Isolation and Characterization of a Bacteriophage for Vibrio fetus

JOURNALOFVIROLOGY,JUly1970, p. 94-99

Copyright @ 1970 AmericanSocietyforMicrobiology

Vol. 6, No. 1 Printed in U.S.A.

Isolation and Characterization of

a

Bacteriophage

for

Vibrio fetus

JOHN H. BRYNER, ALFRED E. RITCHIE, JOHN W. FOLEY, AND DAVID T. BERMAN

National AnimalDisease Laboratory, U.S. Department of Agriculture, Ames, Iowa50010, and Department of

Veterinary Sciences, University of Wisconsin, Madison, Wisconsin 53706

Receivedforpublication 3 March 1970

Bacteriophages were isolated from 22 of 38 strains of Vibriofetus by an

enrich-mentprocess, utilizing the donor and host strains growing together in fluid thio-glycollate medium. One phage, V-45, isolated by the conventional lawn-spot

method, was characterized by stability in broth, growthkinetics, and morphology.

It was sensitive to rapid thermal inactivation, chloroform, andpH values above

6.5.Calcium wasrequired for phage replication and stabilityinbroth. Magnesium

provided the best protection against thermal inactivation at 50C in thepHrange

of 6.5 to7.5. The minimumlatent periodwas 135 min, rise time was 75min, and

average burst sizewas 35 plaque-forming units per infected cell. Phage V-45

re-sembled Bradley's morphological group B, having a long tail without contractile

sheath. Dimensions were: head, about 50 nm; tail, about 7 by 240 nm; and tail

lumen, 2to 3 nm.

The detection of

bacteriophages

for Vibrio

fetus until recently was not

successful, largely

becauseofdifficultiesin

propagating

the

organism

and in adapting

phage-investigation techniques.

The need for actively

growing,

sensitive host bacteria for phage

replication

iswell known

(1).

Manclark and Pickett

(10)

describedamethodfor

growing V.fetus in liquid culture medium on a

gyratory shaker which satisfies this need.

Fire-hammerand Border

(6)

firstisolated

phage

from

old broth cultures of V.

fetus.

Their method of

cross-culture

spotting

onagar

yielded

additional phage from this source.

They

described

plaque

types,

lytic

range, and

morphology

of some of

these isolates and

pointed

out the

difficulty

of

obtaining

phage

by

induction with

mitomycin

C.

This report describes technical

improvements

in the

isolation,

preservation,

and assay of V.

fetus phages, and the characterization of one representative isolate, V-45.

MATERIALS AND METHODS

Media.Fluidthioglycollatemedium(BBL,01-140) wasusedfor maintenance and enrichment cultures of

lysogenicstrainsofV.fetus. Broth for shaker cultures

andphagedilution blanks consisted of Brucella broth

(Albimi:A-114) with 3 gof sodium succinate and 0.1 g

of cysteine per liter (pH 6.9), hereafter designated

modified Albimi broth (MA broth). Brucella agar

(Albimi Laboratories, Inc. A-115) was the nutrient

base for double agar-layer cultures, and thesoft top

layer contained, per liter ofwater: agar (Colab

Ion-agar no. 2), 5 g; NH4Cl, 1 g; KH2PO4, 6.5 g;

Na2HPO4, 3.5 g;sodium succinate, 3 g; andglucose,

1 g. The agar cultures were incubated in a gas mixture

of25% air, 10% C02, and65%N2, whereasliquid

cultureswereincubatedinair (5).

Bacterial cultures. Thirty-eight strains of V. fetus from our stock culture collection were tested, including 30strainsoftype 1 and 8 strains of type 2 identified

previously (5). Type 2strains 436 and 2255 were the

bacterialhostsforprimaryphageisolation, and 2255

was the indicator host for phage assays. Log-phase

host cells, grown in nepheloculture flasks on a shaker as described by Keeler et al. (8), contained 5 X 108

colony-forming units when used in preparing plaque

assaylawns.

Phage isolation. Phage V-45/436 (V45) was iso-lated from lysogenic strain 45 on host 436 by the

lawn-spot method of Matsushiro (11). Attempts to

isolate other phages by conventional methods were

unsuccessful.

The following enrichment method was developed

forisolating phagefromlysogenicstrainsof V.fetus.

Each teststrain was inoculated into separate culture tubescontaining 9 ml of freshthioglycollate medium, and1mlofhost2255culturewasadded to each tube. Themixedcultureswereincubated for3daysat36C

and thencentrifuged at2,500 X gfor 20min to

re-movecellsfrom unadsorbed phage in the supernatant fluid. This fluid was transferred to separate 100-mi

flaskscontaining5 X 109log-phasehostcells in20ml of MA broth and incubated for 5 hr.Samples (l-ml)

fromeachflaskwereplatedinoverlayagarto testfor

plaqueformation.

Characterization ofphage V-45. After three single-94

on November 11, 2019 by guest

http://jvi.asm.org/

plaquepurifications ofthe clearplaqueform ofphage

V45, itwasreplicated insoft-agarlawnsof host2255

to atiterof1010plaque-forming units(PFU)E/ml.The

resultinglysatewashomogenizedin three volumes of

MAbroth, sedimented, andfilteredthrough0.45 g&m membrane filters (Millipore Corp. Bedford, Mass.).

This phagestock waspreservedat4 C and used inthe

followingtests.

Thephagestock wasdiluted 10-1 in tubesofMA broth and tested for sensitivity to heat, chloroform,

andvarious pH values bythemethodof McDuffetal.

(12).

Similar tests were performed to assay for phage

activation, inactivation, or protection by using MA

brothcontaining variousamountsofCaC12, MgSO4, tryptophan, cysteine, NH4-benzoate, or phosphates

(1:4ratio ofNa2HPO4andKH2PO4).Thesetest

sus-pensionswereincubated for 18 to 22 hrat36Cand

assayed for PFU/ml.

Therequirement for calciumas acofactorforphage

replication (7) was tested, by using 180 ml ofMA

brothadjustedtopH6.5 with lacticacid,induplicate

culture flasks, onecontaining CaCl2 (0.01 M) andthe

othercontainingnone. Approximately1010 host cells

infected with phageV45 at amultiplicity of0.01 were

inoculated into each flask, incubated at 36 C on a

shaker, andassayed forPFU andchanges inoptical

density(ODin0) for7hr.

Growthkinetics of phageV-45weredetermined by

methodsdescribedbyAdams (1), withexceptionsas

noted. Thephage-celladsorptionrate wasbased on the number of infected bacteria sedimented after 5-min

incubationperiods.Inthesingle-step growthtest, the

phage tocellratiowas0.06, adsorption timewas 15 min at36 C,andserumneutralization of unadsorbed

phage was10min. After appropriate dilution ofthe

infectedbacteria,thefirst growthtube and the second

growth tube consisted of100 mlofMA broth with

CaCl2 (0.01 M) in 250-ml flasks. These tubes were

assayed forPFUat5-to15-minintervalsduring7 hr

at36 C withcontinuous shaking.

Forelectronmicroscopy, phageand host cellsfrom

abroth culturewereconcentratedbycentrifugationat

30,000 X gfor30min, after stabilization with

glu-taraldehyde (0.25%, 15 min), andnegatively stained

withneutralizedphosphotungstic acidbyusing minor

modifications (14) of the Brenner and Home (4)

method. Grids wereexamined in aPhillips EM-200

electron microscope operated at 60 kv with

double-condenserilluminationand a 20- to25-,umaperture.

RESULTS

Phage isolation. Phages were

isolated

from 22

of 38 V. fetus strains by

enrichment

culture of

phage from

lysogenic

strains with host 2255 in

semisolid thioglycollate medium. Thenumber of

plaques

observed

atprimaryplating varied from

15 in some strains to almost complete lysis in

other strains(average,250). All 22 phage isolates

were derived from V. fetus type 1 strains. This

type generally grew

poorly

in soft-agar overlay

[image:2.487.244.437.69.186.2]

cultures, whereas type 2 strains produced dense

TABLE 1. Effect of temperature

inMAbroth

on phage stability

Temp Time Plaque count

C min

4 60 500 X 105

20 60 122 X 105

30 60 63 X 105

40 60 14 X 105

50 60 0.2 X 105

60 10 0

lawns, and the latter were more adaptable as

hosts.

Thelawn-spot methodyielded onlyonephage,

V-45, from various combinations of V.

fetus

strainsspottedonsoft-agar lawns. Althoughlysis

was often observed in thespots,phageswerenot

recovered from filtrates of thelysed zones.

Characterization

of phage V-45. The crude lysatefromphageV-45 onhost2255 in soft-agar

cultures contained approximately 1010 PFU/ml.

After low-speed centrifugation, the supernatant

fluid contained 109 PFU/ml, and, after filtration

through

0.45-,Am filters, the final titerwas 5 x 108 PFU/ml.

Attempts to extract

phage

from host cell lysates with 10% chloroform at 4 Cresulted in

almost complete inactivation (survivors, 0.008).

Oneper centchloroformin MAbroth inactivated

70% ofphage in 1 hr at 36 C.

About 76% of phage in MA broth was inac-tivated

by

incubation at20 C for 1 hr.

Propor-tionately greater inactivation was observed at

temperatures from 30 to 50 C, and all phage

activitywasdestroyed in 10 minat60 C(Table1).

In tests for phage stability at various pH

values, the

optimum

was aboutpH 5.9, with a

rapid drop in titer belowpH 5 and abovepH 7

(Fig. 1).

Theaddition ofCaCl2toMAbrothsuspensions of

phage

V45 contributed

significantly

to its

stability (Table 2).

One effect of

adding CaCl2

wasthe decrease in pH from 7.2to6.7. However, the addition of

phosphate

salts to buffer

broth

atpH6.7,without

CaCl2,

hadnoeffecton

phage

stability.

When5 to7.5 mgof

CaCl2

permlwas

added to broth containing 5 mg of phosphate.

saltsperml, the titer of

surviving

phage was

in-creased by a factor of103.

Addition

of MgSO4 to

MA broth wasbeneficial

for phage stability at 50 C, the water bath

tem-perature of thetop agar (Table 3). The

optimum

pHwith0.005 MMgSO4 was 7.5.

Tryptophan or cysteine in concentrations of

0.2 to1 mg/mlhad no effect on the phagetiter.

However, 2.5 to 5 mg/ml ofcysteine completely

on November 11, 2019 by guest

http://jvi.asm.org/

BRYNER ET AL.

l0

.

E

3-

40 5 8 9 1

a-I0

5.9

3 4 10

pH

FIG. 1. InfluenceofpHonphagetiter.TubesofMA

brothadjustedtovariouspHvalues from 3to 10 with INHCIorNaOH,containingS X 107 PFU ofphage

V-45/ml,wereincubated 24hrat36 Candassayed.

TABLE2. Effects ofCaCl2, phosphates, andpHon

phage stability inMAbroth with incubationfor 24 hrat36 C

Additiontobroth

(mg/ml) EndpH Plaquecount

CaC12 po4a

0 0 7.2 0.2X 10'

1 0 7.2 2.4X 10'

5 0 6.8 24.1 X 106

10 0 6.7 26.6 X 105

0 1 6.8 0.7 X 10'

0 5 6.7 0.5X 10'

0 10 6.4 0.4 X 10'

0 5 6.7 0.5X 105

0.1 5 6.7 0.5 X 10'

1.0 5 6.5 1.6 X 105

2.5 5 5.9 16.7 X 105

5.0 5 5.6 68.7 X 10'

7.5 5 5.4 107.0 X 10'

aP04 consisted of

parts KH2PO4.

one part Na2HPO4 to four

inactivatedthephage,sincethepHdroppedbelow

4.8.

Preparation of phage for electron microscopy

by washing in water with NH4-benzoate (2.5 to

10 mg/ml) inactivated most of the phage present. Calcium was required for phage replication in

broth cultures of the host. The titer of phage

increased by 103 PFU/ml in 7 hr with CaC12, but decreased in the absence of CaCI2 (Fig. 2).

Culture turbidity throughout the growth period

increased at the same rate in both flasks, and evidence of clearing resulting from phage lysis of

host cells was not observed ineither flask.

Antiphage serum, diluted 1:50, neutralized

90% of the phage in 10min, and the

neutraliza-tion K value was 23.

kwThe

phage-cell adsorption rate was

88%

in 15 min; the adsorption K value was 5.55 X 10-11

[image:3.487.56.245.50.325.2]

ml/min.

TABLE 3.Effect ofMgSO4onstability ofphage V45

inMA brothatvariouspH values, withincubation forI hrat50 C

Plaque count pH

MAbroth MAbroth + Mg2+a

6.0 11 X 104 69 X 104

6.5 9 X 104 72 X 104

7.0 3 X 104 124 X 104

7.5 2 X 104 183 X 104

8.0 0 135 X 104

aAs

0.005

M

MgSO4.

0L

0

In

a .

1 2 3 4 5 6 7

TIME (HOURS)

FIG.2. Comparison of phage V-45 replication in

shaker culturesofhost2255 with and withoutCaC12.

Theopticaldensitycurve(light line),superimposedover

thePFUcurves (heavy lines), was the sameforboth

flasks.

96

J.

VIROL.

on November 11, 2019 by guest

http://jvi.asm.org/

[image:3.487.259.451.252.595.2] [image:3.487.54.247.372.606.2]

U

FIG. 3. Vibrio fetusphage V-45inpotassiumphosphotungstate negativestainafterglutaraldehyde stabilization.

(A) Cellfragmentillustratingmultiplicity of receptorsitesandpredominance of "empty" heads. X 180,000. (B)

Typicalphage with "empty"

hlead,

nonsheathedtail,and ill-defined baseplate (B)associated with aknobof

recep-torsubstance (R). X 400,000. (C) Atypicalphage illustratinghexagonalsymmetry ofitspartiallyempty head membraneandsphericalsymmetry ofitsinternal residuum. X400,000. Each bar represents 100 nm.

on November 11, 2019 by guest

http://jvi.asm.org/

[image:4.487.40.440.61.556.2]

BRYNER ET AL.

In the single-step growth test, the minimum

latent period was 135 min, therise time was 75

min, and the average burst size was 34 PFU/

infectedcell.

In electron micrographs, phage were usually

attached to cells or cell fragments (Fig. 3A) and

the head was "empty" regardless of whether it

had been stabilized with glutaraldehyde.

Mor-phologically, this phage resembled members of

Bradley's group B; i.e., it lacked a contractile

protein sheath on the tail (Fig. 3B). Average

dimensions of the phage werehead, ca. 50 nm,

and tail, ca. 7 by240 nm. When discernible, the

tail appeared to have a 2- to 3-nm lumen and

some form of a base plate (Fig. 3B). Free-lying phage, especially after glutaraldehyde

stabiliza-tion, often had avariable-sized knob or disc at

the tail tip (Fig. 3B, C); these were probably

receptors since they were located at a fixed

distance (ca. 10 nm) belowremnantsofthe base plate ofthe tail.

DISCUSSION

Conventional methodsofphage isolationfrom

lysogenic

bacteria were ineffective when applied

to V. fetus. Characterization of phage V-45 in

this study has permitted the development of improvements in the techniques of isolation, preservation, andassay ofadditional phagesfrom

strains of V. fetus.

Phage isolation from V. fetus was greatly enhanced by the liquid culture of lysogenic strains in the presence of host cells. Growth of

lysogenic

strains was improved by cultivation in

thioglycollate medium; the growth time was

extended, resulting in a greater probability of

spontaneous inductionofalytic cycleof virulent

phage; sensitive host cells were continuously

present with the lysogenic cellstoreplicate

viru-lentphagetoa

higher

titer than couldbeobtained with the. "immune" lysogenic cells alone. The extended growth period was also important because of the slow phage-cell adsorption rate

and relatively long replication cycle in the V.

fetus-phage system. The importance of mixing

the host with thelysogenic strain was shown by

thefact that no plaqueswereproducedbyeither

culture when grown alone.

Phage extractionwith chloroform is not

prac-tical, asshownbythehigh inactivation rate in as

little as 1% chloroform.

The finding that low pH enhanced phage stability led to testing ofphosphate salts in MA

broth for plaque assaydilutions, with little

bene-fit. However, addition ofCaCl2 increased phage

stability byafactorof102, andthere wasalinear

relationship between the amount of

CaCl2

and

plaque counts. Furthermore, when both

CaCl2

andphosphate salts were added, the phage titer

wasincreased103,indicatingthat pH isimportant

in phage stability. Work with coliphage (1)

indicates that phosphates accelerate the rate of

heat inactivation of phage T5, whereas CaCl2

reverses the process by forming a precipitatewith

phosphates.

Although the optimum pH for phage stability

inMAbrothwas 5.9, the greatest stabilityin the

presence ofCaCl2was achieved at pH 6.5. Only

in thepresence of MgSO4 were phages stable at

pH 7.5. Althoughtests withMgSO4 utilized only

the 50 C temperature,the datain Table2indicate

thatphageV-45 should berelatively stable over

thepH range of 6 to 8 in MA broth with 0.005

MMgSO4.

Inadditiontoitsstabilizingeffect on freephage in broth, CaCl2 (probably

Ca2+)

also functions

as an essential cofactor for replication of phage

V-45inliquidcultures ofthe host.The mechanism

ofcalcium action probably involves the transfer

ofdeoxyribonucleic acid fromthephageinto the V. fetus cell as described by Lann (9) for the

Escherichia coli-T5 system. Other similarities

betweenT5 andV-45 arethe low serum neutral-izationKvalue, slowadsorptionrate, andgeneral

morphology.

The adsorption rate of V-45 was almost

identical to that reported by Firehammer and Border fora similarphage, but the latent period

waslessby45 min. Thelongerlatentperiod and

failure oftheir phage to replicate inthe second-growth tube

probably

was due to a calcium deficiency in their medium.

Morphologically, phage V-45 is

indistinguish-ablefromphagesVfi-6 and Vfv-3 ofFirehammer

and Border (6) and should be included, with coliphage T5, in Bradleys morphological group

B (3). All three phages appeared to be

"empty-headed" in electron microscopic preparations,

perhapsbecause ofa

proclivity

toattachtoexcess

receptor sites (2, 15) available in thelysate, with

consequent loss ofnucleicacids. The three phages

shareacommontail

antigen

(A.

E.Ritchieetal., unpublished data), indicating a close genetic relationship between the two

principal

V. fetus biotypes, intestinalis and venerealis. Phage

infec-tion of V.fetus

rarely

leadstocomplete

destruc-tion of the host cell, butinduces selective

altera-tion of their

enveloping membranes, revealing

an

underlying

hexagonal

pattern inthecellwall

(13).

LITERATURE CITED

1. Adams,M. H.1959.Bacteriophages. Interscience Publishers, Inc.,NewYork.

2. Bradley, D. E. 1963. The structureofsomestaphylococcus and pseudomonas bacteriophages. J. Ultrastruct. Res. 8:552-565.

98 J.VIROL.

on November 11, 2019 by guest

http://jvi.asm.org/

3. Bradley D. E. 1967. Ultrastructure of bacteriophages and bacteriocins. Bacteriol.Rev.31:230-314.

4. Brenner, S., and R. W. Horne. 1959. Anegative staining methodfor high resolution electron microscopy of viruses. Biochim. Biophys. Acta. 34:103.

5.Bryner, J.H., A. H.Frank,and P. A.O'Berry. 1962. Disso-ciation studies ofvibrios from the bovine genital tract.

Amer.J. Vet. Res. 23:32-41.

6.Firehammer, B.D.,and M. Border. 1968. Isolation of tem-perate bacteriophages from Vibriofetus. Amer. J. Vet. Res. 29:2229-2235.

7. Kay, D. 1952. Theeffect of divalent metalsonthe multiplica-tion ofcolibacteriophage T5st. Brit. J. Exp. Pathol. 33:228-235.

8. Keeler, R. F., A. E. Ritchie, J. H. Bryner, and J. Elmore. 1956. The preparation and characterization of cell walls

aid the preparation offlagella of Vibrio fetus. J. Gen. Microbiol.43:439-454.

9. Lannie, Y. T. 1954. Infection by bacteriophage T5 and its intracellular growth-a study by complement fixation. J. Bacteriol. 67:640-650.

10. Manclark, C. R., and M. Pickett. 1960. Quantity production of Vibrio fetus cells. J. Bacteriol. 79:752-753.

11. Matsushiro, A. 1961. Isolation ofUV-inducible temperate

phage 80. Biken J.4:133-135.

12. McDuff, C. R., L. M. Jones, and J. B. Wilson. 1962. Charac-teristics of brucellaphage. J. Bacteriol. 83:324-329. 13. Ritchie, A. E., and J. H. Bryner. 1968.Astructuralelement

in theenvelopesystemof Vibriofetus,p.78-79. In C.J.

Ar-ceneaux (ed.), Proc. 26th Annu.Meet. ElectronMicrosc. Soc. Amer.Claitor'sPublishing Division,BatonRouge, La. 14. Ritchie, A. E., and A. L. Fernelius. 1969. Characterization of bovine diarrhea viruses. Arch. Gesamte Virusforsch. 28:369-389.

15. Weidel,W.,andE.Kellenberger. 1955. The E. coliB

recep-torfor15.II.Electronmicroscopic studies. Biochim

Bio-phys. Acta 17:1-9.

on November 11, 2019 by guest

http://jvi.asm.org/