An analysis of the processes of infection and induction of E. coli mutant hfl-l by bacteriophage lambda

VIROLOGY

55, 183-192 (1973)

An Analysis of the Processes of Infection and Induction of E. coli Mutant

hfl-l by Bacteriophage Lambda

MARLENE BEL FORTI

AND

DANIEL L. WULFF

Department of ill olecular Biology and Biochemistry, University of California, Irvine, California 92664 Accepted June

4, 1973

The E. coli mutant hfi-): is lysogenized with almost lOD';i~) efficiency by ;\,+ and by AcIlI mutants and at a three- to fivefold increase in frequency by a AcIl mutant. Elevated levels of lysogenization were also found with other lambdoid phage bearing the cIlI gene of lambda (Aimm 434 and Ail1!1n 21) , but not with more distantly related lambdoid phages (Aimm 8o , .p8D, and 424). Lytic development in the absence of repressSOl' within hfl-1 and hfi" strains is the same with respect to burst size and the expression of /'l-exonuclease, while there is within hfl-]: strains a 3-1O-min delay in the time of lysis and in appearance of lysozyme. This delay in lysozyme synthesis in an hfl-). host is dependent on the presence of the AcII gene product. Lysogens of hfl-l strains behave normally with respect to spontaneous phage release, immunity to superinfection by AcI, and with respect to burst size upon heat and UV induction. The latent period following UV induction of an hfi-): strain is extended by 5-10 min. The reduced immunity of a »rim prophage is largely restored in an hfl-l host. Nonlysogenic hfl-). strains are abortively infected by Ac17 in the presence of functional repressor, but not in its absence. These observations suggest that the E. coli hfl-1 mutant provides an abundance of a AcIII-like product to infecting lambda phage, which, in turn, is responsible for a delay in lytic gene expression. It is this delay which enhances the establishment of repression in hfl-1 strains in the presence of normal levels of repressor, INTRODUCTION

When bacteriophage lambda infects a sensitive bacterium, two courses of development may ensue. One is a lytic pathway leading to the synthesis of progeny phage and culuminating in lysis of the infected cell. The second is a lysogenic pathway leading to the repression of lytic genes and integration of the lambda DNA into the host chromosome. The fate of an infecting lambda particle depends essentially on the balance between synthesis of the repressor and integration enzymes on the one hand, and on the expression of the lytic genes on the other (for reviews, see Echols, 1971a, 1971b, 1972; Thomas, 1971; Eisen and Ptashne, 1971). 1 Present address: Dept, of Microbiological Chemistry, Hebrew Univereity-Hadaseah Medical School, Jerusalem, Israel.

In the establishment of repression, the synthesis of repressor coded for by the cI gene is under the positive control of the ell and eIlI gene products acting at pre, the promoter for repressor establishment, while the era gene product negatively regulates repressor synthesis (Reichardt and Kaiser, 1971; Echols and Green, 1971). In a lambda lysogen, the cro, ell, and eIlI genes are not expressed, and repressor synthesis is maintained by transcription from a second promoter called prm (promoter for repressor maintenance). The prm promoter is activated by the cI gene product itself, and for this reason it is not expressed during the initial stages of lambda infection of a sensitive host (Spiegelman etal., 1970;Reichardt and Kaiser, 1971). The hfi.-l mutation channels infecting

183 Copyright © 1973 by Academic Press, Inc, All rights of reproduction in any form reserved,

184

BELFORT AND WULF F

wild-type X and AclII mu t ants over whelmingly int o t he lysogen ic pathway (Belfort and Wulff, 1971). Uninfect ed Hfi+ and Hfl" hosts show no obvious physiological differences except for an alteration in th e cell surface, as eviden ced by a decreased permeability t o rifamyc in in Hfl" strains, an increased resistance to peni cillin, and a somewhat greate r sensitivit y to sodium dodecyl sulph at e (Belfort and W ulff, J. B acteriol., in t he press). All of t hese properties of Hfl" hosts, as well as t he abortive infect ion of Hfl" strains by Acl7, described in this paper, are due to 11 single mutation situat ed at 82.5 minutes on the E . coli map, extremely tightly linked to the purA locus. In this p aper an analysis of the pr ocesses of infection and induction of hfi -1 strains by lambda and its mut ants is made with t he aim of establishing the possible t argets wit hin the regulatory cont rols of t he phage at which t he host may exert its influence. MATERIALS AND METHOD S

Bacterial and phage strains. Strain UC2014, which carries the hft-l mutation, was obtained by nitrosoguanicline mutagenesis of UC4185 (ser- irp: leu- i lv- lys- strR F-) as previously describ ed (Belfort and Wulff, 1971). UC2014 is also p yr- and pan- (not pda: as err oneously reported in Bclfort and Wulff, 1971). UC 2156 is a pur" hfi» derivative of the E. Siegel st rain ES4 (pu1'A45 mtl-2 xyl-7 mal- galK6 lac- tsx- ton- .p80 R ) obt ained by tran sducing ES4 t o purr with PI grown on UC4185. UC2157 is derived from E. coli strain ES4 by transducing ES4 to pur" with PI grown on UC2014 (Belfort and Wulff, unpublished). The E. coli st rain CGOO su+ was routinely used as the indi cator for plaque forming units. AB1122 (arg- hispro- thi: gaZ-) and a gal- derivati ve of UC20l 4, obtained by ethy l methanesulfon at e mut agenesis, were used as recipients in G al t ransd uctions. The phage strains used in this study are listed in T able 1. Med ia. Trypt one broth (TB ) was rout inely used for b acterial and ph age work (1.0 % tryptone, 0.5 % N aCl, suppl emented with 1.5 % bacto-agar for solid media and 0.7 % agar for top agar) . ElVIB-gal plates used for t ransd uctio ns contained 2.7 % bacto EMB base and 0.4 % galactose. EMBO

plat es cont aining 0.1 % ye ast extract were pr epared as described by Gott esman and Y armolinsky (1968). Dilutions of phage were made int o a Tris-gelatin buffer cont aining 10- 2 M tris, G X 10-6 M MgCb , 5 X 10-4111 (NH4)2S04, 4 X 10-10 )J!J F eS0 4, pH 7.2. Gelatin was added t o a final concentr ation of 0.01 % after auto clavin g. Bact eria at the appropriate ph ase of growt h were centrifuged and resuspended in 2 X 10-2 M M gCh before pha ge adsorption. Isolation of lysogens. Lysogens were isolated from low-multiplicity infections (0.10.3) by plating t he infected cells onto ElVIBO plat es seeded with ca. 109 AcI2Gjplatc. Lysogenic clones, app earing as large, round, pale colonies after an overnight incub ation at 37°, were restreaked, spot-teste d for immunit y t o lambd a, and cross-streaked with sensitive C600 t o check for release of phage. Spec ialized gal transductions. Low frequency t ransducing lysates were prepared by inducing lysogens with an empirically det ermined dose of UV light. St ationar y ph ase gal- cells (AB 1122 or UC 2014 gal- ) resuspend ed in 0.02 111 IVI g2+ were infected at a multiplicity of 10 an d plated onto EMB-gal plat es. Transduced clones appeared dark again st a lawn of pale gal- ba ct eria. Preparation of 3H -labeled T7 DN A. Phage were labeled, and the crude lysat e pr epared b y the method of McM acken ei al. (1970). Bacterial debris was removed by centrifugat ion and the lysat e made 0.002 M in NIgH. P ancreatic deoxyribonuclease I (Worthington) was added t o a final concentration of 8 pgjml, and t he lysate incubated at 37° for 30-45 min. The phage were then collected by centrifugat ion, purified by two rounds of high- and low-speed centrifugation, and suspended in the T7 buffer of Kelley and Thomas (1969). F urther pur ificatio n by banding in CsCI was found not to be necessary. The [3H]DNA was ext rac ted by three phenol treatments as describ ed by Kelley and T homas (1969), and its specific activity found to be 7.7 X 103 cpra / ug. Preparation of cell extracts and measurement oj exonuclease activity. Sonic extracts for the assay of exonuclease were prepared as describ ed by Sly ei al. (1971). The assay, based on t he acid-solubi lization of count s of the 3H -labeled T7 DNA b y {'I -exonuclease,

A BILITY OF DU'FEIlEN T

Gr oup

TABLE 1 PH AGES T O PL ATE ON HFL-1

Phage

Sourc e

BA e 'rERIA"

Plaqu e morphology UC4185 (lifl+)

L Lambda en I mutants

2 . Other lambda clear mutants

i\cIIIco2 XcIII611 (a m )! XcII I6 15 (am)! i\cI857biolOu xcI857bio256 u i\imm 434elts bi o252h XcI857incl-(300)i (40°) i\cI26 XcI14 (a m ) Ximm'''eI Ximm 2l cI XclI08 AcII28 Xc41l; Acy42 Xvir AvIt'3 k

4 . Miscellane ous phage

5 . c 17 deri v a t ives

0'

1. 1. 1. 1. 1. .T .

0

Herskowi tz Herskowitz Herskowitz Herskowitz Herskow itz Parkinson

J . P arkinson H . E oho ls .T . Parkinson 1. H erskowitz J . Parkinson D . Court II . E ch ols M . Lieb M . Meselson W. Sly W. Sly W . Sly L . Reichardt M . Meselsou H. Strack J . Par kinson M . Gottesman H . Echols 1. Herskowi tz 1. Herskowitz 1. Herskowitz J. H oll and R . Edgar R . Edgar C . Y an ofsky 1. H erskowitz 1. Herskowit z L. Reichardt

Av2~

3 . O ther lambdoid phages

J . Parkin son

Xv1vs k XcI8!57s ex-cl'o27 (400) I X+

»rim» ximm ' ''c+ Ximm 21c+5 .p80immx .p80 xi mm so 424 f2 T4 T7 PI Xcl7 Xcl7cHlOp Xc17cI857 (30°) Xc17cI 857 (40° ) Xc17cIIlc 02 i\cl7cIl28 xc17cIl68

L . Re ichardt L . Reichardt L . Reichard t

c e e 0

t c e

UC2014 (lifl-l )

IIfl- l/lrjl+

t", h' t t t t t t, h

0 .1 0.1 0.1 0 .1 0 .1 0 .1 0 .03 1.0 1.0 1.0 1. 0 1. 0 1.0 1.0 1.0 1.0 1. 0 1.0 1.0 1.0 1. 0 0.05 0 .05 0 .07 0 .05 0 .1 1.0 1. 0 1. 0 1.0 1.0 1. 0 1.0

0

0

c c

0

0

c

0

0

c c

c

c

0

c

c c c c

0 0

c c c t t

t t t t t t c

c c t

c c c c

c.n.p. b

e c t, t, t, t, t, t t

h h h h h

t,

c c c t C,

sm"

c

c, sme

0

C,

0

0

c

e

smn

10-'-10-6 • 1.0 10- 3 • 0. 5

10....'· 1.0 1. 0

u Stationary p h ase cultures of hfl+ and hfl-1 strains in 0.02 !If M gCI, were pl a t ed with the phage indica ted , us ing the top-agar method. Plates we re incu b a ted at 37° unless otherw ise indicated. b c .o .p . den ot es the efficiency of plating on nn hfl-l strain relativ e to the plating efficien cy OIl an

hjl+ strain. c c = clear plaques.

"t

=

turbid pl aques .

h = heter ogen e ou s pl aque s iz e . ! A cIl I am bel' mutant (Signer et al., 19(9) . o The errI gene is deleted (Sign er et ai ., 19139 ). e

The cIlI gene is partially deleted. T em per ature -sen s it ive rep ress or. ; A cIl mutant w h ich fails to complement cIl I mutants (Strack and Ziegler, 1969). ~ Par tial virulen t (Ordul, 1971) . I See Reichardt a n d Kaiser (1971) . m T h e rim mutant shows re duce d immunity (Strack et al., 1970). " The p laques which do appe ar are s m all and clear. A purified p laque als o pl at es on hfl-l strain UC2014 wi t h at least 100-fold re du ction in efficiency . • The extent of reduction of e .o.p, with Xc17 is variable . p Viruleut (Sly a nd Rabideau , 19139) . h

i

185

186

BELFORT AND WULFF

was performed by the method of Radding (19()6) as modified by Sly et al. (1971). Lysozyme synthesis. Bacteria were grown to 2 X 108 cells/ml in TB, centrifuged and resuspended at 2 X 10 9 cells/ml in buffer (0.01 111 Tris-CI, pH 7.1,6 X 10-6 111 .MgCb, 5 X 10-4 111 (NH 4)2S04 and 4 X 10- 10 111 FeS04)' After incubation for 10 min at 37°, the bacteria were chilled and an equal volume of phage (2.4 X 1O l D/mi in Tris-Mg buffer: 0.01 111 Tris-CI, pH 7.1, 0.01 M lHgC12) was added. Following adsorption for 15 min at 0° and 6 min at 37°, antiserum was added to a final K = 5 (5 min at 37°). (Under these conditions adsorption was found to be 8090 % complete). The cells were centrifuged and resuspended at 2 X 108 cells/ml in TB at 37°. Extracts were prepared at various times for lysozyme assay by the method of MclVlacken et al. (1970). The lysozyme assay, based on the decrease in OD of an EDTAsensitized bacterial test culture (W3110), was performed as per Jacob et al. (1957). RESULTS

The IIi[jh Lysogenic Response on Injection oj hfl-l II osis

As we have previously reported, :Ac+ and AcIIlc02 lysogenize the E. coli mutant hfl-1 with almost 100 % frequency (BeHort and Wulff, 1971). If the AcIII gene product is indeed not required for lysogenization in an hfl-1 host, all cIlI mutants, including deletions of all or part of the cIlI gene, should lysogenize with high frequency on hfl-l. This expectation is fulfilled (Table 1, group 1). The high tendency towards lysogenization in these infections is manifest by the extremely turbid plaques formed by AcIH mutants, and by the reduced plating efficiency on the hjl-l strain UC2014 relative to the parental hjl+ strain, UC4185. All other clear plaque type phages, characterized by a reduction in repressor synthesis (i\cH, ACy), activity (Xcl), or effectiveness (i\vil', :Av2, Av1v3) (Reichardt and Raiser, 1971, Echols and Green, 1971, Ptashne and Hopkins, 1968), produce clear plaques at 100 % efficiency on both hfl-l and hjl+ hosts (Table 1, group 2). Quantitative measurements show that the low frequency of lysogenization which occurs after infection of hfl+ strain

UC4185 with i\cII is slightly increased (3to 6-fold) in i\cII infections of hfl-l strain UC20l4 (Table 2). The enhanced tendency towards lysogenization is not restricted to imm A phage, but is also seen when i\imm 434 and :Aimm21 infect hfl-): strain UC2014 (Table 1, group 3). Both of these phages have a lambda cHI gene and a promoter for repressor establishment (pre) which responds to the lambda cIlI gene product. However, two other lambdoid phages, 4>80 and 424, show normal levels of lysogeny in lifi-). strain UC2014. Phage 4>80 shows no homology with either the i\cIlI region or the :Apre region (Fiandt et al., 1971). (Phage 424 has not been investigated in this regard.) Investigation of hybrids between 4>80 and i\ show that ¢80imm' has a high tendency towards lysogenization like A, while :Aimm 8D has a normal tendency towards lysogenization like 4>80. The 4>80imm' hybrid phage has the cIlI gene of A, as 'well as the A pre. The Aimm. 8D hybrid phage shows no homology with either the :AcrIl region or the Apre region (Fiandt et al., 1971).

Temperate phage Pl+ also is unaffected by an hfl-l host, and allnontemperate phage tested (T4, T7, £2) plato normally on hjl-l strain UC2014 (Table 1, group 4). The Inability of hft-l Stmin UC2014 to Propagate Ac17

In Ac17 infections there is constitutive expression of the replication genes 0 and P. Upon infection of a homoimmune hfi" lysogen with Ac17, lambda DNA synthesis and killing of the host ensues without the release of infective progeny phage. Infection of a nonlysogenic hjl-1 strain with i\c17 is similar to infection of a lysogenic hfi" strain with Ac17. Lambda c17 plates on hfl-1 strain UC2014 with an efficiency of only 10-2 to 10-5 per input phage (Table I, group 5). In exact analogy to infection of an hft: lysogen, Ac17 synthesizes its DNA constitutively in nonlysogenic hfi-): (unpublished result, this laboratory) and kills the host cell without any appreciable phage release. Kinetics of repressor synthesis as well as shut-off is the same in i\c17 infection of either hjl-1 strain UC20l4 or hfi+ strain

187

INFECTION AND INDUCTION OF E. Coli hjl.-1 TABLE 2 PERCENT LYSOGENY Phage

X+ XellIco2

x-nes n

% Lysogeny on host"

m.o.i.

5 0.1 5 0.1 5 0.1

hjl+

Iljl-l

hjl-l/

(UC4185)

(UC2014)

11jZ+

94 97 87 97 1.8 2

<2 10 10 1003

70 11 8

<1 0.0 0.3

e

One Step Growth

).c126

o 1010

:::>

u,

a,

109

Aliquots of starved stationary phase cultures

(5 X 10 8 cells/ml) were infected as shown. Adsorp-

tion was allowed to proceed for 30 min at 37", antiserum was added (final J( 5), and after 5 min cells were diluted and plated (a) for lysogeus on EMBO plates, each seeded with 10° >..cI2G as described by Gottesman and Yarmolinsky (19G8), and (b) for infective centers on '1'13 with CGOO as indicator bacteria using the soft agar plating techuique. After 4-G hr incubation at 37°, plates were irradiated with a predetermined dose of UV so as to induce all lysogenic clones, so revealing the total number of infective centers. Percent lysogeny is expressed as (a)/(b) X 100. Results obtained using this method agree closely with the bromothymol blne plating technique used previously (Belfort and Wulff, HJ71) , while allowing the measurement of lower levels of lysogeny. b Note that the 100-fold increase in lysogenic response is that maximally attainable under these conditions, since A.cIlI is lysogenizing the !lfl-1 host with almost perfect efficiency. The difference in lysogenic response between hfl-1 and Wild-type cells could therefore conceivably be much greater than the two orders of magnitude indicated. =0

UC4185 (L. Reichardt, personal communication). However, when active repressor cannot be synthesized in an hjl-l strain, as in infection with Ac17cI90 or with Ac17cII68, a normal productive infection ensues. This is not the case with Ac17cIIIco2, presumably because AcIII mutations are not expressed in hjl-l straills. Expvessum. of Lytic Functions

Since it is readily conceivable that an impairment in lytic function might bias an infection toward the lysogenic pathway, the vegetative development of lambda was examined in hjl-l hosts. At the biological level, we measured phage release on infection by

o UC4185 (.h!! t) • UC2014(.h!!-11

20

30

40

50

60

70

so

90

100

MINUTES

FIG. 1. A culture grown to mid log phase in

'1'13 was resuspended at 10° cells/ml in 0.02 M MgCh and infected at a multiplicity of 0.1 with XcI26. Adsorption was allowed to proceed for 20 min at 0°. After treatment with antiserum (K. 5), the cells were diluted 106- and 107-fold into TB at 37° and aerated. Aliquots were removed at the times indicated and plated for plaque forming units (PFU) using C600 as plating bacteria. =0

AcI and 011 induction of various lysogens by temperature and UV. A one-step growth experiment (Fig. 1) shows a normal burst with an hjl-l host and a 5-10 min extension of the latent period. Similarly, the burst size after temperature induction of AcI857 lysogens is normal, as is that after UV induction (data not shown). A 10 min delay in latent period was recorded on UV induction at 37°, and, in order to eliminate the possibility of this effect being attributable to secondary mutations in hjl-l strain UC2014, the UV induction was repeated on lysogens of the isogenic strains UC2156 (hfi+) and UC2157 (hjl-l) with the same result. Lysozyme synthesis was used as a monitor of late lytic function at the biochemical level. Figure 2 indicates that, while the patterns of lysozyme synthesis following AcI857 infection at 38° were similar in the isogenie strains UC2156 (hjl+) and UC2157 (hjl-l), a delay of 3 min in the onset of

188

BELFORT AND WULFF 80

Lvsozyme Syntnesls 70

60

• el857 In UC2156 (!.!!l') o cl857 In UC2157(!.!!l-Il • cra57ell6B in UC2156 (1l!.!'j o cIB57e1l6B in UC2157 (till-I)

l-

0 0::: n,

'"

E

w

SO

£a:

40

Ul

:::>

30

f

<,

IZ

AcL26

z

Z

iii

Exonuclease Synthesis

VI

IZ :::>

30

20

20

o 10

o

UC4185 (]l!..!+)

• UC2014 (!.!!l-Il

5 10

MINUTES

FIG. 2. Parallel cultures of the isogenic strains UC2156 (hfl+) and UC2157 (hft-1) were infected at 39° with t-cl857 and with >.c857cII68 at an m.o.i. of 12 and assayed for lysozyme at various times.

lysozyme synthesis was recorded in the hjl-1 strain. From Fig. 2 it is also evident that this delay in lysozyme synthesis is dependent upon a functional }..cIl product, since lysozyme synthesis following }..cI857eIl68 infection at 390 is identical in hjl+ and hfl-1 hosts. The onset of lysozyme synthesis following 'AcI857eII68 infection is appreciably earlier than following cI857 infection of either host, confirming the results of McMacken et ol. (1968). In a AcI26 infection of strains U04185 (hjl+) and UC2014 (hjl-1) at 37° there was an 8-10 min delay in appearance of lysozyme in the hjl-1 host (data not shown).

20

30

40

MINUTES

FIG. 3. Details of the infection procedure, extract preparation, and exonuclease assays are given in Materials and Methods. One unit of activity is defined as that amount of enzyme which produces 10 nmoles of acid-soluble nucleotide in 30 min.

thesis of exonuclease in }..d infection of UC4185 (hjl+) and UC2014 (hjl-1) are virtually identical, which argues that the cro gene is expressed normally in hfi-1 hosts.

Integration

I t is known that under certain conditions lambda can integrate at random sites on the E. coli chromosome rather than at the usual att' locus, which is closely linked to the bacterial gal gene (Shimada et ol., 1972). In order to verify that the attachment is normal in the hfl-l strain UC2014, lysates Exonuclease Synthesis and the Expression obtained by the UV induction of 3 indeof era pendent isolates of UC4185 hfi+(A,+), UC2014 Measurements of exonuclease synthesis are hfl-1 (}..+), UC4185 hfl+(AcIII), and UC2014 illuminating since, apart from being the only hjl-1(}..cIII) were tested for their ability to assayable "early" protein, exonuclease ex- transduce the gal locus of E. eoli into a pression is controlled by the lambda cro gal- recipient strain (see Materials and gene, and it can therefore be an indicator of Methods). Transduction frequencies in all ero function (Pero, 1970). Furthermore, cases were between 0.2 and 1.0 X 10- 5 genes eIlI, exo, and int are subject to similar pel' input phage, a range well in line with regulatory controls, since they form part of normal frequencies of specialized transducthe same operon (Szybalski et al., 1970). tion (Morse, Lederberg, and Lederberg, Figure 3 shows that the patterns of syn- 1956). Therefore integration occurs pre-

189

INFECTION AND IN DUCT ION OF E . Coli hjl -l

lysogens (Strack, Kayser, and Holder, 1970). In particular, UC4185 hjl+("Xrim) allows plaque production by "Ximm434susN, "Ximm434 Behavior oj hjl-l L ysogens and Interactions susO, and "Xv1v3, all of which do not plate on with "Arim UC4185 hfl+ ("X+) . In extension of these reThe behavio r of hjl-l("X+) and hfi-1("XcIIl) sults we have found that "Ximm484cII68 , but Iysogens is similar to t hat of hfi': lysogens not "Aimm434cIIl co2, produces turb id plaq ues with respect to spontaneous phage produc- on UC4185 hfl+("Xl'im) (Table 3). T his means tion (Belfort and Wulff, 1971). There is also that UC4185 hfi+("Xrim) provides sufficient no difference in phage release following UV N , 0, and cIl functions, but not sufficient induction, nor is there any difference in heat cIII function, to complement superinfecing induction of a ther mosensitiv e lysogen. Both heteroimmune phage. With UC2014 hfi-llifl-). and hfi+ lysogens are equally resistant ("Xl'im) lysogens, the plating characteristics to killing by superinfecting Xcl and "XcIcIIcIII of "Xc phage are reversed, with "XcII producing pha ge (dat a not shown). H owever, hjl-1- clear plaques and "XcIII producing t urbid ("Xrim ) lysogens do differ from their hjl+- plaques. Not only is UC2014 hfi-1("Xrim) less ("Xdm) count erpart s. able t o provide AelI function to a superinfectT he reduced immunit y "Xrirn lysogens are ing het eroimmune phage, but it is also characterized by an increased spontaneous much less able to supply Nand function phage prod uction, by a reduced resistance than is its UC4185 hfi+(Arim) counterpart, to killing by superinfecting "XcI, and by In particular, UC2014 hfi-1("Xrim) allows their sensitivity to certain phages which are plaque development with "Aimm434susN, unable to plaque on normal heteroimmune "Ximm434susO, and Avlv3 with a 10-, 100-, and I ,OOO-fold reduction in platin g efficiency T ABLE 3 (Table 3). There is also a slightly enhanced exclusion of T4rII by UC2014 hfi-1("Xrim) INFKCTION OF Ar i m L YS OGENs a as compared with UC4185 hfi+("Xrirn). These a. Plaqua morphology observations confirm the ability of the hfl-1 Host Inf ecting phage mut at ion to substantially improve the maintenance of repression by a "Xrim prophage. dominantly at the normal site in the chromosome in the hfl-): strain UC2014.

°

UC4185 UC20l4 UC4185 UC20l4

hfl+{)..+) hfl-lC>-'+) hfl+(Arim) hfl-l (xri m)

clear clear turbid clear

clear turbid clear' t urbid

b. P lati n g e.tfici ency Infecting phage

Host-e-e.o.p.s

- - - -

UC4185 (Adm) (1Ijt+)

T4rII105

0.9

Avlv3

0.5

Ailllill 434su sN

Aimm 4' 4susO

0 .5 4 X 10- 2

UC2014 (Adm) (hfl-l)

0 .3 5 X 10- 4 2 X 10- :<

3 .5 X 10-'

a Starved stat ionary phase cultures were in fected with t he appropria te phage and, after Do 20-min absorpt ion period, were plated onto TB , using t he soft-agar t echnique, and incubated at 30° overnight . h e.o .p. denotes the efficiency of plating of phage re la tive to t hat on 11 nonlysogenic host which is as signed a value of 1.0.

DISCUSSION

All of the observations in this paper are consistent with the hypothesis that hfi-l hosts strongly bias infecting phage towar d lysogenization by providing a "XcIII-like function in large amount s and thereby increasing the probability of estab lishing repression. Fir st we shall consider the evidence that hfl-1 hosts provide a "XcIII-like function : (1) All AellI mutants give high frequencies of lysogenization in hfl-l strains, even AcIIl deletions. (2) Related lambdoid phages which contain the lambda AcIII gene, such as Aimm434 and "Ximm21, lysogenize with high frequencies in hfi-1 strains, whereas lambdoid phages which show no homology with the lambda "AcIII region, such as 4>80 and Aimm 80, lysogenize normally in hfl-). strains. (3). Whereas "XcIII mutants lysogenize with very high frequencies in hfi-1 strains, "XcII

190

BELF OHT AND WULFF

mutants lysogenize with only slightly elevated frequencies. (4) Lambda int egrates at th e normal attachment site in hfl-). strains, which argues that the high frequency of lysogenization in hjl-1 strains is not due to a new attachment site. (5) Th e kinetics of is-exonuclease synthesis are normal in hfi-1 str ains, indicating normal transcription of the N-cIII-exo-xis-int operon and normal behavior of the Aero gene . A completely different type of experiment jed Herskowitz (1971 ) to conclude that the primary effect of the hfl-1 mut ation is to provide a AeIII-like function, as opposed to a primary; effect upon era gene expression or repressor activity. Herskowitz noted that AcI857era- phage did not grow at 42° in an hfl+ host unless a secondary ell, elIl, or ey mutation was also present. He int erpreted this t o mean that if a productive lytic infection is to occur, th e ero product must shut off leftward transcrip tion from the promot er for repressor establishment, pre. In ell, elII , and ey mutan ts, there is no leftward transscription at pre ever, and the cro produ ct is not necessary. In an hfi -1 host , AcI857crophage grew at 42° only if a secondary eII or cy mutation was present. In direct contrast to hfi+ hosts, hfl-l hosts did not support lytic growth of AcI857cro- cIlI phage. Since the hfl-1 mutation is t hereby expressed in the absence of both the era gene product and the cI gene product , it is argued that the hfl-1 mutation must directly affect transcription at pre through providing a AelII-like function. In any case, the lack of a requirement for the AcIlI gene product in hfi-1 strains cannot be an indirect consequence of a primary effect of the hfi-1 mutation on either the era gene product or the cI gene product. Let us now consider why the provision by an hfi-1 host of a large amount of a AcIIIlike function should so greatly enhance the frequency of lysogenization in A+ and AcIlI infections. The AcIII gene interacts at pte t o facilitate transcription of the AcI gene. One might imagine th at in hfl-1 strains the onset of repressor synthesis would occur sooner and/ or that there would be a great er net synthesis of repressor. However, the independent experiment s of P . Chadwick and L. Reichardt (personal communications) have shown no gross differences in the

kinetics of repressor synthe sis foll owing infections of hfl+ strains wit h A+ and hfl-l strai ns with 11.+ and AcIII mutants. Infections of hfi" stra ins with AcIII mutants result in greatly reduced amounts of repressor (Reichardt and Kaiser, 1971). A second proposed functi on for the AclI and AcIII gene product s is th at they delay the onset of lytic functions, th ereby facilitating th e establishment of repression by a given amount of repressor (Mc'Macken et al., 1970; Echols, 1972). Since the AcII and AcIlI products are presumed to bind only at th e promot er pte for leftward transcription of the AcI gene, this delay is believed to be caused by an inhibition of th e riqht'Ward transcription necessary for lytic growth, which st arts t o the left of 'pre and proceeds through pre. Direct evidence for a delay in th e expression of lytic functions in hfi-1 strains is present ed in thi s paper: Lambda cI mut ants, which make no repressor in either hfi" or hfi-1 st rains, show a 5-10 min delay in the time of phage release in an hfl-1 host. Most importantly, th e onset of lysozyme synthesis is delayed 3- 10 min in an hfi-1 host infected 'with Xcl, as compared with Ac! infection of an isogenic hfi + host. We propo se that this delay in expression of the lytic functions in hfl-]: hosts favors lysogenization by increasing the functional effectiveness of a given amount of repressor. There is, however, no delay in the onset of lysozyme synthesis following infection of an hfi-1 host with a AcIcll double mutant, which means th at the hfl-1 induced delay is dependent upon a functional AcIl product as well. Therefore, the delay cannot be due to a generally decreased ability of th e hfl-1 host to support lytic growth, but must rather be a consequence of the abundant supply of a AcIII-like function by th e hfi -1 host, acting in conjun ction with the AcII product . This AcIII-like function provided by the hfi- 1 host could be identical to the AcIII fun ction, in which case one would suppose that an increased amount of >.elII function is more effective in inhibiting righ tward tr anscription than in promoting leftward transcript ion at pre. Conversely the AcIlI-like function provided by the hfl-1 host could be an analogue of, but not identical to, the AcIll funct ion. In this case one

INFECTION AN D INDUCTION OF E . Coli hfl- l

might suppose that thi s AcIIr ana logue has a great er efficiency in functioning as a repressor of rightward transcription than as an induc er of leftw ard transcription. Sin ce the molecular action of the AcIIl gene is not known, one can only speculate on t he way in which the hfl-). host pro vides a AcIII-like functi on . On t he one hand, the XcIII an d >-cII pro t eins could bind directl y to th e promot er pre (see review by Echols, 1972). If t his were true, the hjl-l host could b e providing a sub stitute binding prot ein. [This is reminiscent of certain leul) mutati ons, including deletions of the leul) gene, which may be suppressed by gene substitution (Kemper and Margolin, 1969).] On the other hand, the AcIII protein could promote the accumulation of a metabolite which th en binds to the AcII protein at pre. If t his were true, the lifi-): host might have a high endogenous level of this metab olite as a consequence of its genetic lesion. Infection of a nonlysogenic hfl-1 strain with Acl7 is abor tive, just as Ac17 infection of a lysogenic hfl+ st ra in is ab ortive. This nonproductive infection of an hft-l host is dependent upon fun ctional AcI and XcII genes. P ackman and Sly (1968) ascribe the killing of an hfi" lysogen by Xcl7 to constitutive DNA synthesis in the face of repression of some essential pha ge genes. This repression is believed to be facilitat ed by repressor coded by t he prophage. We similarly ascribe the killing of a nonlysogenic hfi-). strain to the channeling of Xc17 into the lysog enic pathway, coupled with constitutive DNA synthesis resulting from th e Ac17 mutation. We believe, however, t hat in Acl7infect ion of nonlysogenic hft-l st rains repression is facilit ated by a delay in lytic expression . These ideas are consistent with the findings that (1) repressor levels are no higher upon Acl7 infection of an I1jl-l. strain tha n upon infection of an hft': strain (L. Reichardt, personal communication) and also that (2) patterns of DNA synt hesis following Xcl7 infection of a nonlysogenic hjl-1 host and upon Ac17 infection of a lysogenic hft+ strain are similar. N orm al lysogens of Arim, U C4185 hfl+(A1'im), exhibit reduced immunity, presumably from a mutation in t he promoter for main taining lysogeny (prm) which leads to decreased cI gene t ra nscripti on . Immunit y

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is largely rest ored in UC2014 hfi-l(Arim) Iysogens, which could either be caused by an increase in the frequency of left ward transcription initiated at the promoter for establishing repressor syn thesis, pre, or by a redu ction in rightward tran scription of the cro-cl l- OiP operon. In eith er case, restoration of immunity would be a consequence of t he AcIII-like function supplied by the I1ft-l host. R egardl ess of th e detailed mechanism by which immunity is restored in hft-I (>-rim ) lysogens, one must postulate the pr esence of a small amount of XcII function in th e hft-1(Xrim) lys ogen. On the one hand, cIl function is required for leftward transcription at pre (R eichardt and Kaiser, 1971). On the other hand, the argument for reduced rightward transcription of the cra-cII-O-P operon rests upon the delay in the onset of lysozyme synthesis which is observed in lytically infect ed hft-I hosts, this delay being depend ent upon AcII function (Fig. 2). We have in fact shown that XcII function is liberally expressed in a normal Arim lysogen, since it complement s a sup erinfscting Ximm434cII phage for lysogenization. Although the AcII function is not expressed in an hft-l(Arim ) lysogen in amounts sufficient to complement a superinfecting phage for lysogenization , it is not unreasonabl e to assume that it is present in quantities sufficient to partially restore immunity, ACKNOWLEDGMENTS

We t hank M. Mahoney for his compete nt technical assista nce. We wish to thank particularly P. Chadwick and L. Reichardt for communicating the results of t heir repressor assays and for liv ely discussi ons. We are also thankful to D. Court, H. Ech ols, M. Gottesman, I. Herskowitz, M. Lieb, J. Parkinson, L. Reichardt, and H. Strack for discussions and for providing phage stocks. Thanks ar e als o due to 1. Herskowitz , II. Strack, and B. Rolfe for communicati ng t heir unpublished reo suits. Th is research was supported by NSF grant No. NSF·GB 32194 to D.W. REFERE NCES

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P ACKMAN, S., and SLY. W. (1968). Cons titutiv e lambda DNA repli cation by hel7 , a regulatory mu tant related t o virulence. V irology 3't, 778789. PERO, J. (1970). L ocati on of phage lambda gene responsible for turning off tJ-exonuol ease synthesis. Vir ology 40, 65-71. PTASH NE, M., and HOPKI NS, N . (1968). The operators controlled b y th e lambda ph age repressor. Proc. N at . A cad . S ci. U.S .A. 60, 1282-1287. R ADDING , C. (1966). Regulation of lambda exo uuclease . I . Properties of lambda exonuclease purifi ed fr om lysogens of Tll and wild- type. J. Mol . Biol. 18, 235-250. REICHARDT, L. , an d KAISER, A. (1971) . Control of lambda repressor synthesis. Proc. Nat . A car/. Sci. U.S.A . 68, 2185-2189. SHIMADA, K ., WEISBERG, R., and GOTTESMAN, M, (1972). Proph age lambda at unusual chr omosome locations. 1. Location of the secondary attachment sites and the properties of the lysogens. J. Mol. B iol. 63, 483-503. SIGNER, E ., MAN LY, K. , and BRU N I>TETI,~;R , M , (1969). Delet ion mapping of the cIII-N reg ion of the b ac ter iophage lambda. V irology 39, 137141. SLY, W., a nd R ABIDEAU, K . (1969). Mechanism of e17cl vir ulence. J. !rIaL B ioi. 42, 385-4 00. SLY, W., R ABIDEAU, K. , and K OLBER, A. (1971). T he mec han ism of lambda vir ule nce. II . Re gulat ory mutations in cla ssical virule nce. I ll, " The Bacteriophage L ambda" (A. D. H ershey , ed .), pp . 575-588. Cold Spr ing Harbor L aborat orv . Cold Sp ring Harb or, Ne w York. SPIEGELMAN, W. G ., HEINE~{AN, S. F., B RACH ~T, P. , PEREIRADA SILVA , L. , and EISEN, H . (1970). Regul ation of the synthesis of phage lamb da repressor . Cold S prin g H arbor Symp . Qua nt . Bioi. 35,325-330. STRACK, H., KAYSER, M., and HOLDER, S. (1970) . Reduced immunity in Iysogens of bacteriophage lambda due to a mutation in the prophage. Virology 42, 707-716. STRACK, H. , and ZrmGLER, R. (1969). A new class of clear mutan ts fr om coliphage 434hy not complementing ell and clII mutants for lysogenization. Mol. Gen . Genet . 106,80-88. SZYBALSKI , W., BOVRE, K ., FI ANDT, M., HAYES, S., Hil A-DEeNA, Z., KU}[AR, S., LOZERON, H ., NI.JKAMP, H ., and STEVENS, W. F . (1970) . T ranscripti onal unit s and their con trols in E scherichi a coli ph age lambda: Operons and script ons. Cold Spring H arbor S ymp. Quant . B i oi. 35, 341-353. THOMAS, R. (1971). Control circuit s. 111" T he B act eriophage Lambda" (A. D . Hershey , ed .), pp. 211-220. Cold Spring H ar bor L ab oratory, Cold Spring H arbor, N ew York.