PBP1: A flagella specific bacteriophage mediating transduction in Bacillus pumilus

47, 743-752 (1972)

VIROLOGY

PBPI: A Flagella

Specific

Transduction

in Bacillus

PAUL Department of Biological Sciences, University

Bacteriophage

Mediating

pu~i~us

S. LOVETT

of Maryland

Baltimore

County,

Catonsville,

Maryland

R1288

Accepted October $1, 1971

Bacteriophage PBPl was purified by dilferential and equilibrium centrifugation and characterized with respect to morphology, adsorption properties, and host range. PBPl is a phage witch 5 flexible non~ontractile tail. The adsorption of PBPl to cells of Buciltus ~u~~ZU~ NRRL B-3275 (BpBl) in penassay broth follows first-order kinetics with an average adsorption velocity constant at 37” of 6.1 X 10-Qml-l min-I. The activation energy for the adsorption reaction is approximately 24 kcal. On the basis of the following properties, it is concluded that PBPl interacts with the bacterial flagellum during the adsorption process: (1) PBPl does not adsorb to nonflagellated mutants derived from three phage-sensitive strains of B. pumilus. (2) Mechanical deflagellation of BpBl renders the cells incapable of adsorbing PBPl until the flagella have regenerated. (3) Nonfla~ellated mutants of B. punli~~s ATCC 7065 can be isolated by selecting for resistance to PBPl. (4) Addition of a high multiplicity of PBPl particles (>20) to motile BpBl cells results in immobilizatio~l of the cells. (5) Electron microscopical examinations of mixtures of PBPI and BpBl cells demonstrate that the phage are capable of attaching to flagella. Comparison of the host ranges of PBPl and PBS1 on thirty-two strains of B. pumilus divides the strains into three groups. One group is sensitive to PBPl and PBSl, one group is sensitive t,o PBPl but resistant to PBSl, and one group is resistant to both viruses. The possible use of PBPl and PBS1 for distinguishing flagella types in 3. pumilus strains is discussed. PBPl mediates generalized transduction in BpBl at a frequency on t,he order of 10-8 transd~~~tants per plaque-forrniI1~ unit,. Preliminary genetic studies indicate that cotransducible markers in BpBl are more weakly linked by PBPl transduction than by PBS1 transduction. INTRODUCTION

Two bacteriophages have been described which do not infect nonflagellated mutants of phage-sensitive bacteria. One of these viruses, chi, infects certain motile represent,atives of the Enterobacteriaceae, includingstrains of Salmonella (Meynell, 1961), Serratia (Iino and Mitani, 1967), and Escherichiu coli (Schade and Adler, 1967). The other phage, PBSl, infects strains of Bacillus subtilis (Frankel and Joys, 1966; Joys, 1965; Raimondo et at., 1968; Takahashi, 1963), B. ~~~~Z~~ (Lovett. and Young, 1970), and at least one strain of B. l~~he~i~~r~is (ATTC 8480; unpublished observations). Alt’hough t,he mechanism of infection by

t’hese viruses has not been completely elucidated, several lines of evidence support the idea that both viruses interact with the bacterial flagellum during the adsorption process (~~eynell, 1961; Raimondo et al., 1968; Schade et ab., 1967). The potential usefulness of these so-called “flagellatropic” viruses for detecting differences bet.ween flagella t’ypes is suggested by t,he correlation between Salmonella strains which are sensitive to chi and the antigenic composition of their flagella (Meynell, 1961). During an examination of the transdu~ix~g properties of a bacteriophage designat~ed PBPl, it became evident t,hat. t$headsorption properties of this virus were similar to those 743

Copyright

0

1972 by Beademic

Press,

Inc.

744

LOVETT

reported for chi and YBSl. Because of the current interest in the structure and composition of the flagella of B. pumilus (Abram et al., 1970; Mitchen and Koffler, 1969; Sullivan et al., 1969; Suzuki and KoWer, 1970; Oiler et al., 1971) a detailed examination of the adsorption characteristics of PBPl was undertaken. The results of this investigalion demonst,rate that PBPl interacts with the bacterial flagellum during the adsorption process. The availability of two dist,inct flagellatropic phages, PBS1 and PBPl, which have overlapping host ranges among strains of B. pumilus may provide a convenient method for distinguishing flagella types in this species. A preliminary report of t)hisstudy has been presented (Love& 1971). MATERIALS

AND

METHODS

Bacteria. Bacillus pumilus NRRL B-3275

(BpBl) was the propagating host and indicator for PBS1 and PBPl unless otherwise specified. Physiological and genetic characteristics of BpBl have been described (Lovett and Young, 1969, 1970, 1971). Two nonmotile mutants of BpBlO (a tryptophanrequiring auxotroph derived from BpBl; Lovett and Young, 1970) were isolated after ethyimethanesulfonate treatment of BpBlO spores (Lovett and Young, 1971) as colonies unable t,o swarm on semisolid agar. The nonflagellated character of these mutants, designated BlO NFl and BlO NF2, was determined by electron microscopy. Two nonflagellated mutants of B. pumiZus ATCC 7065 were isolated and characterized in an identical manner and are designated 7065 NFl and 7065 NF2. The other B. pumilus strains used in this study were acquired from the following sources: BD-2002 from D. Dubnau; American Type Culture Collection (ATCC) strains 7065, 1, 18, 70, 71, 72, 98, 945, 4510, 4520, 4522, 6631, 6632, 7061, 14884 and National Collection of Industrial Bacteria (NCIB) strains 8600, 8738, 8982 from V. Gage; Nathan R. Smith (NRS) strains 331, 637, 706, 939, 337, 333, 576, 896 and ATCC strains 19646 and 12140 from R. E. Gordon; B. pumilus M from M. Simon; B. pumi2us 101 and its nonflagellated mutants NF2, Pl, P2, P3 and P8 from G. Fitzgerald and H. Koffler. B. subtilis 168 and W-23

were obtained from F. E. Young and B. 9945 A from V. Gage. The auxotrophic mutants of BpBl have been described (Lovett and Young, 1970, 1971). Bacteriophage. The bacteriophage B 12140 was obtained from the ATCC, Rockville, Maryland. The phage was initially assayed on BpBl. A slllgle, morphologically typical plaque was starting material for the virus preparations used in this study. This phage is referred to as PBPl. Media and growth conditions. Cells were grown in antibiotic medium No. 3 from Difco (penassay broth; PB) at 37” with rotary shaking. Viable counts were plated on tryptose blood agar base (TBAB; from Difco) and counted after 18 hr at 37’. The minimal medium was that previously described (Lovett and Young, 1970). licheniformis

Bacteriophage techniques and puri$cation.

The plaque assay used for PBPl and PBS1 was that previously described for PBS1 (Lovett and Young, 1970) except that the agar overlays were incubated at 25’. The basal agar layer used in t’he plaque assay was poured and allowed to solidify at room t,emperature, held at 37” for approximately 20 hr, and stored at 4’ for no longer than 4 days prior to use. PBS1 was propagated as previously described (Lovett and Young, 1970) and was not further purified. PBPl lysates were prepared by scraping the semisolid agar overlays from confluently lysed plates into TMA buffer (0.05 M Tris-acetabe + 0.01 M magnesium acetate i- 0.5 % bovine serum albumin, pH 7.15; Lovett and Shockman, 1970a). Material pooled from 20 plates was centrifuged at low speed (5000 g, 10 min) and the supernat’ant fraction (30 ml) was incubated at 37” for 1 hr with DNase and RNase (each at 20 clg/ml). After centrifugation of the lysate (44,000 a. 1.5 hr), the pellet was overlaid wit’h 2 ml of TMA buffer held at 4” for 18 hr, resuspended and centrifuged at low speed. The supernatant fraction (2 ml) was mixed with a cesium chloride (Harshaw) solution in TM buffer (TMA minus albumin) to give an average buoyant density of 1.50 g/cm3 (determined by refractometry; Vinograd and Hearst, 1962), and centrifuged at 30,000 rpm for 36 hr at 5” in the SW-50 rotor. Fractions were collected, their refractive

FLAGELLA-SPECIFIC

TRANSDUCING

indices determined and each was processed as previously described (Lovett and Shockman, 1970a). Recovery of virus averaged approximately 50 %. Purified virus stored in TMA over chloroform at 4” retained 85% of its infectivity for 2 months. Unless otherwise specified, only purified PBPl was used in the present experiment’s. PBPl adsorption was assayed by mixing 1 ml of exponentially growing bacteria (grown at 37” in PB) with lml of PB suspended phage (m.o.i. =
PHAGE

745

ATCC 7065 on which PBPl produces clear plaques. The plaque assay for PBPl is sensitive to variat’ions in the moisture content of the basal agar layer. Plates which are too dry cause a reduction in the apparent phage titer and a reduction in the diameter of the plaques. Plates containing excess moisture cause smearing of the plaques. The regimen for drying plates prior to their use (see Materials and Methods) was found to give optimal results in the plaque assay for PBPl and PBSl. PBPl infect,ivity bands in a single peak in CsCl gradients at a buoyant density of 1.490 (f0.005) g/ cm3.The morphology of a typical phage particle purified through gradient centrifugation is shown in Fig. I. PBPl has a flexible tail of approximately 200 nm in length. The t’ail has never been observed in a shortened or contracted state. The head diameter is approximately 65 mn. Based on these characteristics, PBPl is similar to the morphologic phage type designated as group B by Bradley (1967). A structural characteristic of PBPl is the flexible tail fiber(s) which generally exhibit’s an apparent helical B. pumilus

RESULTS

Properties of PBPI.

Bacteriophage PBPl produces turbid plaques on BpBl and on most phage-sensitive bacteria (see below). An exception is

FIG. 1. Morphology of bacteriophage PBPl stained with 1% many1 acetate. The bar represents 0.1 &f.

746

LOVETT

O.l32

36 l/T

"K (x10+)

FIG. 2. Temperature dependence of the adsorption of PBPl to BpBl. Each point represents the average of at least two separate determinations performed at cell concentrations ranging from 4 to 7 X 107/ml. Replicate determinations were within &15% of the average. Numbers within the figure indicate the temperature (in degrees centigrade) at which the adsorptions were performed. The activation energy calculated between 10” and 30” is 24 kcal.

arrangement. I have not determined whether the tail fiber assembly consists of one or more individual fibers. Adsorption of PBPl to BpBl follows first-order rcact)ions kinetics with an average adsorption velocity constant (K value) of 6.1 X 1O-g ml-l min-l. This K value is valid within &15 % over the range of cell concent>ration tested (1 to 8 X lO’/ml). The rate of adsorption of PBPl is highly temperature dependent. The K values determined at temperatures ranging from 10” to 37” are shown in the form of an Arrhenius plot (Fig. 2). The activation energy calculated using the K values determined bet,ween 10” and 30” is 24 kcal. In attempted one-step growth determinat,ions performed at 37” in PB no phage bursts were detected during a 2-hr incubation of PBPl infected cells (2000 PFU/ml). Treatment of infected cells wit,h chloroform at, 30-min intervals during such incubations destroyed over 99.9 % of the

PFU. It is concluded that under the conditions employed, phage bursts did not interfere with the adsorption assays. Approximately two-t,hirds of the B. pumilus strains examined are sensitive t’o infection by PBPl. Comparison of the host ranges of PBPl and PBS1 on 32 strains of B. pumilus (Table 1) demonstrates t,hat those strains which are sensitive to PBS1 are also sensitive to PBPl. In addition, PBPl infects several strains which are resistant to PBSl. Approximately one-third of the B. pumilus strains examined are resistant to both PBPl and PBSl. PBPl does not form plaques on B. subtilis 168, W-23 or B. licheniformis 9945 A. PBPl and PBS1 are serologically dissimilar. Antiserum prepared against PBS1 or PBPl (each diluted in PB to a K value of 300) did not reduce the infectivity of lo6 PFU/ml of the het,erologous phage during a 2-hr incubation at 37”. Correlation between, Motility sorption

and Phage Ad-

Nonflagellated mutants derived from three PBPl sensit,ive strains of B. pumilus are incapable of adsorbing PBPl (Table 2). Identical results ho those shown in Table 2 were obtained with 4 other nonflagellated TABLE SENSITIVITY

1

OF 32 STRAINS OF Bacillus TO PBPl AND PBS1

pumilus

Strains of B. pumilus

Phage sensitivity” Sensitive PBS1

to PBPl

and

Sensitive sistant

to PBPl, to PBS1

re-

Resistant PBS1

to PBPl

and

BpBl, 101, M, Bl>2002; ATCC strains 98, 4510, 4522, 6631, 19646; NCIB strains 8600, 8738 ATCC strains 7061, 1, 72, 945, 4520, 7065, 12140; NBS strains 939, 331, 576, 337, 333 ATCC strains 18, 70, 14884; 6632, 71, NCIB 8982; NBS strains 706, 896, 637

a Resistance to PBPl or PBS1 indicates an efficiency of plating (e.o.p.) below lo-r0 or lWg, respectively; e.o.p. values are relative to the titer obtained on BpBl (e.o.p. = 1).

FLAGEI,LA-SP~~~CIFI~ TABLE

TRANSDUCING

2

ADSORPTION OF PBPl TO Bacillus pumilus STRAINS BpBl, 101, ATCC 7065 AND THEIR NONFLAGELLZTICD MUTANTS~

Bacteria Control (Pl3) BpBl 101 ATCC 7065 BlO NFl NF2 7065 NFl

Phage titera (PFU/mlj 4.2 5.4 6.4 5.9 4.3 4.7 4.4

x 106 x

104

x

104

x x x x

IO” 106 106 106

Efficiency of plating 1 1 1 < 10-10 <10-J < 10-10

747

PHAGE

ot’hers had regained sensitivity to PBPl and were motile. Five of the nonmotile variants were examined by electron microscopy for the presence of flagella and none were observed. Isolation of PBPl resistant mut,ants of BpBl was not att,empted due t-0 the inability of PBPl to produce complete lysis of BpBl lawns, even at, high phage concentra-

c*Cells (1.0 & 0.5 X lO*/mlf were incubated with PBPl (4.3 X 106PFU/ml) for 10 min at 37”. b Unadsorbed phage after the adsorption period.

mutants of 101 (PI, P2, .P3 and PS), one additional nonflagellated mutant of BPBl (BlO NFZ) and one other non~ageIlat,ed n~L~tantof ATCC 7065 (7065 NF2). Mechanical deflagellation of BpBl, performed by shearing cells in a Waring Blendor, renders the cells temporarily nonmot’ile and incapable of adsorbing PBPl, The results of an experiment in which sheared BpBl cells were allowed to regenerate flagella (at 37”) in the presence of a low m.o.i. (0.01) of PBPl are shown in Fig. 3. During the first 10 min of incubation of sheared cells with phage virtually no motile cells were observed and no phage adsorption occurred. Between 10 and 1.5min of incubation a high percentage (approximately 60%) of the cells regained mot,ility. During this time period, the first significant increase in the number of adsorbed phage occurred. The addition of lOlo infectious PBPl part,icles (in 1 ml PB at 37”) to 5 X lo* activeiy mot,ile BpBl cells (also in 1 ml PB at 37”) causes complete paralysis of the cells within 4 min. Examination of the cells by phase microscopy at intervals following the addition of phage showed that the cells undergo erratic spinning and tumbling rnoveI~~~nts prior to their complete imxnobilization. PBPl resistant variants of ATCC 7065 are readily obtained by prolonged (48-72 hr) incubation of confluently lysed plates. Thirt,y such variants were cloned and examined for motility, Eighteen were nonmotile. The

l

-5’

2 0

5

1 10

1 15

I 20

8 25

I 30

1 35

MINUTES

FIG. 3. Adsorption of PRPl to mechanically deflagellated BpBl cells during t,he regeneration of flagella. BpBl cells were grown to a density of 6 X 10’ cells/ml. The culture was chilled in ice and 20 ml was transferred to a Waring Blendor maintained at 4”. The culture was sheared (at the maximum setting) at 15-see intervals (with 30-see rest periods) for a total shearing time of 1 min. Shearing for this time had no effect on the viability of the cells, but rendered lOOgo of the population nonmotile. The sheared cells and an unsheared cont’rol were concentrated by centrifugation (10,000 g, 15 min, 4”). The supernat,ant fraction was discarded and the Kell-drained cells were resuspended to t,heir original volume (10 ml) in PB prewarmed to 42”. One-milliliter portions of the two cell suspensions were preincubated 3 min on a shaking water bath at 37” prior to the addition of one ml of phage (also at 37”). The time of phage addition is taken as zero time. The final cell concentration in both adsorption mixtures was 3.1 X 107/ml. At, 5-min intervals, both adsorption mixtures were assayed for free phage (by dilution into PB-chloroform dilution blanks) and for the number of mot,ile cells (estimat,ed by phase cont.rast microscopy). Greater than 95% of the unsheared cells exhibited translational motility throughout the experiment. The percent of the sheared cells which were motile at the int,ervals tested is shown in parent,heses withint he figure.

748

LOVETT

FIG. 4. (A and B). Interaction of PBPl with flagella of BpBl. Purified PBPl particles were incubated with exponentially growing BpBl cells (M.O.I. = 50) for 15 min at 37” in 1 ml of PB. The cells (5 X 108) were concentrated by centrifugation at room temperature (10,000 g, 5 min), resuspended in 0.5 ml of TMA, applied to grids and stained with lGT0 uranyl acetate. In this experiment approximately 30% of the phages associated with the cells were attached to the filament portion of the flagella. The remaining 70% of the particles appeared to be associated with the cell orientation. The morphologic details of phage attachment to the cell body in a “tail-first” body are currently under study (Lovett and Nauman, in progress). The bars represent 0.1 rlM.

tions. This appears to be due to the ability of BpBl cells to carry PBPl in an unstable, probably “pseudolysogenic,” state as has been reported for other Bacillus phages ineluding PBS1 (Bott and Strauss, 1965; Romig and Brodetsky, 1961; Takahashi, 1964). Attempts to demonstrate inactivation of PBPl with flagella sheared from BpBl have been unsuccessful. These experiments were performed by incubating phage (lo4 PFU/

ml) with flagella sheared from lo9 BpBl cells/ml in PB for 1 hr at 37”. Similar observations with PBS1 (Raimondo et al., 1968) and chi (Schade et al., 1967) suggested that the interaction of these phages with flagella was reversible. Two techniques have been used to demonstrate the reversible binding of a bacteriophage to an adsorbing substrate (Lovett and Shockman, 1970b). I have been unable to demonstrate reversible

FLAGELLA-SPECIFIC TABLE

TRANSDUCING

3

EFFECT OF HOMOLOGOUS .~ND HETISROLOGOUS ANTISERUM ON THE TR.INSDUCTION MEDIATED UY BACTERIOPH~XGES PBPl AND PBSla Bacteriophage Addition

PBS1

PBPl

(transductants/ ml) Minimal glucose PBS1 antiserum PBPl antiserum Deoxyribonuclease

medium (K = 20) (K = 20) (1 mg/ml)

1840 (10 1760 1800

1100 1060
a Half-milliliter volumes of PBPl (5 X lOlo PFU/ml) and PBS1 (5 X 10’ PFU/ml) were incubated 15 min at 37” with 0.1 ml of the above additions. Then 0.5-ml volumes of BpBlo (lo9 cells/ml) were added and the remainder of the transduction was performed in a routine manner (Lovett and Young, 1970). The PBPl lysate had previously been exposed to ultraviolet irradiation for 5 min by the procedure described in Table 4. The infectious titer of the PBPl lysate shown above was determined prior to irradiation. Irradiation reduced the PFU over 99.9yc.

binding of PBPl to flagella using one of these techniques, the competition assay (Lovett and Shockman, 1970b). These experiments were performed by determining the rate of phage inactivation by a standard concentration of BpBl cells in the presence and absence of an excess of added flagella. (Excluding phage, the adsorption mixtures contained 5 X 10’ BpBl cells/ml. The flagella added to the cells was not quantitated, but was that amount mechanically sheared from lo9 BpBl cells.) The rate of phage inactivation by cells wiith or without added flagella was ident’ical. Electron Microscopy The results of the preceding experiments establish a correlation between motility and phage adsorption. In order to directly determine whether or not PBPl particles were capable of attaching to flagella, phage were incubated with exponentially growing BpBl cells (m.o.i. = 50) in PB for 15 min at 37”. The adsorption mixture (1 ml) was concentrated by centrifugation at room temperature, resuspended in 0.05 ml of TMA, stained

749

PHAGE

with 1% uranyl acetate, and examined by electron microscopy. The majority of phage particles seen on the grids (i.e., 60-90% in different, preparations) were found in association with the cells. In virtually all preparations examined, phage particles were associated with both the filament portion of the flagella and the cell body (Nauman and Lovett>, in progress). The attachment of phage to flagella is accomplished by wrapping the PBPl tail fiber around the filament (Fig. 4, A and B). D&ailed examination of the association between PBPl part,icles and the cell body is in progress. However, phage particles appear to attach to the cell body in “tail-first,” orientation (Nauman and tovett, in progress). Transduction PBPl mediates transduction in auxotrophs of BpBl at a frequency on the order of lo-* transductants per PFU. The genetic activity is unaffected by preincubation of phage lysates with DNase or PBS1 antiserum but is neutralized by preincubation with PBPl ant’iserum (Table 3). Ultraviolet irradiation of PBPl lysates sufficient t’o reduce the infections t,iter 99.9 % increases the transducing activity approximately lo-fold (Table 4). Four mut’ant loci in BpBl which are unlinked as determined by PBS1 transduction (Lovett and Young, 1970,197l) can be t’ransduced to prototrophy by PBPl TABLE

4

EFFECT OF ULTRAVIOLET TRANSDUCING ACTIVITY Irradiation” (min) 0 1 2 3 4 5

IRRADIATION ON THE OF .4 PBPl LYSATE

PFU/ml 1.7 1.4 9.9 2.0 3.0 2.5

x x x x x X

10” 10’0 108 10’ 106 lo6

Transdu$ants/

140 250 460 1350 1200 1270

a A PBPl lysate in 15 ml PB was exposed to ultraviolet irradiation (two 15-W General Electric Germicidal Lights) at a distance of 50 cm. At the times indicated, samples were removed and assayed for PFU and transducing activity using BpBlO trp-2.

750

LOVETT ‘I1IsCuYsIo~

TRANSDUCTI~S ob‘ FOUR Un~,Irclim PTW TRASSDVCTIOS) (;ISETIC IS TSpBl IIP 1’13Pla

(I)EFINED hIn~um~s

..-

HY

-.

Kecipicnts Donor

urg Al

trp-2

IlWl-1

wet-2

(transductants!ml) HpHl

910

1250

1120

780

o I)onor lysate was that shown in Table -I after 5 nlin of ultravioh~t irrndiation.

(Table 5). It is concluded that PHI’1 modistcs gencralizcd transduction. Four linkage groups have been established in BpRl by PBS1 transduction (Lovett and Yourtg, 1970, 1971). 1,inkagc values obtained between markers in linkage group 0 detcrmined by 1’13Pl transduction are shown in Tnblc 6. Comparison of thcsc linkage valuw (Table 6) with those previously obf aincd b) by PBS1 transduction (I,owt t and Young, 1970) demonstrates that wtransduciblo markers arc more weakly linked by PBl’l transduction than bv PBS1 tratwdu(‘fion. This difference tnav’ indicate: that PHI’1 particlrs kontain a ‘lowr molecular wiglit. 1)KA than PBS1 part i&s. IXrcct mcasurcmonts of.thc molecular weight of PBI’l I)X,r,\ have nOt been tnadr. However, t ho hcad diameter of I’HT’I particles (ap~roxintatcly 65 ntn) ‘is about half as Inrgc: as thca hwd diameter of PI3Sl (Eiwrling, 1967).

hSh:.\~,.:

~.\I.~LS

-..

-. trp-2

trp-2 ly.u-1 ser-1 _

iIllile-I .-.

evi&ric:e supgt:stitig th:lt, flngcllatropic viruws adsorb to scwsitiw wlls in a multistcp prowss. The irlit i:LI int rraction bctwrn

()W~.\INEI) YHOM T\\ o-&~\c:~rolt ~ttoSSt:s OF :~I~SO1.IWI~HS OF RpT3, BY PHI’1 T1L\sSUI:C1.1oS’ -.

Donor

Tltc cvidcncc: prcstntc~d in this and a prwious comtnuniwt ion (T,ovc~t and Young, 1970) dcmonst ratw that wvcral strains of R. pumilus arc wtteitiw to infection by IWO distinct flagella spwific t rawducing vtruscs, PBS1 and PBl’l. Thcl infwiivity of thaw viruses on thirty-l \VO strains of B. punrilus providw a basis for dividing thtw strains into thrw groups. One group is sensitive to both viruses, anothw group is resistant, to PBS1 and wnsitivc to 1’13P1 and a third group is resistant. to both viruses. As prcviOUSI~ rrlcrlt ioticxl, n~rllyllctll (1961) IKLSS~OWII :I cwrrelation bctwwt Saho~~ellu st.rains whit+ arc stwsit ivc to c-hi and the antigenic c*otiipf~sil ion of ttiflir flng~lla. It is not yet possible to suggest a wrrclat ion bet wwn thr host, ranges of P13Pl :mtl PBS1 and differcnws (or similarit iw) among the flsgrlla of B. pumilus strains for at lcast two rcawns. l’irst , cnmparisons of the chemical and serologic proprrticls of thr flagell:l of wvrral strains of B. pw~~ihs IIUW not brrn wported although such studies arc prcscnt ly in progress (14’. lCoc:ha, personal wmmunication). Sw)nd, a c:nrrrlation between rcsisfanw to PBPl or PHSl and PBPl and a lack of virus adsorption has not brrn dcmonstratcd in most (*asrs. Studies wit.11 clti and PBS1 huvc providtd

-

.--

-. lys-1 --1(8/21r,)

-

Recipient -.. -.._ ser-I ilp- 1 ..-. ~ -. ~ 19(21/117) 3(6;1i8)

-..

-.

_

_

_

de-1 0(0/156)

5(10,208) 21 (W208) 4 (9 .‘208) 0(0.‘250~ .-

~.

-.-

..~

* Values in the parentheses indicate the number of donor type transducttlnts (numerator) and the total number of tranaductants examined (denominator). The figuws outside the: parentheses indicate the percent of the tot,al number of trRusductants examiucbd whirh carry the donor auxotrophic marker (‘y‘ linkage).

FLAGELLA-SPECIFIC

phage and host appears to involve an association of the virus tail fibers with t’he flagellum (Raimondo et al., 1968; Schade et al., 1967). This process, in itself, probably does not result in cellular infection since isolated flagella do not inactivate chi or PBS1 (Raimondo et al., 1968; Schade et al., 1967). Schade et al. (1967) provided morphological evidence suggesting t’hat the ultimate receptor for chi (i.e., t’he cellular receptor which result’s in phage inactivation and nucleic acid injection) is located at the base of the flagellum. The evidence for this is 2-fold. First, immediately following the addition of chi to E. coli, those phage particles seen associated with t.he cells were virtually exclusively attached to the filament portions of flagella. As the time of incubation continued, t,he number of phages attached to flagellar bases increased. Second, the majority of phages attached to filaments retained their DNA, whereas the majority of phages attached to the bases had lost their DNA. It was suggested (Schade et al., 1967) that aft’er the initial interaction of a phage with the flagellum, the virus slides down the filament to Dhe base where infection occurs. The preliminary results of the morphological studies of PBPl adsorption to BpBl cells arc consistent wit’h this hypothesis (Nauman and Lovett, in progress). The interaction of PBS1 and chi with flagella does appear to play an import,ant (perhaps mandatory) role in the infection process based on the following observations: (1) nonflagellated mutants of phage-sensitive cells do not, adsorb the phages (Frankel and Joys, 1966; Meynell, 1961; Raimondo et al., 1968; Schade et al., 1967); (2) cells wit’h genetically paralyzed flagella eit’her do not adsorb phage (Frankel and Joys, 1966; Schade et al., 1967) or do so at a reduced rat’e (Raimondo et al., 1968); (3) a correlation exists between cells which are sensitive to infect,ion by chi and the antigenic composition of their flagella (Meynell, 1961); (4) mechanically deflagellat,ed cells adsorb phage poorly or not at, all (Raimondo et al., 1968; Schade et al., 1967) ; (5) morphological evidence indicates that the phages are capable of attaching to flagella (Iino and Mit,ani, 1967; Meynell, 1961; Raimondo et al., 1968;

TRANSDUCING

PHAGE

751

Schade et al., 1967); (6) mutants of E. coli selected for resist,ance to chi are either nonflagellated or possess paralyzed flagella (Armstrong and Adler, 1967); (7) t’he addition of a high m.o.i. of chi or PBS1 to sensitive motile cells immobilizes the cells (Frankel and Joys, 1966; Meynell, 1961; Raimondo et al., 1968); (8) adsorption of PBS1 by cells is inhibited if the cells are pret’reated with antiflagellar antiserum (Raimondo et al., 1968). Five of these eight observat,ions have been confirmed with PBPl in t’he present study. Despite the large number of viruses isolated which infect members of the Enterobact,eriaceae, chi (and phages morphologically and serologically relat’ed to chi; Edwards and Meynell, 1968) is the only flagellat,ropic phage reported for this group of bacteria. By cont’rast, within the past, few years three of the viruses which infect members of the genus Bacillus have been shown to be flagellatropic. These include PBSl, PBPl, and SP-15 (J. Mele and C. B. Thorne, cited as a personal communication in Tyeryar et al., 1969). SP-15 infects B. subtilis W-23 and B. Zicheniformis 99458 but does not form plaques on BpBl (unpublished observations). All three of t,hese Bacillus phages mediat’e generalized t’ransduction in their respective host,s (Lovett and Young, 1970; Takahashi, 1963; Taylor and Thorne, 1963; Tyeryar et al., 1969). Chi has not been reported to mediate transduction. ACKNOWLEDGMENTS This investigation was supported by Public Health Service Grant AI 10331 from the National Institute of Allergy and Infectious Diseases. REFERENCES

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