function and structure of microvirid phage α3 genome: i. electrophoretic characterization of...

Biochimica et Biophysica Acta, 788 (1984) 333-338 333

Elsevier

BBA 31966

FUNCTION AND STRUCTURE OF MICROVIRID PHAGE a3 GENOME

I. ELECTROPHORETIC CHARACTERIZATION OF PROTEINS ENCODED BY WILD- TYPE PHAGE

KEN-ICHI KODAIRA and AKIRA TAKETO

Department of Biochemistry I, Fukui Medical School, Matsuoka, Fukui 910-11 (Japan)

(Received February 16th, 1984)

Key words: Phage protein," Gel electrophoresis," a3 genome; (Bacteriophage)

The proteins encoded by microvirid phage a3 were characterized by electrophoresis on polyacrylamide gels. Virion of a3 was composed of four major proteins F (capsid: 48.5 K), G (spike: 22.0 K), H (spike tip: 37.3 K) and J (core: 2.8 K). Besides these, three major proteins A (56.5 kDa), B (18.2 kDa) and D (14.6 kDa) were detected in ultraviolet-irradiated uvrA cells of Escherichia coil infected with a3. Protein A was resolved into a doublet and protein B was accompanied by a band with slower mobility. Heterogeneity was also observed in proteins H and G, on S D S / u r e a polyacrylamide gel or isoelectric focusing gel. In addition to these products, several low-molecular-weight proteins were induced by the phage. Protein composition of a3 intermediate complexes was analyzed as well. Comparison was made with the proteins encoded by thK, GI4, G4 and ~X174.

Introduction

Bacteriophage a3 [1] is a member of Microviri- dae (isometric single-stranded DNA phage family), which includes more than 20 viral species. Unlike the ~X174 group (~A, ~B, ~R, G6 and $13), a3 can replicate without host functions directed by dnaB and dnaC(D) genes [2,3] and infects Escherichia coli B as well as E. coli C [1]. Immuno- logically, a3 is somewhat related to ~K-St-1 group, whose replication is also independent of host dnaB and dnaC(D) activities [4,5]. Replication of such microvirid phages as G4, G13, G14, ~C and U3 is restricted at 41-43°C, even in the wild-type host [6], whereas a3, ~,K and St-1 efficiently grow over this temperature range [2,4,5].

Despite these properties, its higher yield and stability, neither biochemical nor genetic analyses have been performed with a3 proteins. In this paper, we describe properties of a3-encoded pro-

teins as revealed by electrophoresis on polyacrylamide gels. Intracellular proteins induced by d?K-h3 have also been analyzed and compared with those of other microvirid members.

Materials and Methods

Phage and bacterml strains. Bacteriophages used in the present investigation were a3, ~X174, G4, G14 and ~K-h3. Strain ffK-h3 was a host range mutant of ,~K and, like ~Kh-1 [5], infective to E. coli C and HF4704 as well as to K12. Sources of other phage species have been described previously [6]. For propagation and titration of these phages, E. coil C was used, whereas strain HF4704 uvrA thyA was employed for radioactive labeling of phage-induced proteins.

Preparation of virion proteins. Cells of E. coli C were grown in a Tr is /casamino acids/glucose (TCG) medium [7] at 37 °C, with shaking. When

0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.

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the absorbance at 660 nm (A660) reached 0.7, 1/100 vol. of phage (input multiplicity about 0.001) were added together with 1/200 vol. of 1 M CaC12. After lysis, phage particles were extracted as described previously [8] and purified by two cycles of CsCI equilibrium centrifugation. After removal of CsC1 by dialysis, the particles were heated at 100°C for 2 min, in 62.5 mM Tris-HCl (pH 6.8)/3% SDS/5% mercap toe thano l / l% glycerol (buffer D).

Labeling of phage-induced proteins. Cells of E. coli HF4704 uorA thyA were grown, at 37 °C with shaking, in TG medium (TCG medium deficient in casamino acids) supplemented with 2 /~g/ml thymine. When A660 reached 0.3, the culture was irradiated with ultraviolet light, at a dose of about 4000 e rg /mm 2. After incubation at 37°C for 15 min, phage (input multiplicity about 10) and CaC12 (final concentration 10 mM) were added to the irradiated culture. At 20 min post-infection, uni- versally labeled L-[14C]leucine was added to 50 /~Ci/ml, and labeling was continued for 20 min. The culture was chilled at 0 °C, centrifuged and the cells were heated in buffer D at 100 °C for 2 min, for protein extraction.

SDS-polyacrylamide gel electrophoresis. The denatured protein prepared as above were electrophoresed through a slab gel (14 × 13 × 0.1 cm), at 15 mA for 6 -7 h, with an electrode buffer composed of 25 mM Tris-HC1 (pH 8.3)/0.1% S D S / 1.44% glycine. A discontinuous SDS-polyacrylamide gel system similar to that described by Laem- mli [9] was used. The separating gel contained 10 or 15% polyacrylamide (the ratio of acrylamide to N,N'-methylenebisacrylamide was 37.5), 0.375 M Tris-HC1 (pH 8.8) and 0.1% SDS. The stacking gel was composed of 4% acrylamide, 0.8% N, N'-methylenebisacrylamide, 0.125 M Tris-HC1 (pH 6.8) and 0.1% SDS. For gel polymerization, 0.05% N, N, N ', N ' - t e t ramethy le thy lened iamine and 0.075% ammonium persulfate were used. Electro- phoresis on 18.5% polyacrylamide gel containing 0.5 M urea and 0.1% SDS was carried out as described by Pollock [10]. After the electrophoretic run, non-labeled virion proteins and marker proteins were detected by staining with Coomassie brilliant blue in 40% methanol/10% acetic acid at 37 °C for 30-60 min. The gel was then destained in 20% methanol /10% acetic acid, dried at

60-70 °C in vacuo and exposed directly to Kodak NS-5T X-ray film at room temperature for 4-5 days.

Two-dimensional gel electrophoresis. Two-dimensional (isoelectric focusing-SDS polyacrylamide slab gel) electrophoresis was performed by the method of Ames and Nikaido [11], with minor modifications. Ampholines covering pH ranges 3.5-10 and 9-10 were mixed in the ratio of 4 : 1. Total concentration of ampholines was 2%. The foregoing 15% polyacrylamide containing 0.1% SDS was used as the second-dimensional gel.

Other details. L-[14C]Leucine (uniformly labeled, 324 mCi /mmol) was purchased from Amersham International. Trypsin inhibitor (molecular weight 21 500), cytochrome C (12500), aprotinine (6500) and insulin chain B (3400) were obtained from Boehringer-Mannheim GmbH. Chymotrypsinogen A (25000) and myoglobin (17800) were from Sigma Chemical Co. and albumin (67000) from Nakarai Chemicals, Kyoto. These proteins were boiled for 2 min in buffer D, and used as the marker in electrophoresis.

Results and Discussion

Proteins of a3 virion Purified a3 particles were disrupted by heating

in buffer D (containing SDS and mercaptoethanol) and the virion proteins were characterized by electrophoresis on 15% polyacrylamide slab gel containing 0.1% SDS. Proteins of ~X174 and G14 particles were run in parallel, together with authentic marker proteins. As shown in Fig. la, four major proteins were detected in a3 virions, as in 4,X174 and G14. These proteins were respec- tively assigned the cistron letters F (capsid), H (spike tip), G (spike) and J (core), as those of q~X174, $13 and G4 [12]. Electrophoretic mobilities of the proteins F, H and G were lower in a3 than in 4,X174. In this regard, it seems noteworthy that a3 sediments slightly faster than q, X174 [13]. If mercaptoethanol was omitted from buffer D, the mobility of protein G decreased substantially. The apparent molecular weights of a3 virion proteins, estimated by comparing each relative mobility with that of q~X174 proteins and authentic markers, are presented in Table I. Besides the four proteins, minor bands (P1-P3) were observed be-

335

( A )

• .,= ~

( S )

F

" Pz

A"

- - G

- - .J

- - F

- - H

~ Gt Gz

G3

- - J

Fig. 1. Electropherograms of a3 virion proteins. (A) a3 particles purified by CsC1 equilibrium centrifugation were denatured and electrophoresed on a 15% polyacrylamide/SDS gel. Virion proteins of epX174 and G14 were coelectrophoresed together with marker proteins (not shown). Letters at the fight side indicate positions of a3 proteins and not of G14 proteins. (B) Denatured a3 virion proteins were electrophoresed on a 18.5% polyacrylamide/SDS/urea gel. After electrophoresis, proteins were visualized by staining with Coomassie brilliant blue.

tween F and H. Although significance of these minor bands is at present unknown, P3 is probably related to protein H, because both P3 and H specifically disappear from the extract of cells infected with amber gene H mutant (unpublished data). A protein band similar to A* of ~X174 was also detectable between H and G. When 18.5% polyacrylamide gel containing SDS and urea was used, protein G was resolved into one major band G 1 and two minor bands G 2 and G 3 (Fig. lb). Pollock et al. [15] obtained similar results with G protein of ,~X174 and S13, and suggested that the multiple gene products might arise from multiple sites for initiation or termination of translation [15]. Upon two-dimensional electrophoresis, a3

protein H, in addition to G, exhibited heterogeneity (Fig. 2). In protein H, the observed heterogeneity may be due to protein modification, because its occurrence is markedly affected by conditions for phage preservation, e.g., ionic strength of buffer. On the other hand, multiple spots or doublets were not detected in protein F. Reflecting its basicity, a3 protein J ran off the first isoelectric focusing gel (Fig. 2 and unpublished results). The J protein of ,#X174 is selectively extractable from capsid fraction, with formic acid [16]. When a3 particles were treated with 5% formic acid, bulk of the J protein and a small amount of F were released into the soluble fraction. Using the molecular weights presented in Table I and protein con- centrations calculated from densitometer scans of electropherograms, the number of molecules of J protein per a3 virion was estimated, assuming that a3 virion, like ¢X174 [17], contained 60 molecules of protein F. Depending on each SDS gel (15-18.5% acrylamide with or without urea), the number of J varied from 50-200 molecules per virion. This fluctuation may be due to partial loss of the basic and low molecular weight protein, during staining and /o r destaining steps.

Fig. 2. Two-dimensional electrophoresis of a3 virion proteins. Purified a3 particles were denatured and subjected to two-dimensional electrophoresis: first, on isoelectric focusing gel (IEF); second, on SDS gel.

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TABLE I

MOLECULAR WEIGHT OF PROTEINS ENCODED BY a3 AND RELATED PHAGES

The molecular weight of each protein (× 10 -3) was estimated from electrophoretic mobility (a3 and q~K-h3) or from nucleotide sequence (~X174 and G4) reported by Godson et al. [14]. n.d., not determined.

Cistron Virion proteins Intracellular proteins encoded by:

a3 ~,X174 a3 ffK-h3 q~X174 G4

A - - 56.5 54.0 58.7 63.4 F 48.5 48.4 48.5 52.3 48.4 48.7 H 37.7 34.4 37.7 35.8 34.4 35.7 G 22.0 19.1 22.0 21.8 19.1 19.2 B a _ 18.2 18.2 13.8 13.8 D a - - 14.6 14.6 16.5 16.9 E - - n.d. n.d. 10.6 10.5 C - - n.d. n.d. 10.1 9.9 K - - n.d. n.d. 6.4 6.3 J 2.8 4.2 n.d. n.d. 4.2 2.8

a In proteins B and D, the molecular weight estimated from their electrophoretic mobility differs considerably from that calculated from DNA sequences.

2 ~z

F -

6 - -

Fig. 3. Electrophoresis of intracellular phage-encoded proteins on a 10% polyacrylamide/SDS gel. Ultraviolet-irradiated cells of HF4704 uvrA thyA were infected with the indicated phage

Intracellular proteins induced by a3 and ¢oK-h3 I n o r d e r to c h a r a c t e r i z e t he i n t r a c e l l u l a r v i ra l

p r o t e i n s , u l t r a v i o l e t - i r r a d i a t e d H F 4 7 0 4 uvrA cel ls

w e r e i n f e c t e d w i t h a 3 a n d l a b e l e d w i t h [14 C] leuc ine .

T h e r a t e o f r e s i d u a l s y n t h e s i s o f h o s t p r o t e i n was

n e g l i g i b l y low in t he i r r a d i a t e d b a c t e r i a . T h e in-

f e c t e d cel ls were lyzed a n d p r o t e i n s we re e l ec t ro -

p h o r e s e d o n S D S - p o l y a c r y l a m i d e gels, f o l l o w e d b y

a u t o r a d i o g r a p h y . A s s h o w n in Figs . 3 a n d 4, a b o u t

t e n m a j o r b a n d s w e r e d i s c e r n i b l e in t he a u t o r a d i o -

g r a m s . A m o n g t h e s e b a n d s , six p r o t e i n s we re i d e n -

t i f ied , b y r e f e r r i n g to i n t r a c e l l u l a r p r o t e i n s en-

c o d e d b y q, X 1 7 4 a n d G 4 as wel l as to a 3 v i r i o n

p r o t e i n s . I d e n t i f i c a t i o n o f p r o t e i n J was n o t easy

in e x t r a c t s o f a 3 - i n f e c t e d cells, e v e n 18.5%

a c r y l a m i d e gel c o n t a i n i n g 0.5 M u r e a a n d 0.1%

S D S was used . I n 15% gel a n d 18.5% gel ( c o n t a i n -

i ng urea) , s eve ra l p r o t e i n b a n d s we re c o n s t a n t l y

o b s e r v e d w h i c h m i g r a t e d f a s t e r t h a n D, a n d m i g h t

c o r r e s p o n d to E, C a n d K. I d e n t i f i c a t i o n o f t h e s e

and labeled with [14C]leucine (from 20-40 min postinfection). The cells were lyzed and the extracted proteins were electrophoresed on a 10% gel. After Coomassie blue staining of coelectrophoresed a3 virion proteins (each position indicated by arrow) and marker proteins, radioactive proteins were detected by autoradiography. Letters at the left-hand side indicate positions of intracellular q, X174 proteins and letters at the fight-hand side locate the a3-encoded proteins.

337

F - -

H - -

B

D- -

E m

C--

K ~

7

>

--gt; F Pt ~-- P2 P3 H

A •

G

B

D

Fig. 4. Electrophoresis on 15% polyaerylamide-SDS gel of intracellular proteins encoded by ,*3 and related phages. Condi- tions and indications are the same as in Fig. 3, except that 15% gel was used.

low-molecular weight proteins is difficult at present stage. Table I summarizes the apparent molecular weights of the intracellular a3-encoded proteins. Proteins A and D of a3 were smaller than those of ~X174 and G4. As observed in virion proteins, minor bands (P1-P3) were also detected in electropherograms of proteins extracted from a3-infected cells. In addition, protein A was separated into doublet A 1 (minor) and A 2 (major), whereas protein B was accompanied by a band with slower mobility (Fig. 4). Although this slower component is absent in extracts from uninfected cells, its cistron assignment is not feasible as yet. Intracellular protein G of a3, unlike those of g, X174 and G4 [15], did not exhibit any heterogeneity in 10-15% gel.

Cells of HF4704 uvrA, infected with a3 after ultraviolet irradiation and radiolabeled as above,

were gently lyzed with lysozyme-EDTA treatment and the extract was centrifuged in a linear sucrose gradient [18]. Protein aggregate equivalent to the 12 S complex of ~X174 [18] was detected in a3-infected cells as well. Only two protein bands, F and G, were generally observed in electropherograms of this aggregate. In some cases, however, this '12 S complex' of a3 was accompanied by small amount of protein D. In addition, a DNA protein aggregate having an S value of about 50 was isolated, according to the method of Fujisawa and Hayashi [19]. Upon electrophoresis, the complex revealed four virion proteins (F, G, H, J), A, and two minor bands (B and D). Further characterization of these intermediate complexes is in progress, using a3 mutants.

From the results presented above, it seems clear that at least seven major proteins (A, B, D, F, G, H and J) are encoded by a3. Genetic and biochemical analyses of amber or missense mutants have proved the validity of the cistron assignment (unpublished data). Although three additional proteins (C, E and K) are known in ~X174 and G4 [12], and candidates with faster mobilities are cer- tainly seen in the autoradiograms of a3-induced proteins, physical identification of these bands seems to be impracticable. The presence of cistron C in a3 has, however, been confirmed by the isolation of distinct amber mutants, whereas anal- ysis of the nucleotide sequence has detected a DNA structure closely resembling gene E of ~ X174 (unpublished data).

The electrophoretic profile of intracellular proteins induced by ~K-h3, a host-range mutant of ~K [4] infective to E. coli B, C and K12, was essentially similar to that of other microvirid members. As shown in Fig. 3 and 4, six major protein bands (A, B, D, F, G and H) were distinguishable in 10% a n d / o r 15% gel. Protein A of ~K-h3 was the smallest, whereas protein B of this phage was as large as that of a3, and the F protein was the largest among the microvirid species thus far analyzed. Protein G of ,~K-h3 was separated into two bands on 15% gel.

Regardless of differences in antigenicity, host range and host factor requirement, microvirid phages seem to encode for similar spectrum of proteins. In addition, the genetic map of a3 closely resembles those of ~X174 and G4 (unpublished

338

data). Corresponding proteins, however, somewhat diverge from each other, at least in size. The nucleotide sequences of several a3 genes have been determined completely (H and J) or partially (A, D, E, F and G), and amino-acid sequences of these proteins deduced from the DNA sequences also exhibit a certain divergence from those of ~X174 and G4 (unpublished data). The double-stranded replicative form DNA of a3 is several hundred basepairs longer than those of epX174 and G4, having capacity for encoding additional protein(s). Thus, unlike e#X174 and G4, a3 has several poten- tial coding frames in the intracistronic region between A and H. Further studies are underway to elucidate the relationship between these DNA re- gions and unidentified protein bands in electropherograms.

Acknowledgements

Most of this work was performed at Depart- ment of Biochemistry, Kanazawa University School of Medicine. We are grateful to Dr S. Kuno for his generosity and support.

References

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(1978) in The Single-Stranded DNA Phages (Denhardt, D.T., Dressier, D. and Ray, D.S. eds.) pp. 51-86, Cold Spring Harbor Laboratory, New York

15 Pollock, T.J., Tessman, I. and Tessman, E.S. (1978) Nature 274, 34-37

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