Host-phage interaction on Agrobacterium tumefaciens. IV. Phage-directed protein synthesis.

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Host-Phage Interaction

in

Agrobacterium tumefaciens

IV. Phage-Directed

Protein

Synthesis'

PATRICIA A. WALLS2 AND CHRISTINE F. POOTJES*

Department ofMicrobiology, ThePennsylvania StateUniversity, UniversityPark, Pennsylvania 16802

Received forpublication 10 September 1974

Gel electrophoretic and autoradiographic techniques were used to detect the

temporal sequence of protein synthesis after infection of the sensitive strain

Agrobacterium tumefaciens with phage LV-1. Three classes of protein were

detected: early proteins, class I, which include a protein capable of shutting off

hostproteinsynthesis;class II, proteins which are detected after 30min;and late

proteins, class III, which include the phage-directed endolysin and five

addi-tional proteins that appear 45 min after infection

Agrobacterium tumefaciens is a

gram-nega-tive rod which causes crown gall, a neoplastic

disease affecting avariety ofplants.

Lysogeny has been shown to be relatively

common in strains of A. tumefaciens (12).

Phage LV-1,atemperatephageisolated fromA. tumefaciens V-1,issimilar in size and

morphol-ogy to coliphage X. DeLey et al. (4) havefound

that phage LV-1 is virtually indistinguishable from three other agrophage, including omega,

the first temperate agrophage to be described

(2). Phage LV-1 has a polyhedral head and a

longflexibletail (12), which places it intoclass

B of Bradley's phage classification (3). The linear, double-strandedLV-1 DNA hasa

molec-ularweightof 31 x 106(5).Agenomeofthissize should be able toencode 30to40proteins. The phage is composed of four major structural

proteins and possibly as many as seven minor

ones.It isestimated that the structuralproteins

constitute less than 25% of the total coding

capacity of thephage DNA.

One

phage-coded

protein,

endolysin,

a late

enzyme responsible for lysis of the host cells

afterphage infection, has been described

previ-ously (10).

Synthesis

of

endolysin

can be

de-tected at 45 min after infection, halfway

through the latent period.

In the present study, gel electrophoretic and

autoradiographic techniques were used to de-tectthe temporal sequence ofprotein synthesis

after infection of the sensitive strain A.

tumefaciens B6 withphageLV-1.

MATERIALS AND METHODS

Bacterialandphage strains. A. tumefaciens B6, a phage-sensitive strain, originated from R. Klein,

'This is paper no. 4658 in the journal series of the Pennsylvania Agricultural Experiment Station.

'Presentaddress: Blair Memorial Hospital, Huntingdon,

Pa. 16652.

University of Vermont. Phage LV-1 was obtained by mitomycin C induction of the lysogenic strain A. tumefaciens V-1, which wasoriginally isolated by R. Hamilton, the Pennsylvania State University. Phage wereconcentrated and purified by CsCl density gradi-entcentrifugation prior toelectrophoresis.

Media and growth conditions. Labeling experi-mentsutilized modifiedStonier medium consisting of (perliter): 0.1 g ofCaSO4,0.2gofMgSO4 .7H2O,0.2g ofNaCl, 2.7 g ofNH4NOS, 8.4 mgofFe(NO3) .9H2O,

0.1 mgofMnCl2, 0.5 mg ofZnCl2, 10 g of potassium citrate, 2 g of sodium glutamate, 0.34 g of

NaH2PO4.H2O, and 0.9 g of K2HPO4 (8). Glucose,

sterilized separately, was added to a final

concentra-tion of0.2%. Cultures were incubated at 30 C in a gyratory waterbath.

Aglucose-yeast extract broth was used for all other experiments(7). These cultures were grown at 30 C on areciprocalshaker.

Radioactive labeling of proteins. A. tumefaciens B6 was grown to mid-log phase (3 x 108/ml) in Stonier medium. Before and after infection with phage LV-1 (multiplicity of infection of 15), portions of the culture were pulse-labeled for 3 min with "4C-labeled mixed amino acids (New England Nu-clear) at adosage of2uCi/ml.Incorporationoflabel was stopped by adding chilled Casamino Acids (Difco) to a final concentration of 3% and immedi-ately placing themixture onice.The cells were then harvestedby centrifugingfor 10minat 4300 x g.

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. (i) Preparation of samples for disc gels. The cells from 2.5-ml portions of culture

were harvested and lysed with 50 gliters of sample buffer. The sample buffer, a modification of that described by Laemmli (6), consisted of 0.0625 M sodiumphosphate buffer, pH 7.2, containing 2% SDS,

10% glycerol, and 5% f,-mercaptoethanol. The sam-ples were heated for 1 min at 100 C to facilitate dissociationofprotein subunits.

(ii) Disc gels. Acrylamide gels (10%) were pre-paredandelectrophoresis performed accordingtothe

methodsof WeberandOsborn (11).Samples (25to 40

,uliters)wereappliedtoeachgel.

Afterelectrophoresis, gelsforscintillationcounting

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PROTEIN SYNTHESIS IN A. TUMEFACIENS

were frozen and sliced into 2-mm fractions. Each fraction wasplaced into a vial and solubilized in a 3% solution of Protosol (New England Nuclear). After incubation at 37C for 48 h, the samples were counted in aliquid scintillation counter.

(iii)Slab gels forautoradiography.

Polyacrylam-ide gels andbuffers were prepared using the methods of Studier (9). Samples (10 to 20

gliters)

were placed into each well of the slab gel. Electrophoresis was carried out at a constant voltage of 35 V for 12 to 14 h.

Stainingand destaining werethe same as for the disk

gels. The slab gels were dried under vacuum and placed on Kodak Blue brand medical X-ray film for 10 to 14days before being developed.

RESULTS

Synthesis of phage LV-1 structural

pro-teins in infected cells. The four major

struc-tural phage proteins, Al, A2, A3, and A4,

described by De Ley et al. (4) are readily

detected by SDS-polyacrylamide gel electro-phoresis ofpurified phage LV-1 (Fig. 1). Only

two of these proteins, A2 and A3, can be detected in infected cell extracts against the heavily strained background of host protein

(Fig. 2).Thesetwoproteins arefirstobservedat 45 min after infection, halfway through the latent period.

Synthesis of majorphage-directed proteins

in infected cells. Pulse labeling of cells for 3 min with

"4C-labeled

mixed amino acids (2

iACi/ml)

atintervalsafter infection revealed the

pattern ofphageproteinsynthesisininfectedA.

tumefaciens B6. Attemptsweremadetoshutoff

hostproteinsynthesistoinsure incorporation of

the labeled amino acids into phage protein rather than into host protein. UV irradiation,

evenin low

doses, rendered

A. tunLefaciens B6 virtually incapable of supporting phage-directed protein

synthesis.

Another indirect

attempt to suppress host

synthesis

was to treat

the cells with nalidixic acid (10

ug/ml) shortly

before phage infection, but nalidixic acid only partially suppressed host cell

synthesis.

Fortunately,

one of the early functions of

phage LV-1istoreduce the proteinsynthesisof

the hoststrain B6. Thisinhibitionisevidentas early as 1 min after infection and is quite pronouncedby30min(Fig. 3), asdemonstrated

by gel electrophoresis

ofthelabeled

samples. By

60 min postinfection, 10 to 12 protein peaks

appear, and the major structural proteins are

evident.

Synthesis

ofminor

phage-directed proteins

ininfected cells.Autoradiographywasusedto detect phage-directed proteins which are

syn-thesized in smaller quantities than could be

readily detected by scintillation counting of fractionated disk gels.

"4C-labeled

extracts of

uninfected and phage-infected A.

tumefaciens

B6were run on 10 and 15%polyacrylamideslab

gels accordingtotheprocedures ofStudier (9).

An autoradiogram of a 10%gel isshown in Fig. 4. Uptake oflabel was lowest at 15 min after

infection, and it was slightly higher in later

samples. The intensity of the exposure of the

autoradiogram (Fig. 4) indicates that protein

synthesis did not return to the preinfection level.

Since hostproteinsynthesis was not entirely stopped, it was sometimes difficult to

distin-guish phage proteins from residual host

pro-teins. Thirteen new proteins appear to be

defi-nitely of phage origin. An additional seven

proteins, P1,P3, P5, P8, P10,P11, andP15 (Fig.

5a), could be either phage or host directed. Figure 5a provides an analysis of the time

sequence ofappearance ofthe phage proteins.

Synthesisofeightofthe 13phage proteins had begun by 15minafter infection. Phage proteins

P4, P7, P9, P13, P17, and P18 exhibited

inter-valsofmaximal synthesis followedby decreased synthesis, which suggests the existence of

con-trol mechanisms.

The majorphage structural proteins, A2and A3, areindicated inFig. 4. Synthesis of A3 had begun by 15min,whereas A2wasfirstdetected

inthe30-minsample. Either P12orP13(Fig. 4)

probably corresponds to phage structural pro-tein Al. A4 could not be located because the Tris-glycine buffersystem doesnot allow

sepa-ration ofproteinshavingmolecularweights less

than 25,000 on a 10%gel (9).

When theproteinswere

separated by

electro-phoresis on a 15% slab gel, four new proteins

migrating in advance of P18 could be

distin-guished on the autoradiogram (Fig. 6). These

weredesignated P19a, P19b, P20, andP21.The

times oftheir appearance are

presented

in

Fig.

5b. Protein P19a is an

early protein,

which is

being synthesized

by

15 min

postinfection.

The other three proteins did not appear until

half-waythrough the latent

period.

DISCUSSION

We have found that, after infection of A.

tumefaciens B6 with phage LV-1, one of the

early functions of the phage is to reduce host protein synthesis. This suppression is evident as

early as 1 min after infection, and it is very

pronounced by 30 min (Fig. 3). This was a

fortuitousdiscoverysince ourattempts to

artifi-cially suppress host synthesis either had little

effect or were so effective that phage protein

synthesiswasalsoblocked.However, since this

phage-directed suppression was not complete,

some of the proteins being synthesized after

infection could still represent residual host

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A

I

_Ip

A2

tulip

A3

MP

A

4

synthesis. Such doubtful proteins are desig-nated by a question mark in Fig. 5.

Sixteen, and possibly asmany as 24, phage-directed proteins weredetected by autoradiog-raphy. This represents a substantial portion of

theestimated30 to 40proteinswhich the phage

DNA should be able to encode. The major

structural proteins described by De Ley et al. (4) are tentatively identified on the

autoradio-grams (Fig. 4 and 6).

A plot of the times of appearance of new

proteins suggests that control mechanisms are

important in directingsynthesis ofphageLV-1

proteins (Fig. 5). Proteins P17, P18, and P19a

areactivelysynthesized from 15 to45min after

infection, afterwhichtheir synthesis appears to

decline steadily. P14and P16 appear at 30 min

whereasP2, P6, P20, and P21 are delayed until

45 min. It isinteresting to notethatonstained

gels (Fig. 2), the two mostabundant structural proteins, A2 and A3, were not

distinguishable

until 45minafter

phage

infection.However,

by

autoradiographic

methods,

A3 can be detected

at 15 min and A2 at 30 min (Fig. 4and6).

Proteins P19a andP19b, seen inFig. 6, may

betwodistinct proteins, or

they

mayrepresent

different forms of the same protein. P19b is

vaguely discernible at the leadingedge ofP19a

in the 45-min sample. By 60

min,

P19b is

predominant, and P19aisveryindistinct. Only P19b is evident at 90 min. This suggests that P19b may be a cleavage product of P19a. As

P19a is being cleaved at 45 min, the new,

smaller protein,P19b,isabletomigrate

farther,

thus appearing at theleading edgeofP19a. As

more P19a is cleaved and no more is

synthe-sized, it disappears and

P19b

predominates.

EitherP19borP20 isprobably equivalenttoA4,

oneofthephage structural proteins.

Although the exact size of agrophage

en-dolysin

hasnot yet been determined (10), it is

probably similarto thelyticenzymescoded by the T phage and by coliphage X. Ifthis is the

case, the enzyme

probably

has a molecular

weight of 15,000 to 20,000, and it would be expected to migrate in the lower third of the

15% slab gel. The in vitro assay revealed that

endolysin begins to be synthesized at 45 min

after phage infection (10), the same time that

P19b, P20, and P21 are firstdetected(Fig. 6).

Different classes of protein synthesis can be

detected in phage-infected cells. The early

proteins, class

I,

include one capable of shutting

FIG. 1. Structuralproteins of phage LV-1. Phage

particles, purified by banding in CsCl, were

dena-tured, and thecomponent proteins were separated by

electrophoresison 10%SDS-polyacrylamidegels.The

gels were stained with Coomassie Blue. MP, Minor

protein.

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PROTEINSYNTHESISINA. TUMEFACIENS 375

~ _

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_

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~~~~~A2

4

A 3

FIG. 2. Theappearanceof phageLV-l structuralproteins inextractsof infected A. tumefaciens B6. Proteins inthe crudecellextractswereseparated byelectrophoresison10%SDS-polyacrylamidegels and stainedwith Coomassie Blue. (a) Uninfected; (b)45minafterinfection; (c) 60min; (d) phageLV-1 structural proteins.

VOL.15, 1975

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offthe host protein synthesis as early as 1 min

after infection. Fourteen proteins can be

de-tected by radioautography within the first 15

min after infection. Anadditional five proteins

appear in a second class, class II, which are

detected after 30 min. The late proteins, class

III, which include the endolysin, consist ofan

additional five proteins thatappear45minafter

infection and these continue to be synthesized

until the end of the latent period. One of the

class I proteins and one ofthe class II proteins

appear to be shut off after 48 min which

indicatesaregulatory mechanism for turning off

protein synthesisaswellasinitiation. Fromthe

concentrations of the bands, periods of

in-creased and decreasing amounts or protein

synthesis are observed with the suggestion of

the cleavage ofone ofthe proteins, P19.

Further experiments with temperature-sensi-tive mutants will be needed to determine the function of these proteins.

LITERATURE CITED

1. Beardsley, R. E. 1955. Phage production by crown-gall bacteria and the formationofplanttumors. Am.Nat. 89:175-176.

2. Beardsley, R. E. 1960. Lysogenicity in Agrobacterium

tumefaciens.J.Bacteriol.80:180-187.

3. Bradley,D.E. 1967.Ultrastructure ofbacteriophagesand bacteriocins. Bacteriol. Rev. 31:230-314.

4. DeLey, J., M. Gillis, C. F. Pootjes, K. Kersters, R. Tytgat,and M. VanBraekel.1972.Relationshipamong temperate Agrobacterium phage genomes and coat proteins.J.Gen. Virol. 16:199-214.

5. Korant, B. D., and C. F.Pootjes. 1970.Physiochemical properties ofAgrobacterium tumefaciens phage LV-1 and itsDNA.Virology40:48-54.

6. Laemmli, U. K. 1970. Cleavage of structural proteins duringtheassembly of the head of bacteriophage T4. Nature(London)227:680-685.

7. Pootjes, C., and B. Stemberger. 1974. Host-phage in-teraction inAgrobacterium tumefaciens.II.Hoststrain

responsetomitomycin C induction.Can. J. Microbiol. 20:367-370.

8. Stonier,T. 1956.Labelingcrowngall bacteriawith P32 for radioautography. J. Bacteriol. 72:259-268.

9. Studier, F. W.1973. Analyses ofbacteriophage T7early RNA's and proteins on slab gels. J. Mol. Biol.

79:237-248.

10. Walls, P. A., and C. F. Pootjes.1974.Host-bacteriophage interaction inAgrobacteriumtumefaciens. III.

Phage-codedendolysin.J. Virol. 13:937-938.

11. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determinations bydodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412.

12. Zimmerer, R. P., R. H. Hamilton, and C. Pootjes. 1966. Isolation andmorphologyoftemperateAgrobacterium tumefaciens bacteriophage.J. Bacteriol. 92:746-750.

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J.VIROL.