DNA replication studies with coliphage 186

J. Mol. Biol. (1989) 206. 59-68

DNA Replication Studies with Coliphage 186 II?. Depression of Host Replication by a 186 Gene Helena Richardson Scripps Clinic and Medical Foundation La Jolla, GA 92037, P.S.A.

and J. Barry Egan Department of Biochemistry University of Adelaide Adelaide, South Australia 5001, Australia (Received 30 March 1988, and in revised form 21 October 1988) IJsing pre-labelling rather than pulse-labelling studies to determine rates of replication, we have shown that coliphage 186 infection is accompanied by a depression in host DNA replication. We have isolated mutants of the phage gene involved and mapped them in the early region of the phage genome. Sequencing the mutants ultimately led us to the identification of the gene that we have named the dhr gene.

1. Introduction

primer (Gilbert & Dressler, 1968), whereas the postulated role for DnaA in E. coli replication associates it with the RNA primer required for initiation of replication (Fuller & Kornberg, 1983; Lindahl & Lindahl, 1984). A phage replication gene has been described (Hocking & Egan, 1982b) and, by parallel with the related phage P2 (Geisselsoder, 1976; Chattoraj, 1978), it is presumed to encode an endonuclease that nicks the DNA for the start of rolling circle replication. The subject of this paper is a phage function involved in the interaction of 186 with host replication. Hocking & Egan (1982b) reported, from pulse-labelling studies, that’ bacterial DNA

Coliphage 186 is a temperate phage, with the rare property that it reyuires the host DNA replication initiation functions DnaA and DnaC for infection (Hooper & Egan, 1981). Coliphage 186 shows a further rare property, in that its replication is transiently delayed when the phage infects a host cell that has been u.v.$-irradiated (Hooper et al., 1981). Interrelating these two properties led to the prediction that U.V. irradiation inhibits initiation of Escheriehia coli replication, which has now been confirmed with experimental data (Verma, 1985; Verma et al.. 1989). These facts have increased our interest in characterizing how U.V. irradiation inhibits initiation of E. coli replication and 186 replication itself. and its interaction wit’h E. coli functions and replication. Little is known about 186 replication. A direct involvement of DnaA in 186 replication is yet to be established, but its requirement for a successful infection is intriguing because 186 replicates by a rolling circle mechanism (Chattoraj & Tnman, 1973), a mechanism

that

classically

synthesis

induction. labelling

is inhibited

In

this

studies

soon

work,

that

after

186

we confirm

host

replication

prophage

with

pre-

is indeed

depressed, and t,hat this depression is due to the expression of an early phage gene, the dhr gene, but that this function, not, essential.

needs no RNA

while

2. Materials

of value

to the phage,

is

and Methods

(a) Bacterial strains t Paper I in this series is Hooper & Egan (1981). 1 Abbreviations used: u.v.. ultraviolet light; kb, lo3 bases; r.f.u., colony-forming units; moa, multiplicity of addition.

All bact,erial strains used were derivatives of E. coli K-12. The Su+(supE) strain C600 (Appelyard, 1954) was used for phage assays. The Su- strains E251 (Hocking & Egan, 19826) and 594 (Weigle, 1966) are StrR derivatives 59

0

1989 Academic

Press Limited

60

H. Michardson

of W3350. E251 thyA thytl (low-thymine requiring strain) used in the pre-labelling studies was constructed by trimethoprim selection (Miller, 1972). 594 $Awas constructed by I’1 transduction of aji: :Tn5 from the strain GC4540 (D’Ari & Huisman, 1983), and 594 recAwas constructed by transduction of recA56 srl300 : : TnlU from the strain JC10240 (Csonka & Clark, 1980). 186 int lysogens were constructed by using 186 ~110 as a helper phage to supply the Int function needed for integration. AH1 is the designation for the K-12 strain M72 StrR la&am Abio-uvrl? AtrpEAZ (1Nam7NamS3 cIts857). AH1 deletes i genes cro-tl-A-J-b8. The strain M72 was used by Remaut et al. (1981) to provide temperature sensitive repression for their expression vector pPLc236. (b) Phage strains All 186 bacteriophage used carried the cItsp allele (Baldwin et al., 1966; Woods & Egan, 1974). 186 Aamll and 186 dell have been described (Hocking & Egan, 1982a; Finnegan & Egan, 1981). 186 Aamll int was constructed by re-section in vitro of the 4.4 kb XhoIRgZII fragment from the Intphage 186 cItsp ins3 (Bradley et al., 1975), containing an IS3 element in the int gene (Younghusband et al.; 1975; Saint, 1979). into 186 cItsp Aamll at the unique XhoT (67.6%) and BgETT (79.6%) sites (Finnegan & Egan, 1979). 186 CP77am and 186 CP78am were constructed as follows: the PstT-BgZII (77.4% to 79.6%) fragments from the CP77am and CP78am derivatives of mEC401 (see below) were recombined with the Xho-PstI (67.60/; to 77.4%) fragment from 186 cItsp and each resulting XhoI-BgZII fragment was ligated into 186 cItsp using these unique sites (Finnegan & Egan. 1979) to form 186 cTtspCP77amn and 186 cItspCP7llam. These phage were shown to carry their respective CP77am and CP78am mutations by plaque hybridization with the oligonucleotides (now radioactively labelled) used for their creation. 186 de13 is a mutant of 186 cItsp carrying a deletion of the 0.25 kb HaeIII-H&II (77.8% to 78.7%) fragment (sequence coordinates 583-830, Fig. 6). This deletion removes the 3 end of the CP77 gene (54 amino acid residues), and the 5’ end of the CP78 gene (33 amino acid residues). The 5’ end of the CP77 gene (22 amino acid residues) is fused out, of frame to the remainder of CP78 and from sequence translation terminates 60 bases earlier than CP78. mEC401 is an M13mp8 clone containing the r-strand of the 0.69 kb PdI-Bg1IT fragment (77.4% to 79.6%) from 186 dell cloned into the PstI--BamHI sites of M13mp8. (c) Plasmids The plasmid pKC7 is a derivative of pBR322 (Rao & Rogers, 1979). pPLc236 is a plasmid containing the bacteriophage 1 promoter pL for the cloning and controlled expression of genes in hosts encoding the J. ~I857 gene (Remaut et al., 1981). ~~1857 is a plasmid derived from pACYC177, and therefore compatible with ColEl-derived plasmids, which encodes the 1cIts857 gene (Remaut et al., 1983). pEC400 contains the 1.78 kb XhoIBglII (67.6% to 79.6%) fragment from 186 dell, cloned into the XhoI and BgZII sites of pKC7. This clone encodes the 186 early lytir genes, including a lethal function under 186 c1 control. pEC401 (pEC402. pEC403) is pEC400 containing the Let1 (Let2, Let3) mutation which allows cell survival in a non-lysogen. pEC404 is pPLc236 containing the 0.4 kb P&-H&II (77.4% to 78.7%) fragment from 186 cIt.sp in the orientation such that rightward genes are expressed from IpL. The clone was

and J. H. &an obtained by replacing the I.58 kh BamHI- /‘vu11 fragment, from pPLc236 wit,h the 0.4 kh Kant HI (in tht> cloning site of pPLc236 DNA) to HincIT (in t.he 186 cloned DIVA) fragment from pEC503. This clone rnrodrs the CP77 gene. pEC420 is pPLc236 containing the 0.53 kb SaaITTA~BglII (77.9oi, to 79.6:&) fragment, from CP78am DNA in t,he orientation such that rightward genes are expressed from ApI,. The clone was obtained (as described for pEC421) from the CP~XU.WLderivative of mEC401. This clone encodes t,he (‘P7Nam gene. pE(J421 is pPLc236 containing the 0.53 kb RauIIIA- 11glIT (77.9”,,, to 79.6%) fragment’ derived from wild-type 186 in the orient’ation such that rightward genes are expressed from 2 pL. The clone was obtained hy isolating the SauTIIAHgZIT fragment from mEC4OI after digestion with SuuIIIA, and ligating this fragment into the BarnHI site of pPLc236. This clone encodes the (IP7X gene. pEC422 ix pPLc236 containing the 0.4 kh Pstl-Him11 (77,4y;, to 78.7Oh) fragment from (‘P77am, DNA in the orientation such that rightward genes are expressed from i pL. The clone was obtained by replacing the 0.6 kh NindIII #ruI fragment from pPLc236 with the 0.4 kb HindITTHincII fragment frotn the (‘P77am derivative of mEC401 (the Hind111 site is in the rloning sit’e of Ml3). This clone encodes the CP77am gene. pEC503 contains thr 0.69 kh f%-BglII (77.49; to 79.6%) fragment isolat)ed from pEC400. end-filled and cloned into the end-filled Hind111 site of pPT,c236 in the orientation sucah that rightward genes are expressed from 1 pL (A. Puspurs. this laboratory). This clone encodes the CP71 and (‘1’7ri genes. (d) Other materials All media and solutions used have heen described (Hocking & Egan, 1982a). Restrict,ion enzymes were obtained from Bethesda Research Laboratories, and phage T4 DNA ligase was obtained from HoehringerMannheim. (e) DNA synthesis

measurewbents

Pulse-labelling and pre-labelling experiments carried out as described in the Figure legends.

were

(f) Site-directed mutagenesis Oligonucleotide site-directed mutagenesis, to form the amber mutants of CP77 and CP78, was performed by the method of Zoller & Smith (1983) on the PstI-BglIT (77.4%) to 79.6%) region cloned into Ml3 (mEC401). Synthetic nucleotides were as follows: (‘P7i’ amber. 5’.CGCCGAAAUTCAGGT-3’ (sequence co-ordinates 669-685). TAG replaces TCG. CPIX amber, 5’.GAATTGTTWGGTGCC-3’ (sequence co-ordinates 751.~767). TAG replaces TTG. Screening for mutant phage was performed by in .situ hybridization (Benton & Davis. 1977) using each of the 3’P-labelled mutagenesis oligonucleotides. Plaques that hybridized strongly relative to parental controls, following high-temperature washes in 3 M-tetramethyl ammonium chloride (Wood et al., 1985), were picked and single strand Ml3 preparations sequenced with the Ml3 universal sequencing primer to confirm the mutant genotype. (g) Other methods Bacterial and phage assays, phage stock preparations, and nitrosoguanidine mutagenesis of bacteria were

61

The dhr Gene of Coliphage 186 carried out as described by Hocking & Egan (198%). Nitrosoguanidine mutagenesis of plasmids was carried out by plasmid isolation from mutagenized host cells and transformation of 594 for the detection of plasmid mutants. DNA sequencing was by the dideoxy chain terminating method of Sanger et al. (1977).

3. Results (a) The Dhr Effect Figure l(a) (in part) records a repetition of the Hocking b Egan (19823) experiment and is presented here to aid definition of the Dhr Effect. It shows that, after heat induction of a 186 cItsp lysogen, the rate of DNA synthesis, as monitored by pulse-labelling with [3H]thymidine, was depressed. The depression began approximately five minutes after the temperature shift and the rate continued to fall to a level about 60% the initial value at 25 minutes before rising again and passing through a maximum at 35 minutes due, in large part, to DNA synthesis of the 186 phage (Hooper & Egan, 1981; Hocking & Egan, 19823). The same depression in rate was seen (Fig. l(a)) after heat induction of a culture lysogenic for the phage replication mutant 186 cItsp Aaml 1, and the rate remained depressed with no peak at 35 minutes, although DNA synthesis did gradually increase from the 45th minute. This apparent reduction in

the rate of host DNA synthesis was referred to as the Dhr (depression of host replication) Effect, a term we later show more accurately described the phenomenon than the acronymn Dho (DNA host off) we originally used (Hocking & Egan, 1982b). The increase in DNA synthesis seen at 45 minutes after induction of the Aam mutant corresponded in time with the appearance in the culture of nonlysogenic cells, which presumably arose by segregation after prophage excision of a phage template that could not itself replicate. We therefore suspect that this gradual increase reflected the replication of non-lysogenie cells rather than a true recovery of DNA synthesis with time. (b) Isolation of a presumptive dhr ,mutant A possible rationale for the isolation of a dhr mutant, a phage mutant unable to depress host replication, arose from the work of Finnegan (1979). From the behaviour of different clones in transformation, she postulated that 186 encoded a lethal gene under c1 repressor control that mapped between the PstI site at 77.4% and the BgZII site at 79.6% (Fig. 2). As the dhr gene might well be expected to behave as a lethal gene when cloned, and the timing of the Dhr Effect suggested that the gene involved was an early gene, we entertained the possibility that the lethal gene and the dhr gene

0 Time after heat inductnn (a)

(mln)

20

40

Time after Infection

60 (mm)

(b)

Figure 1. Pulse-label incorporation of [3H]thymidine. (a) Heat induction of Su- lysogens of phage 186 cItsp and 186 cItsp Aamll. Overnight cultures, grown at 30°C in TPG-CAA medium, were diluted into the same broth and incubated with aeration at 30°C to a density of 2 x 10’ colony-forming units (c.f.u.)/ml. Cultures were transferred to 40°C at 0 min, incubation with aeration continued, and samples (200 pl), taken every 5 min, were added to 50 ~1 of prewarmed [3H]thymidine in TPA-CAA (final concn 20 &i/ml) and incubated without aeration for 2 min at 4OO”C.Then lOO+I sample was withdrawn, placed on a GF/A filter, and the pulse terminated by immersing the filter in ice-cold 10% (w/v) trichloroacetic acid. The filters were bulk washed 4 times in ice-cold 10% trichloroacetic acid, twice in ethanol, and dried overnight at 65°C. Radioactivity was assayed using a Packard scintillation spectrometer after adding toluene scintillation fluid to the filters. Symbols: (A) E251 (186 cItq A’); (e) E251 (186 cItsp Aamll); (m) E251 (b) Infect.ion of Su- bacteria with phage 186 dell Aamll Let+ and 186 dell Aamll Letl. An overnight culture of E251, grown at 39°C in TPG-CAA medium, was diluted into the same broth and incubated with aeration at 39°C to a density of 2 x lo8 c.f.u./ml. At time zero, cells were infected at a multiplicity of addition (mou) of 20 (with CsCl-prepared phage stocks) and samples taken with time were pulse-labelled and processed as above. Phage 186 is slow to absorb (50% in 30 min at 37°C) and a high 71u)ais necessary in order to have every cell infected within the first 5 min of the experiment. Symbols: (0) 186 dell Aamll Let+-infected cells; (A) 186 de21 Aaml I Letl-infected cells; (m) E251 uninfected control. a

62

H. Richardson and J. H. h’gan EL Es E

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Figure 2. Physical genetic map of phage 186. The functions of the genes (Hocking & Egan, 1982a) and the physical mapping (Finnegan & Egan, 1979) have been described. The lower Figure is an expanded view of the Xho-BgZII region (Richardson et aE.,1989) showing genes CP69, CP75, CP76, CP77, CP78. int and CT,and the early lytic transcript from pR. and the lysogenic transcript from pL. were the same gene. Consistent with the prediction by Finnegan (1979), the XhoI-BgZII (67.6% to 79.6%) fragment from the c1 deletion phage dell (Kalionis et al., 1986), when cloned into the plasmid vector pKC7 (clone pEC400), could transform cells only if they were lysogenic (7 x 10’ transformants/ pg DNA for lysogenic recipients versus < 10 non-lysogenic transformants/pg for DNA recipients). We next determined that pEC400 encoded the dhr gene by showing an enhanced and extended Dhr Effect for ‘lysogenic cells induced by a temperature shift when the cells carried pEC400 with an (data not shown), results compatible increased dhr gene dosage. We therefore concluded that it was quite possible that the lethal gene was in fact the dhr gene and sought to isolate non-lethal mutants of the plasmid as presumptive dhr mutants. As stated above, pEC400 was unable to transform non-lysogenic recipients. However, after in vivo mutagenesis of pEC400 DNA with nitrosoguanidine, it was found that the treated DNA gave rare transformants among the non-lysogenic recipient cells, indicative of potential mutations in the lethal gene. Two of these transformants were purified through single colony isolations, and the plasmids carried (pEC401 and pEC402) were confirmed (1) to possess the XhoI-BgZII fragment by restriction analysis and (2) to display a nonlethal phenotype by their ability to transform both non-lysogenic and lysogenic cells at equal efficiency (7 x lo5 transformants/pg DNA). The mutations carried by these plasmids were referred to as the Let1 and Let2 mutations, respectively. The next step was to test for the Dhr phenotype. The XhoI-BgZII (1.75 kb) fragment was recovered from pEC401 and resected into 186 dtsp Aamll DNA via its unique XhoI-BgZII sites. The Aam, mutant background was used in order to minimize the contribution of phage DNA synthesis in the experiment. The Let- recombinant phage, 186 dell Aamll Letl, was recovered as a plaque after

transfection, and tested for its Dhr phenotype in pulse-labelling studies with [3H]thymidine after phage infection, As a control, the same restriction fragment from pEC400 was resected into 186 deZ1 Aamll to give the appropriate Let+ phage (186 dell Aamll Let+). The results in Figure l(b) record the fact that the rate of DNA synthesis in the Let’infected culture, compared with that of the uninfected culture, dropped soon after infection, then remained constant until the 45th minute, when it, again increased at a rate of increase that paralleled that of the uninfected culture. In contrast, no Dhr ESfect was seen after infection by the Let1 phage, as the rate of DNA synthesis increase essentially followed that of the uninfected culture. To test that the dhr mutation was located between the PstI (77.4%) and BgZII (79.6%) sites, as suggested by Finnegan (1979), this fragment was recovered from the Let1 phage mutant, recombined in vitro with the XhoI-PstI (67.6% to 77.4%) fragment from a Let,+ phage, and the XhoI-BgZII fragment formed then resected into 186 DNA via these unique restriction sites. The resulting recombinant phage was not able to display the Dhr Effect (data not shown). The Pstl-RgZII fragment from pEC402 was similarly resected into 186 cItsp Aamll DNA to give the recombinant phage 186 dtsp Aamll Let2, which was also found to be unable to display the Dhr E@ect (data not shown). The likelihood then was that the lethal gene and the dhr gene were the same gene encoded in the 77.4% to 79.6% region but, until genetic analysis is presented in a later section, we will continue to refer to the mutants as Let mutants to emphasize the nature of their isolation. (c) The eSfectof the Dhr function

on the host

(i) DNA replication The Dhr Eflect as demonstrated by pulse-labelling studies might not be a true depression of host replication but might reflect some restriction in

The dhr Gene of Coliphage 186 entry of the labelled thymidine into the intercellular DNA precursor pool. To avoid this possibility, pre-labelling studies were pursued. With pre-labelling, the radioactive thymine is added many generations before infection such that the host DNA and precursor pool will essentially maintain a constant specific activity throughout the infection regardless of perturbations. After prelabelling Su- thy cells with [‘Hlthymine for three generations, the cultures were infected with either 186 dell Aamll Let+ or 186 dell Aamll Let1 phage and DNA synthesis monitored. Figure 3(a)

,I0 ,5

. x

7-i-

f r” II

I-

x,(I ,’ Time

,-

*t+*r

mtection

(min)

I,tr I IL

(b) I -60

,I

I 0 Time

I 60 after

II‘1

I1 I20

temperature

180 shift

I1 240

300

(Inin)

Figure 3. Accumulated incorporation of [3H]thymine. (a) Infection of Su- thyA thyR bacteria with phage 186 dell Aamll Let+ and 186 dell Aamll Letl. Overnight cultures, of E251 thyA thyR, grown at 39°C in TPG-CAA + thymine (2 pg/ml) were diluted into the same medium containing [3H]thymine (4 pCi/ml). The cultures were grown at 39°C until they had reached a density of 2 x lo* c.f.u./ml and then were infected (0 min) at a moo of 20 (with CsCl-prepared phage stocks). Samples (100 ~1) were taken with time and treated as described for Fig. l(a). Symbols: ( x ) 186 dell Aamll I.&+-infected cells; (0) 186 dell Aamll I&l-infected cells; (0) uninfected cells. (b) Heat induction of Su- thyA thyR (186 cItsp Aamll int Let+). Overnight cultures, grown at 30°C in TPG-CAA + thymine (2 pgglml), were diluted into the same medium

containing [jH]thymine

(4 &i/ml) and incubated at 30°C

to a density of 2 x lo* c.f.u./ml. The cultures were then transferred to 40°C (0 min) and incubation with aeration continued. At 105 min after induction, the cultures were diluted lo-fold (1) into pre-warmed TPG-CAA + thymine (2 pg/ml) + [jH]thymine (4 &i/ml) and incubation continued. Samples (100 ~1) were taken with time and treated as described for Fig. l(a). Symbols: ( x ) E251 thyA thyR (186 cItq Aumll int Let+) lysogen; (0) E251 thyA thyR. The lines drawn before and after the temperature shift represent the lines of best fit, as established by linear regression analysis.

63

shows that the rate of DNA synthesis in Letlinfected cells was approximately the same as the uninfected control, whereas that in Let’ infected cells was reduced. Although not shown in Figure 3(a), the repression of replication did not persist but, after some 40 minutes, the rate increased in parallel with the control rate much the same as seen in Figure 1(b). This increase probably reflected replicating phagefree cells in the infected culture that arose due to unilinear inheritance of the non-replicating phage chromosome. Whatever the reason, the pattern made it difficult to quantify the depression confidently. To extend the depression and enable some measurement of its magnitude, we heatinduced a cIts Int- lysogen. The Int function is required by 186 for prophage excision (unpublished results); thus, with an int lysogen, the prophage is unable to excise from the host chromosome and, when depressed, the dhr gene will be expected to be expressed in all cells indefinitely. The prophage carried a mutation in the replication gene A to eliminate phage replication contributing to the counts incorporated. A thy lysogen of 186 cIts int Aum Let+ was pre-labelled with [3H]thymine for three generations, heat-induced, and the rate of replication assayed and compared with that of the non-lysogenic control. The results are recorded in Figure 3(b), and represent the lines of best fit as established by linear regression analysis. In the non-lysogen control, the rate of DNA synthesis increased as expected with the change in temperature, giving a generation time, as calculated from the slope, of 40 minutes. In contrast, the replication rate of the lysogenic culture remained essentially unchanged with the change in temperature,

giving

a calculated

generation

time of

59 minutes. The depression in rate of DNA synthesis in the presence of activated prophage, and the results in Figure 3(a) showing that this ability to depress host replication was lost with the Let- phage, were not results that could be explained by variations in the precursor pool composition and, taken together, lead to the conclusion that the phage encodes a function that acts to depress host DNA replication. (ii) Cell division

During experiments investigating the lethality of the Dhr function encoded on a plasmid, we observed that the change in the viable count of a culture was more sensitive to Let’ expression than was the change in optical density. In the experiment, the expression of the Let+ plasmid pEC400 was controlled by a prophage encoding a temperature-sensitive mutant repressor. After depression by a shift in temperature, the viable count and optical density were followed with time. It can be seen in Figure 4 that, whereas the optical density continued to increase until the 180th minute and then gradually decreased, the viable count dropped

rapidly

from the 30th minute

and by

240 minutes was reduced to 0.03% the number of

64

H. Richardson

i ’ /

and J. B. E’gan

/. /.

\ I t \ I)il”“” -120

0

120 Tme (a)

240 after

,I

L 3 60 heat

0 inductm

. I,,

120

240

I

36C

(min)

(a) (b)

Figure 4. Relative

cell mass and viable count after heat induction of Su- 186 lysogen carrying the Let+ plasmid pEC400. Overnight cultures were diluted into Luria broth and incubated with aeration at 30°C to a density of 2 x lo8 c.f.u./ml. Cultures were transferred to 41.5”C at 0 min and incubation continued. At 60 min after induction, the cultures were diluted l/l00 (7) into pre-warmed Luria broth and incubation continued. (a) Absorbance at 600 nm was followed for 180 min before heat induction, and for 360 min afterwards.

(b)

Figure 5. Phase contrast micrographs of the Sue (186) lysogen containing the Let+ plasmid pEC400. 594 (186 cItsp Aamll Let+), pEC400 was grown at 30°C in Luria broth to 2 x 10s c.f.u./ml then heat-induced at 42°C’ with aeration for 4 h. A control culture was grown for 4 h at, 30°C. Cells were then viewed in the light microscope under phase contrast at a magnification of 400 x (a) 594 (186 cItsp Aamll Let+), pEC400, grown at 30°C. (b) 594 (186 cItsp Aamll Let+), pEC400. 4 h after heat induction at 42 “C.

(b) The viable count, as c.f.u./ml at 3O”C, was determined with time. Symbols: (A) 594 (186 cItsy, Aamll pEC400; (a) 594 (186 cItsp Aamll Let+), (0) E251, pKC7.

Let+), pKC7;

cells at time zero. This disparity between the changes with time in optical density and viability suggested to us an interference with cell division and indeed filamentation was observed when the culture was viewed in the microscope under phase contrast (Fig. 5). Thus the lysogen carrying pEC400 four hours after heat induction showed filaments 20 to 50 times the length of normal cells (Fig. 5(b)), whereas the uninduced lysogen carrying pEC400 showed normal cells with a few filaments (Fig. 5(a)). Induction of a lysogen carrying the Let1 plasmid pEC401 also gave normal cells and no filaments (data not shown). Filamentation was therefore part of the Let phenotype and might well be associated with the Dhr function. We made another interesting observation during lethality studies on cells lysogenic for the nonexcisable prophage 186 int. After prophage induction we had expected cells to die, due to in situ replication of the non-excised prophage within the host chromosome. Indeed, the viable count of the 186 cIts int lysogen after overnight incubation of the inoculated plates at 41*5”C was 10e6 (compared with 30°C). However, we found that this lethality could be accounted for entirely by the Let phenotype as the 186 cIts int Let1 lysogen showed 100°?&survival under the s&tie con-ditions. Survival was lo-’ in the case of the replication-defective 186 cIts int Aamll Let + lysogen. It therefore appeared that excess Let function(s) resulting from the

increased gene dosage was the reason for lethality, and not in situ replication, when a 186 prophage unable to excise was induced. (d) Identifying

the dhr gene

In the accompanying paper (Richardson et al., 1989), we report the sequence of the P&I-BgZII (77.4% to 79.6%) region of 186. As we had mapped the Let mutation to this region, sequencing of the DNA of the Let mutant was expected to locate the dhr gene. The region encodes the carboxy terminus of the ~11 gene, genes CP77 and CP78 of unknown function and therefore candidates for the dhr gene, and the amino terminus of gene CP79. The DNA sequence of the PstI-BglII region of the Let1 mutant was determined (Fig. 6) but was found to differ from the wild-type sequence at three positions, each involving a C to T base change; namely, at base 551 in CP77 to give a missense mutant of Leu to Phe, at base 834 in CP78 creating another missense mutant of Pro to Ser, and at base 1105 in CP79, to give a third missense mutant of Ala to Val (not shown in Fig. 6). To resolve the dilemma in the identification of which of the three genes was the dhr gene, the PatI-BglII region of a second mutant (Let2) was sequenced but this too was found (Fig. 6) to contain a mutation both in CP77 (a G to an A base change at base 619 resulting in a Trp to opal nonsense mutant) and CP78 (a G to an A base change at position 790 resulting in a Gly to Asp change). A third mutant, Let3, was then sequenced (Fig. 6) but it proved to have the insertion element IS1 (Johnsrud, 1979)

65

The dhr Gene of Coliphage 186

CIGASFGLI* GGTAlTGGCGCATC~ CE 490

NLKSEPSFASLLVKPSPGHHYGHGUIAG CTGATTTGA-A-~~CA~AA 500 510 520 530 540

T Let1

CP77 KDGKRUHPCRSQSELLKGLKIKSPKSSGFLIlRIVHFVIK TMGGACGGCMGCGCIGGCACCCGTGCM;CTCACAGTCCGM 610 A 630 640 L&Z

o-77

GVKHVTR* NSRDELRIVLGANIPNHEEGFEIKIR-DGAILRVDPE AGGAG7CAM(3AIGIUCGC(~~M~~~~~~ 730 740 750 A CP78 CP78am

_

CP78

650

770

660

6M

-A-n--m= 560

A CP77am

HPUII ?i=S%

690

580 Hoem

700

lTCCMATATGGAGGAA~TTAAAACCCGCGACGGCGCMTACTTCGC~GT 780 A 800 810 820 Let.2

YECCKEFKDGLKAEIIKQLKSKPAVVFGYS* GGGAcTGCTGC~GMT77M~TffiA~~~~~~~~~~~~~A~~TATA~M~M~~C~M~A~~~ 850 860 870 880 890

900

910

920

930

940

SOuDdtA 77.9%

720

ISf L&3

HindI 78.7%

T L&l

950

840

960

I

Figure 6. The sequence of CP77 and CP78 mutations. The sequence of CP77 and CP78 is reproduced from the accompanying paper (Richardson et al., 1989). The base-pair changes associated with the Letl, Let2, CP77am and CP78am mutations, are as shown and discussed in the text. The Let3 mutation resulted from an IS1 insertion, underlined is the 9 base sequence repeated as a result of the insertion.

inserted at position 711 in CP77. IS1 is extremely polar in both orientations (Besemer, 1977), so the dilemma of which was the dhr gene remained. The phenotype of clones of CP77 and CP78 in the expression vector pPLc236 (Remaut et al., 1981) enabled us to make the dhr assignment. The expression of a clone of CP78, pEC421, was lethal to the host and resulted in a depression of E. coli

DRiA replication, while the CP77 clone, pEC494, was not lethal and failed to depress host DNA replication when expressed (Fig. 7). An amber mutant of CP78 (Fig. 6) was constructed by oligonucleotide mutagenesis (see Materials and Methods) to yield the CP78am clone pEC420. This was unable to depress host replication. Therefore, CP78 was identified as the dhr gene. Cell filamentation was also described as part of the Let phenotype but we found that the CP78 clone when expressed did not generate filaments (Fig. S), whereas the CP77 clone did. This ability was lost with the clone of the amber mutant of

pPLc236

I

I 30

I 90

I 60 Time

after

pEC404

(CP77)

EC421

(CP78)

, 120 heat

induction

I I50

I 160 (min)

Figure 7. Effect of CP77 and CP78 gene products on DNA synthesis. Cultures of the AH1 lysogen carrying the plasmid ~~1857 together with the parent vector pPLc236, or with clones of CP77 (pEC404) or of CP78 (pEC421), were grown overnight at 30°C in TPG-CAA medium (containing the appropriate growth supplements and antibiotics), diluted into the same broth and incubated with aeration at 30°C to an absorbance at 606 nm of 0.2 (2 x 10s c.f.u./ml). Cultures were transferred tb 41.5”C at 0 min and incubation with aeration was continued. Samples (20 ~1) were taken at the indicated times and the rate of DNA replication was determined by pulselabelling with [3H]thymidine. Symbols: (0) pEC421; (m) pEC404; (A) pPLc236.

(b)

Figure 8. Cell morphology

associated with expression of CP78 (pEC421) and of CP77 (pEC404). Cultures of the AH1 lysogen carrying either the vector pPLc236, or the clones pEC404 or pEC421, were grown in L broth at 30 “C to an absorbance at 600 nm of 0.2. The cultures were diluted IO-fold into fresh media and grown for 4 h at 30°C (control) or at 42°C. Cells were photographed at a magnification of 400 x under the microscope using phase contrast optics. (a) Cells carrying the CP78 clone (pEC421) at 42°C. (b) Cells carrying the CP77 clone (pEC404) at 42°C. The cultures grown at 3O”C, and the cells carrying the vector without insert at 42”C, presented morphologies as seen in (a).

66

li. Richardson

PP77, pEC422 (see Materials and Methods). Therefore, we have named CP77 the $1 gene. We note that these two genes overlap by 14 bases and future work will be directed towards determining whether there is any significance in this arrangement with regard to the expression and function of ,fil and dhr. (e) The effect of Fil and Dhr functions 186 infection

on

The 186 Let1 and Let2 mutants have mutations in both $1 (CP77) and dhr (CP78). These mutants gave a reduced burst size (70% of wild-type), suggesting that either function was of some minor importance to 186 lytic development. However, these mutants might not be completely functiondefective and the possibility remained that either function (or both) was indeed essential in 186 infection. To assess the importance of $1 and dhr, phage carrying amber mutants in these genes were studied after resection of the mutated DNA from the respective plasmids, pEC422 and pEC420, into 186 cIts DNA (see Materials and Methods). When plated on a non-suppressing strain (E251), the 186 JiE am mutant gave plaques indistinguishable from wildtype plaques in their size and appearance. The dhr am mutant gave very small plaques, particularly at high temperatures (37 to 41.5”C), even smaller than those plaques obtained for the 186 Let1 and Let2 mutants. The degree to which the 186 $1 am and dhr am mutations affected development was 186 investigated by determining the burst size after the heat-induction of the corresponding lysogens. 186 $1 am showed a slightly reduced burst size (80 to 90%), whereas 186 dhr am gave a significantly reduced burst size (25%) as compared with the wild-type lysogen (data not shown). These results suggested that the dhr gene was important but not essential to phage infection, while the $1 gene was not important. This was confirmed by the study of 186 del3, in which the deletion created removed 54 (of 75) carboxyterminal amino acid residues of the Fil protein and 33 (of 66) amino-terminal amino acid residues of the Dhr protein. The burst size after heat induction of t’he 251 lysogen of 186 cItsp de13 was approximately 30% that of wild-type, and similar to that of 186 cTtsp dhr am.

We concluded that only the Dhr function important for 186 infection, but not essential.

was

4. Discussion Replication of host DNA is reduced soon after infection of E. coli by the bacteriophage 186. The depression in the rate of host replication amounts to about 60°% as judged by pulse-labelling, and commences about five minutes after infection (or prophage induction). The possibility existed that the drop in radioisotope incorporation seen with

and .J. H. Egan

pulse-labelling reflected restricted entry of t,hth radioisotope into the precursor pool, as found with the Hin phenotype of coliphage i (Court rt (xl.. 1980), but we concluded that, it truly reflected a depression of DNA synthesis as infection under prelabelled conditions also led to a depressed rate of radioisotope incorporation. Expression of the phage early region cloned on a multicopy plasmid caused a dramatic depression of host replication, which confirmed our expectation of which region in the chromosome would encode the property. This depression of host replication was accompanied by cell filamentation and cell lethality. We mltially considered that this may have reflected induction of the SOS functions consequent upon the inhibition of DNA synthesis (for a review, see Walker. 1987) but found t’hat cell filamentation persisted in +A and recA host (data not shown), which eliminated the possible inrolvrment of the SOS regulon (Walker, 1987). In fact, as we later discuss, cell filamentation is exhibited independently of depression of host, replication and cell lethality. The lethality shown by the cloned early region enabled non-lethal (Let-) mutants to be isolated as surviving colonies after in vivo mutagenesis of the plasmid followed by plasmid isolation and transformation of non-lysogenic cells. The association of the Dhr and Fil phenotypes with the Let phenotype was shown by their simultaneous loss in st,udies with the mutant plasmid and with the mutant, phage formed by resection of the J’stI-BglII (77.4% to 79*60/b) region into phage DNA. We therefore expected the three phenotypes to be identified with mutation of a single gene, but DNA region of the Let sequencing the PstT-BgIIT mutants in order to locate the gene presented us with mutat,ions in both (,‘P77 and (rl’i+X. f{y c*loning these genes independently into an expression vector, we were able t,o associate the function to depress host replication and lethality with (‘/‘7X. not with CP77. Cell filamentation, on the other hand, was not) a property of CP78 expression, but proved to be that of CP77. We have therefore named Cl’78 the dhr gene and CP77 the $1 gene. The assignments were confirmed by the facts that the clone of a (‘I’77 amber mutant lost only the ability for causing filamentation, while the clone of a Cl’7X amber mutant lost only the ability to depress K. roli replication. The role of these functions in the phage life-cycle is unknown. The burst size for the dhr(CP78)am mutant phage was reduced to about 25% and for the jil(CP77)am mutant phage to approximately 85%, suggesting that the Dhr function is more important to the phage infection than is the Fil function, but that neither is essential. The simultaneous loss of both functions, as seen in the de13 mutant, does not exacerbate the loss of the Dhr function. As 186 requires every host’ replication function we have tested (Hooper & Egan, 1981; and unpublished observat.ions) the Dhr function, by

The dhr Gene of Coliphage

may lessen the inhibiting E. coli replication competition from the host for some limiting components needed for 186 replication. Host DNA replication is inhibited during infection by a variety of coliphages. Inhibition occurs 10 to 15 minut#es after infection by the virulent single-stranded DNA phage 4X174 (Lindqvist) & Sinsheimer, 1967) and the A* function of the phage has been implicated as being responsible for this inhibition (Martin 6t Godson, 1976; Eisenberg & Ascarelli, 1975; Funk & hover, 1981). 1t has been suggested that this protein plays a role in the transition from the semi-conservative replicative form DNA replication to viral singlestrand DNA synthesis. Tnfection wit’h 4X174 also results in cell division inhibition (Stone, 1970). which is most likelv as a result of the inhibition of host, DNA replication by A*, since it has been shown that the expression of A* from a plasmid clone both inhibited host DNA replication and caused cell division inhibition and cell death (Colasanti & Denhardt, 1985). The virulent phage T4 inhibits host replication four minutes after infection due to the act,ivity of the ndd gene, which causes nuclear disruption by moving the host chromosome to a position closely associated with the cell membrane (Snustad & Conroy, 1974; Snustad et al., 1974, 1976). Host DNA is t)hen degraded tJen minutes after infection to supply precursors for phage DNA synthesis. In the case of the t,emperate coliphage 1. whether host replication is inhibited or not is still in doubt. Cohen & Chang (1970), from pulse-labelling and hybridization DNA-DNA studies following infections with phage conditional lethal mutants, concluded that j” depressed host’ replication but indicated in their discussion the divergent views on this point recorded in the literature. Court et al. (1980). from their induction studies of the extensively deleted (AHl) prophage that includes deletion of the cro gene, suggested that this inhibition of host replication may be more apparent than real due to the influences in pulse-labelling experiments of the 1, Hin function (encoded on the t’hat could restrict entry of PlJ transcript) exogenous thymidine into the intracellular pool. On the other hand, Georgiou et al. (1979) concluded that thymidine entry int,o the pool was not a problem (but no data shown) and described the inhibition of host replication as part of the Tro phenotype displayed by a cI cro mutant of A. The Hin study of Court et al. (1980) emphasizes the dilemma in interpretation associated with pulselabelling studies. indeed, this motivated our choice of pre-labelled cells for the present study. Coliphage A also encodes a gene kil (Greer, 1975a), on the pL transcript along with hin, the expression of which leads to cell death, apparently due to damage to the host cell envelope (Greer, 19758: Volpi et al., 1983), with inhibition of E. coli replication and filamentation as delayed secondary effects. The temperate phage Mu also encodes a kil gene, which causes cell death (Van de Putte et al.,

186

67

1977). When Mu kil is expressed from a multicopy plasmid clone, it results in cell death (GiphartGassler &’ Van de Putte, 1979) with survival kinetics similar to the expression of 186 Dhr+ from the plasmid clone pEC400. However, Mu kil does not, appear to act by inhibiting E. coli DNA synthesis or cell division (personal observation). We made an interesting observation in our original viability studies on the effect of the clone pEC400 that carries the 186 early region. Expression of this clone was controlled by the repressor present in a non-excisable (id) heatinducible lysogen. We predicted it necessary t’o avoid the use of any replication competent (A+) lysogen, because we expected lethality, due to in situ replication of the non-excisable prophage (id), would confuse interpretations. However. we subsequently found that the survival frequency after overnight incubation at 41.5”C of the 186 cItsp int A+ Let+ lysogen changed from O.OOO1~o to lOOo;, when the 186 cItsp int A+ Let1 lysogen was studied. In the case of the non-replicating mutant 186 cItsp int Aam Let+, the survival frequency was 1%. Given the association of the lethal phenotype with the dhr gene, we surmise that it is the dhr gene dosage rather than in. situ replication that is the cause of cell death in the case of 186. It is interesting to speculate that if the closely related phage P2 encodes a similar gene, as appears likely (Saha et al., 1987), then derepression of the non-excisable P2 prophage during integrative suppression would lead to cell death unless the gene is inactivated. Integrative suppression refers to the suppression of the replication defect of a host dna.4 mutant by the presence of an alternative origin of replication such as the integrated P2 prophage (Lindahl et al., 1971; Chattoraj et al., 1975). However, Lindahl et al. (1971) found that only certain mutant,s of P2, such as sig5. could integratively suppress and we now suggest that the role of a mutation represented by sig5 in suppression of the host dnaA mutant by the P2 prophage is to inactivate a dhr-like gene in P2. Our future experiments will aim to identify the mechanism by which the Dhr function depresses host replication. The immediacy of the effect suggests to us that elongation rather then initiation is depressed. Our current approach is t’o isolate host mutants resistant to the Dhr function and then map the mutation on the E. coli chromosome, with the possibility that its location might coincide with the gene of a known function and so possibly ident’ify a host, function associated with Dhr. Dhr resistant mutants, that completely eliminate t.he Dhr &feet in 186 infection, have now been isolated as survivors to overexpression from the plasmid pEC400. These mutants are being characterized and will be mapped on the bacterial chromosome in the near future. The authors appreciate the financial support of the University Postgraduate Research grant (H.R.) and of a Program grant from the Australian Research Grants

6X

H. Richardson

and thank Ioanis Anargyros for Scheme (J.B.E.), excellent technical assistance and photographic work. References Appelyard, R. K. (1954). Genetics, 39, 440-452. Baldwin, R. L., Barrand, P., Fritsch, A., Goldthwait, D. A. & Jacob, F. (1966). J. Mol. Biol. 17, 343-357. Benton, W. D. & Davis, R. W. (1977). Rcience, 196, 180-182. Besemer, J. (1977) In DNA Insertion Elements, Plasmids (Bukhari, A. J., Shapiro, J. A. & and Episomes Adhya, S. L., eds), pp. 1333135, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Bradley, C., Ong, P. L. & Egan, ,J. B. (1975). Mol. Gen. Genet. 140, 123-135. Chattoraj, D. K. (1978). Proc. Nat. Acad. Sci., U.S.A. 75, 16851689. Chat’toraj, D. K. & Inman, R. B. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 1768-1771. Chattoraj, D. K., Younghusband, H. B. & Inman. R. B. (1975). Mol. Gen. Genet. 136, 139-149. Cohen, 8. N. & Chang, A. C. Y. (1970). J. Mol. Biol. 49, 557-575. Colasanti, J. & Denhardt, D. T. (1985). J. Virol. 53, 807813. Court, D., Gottesman, M. & Gallo, M. (1980). J. Mol. Biol. 138, 715729. Csonka, L. N. & Clark, A. J. (1980). J. Bacterial. 143, 529-530. D’Ari, R. & Huisman, 0. (1983). .I. Bacterial. 156. 234250. Eisenberg, S. & Ascarelli, R. (1981). Nucl. Acids Res. 9, 1991-2002. Finnegan, J. (1979). Ph.D. thesis, University of Adelaide. Finnegan, J. 6 Egan, J. B. (1979). Mol. Gen. Genet. 172, 287-293. Finnegan, J. & Egan, J. B. (1981). J. Viral. 38, 987-995. Fuller, R. S. & Kornberg, A. (1983). Proc. Nat. Acad. Sci., C’.S.A. 80, 5817-5821. Funk, F. D. & Snover, D. (1976). J. Viral. 18, 141-150. Geisselsoder, J. (1976). J. Mol. Biol. 100, 13-22. Georgiou, M., Georgopoulos, C. P 6 Eisen, H. (1979). Virology,

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by N. Sternberg