Role of MotA transcription factor in bacteriophage T4 DNA replication

Role of MotA

Transcription Factor in Bacteriophage T4 DNA Replication

Kim H. Benson and Kenneth N. Kreuzert Departmen,t of Microbiology and Immunology Duke Vnkwaity Medical Center Durhcm, Xl' 27710, I'.,S.A. (Received 2 &!nrch

1992; crccepted 3 .July 1992)

T-C replication origins. ori(/scs Y) and ori( caontain a T1 At least two bacteriophage middle-mode promoter t,hat is necessary for origin fun&ion. \I’e wanted to analyze the requirement of these t)wo replication origins for the MotA protein. which is the phagt,rnroded activator of middlr-mode promoters. To ensure the complete absence of JlotjA protein, we deleted the nzotA gene from the T4 genome. L-nexpectedly. the deletion mutant was not viable unless t’he MotA protein was provided from a recombinant plasmitl. Therefore. MotA is an essential protein for TLC growt’h. The ~rofd~ mut,ation reduced the synthesis of several proteins that are encoded by genes with middle-mode promoters. delayed and reduced t)he synthesis of late proteins, and substantially reduced phage genomita replication. The W&A* mutation also reduced the replication of an ori(urTs Y)-containing plasmid. The replication plasmid and virtually abolished replic*at)ion of an ori(34)- containing defects of t,he two origins correlated w&h transcriptional defect’s: the rrrotA* mutation modestly reduced t,ranscription from the plasmid-borne ori(zcr-s Y) promoter and stronglq reduced transcription from the ori(.34) promoter. These resultIs provide strong evidence that MotA protein is normally involved in origin-dependent replication. However. Mot A is not required for origin-directed replieatiotl as long as transcription can occur from t’he origin promotor. Kryuvrds:

bacteriophage T4; initiation MotA; replication

1. Introduction Replication of bacteriophage T4 DSA initiat*es b? at least two distinct strategies (for reviews, see Keppel et al., 1988 and Mosig & Eiserling, 1988). At sequences art origin earl\- times of infection. rec&ired for the initiation of replication. Based on a variety of approaches, multiple T4 origin locations have been proposed and three origins have been characterized at the nucleotide seyuence level (Halpern et al., 1979. 1982: King & Huang. 1982: Macdonald e:t al., 1983; Macdonald $ AMosig, 1984: ‘L’ee & Marsh, 1985; Kreuzer 8r Alberts, 1985; Menkens 8r Kreuzer. 1988). At later times of inferstion, a recombination-dependent initiation mechanism obviates the need for origin sequences (Luder 8r recombination-dependent 1982). The Mosig, strat’egy requires a number of T4encoded proteins. including a synaptasr (UvsX), a synaptase accessory protein (Vvs\r), an exonuclease (gp46/47). a type IT I)SA topoisomerase (gp39/52/60). and the

of replication: origins

transcript~ion:

product of gene 59 (for reviews. see Mosig. 1983. 1987). i-\ plasmid model q-stern has been established to study origin-dependent replication ix c>i,~o(Krruzer & Alberts. 1985. 1986: Kreuzer et al., 1988). Derivatives of pRR322 containing either of two T4 origins. ori(zlcs Y) or ori( replicat)e extensiveI> during phage infection. The replication of’ origincaontaining plasmids requires the T4 replication fork proteins. but is independent of T4 rec~ombination proteins and does not require homology with the phage genome (Kreuzer rt al.. 1988: Benson & 1992). Deletion analyses of the c~loned Kreuzer, ori( ues I’) and ori(34) regions have defined minimal origin sequences of less than 100 bpf (Slenkens Q Kreuzer, 1988). Each minimal origin contains a ‘1’4 middle-mode promoter sequence. implicating transcription in origin initiat,ion, and a region of about t =Ibbrrviations usrd: hp. base-pairs: I EC.iu~niediatc~ early; DE. delayed early: kb. IO3 hasr-l)airs; IIS. insertjion/suhstitution; tn.0.i.. rnult,iplicity of infection: p.f.rr., rjlaqur-forming units.

Hole of Motil

50 bp just downstream from the promoter. This downstream region is required for optimal replication but not for transcript’ion, and probably acts as a DXA-unwinding element’ during the initiation process (R. P. Schmidt et al., unpublished results: Kowalski et al.. 1988). The precise roles of the middle-mode promoter and downstream region in the initiation of replication have not yet been clarified. of drigin-containing plasmids is R)eplication markedly increased by mutations that eliminate the phage-encoded L:vsTLTprotein (Kreuzer rt al., 1988). This enhancement may be caused by an increa,sed frequencay of replication initiation or an enhanced production of rolling-circle forms that yield many c*opies of product, per init’iation event. Regardless of the mechanism, this enhancement of plasmid replication in the absence of UvsE’ protein complicated our analysis of the MotA requirement for origin function. As will be described in more detail below. motA mut,ations apparently cause a deficiency in VvsY protein dur to the defect in middle-mode transcaription. and this deficiency in turn can replication of origin-containing enhance thr plasmids. T4 transcription involves three promoter types: early, middle and late (for a review, see Rabussay 1983: Rrody et al., 1983; Geiduschek rt al., 1983). Transcription from early promoters results in immediate-earl(IE) transcripts during the first few minutes of infection, followed shortly thereafter b> polycistronic transcripts that extend into the delayed-early (DE) genes. At about the time when polyc>ist,ronic transcription extends into DE genes, middle-mode promoters are also activated. Most middle-mode promoters lie within early transcription units, resulting in a considerable overlap between l>E and middle-mode transcripts. T1 middle-,mode promoters incorporate a - 10 region that is simila,r to that in host promoters and a unique -30 consensus sequence (Brody rf a,l., 1983: Guild et al.. 1988). This -30 region sequence differmiddle-mode promoters from early entiates promoters. which generally conform to the - 10 and - 35 czonsensus sequences of h’sch~rrichia coli promot’ers (Brady ef al., 1983; Liebig and Riiger. 1989). =\ctivation of middle-mode promoters in t’he T4 penome requires bhe phage-encoded MotA protein (Mattson et al.. 1974; Hercules and Sauerbier. 1974). which has recently been shown to bind to the -30 consensus region (Hinton. 1991; Schmidt and Kreuzrr, 1992). Furthermore, the activation of in Gtro middle-mode transcription by T4-modified RNA polymerase requires the MotA protein (drFranciscis & Brody, 1982; deFran&cis et al.. 1982: Hinton, 1991: Schmidt & Kreuzer. 1992). Although middle-mode transcription from the T1 genorne requires MotA protein, some middle-mode promoters art’ also capable of directing MotA-independent transcript’ion. In particular. transcription has been detected from recombinant plasmids in uninfected cells (which cannot have any MotA prot,ein) and aft)er infection by it motAam

in T4 Replication

89

mutant (Shinedling et al., 1986; Guild et al., 1988). Thus, certain promoters (including that wit’hin ori(u%lsY)) require activation by MotA protein when they are located in T4-modified linear DNA but not when they are located in a recombinant plasmid (Shinedling ef al.. 1986; Guild et al., 1988; Menkens & Kreuzer. 1988: also see below). Perhaps extensive supercoiling or the lack of T4 modifications reduces or rliminates the requirement for Mot.4 in transcription from such promoters. Several motil mutants (t&l, amCiI, aiyl. sip2) ha\-e been isolated by selecting for survival of an t-11 phage in a i lysogen Mattson et al., 1974. 1978: Homyk et al.. 1976). The rI/- mut’ation normally prevents phage survival in a 1. lysogen. but a delay in T4 gene expression by a mot,4 mut,ation was proposed to alleviate this block (Mattson et al.. 1978). Two other motA mutations. farP14 and farP85. lead to overproduction of dlhydrofolate reductase. a T4 early protein. a.nd thereby provide resistance to folate analogs (,Johnson and Hall, 1973). Because all of these smotrl mutants arc viable in wild-type strains of E. coli. the mot.4 gene was thought to 1~ non-essential. HowevrJr. most of the mutants were isolated bv selections that required growth and/or survival oi the mutant. Therefore, it is possible that the motd gene is essential and that all previously isolated m.otd mutants retain residual MotA function that allows growth. The growth of mot.4 mutants is restricted by a mutant strain of E. coli. TahG. for reasons that arc not underst,ood (Pulitzer et al.. 1979). The primary phenotype (*aused 1)~ mot,4 mutations is an inability to act,ivate transcription of middle-mode promoters (deFranc&is 6 Brady. 1982: deFranc*iscis et al., 1982: Kroida 8 Abelson, 19%; Pulit)zer rf al.. 1985: (build ef nl.. 1988) ant1 a commensurate reduction in the expression of proteins produced from middle-mode promoters (1slattson rt ml.. 1974. 1978: Hercules 8 Sauerhier, 1974: Halt B Snyder, 1981). However. previously isolated motA mutations also prolong t hr expression of caclrtain early proteins and delay the production of late proteins (Ma,ttson et al., 1971. 1978: Hercules 6t Sauerbier: 1974: Hall Rr Snyder. 1981). This pleiotropic phenotype suggests that the temporal progression of T4 prot,ein expression is tight]?- rrgulatrti. For example. the prolongetl rxpression of early proteins indicates that one or more moth del)r-indent transcript encodes a protrin t,hat reltresses early gene expression. Further. the delay in I;\t’e protein expression suggests that lat,e transcAption is dependent on proteins expressed from middle-mode promoters. In addition to t’hrse effects on protein synthesis, mot,4 mutations delay and rrdutze T4 grnomic DXA replication, perhaps bwause of defkiences in expression of rrplication proteins (hlattson et al.. 1974. 1978). Thus, all of the rn)-riad eRects of nbotil mutations could p4entially br explained as indirect consequences of the defect in middle-mode transcription. In this study. we analyzed the roles of’ t.he >lotA protein in the growt’h and J)NA replic.ation of 7’4.

1Ve generated a null mot.4 mutant (ttwt,lA) and thereby discovered that mot/l is an essential g,rerw. l’hr effects of the motAA mutat,ion on transcription and replication of T4-origin-containing plasmids implicates the MotA protein in the init,ia,t,ion of ‘I’1 I)XA replication.

2. Methods (a)

and Methods Natrrials

Restriction enzymes. T4 DSA ligase, T4 polynucleotide kinasr. (a-32P]dATP, ]y-32P]ATP. (35S]met,hionine. dideoxynucleotide sequencing reagents. and avian myeloblastosis virus (AMY) reverse transcriptase were purchased from commercial sources. The synthet,ic oligonucleotide used in constructing pKB17 (m&AA) was a generous gift from R.M. Alberts (Cniversit,y of California. San Francisco). Plasmids pGJB1. pKKO61. pKKO61-6 and pRS31 were as described elsewhere (Kreuzer 8r. Menkens. 1987; Menkens & Kreuzer. 1988: Schmidt & Kreuzer, 1992). L broth contained NaCl (10 g/liter). Hacto-Tryptone (10 g/liter) and yeast extract (5 g/liter). and was supplemented with ampicillin (25 mg/liter in liquid or 40 mg/liter in solid media) for selection of pHR322-derived plasmids.

(b) Strains h’scherichiu coli host strains B, (non-suppressing) and (‘R63 (carrying a su@ amber suppressor) were described I)?; Edgar rt al. (1964). MCS 1 (supD and transformat,ioncaomprtent) and ABl (non-suppressing and transformationrompetent) were described by Kreuzer et al. (1988). XapTV (non-suppressing and transformation-competent: see h’elson et al.. 1982) was kindly provided by L. Cold (ITniversitv of Colorado. Boulder). T4 strain KlO has the following mutations: amB262 (gene .38). amS29 (gene al). nd28 (denA), rI1PT8 (rZ1den.!3 deletion) (Selick rt al.. 1988). Strain KIO-uvsY* is isogenir with K10 excaept for the presence of a 0.12.kb deletion that) removes ori(uosY) and renders the phagr IL/‘sY~ (Kreuzer rt al.. 1988): K~O-UVSI’~ has also been referred to as KIO-608 by Derr & Kreuzer (1990). The motd mutation amG1 (Mattson et al.. 1978) was crossed into the KlO genetic background by % successive rrossw with an unequal parental input, (1 am:10 K10) (see Kreuzer et al. (1988) for analogous constructions).

(v) (‘onstruction

of motA

deletion

mutant

The motA deletion mutant was generated by in vitro construction of plasmid pKB17 (motA*), followed by deposition into the T4 genome using the insertion/substitution (I/S) syst’em (Selick et al., 1988). pKB17 is a derivative of the insertion/substitution vector pBSPL0 f with a cloned insert derived from a synthetic oligonucleotide (see Fig. 1). We designed a single-stranded oligonucleotide which fused 2 segments of T4 sequence: the iirst (48 bases) contains the sequence from the 36th nucleotide upstream from the wbot4 gene through the 12th nucleotide of the gene, and the second (51 bases) begins at the 12th nucleotide before the end of the motA gene and continues through the 39th nucleotide downstream from the stop codon. The oligonucleotide thus represents a 609 bp deletion of the 633-bp motA coding sequence (Ilzan et al.. 1990; R. P. Schmidt & K. pi. Kreuzer, unpublished results). The synthetic oligonucleotide was

Figure 1. Schemat’ic diagram of nmtA deletion and The locations of the gene 52, mot,4 mutation. nlotA - 1 coding regions and the early promoter upstream from gene motA (P,) are indicat,ed. The 2 segments (48 and 51 nucleotides, respectively) of the synthe?tir oligonuc~leot~ide comprising the mot,4 deletion mutation are shown below the gene map. which is not to scale.

converted to a duplex by priming DXA synthesis with self-complementary 3’ ends according to t#he procedure of Oliphant rt al. (1986). The duplex oligonucleotidr was then treated with HindITI and XhoT. which cleave at sites just outside the T4 sequence. The T4 I/S vector was cleaved in the polylinker region with Hind111 and Xhol and then ligated with purified duplex oligonucleotidc to generate plasmid pKB 17.

E. coli MC61 with or without pRS31 (MotA supply plasmid) was incubated with vigorous shaking at 37°C in L broth to a density of 4 x lo8 cells per ml and then infected with the indicated T4 strain at a multipli&y of infection (m.0.i.) of 005 plaque-forming units (p.f.u.) per cell. The infected cultures were immediately diluted 2509 fold and then another 4-fold into pre-warmed L br0t.h. Unattached phage were measured by chilling a sample from the 2500.fold dilution and adding chloroform. The infected cells were incubated for 1 h with vigorous shaking at 37°C’. The infections were terminated by decanting into chilled tubes containing chloroform. and p.f.u. were determined by plating on MCSl-pRS31 and ,MCSl cells. Sverage burst sizes were calculated by dividing the tot,al p.f.u. by the number of infected ~11s.

(e) DNd

replication

assays

E. coli M(!Sl or ABl with the indicated plasmid was incubated with vigorous shaking at 37°C’ in I, broth to a density of 4 x 10’ cells per ml and then infected with the indicated T4 strain at an m.o.i. of 3 p.f.u. per cell. After a 3min incubation without shaking to allow phage adsorption, the infected cultures were incubated for 1 h with vigorous shaking. Total DK;,4 was then prepared as et al., 1988). Briefly. the previously described (Kreuzer infected cells and any released phage particles were collected by centrifugation and pooled, treated wit,h SDS/proteinase K. extracted sequentially with phenol. phenol/chloroform/isoamyl alcohol (25 : 24 : 1, by vol.). and chloroform/isoamyl alcohol (24 : 1. v/v). and finally dialyzed against TE buffer (LO mw-Tris. HCI (pH 7.8). 0.5 mM-ru’a,EDTA).

(f) Analysis

E. coli AR1 broth at 37°C’ infected with p.f.u. per cell. 4 min without

of phage

proteins

was incubated with vigorous shaking in 1, to a density of 4 x lo8 cells per ml and then the indicated T4 strain at an m.o.i. of 3 The infected cultures were incubated for shaking to allow phage adsorption and

Role of MotA

then at 37°C’ with vigorous shaking. Samples (625 ml) were removed at the indicated times and incubated with 20 p(‘i of [3sS]methionine (1000 Ci/mmol). The pulselabelling was terminated after 4 min by adding 675 ml ice-cold L broth. centrifuging for 2 min at 4”C, and freezing the pellets at - 70°C. The pellets were dispersed in 16 ~1 of SDS sample buffer (60 m&r-Tris . HCl (pH 68). I (+. Zmercaptoethanol, 1% SDS, 10% glycerol, 001 c,, bromphenol blue) and heated for 5 min at 100°C’. Polyacrylamide gel electrophoresis was performed as described by Laemmli (1970), and the gel was dried and subjected to autoradiography. (g) FLYA assays Total in oivo RNA was isolated as described by NcPheeters rt al. (1986), with the following modifications: the NapIV hosts were grown in L broth at 37°C; the cells were infected at an n1.o.i. of 3 p.f.u. per cell. with adsorption without shaking for 3 min; the centrifuge tubes did not, contain ice; and the resuspension solution did not contain gelatin. Primer-extension and sequencing analyses of the RNA samples were conducted by reverse transcriptase extension of a -5’ end-labelled pBR322 HindITIccw Site Primer (New England Biolabs). using the procedure of Zaug et al. (1984) as modified by Guild et al. (1988).

3. Results (a) D&ion

of gene motA

is lethal

Roth ori(u~s Y) and ori(34) contain middle-mode promoters, suggesting that the T4 MotA protein is involved in origin-dependent replication initiation. We t’herefore began by testing the effect of a motABm mutation (u&l) on replicat’ion of an ori(uvs Y)-containing plasmid. Surprisingly, replication of t,he origin-containing plasmid appeared to be moL4”” unaffected the (non-suppressed) 1)) mutation: suggesting that MotA is not required for origin-dependent replication initiation. However. the motA”” mut,ation might not eliminate MotA protein act’ivity. For example, an amber peptide could retain activity, an active C-terminal peptide could be produced by translation initiating downstream of the amber codon, or a low level of fulllength protein could be produced by read-through of the amber codon. Recause anaG 1 and other available motA mutat*ions might not eliminate MotA activity, we chose to delete the motA gene from the T4 genome. A ,motA deletion mutation was constructed in a recombinant plasmid and then substituted into the T4 genome using the T4 I/S system (Selick et al.. 1988). The deletion mutation was created by designing and cloning a synthetic oligonucleotide that fuses two sequence elements from the T4 genome: a 4%nucleotide segment that includes the motA initiation codon and a 5lnucleotide segment that includes the motA termination codon (Fig. 1: see Materials and Methods). The oligonucleotide creates an internal 609-bp deletion of t’he mot.4 gene. but’ maintains the reading frame such that a peptide of seven amino acids should be produced.

in T4 Replico!ion

91

Table 1 C’on,ditionaE lethal motA

deletion mutant

Host

Phage

MC61 -pKS31 KIO hl(‘S I K10-WKh4A M(‘s1-plis31

KIO

KIO-mofAA

M(‘S1

t p.f.u.~‘infectrti

wll.

The motA reading frame was maintained wit’h the hope that transcription and translation of the downstream reading frames (Uzan et al.. 1990) would be unaffected. Sequencing across the cloned oligonucleotide revealed a single base change from t’he expected sequence, altering the fourth c-odon of the reading frame from GTA to ATA and thereby substituting the fourth amino acid of’ the short peptide; this alteration was considered insignificant. A phage K10 derivative with the integrated motAA plasmid was obtained by selecting for homologous recombination between the plasmid and phage genome. The length of homology on each side of t’he deletion was only about 50 bp. but this was sufficient t’o allow plasmid integration at a frequency about sixfold greater than t,hat of the vector control (which integrates by illegitimate recaombination: also see Selick et nl.. 1988). T4 K16 Slam) with the integrat,ed supFphage (38”” containing plasmid were selected on a nonsuppressing host (Selick et al.. 198X). and the DNA of several integrants was isolated and analpsed by digestion with appropriat’e restriction enzymes. As expected. most integrants had incorporated the plasmid by homologous recombination in the targeted motA region of the phage penome. One such integrant was grown in a suppressing host. allowing propagation of plasmid-free segregant, phage that resulted from a second recombination event. This second event should OWUI‘ with similar frequencies on either side of the deletion mutation, resulting in similar frequencies of mot.4 + and motAA sepreganm (see Selick et al.. 1988). However, only mot;l + plasmid-free segregants were obtained, suggesting that segregants carrying the motA4A mutation were inviable. If the motA gene is essential, then not-4” segregants could only be isolated by providing the MotA protein irk trans. A MotA supply plasmid. which complements the growth defect of amG1 phage on TabG cells (pRS31; Schmidt & Kreuzer. 1992), was therefore introduced into the suppressing host used for the isolation of segregants. Rhen the integrant phage was grown in cells with this NlotA supply plasmid. both motA + and motA A segregants appeared. A K IO-motAA segregant’ was thereby isolated and found to be a conditional lethal mutant that forms plaques only on hosts with the MotA suI)ply plasmid. The MotA supply plasmid provides 12.bp and 96.bp segments that are homologous to the DSA adjoining the deletion mutation in

K IO-motA A* but presumptive mot,4 + recombinant s wxw found infrequently (less than 3 x 1O-’ per p.f.u.) and should not affect, most experiments. The average burst sizes of T4 KIO and KlO-motAA were compared in host cells with and without the IVIotA supply plasmid (pRS31). Tn t,he absence of the supply plasmid. KlO-motAA produced no detectable progeny phage: the apparent burst) size was not significantly different from the number of unattached phage (Table 1). Progeny phagc production was restored by the presence of t,hc YTotA supply plasmid (Table 1). Thesci result)s. together with the following considerations, strongly argue that MotA is an essential T4 protein. First, the open-reading frame immediately downstream from mot/l. motA. - 2 (Uzan et al.. 1990), is left intact in the KlO-motA* genome and the deletjion was designed to allow transcription and translation of downstream reading frames (see above). Second. thr MotA supply plasmid, which complements the growth defect of K lo-motAA, contains the entire mot,4 gene but only the first 26 amino acaids of the X&amino acid mot.4. - 7 reading frame. Finally. the was possibility remained that thr ,motA* mutation not unique, but rather that r&AA and previously mot4 mutations caause lethality onlv in isolated combination with one or more of the K 10 mutakons (dW.4. (&nR-rZ/) A. 3X”“. .5Prn), LVfJ could not possibly det,ec:t a lethal effect, of mot.4““’ iu tllr K 10-motA”” strain. since two of the K 10 mutations (.H”” and Slam) are themselves c*onditional lethal ambers. To compare directly the viabil&y of mot,lam and tnotAA mutants, we therefore generated ZY+:il* derivatives of KIO-motA”” and K IO-mofd' by marker-rescue recombination with plasmids carrying the 2X+ and :if + alleles. Tn these isogenic& st’rains. the motil* mutation causes conditional lethalit)\ but the motAam does not. We conclude that t,he &f/l’ mutation causes lethality because MotA is an essrnmot,4 tiwl protein. and that previously isolated mutants produce enough MotA protein to survival.

(b) Protein

expression

b?yt?w mot’AA mutant

Previously isolated motA mutations cause pleiotropic alterations in T4 protein synthesis, including prolonged expression of certain early proteins, reduced levels of middle-mode proteins, and delayed production of late proteins (see Tntroduction). (including Nonetheless. these motA mutations amG1) do not, cause lethality as does the srnot8’ mutation. To begin the search for an essential activity of the MotA protein, we compared the time course of protein expression in infections by KIO. K lo-motAA, and non-suppressed Kl@motA”“. Proteins were pulse-labelled with [35S]methioninr and analyzed by SDS/polyacrylamidr gel electrophoresis (Fig. 2). The first notable result’ is that protein expression by both motA mutants was similarly affected through the first 14 minutes of infection. Tn particular, the motA”” and m&A* mutations both resulted in prolonged expression of certain early

I MW 66-

45-

2

3

4

5

6

7

8

910

Ill213

141516

-9p43

--9P23

-

gp23*

3629-

--9P32

24-

- 9P45 -A33 ml-

14?-

proteins (e.g. compare band A33 in lanes 2 to IO). In mot.4 mutat,ions reduced thr both addition, expression of gp45 and gp43 (compare lanes 5 t 0 7). which are t’wo proteins that are mainly expressed from middle-mode promoters (Guild ~‘1 trl., 198X; Hsu 02 Karam. 1990). Finally, late protein synt,hesis was evident by 10 to 14 minutes in the nad.4+ control (e.g. gp23; lane 8). but was not dr+c*tetl in either motA-mutant, infrcstion until after 14 minutes of infection (lanes 13 and IS). Although thus expression of gp32 appeared t.o be reduced by both motA mut’ations at early times (lanes 5 t’o 7). thta protein was overproduced at later times in t hr. mut,ant infections (lanes t I to 16); this is psunably due to autogenous derepression of gp32 c*aused by excess single-stranded IIIXA in the motA infections (for a review, see von Hippel et al.. t 9X3). To summarize the early stages of infection. t.he motAam and motA* mutations resulted in apparcant Iy itlvntical patterns of protein rxpression. Thus. t,he IetJhalit-y caused by the rtzotffA rnutat,ion did not c*orretatc~ detectable difference in pre-late with an> expression, although it is possible that one or more proteins not visible in this assay might be expressed by the amber hut not the deletion mutant.

Role of MotA

kb

1234

1234

in T4 Replication

1234

93

1234

543-

Plosmid DNA

2-

l-

(a)

(b)

(cl

Figure 3. Replication of phage and origin-containing plasmid DNA (d)) and MCSl (Cam@: (b)). each with the ori(uvsY)-containing plasmid ((b) and (c)), or K10-)1~otA~ (d). Samples were harvested 5 min (lanes (lanes 4) after the :l-min adsorption period, and total DNA was isolated

(d) by mot.4 mutants. E. coli AK1 (sup”: (a), (c), and pGJB1, were infected with KlO (a), K IO-motA’” 1). 10 min (lanes 2), 20 min (lanes 3), or 60 min

and cleaved with SspI and HapITT. The resulting and visualized by st’aining with ethidium bromide. Phage DNA DIVA is indicated by an arrow on the right. The scale (in kh) on the left was

fragments werp separated by agarose gel electrophoresis is indicated by a brace. and plasmid generated from the migration of XbaT fragments

of unmodified

The deletion-mutant infection displayed a distinct phenotype at later times of infection; namely. a more severe delay in late protein (e.g. gp23) expression than in the motAa” mutant (compare lanes 12, 13, 15 and 16). The more severe delay in late protein expression is presumably an indirect consequence of the absence of MotA protein. One possible explanation is that a late-mode transcription factor requires a MotA-dependent promoter for expression. A second possibility is that the more severe delay is caused by the replication defect of the ,motA* mutant (see below); late T4 transcription is greatly stimulated by DNA replication (Epstein et al., 1963; Bolle et al., 1968; Lembach et al., 1969; Riva et al., 1970). In either case, the more severe defect in late protein expression caused by the deletion mutation provides one possible explanation for t,he essential nature of the MotA protein. (c) Replication

defects in motA*

mutant

The presence of a middle-mode promoter within at least two T4 origins suggested that MotA protein is directly involved in T4 DNA replication. We therefore examined the replication of both phage and ori(uvsY)-containing plasmid DNA as a function of time after infection by KlO-motA* (Fig. 3(d)), KlO-motA”“’ ((b), (c)), and KlO (motA+. (a)). The DNA samples were digested with Sap1 and HaeIII. SspI cleaves T4 DNA into a series of fragments that are approximately 3 kb and smaller

T-4 DR’A (Kutter

it al., 1990).

while cleaving replicated plasmid USA into an approximately 45kb fragment. Hoe111 increases the sensitivity of the replication assay because it cleaves unreplicated (unmodified) plasmid DNA into small fragments but does not’ cleave T4-replicated plasmid DNA (which contains the cytosine modifications characteristic of T4 DNA). Phage genomic DNA replication was markedly delayed in the non-suppressed motA”” infection, although wild-type levels of DNA accumulated by 60 minutes (compare Fig. 3(c) with (b) and (a)). Similar results were obtained by Matt’son et al. a continuous-labelling replication (1978) using assay. The motA* mutation had a much more severe effect, with very little phage genomic replication even at late times of infection (Fig. 3(d)). Therefore, the MotA protein is necessary for most phage genomic DNA replication. MotA could be necessary for activation of T4 replication origins and/or for transcription of T4 genes that encode certain replication protein(s). Recause ori(uvs Y) contains a middle-mode promoter that is required for plasmid replication, we were somewhat surprised to find that the ori(uvsY)-containing plasmid replicated in the motAA-mutant infection (lane (d)4). Clearly, replication of an ori(uvsY)-containing plasmid does not require MotA. Replication of this plasmid was delayed in the motAA infection, but was also substantially enhanced. As discussed above, origincontraining plamids replicate more extensively in the absence of the recombination protein ITvsY (see

kb 5-

3-

2-

Figure 4. Replication of phagr and origin-c~ontairling plasmid I)Si-\ by n~otAAua.sYA mutant. E. roli MS(‘1 (srrpl)) with the ori(ctr,s Y)-containing plasmid l)(:qI HI were infected with KIO (lane 1). KlO-nc0t.4~ (lam1 2). K IO-rr~~til~u~s YA (lane 3). or K IO-~7,sIFA(lane 4). Samples were harvested 60 min post-infection. and total l)XA wax cleaved with A’spI and HarIIT. The resulting fragments were separated by agarose gel clrctrophorrsis and visual ized by staining wit,h rthidium bromide. I’lasmid 1)X,1 is indicated by an arrow on the right, and the scale (in kl)) on t,hr left was generated as for Fig. 11.

Introduction). The enhanced plasmid replication in be the motA’-mutant infection might therefore caused by a deficiency in I:vs\’ protein, because transcriphon of the UVSY gene is primarily deprndent on a MotA-dependent middle-mode promoter (Gruidl &. Mosig, 1986). To explore further the replication phenotypes we constructed a caused by the motAA mutation, Replicated DNA was double motAAuvx YA mutant. then examined 60 minut’es after infection of plasmid-bearing cells by K10 (Fig. 4, lane I ). KlO-motAA (lane 2), KlO-motAAuvsYA (lane 3), and KIO-UZXY~ (lane 4). The motAAuvsYA double mutant replicated the ori(uvs Y)-containing plasmid mutant t,o the same extent as did the single motrl’ (compare lanes 2 and 3). This result is consistent with the possibility that enhanced plasmid replication in the rnotAA background is caused by a deliciency in CvsY protein, because the ws Y mutation did not further increase plasmid replication in this background. Perhaps the most) notable conclusion is that ori(uvs Y)-containing plasmid replication is pa,rtially dependent on the MotA protein in the UUAYA background (compare lanes 3 and 4). Apparently, by eliminating the complication of altered levels of Ilvs?’ protein. a partial dependence on the MotA protein was revealed. This raises the possibility that t,he action of MotA on the middlemode promoter of ori(uvxY) can lead t,o the initiation of replication (see below). The small amount of phage genomic replication in the motAA-single-mutant infection (Fig. 4, lane 2)

\vas virtually t~liminat6~tl in t IIt) n~ot.-l%~~s 1”’ irlftlc.t.ion (lanr~ 3). This IJI‘~ I7 tnrrtation is i1 O.ll’-kl~ ant1 1Irtl deletion that removes the ucs I’ promoter phage chromosomal ori( uvs I’). Therefore, thc3 t4ilnination of phage genomic replication t~onlti lw explained either bv a loss of recombinatiorr-tlt.~J~,~~ dent genomic rephoation (which depends on I .vsi\r protein) or by a loss of ori(u,ls Y)-directed gt~llt~niic replication. To distinguish between these two ~)ossibilities. WP constructed anot her KIO-mofA”rcr,s 1. mutant using a ws Y linker-ir~st~rtion double but tlot~ not mutjation that blocks I’vsY production affect ori(nlts Y) (Krruzer f’t cd.. 19X8). This tlouhlr mutant produced about t,he same amount 01’ rrplit3tetl genomic I)SA as did K 10-~nwtAA~wsF” (clata not shown). Therefore. the low lewl of gctnotnic, replication occurring in tht, absentbe of Mot A ih I‘\-F.l’)predominantly recombination (e.g. dependt~nt rather than ori( ((vs Y )-dependent ‘t‘his result iLlSO implies that t ht, tnot.4’ sinylr tllnt alit IJrdlI(Ys itt least a small itrrIcjU~lt of’ I’\.hY r)rott~iti. I’erha~~s the I’vsY f)rotvirr Itzvcl is I~ILV enongh so that origirl-c.ontaining plasmid rc~plication is enhan& but rrc:omhinatiorl-tirprndt,llt rr~plit~ation of the genom~ is still possibkt.

middle-mode promoters that arf’ required for rtkplication, and yet replication of an ori( /(?t.‘:Y)containing plasmid does not, require MotA. which activates middle-mode promoters (Menkrns & Kreuzer, 1988: Schmidt d nl.. unpublished results: see above). A possible solution t,o this ~~uzzle is suggested by the fact that~ some middle-mode promot’ers, including the ori( ws I’) promoter. are capable of directing MotL+independent transc*rip tion from recombinant plasmids (see Int’roduc+tion). It is therefore possible that t.he MotA-inctepentlent replication of ori( u/-s I’)-containing plasmids requires blotA-independent transcription. Furthermore, transcription and replicat~ioll from ori(d4) could have a different, tlc~pendenc~r on &l:lot,A. LZ’tl t hereforr compared replication from ori (UVX Y j and ori(containing plasmids in wild-type and various rnutant infections (Fig. 5). As in the experithe ori( /At’s Y)-containing plasmid ment’s above. displayed enhanced rcplic~ation in the motilA infetation (Fig. 5. lane (a)4) (~ornpared to thr r~1t.4 + control (lane (a)l). 1~111thr motAA mutation modestly reduced plasmid replic~ation in t.hc uvs 1. background (compare Fig. 5 lanes (a)5 and (a)fi). was the The most interesting result that ori(34)-cxontaining plasmid displayed a muc4l stricter requirement for the transcription fat:t>or. The greater Mot,A dependence of ori(3-I) is most’ obvious when comparing the motAAuus YA infection to that of U/V PA ((tompare Pig. 5, lanes (t))f, and (b)(i). but is also evident in the 11~sY+ background for either thta motnm or rr~t.4~ mut’ant, infections (Fig. 5. la,nes (b)3 a,nd (b)4).

Role of MotA in T4 Replication

95

12345

123456123456

12345

kb 543-

(a)

(b)

Figure 5. Replication of origin-containing plasmids by ~~ot.4 mutants. E. ~oli MCSI (supD: lanes I.-g. 4. 5. 6) and ABI (sup’: lane 3). each with the ori(ul:s Y)-containing plasmid p(LJ El (a) or the ori(33)-containing plasmid pKKO61 (b). \vpre infected with KIO (lanes 1). KIO-rnot.4”” (lanes 2, 3). KIO-nlotAA (lanes 4). K 10-n/ofilA~~?-.s JVA (lanes 5), or KIO-ups I” (lanes 6). Samples were harvested 60 min post-infection. and t,otal I)IvA w-as isolat’rd and cleaved with ~%pl and HaeITI. The resulting fragmrnts were separated ,b.y agarose gel electrophorrsis and visualized by s&rung with ethidium bromidr. Plasmitl DSA is indicated by an arrow on the right. and the sc~lr (in kb) on thr left was generated as for Fig. :I.

(a) The above results are consistent with a model in which origin-containing plasmid replication is strictly dependent on transcription from the origin promoter. However, for this model to be true. t&nori(uvs I’) the plasmid-borne scription from promot)er must be much less dependent on Mot)A t,han is transcription from the ori(34) promoter. Menkens $ Kreuzer (1988) found that transcription from a plasmid-borne ori(uwsY) promoter was conpletely MotA-dependent, but that experiment involved T4-modified plasmid concatemers which apparently exhibit a stricter MotA dependenc?e than does unmodified circular plasmid (see above). We therefore analyzed the MotA dependence of transcription from the origin promoters located in unmodified circular plasmids. Total RNA was isolated without infection (Fig. 6, lanes 1) and at’ X and 40 minut,es aRer infection by KIO-uus YA (lanes 2 and 4, respecbtively) or KIO-uvs YAmotAA (lanes 3 The RNA samples were and :?. respectively). of a primer t’hat analyzed hy in nitro extension hybridizes to vector sequence downstream from either the ori(uc>s Y) (Fig. 6(a)) or ori(34) (Fig. 6(b)) promoters. Sequencing reactions were co-etectrophoresed to confirm the correct transcript start sites (data not shown). DNA samples were also isolated from these infected cells at 60 minutes after infertion. and the levels of replicated plasmid DNA were

(b)

Figure 6. kIot.4 dependenc*e of thr ori(cr7l.sY) and ori(34) promoters. E. coli XapT\’ ~~11s with the ori(ul!s l-)-containing plasmid pG,JHl (a) or thr ori(,34)-c~ontaining plasmid pKKO61-6 (b) were’ uninfecated (lanes 1) or infrcated with KIO-ursYA (lantas 1. 4) 01 K IO-rn0ti2~uc~ lTA (lanes 3. 5). Total RNA was harvested 8 min (lanes 2. 1%)or 40 min (lanes 4. 5) post-infection (timps includr the Y-min adsorption period). The transcsripts were analyzed by primer extension with reverse transcript)ase. using radloact’ively labpllrd HindIT Site Primers ((Bountrrelockwise: h-ew England Biolabs). Samples were subjected to electrophoresis through an X?b denaturing polyacrylamide grl. and a photograph of the resulting autoradiogram is shown. Standard c.hain-termination sequencing reactions with t,ht: same primer and RN.4 t~rmplatrs were also analyzed on this gel. The originpromottlr t)ranscript,s, which hare t,hr same 5’ ends as previously reported (Gruidl 8: ylosig. 1986; (build rt 01.. 19X8; Jlrnkrns & Krruzer, 1988). arr indicated hy arro\vs. -

found t’o be very similar to those shown in Figure 5 (data not’ shown). Roth plasmid-borne origin promoters w-(Are active in uninfected cells that contain no MotA protein (Fig. 6. (a)1 and @)I). and therefore neither promoter is strictly ,MotA-dependent. However, the ori(?~z~sY) promoter provided much higher levels of transcript than did the ori(34) promoter. The two transcripts differ only in t’heir 5’ ends, suggesting

that their stahilitiw mwy bcb similar: it’ ho. the, ori( ///T.s I’) promott~r~ is mu& stronger t ban 1 lit, ori promoter in uninfected cells. Transt~ripls f’rom both promotrrs \VPW also detwtt~tl at Iwt h t inrc points aft)er inf&tion by c~it~hrr phage, ‘i’trc~ amounts of trunwript from the ori( 11(‘s I-) proino1~~r ill raight minut.es aft’txr infection by either phagcs (Fig. 6. lanes (;t)L’. (a)3) ant1 at 10 minutw afttlr infwtion by thrl r,rot.-l’ phagf~ (I:uw (a).‘;) w(‘rt similar to that tlr>tet+ed in uninfected wtls (Iant\ (a)1 ). I~rcause t)htk total amount of ori(trw j7) trwstaript is unchangrd b;\, infection. it is not possihltx to ,judgtB t,hr proportion of ori( ut-s 1’) transcripts that UYW nc\vly synthesized aft,er infection: this Assam measures tOti KSr\ and \vc’ do not kllont Irtl stability of the t ranswipts produwd prior to inft,c,tion. Xtt,hough drtrc~tion of MotA-induwtl traw st~ription is prwluded I)? t)hc high Iwrl ot Mot r\-indeptwdent t ranstYipt ion. wt’ hclievta that some transcription of the ori(llt3 Y) promotrr in tht wof,-l + infection is induwd by binding of the Mot A prott,in (see I)iscussion). In cont,rast. the levtlt of tranwript~s from thr ori(34) promoter was markedI> intareased by right minutes of the rrwt,-l+ (Fig. fi. larw (b)d) but not t ht. mot/l* (lane (b)3) infet~t ion. Thr~ relative levels of’ transcr$As presrnt at right minutes were estimated by primer-extension anal>.sis of dilution scrirs. In t’hree separate exprrimcnts, t bra ori( 1’) transcript levels in thr rrrot.-l :lnd tnot.-l* infections wew about the same. but the ori(54) tranwript) level was between 6- iktld 1 %-fold yrratw in thta rrrof.-l* than in the wot.-1’ infrtbtiorl (data not shown). tieplit~ation of ori~in~t.ontairritip plasmids owurs at relatively early timw in roof.1 + infwtions (Fig. 3(a)): therefort,. trnns~~ripts from hot h the ori(z1t.s Y) and ori(S4) promoters are prcsrnt at t hr time of plasmid DNA repli&,ion. At that time. atstive YlotX-dependent tranwription also l”rsumabl~~ occurs from both origin promoters. Pithtar pre-exist,iny t ranscrif)ts or iLt’tiVt> ‘I’ht~reftwr. tra~lst~ription t*ould be involved in the initiation of plasmid 1)X;\ replicaiion. Ttrpticatioll of an origin-t~ontainillg plasmid in a ,not.-l” infect~ion is severely drla+ (SW Fig. 3(d)): thus the #-minute rnotL~* RSA samples ma\’ be wlrrant in t~onsidrrinp r)lotL-lA ptasmid replica’tion. Tn this tease. an exwltent wrrelatitm is obtained: the ori(uc,s 1’) ttanstbript was detected at much higher It~vrls than the ori(.?-/) tranwripl (Fig. 6. wmparf~ lam23 (a)5 and (1))s). and ori( t/1’s Y) directed much higher Irvels of plasrnid replication than did ori(J4) (set’ Fig. 5). The RNA samplrs from 10 minutes afttbr rrrofA + infection (Fig. 6. lane 1) rrsultetl in a t~ollec~tion of hrterogeneous t)rilrit,r,~rxtrrisioli produrt~s. which might represent degradation products grnerat~etl by a nuclease whose expression is dependent on MotA transcriptional activation. These resutt)s support a role for transcription in origin-dependent rrplit~ation. I’ndcr twsh of the twntlititms in which an origin-containing plasmid ran replicaate. rrlativrly abundant levrls of 1 railscript art‘ present and active transcription may also br occurring. Thea possible import,ancc of at*tivr

1 ranwription sht~utti bta atlrlrtw+3l iir thf, t’r11 urf’ \‘I 111; Nl,nrtheles;h. II i?. f)ulse-labrlling ap~)roathw. already clear t>hat t,he Mot.-2 dependent-tt ot’ i riili swipt’ion and replication from plasmi&borne origirw are wrrrlated: Slot,A prot~tin strongly stinlllliltt~s both transcription and replit~ation from ori( Ilut has much less effwt. on t,1.a~lst~r,if)tior1 and rt~ptic.;~i ion from ori(/r/~s >-).

4. Discussion The nwtA* mutant describetl in this report is a wnditional lethal that’ grows only when Mot A prot.ein is supplied from a ptasmid. Thereforc~. Mot A is an essential protein. The previously t~harwtrrixrtl rnot.4 mutants. which do not t’auw k>thalitv. must ret)airi rwidual Mot A achtivit \ Ii is t~wious that swe’cns for cwr~ttitiorial It+ hal 7-C mutants (t~~pstt~in uf al.. 1963: Edgar 8~ \Vood, I!Mi) ditl not u11f9vf’r rnotil mutants. However. t ht. viahilit!, of t hta rrrnC1 mutant suggrsts that i\ \-rry lo\\ It>\-r*l of MotX activity may lJt\ sufficient for gro\\ tlr. I’clrhaps a majorit)y of all possiblr rriisstww and nonwnw rnutations ill t hr rnot,-l grnr l?itVt’S suffit*it~~it wsitlua,l activity for survival. Why is t.hr wot.l gene wstwtial for ‘I’4 grou’t h! One possibilities is that expression of ont or more essential T1 prot.t‘ins is drpendrnt on t rallstaript ion from rnitltfle-rnodc promotws. Howevt>r. most T-C genes with rnitldle-mode promoters arc also tr’aw npst ream t%arly ~~W~l~tltt~~~S. iLIlt scribed front SlotA-deI)t,iitIt,ilt transcription of thtw gt~rws shoriltl thrrrfow not he wtwsary. Furthcrrnore. \VP cwultl detrct 110 tlifft~rrnw in t trtx l)rtb-liltcl l~rr~tt~ins ~~rottucctt in thtt rrcol.-l* and w~f.~Um infwtions. 0vt~11 though only t’lre rwt.l * infet%ion is iloir-~)t~odnt~ti\-t~. Nevertheless. it is Iwssible that a t‘rititaat phag protein. not detwtt~d in the ~)oI~at:r~lamit~~~ gel. was produced at a low levrl in t,he ~w~t.-l~~ infect.ion. but was absent in the motAA infwtion. An intewsting possibility is that synthesis of an essential late transcription f’wtor depends on middle-mode t ranstLrif)tion. Such a defect ~outd explain thti ~~ronp inhibition of lat,e protein rxpression in thrb r,~ofrl* mutant. All alternate explanation ftrr the wsrlitial nature of t.hr MotA protein is that ori~in~tie~)erltit~~~t replication. atativat ed by Mot A. is nwrssary for phage growth (also see bc%w). LVr have shown that the rnot.lA mut’ation results in a Iargt~ rt~ttut~tio~r in phage genomit* replication (Figs 3 to 5). arrtl havt, also imptit3ted MotA in thra replication of two specific T1 origins (see below). Oriffin-tir~)rntlent replication might be nrcessary for genomr duplication or for a&ration of t&r transt:ript,ion, tvtiich normall~~ requires l)K\‘A replicat~ion (for a rrvitw, we Geiduschck et trl.. 1983). The n&A gene product is rrquiretl for the ac*t,ivation of T1 middle-mode promoters during ‘I’4 inf&tion (Matt,son it ~1.. 1971. 1978: Hwtwles & Sauerbier. 1974: Hall 8 Snyder, 1981: drFrancisc~is Kr Brotly. 1 !M2; drFrailciscis c/, III.. I !%2). Nonetheless, at least some middle-mode I)romot~ers are activta on recombinant, plasmids in uninfet:tt?d

Role oj MotA

~11s which have no MotA protein (Shinedling et al., 1986: Guild et al., 1988; this study). Therefore, certain T1 middle-mode promoters must resemble E. ~1; promoters sufficiently to he recognized l)y the host’ RNA polymerase without the viral activat.CJr protein. This recognition may he largely due to thtl - 10 region, which follows the consensus srquen~e of host promot’ers (Brady et al., 1983; (build rf al.. 1988). Recent in vitro studies with purified ;CfotA protein have c*larified t,he requirements for middle-mode promoter activity: Mot,A protein nas shown to hind t)o the middle-mode promoters for genes WSS and uw I’ (Hinton, 1991: Schmidt’ & Kreuzrr, 1992). Tn each case, MotA activated in ~*ifro transcription hy T&modified RSA polythe template DNA merasv. whether or not caontained the cytosine modifications characteristic of T1 DNA. However, unmodified RSA polymerase uninfec$ed (sells was capable* of Mot,\from independrnt t ranseription from the middle-mode promoters. Thus. one or more of the R?r’A polymerasr modificxations induced hy T4 infection restricts t.he a,ctivity of RXA polymerase so that the b1ot.A activator protein is required for transcription from middle-mode promoters. These results suggest a model for regulation of T1 transcription: one or mart’ of the T-binduced modifications of RNA polymeras(’ alter the polymtlrase specificity so that host (and perhaps T3 early) promoters can no longer he IJecWJse certain T4 recognized. middle-modr promoters can also he recognized as host promoters (see ahove). t,his RNA polymerase modification also l~lo~ks the Mot,-\-independent transcription from TI middk-mode promoters, leading to a requirement for the transcriptional activator MotA. Perhaps t,he 7’4 prot,rin that modifies the RNA polymerase is expressed from a middle-mode promoter that has no activity wit,hout SlotA. This could explain the ovel’production of certain early proteins in the absencae of Yet A. and might complete a feedbck loop that shifts ‘1‘1 transcsription from the early to the middle motfr.

The rrsultJs of this study clarif:y the in Vito rfsquirtments for transcription from two middlemode promoters contained within Ta replication origins. B&h origin promoters allowed some transcbription in uninfect
in T3 Replication

97

RSA polymerase in vitro (see above). The ori(34) promoter directed MotA-dependent transcription from the recombinant plasmid: the total amount of transcript was much higher af%er T4 mot,4 + infection than it was prior to infection or after T1 rnotdA infection. Overall, we conclude that, the ori promoter has a stronger dependency on t,he MotA protein than does the ori(u,/:s Y) promoter. at least n-hen tested in the form of unmodified circular 1)NA. The sequence elements of’ a middle-mode promoter that determine the stringency of the >lot’A are unknown. However. we have recluirement rt,centty found that a single-base suhstitut,ion in the ori(u/ls 1.) promoter can render t rariscriptjion much more dependent, on the .IIot.1 prot,ein (It. P. Schmidt. A. E:. Yenk~ns & K;. S. Krruxer. unpublished results). Thr original aim of constjruc*ting the rnotAa mutant was to help clarify the possible relationships betwren Slot,A action. transcription and DNA replication from the middle-mode promoter-origins. [‘sing the null mubant. we were able to show that plasmid replication direct,ed hy eit htar origin is stitnulated t’y t,he presence of Mot A protein in t,he /I /‘S 1 background. However. plasmid replication did occur in the absence of M&i\. particularly in the The relative amount, of case of ori( uf:s I’). nlot.L\-irider)etitlerlt replication from the two origins cborrelated with th roughly amount of transcription. hl~,tA-2ndependent tc’nrthermore. relatively large amounts of origin trwnsc.ript,s were present under each of the conditions that allowed r)m replication (e.g. ori(urls I-)-containing plasmid in motA ’ or motAA infections and ori(.‘ll)~c,ont’aining and a rnuc*h smaller ptasmid in mofd + infection). amount of transcript was present in tlit~ caondition that did not allow plasmid replic~ation (ori(S4) in rrtofrl” infection). The conditions under which the origin-caontaining plasmids replicated wtbre also presurnahly conditions under which activtl transctription was occurring at the time of replic~at ion. These results theret)? provide evidencr that ttit her preexisting origin transcripts and;‘or active transcaription is necessary for replication initiation t’rorn these two T3- origins. Replication initiation in ot)her si;\.stems (e.g. E. coli ori(‘, 2. 77 ant1 (‘~blII:l) also requires transcription, although the prrcisr role of transcription is va,ried (for a review. SW Kornherg 8 Baker, 1992). Jlodels for the role of transc>ription in repticatjion from t,he two T4 origins must explain the rifampicin resistanche of origin-dependent replication (Kreuzer & ~\lhert~s. 1986). Rifampicin inhibits thr host RNA polymrrase and thereby tjlocks transcription from all T1 promoters (Haselkorn rf (I,/.. 1969; O’Farrell 8 (iold. 1973). T+modified origin-c,ont,aining plasmid concatemers were found to replicaat e in the presence of rifampicin that had been added 12 minut,es after infection (Kreuzer Br Alherts, 19&i). Furthermore, unmodified circular origin-containing plasmids also replicated after the addition of rifampicin at, six minut.es following infection (K. H. Benson, unpuhtished results). The rifampicin resistanc*tJ indicates

that origin init,iation has no nerd for, ~9nt*urrt~rlt t ranst~ription. or that a putativct rifampit,irr-s~~I~sitivta step in t)he initiation ~J~CJC~SS had alrratiy ot~c~urrrtl Kay the tirncx of drug addition. On? ptrssibility is that, origin transt~ripts van hv ~)KJdUiTVl t~arly in infection or evtW prior to infktion. iLlId 1 hf3 stsrve a8 primers or allow unwinding of t hta origin l)NA at later Cmes. Alternatirrly. I hv role of I htx RX;.4 polymerasv may hr to ~~~wlucr ahort.ivr transt+pts or to unwind thr helix tiirrctly. both of which are rifampitin-resk&ant activities (St~hulz & %illig. 1981: (‘arpousis & (iralla. 1985). of phage genomit, I)KA is rxt,remely i<.rplication tkiayetl and significantly reducWf in motAA infetatiotls. intlicating that t>he rnerhanisrns of’ replication initiation may lye t~onsidera.bly altered from those in tt/of.I + infec4ions. Sirigk-stranded I)NA is prohahl> present at an a,l)normally high level. because synthesis of autoyenously regulated gp32 (SSK) procerds for much longer times in the m0t.4~ infec tion than in the ttmtA+ infection (Fig. 2). The taxtremr delay in gc~nomit+ replication coultl k~v t~~usrti Kay reduced cpmntities of ‘I‘4 rrplicat~iorq tvornhination protein(s) (e.g. gpl3. gp45. gp(i1. lTv~Y) and/ or by a defe& in origin-clrpcnderlt rrplit~ation. Iteplication diret+ed hy ori(U/.s I’) anal ori(31) lovatetl in the phage genomv may st,rietJ from rcyuirv the MotA protein. as does transcription t hc origin promoters in thtl phagr g:rxnornr (SW ;LIJtw).

In summary. a ttrotAA mutation causes defects in tnitldle-rnotle ant1 late protein expression, as well as in or,iQin-dependent, DNA replicaatdon. One or mot-r ol’ these defects must result, in a norl-FJt,t~du~ti~~~ ink4ion. because a tleletion of t,hr v&A gene is a Irthal mut,ation. The results of this stud\also provide st rang evident~e for a role of t,ranscription in rtJI)lication from t,wo ‘1‘4 origins that tvnt.ain middkriiotl~~ promoters. Transcription anct rrplicatjion from ori(li4) are more dependent on t’he MotA protein t,han ilrtl transcription and replication from ori(rtv,s Y), apparently because only the ori( uv.4’ I-) as a relat’ively strong Mot& promot,er functions itidept3ritknt promoter. Kecausr ori( UPS Y) can dirrtat plasmid replication in the tnotAA-mutant infection. !FlotA protein must not. IJ~ required for oriyin-tieprtltknt replit~at,ion init iat,ion under tvndit,ions in which active. Sonethrless. the origin promoter is Jlot.-\-dt,~Jerltlenl transcsription may normally play a tarit icxl role in replit~ation from the IJhagc gvnorrrit~ origins.

References Iit~nson. K. 11. & Krruztsr. K. N. (1!1!kZ). I’fasmiti trrcl
Wart~riophm!/r

I)uke

Medical

Schmidt and A.

rrlot.4am.

(‘en&r

for providing E. Menkrns

f’or important

discussions.

the MotA supply pfasmid for preliminary experiments

11. I’. (pRSS1). with T4

(Mathrus. (‘. ii..

Kuttrr..

I’.

11.. IX{. IN’. ion T-l

trim

(‘arpousis. .I. .J. k (:ralfa. .1 I). (l985). RX,\ pof~mt~rast~ \rith Incl’\y.i f~rottiotrr tnRNAA ‘itlitiittiotl itt1tt rfotigatiotr.

Intrrat~tiotl

ot

l)K:L\ tluritig I~oc~tf~rintitrg. l~tr;rtlg~!s ./. .Ilo/. HirJl.

mrthylatiotr. t~ifbrrrf)it~iti~st~tlsiti\.it~ anti at~wnrl)aii~ing tratist~rif~tiotr initiatiou. 183. I65 I7i. (‘ilrUs0. J’I.. (‘0l)f)o. .\.. J’lanxi. .\. k t’ufitzrr. strtrr fcbt, midtlft~ ‘T’1 RX.4. I Stutliw \vith /~‘w/~w~ichirr co/i RX.1 pofymc~rasr. .I. Rid. (‘hrn/. 257. 1087 lO!Ni. tleFrant.ist.is. \‘.. Favrt,. I<.. I.zatl. >I.. I,t~aut.tc~>~. .I & Brotly. I<. (I9W). It/ ,.i/ro svstrm for tnitltllt~ Ti RX;\. I I. Studies with T+tnt)&tit~tl RSA pof~mt~r;~sc~. J. Niol. (‘hrrt,. 257. 1OHi 4101. I)rrr. I,. K. & Krruzrr. K. S. (f!l!tO). Exprrssiotr antf fitnt*tiotr of’ the /cc~.slC’ get,+’ of l,at.trriol)h;ltlt~ ‘I’$.

.I. Jlol. Edgar.

/Co/.

I<. S. & bactrriof~hapt~

I’ror.

Sul.

214. 6l:lb656. lVootl. \v. II. T-C in vstrat+s

.4urrl. Sri..

( .S..

(I!NX). ~lorpho#tlnrsis of nllltarrt-itlfi,t.t~tl

of t~c~lfs.

I 55. 1!M X:5.

Edgar. R. S.. I)rtthartlt. (:. H. & Epstein. K. H. (I!W). .l cotnptrat~ivf~ study of’ twntlit~ionaf frthal mutations of fw.teriophagc, T41). (&nrtics. 49. 6%~ 64X. Epstrin. I<. H.. l%olfr. .\.. SttLinberg. (‘. kl.. K~ff~wf~t~rgrr. E.. I, tfr la Tour. E:.. (‘hrvalfey, I<.. Edgar. I<. S.. Sosma~r. >I.. I)rnhartft. (:. H. Kr T,irlausis. .\. (l!Ni:i). I’hysiofogit~af stutliw of c~ontlitiotlal ft~thaf tlrutalrts of f)actrriopfragc~ T4f). (‘o(rl ,Sprit/g Hnrhrn .V!/ttip. Quart. Hid. 28. X.5 392. (:riduwhrk. IC. I’., lCfliott. T. & Kitssavetis. (:. L\. (f!W). Ktyulatiorr of fatr grrw t~xl)rtwitm lrr Rrrcf/,~iophrryr 7’4 (Mathews. (‘. K.. Kutter. E:. &I.. Mosig. (:. & ISergt~t I’. I%.. ~1s). pi), IX!)- 192. .~tnrrit~wn S(wit+> t’or Mi~rol~iofog~~. LVashinyton. IX’. (iruidf. 11. 1.:. & Rlosig. (:. (1986). Srqutmt2~ iirltl tr;tnst.t,ipts of’ the bat*tt~riol)hage ‘I’4 I)SA rt’f)air gtatrt’

ursl’. (~uiftl.

This work was supported by grant CJM3462Z from thr Xationaf Institutes of Health. K.H.H. was supported b> thta Xational Research Service Award .?TSP CA0911 I ~14. Tfris work was done during the tenure of an Established lnvrstigatorship f’rom the American Heart Association to K.B.K. We thank H. M. Afbrrts and our coffeagues at

7’4

Mosig. (;. & IG~rgrt. I’. IL tltfs). ftfj. 17-1 :imeritwi Sot,iety for Mi~~rof)it~fog~~. 12’ashingtotl. f
I:Pupfic.s,

114. IO(il- IOi!3.

S.. (:a~ ft.. 31.. Swtvnry. IC.. Hoffingswortf~. ‘I’.. Motfrrr. T. & (:oltf. I,. (198X). Transvrif)tit,nal at$ivatiotl of’ l~at~tt~riol~hagt~ ‘I‘4 mitldft~ promott~rs I),\ thr nto/.1 protrin. -1. Jlol. Bin/. 199. 241 25X. Half. I). H. & Snyder. IC. I) (IWI). Supprrxsot~s of mutat,ions iti the rII gene of’ bat~tt~riophagr ‘I’4 afftvt promottlr utilization. C&tLdic.v. 97. I $1. Hafpern. n1. E:.. Jlattson. 7’. 6 Kozinski. A. \I’. (ICYi!)). Origins of‘ f)hagr T-C I)i’C;:\ rrf)fication as rt~vtv~fetf b>hyf)ritlization to t*font~f grows. Plvc Smt. .-Icwrl. ~Yci.. I..$.,-I. 76. 6137-6141 Hafpern. RI.. Mattson. T. Kozinski. A. IV. (IN?). l,ate ill T4 bac.teriophap I)XA rc~pfication t~vrrlts

Role

qf MotA

III. Specificity of DNA reinitiation as revealed by hybridization to cloned genet)ic fragmentas. J. Viral. 42, 422-43 1. Hasplkorn. R.. Vogel. M. & Brown, R. D. (1969). Cbnservation sensitivity of of the rifamycin transcription during T4 developmmt. X~at~rr (London) 1 221 1 836--8.X Hercules, K. 8r Sauerhier, W. (1974). Two modes of in /silo transcription for genes 45 and 4.5 of phage T4. ./. I’irol. 14. 341-348. Hinton. T). M. (1991). Transcription from a bacteriophage T4 middle promoter using T4 motA protein and phagr-modified R,KA polymerase. J. Hiol. C’henL.266, 18034-18044. Honryk. T.. Jr, Rodriguez, A. & Wril. J. (1976). (‘haracterization of T4 mutanb that partially suppress the inability of T4rlZ to grow in lambda Iysogens. (&n&x, 83, 477-487. Hsu. T. $ Karam. ,J. I). (1990). Transcriptional mapping of a DNA replication gene cluster in bacteriophage T4. Sites for initiation, t’ermination. and mRSA processing. J. Riol. (‘hem. 265. 5303-5316. .lohnson. .I. R. Ur Hall, D. H. (1973). Isolation and caharactcrization of mutants of hactjeriophage T4 resistant t,c)folat,e analogs. Virology. 53. 413-426. Krp~wl. F.. Fayet. 0. & Georgopoulos. c’. (1988). Strategies of bacteriophage DNA replication. In The Hmrtrrioph,agrs ((‘alendar. R.. ed.). vol. 2. pl). 145-262. Plenum Press. NPW York. King. (:. .I. Sr Huang. 1%‘.M. (1982). Tdentificat)ion of the origins of T-C DSA rrplivation. Proc. Snt. Acad. Sri., 1-..S.A. 79. 723X~ 7%“. Kornhfirg. A. & Baker. T. 4. (1992). L),v,-l Replication. \V H. Frrrman and (‘ompany, Xew 1’ork. Kowalski. J).. Satale. I). & Eddy. M. ,J. (1988). Stable IJX.4 unwinding. not “breathing” accounts for single-strand-spe~itir nucleasr hypersensit’ivity of sl)Gfic> At T-rich sequences. Proc. SC/~. Acad. Sri., l’..S.d. 85. 9464-9468. Kreuzrr. K. S. & Nbrrts. IS. 11. (1985). A defective phagr systrm rrvrals bacteriophage T4 replication origins that coincidtl with recombination hot spots. Proc. .Vot. A rod. A’ci., I’.LJ.A 82, 3345-3349. Krrrrzrr. K. N.. d .ilherts. K. 11. (1986). Characterization of a clefbc~tivc~ phagr system for thr analysis of hac%rriophagtL T4 DSA replication origins. J. Mol. lfiol. 188. I X5-- 19X. Kreuzrr. K. 9. ~1:Menkens. A. E. (1987). Plasmid model systrms for the init,iation of bacteriophage T4 I>r\‘A rrplicaation. In /ISA Replication and Recombination (Mc~Mac~ken. R. Xr Kelly, T. J., eds). pp. 451-473. Alan Ii. I&s Inc... X1-. Krruzer. K. S.. Engman. H. W. &, Yap, \V. Y. (1988). Trlrtiary initiation of rr+ication in bacteriophage T4. I)rletion of thr overlapping ~1~sl- promoter; rthplication origin front the phage genomr. .I. Hiol. (‘hem. 263. 11348-l 1357. Kuttrr. E.. (:uttnran. B., Mosig, C. & Riiger. \I;. (1990). (:rnomic map of hactrriophage T4. In Genetic Maps (O’fSrien. S. *J.. ed.). pp. 1.24-I .51. Cold Spring Harhor Press. Cold Spring Harbor. h’s’. Laemmli. Cr. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. .Vutnw

(Lovvdon).

227.

680-685.

T,ernbacah. K. .J.. Kuninaka. A. & Buchanan. .J. M. (1969). The relationship of DXA replication to t,he control of protein synthesis in protoplasts of T4-infected h’srherirhio roli I{. t’ror. Nat. Acad. Aci., I’.S.A. 62, -c-k6-453.

in

T4 Replication

99

Liebig. H.-D. B Riiger, W. (1989). Bacteriophage T4 early promoter regions. Consensus sequences of promoters and rihosome-binding sites. J. .Zfol. Biol. 208. 517-836. T,uder. 4. & Mosig. Q. (1982). Two alternat’ive mt~chanisms for initiation of I>XA replication forks in bacteriophage T4: Priming by RNA polymrrasr and by recornhination. Proc. Nut. :lcd. Sci.. C’.S.A. 79. 1101-1105. Rlacdonald. 1’. &I. & Mosig, G. (1984). Regulation of a ne\\ bacteriophage T4 gene, 6.9. that spans an origin of replication. EMBCJ J. 3. 2863-2871. Macdonald, P. M., Seahy. R. M.. Brown. W. CyrMosig, U. (1983). Initiator D8A from a primary origin and induction of a secondary origin of hact)rriophage T4 1)S.A rf,plication. In ~Wicrohioloyy~ 1983 (Schlessinger. I).. rd.). pp. 1 I I- 116. American Society for Jlicrobiology. Washington. IN’. Mathews, C’. K.. Kutter. E. M., Mosig. (:. $ Herget. 1’. B. (1983). Bacteriophage 1’4. American Society for Microbiology, 1Vashington. IX”. Mat,tson. T.. Ric.hardson. *I. & Coodin. I). (1974). Mut’ant of hactrriol)hage T41) affecting expression of many 250, 18-50. early genes. Snture (London). Mattson. T.. \-an Houwr. (:. & Epstrin. R H. (1978). Isolation and charactrrizatjion of conditional lrthal mutations in the mot gene of bactrriophagr T4. ture and In The Bnrteriophages ((‘alendar, FL. mrtaholism. etl.). vol. r’. pp. .52f -606. Plrnum l’rt’ss. SW York. Xc~lson. M. .i.. Erirson, &I.. (iold. T,. & Pulitzer. .J. F. (19X2). The isolation and charac+rization of TabR bacteria: hosts that restrict bacteriophage T4 rTT mutants. Mol. C&n. Oenat. 188, 60 -68. O’Farrell. I’. %. & Gold. I,. hl. (1973). Bactrriophagr ‘I’4 gene exprrssion. Evidence for tuo cslasses of prt’replicative cistrons. .J. Bid ( ‘hem. 248. ,5.50”- 5.51 I Oliphant. .-\. R.. Xusshaum. ,A. I,. 8 Struhl. K. (1986). (‘loning of random-sequence oligodeoxvrruc~leotitles. (knc. 44. 177-183. Pulitz;rr. .I. F., (‘oppo. -4, 8: (laruso. M. (1979). Host,-virus interactions in the control of T4 prereplicativr transcription. II. Interaction hetwrtan tuti’ (rho) mutants and T4 mot mutants. ./. No/. Biol. 135, 979

~9!)7.

Pulitzer. .J. F.. (‘olumho, M. dt (‘iaramrlla, >I. (198.5). ?rje, control elrrnrnb of hactrriophage T4 pre-replicativr transcaription. J. Mol. Biol. 182. 249-m”6:3. Rahussay. D. (1983). Phage-evoked changes in RNA polymerasr. In Bacteriophage T4 (Mathews, (‘. K.. Kutter. E. JI., Mosig. G. 8r. Krrget. P I%.. rds). pp.

I67 15:1. .\tnrric.att Swirt>for Yfic~robiologj \Vashington, IX’. Riva. S.. (‘a .scLino.A. & (:ricfuwhrk, E. f’. (1970). C’ouf)lit~g of’ late transcription to viral 1)X,-\ rrplication tn tttwtrriofthage ‘I’d drvelopmrnt. ./. Mol. Hid. 54. ST,- IO:‘. Schmidt. 1~. f’. Kr Krruzer. K. S. (1992). f’urfied 1lot.d f)rt&in binds tht> -30 region of a bactwiophagc Td tniddlr-tnodr promoter and activates t~ranwription in vitro. ./. Rid. (‘hem. 267. 11399-f 1407. Schulz. W’. X: Zillig. \I’. (1981). Rif’ampicin inhibiti of RSA synthesis by dextabilisation of f>N:\-RSA ftof~tnerase-oligon~t~l~otidr-c:omplrxes. Strcl. .-Icifls Krs. 9. 6889-6906. Slick. H. E., Kreuzer. K. iX. & Albrrts. K. 41. (1988). Thv vt’c’tor bac%eriophagr T+ insertionjsubstitution site-spwific systrm. A mvthotf for intjroducing tnutations into thr virus c~hromosome. .J. Hid. (‘hrm. 263. 1133li-I 1347. Shittrtifing. S.. Tamaralayne LValker. T,. 8i (:old, I,. (19%). (‘lotring thr c,otufwte ~1113genv of bacteriophagc~ T-C

van Hipfwl. f’. f-1 Kowalt~zykowski. S. C’.. f,onbrrg:. N Sewport, .J. \V.. Paul. 1,. S.. Stormo. (:. I). & (:old. Ia. (1983). Autoregulation of ~xpwssion of T-C c(~nc’Z?: ii quantitative analysis. In Kncf~riophqz 7’4 (Mathrws. (‘. K.. Kutter, E. 1I.. Mosig. (:. & Retget. I’ I< ds), f)p. ZO:! 207. Anit~rican Swietv for Micrcil)iology. \Vashington. IN’. Yrr. ,J. K. & kfarsh. Ii. (‘. (l!M). f,oc,atiotts ot bwtrriophqq T1 origins of’ replicatiott. .I l’irol. 54. “7l-“77. %aug. A. ,I.. Krnt. .I. I<. & C’wh, T. K. (1984). .I lal)ilt~ f)t”)sJ)hodirstt,r bond at the ligation junctiott itt a cirwlar itrtrrvcning sryurnc~r~ RX&Z. Scicjtwr. 224, 571

57X.