage.

Protoplasts of B. subtilis retain the ability toadsorb PBS1, yet are not susceptible to phageSP8. The rate of adsorption of PBS1 by theprotoplasts is slightly slower than that observedwith vegetative cells. This may be ascribed to thefact that some of the flagella are lost in the processof preparing the protoplasts and, further, theincubation of the phages with protoplasts wascarried out under suboptimal adsorption condi-tions, i.e., gentle shaking in 20% sucrose ratherthan under vigorous aeration. Nevertheless,protoplasts are nonmotile and are devoid of cellwall. This suggests that cell wall material, as such,does not play an essential part in the adsorption

262 J. VIROL.

ADSORPTION SITE OF PHAGE PBSl

process. It also lends further support to the ob-servation that nonfunctional flagella can serve asattachment sites for PBS1.These data, then, lend credence to the conclu-

sion that it is on the flagellum that initial contactis made between PBS1 and the bacterium. Thedata also demonstrate that functional flagellaare not prerequisite for phage adsorption, sincecells rendered nonmotile by cyanide, by produc-tion of nonfunctional flagella, or by protoplastingall served as suitable adsorbers of the phage. Thisis contrary to the report by Frankel and Joys(7) and the observations of Meynell (11) andSchade and Adler (13), who proposed that func-tional flagella were requisite for phage adsorption.Further studies on the chi phage by Schade, Adler,and Ris (14) revealed that this phage alsoadsorbs to nonfunctional flagella.The time required for infection of B. subtilis by

PBS1 appears to be considerably longer thanSalmonella infection by phage chi (11). No in-fected bacteria were observed prior to 4 min(Table 1) after mixing PBS1 with SB19, and yetMeynell (11) reported that chi infection of Sal-monella takes place within 1 min after mixing.The time discrepancy may be due to the relativelylarge amount of DNA that must be transferredby PBS1 in comparison to the DNA in chi (2 x108 versus 4 X 106 daltons, respectively).

It is tempting to propose from the data pre-sented in Table 4 that DNA injection by PBS1may require cellular energy. It is clear that injec-tion is inhibited by cyanide, since the cyanide-treated bacteria did not become infected if theywere deflagellated prior to plating. Further, thecyanide experiments also suggest that there maybe two steps to the adsorption process: a cyanide-insensitive reversible step, after which infectivephage may still be removed by a chloroformtreatment, followed by an irreversible step thatis prevented by cyanide. It would appear that thereversibility of the initial adsorption step is notmerely due to lack of translational motility in thecyanide-treated culture, since phage adsorptiononto other nonfunctional flagella (protoplasts,paralyzed mutants) was measured by the chloro-form method and was not found to be reversible.A prime question which has not been resolved

by these studies is the route by which PBS1DNA enters the bacterial body. There are severalmechanisms which may be considered. (i) Directinjection of PBS1 DNA into the flagellum is onepossibility. If we consider, however, that a centralcanal has not been observed in the flagella ofB. subtilis (although such a canal might in-deed exist), and that phages with contractedsheaths and empty heads are rarely seen attachedto the flagella, then this proposal seems unlikely.

Further, one would expect that injection ofDNAinto the flagellum might result in a morphologicalalteration of the flagellum at the site of injection,and no such changes have been observed. (ii) ThatDNA injection does not occur via a modifiedtransfection mechanism (12) after phage adsorp-tion was ruled out by the fact that the infectiousprocess is completely deoxyribonuclease- resistant(W. B. Pritikin, Ph.D. Thesis, Univ. of California,Los Angeles, 1967). (iii) The fact that non-flagellated cells do not become susceptible tophage infection also suggests that initial adsorp-tion to the flagellum does not alter the attach-ment mechanism so that a secondary adsorptionto cell walls can follow. This experiment does notrule out the possibility that initial attachment tothe flagellum of one cell may be followed bytransfer of the phage to the wall of the same cell.Indeed, this is in essence the mechanism proposedby Schade, Adler, and Ris (14) for chi phageDNA transfer. These workers suggested thatchi phage attaches to the flagella of E. coli andthen slides along the flagellum to the base, whereDNA injection occurs. They suggested that themovement of the flagellum is responsible for, oraids, in the sliding of the phage down the lengthof the flagellum, although they presented evi-dence demonstrating adsorption to nonfunctionalflagella. Although we have no evidence whichsupports this hypothesis, it is the only tenableone at present. We would, however, expect thatthe shorter the flagellum, the more rapid the in-fection, since the phage would have a shorterdistance to travel from the site of attachment.This does not appear to be the case (Table 6).Further, there are arguments which may be in-voked against such a mechanism. A moving bodyproduces a column of fluid which moves in adirection opposite to that of the direction ofmotion of the cell. This moving column of fluidwould create an impedance against the sliding ofthe phage particle toward the cell body. However,it is also known that there exists a fixed, non-moving boundary of fluid which surrounds mov-ing objects. Although the functional area coveredby such a boundary has not been clearly measuredfor bacterial systems, it may be sufficiently largeto eliminate the argument of impedance posedabove. Thus, the question still remains: how doesPBS1 infect B. subtilis?

Finally, the phenomenon of immobilizationof B. subtilis by CsC1-purified phages merits dis-cussion. It appears from the data that adsorptionof one phage particle to one flagellum of B.subtilis renders the entire complement of flagellanonfunctional. This is a rather dramatic observa-tion, especially when one considers that thisbacterium possesses 15 to 20 flagella and that the

263VOL. 2, 1968


time required to immobilize the cell is less than30 sec. Although infected bacterial centers donot appear before 4 min after addition of phage,we considered the possibility that the cessationof motility might be due to the initiation ofDNA injection with some concomitant channel-ing of energy. To test this possibility, phage ghostswere prepared by osmotically shocking PBS1 byrapid dilution from 35% glucose. The deoxy-ribonuclease-treated ghosts immobilized the bac-terial cells as effectively as did intact phages.The fact that the entire complement of flagellais immobilized suggests that the functioning of allthe flagella of B. subtilis is under the control ofone "motor."

ACKNOWLEDGMENTS

This investigation was supported by NationalScience Foundation grant GB 4633. One of us(L. M. R.) was supported by National Science Foun-dation Undergraduate Research Participation grants(GY 3102, GY 938, GE 6135); N. P. L. holds aNational Institutes of Health predoctoral fellowship(5-F1-GM-29,834).

LITERATURE CITED

1. ADA, G. L., G. J. V. NOSSAL, J. PYE, AND A.ABBOT. 1964. Antigens in immunity. I. Prepara-tion and properties of flagellar antigens fromSalmonella adelaide. Australian J. Exptl. Biol.Med. Sci. 42:267-282.

2. ADAMS, M. H. 1959. Bacteriophages. Inter-science Publishers, Inc., New York.

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Primary Adsorption Site of Phage PBS1: the