JOURNAL OF VIROLOGY, Jan. 1974, p. 211-221Copyright i 1974 American Society for Microbiology

Vol. 13, No. 1Printed in U.S.A.

Analysis of Bacteriophage 029 Gene Function: Protein Synthesisin Suppressor-Sensitive Mutant Infection of Bacillus subtilis

DWIGHT L. ANDERSON AND BERNARD E. REILLY

Department of Microbiology and School of Dentistry, University ofMinnesota, Minneapolis, Minnesota 55455

Received for publication 2 July 1973

Phage 029 suppressor-sensitive (sus) mutants of 14 cistrons have beenexamined for production of "C-labeled viral-specific proteins in restrictiveinfections of Bacillus subtilis. Proteins specified by four cistrons (H, J, L, and N)have been resolved and identified by sodium dodecyl sulfate gel electrophoresisand autoradiography, and fragments of the normal polypeptides were detected.Mutants of six cistrons (C, D, E, F, I, and M) demonstrated two or more missingbands in the gel profiles, and thus some of these gene products may haveregulatory functions. Mutation was detected in at least five genes codingfor low-molecular-weight proteins, but a conditionally lethal mutant in only oneof these genes has been isolated. Preliminary evidence that a precursor protein iscleaved to generate the neck appendage structural protein and a low-molecular-weight product has been obtained.

The Bacillus subtilis phage 029 has a uniquecomplex morphology (3), and seven viral struc-tural proteins resolved by electrophoresis havebeen identified as components of the head, neck,and tail (2, 15). The genome of 029 is a linearDNA duplex with a molecular weight of 11 x101 (3). A genetic map has been constructed bythree-factor crosses with suppressor-sensitive(sus) mutants that defines the order of 10 of 13cistrons (20). We have resolved 23 'IC-labeled429-specific proteins in lysates of UV-irradiatedB. subtilis by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis and autora-diography (8). We have described the temporalsequence of protein synthesis and concludedthat the combined molecular weights of theproteins reflect about 90% of the coding capac-ity of the 429 genome (8).We have examined the patterns of

4)29-specific protein synthesis after infection bysus mutants of 14 cistrons under restrictiveconditions. Sus mutants, viable in su+8 cells (7),produce fragments of specific polypeptidechains in the nonpermissive host. For four viralcistrons we have established the identity of theprotein coded by the cistron, and for eachcistron we have identified at least one polypep-tide fragment. Mutation in six additional cis-trons results in the loss of two or more proteins.The regulation of protein synthesis during 429development is thus quite complex, and thenumber of cistrons committed to this function ismore than we had imagined.

MATERIALS AND METHODSPhage and bacteria. Phage 029 (19) and a clear-

plaque mutant of spontaneous origin, 429c, wereemployed in this study. Suppressor-sensitive (sus)mutants of 429 or 029c were isolated after mutagene-sis with hydroxylamine (20) or bromodeoxyuridine(see below).The properties of the permissive host B. subtilis

L15 and the nonpermissive host B. subtilis SpoA12have been described (20). B. subtilis trpC2 thyAthyB, a tryptophan- and thymine-requiring auxo-troph, was employed in the isolation of bromodeoxyu-ridine mutants (24).

Bacteriophage assay. Our methods and media forlysate preparation have been described (4, 19), andstandard phage techniques were used (1). The phagewere stored in Difco antibiotic medium 3 (PB).Mutagenesis with bromodeoxyuridine. Phage

4)29 replication occurs in the presence of 6-(p-hy-droxyphenylazo)-uracil (HPUra), but incorporation of['H]thymidine into B. subtilis DNA is blocked (14,24, 27). HPUra was obtained from Bernard Langley ofImperial Industries, Ltd., and was supplied to us by J.Pene. B. subtilis trpC2 thyA thyB was grown in theminimal medium of Spizizen (25), supplemented with0.5% casein hydrolysate (Nutritional BiochemicalsCorp., Cleveland, Ohio), tryptophan (10 ;sg/ml), andthymidine (10 ug/ml). Phage 029 was added, at amultiplicity of infection (MOI) of 5, to cells in thesupplemented minimal medium containing bromode-oxyuridine (40 pg/ml). HPUra was added to oneculture to give a final concentration of 250 jg/ml.After 90 min at 37 C with aeration, the infected cellswere lysed with egg white lysozyme (Calbiochem, 10jsg/ml). The burst size varied from 25 to 50 phage percell. These mutagenized stocks were used to infect the

211

ANDERSON AND REILLY

permissive host (107 cells/ml) in PB at an MOI of 5.After 30 min at ambient temperature, samples werediluted in PB to give about 100 infected cells per tubeand incubated at 37 C until lysis occurred. Only onemutant was isolated per tube, and the mutants wereassigned to a cistron by the use of qualitative andquantitative complementation (20).

Backeross methods. To reduce the frequency ofmultiple mutation in phage stocks, representativemutants of each cistron were backcrossed with refer-ence phage. This was especially useful when the susmutant was temperature sensitive or the plaquemorphology of a revertant virus differed from thewild-type 429. One method employed the standardtwo-factor cross (20) to generate recombination be-tween the sus mutant and a reference phage, 429 or429c, that differed only in the allele present at theclear (c) locus. Alternatively, a recombinant of the susmutant and a reference sus virus was constructed, andthe recombinant was then crossed with 429 to regener-ate the initial sus mutant. On occasion these twomethods were employed in sequence.

Conditions for infection and isotopic labeling.Our methods for infection and isotopic labeling havebeen reported in detail (8). In brief, B. subtilisSpoA12, grown at 37 C in M40 medium (17) to 2 x 101cells per ml, was sedimented (5,000 x g, 5 min, 25 C)and suspended at 2 x 109 cells per ml in M40. A 2-mlsuspension of cells was irradiated with UV light for 10min at a dose of 50 ergs per mm2 per s in a petri dish(35 by 10 mm), with mixing every 15 s. Wild-type ormutant 029 was added at an MOI of 50; after 10 minat 25 C, 0.1 ml of the culture was added to 0.9 ml ofM40 at 37 C containing a mixture of "4C-labeledamino acids (New England Nuclear Corp., NEC 445,2 pCi/ml, about 200 MCi/mmol); and incubation wascontinued at 37 C for 35 min. The labeling wasterminated by pipetting the infected culture into anequal volume of an iced solution containing 0.1 MNaCl, 0.05 M sodium citrate, 0.01 M sodium azide,chloramphenicol (200 ;ig/ml), and the serylproteaseinhibitor, phenylmethysulfonylfluoride (Sigma Chemi-cal Co., St. Louis, Mo., 600 jsg/ml).

Preparation of "4C-labeled 429 proteins and gelelectrophoresis. The "4C-labeled proteins of the cellpellet and supernatant fractions were prepared forSDS gel electrophoresis as described previously (8).The proteins were separated on linear gradient gels of12 to 16% or 16 to 20% polyacrylamide. The methodsemployed for autoradiography and microdensitome-try have been reported (23).

RESULTSTwenty 029-specific proteins were detected in

UV-irradiated B. subtilis when the culture waslabeled with a mixture of "4C-labeled aminoacids 10 to 45 min after infection and analyzedby SDS gel electrophoresis and autoradiogra-phy. These proteins are demonstrated in Fig. 1,as presented previously (8). Because someproteins begin to enter the medium 8 min afterinfection and others are exclusively retained bythe cells, both the cell pellet and the trichloroa-

P(J)-

Ap- - a -AD

P(H)- _ _ -P(H)

Hd-Hd

Ai~~~~~~~~~~c

C2(L) P(L.)M2-SC2

M4 -

P(N)

Al- _4A2 _

LM2

LM1 3 - z

LM5B-

_M6 -

LM7-

LM8- _LMS-L_M9 -

a b c d e

Fia. 1. Autoradiograph of "4C-labeled 429-specificproteins produced in UV-irradiated B. subtilis andseparated by SDS polyacrylamide gel electrophoresis.Cells were infected with wild-type 429 (MOI of 50)and labeled with a mixture of "4C-labeled amino acids10 to 45 min after infection. (a) Cell pellet fraction ofinfected cells; (b) trichloroacetic acid precipitate ofthe supernatant from the infected culture; (c) cellpellet fraction of an irradiated, uninfected control; (d)trichloroacetic acid precipitate of the supernatantfrom the irradiated, uninfected control; (e) purified4)29 virions. Profiles a, b, c, d, and e were from a 12 to16% linear polyacrylamide gradient. Insert profiles fand g from a 16 to 20% linear polyacrylamide gradientshow the collar region more clearly. (f) Purified 029virions; (g) trichloroacetic acid precipitate of thesupernatant from the infected culture. Symbols,names, and molecular weights (8), respectively, of the029-specific proteins are as follows: P(J): (87,500);Ap: neck appendage (75,300); P(H): (62,300); Hd:major head protein (45,000); Cl: neck upper collar(36,400); P(L): (36,000); C2: neck lower collar(35,200); F: head fiber (28,500); and P(N): (25,100).Proteins Al, A2, and those with the LM (low molecu-lar weight) prefix are of unknown function and havethe following molecular weights: Al (22,400), A2(21,800), LM2 (18,800), LM3 (16,700), LM4 (15,200),LM5 (14,700), LM5B (14,200), LM6 (13,000), LM7(8,500), LM8 (5,300), and LM9 (4,500).

212 J. VIROL.

BACTERIOPHAGE 029 GENE FUNCTION

cetic acid precipitate of the supernatant wereexamined by electrophoresis. Although somehost protein synthesis could be detected in thecell pellet fraction of uninfected cells (Fig. lc),,V irradiation was considered an effective sup-pressor of host protein synthesis (8). Single,irradiated cells, when examined by an in situlysis technique and electron microscopy, pro-duced more than 100 DNA-containing )29 par-ticles and an average of 10 PFU per cell (8). Thesymbols employed to label protein bands (Fig.1) reflect, in part, our current understanding of429 gene function and the genetic map. The 13viral cistrons have been named by letter inconstructing our map with sus mutants (20).The polypeptide product of cistron H, for exam-ple, is listed as P(H). Other bands representcomponents of the virion (e.g., Ap for neckappendage, etc.) or are named by position in theprofile (e.g., LM3 for low-molecular-weight pro-tein 3). The insert in Fig. 1 illustrates theresolution of neck proteins (15) needed to visu-alize the P(L) band. A summary of this ter-minology and the molecular weights of theproteins are included in the legend of Fig. 1.The sus mutants used for studies of protein

synthesis in the nonpermissive host are given inTable 1. Several independent isolates wereused, when available, to define the phenotype ofa cistron. The isolation and characterization ofthe hydroxylamine mutants have been de-scribed (20), and the bromodeoxyuridine mu-tants have been placed in cistrons by the samemethods. Mutagenesis resulted in multipledamage in several mutants, and we employedbackcrossed phage as they became available.

UV-irradiated nonpermissive cells were in-fected with sus mutants (MOI of 50) andlabeled with a mixture of "C-labeled aminoacids 10 to 45 min after infection. Labeled viralproteins in both the cell pellet and supernatantfractions of these cultures were separated bySDS gel electrophoresis and identified by auto-radiography (Fig. 2-5). Three 4)29-specific pro-teins, AF, BF, and LM6B, are not identifiedeasily in these gels since they are synthesized ata maximal rate early in infection and arelabeled poorly after 10 min (8). Also, proteinLM5B obscures the band of protein LM5, par-ticularly in the supernatant fraction. Ambermutants of phage T4 produce a fragment of thenormal polypeptide in the nonpermissive host(21); 429 mutants behave similarly.Protein synthesis with mutants of cistrons A

through F is illustrated in Fig. 2 and 3. Allproteins are present in the profiles of mutantsA628 and A752 (Fig. 2a, a'; b, b'), except that afragment of protein LM5 can be observed in theA752 gel (Fig. 2b).

TABLE 1. Suppressor-sensitive mutant roster

Clear" No. of MutagenCistron Mutant allele back- BUdr +

status crosses _ BUdre HPUra+

A

B

C

D

E

F

G

H

I

J

K

L

M

N

628'752

629"

713732

369"711737

626"727748

769674788805827

614'701

756'790807823

302"703810

305e602662716

330"817

300'700809

741"

724726

+

+

++

+++

+

+

+

+

CCC

C++

C

CC

+

50

2

40

622

402

40000

40

2000

620

4000

40

400

1

00

++

+

++

+++

+++

+

++

+

++

++++

+

++

+

+

aClear or turbid.b Hydroxylamine.c Bromodeoxyuridine.d 6-(p-hydroxyphenylazo)-uracil."Reference mutant (20).

+

+

+++

+

+

+

+

Mutant B629 does not synthesize proteinLM7, and the synthesis of proteins LM8 and

213VOL. 13, 1974

ANDERSON AND REILLY

Cell PelletsA B C D

SupernatantsA B C D

e_ _ _

- "1 V

_4_ C_ Hd

* -aen w-

AF

BPflflfl ee 0* _ e 0

-~~~~~~~~~~~~~*-0,-n-iilk-0

_. _ _~~~~

4mew 4w4

1MOIP -C2

__ e -P(N)

-AlIw_ WM", -A 2

-o .-' -aw -LM2

K.

===:-44

'_ -__4__~ ~~~~ ~~~....IW4tU;-F ~ s

*..

a b c d e f g h k m n 0 p q a' b' C' d' e' f' g' j' k' I' m' n' ol p, q

FIG. 2. Autoradiograph of "4C-labeled +29-specific proteins produced by sus mutants in UV-irradiated B.subtilis SpoA12. Cells were infected with sus mutants (MOI of 50) and labeled with a mixture of "4C-labeledamino acids 10 to 45 min. Labeling was terminated as described in the Materials and Methods, and both the celland supernatant fractions were analyzed by SDS gel electrophoresis on 12 to 16% linear polyacrylamidegradients. Profiles a through o represent pellet fractions of mutant-infected cells, and profiles a' through o' are

of corresponding trichloroacetic acid precipitates of supernatants of the infected cultures. Profiles p and p'represent cell pellet and supernatant fractions, respectively, of uninfected control cultures; and profiles q and q'are of purified 029 virions. (a and a') A628, (b and b') A 752, (c and c') B629, (e and e') C713, (g and g') C732, (h)D369, (i) D369, (j and j') D711, (k and k') D737, (1 and 1') E626, (m and m') E626, (n and n') E748, (o and o')E727.

LM9 is ambiguous (Fig. 2c and c'). Analysis ofother gels, however, leads us to conclude tenta-tively that proteins LM8 and LM9 are synthe-sized (data not shown). Additional mutants incistron B are needed to confirm this phenotype.

In the cell pellet profiles (Fig. 2a through o),the head fiber protein F is nearly absent and theproteins AF and BF, which band above andbelow F (8), are visible. Gels of cistron Cmutants C713 and C732 (Fig. 2e and g) lackthese bands. To confirm the C cistron pheno-type, we have infected UV-irradiated nonper-

missive cells with mutants C713 or C732 andlabeled them with the "C-labeled amino acid

mixture 2 to 8 min after infection, a period inwhich the AF and BF proteins are synthesizedmaximally and the fiber protein F is not made(data not shown).

Synthesis of the structural proteins Ap, P(H),Hd, Cl, C2, and F among others is virtuallyabsent after infection by cistron D mutants(Fig. 2h, i, j, j', k, and k'). The microdensitome-ter tracing of the SDS gel profile of mutantD369 presented in Fig. 3 illustrates this point.Mutants of cistron E do not produce proteins

LM3 and LM8 (Fig. 2 1-o; 1'-o').Gels of cistron F mutants (Fig. 3b) are

characterized by the absence of the major head

214

A p - _ e

P(H) -

J. VIROL.

e_ -

E

Hd-

_ n _

- P(J)

p - Ap

- -P(H)

Al--.

LM3 - _

LM4 _ _LM5 -LM6 -

LM 7 - ,

LM8-

_11_LM9 -

- LM3

-LM6

-LM7

- LM8-LM9

BACTERIOPHAGE 029 GENEFUNCTION2

protein (Hd) and head fiber protein (F).Protein syntheses with mutants in cistrons J,

L, M, and N are presented in Fig. 4. We havepresented three profiles for J305. The mutantJ305 as isolated (Fig. 4c and c') was defective insynthesis of proteins P(J), Ap, LM2, LM3,LM5, and LM9. After being backcrossed twice,J305 could synthesize LM5 (Fig. 4b and b'), andtwo additional backcrosses restored bands LM3and LM9 (Fig. 4a and a'). After these andsubsequent backcrosses, mutant J305 lackedproteins P(J), Ap, and LM2 (Fig. 4a and a'), a

phenotype characteristic of other independentcistron J mutant isolates (Table 1). If the gelprofile made after infection by the clear-plaquemutant 029c (Fig. 4d and d') is compared to theprofile of mutant L700 (Fig. 4g and g'), theprotein P(J) band is more dense after 029cinfection and there is relatively less material inthe appendage (Ap) band. This is also evidentin the clear-plaque mutant M741 profile (Fig.

FIG. 3. Microdensitometer tracings of an autoradi-ograph of 'IC-labeled +29-specific proteins producedunder restrictive conditions by sus mutants in UV-irradiated B. subtilis. Cells were infected and labeledas described in Fig. 2, and proteins in the supernatantfractions were analyzed by SDS polyacrylamide elec-trophoresis on a 16 to 20%Yo linear gradient. (A) MutantE626, (B) mutant F674, (C) mutant D369, (D) unin-fected control cells.

4i') and occurs in all infections by sus mutantsobtained by mutagenizing 429c.

Protein P(L) is absent in cistron L mutantinfections (Fig. 4g'); P(L) is not present in thevirion and appears in the collar region of the gelautoradiograph. This region of the profile isre-examined in Fig. 5. Mutants L700 (Fig. 5j)and L809 (data not shown) do not synthesizeP(L) but produce a fragment that appearsbelow the head fiber protein.Mutant M741 cannot synthesize proteins Al

and A2 (Fig. 4h, h'; i, i'). The profiles of mutantN724 (Fig. 4j and j') lack the protein P(N) band.Sus fragments in mutants N724 and N726 infec-tions are tentatively identified below the LM5band of the gel autoradiograph (Fig. 4j for N724;data not shown for N726).The proteins Cl, P(L), and C2 that band in

the collar region of the gel are best resolved on a16 to 20% linear gradient of polyacrylamide.The high-molecular-weight proteins producedby mutants in cistrons H, I, J, L, and M areresolved in the 16 to 20% gel shown in Fig. 5.Typical synthesis is illustrated with mutantM741 and a clear-plaque mutant M741c infec-tion (Fig. 5k and 1); infection by the clear-plaque mutant 429c (Fig. 5f0 gives a similarpattern. Protein P(J) is prominent with theclear mutation, and protein Ap is reduced.Proteins P(J) and Ap always appear as a doubleband in this type of gel. Mutant H756 (Fig. 5a)does not make P(H), but a fragment appearsabove protein F. With cistron I mutants (Fig. 5band c), the collar proteins Cl and C2 of the virusare not synthesized. Two cistron J profiles arepresented (Fig. 5d and e). Proteins P(J) and Apare absent in the profile of mutant J602 (Fig.5e), and a double-band fragment appears justabove protein P(H). The protein P(L) is notpresent after infection by mutants L300 andL700 (Fig. 5i and j), but mutant L700 producesa fragment present above the protein P(N)band.A summary of the cistron phenotypes re-

vealed by mutant analysis is presented in Table2.

DISCUSSIONHydroxylamine and bromodeoxyuridine mu-

tagenesis has yielded sus point mutations in 14cistrons of the 029 genome. Ten cistrons havebeen placed on a linear genetic map by three-factor crosses (10). Cistrons were designated byletter, from the left, in the series A, C, D, E, G,H, I, J, K, and L. Cistron B is near cistron A,cistron F is near cistron G, and the loci ofcistrons N and M are unknown. If we obtain a

P(N)

c

D

+ --_

215VOL. 13, 1974

ANDERSON AND REILLY

Cell PelletsJ cl L M Nf lr---

Su pern atantsci ($29 L. M N

I Ii mr ml,--

P(J) -

Ap -

00up

m

P(H)

Hd --

P(N) - _

Al -_

A2a-

_0

e>..lo

Hd

- P(J)

- Ap

P(H)

r - C2

- F

*_ -P(N)

- Al

- LM2

LM3 _.ww .-

LM4LM5 -

*

LM6

_.S

t_

i_

L M 7 - _4*;0

LM8 -

LM9 -

_s_- L M 3

l -L-LM6

- LM7

LMS

- LM9

a b c d f g h a' bl c' d' e' f g' h'

FIG. 4. Autoradiograph of "4C-labeled 029-specific proteins produced by sus mutants in UV-irradiated B.subtilis SpoA12. Cells were infected and labeled as described in Fig. 2, and proteins in both the cell and super-natant fractions were analyzed by SDS gel electrophoresis on 12 to 16% linear polyacrylamide gradients. Pro-files a, b, c, d, g, h, i, and j represent pellet fractions of mutant-infected cells. Profiles a', b', c', d' ,g, h', i' andj' are corresponding trichloroacetic acid precipitates of supernatants of the infected cultures. The pellet fractionof uninfected control cells is shown in profile f, and the corresponding supernatant is shown in profile f'. Profilee' is of purified 029 virions. (a and a') J305, (b and b') J 305, (c and c') J305, (d and d') q29c, (g and g') L700, (hand h') M741, (i and i') M741c, and (j and j') N724.

216 J. VIROL.

H I cl (029m-r I I I rI *

P(J) -_ _

Ap -.iM SE

P(H) -

L MI n mI

- P(J)

-Ap

- P(H)- .a_.,._

Hd - _- -e S U -Hd

Cil)

C2 -

so -ClU- P(L)- C2

- Fno0om

_mp - P(N)

c d e f g h i j k I

FIG. 5. Autoradiograph of "4C-labeled 029-specific proteins produced by sus mutants in UV-irradiated B.subtilis SpoA12. Cells were infected and labeled as described in Fig. 2, and proteins in the supernatant fractionwere analyzed by SDS gel electrophoresis on a 16 to 20% linear polyacrylamide gradient. (a) H756, (b) I302c,(c) I703, (d) J305, (e) J602, (f) +29c, (g) purfied 029 virions, (h) uninfected control cells, (i) L300, (i) L700, (k)M741, and (I) M741c.

217

F-

P(N) -en

a b

ANDERSON AND REILLY

common pattern of protein synthesis underrestrictive conditions with three or more inde-pendently isolated mutants, we conclude thatwe have defined the pattern of protein synthesisby the mutants of a given cistron under theconditions employed in this investigation. Be-cause multiple mutation is common in chemicalmutagenesis and we have isolated mutants withsus mutations in three or more genes (e.g., D369and J305), we believe extensive backcrossing ofrepresentative sus mutants employed in thisstudy (Table 1) gives additional support to ourclaims. Interpretation of our results is compli-cated because point mutation in at least fivecistrons is characterized by the absence of twoor more protein bands from the SDS gel profileand because at least 10 of the 23 proteins thatwe detect during 029 infection have a molecularweight of 20,000 or less (8).Mutation in cistrons H, L, and J results in the

absence of single proteins in the gel profile. Theproduct of cistron H is protein P(H), and itcorresponds to the virion tail protein, TP1,described by Mendez et al. (15). Lysates ofmutant H756 contain the serum blocking powerof the wild-type 029 lysate (28). Protein P(L),the product of cistron L, is not present in thevirion and appears between the collar proteinsCl and C2 in the gel autoradiograph. Pointmutation in cistron J of 029 results in theabsence in SDS gels of proteins P(J), theappendage Ap, and LM2. The cleavage ofprecursor proteins during maturation in phageT4 infection has been demonstrated (5, 9, 11,12). Our data indicate that P(J) is a precursorprotein and is cleaved. In the gel profile of 429c,a spontaneous mutant with a clear-plaque phe-notype, P(J) accumulates, and Ap and LM2 arereduced in quantity accordingly. In the 16 to20% polyacrylamide linear gradient gel, bothproteins P(J) and Ap appear as double bands(8). The fragments that are present after infec-tion by mutants J602 and J662 also give adouble band. Preliminary pulse-chase experi-ments with wild-type 029 indicate that proteinP(J) is the precursor of the appendage proteinAp and the protein LM2 (data not shown).Mutant J305 does not produce the serum block-ing power of 029 during the restrictive infection(28).Mutation in cistrons D, E, F, and I results in

a more complicated change in the phenotype.Three or more mutants have been examined foreach cistron. The frequency of reversion forthese mutants does not differ from that ofmutants in cistrons H, L, and J (data notshown).Mutants of cistron I fail to synthesize the

collar proteins Cl and C2, previously termedNP2 and NP3 (18). A precursor of proteins Cland C2 having a molecular weight of at least70,000 would band near protein Ap. We havenot detected a precursor in the wild-type 029infection or in the autoradiograph of any cistronJ mutant infection. Peptide analysis of NP2 andNP3 indicates that NP3 cannot be a cleavageproduct of NP2 (18). Therefore, we have notnecessarily identified protein P(I).

Cistrons D and F may have a positive regula-tory function during 429 infection. Mutants ofcistron D do not synthesize proteins P(J), Ap,P(H), Hd, Cl, P(L), C2, F, P(N), and LM5B.We believe that the appearance of these pro-teins reflects late mRNA synthesis and Hstrand transcription (8, 13, 14, 16, 22). Twomajor species of late mRNA have been identi-fied (13) that reflect about 50% of the transcrip-tion potential of the 029 genome. The proteinP(D) may regulate transcription of the theselate mRNA species.

Five mutants have been examined for cistronF. Each is a point mutation by the criterion ofreversion, and three-factor crosses place eachmutation between mutants G614 and H756(data not shown). The major head protein, Hd,and the head fiber protein, F, (combined molec-ular weight of 73,000) are not synthesized bycistron F mutants, sus fragments are not found,and there is no evidence for a precursor protein.The SDS gel profiles of cistron E mutants lack

proteins LM3 and LM8. As for the case ofmutation in cistrons D and F, we can predictthe gel profile but cannot identify the primaryprotein gene product.

Typical mutants of cistrons A, G, and Kmake all the proteins visible in the wild-type gelautoradiograph. Mutants A752 (Fig. 2b) andK330 (Table 2) do not synthesize protein LM5,and a fragment band is present in both cases.We believe that protein LM5 is not a product ofeither cistron A or cistron K because mutantA752 complements K330 in mixed infection of arestrictive host. We are continuing to backcrossthese mutants in an effort to restore synthesis ofprotein LM5 as we did with mutant J305. Wehave examined additional mutants that map incistrons A, G, and K and are using a differentlabeling protocol and different polyacrylamidegradients in our attempt to identify the prod-ucts of these cistrons.

In wild-type 029 infection two "pairs" ofproteins appear, and each pair could result fromslight alteration of one member (8). Proteins AFand BF appear early in infection, and in timethe head fiber protein, F, appears and obscuresthis region of the gel (8). The early proteins Al

J. VIROL.218

BACTERIOPHAGE 029 GENE FUNCTION

++++++++ ++ ++ ++++

++++++I+ ++ ++ ++++

z +++I++++ ++ ++ ++++

~ ++++ +++ + + + + ++++

++++++++ + + + + ++++

2 ++++++++ ++ ++ ++++

++++ ++I+ ++ + + ++ ++

2 ++++++++ ++ ++ ++++

+++++++ ++ ++ ++++

rz +++++I+I+ ++ ++ ++++

_-I

+ + + +I++ + + + + ++ +

:+++++I++ ++ + + + ++

z +++++I++ ++ ++ +I++

r +++++I++ + +++ ++++

go

m + + +++ ++ + + + + + + + +

+++++I+ ++ ++ ++++

< ++++l+++ ++ ++ ++++++ + + +I+ + + + + + +I + +

C;+ + + + + I++ + + + + + + +

+ + + + + +I++ + + + + + +

+++++I+ +I ++ +++ +--

+ + + + + + + ++ +

t-t-0 _ 0 -C O

e > > -4i0 00Q _t- t- ^- ^ ^ Ct- _- CD t-S CS_0 CD 0 _O CD0C O t- _ O CU4:4 Cq _i C-4 0CD 0 0 _0 0l C41CSC C CDt- CD -e °

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VOL. 13, 1974 219

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ANDERSON AND REILLY

and A2 are synthesized in quantity (8). Duringinfection protein A2 remains associated withthe cell, while protein.Al is found primarily inthe supernatant fraction of the infected culture.The function of these proteins during infectionis unknown. Proteins AF and BF are not synthe-sized by mutants of cistron C in the nonpermis-sive infection. We have examined additionalcistron C mutants, used a 2- to 8-min pulse with"C-labeled amino acids to avoid labeling thelate F protein, and constructed recombinantsthat cannot synthesize protein F. In each in-stance, AF and BF are not present after Cmutant infection (data not shown). MutantM741, our single representative of cistron M,does not synthesize proteins Al or A2. Whateverthe relationship of protein AF to BF, we con-clude that sus mutation in cistron C precludestheir synthesis. As we isolate more cistron Mmutants, we will seek to confirm our observa-tions with the mutant M741 infection.Mutations in cistrons B and N are rare in our

collection. Mutants N724 and N726 do notsynthesize protein P(N). Mutant B629 does notsynthesize protein LM7 and may not synthesizeproteins LM8 and LM9. These conclusions aretentative.We have defined the relationship of cistrons

H, J, and L to proteins P(H), P(J), and P(L). Asimilar relationship may exist for cistrons C, I,M, and N. At least 10 of the 23 proteins that wedetected during 029 infection have a molecularweight of 20,000 or less (8). We will concentrateon the resolution of these proteins in an effort toidentify the regulatory proteins our data leadsus to seek, e.g., P(D), P(E), and P(F).Host DNA synthesis, transcription, and pro-

tein synthesis continue throughout the 429 in-fectious cycle (3, 23). Regulation of biosynthesisby low-molecular-weight proteins might be ex-pected. With bacteriophage T4, there is evi-dence that low-molecular-weight proteins in-teract with the ribosome (6), the transcriptioncomplex (26), and the pool of replicating viralDNA (10). Our gel analysis indicates mutationin genes coding for at least seven low-molecular-weight proteins. We have not isolated a condi-tional lethal mutant blocked in the biosynthesisof any single polypeptide of this group.Our analysis has defined the function of

several cistrons of 429, indicated that the com-plex pattern of viral transcription is reflected indiscrete patterns of protein synthesis, and iden-tified mutants that have specific damage butwere not recognized because they were notconditionally lethal. The latter mutants will beuseful in our analysis of the regulation of

macromolecular synthesis during viral infec-tion.

ACKNOWLEDGMENTS

This work was aided by grant GB-29393 from the NationalScience Foundation, by Public Health Service grant DE-3606from the National Institute of Dental Research, and bygrant-in-aid 494-0303-4909-02 from the Graduate School,University of Minnesota. D. L. A. was the recipient of PublicHealth Service Career Development Award K3-DE-10, 934from the National Institute of Dental Research.

LITERATURE CITED

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3. Anderson, D. L., D. D. Hickman, and B. E. Reilly. 1966.Structure of Bacillus subtilis bacteriophage 429 andthe length of 429 deoxyribonucleic acid. J. Bacteriol.91:2081-2089.

4. Anderson, D. L., and E. T. Mosharrafa. 1968. Physicaland biological properties of phage 429 deoxyribonucleicacid. J. Virol. 2:1185-1190.

5. Dickson, R. C., S. L. Barnes, and F. A. Eiserling. 1970.Structural proteins of bacteriophage T4. J. Mol. Biol.53:461-474.

6. Dube, S. K., and P. S. Rudland. 1970. Control oftranslation by T4 phage: altered binding of disfavouredmessengers. Nature (London) 226:820-823.

7. Georgopolous, C. P. 1969. Suppressor system in Bacillussubtilis 168. J. Bacteriol. 97:1397-1402.

8. Hawley, L. A., B. E. Reilly, E. W. Hagen, and D. L.Anderson. 1973. Viral protein synthesis in bacterio-phage +29-infected Bacillus subtilis. J. Virol.12:1149-1159.

9. Hosoda, J., and R Cone. 1970. Analysis of T4 phageproteins. I. Conversion of precursor proteins into lowermolecular weight peptides during normal capsid forma-tion. Proc. Nat. Acad. Sci. U.S.A. 66:1275-1281.

10. Huberman, J. A., A. Kornberg, and B. Alberts. 1971.Stimulation of T4 bacteriophage DNA polymerase bythe protein product of T4 gene 32. J. Mol. Biol.62:39-52.

11. Kellenberger, E., and C. Kellenberger-Van der Kamp.1970. On a modification of the gene product P23according to its use as subunit of either normal capsidsof phage T4 or of polyheads. FEBS Lett. 8:140-144.

12. Laemmli, U. K. 1970. Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4.Nature (London) 227:680-685.

13. Loskutoff, D. J., and J. J. Pene. 1973. Gene expressionduring the development of Bacillus subtilis bacterio-phage 429. II. Resolution of viral-specific ribonucleicacid molecules. J. Virol. 11:87-97.

14. Loskutoff, D. J., J. J. Pene, and D. P. Andrews. 1973.Gene expression during the development of Bacillussubtilis bacteriophage 429. I. Analysis of viral-specifictranscription by deoxyribonucleic acid-ribonucleic acidcompetition hybridization. J. Virol. 11:78-86.

15. Mendez, E., G. Ramirez, M. Salas, and E. Vinuela. 1971.Structural proteins of bacteriophage 429. Virology45:567-576.

16. Mosharrafa, E. T., C. F. Schachtele, B. E. Reilly, andD. L. Anderson. 1970. Complementary strands of bac-teriophage 429 deoxyribonucleic acid: preparativeseparation and transcription studies. J. Virol. 6:855-864.

17. Polsinelli, M., and M. Beretta. 1966. Genetic recombina-

220 J. VIROL.

BACTERIOPHAGE 029 GENE FUNCTION

tion in crosses between Streptomyces aureofaciensand Streptomyces rimosus. J. Bacteriol. 91:63-68.

18. Ramirez, G., E. Mendez, M. Salas, and E. Vinuela.1972. Head-neck connecting protein in phage 029.Virology 48:263-265.

19. Reilly, B. E., and J. Spizizen. 1964. Bacteriophage de-oxyribonucleate infection of competent Bacillus sub-tilis bacteriophage 029. J. Bacteriol. 89:782-790.

20. Reilly, B. E., V. M. Zeece, and D. L. Anderson. 1973.A genetic study of suppressor-sensitive mutants of theBacillus subtilis bacteriophage 029. J. Virol. 11:756-760.

21. Sarabhai, A. S., A. 0. W. Stretton, S. Brenner, and A.Bolle. 1964. Co-linearity of the gene with the polypep-tide chain. Nature (London) 201:13-17.

22. Schachtele, C. F., C. V. DeSain, and D. L. Anderson.1973. Transcription during the development of bacteri-ophage 029: definition of "early" and "late" 029ribonucleic acid. J. Virol. 11:9-16.

23. Schachtele, C. F., C. V. DeSain, L. A. Hawley, and D. L.Anderson. 1972. Transcription during the development

of bacteriophage 029: production of host and 029-spe-cific ribonucleic acid. J. Virol. 10:1170-1178.

24. Schachtele, C. F., B. E. Reilly, C. V. DeSain, M. 0.Whittington, and D. L. Anderson. 1973. Selectivereplication of bacteriophage 029 deoxyribonucleic acidin 6-(p-hydroxyphenylazo)-uracil treated Bacillussubtilis. J. Virol. 11:153-155.

25. Spizizen, J. 1958. Transformation of biochemically defi-cient strains of Bacillus subtilis by deoxyribonucleate.Proc. Nat. Acad. Sci. U.S.A. 44:1072-1078.

26. Stevens, A. 1972. New small polypeptides associated withDNA-dependent RNA polymerase of Escherichia coliafter infection with bacteriophage T4. Proc. Nat. Acad.Sci. U.S.A. 69:603-607.

27. Talavera, A., M. Salas, and E. Vinuela. 1972. Tempera-ture-sensitive mutants affected in DNA synthesis inphage 029 of Bacillus subtilis. Eur. J. Biochem.31:367-371.

28. Tosi, M., and D. L. Anderson. 1973. Antigenic propertiesof bacteriophage 029 structural proteins. J. Virol.12:1548-1560.

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