Heat-stable and Density Mutants of Phages T1, T3 and T7

Transcription

1 J. gen. Vrol. (97), 9, Prnted n Great Brtan 35 Heat-stable and Densty Mutants of Phages T1, T3 and T7 By D. A. RTCHE AND FNA E. MALCLM nsttute of Vrology, Unversty of Glasgow, Glasgow W., Scotland (Accepted 29 May 97o) SUMMARY The colphages T, T3 and T 7 rapdly lost nfectvty when ncubated at 60 n salne-ctrate medum. From the survvors of heat-nactvated wld-type phage stocks heat-stable (st) mutants were solated, whch all had a reduced buoyant densty. The st mutant phages of T3 and T7 contaned 3 to 5 ~ less DNA than ther wldtype parent, whle no loss of DNA could be detected for T~ st mutants. Ths correlaton between heat stablty and reduced densty was confrmed by showng that a mutant of T3 selected for ts lghter densty had also mutated to heat-stablty. NTRDUCTN Wld-type phage T 5 loses nfectvty on ncubaton at elevated temperatures n salnectrate medum (Adams & Lark, 95o; Lark & Adams, 1953). From the survvng phage, mutants have been solated whch are heat-stable (Adams & Lark, 195o; Hertel, March & Mller, 96Z; Rubensten, t968). These heat-stable mutants (called st mutants) are less dense than wld-type phage (Lark, 96Z; Hertel et al. 96Z; Rubensten, 1968). Rubensten (968) showed that the reduced densty s assocated wth a reduced DNA content of the st phage partcle; the most heat-stable mutants are the lghtest and have lost most DNA. Ths nterestng correlaton between mutaton to heat stablty and reduced densty and DNA content n T5 suggested to us that the smple process of selectng heat-stable mutants mght be a general method, applcable to a varety of phages, for solatng varants wth altered DNA contents. Such a method allowng the solaton of vable phages wth smaller DNA molecules would permt studes of the sze, dstrbuton and nature of the apparently non-essental parts of the genome of ths group of phages. For these reasons we have nvestgated whether the ablty to sport heat-stable mutants s a general property of several phages, and whether such mutants also consequently have decreased denstes and smaller DNA molecules. f heat-stablty results from the deleton of part of the DNA molecule, the selecton of heatstable mutants should also select for partcles wth a reduced densty and less DNA. n ths paper we present results obtaned wth the colphages T, T3 and T7. These phages were chosen because ther DNA molecules, lke those of T5, have non-permuted base sequences (llgs, 1967; Thomas & MacHatte, 1967). t has been suggested that the maturaton of phage DNA molecules n some cases nvolves the excson of phage-equvalent lengths of DNA from larger DNA structures known as concatemers (Stresnger, Emrch & Stahl, 967; Thomas, Kelly & Rhoades, 1968). For phages wth non-permuted DNA molecules the unt lengths of DNA would be expected to be cut from concatemers by a mechansm whch recognzes specfc termnal nucleotde sequences (Thomas et al. 1968). f ths s so, the deleton of DNA whch does not nclude those specfc sequences would lead to the producton of phage partcles wth less DNA and a lower densty than ther wld-type. P:

2 36 D.A. RTCHE AND F. E. MALCLM METHDS Phage and bactera. The wld-types of phages T, T3 and T 7 and ther mutants were grown and assayed on Eschercha col stran e. solaton of heat-stable mutants. The expermental technque adopted was to expose wld-type phage populatons to successve heat nactvaton cycles. Followng each heat nactvaton the survvng phage populaton was grown to gve a further hgh-ttre stock. Preferental heat nactvaton of the parent phage would progressvely enrch the proporton of any heat-stable mutants among the survvors. For each nactvaton cycle the phage suspenson (about 5 x l 9 p.f.u./ml.) was dluted ~oo-fold nto 2 x SSC (o. 3 M-NaC, o'o3 M-sodum ctrate) and ncubated at 6o to reduce the nfectvty to about ~oo of the ntal ttre. ne ml. of the nactvated phage suspenson was added to 5 ml. culture of Eschercha col B (5 x v cells/ml.) growng at 37 n H-broth (Kreg, 959) and the culture ncubated untl lyss was complete. Ths process was repeated 8 to tmes. From the last lysates of each phage type, 2o to 5 plaques were solated and dluted to a ttre of ~o 6 p.f.u./ml, n 2 x SSC; samples were removed before and after ncubaton at 6o for ~ hr. Part of each sample was assayed for nfectvty. A plaque stock showng an enhanced heat stablty was gven an st symbol and plaque solaton number and used to prepare a hgh-ttre phage lysate. Growth and purfcaton of radoactve phages. Cultures of Eschercha col B were grown wth aeraton at 37 n synthetc TCG medum (Koznsk & Szybalsk, 959). At a densty of 108 cells/ml, the medum was supplemented wth ether [ah]thymdne ( o #c/ml.) or [a2p]orthophosphate (4/zc/ml.) and further ncubated to a ttre of 3 x o 8 cells/ml. Phage was then added to an m.o., of 2 p.f.u./cell for T or 0.25 p.f.u./cell for T3 and T7, and the nfected cultures ncubated for one hr at whch tme lyss usually occurred spontaneously. Routnely a few drops of chloroform were then added and ar was bubbled through the culture untl lyss was complete. The phage partcles were mmedately purfed and concentrated by two cycles of dfferental centrfugaton wth low speed runs at 7,ooog for ~o mn. and hgh speed runs at 5o,ooog for 3o mn. Phage pellets were resuspended n phosphate buffer ph 7 (Hershey & Chase, 952 ) by gentle mechancal shakng, and the suspensons stored at 4. Assay methods. Phage nfectvty was assayed on nutrent agar plates by the top-layer method (Adams, 1959). Radoactvty was counted n a Nuclear Chcago scntllaton counter. Lqud samples were dred on 2. 5 cm. squares of Whatman GF83 glass fbre paper and each square suspended n ~o ml. lqud scntllant (4 g. 2,5-dphenyloxazole and o'05 g. 1,4- bs[z-(5-phenyloxazolyl)]benzene/1, of toluene). Fractons from both caesum chlorde and sucrose gradents were usually collected drectly on glass fbre paper. Extracton of DNA molecules. Unbroken DNA molecules were extracted by rollng equal volumes of purfed phage suspenson and freshly dstlled water-saturated phenol on a test-tube roller wth the tubes rotatng about ther long axs at 75 rev./mn. The detals of ths method were descrbed by MacHatte et al. (1967). The extracted DNA was dalysed 4 to 5 tmes aganst a large excess of 0"05 M-NaC1, o.o~ M-trs, ph 8"0 to remove resdual phenol and stored at 4. Densty gradent centrfugaton. Anhydrous CsC1 was dssolved n H-broth at a concentraton of 45"1 ~ (w/v) and a densty of about 1.5 g./ml. Three ml. volumes of CsC1 contanng o.~ to o.2 ml. of phage suspenson were overlad wth paraffn ol and centrfuged n the SW39 rotor of a Spnco Model L ultracentrfuge. After centrfugaton at 25,ooo rev./mn. for 2o hr at 2o the gradents were unloaded by collectng fractons dropwse from the bottom of the centrfuge tube through a No. 18 steel syrnge needle held n a rubber stopper. P:

3 Heat-stable and densty mutants of phage 37 Refractve ndces of undluted gradent fractons were read at 25 mmedately after collecton. Usually the gradents showed a lnear varaton n refractve ndex. Denstes of phage partcles were calculated from the lnear refractve ndex curves usng the emprcal relatonshp of Thomas & Berns (2961). Because of the relatvely small densty dfferences between wld-type and st mutant-phage partcles, the buoyant denstes of st phage were always determned relatve to ther wld-type n densty gradents contanng both phage types. For ths calculaton the centre of a unmodal band of radoactvty n a densty gradent was determned by a method frst descrbed by Rubensten (968). Peak fractons are selected to obtan a symmetrcal dstrbuton of label and to nclude about 80 ~ of the peak materal. The band centre s calculated by summng the products of the counts n each fracton and the dstance of that fracton from one end of the gradent and dvdng ths product by the total number of counts n the selected fractons. For a gven peak of label the band centre s the same regardless of whether the dstance s measured from the top or the bottom of the gradent. Ths method was used by Rubensten to calculate the average dstance that DNA molecules sedment n a sucrose gradent, but we have found that t s also sutable for equlbrum densty gradents. Sucrose gradent centrfugaton. The sedmentaton of natve DNA molecules was conducted accordng to the methods of Burg & Hershey (1963). Dfferentally labelled mxtures of wld-type or wld-type and st mutant DNA were layered on 5 m. lnear gradents of 5 to 20 ~ (w/v) sucrose dssolved n o. M-NaC1 0"05 M-phosphate, ph 6.8. The gradents were centrfuged at 2o n the SW65 rotor of a Spnco Model L ultracentrfuge. After centrfugaton the gradents were unloaded by the method descrbed above for densty gradents. The average dstance that a band of DNA had sedmented was agan calculated by the method of Rubensten (1968). From several ndependent determnaton s on a gven par of DNA s, the average dstance that DNA-a had sedmented relatve to DNA-t (D2/D) was used to calculate the relatve molecular weghts (M2/M) from the relatonshp: D2/D = (Mz/M ) 'z5 (Burg & Hershey, 963). The molecular weghts of the wld-type DNAs were taken from those lsted by Thomas & MacHatte (967) and used to calculate the molecular weghts of the st mutant DNA molecules. The solaton of heat-stable mutants RESULTS The wld-types of phages T, T3 and T7 were nactvated when heated at 6o n 2 SSC (Fg. ). After exposure of these wld-type phages to a seres of 8 to successve heat nactvaton cycles, 3o to 5o ~ of the phages solated from the fnal lysates of each phage type showed an ncreased heat-stablty. From the heat nactvaton curves of several heatstable (st) mutants derved from each parent phage, the greatest dfference n nactvaton rates was between T3 + and ts st mutants (Fg. ). Tlst mutants showed the least relatve change n heat-stablty. Densty gradent analyss of the heat-stable mutants The wld-type phages were routnely labelled wth 3H or 32p and the st mutants wth z2p. The 3H and z2p-labelled phages were then centrfuged n pars n the same densty gradent. When the phages were dentcal, except for the label, they banded at dentcal postons n a densty gradent (Fgs. 2 a, 3 a, 4a). However, whenever the par of phages conssted of the wld-type and one of the several st mutants solated, the st mutant nvarably banded at a lower densty (Fg. 2b to 4b). The st mutants oft3 showed the largest densty reducton whle Tlst mutants were only margnally less dense than T1 + (Table ). P:

4 38 D.A. RTCHE AND F. E. MALCLM Sucrose gradent centrfugaton of wld-type and st mutant DNA molecules The smlar behavour of T, T3 and T7 to that of T5 regardng mutaton of heat-stablty and reduced densty led us to look for dfferences n the molecular weghts of wld-type and st mutant DNA molecules. The sedmentaton behavour of wld-type and st mutant DNA Mnutes ] ~--'---~'~ e. ~ ~- [~"~ J l.~-"~--r._"--m r3st18 \\~. ] - ~A~------'l:r3st11 ~ ~ \ -1 T3st16 T7stl mo ' T1 st19 T ~T1 + ~ 1 Fg.. The knetcs of heat nactvaton of the wld-type and some st mutants of phages T[, T3 and T7. Phage suspensons were ncubated at 60 n 2 x SSC and at ntervals samples were assayed for nfectvty. b o 15 t e~ _, 10 K ~ L "7 10 "~ o s. o ~- :6 l 5 t~ ~ s p- L ~-'"J 0 o oj Fracton no. Fg. 2. The CsC densty gradent dstrbutons of T[ + and Tst mutant phage partcles. (a) Mxture of [~H]T + and [32PlT+. 05, two-drop fractons were collected. (b) Mxture of [2H]T[+ and [3zP]-T]st2; ] o, two-drop fractons were collected. The bottom of the gradent s to the left.,3h label;, ~2p label. }- P:

5 Heat-stable and densty mutants of phage 39 was examned by centrfugng dfferentally labelled DNA mxtures n the same sucrose gradent (5 to zo ~ (w/v) sucrose ph 6.8). When ntact molecules solated from the wldtype and st mutants of phages T 3 and T7 were sedmented through sucrose gradents, the st mutant DNA always sedmented more slowly than the correspondng wld-type DNA (Table 2). Control mxtures of wld-type DNA dfferng only n ther label sedmented at a 15 b "1-20 > r~ ~" 10 3 l~. ~_, ~, 20.~.> 5 L '-.3 E t~, t t L_ E" el m. > r~ 3 b- - l - o ~ - g Fracton no. C J J 0 Fg. 3- The CsC1 densty gradent dstrbutons of T3 + and T3st mutant phage partcles. (a) Mxture of [3H]T3 and [z~p]t3+; 73, three-drop fractons were collected. (b) Mxture of 13H]T3 + and [3~P]T3st6; 67, three-drop fractons were collected. The bottom of the gradent s on the left.... 3H label; a2p label. 3-1-,n..>..~ 20 8 ~ 10 J3o /, 30.b ',-~ l : f3 " v r J t 20 - ', 20 ~ lo _fl 8 8 la _~ lo 20 g T, 10 ~ 0 - ', r ~ L- r_. ~_~_ - ~ J t Fracton no. Fg. 4. The CsC1 densty gradent dstrbutons of T7 + and T7st mutant phage partcles. (a) Mxture of ph]t7 + and [82P]T7+; 75, three-drop fractons were collected. (b) Mxture of [3H]T7 and [3~p]_ T7st 15.7o, three-drop fractons were collected. The bottom of the gradent s on the left. - 3H label;, z2p label. 0 P:

6 4 D.A. RTCHE AND F. E. MALCLM dentcal rates (Table 2). The slower sedmentaton oft3 and T7 st DNA ndcated that these DNA molecules had smaller molecular weghts (Burg & Hershey, 1963). Ths concluson only assumes that mutaton to heat-stablty dd not alter the confguraton of the st DNA molecule. The calculatons of molecular weghts ndcated that for T3st mutants there was an apparent deleton of about 5 % of the DNA molecule, whle for T7st mutants the amount of DNA lost was about 3 % (Table 2). The sedmentaton profles of T -~ and Tlst DNA molecules overlapped almost completely showng no detectable dfference n sedmentaton rate. Table. Buoyant denstes of wld-type and st mutant phage partcles Phage Densty n CsC1 T + 1"494 + o'oo (9) Tst o'ool (3) Ttst3 '493 +o'ooo (3) T3 + '52_+o'oo (5) T3st "496 + o'oo (2) T3st 6 1'496 () T3st 8 1 ' o'oo2 (2) T7 + '5 + o'oo (3) T7st 5 "497 () T7st ~8 1'497 (x) T7st26 1"497 () Each value s the average of the number of ndependent measurements gven n parentheses. Table 2. Relatve sedmentaton rates and molecular weghts of wld-type and st mutant DNA molecules DNA-* DNA-2* D2/D + M2/M Ml x 10 6 M211 x 0 6 T + T + ' +o'ooo (4)~ ' 3 "2 3"2 T1 + Tst2 1'ooo2-t-o"ooo2 (4) ' 3'2 T + Tlst 19 ' + 0"oo7 (2) "oo3 3 '3 T1 + Tlst3 '6_+ o'ooo6 (3) 1"oo2 3"3 T3 + T3 + o' o'ooo2 (3) o'999 23"3 23"3 T3 + T3st 0"9799 _+ 0"0003 (3) 0" T3 + T3st6 o'981o -b o.ooo (4) 0" T3 + T3stt7 o'9856+o'oo13 (4) o'959 22'3 T3 + T3st 18 o'9827 -} (4) T7+ T7 +. _+ o.ooo2 (4).ooo 24"7 24"7 T7 + T7st 5 o'9889 _+ o'ooo5 (3) o'969 23"9 T7+ T7st8 o' o'ooo3 (3) o'958 23'7 T7+ T7st26 o'99o3 +o'oo3 (3) o'973 24"o * DNA types sedmented as a mxture through a partcular sucrose gradent. t Average of the ratos of the dstances that DNA-2 had sedmented relatve to DNA-. Each average based on the number of ndependent determnatons gven n parentheses. Mol. wt. of DNA-. [ Mol. wt. of DNA-2. The non-revertblty oft3 and T7 st mutants Stocks of T3st16 and T7st15 were each centrfuged n a densty gradent and the fractons from the heavy sde of the band were pooled and the phages regrown. (The selected heavy fractons ncluded the frst fracton on the heavy sde of the peak down to the fracton contanng about 1% of the peak nfectvty.) Ths process was repeated sx tmes. The fnal phage stocks were labelled wth a2p, mxed wth ther correspondng [ah]trtated parent st mutant and centrfuged n a densty gradent to compare ther denstes. There was no P:

7 Heat-stable and densty mutants of phage 4 dfference n ether case. ur nablty to solate phage partcles wth a greater buoyant densty followng extensve selecton from the heavy sde of densty gradent bands of T3 and T7st mutants suggested that full or partal reverson dd not readly occur. Drect selecton of densty mutants Snce densty mutants of T, T3 and T7 exsted and could be solated by heat selecton, we thought t should also be possble to solate them by the drect, but more laborous process of repeatedly selectng phage partcles from the lght sde of a densty gradent band of wld-type phage. A T3+ stock was centrfuged n a CsC1 gradent and the fractons on the lght sde of the peak were pooled and grown to a hgh-ttre stock. Ths second lysate was n turn banded n a densty gradent and the lght fractons solated and regrown. After fve selecton cycles, a plaque was pcked from the ffth lysate and the densty of the phages compared to that of T3 +. The selected T3 phage showed a unform shft to a lghter densty very smlar to that of the T3 st mutants. Ths lght phage stock was heat-stable to about the same degree as the T3 st mutants obtaned by drect heat selecton. Mutaton to heatstablty and lghter densty were thus correlated and a consequence of the same process. DSCUSSN ur results demonstrate that the ablty to mutate from the heat-senstve wld-type to a heat-stable form s a general property of phages T, T3 and T7. Prevously ths had been shown for T 5 (Adams & Lark, 95 o) and more recently for phage A (Parknson & Huskey, 197o). For all fve phages mutaton to heat-stablty s correlated wth a reducton n the densty of the phage partcle and, wth the excepton oft1, the reduced densty has been demonstrated to result, as predcted, from the loss of some DNA from the phage partcle. The falure to detect reverson of T3 and T7st mutants supports the deducton from the sedmentaton data whch ndcate that st mutants have lost DNA sequences. The Tlst mutants show only a slght densty reducton compared to T1 + phage. The dfference, however, s reproducble and was observed throughout a seres of eleven experments comparng the densty of T + phage wth each of four T st phages examned. The sedmentaton rates of T + and Tst DNA molecules were vrtually dentcal, Therefore we cannot dstngush between the possblty that the two DNA types have dentcal molecular weghts or that Tst DNA molecules are shorter by a small amount not resolved by sedmentaton analyss. (Snce molecular weght and sedmentaton rate are logarthmcally related, two DNAs wth molecular weghts dfferng by 2 % would dffer only by 0"7 % n ther sedmentaton rate. Smaller molecular weght dfferences would not be dstngushable by ths method.) ur fndngs, taken together wth those of Rubensten (968) and Parknson & Huskey (97 o) suggest that for phages wth non-permuted DNA molecules the solaton of heatstable mutants can be used as a standard method for selectng vable phages wth reduced denstes[ The procedure s smpler and qucker than the drect method of selectng densty mutants from densty gradents. The st mutants of each phage type were solated by repeated heat selecton from a sngle ntal wld-type stock. Thus t s possble that the st mutants solated from a gven phage type are clonally related. n ths study we were nterested n tryng to establsh the exstence of heat-stable mutants and were not concerned wth ther possble genetc relaton, so dd not attempt to ensure that each st mutant was of ndependent mutatonal orgn. Smple modfcaton of the heat selecton technque would permt the solaton of genetcally dstnct st mutants. P:

8 4 2 D.A. RTCHE AND F.E. MALCLM Why there should be the observed correlaton between heat-stablty and DNA content s not fully understood. Heat nactvaton causes the DNA to be released from the partcles of several phages ncludng T5 (Lark & Adams, r953), T7 (Frefelder, 965),,~ (Parknson & Huskey, 97), T and T3 (D. A. Rtche, unpublshed results). n fact, we fnd that for T, T3, T7, T2, T4 and P22 the DNA s released as full-length molecules. But why the DNA release mechansm should be so senstve to small varatons n DNA complement we cannot yet explan. As yet there s no nformaton about the locaton of the deleted regons n the st phages of T3 and T7. We are attemptng to dentfy whch regons of the DNA are mssng and whether the DNA s deleted from only one or several dfferent locatons n a gven DNA molecule. These deleted sequences must be genetcally non-essental, at least for growth n Eschercha col B. Such regons mght represent duplcate phage genes or genetc nformaton also carred by the host cell, or perhaps phage functons whch are not always requred for vegetatve growth, such as the rl gene products of phage T4. The stuaton s clearer n temperate phages, for t s known that DNA regons nvolved wth lysogeny can be deleted wthout hnderng lytc functons. No doubt ths s why as much as 23 ~o of the ;~ DNA molecule can be deleted (MacHatte & Thomas, 1964). The fve phages capable of producng heat-stable densty mutants all have DNA molecules wth non-permuted DNA sequences (llgs, 967; Thomas & MacHatte, 967), and for several of them a concatenated structure has been mplcated n the DNA replcaton process (Smth & Skalka, 966; Salzman & Wessbach, 967; Thomas et al. 968). A suggested mechansm for cuttng non-permuted DNA molecules from concatemers nvolves the recognton of specfc termnal nucleotde sequences (Thomas et al. 968) so that the deleton of DNA would lead to the excson of shorter DNA molecules. For phages wth permuted DNA sequences, such as the T-even phages, t has been suggested that phage-szed DNA molecules may be excsed from concatemers by a reacton whch recognzes a fxed length of DNA and not specfc DNA sequences (Stresnger et al. 967; Thomas et al. 968). n ths case, a deleted DNA regon would be compensated for by the DNA molecule havng a longer termnal repeat regon (Stresnger et al. 967; Kvelland, 1969). Thus for permuted phages, mutaton to heat-stablty and reduced densty, f t does occur, may not reflect a change n the sze of the DNA molecule but some other mechansm. ur prelmnary results wth T2 and T4, to be the subject of a further paper, show a dfferent pattern from that wth the T-odd phages; repeated heat selecton (5 to 6 successve heat-nactvaton cycles) has revealed phages wth only a margnally ncreased heat-stablty, compared to the parent phage. The major fndngs wth the T2 and T4 heat-selected phages relevant to the present dscusson are the followng: () the DNA content s very smlar or dentcal to the parent phage, (2) the phage partcles show a bmodal densty dstrbuton wth o to 3o ~ bandng wth the parent phage, the remander beng of very lght densty, (3) both densty bands contan fully nfectous phages, (4) the two densty types are genetcally dentcal snce the progeny of phages taken from ether densty band reproduce the bmodal densty pattern, (5) osmotc shock evdence wth the T4 heat-selected phages ndcates that the confguraton of the capsd proten subunts s altered and that these changes result from a sngle reversble mutaton. We wsh to thank Mr R. A. Elton for hs help and advce n processng the data and also Professor J. H. Subak-Sharpe and Dr Mary R. Lunt for ther crtcsms of the manuscrpt. P:

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