Isolation and Preliminary Characterization of Bacteriophages for Bdellovibrio bacteriovorus

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Isolation and Preliminary Characterization of

Bacteriophages for Bdellovibrio bacteriovorus

MARY ALTHAUSER, WILLIAM A. SAMSONOFF, CAROL ANDERSON, AND S. F. CONTI Thomas Hunt Morgan School of BiologicalSciences, University of Kenttucky, Lexinigtoni,Kenitucky40506

Received forpublication 15 June 1972

Tenbacteriophages that attack and lyse saprophytic strains of Bdellovibrio bac-leriovoruswereisolated.Morphological, serological, and host-range studies revealed that there werefour different bdellovibrio phagespresentamongthe isolates. One of thephages lysedastrain ofB.bacteriovorusthatrequires thepresenceofasuitable bacterial host forgrowth.Thephageattachedtothe bdellovibrio cells in the absence of the bacterial host cells; lysisoccurred onlyin the presenceof host cells. The 19

saprophytic bdellovibrio strains employed in the phage host-range studies were

groupedonthe basis of theirsusceptibilitytophage lysis.

Bdellovibrio bacteriovorus was initially de-scribed as an ectoparasitic and bacteriolytic

or-ganism that attacks gram-negative bacteria (18). Subsequently, saprophytic strains were isolated (11, 13, 15, 17); some of these strains were capableofgrowth onartificial media andinitially

retained their parasitic abilities. A facultative strain designated UKi2, which is capable of growing in host-free media or endoparasitically onEscherichiacoli B/r, wasreported (5). Studies

of these parasitic, saprophytic, and facultative strains have considerably expanded our knowl-edge ofthe unique bacteria (14, 16). Neverthe-less, the classification ofB. bacteriovorus andthe interrelationships of the various strains have not

been clearly established.

Sincebacteriophages for bdellovibrio could be usedtoclassify thevarious strains of B.

bacterio-vorusand asprobes for elucidating someaspects

ofthehost-endosymbiont relationship, the avail-ability of phages could prove to be useful. We have reported (6) theisolation ofabacteriophage which attacks and lyses B. bacteriovorus strain UKi2; this bdellovibrio can grow either in the presence or absence of host cells (5). We have

isolated additional bdellovibrio phage which not

only lysed saprophyticstrains of B.

bacteriovorus,

but alsowereinfective for obligately endosymbi-oticstrains of bdellovibrio inasystem consisting ofbdellovibrio, bacterial host, and phage (Mary Althauser and S. F. Conti, Bacteriol. Proc., p.

173, 1971; Abstr. Annu. Meeting Amer. Soc. Microbiol., p. 220, 1972). Varon and Levisohn

(19) have recently also reported the isolation of bacteriophageswhich will attackaparasiticstrain

ofB. bacteriovorus.

This paper describes the isolation and

pre-liminary characterization offour different addi-tional bacteriophages forB. bacteriovorus.

MATERIALS AND METHODS

Media. The liquid medium used throughout this investigation for growing the bacteria and diluting the phage suspensions was peptone-yeast extract (PYE) whichcontains iS0 (w/v) peptone (Difco) and 0.3% (w/v) yeast extract (Difco). The pHwas adjustedto

7.2with 1 M NaOH. PYE agar plates for enumera-tion ofphage and parasiticbdellovibriowereprepared by adding1% (w/v)agartoPYE broth for the bottom layer and 0.7%agarin theoverlay.

Bacterialstrains and cultural conditions. The sapro-phyticB.bacteriovorusstrains (100,109, 110, 114, 118, 120, B, D, E, OX9-2, OX9-3, A3.12, 2484-2, 2484-3,

Sa 109, SaD3Smr, Xty) werekindly supplied by R. J. Seidlerand M. P.Starr. AfacultativestrainUKi2(5) and an additional saprophytic strain (UKil) were isolated fromraw sewage.Erwiniaamylovora, Proteits mirabilis, Serratia marcescenis, Spirillum serpenis, Sal-monlella typhimurium, and E. coli (ATCC 15144), furnishedby D. AbramorM.Starr,wereemployed in thehost-range determinations.

The saprophytic bdellovibrio strains were

trans-ferred every 12 hr using a 10% (v/v) inoculum. The members of the othergenera weregrowninasimilar manneremployinga 1% (v/v) inoculum for transfer. B.bacteriovorus 114 (ATCC 15362),aparasitic strain, was grown onE. coli (ATCC 15144) in PYE broth. Forstartercultures of theparasitic bdellovibrio, 9 ml

of a 12-hr PYE broth culture of E. coli (4 X 109)

cells/ml) ina16by120mmtesttubewasinfected with

1 ml ofan 18- to 24-hr B. bacteriovorus 114 culture

(108 cells/ml).

All liquid cultures were incubated at 30 C in a

gyratoryshaker (New Brunswick Scientific Co., New

Brunswick, N.J.).

Enumeration of bacteria. The saprophytic cells of

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B. bacteriovorus and the bacterial host cells were enumerated by colony counts on PYE agar and the parasiticcells byplaque assayaspreviously described (5).

Isolation of bacteriophage. Two procedures were used to isolate bacteriophage. In the first isolation procedure 100ml of raw sewagefrom the Lexington, Kentucky, sewage treatment plant were clarified by lowspeed centrifugation, and the supernatant fluid

waspassed throughdisposable filters of 0.45- and

0.2-/Am

poresize(NalgeCo., Rochester, N.Y.). The filtrate

wascentrifuged at40,000 X g for 1 hrinaBeckman modelL-2ultracentrifuge. Thepelletwasresuspended in 10.0ml ofPYEbroth, and 1 mlof thesuspension

was plated with 0.05 ml ofa 12- to 14-hr culture of each of thefollowing strains ofB. bacteriovorus: 100, 109, 118, D, E, UKil, UKi2, 2484-2, and Xty, using thedouble-layer platingproceduredescribedbelow.

The secondmethod usedwasavariation of the

con-ventional enrichmenttechniquedescribed forisolating bacteriophage. A 2-liter flaskcontaining 1 liter ofraw

sewage was inoculated with 20 ml of an overnight culture of each of the nine strains and incubated at

25 Cfor72hr.The flaskswereoccasionallyshakenby handduringthisperiodof time. Afterincubation, the suspensions were clarified and filtered as described above. A 10-ml amount ofa 12- to 14-hr culture of each saprophytic bdellovibrio was added to a 1-liter flask containing 500 ml of filtrate from thefirst

en-richment.A5-mlamountofa20%- (w/v) yeastextract

(Difco) solution was then added to the flask. After incubation at 25 C for 72 hr,the suspensions were

clarified, filteredthroughadisposablefilter of0.45-jim pore size, and plated as described in thefirst enrich-ment procedure.

Plaques which appeared to be ofdifferent size or

morphology, or both, were picked separately, and if subsequent platings showed these differences were

consistent they were treated as different isolates. Plage werepurified by atleastthree successivesingle plaque isolations. High-titered stocks of each of the phagewereprepared by the plate lysis technique (1). Enumeration ofphage. Forthe phage isolation

ex-periments, petri dishes containing approximately 20 ml ofPYEagarwereoverlaid withamixture of2ml ofPYEoverlay agar, either 0.05 ml or0.3 mlofan

exponential-phase culture oftheorganism,and 1.0 ml

of the material being tested for phage. The plating procedurewas modified in the experiments to deter-minethe hostrange ofthephageandphagetitersby mixing0.1 mlofanappropriatedilution ofthephage suspension with the overlay agar. All plates were

incubatedat30Cfor24to48hr. Theorganismsused

inthephage host-range studies included allthestrains

ofbdellovibrio and the members ofthe othergenera

listed underbacterialstrains.

Effect ofphageMAC-3 inathree-memberedsystem.

The effect ofone ofthe phages (MAC-3) on the B.

bacteriovoruis 114-E. colisystemwastested by

adding

4 X 108

plaque-forming

units

(PFU)/ml

to a 12-hr

culture ofE. coli (4 X 109/ml)

immediately

after the

addition of the bdellovibrio (108

cells/ml). Samples

were taken from the tube with the three-membered system at zerotimeandafter 12 and 24 hr of

incuba-tion at 30 C and assayed for numbers ofbdellovibrio, E.coli, and phage. Control tubes, lacking one or more components of the system, were treated similarly; ex-periments were repeated a number of times with equiv-alent and reproducible results.

To determine the adsorption kinetics of MAC-3 to to B.bacteriovorus 114, 3 X 109 PFU of bdellovibrio cells per ml were infected with MAC-3 at a multi-plicity of infection (MOI) of 0.2. Samples (0.1 ml)

were removed at various time intervals and diluted with 9.9 ml of cold PYE broth containing a few drops ofCHCl3. The mixtures were shaken on a Vortex shaker for 20 sec and titrated after 1 hr.

Totestdirectly the effect of MAC-3 on the motility of thebdellovibrio cells, 2 X 109 B. bacteriovorus 114 cells/ml were mixed with MAC-3 at ratios of phage to bdellovibrio of 30, 3, and 0.2 and incubated at 30 C. Control tubes contained bdellovibrio cells without phage. Samples were removed after 15, 30, and 60 min of incubation, and the motility of the cells was immediately checked by observation with a Zeiss Universal phase microscope.

Todetermine the effect of the phage on the ability of thebdellovibrio to attach to and penetrate the host cells, samples from the two-membered system des-cribed above and samples from the control tubes were added to 4 X 109 E. coli cells. The attachment and penetration of the parasites within the host cell were observed by phase-contrast microscopy over a 2- to 3-hrperiod.

Serology. Antigen preparations were obtained by propagating each phage in PYE broth cultures of an appropriate saprophytic strain of B.

bacteriovorius.

The phage from the broth lysates were then purified by differential and sucrose density gradient cen-trifugation, followed by dialysis against phosphate-buffered salineasdescribed by Bradley (2). The purity of each phage preparation was examined by direct electron microscopy, the amount of protein was determined by the procedure of Lowry et al. (7), and plaques were assayed. Six intravenous injections, each containing30 to 50

jig

ofviral protein, were given to rabbits at 3- and 4-day intervals. Rabbits were bled fromtheheart 10 days after the last injection.

Each antiserum was tested first against the homol-ogousphageand thenagainsttheremaining phagesto

detect cross-neutralization reactions. In both

neu-tralization tests, heat-inactivated (56 C, 30min) anti-serum wasdilutedandmixed withanequal volume ofa

phage preparation containing 107 PFU/ml. At

10-min intervals, 0.1-ml samples of the phage-serum mixture were withdrawn and added to 9.9 ml of diluent to stop the neutralizing action. From these tubes, further dilutions were madeand samples were

plated bythedouble layer procedure. Neutralization

velocityconstants (k min-') foreach antiserum were determined as described by Adams (1). The per-centage of phage neutralized was determined, and a

velocity constant,k,wascalculatedat

99'%

neutraliza-tion by using the relationship k = 2.3 D/t X log Po/P ; where D is the dilution ofantiserumused in the determination; t, the time required to obtain 99% neutralization; pa, the plaque count before the

addi-tion of antiserum; and p, the plaque count after neutralization.

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ALTHAUSER AL. Electron microscopy. Phagespecimens for negative

staining were obtained by placing a drop of the purified phage preparation on a carbon-coated grid or by lightly touching the grid to an isolated phage plaque; specimens were stained with 0.2% uranyl acetate.

Thin sections were prepared by using previously described procedures (4); all specimens were

ex-amined in a Philips EM 200 electron microscope operatingat 60kv.

RESULTS

Phage isolation and morphology. Ten bacterio-phages that attacked and lysed saprophytic bdellovibrios were isolated from raw sewage (Table 1). Two different

morphological

plaque types were observed on the plates of

the

phages initially designated as MAC-1 and MAC-4; each plaque was

subsequently purified,

and

the

second typewas designated with a prime notation.

Microscopic

examination of all ten phage iso-lates revealed four distinct

morphological

types. Seven of the

phage

isolates

(MAC-1,

1', 2, 4, 4', 5, and

7)

were ofidentical structure and corre-spond to Bradley's group E (3). All are tailless, have a regular

hexagonal outline,

and are

ap-proximately

25nmin

diameter (Fig.

1).

The

remaining

isolates were

typical

of Brad-ley's type A

bacteriophage, possessing

hexagonal

heads,

long

tails,

andcontractile

sheaths.

MAC-3 (Fig. 2a) has an elongated

hexagonal

head 75 nm

by

50 nm in

diameter,

and an overall length of150 nm. Subunits of

the

tailare

clearly visible

(Fig. 2a), and its double

base

plate and four tail pins are best seen in Fig. 2b.The apparent

swell-ing

or

collapsing

of

the

head

frequently

seen when this phage attaches toits host is

shown

in

TABLE 1. Neutralizilg velocitiesaofB.

bacteriovoruis

by phageanitiserutm

Phage AIAC-36 -MAC-6 TIDC-2 HDC-1 AIAC-1

MAC-3 ... 698 -C

MAC-6 ... - 115

_-HDC-2 -- 41

HDC-1'1.

70

MAC-1 2,705

MAC-1'

.

- 1,468

MAC-2 2,188

MAC-4

._

- 2,482

MAC-4' -- - -- 1,889

MAC-5 .. 2,610

MAC-7 2,595

a Expressed as k values (min-'); k values

deter-mined as described in Materials and Methods. h Phage antisera tested.

ck values ofless than I arerecorded as -dThis phage was previously isolated and described (6).

Fig. 2c. MAC-6 hasan overalllength of 180 nm

and a head diameter of 65 nm (Fig.

3a).

The complex tail assembly, consisting ofasingle base plate and three or four tail pins with spherical structures on their distal end, is shown in Fig. 3b. HDC-2 hasahead of 70to 75nmand is 150

to 160 nm

long.

A complex tail assembly as

seen on MAC-6 was not seen on the intact or

contracted HDC-2particles (Fig.4a,

b).

Table 1 summarizes the results of cross-neu-tralization tests conducted to detect serological relationships among the various phage isolates. Antisera to phages MAC-3, MAC-6, HDC-2, andHDC-1 had neutralizing activity onlyagainst the homologous phage. The MAC-1

antiserum,

however, neutralized the homologous phage, MAC-1, and also neutralized phages MAC-l', MAC-2, MAC-4, MAC-4', MAC-5, and MAC-7. Similar first-order inactivation kinetics were observed when each of the seven phages was exposedto MAC-1 antiserum, indicating that the seven spherical phage are closely related, if not identical.

Host range of the phage isolates. The host ranges of the phages among the saprophytic bdellovibrio strains are summarized in Table 2. The spherical phage MAC-1, like all ofthe other spherical phage isolates had a rather broadhost range andlysed 12 ofthe 19 bdellovibrio strains tested. MAC-3, one of the tailed phages, had a more narrow host rangelysing7 of the 19strains and was the only isolate to lyse strains 110 and 120. MAC-6, HDC-2, and HDC-1 had very specific host ranges. None ofthe phage isolates lysedstrainsUKil, A3.12, oranyofthebacterial hosts used topropagatethe bdellovibrio.

Effect of MAC-3 on a host-dependent B. bac-teriovorus. The effect of MAC-3 on a parasitic strain ofB. bacteriovorus 114 grown on E. coli is shown in Table3. Inthetestsystemcontaining bdellovibrio and host cells, there was a 500-fold increase in bdellovibrio cells after 24 hr of incu-bation. In the presence of MAC-3 phage, the number of bdellovibrio cells decreased 100-fold in 24 hr, with an

accompanying

1,000-fold increase in the phage titer. -he MAC-3 phage did not multiply on either the parasitic bdello-vibrio cells in the absence of host or on E. coli. Inaccordance withthese

observations,

therewas an increase in the number ofviable E. colicells remaining after incubation with the bdellovibrio in thepresenceofphage.

Since thefrequency ofmutation ofparasiticto

saprophytic cells is relatively high (11), during thecourse ofthe experimentation weassayed for saprophytic cells in the B. bacteriovorus strain 114cultureemployed; none weredetected.

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2a

2b

:

2c

[image:4.493.49.439.73.380.2]

4b

FIG. 1. Negativestainpreparationofthetailless, hexagonallyshapedphageisolate MAC-i. Scalemarker rep-resents200irmandappliestoFig. Ithrough4.

FIG. 2. Negativestainpreparationsofphage MAC-3. Theelongatedhead and subunits ofthetail assemblyare

shownt(a);the double baseplate (arrows)and small tail pinsareevident(b).The headsof MAC-3areusually spher-icalorcollapsed after attachmenit toits host(c).

FIG. 3. Negativestainpreparationsofphage MAC-6. The subunitstructureof the uncontracted tail (a) and the complexstructureofthetailpinsotn the contractedphage particles(arrows, b)areillustrated.

FIG. 4. NegativestainpreparationsofphageHDC-2. Thephagehasahexagonalhead anddistinct tailsubunits

(a). The contractedphage particle (b) doesnotshowanycomplex tailstructures.

TABLE 2. Host range oJfive B. bacteriovorusphages

LysisofsaprophyticB.bacieriovoruisteststrai.'sb

Phage__

100 109 114 SaD3Sm' 118 D |x9-2 (x9-3 2484-2 2484-3 Xty Sa109 110 120 B E UKi2

MAC-I +r + + + + ++ + + + + +

MAC-3 + + + + ±-- - - +

--MAC-6 - -- - --- - -

-+

+-HDC-2 - T- 't - _ - .._ +

HDC-1 - --

-

.+

aThehomologous host (saprophytic bdellovibrio strains used for initial phage isolation) ofphage

MAC-1, MAC-3,andMAC-6areB.bacteriovorus strainsXty, 118,and E,

respectively.

Thefacultatively parasitic bdellovibriostrain UKi2was usedfor initial isolation ofphagesHDC-1 andHDC-2.

bB.bacteriovorusstrains UKil and A3.12 were notlysed byanyofthe phages.

c+, Lysis ofcells; -,no lysis of cells.

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TABLE 3. Effect ofphage MAC-3 o01 the host-depenzdent Bdellovibrio bacteriovorus straini 114a

Testsystemb

1. Escherichia coli

B. bacteriovorus 114 MAC-3

2. E. coli

B. bacteriovoruts 114

3. B. bacteriovorus 114

MAC-3

4. E. coli MAC-3

5. MAC-3

6. B. bacteriovorus 114

7. E. coli

to

4 X 109

1x 107

4 X 107

4 X 109

1 X 107

I X 107 4 X 107

4 X 109 4 X 107

4 X 107

1 X 107

4 X 109

aThe test systems which contained bdellovibrio cells were assayed for the presence of saprophytic

bdellovibrio cells atzero time and after 12 and 24 hr ofincubation; none weredetected. E. coli viable

cellcounts per milliliterwere enumerated byplating onPYEagar, and parasiticcells by plaque assay

asdescribed inMaterialsandMethods;phagecountspermilliliterweredeterminedby thedouble-layer technique (1).

bPYE brothwasaddedtothe testsystems which containedone ortwomemberstomaintainavolume of 10ml. All cultureswereincubated at 30 Con agyratoryshaker.

Whenthe bdellovibrio cellsandthephagewere mixedinPYE brothataratio of 50 to 1,

99%/

of MAC-3 adsorbed to B. bacteriovorus 114 after 1.5 hr of incubation at 30 C (Fig. 5). Electron micrographs of negatively stained preparations from thethree-membered system ofphage, bdel-lovibrio, and E. coli (Fig. 6) revealed the

attach-ment ofa bdellovibrio cell to host after 1 hr of

incubation of the bdellovibrio and MAC-3 in a

ratio of1 to 30. A section ofa sample from the three-membered system of phage, bdellovibrio, and host which was incubated at 30 C for 4 hr

shows MAC-3 particles insidea bdellovibrio cell which is withinanE. colicell(Fig. 7).

MAC-3 didnotaffecteither themotility of the bdellovibriocells in the two-membered systemor the attachment and penetration ofthe parasites into the hostcells,evenwhenratiosashighas 30

phageto 1 bdellovibrio cellwere employed.

These data and observations support the

con-clusion that MAC-3 adsorbs to and multiplies

on B.bacteriovorus strain 114growingon E.coli.

DISCUSSION

Although ten bacteriophages wereinitially iso-lated for various strains of B. bacteriovorus our

results indicate that only four different phages

were present among the initial isolates. he

morphological and serological data and the host

30 60 90

FIG. 5. Adsorption of'MAC-3 to B. bacteriovoruis 114.Bde/lovibrio cells (3 X 109PFU/ml inP YEbrotlh)

wereilifected wvithMAC-3ataiiMOIof0O.2.Samnplesof

0.1 m/l were renmoved at various time intervals and

diluted in9.9 nil of coldPYEbroth conitaininlg a

fiew

dropsofCHC/3. Themixtures wereslhakeliina Vortex

mixerfor20secanidassayed/clrphagealterIhr.

12hr

3 X 109

7 X 107 6 X 108

5 X 109

7 X 108

1 X 107

8 X 107

4 X 109

1 X 108

1 X 10,

1 X 107

5 X 109

24 hr 3 X 109 1 X 105

3 X 101°

5 X 103

5 X 109

3 X 101 5 X 107

4 X 109 1 X 10t

1 X 108

5 X 106

4 X 109

E

CL

0 a, n

CL.C

-2

c

n

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BACTERIOVORUS

P

.A

El,

/B

Bd

0.5 pm

[image:6.493.46.438.64.606.2]

hWA.W.

FIG. 6. Negativestainlpreparationlofathree-memberedsystem. C0ontractedMAC-3phlageparticlescanlbeseen

adherinigto thehdellovibriocell, which in turntisattachedtoani E.colicell. Uniattached, iiliconitractedphageare

presenit

(triplearrow).

FIG. 7.

Thlinl-sectionz preparationz

illustratinlgMAC-3phage particles (P) withinabdellovibriocell(Bd),w1hic1his. locatedintheperiplasmicspace ofaniE.coli cell(E).

52-1

1972

.i,,..A"

N.,

II

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ranges of the phage support the conclusion that phages MAC-1, 1', 2, 4, 4', 5, and 7 are very

similar, ifnotidentical; MAC-1 has been selected astherepresentative of thisgroup.The data also

support the conclusion that MAC-3, MAC-6, and HDC-2areseparateand distinctbdellovibrio phage types. It thus appears that seven dif-ferent phages now havebeen isolated which are

infective forsomestrains ofbdellovibrio: MAC-1,

MAC-3, MAC-6, HDC-2, the previously de-scribed HDC-1 (6), and the VL-1 and VL-2 phages recently isolated by Varon and Levisohn (19). The apparent ease with which phages for B. bacteriovorushave been isolated indicates that utilization ofconventional procedures, or minor modifications thereof, should result in the isola-tion ofadditional phages from natural habitats containing B. bacteriovorus.

Themost extensive studieson thesaprophytic strains of bdellovibrio (9, 11, 12) indicatea very close similarity between all of the saprophytic strains tested with the exceptionofstrains A3.12 and the facultative B. bacteriovorus UKi2. Fur-thermore, there are no completely satisfactory methods available for differentiating between the 20 or so saprophytic strains used in various laboratories, or for determining the number of truly distinct saprophytic strains. Our data (Table 2) on the susceptibility of the various saprophytic strains to the phage isolates illus-trates that some of the strains can be differen-tiated from others. On the basis of phage

sus-ceptibility,

thesaprophyticstrainscanbegrouped as follows: Group I, strains 100, 109, 114,

SaD3Smr, 118; Group II, strains D,

Ox9-2,

2484-2, 2484-3, Xty, Sa 109; Group III, strains 110 and 120; Group IV,strains B andE; Group V, strains UKi2; and Group VI, strains A3.12 andUKil, whichare strains notinfected byany

of the phage isolates. The availability of addi-tional phages and appropriate antiserum,

com-bined with otherapproaches, should allow differ-entiation between strains within a particular

group, and perhaps establish more definitively

whetheraseparatestraindesignationiswarranted

for allofthesaprophytic bdellovibrio isolates. TherecentreportbyVaron and Levisohn(19) andthe datapresentedin thispapersupport our

previous contention (Mary Althauser and S. F. Conti, Bacteriol. Proc., p. 173, 1971) that some phage which attack, lyse,andreplicateon sapro-phytic strains of bdellovibrio are also infective

for parasitic strains, provided that a suitable

host bacterium is present inthetestsystem. The requirementfor thepresenceofahostbacterium ispresumably duetotheinability of the parasitic bdellovibrio togrowand divideintheabsence of

hostcells, although they are metabolically active

(14).

The

three-membered

system (host,

bdello-vibrio, and phage)

described

by Varon

and

Levi-sohn

(19)

is

similar

to our system,

but there

are somesignificant

differences.

In

their

system, and undertheir conditions, host-free cultures of bdel-lovibrio which are unable to support

phage

growth can

adsorb the

phage particles and are

subsequently

killed.

As a consequence, even a

delay of

a

few

minutes in the addition

of

the host

cells

to

the

bdellovibrio-phage

system results

in

a

diminished

phage yield.

In our system

the

incu-bation of the

phage-bdellovibrio

mixture for as long as 6

hr before addition of host

cells did not

have

any noticeable effect on

the

phage yield. Varon

and

Levisohn

also

observed that the

motility

of

the

bdellovibrio cells

ceased 10 to 20 min

after

addition of

the phage; this

did not occur in our system.

The observations that

phage

which can infect

saprophytic strains

can also attack

bdellovibrio

which

require suitable bacterial

host cells for

growth

is not

unexpected in

view of the

observa-tion of

Reiner and

Shilo (8) that

so

called

host-dependent

or

wild-type strains

of B. bacteriovorus can

elongate in

the absence of host cells when

incubated in cell-free microbial

extracts.

Subse-quent work

has resulted in the isolation

of a

factor

which, when added

toa

parasitic strain

of B.bacteriovorus,

enables

cells

togrow

and

divide,

i.e.,

cells

go

through

at

least

one

cycle of

elonga-tion and

fragmentation (Shilo,

personal

com-munication).

It

would

therefore

appear

that the

major difference

between

saprophytic and

para-sitic

bdellovibrios

is

their

ability

to grow

and

divide in the absence of

a

"factor"; parasitic

bdellovibrio

can

obtain the "factor" from

suit-able host bacteria whereas

saprophytic

strains

can

either

synthesize

this

factor

or

do

not

require

it.

Presumably, all so-called host-dependent

bdellovibrio

can

be "transformed" into

host-independent

cells

by addition

of

the "factor"

to

the medium under

appropriate

conditions. We

are

presently

testing

the

infectivity of

our

five

phage isolates

against

a

number

of

parasitic

bdellovibrio

with

the expectation

that

phage

infective

for

saprophytic

strains will infect the

closely related

parasitic strains.

ACKNOWLEDGMENT

This researchwassupportedbyNational Science Foundation grantGB 7972.

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