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MICROBIOLOGICAL REVIEWS, June 1979, p. 199-223 Vol. 43, No. 201460749/79/02-0199/25$02.00/0

Shutoff of Host Macromolecular Synthesis After T-EvenBacteriophage Infection

JAMES F. KOERNER'* AND D. PETER SNUSTAD2

Department of Biochemistry, Medical School, University ofMinnesota, Minneapolis, Minnesota 55455,1 andDepartment of Genetics and Cell Biology, College of Biological Sciences, University ofMinnesota, St. Paul,

Minnesota 551(82

ITRODUCTION ............................... 199Overview of Problem ....................................................... 199Historical Background......................... ........ 200

POTENTLAL SITES OF SHUTOFF...................... 200Degradation of Host Deoxyribonucleic Acid 201Nuclear Disruption ............. ............................. .. 204Unfolding of the Host Nucleoid ........... ........... 209Ribonucleic Acid Polymerase Modifications ............... 213Ribosome and Transfer Ribonucleic Acid Modifications ........... .... 213

MECHANISM OF SHUTOFF .................................. 214CONCLUSIONS................ 216LI`I`ERATURE CITED .................................. 217

"Despite all that is known about T4 infection,there is still no explanation why host macro-molecular synthesis stops so promptly and com-pletely. This remains one of the major unsolvedquestions of molecular biology. One suggestionis that an effect on the membrane by phagepenetration secondarily influences the replica-tion and transcription of the host chromosometied to the membrane. Other evidence invokes aphage-induced protein whose effect is not de-fined. The attraction of this problem is not somuch a compulsive need to complete the cata-logue of events surrounding a phage infection,but more the promise of clarifying the spatialorganization and control of the E. coli chromo-some and with it another major insight intomolecular biology."

-A. Kornberg (72)

INTRODUCTIONOverview of Problem

The T-even bacteriophages (T2, T4, and T6)of Escherichia coli are among the most complexand also the most virulent bacterial virusesknown. It is only partly historical accident thatthey were among the earliest phages selected forintensive study by virologists and molecular bi-ologists; their dramatic life cycle demanded at-tention when the slower or more subtle changescaused by other viruses were easily misinter-preted or overlooked. Today other phages maybetter illuminate specific steps of certain proc-esses, but the T-even phages remain the modelfor viruses which promptly and completelyusurp host metabolism, assemble much of the

machinery for viral synthesis using instructionsfrom their own genome, and drastically altermuch of the host apparatus that they do use.The earliest studies on these viruses disclosed

that they shut offhost macromolecular synthesiswithin a few minutes after infection. Today theyremain quite unusual among known phages withrespect to the promptness and completeness ofthis shutoff. At the opposite extreme are phageslike 429 of Bacillus subtilis, for which hostmacromolecular synthesis continues untilshortly before lysis. Thus shutoff is not a uni-versal phenomenon but a specialized group ofmechanisms which apparently confer biologicaladvantages to certain viruses.The events surrounding shutoff after T-even

phage infection are exceedingly complex. Manyof the components involved in macromolecularsynthesis undergo change shortly after infection.These include modifications of the bacterial ri-bosomes and ribonucleic acid (RNA) polymer-ase, changes in spatial organization of the hostchromosome, and enzymatic degradation of thehost deoxyribonucleic acid (DNA). A majorchallenge today is to determine exactly which ofthese changes are primarily responsible for shu-toff. Of broader, and perhaps even more exciting,concern is to discover the real physiological rolesfor processes which have been uncovered in thesearch for mechanisms for shutoff but whichseem to lack a direct role in this process. Thus,directly or indirectly, the pursuit of mechanismsof shutoff still holds the promise, suggested inthe quotation above, of providing further insightinto the organization and control of the bacterialchromosome.

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200 KOERNER AND SNUSTAD

Historical BackgroundThe cessation of host macromolecular synthe-

sis after infection with T-even bacteriophagewas first observed more than 30 years ago. Al-though many other biochemical problems ofphage infection have subsequently been definedand worked out, information on the mechanismof shutoff remains very incomplete. In one of thefirst biochemical experiments done on phage-infected cells, Cohen and Anderson in 1946 (13)showed that respiration continues unabatedafter T2 infection. The following year, Cohen(11) reported the first observations on DNA,RNA, and protein synthesis in this system.Within a few minutes after infection RNA syn-thesis appeared to stop. There was a brief pauseof DNA synthesis shortly after infection, afterwhich this synthesis resumed at a greater ratethan in the uninfected cell. Net protein synthesiscontinued unabated after infection. In the sameyear, Monod and Wollman (90) reported that aninduced bacterial enzyme, ,8-galactosidase, is notsynthesized after T-even phage infection. Thisobservation and many subsequently reported onother enzymes suggested that at least some ifnot all bacterial protein synthesis stops shortlyafter infection. Thus, the proteins being formedat this time are presumably specified by thebacteriophage genome. Pulse-labeling experi-ments showed that much of the protein synthe-sis later than 10 min after infection could beattributed to the capsid proteins of the virus,but the roles of the proteins synthesized beforethis time were unknown (50). The nature ofsome of the early proteins became clear with thediscovery by Flaks and Cohen in 1957 (29) of thesynthesis of a phage-induced enzyme.These early data all suggested a model in

which host protein synthesis ceases entirelywithin a few minutes after infection and theprotein synthesis occurring after this time isentirely phage directed. More recently, this hy-pothesis has been rigorously demonstrated bytechniques using gel electrophoresis (83); theonly exception seems to be a few bacterial pro-teins associated with the outer membrane of thecell envelope (5, 57, 102).

Cohen's early suggestion that, within the sen-sitivity of his experiments, RNA synthesis stopsafter T-even infection was subsequently modi-fied by the important observation of Volkin andAstrachan (157) that a small analytically distinctfraction of RNA is synthesized after phage in-fection. This was one of the early observationsthat led to the hypothesis that short-lived mes-senger RNAs (mRNA's) are involved in proteinsynthesis. Nevertheless, Cohen's original obser-vation that the bulk of the RNA synthesis is

turned off at the time of phage infection remainsvalid to this day, and the mechanism of thisshutoff, like the mechanism responsible for theshutoff of host protein synthesis, remains un-known.The discovery of Wyatt and Cohen (177) that

T-even bacteriophage contain the unusual py-rimidine hydroxymethylcytosine provided a newtool for distinguishing host and viral DNA. Her-shey et al. (49) demonstrated that the only DNAsynthesized in T2-infected cells is hydroxy-methylcytosine-containing viral DNA. Thus, thenet DNA synthesis observed after T2 infectionby Cohen in his kinetic analysis includes twocomponents: the prompt cessation of host DNAsynthesis after phage infection and the subse-quent initiation of viral DNA synthesis. Likehost protein and RNA, host DNA synthesis isshut off after infection.

POTENTIAL SITES OF SHUTOFF

Because of the nearly simultaneous occur-rence of many events, early physiological exper-iments could provide little insight into the tem-poral sequence of reactions leading to shutoff ofhost macromolecular syntheses. The altemativeapproach of blocking shutoff at specific stepsbecame feasible with the isolation of mutantsrepresenting a large fraction of the phage T4genome. The first major step in this directionwas production of amber and temperature-sen-sitive conditionally lethal mutants by Epstein etal. (28). Ingenious procedures such as use ofhydroxyurea to block de novo deoxyribonucleo-tide synthesis (162, 163) provided a wider rangefor conditional lethality. Finally, a procedure forefficient mutagenesis of T4 with hydroxylamine(151) coupled with assay procedures for screen-ing by "brute force" have almost completed thetask of defining the T4 genome (172). The mappositions of genes of phage T4 discussed in thisreview are shown in Fig. 1.A fundamental fact emerging from study of

these mutants is that infection with T-evenphages initiates a multitude of events potentiallycapable of terminating synthesis of one or moreclasses of host macromolecules. These includesubunit modifications of the bacterial ribosomesand RNA polymerase, unfolding of the hostchromosome (loss of the RNA-maintained, neg-atively supercoiled loops or domains), nucleardisruption (movement of the host DNA intojuxtaposition with the cell membrane), and en-zymatic degradation of the host DNA. However,whether or not a given event is the primary oneresponsible for shutoff after infection with wild-type phage, or whether the event occurs afterhost synthesis has already been terminated by

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T-EVEN BACTERIOPHAGE INFECTION 201

FIG. 1. Map of T4 genes discussed in this review. This map is based on the detailed T4 map of Wood andRevel (172). Gene functions are designated as follows: (......) nucleotide metabolism and viral DNAsynthesis; (-----) DNA degradation; (F.) unfolding of the host chromosome, nuclear disruption; ( ) RNApolymerase modification; (no designation) marker genes and adjacent genes mentioned in the text.

another mechanism, is often less accurately de-fined. Thus, this discussion must presently onlydefine components with apparent potential forshutoff. Those which seem to be manifest atlater times after infection will be discussed first;thus the order of presentation will be the reverse

of the list given above. After these componentsare defined, current information on the hier-archy of their action in cells infected with wild-type phage will be presented.

Degradation of Host DeoxyribonucleicAcid

Early experiments, using isotopic labels,showed that a fraction of the viral DNA precur-

sors arose from degradation of the host DNA(12, 68, 75, 76, 166). The option of obtainingprecursors for viral DNA from the host DNArather than the medium is of clear evolutionaryadvantage to a parasite which can thereby pro-

liferate under circumstances limiting the supplyof nutrients. The act of degrading the host DNAmay also provide a mechanism for shutoff ofhost macromolecular synthesis. Whether or notthis is true, a parasite which evolves a mecha-nism for degradation of the host genetic materialmust also avoid self-destruction by evolving amechanism by which it can distinguish its owngenetic material from that of its host. In the caseof bacteriophage T4, a genetic system hasevolved which assures that its DNA is chemi-cally distinguishable from that of its host, E.coli. Shortly after infection of E. coli with phageT4, two viral genes are expressed which code forthe enzymes deoxycytidine triphosphatase anddeoxycytidylate hydroxymethylase. Deoxycyti-dine triphosphatase (product of gene 56) rapidlyremoves all deoxycytidine triphosphate from theintracellular pool (42, 70, 73, 158, 168, 181), anddeoxycytidylate hydroxymethylase (product of

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gene 42) catalyzes the conversion of deoxycyti-dine monophosphate (the product of deoxycyti-dine triphosphatase) to deoxy-5-hydroxy-methylcytidine monophosphate (19,29,30, 169).These two enzymes assure that all T4 phageDNA will contain hydroxymethylcytosine,whereas the host DNA contains cytosine. Inaddition, phage T4 codes for two glycosyl trans-ferases, a- and 8- (products of genes agt and,8gt), which catalyze the transfer of glucose fromuridine diphosphate-glucose to hydroxymethyl-cytosine bases in nascent phage DNA with theresult that 70% of the hydroxymethylcytosineresidues in T4 DNA are a-glucosylated and 30%are ,B-glucosylated (35, 36, 54, 60, 73, 74, 107,182). Thus, all of the cytosine in T4 DNA is bothhydroxymethylated and glucosylated. Thesechemical differences provide a simple mecha-nism for distinguishing between the DNA of thehost (E. coli) and the DNA of the parasite(phage T4) by means of enzyme specificity.The evidence available at present indicates

that at least one function of the glucosylation isto protect the viral DNA from the action of oneor more host nucleases (44), whereas the hy-droxymethylation protects the phage DNA fromself-destruction, i.e., from degradation by T4-induced nucleases responsible for degradation ofthe DNA of the host (69, 78, 161, 167, 168). Twocytosine-specific endonucleases induced byphage T4 have been identified and character-ized. One, T4 endonuclease II, induces single-strand breaks in cytosine-containing, double-stranded DNA (113). The second host-specificendonuclease, T4 endonuclease IV, induces sin-gle-strand breaks in exposed single-strand re-gions (gaps) of cytosine-containing double-stranded DNA (114).Mutants of phage T4 have been isolated and

mapped which are deficient in the ability toinduce endonuclease II (47, 106, 117, 163). Theseendonuclease II-deficient mutants (mutation ingene denA) induce little, if any, degradation ofthe host DNA (47, 134, 163). It appears likelythat T4 endonuclease II catalyzes the first stepin the degradation of the host DNA. T4 mutantswhich fail to induce the synthesis of endonucle-ase IV have also been isolated and mapped(mutation in gene denB; formerly D2a) (8, 116,156). The precise role of endonuclease IV is lessclear, however, since endonuclease IV-deficientmutants exhibit at most a minor block in thedegradation of host DNA (139, 156). One possi-ble role is to assure exclusion of cytosine fromviral DNA. It has been demonstrated that spe-cific degradation of cytosine-containing viralDNA occurs, and that endonuclease IV has arole in this process (77). However, a role in host

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DNA degradation, at least under certain circum-stances, is observed during infection with phagemutants deficient in inducing nuclear disruption(discussed in greater detail below). Althoughvery little, if any, host DNA is ordinarily de-graded to acid-soluble form in cells infected withendonuclease II-deficient phage (134), approxi-mately 50% of the host DNA is degraded to acid-soluble form by 90 min after infection with mul-tiple mutants deficient in inducing both endo-nuclease II and nuclear disruption (134). Deg-radation under these circumstances is dependenton endonuclease IV (99). The effect of nucleardisruption on host DNA degradation in the ab-sence of endonuclease II may be indicative of acompartmentalization effect, the host DNA sim-ply being more accessible to endonuclease activ-ity when it is distributed throughout the cyto-plasm than it is when present in close juxtapo-sition with the cell membrane (99, 134).Amber and temperature-sensitive mutants in

genes 46 and 47 of phage T4 are also blocked inthe complete degradation of host DNA, accu-mulating host DNA fragments of size 106 to 107daltons under restrictive conditions (78, 167).The gene 46 and 47 products have not beencharacterized; thus, it is not clear whether thesegenes play a direct or indirect role in host DNAbreakdown. It is unlikely that the conditionallethality exhibited by the gene 46 and 47 mu-tants is directly related to their block in hostDNA breakdown, since earlier blocks in thedegradation of host DNA have been shown tobe nonlethal (47, 163). Mosig and Bock (91) haveshown that T4 gene 32 protein (the T4 DNA-binding protein [2]) is necessary to moderate theactivity of the "gene 46-47 nuclease" and the E.coli recBC nuclease so that intermediates in T4DNA recombination are not degraded.The alternate slow pathway of breakdown of

host DNA, which occurs after infection withendonuclease II-nuclear disruption-deficientmutants, does not bypass the block imposed bygene 46 or gene 47 mutations (99, 134).These relationships are summarized in Fig. 2,

which is an updated version of a pathway fordegradation of host DNA proposed earlier (163).The diagram is clearly an oversimplified schemeintended to illustrate only the best establishedsteps and interactions. For example, there mustbe at least one other enzyme capable of catalyz-ing the step shown to be catalyzed by endonu-clease IV, since T4 mutants deficient in theability to induce endonuclease IV show, at most,a minor block in host DNA breakdown.No phage-induced or bacterial enzyme has

been shown to participate in either step of Fig.2 postulated to be catalyzed by an exonuclease.


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Gene denA(nd2 8)

Endonuclease

nuclear disruption)

Intact E. coliChromosomek2.5 X 109 daltons)

Gene denB Gene 46,47

Endonuclease IY-L Exonuclease? Nucleotides

d16 -107Cdaltons

FIG. 2. Proposed pathway for degradation of host DNA.

Although T4 exonuclease A, because of its spec-ificity and high activity, seemed a likely candi-date, phage mutants deficient in inducing thisenzyme (dexA mutants) (164) induce normaldegradation of host DNA in E. coli B underlaboratory conditions. The possibility that thedexA mutation is suppressed by E. coli exonu-

clease IV (59), an enzyme with specificity similarto T4 exonuclease A, cannot be demonstratedbecause bacterial mutants lacking this enzyme

have not been isolated. Another subject of un-

certain status is the relationship between genes

46 and 47 and the postulated exonuclease re-

quired for the final stage of degradation (78,167).Other T4 phage-induced nucleases have been

described. Endonuclease V (89, 178, 179) andexonuclease B (33, 97) have a demonstrated rolein repair of DNA which was damaged by ultra-violet light. Endonucleases I (3, designated en-

donuclease I later [172]), III (112), and VI (65)and the exonuclease activity associated with T4-induced DNA polymerase (41, 140) all attackboth phage and bacterial DNA. Roles for proc-

essing of the viral DNA have been suggested,but their possible action on host DNA in vivohas not been rigorously excluded.The validity of the proposed pathway is de-

pendent on the assumption that gap formation(the first exonuclease-catalyzed step) does notproduce measurable quantities of acid-solublematerial in the absence of endonuclease II,

either in the presence or in the absence of nu-clear disruption. Since there are fewer than fivebreaks per original bacterial strand at 5 minafter infection with endonuclease II-deficientphage, and only about 15 to 20 breaks by 30 minafter infection (134), this assumption seems rea-sonable.

Despite the complexity of patterns of DNAdegradation observed in cells infected with var-ious mutant phage, one fact has become clear:the shutoff of host DNA synthesis, RNA syn-thesis, and protein synthesis occurs normally inthe absence of host DNA degradation. This wasoriginally suggested by Nomura et al. (94) andhas been more rigorously documented using nu-clease-deficient mutants and techniques formeasuring integrity of genome-sized bacterialDNA (134). The only qualifications to this state-ment are: (i) one or two breaks per DNA strandmight have escaped detection under the experi-mental conditions described, and (ii) highly lo-calized damage, e.g., to a replicating fork, by anuclease releasing small amounts of mono- oroligonucleotides would not have been detected.Although host DNA degradation in cells in-fected with wild-type phage has a demonstrablerole in providing precursors for phage DNA, itspotential capability for shutoff has not beendemonstrated. This will only be possible whenmutants are found with defects in the earlierprocesses normally causing shutoff.The extent and role of host DNA degradation

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differs widely in various phage systems. Thus inB. subtilis phage 429, host DNA and proteinsynthesis continues until shortly before lysis(118, 119). Coliphage T7 is dependent on theproducts of host DNA degradation as precursorsfor phage DNA (79, 115). In contrast, coliphageT5 initiates prompt and drastic degradation ofhost DNA but discards the products of degra-dation into the medium (14, 160, 183). It isconceivable that DNA degradation by thisphage is responsible for shutoff of DNA synthe-sis.

Nuclear DisruptionDuring the early 1950s, when the initial bio-

chemical information was being accumulatedpertaining to degradation of the host DNA, cy-tological studies of infected cells provided an-other line of evidence with bearing on the phe-nomenon of host DNA synthesis shutoff. Within2 to 3 min after infection of E. coli cells with T-even bacteriophages, the nucleoids of the bac-teria undergo nuclear disruption, during whichthe host DNA moves from a largely centrallocation in the host cell into tight juxtapositionwith the cell membrane (6, 63, 84, 92). Duringthis dramatic virus-induced morphological rear-rangement ofthe host cell, the DNA moves fromthe center of the cell to the membrane and theribosomes move from the periphery of the cellto the center (Fig. 3).Nuclear disruption is induced by ultraviolet

light-inactivated T-even phages (84) but not byphage ghosts (6). It is blocked by the addition ofchloramphenicol at the tine of infection (64).These results indicate that nuclear disruption isunder the control of a phage gene or genes.However, all of the classical conditional lethalmutants of phage T4 isolated by Epstein et al.(28) were found to induce nuclear disruptionnormally.

It had often been assumed (142) that nucleardisruption is involved in host DNA degradation.However, it is now known that nuclear disrup-tion occurs normally in E. coli infected with aphage T4 mutant deficient in the ability to in-duce endonuclease II (136). These results indi-cate that either (i) the endonuclease II-catalyzedreaction is not the first step in host DNA break-down or (ii) nuclear disruption is independent ofnucleolytic cleavage of the host chromosome.Snustad and Conroy (133) isolated mutants of

phage T4 that are deficient in the ability toinduce nuclear disruption. The key to success inisolating such mutants was the development ofa simple procedure for examining bacterial nu-clear morphology and nuclear disruption after

phage infection by phase microscopy. This tech-nique involves simply spreading the cells on athin layer (approximately 0.7 mm) of 17.5% gel-atin. Photomicrographs (Fig. 3) do slight justiceto the spectacular view of the distinct nuclearmorphology observed visually in living prepara-tions under these conditions. Nuclear disrup-tion-deficient mutants (termed ndd mutants)were identified by visual screening of clones fromsurvivors of brute-force mutagenesis (151). Thendd mutations were found to map in a phagegene, D2b, previously identified by physicalmapping (123). Burst size and growth rate ex-periments demonstrated that ndd mutants growas well as wild-type T4 does in E. coli strain B/5 (am su-). Thus, nuclear disruption is not es-sential for phage T4 growth. Also, degradationof host DNA to acid-soluble products occurs atthe same rate in the absence of nuclear disrup-tion as it does in its presence, providing endo-nuclease II is present (134).Snustad et al. (134) examined the state of the

host DNA in cells infected with strains of phageT4 (a) deficient in endonuclease II, (b) deficientin both endonuclease II and nuclear disruption,and (c) multiply deficient in endonuclease II,endonuclease IV, and nuclear disruption, usingseveral experimental procedures, as follows. (i)The number of single-strand breaks in the hostDNA at various times after infection was ana-lyzed by alkaline sucrose density gradient cen-trifugation. (ii) The number of double-strandbreaks was estimated using neutral sucrose den-sity gradient centrifugation. (iii) The proportionof the host DNA remaining membrane bound atvarious times after infection was studied by theM-band technique (formation of sedimentablemembrane complexes with crystalline magne-sium lauroyl sarcosinate [27]). (iv) The degreeof "folding" present in host chromosomes ofinfected cells was measured by sucrose gradientsedimentation (146, 173). (v) The distribution ofhost DNA in infected cells was examined bythin-section electron microscopy.

Alkaline sucrose density gradients showedthat the presence or absence of nuclear disrup-tion had little, if any, effect on the rate of accu-mulation of single-strand breaks. At 5 min afterinfection there was an average of five or fewerbreaks per original bacterial strand. By 30 minafter infection with these mutants, each bacte-rial strand had accumulated about 15 to 20breaks. The results of neutral sucrose densitygradient centrifugation indicated that there wasno detectable change in the size of the host DNAat 5 min after infection. In all cases, the majorityof the host DNA sedimented at a rate corre-sponding to a molecular weight of greater than

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a mFIG. 3. Nuclear disruption. (a) Uninfected E. coli B/5; (b) E. coli B/5 10 min after infection with viral DNA

synthesis-deficient, endonuclease II-deficient T4phage (amN82 [gene 44]-nd28X6 [gene denA]); (c) E. coli B/5 10 min after infection with viral DNA synthesis-deficient, endonuclease II-deficient, nuclear disruption-deficient T4 phage (amN82 [gene 441-nd28x6 [gene denA]-ndd98 [gene ndd]). Top row: photomicrographsof E. coli cells spread on a thin layer of 17.5% gelatin and viewed by phase-contrast microscopy. Under theseconditions, the DNA pools appear light and the ribosome-containing regions appear dark (133). Magnificationmarker: 5 ,un. Bottom row: electron micrographs of thin sections of E. coli cells stained with uranyl acetateand lead citrate (133). Under these conditions, the DNA pools appear light and the membrane and ribosome-containing regions appear dark. Magnification marker: 1 ,.m. (Fig. 3c is on p. 206.)

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FIG. 3c.

109. A few double-strand breaks did accumulatein the host DNA by 30 min after infection withendonuclease II-deficient phage and with endo-nuclease II- and nuclear disruption-deficientphage, although the DNA still remained large

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(>108 daltons). Fewer double-strand breaks(possibly none) were detectable at 30 min afterinfection with endonuclease II, endonuclease IV,and nuclear disruption-deficient multiple phagemutants. These results exclude any mechanismfor nuclear disruption that is dependent on theoccurrence of extensive nicks or breaks in thehost DNA.Nuclear disruption could occur as either an

active process, in which the ndd gene product isresponsible for some type of interaction betweenthe host DNA and the cell membrane, or apassive process, which occurs whenever theforces and/or structures that maintain the nor-mal state of the host nucleoids are destroyed. Inthe latter case, the ndd gene product wouldfunction in disrupting the forces and/or struc-tures that hold the nucleoid of E. coli in its"folded" state in uninfected cells. The gene re-sponsible for nuclear disruption does not appearto be required for the unfolding process, how-ever, since it was shown that the chromosome ofcells infected with endonuclease II, endonucle-ase IV, and nuclear disruption-deficient phageare unfolded by 5 min after infection (134). Inaddition, it was demonstrated by thin-sectionelectron microscopy that the host DNA becomesdispersed throughout the cytoplasm in cells in-fected with endonuclease II- and nuclear disrup-tion-deficient phage. These results indicate thatnuclear disruption is not merely a passive proc-ess that occurs whenever the forces and/orstructures which maintain the integrity of the E.coli nucleoid are altered.M-band analysis demonstrated that the host

DNA starts to be released from the cell mem-brane at about 10 min after infection with wild-type T4 phage or mutants deficient in inducingboth endonuclease II and nuclear disruption(134). By 30 min after infection, about 75% ofthe host DNA is no longer membrane bound. Incells infected with endonuclease II-deficient T4phage or endonuclease II-, endonuclease IV-,and nuclear disruption-deficient T4 phage, onthe other hand, over 80% of the host DNAremains in the M band, even at late times afterinfection.The simplest interpretation of the results

summarized above is that nuclear disruptionafter T4 phage infection involves multiple at-tachment of the host DNA to the cell membrane(Fig. 4) (134). In ndd-infected cells, this multipleattachment would not occur, with the result thata limited number of double-strand breaks releasemuch of the host DNA from the cell membrane.The absence of release of host DNA from thecell membrane after infection with phage mul-tiply deficient with respect to endonuclease II,endonuclease IV, and nuclear disruption is prob-


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Endonuclease II-deficient T4

Endonuclease II, nuclear Endonuclease II, endonucleaseIY,disruption-deficient T4 nuclear disruption-deficient T4

FIG. 4. Diagrammatic representation of the pro-posed state of the host DNA in uninfected E. coli andin cells after infection with the respective T4 phagemutants. Attachment sites for the DNA (assumed tobe five in number) on the cytoplasmic membrane(stippled oval) of uninfected cells are indicated byopen circles. The additional phage-induced attach-ment sites that are postulated to be necessary fornuclear disruption are indicated by filled circles.Double-strand breaks in the host DNA are indicatedby discontinuities in the lines representing the hostDNA. (From Fig. 1, reference 71, and Fig. 6, reference134, reproduced with permission.)

ably explained by the fact that few, if any, dou-ble-strand breaks occur in the host DNA afterinfection with these phage. This interpretationis consistent with all of the available data, butmust be considered tentative until the attach-ment of the host DNA to the membrane can beobserved more directly. The ndd gene productcould mediate such multiple attachment of thehost DNA to the cell membrane either by cata-lyzing the binding of host DNA to a componentof the cell membrane or by serving as a struc-tural bridge, binding to both host DNA and a

component of the membrane. The results ofgene dosage experiments suggest that the nddgene product is required in stoichiometricamounts (at least in the ndd-restrictive E. colistrain CT447), thus supporting the second alter-native (132).Majumdar et al. (86) demonstrated that E.

coli DNA polymerase I becomes membranebound shortly after infection with phage T4.One might expect that the nicks and free endsproduced by endonucleolytic cleavage of hostDNA would subsequently bind to the mem-

brane-associated DNA polymerase I (41), result-ing in multiple attachment of host DNA to thecell membrane. However, the ndd gene is not


responsible for the attachment of E. coli DNApolymerase I to the cell membrane (132). Inaddition, the gene controlling this attachment ofpolymerase I to the cell membrane is not rIIA,rIIB, Dl, D2a, pla-262, or any gene within theapproximately 10,000 nucleotide pairs betweengenes 39 and 56 deleted in del(39-56)12 (132).The biological significance of nuclear disrup-

tion is still uncertain. Nuclear disruption mustprovide a selective advantage to T4 bacterio-phage. Otherwise, the gene responsible for nu-clear disruption (ndd) would surely have beeneliminated via spontaneous deletion and naturalselection. Three possible roles can be suggestedfor nuclear disruption (132): (i) early shutoff ofhost DNA biosynthesis, (ii) ability to grow instrains of E. coli carrying certain extrachromo-somal genetic elements, or (iii) compartmental-ization of the phage DNA biosynthesis pathwayfrom the host DNA degradative pathway.Host DNA synthesis does fail to shut off at

the normal time in cells infected with nucleardisruption-deficient (ndd) mutants (132), andthis delay of shutoff, from 4 to 10 min afterinfection, may be sufficient to explain the selec-tive advantage of ndd+ T4 phage. The energywasted on host DNA synthesis after infectionwith nddc phage would certainly be more effi-ciently used if expended on phage-specific bio-synthetic processes. It should be noted thatthere is no measurable difference in the growthrates of ndd+ and ndcl phage growing in thecommonly used laboratory strains of E. coliunder optimal conditions (133). This, of course,may not be the case under other conditions, forexample, conditions encountered by T4 in nat-ural p'opulations. A group of such strains, iso-lated from hospital patients, has been distrib-uted by Wood (see Wilson [170]). In one of thesestrains, CT447, the growth of the ndd mutantsis restricted (18, 132). Thus, in natural popula-tions, T4 phage encounter host strains in whichnuclear disruption is required for normal growth.The interesting question, of course, is why nu-clear disruption is essential in CT447 and not inother strains of E. coli.

In a standard one-step growth experiment(using early-log-phase cells and diluting to about2 x 10' cells per ml after adsorption), CT447cells infected with wild-type T4D yield 150 to200 progeny phage per cell, whereas CT447 cellsinfected with nuclear disruption-deficient T4mutants produce only about 15 phage per cell.If, however, CT447 cultures are grown to >108cells per ml and infected with T4 phage, evenwild-type T4, without dilution, no progenyphage are produced. Such infections of CT447abort early in the latent period. Nuclear disrup-tion occurs, but host DNA degradation does not

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occur. This cell density-dependent inhibition ofT4 phage growth is undoubtedly responsible forthe very poor growth of T4D+ on CT447 indi-cator lawns. The growth of CT447 cells them-selves does not exhibit this exaggerated densitydependence; rather, the effect is specific to phagegrowth.The cell density-dependent inhibition of T4

phage growth exhibited by CT447 made com-parative biochemical analysis of T4D+ and ndd-infected CT447 cultures impossible by standardprocedures. However, a nitrosoguanidine-in-duced mutant of CT447 has been isolated (via"nibbling" experiments) which supports thegrowth of wild-type T4D at higher cell densitiesand still restricts the growth of ndd mutants(132). Since wild-type T4 plates on this deriva-tive of CT447 with a high efficiency, it has beendesignated CT447 T4 plq+ (for T4 plaque').

Several parameters of T4 phage growth inT4D+- and ndd-infected CT447 T4 plq+ cellshave been examined in an attempt to determineat what stage the growth of the nuclear disrup-tion-deficient mutants is blocked (132). Nucleardisruption occurs normally after infection withndd+ phage and fails to occur after infectionwith ndcl phage, just as it does in other strainsof E. coli that are permissive hosts for the nddmutants. Host DNA degradation appears to oc-cur more rapidly in nddc-infected CT447 T4plq+ cells than in ndd+-infected CT447 T4 plq4cells. CT447 T4 plq+ cells infected with nucleardisruption-deficient T4 phage show the samedelay in the shutoff of host DNA synthesis as isobserved after infection of E. coli B/5 with nddmutants. Surprisingly, the rate of phage DNAsynthesis after infection of CT447 T4 plq+ withndd98x5 is about 75% of the rate observed afterinfection with T4D+. The burst size of ndd98x5is less than 5% of that of T4D+ in CT447 T4plq+, however. These results indicate that eitherthe phage DNA synthesized in CT447 in theabsence of nuclear disruption is defective insome way, or the function of the ndd gene prod-uct is essential in CT447 for some later stage inT4 development (such as late transcription ormaturation).

Alkaline sucrose gradient analysis of the DNAsynthesized in CT447 T4 plq+ cells after infec-tion with ndd mutants indicates that the DNAis not extensively nicked and concatemers (asindicated by single strands longer than matureT4 chromosomes) are formed (D. P. Snustadand R. C. Miller, Jr., unpublished data). Parallelelectron microscope studies of ndd-infectedCT447 T4 plq+ cells indicate that few filled orempty heads are formed (D. P. Snustad and C.J. H. Bursch, unpublished data). RNA synthesis

is quantitatively normal in ndd-infected CT447T4 plq+ cells. This was demonstrated by pulse-labeling with [3H]uracil, extracting nucleic acidsand removing DNA with ribonuclease-free de-oxyribonuclease, and showing that the labeledRNA was T4 specific by DNA-RNA hybridiza-tion using purified E. coli and T4 DNA prepa-rations (Snustad, unpublished data). A workinghypothesis is that the switch from early to lategene expression is defective at the level of eithertranscription or translation in ndd-infectedCT447. This possibility has not yet been tested.A possible explanation of why nuclear disrup-

tion is essential for T4 growth in CT447 hasbeen provided by the demonstration that theintroduction of F' factors into CT447 results ina loss of ndd restriction (S. Hefeneider, M.S.thesis, University of Minnesota, St. Paul, 1975).This has been done with two different F' factorscarrying nonoverlapping segments of the hostchromosomes. Thus, the loss of ndd restrictioncannot be explained by the simple dominance ofa gene carried on the F' factors (a gene resultingin lack of restriction of ndd mutants) to a chro-mosomal gene in CT447 responsible for nddrestriction. Moreover, the loss of ndd restrictioncannot be explained by an epistatic effect of thesex factor on the expression of chromosomalgenes, since subsequent removal of the F' factorby acridine orange treatment does not result inrecovery of ndd restriction. These observationsand the fact that CT447 males originate with alow frequency (only 1 to 2% of the CT447 cellsthat acquire the donor F' markers becomemales) are the most easily explained if the nddrestriction gene(s) of CT447 is located on a plas-mid that is incompatible with the F' factor. Itmay well be that the major selective advantageprovided by nuclear disruption has nothing todo with the shutoff of the expression of the hostchromosome, but results from a role in the earlyshutoff of the expression of T4-incompatibleplasmids, in the presence of which T4 could nototherwise reproduce.There is also some evidence that indirectly

supports the hypothesis that nuclear disruptionmight play a compartmentalization role, sepa-rating the pathway of degradation of host DNAfrom the pathway of biosynthesis ofphage DNA.Since both processes are occurring at the sametime in infected cells, some mechanism for sep-arating intermediates in the degradation of hostDNA from intermediates in the replication ofphage DNA would seem desirable. The T4 DNApolymerase (gene 43 product) binds to nicks andends of both host and phage DNA and alsoreplicates both cytosine- and hydroxymethylcy-tosine-containing DNA in vitro (41). If T4 DNA

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polymerase exhibits these properties in vivo andif the pool of intermediates in host DNA degra-dation is not separated from the pool of repli-cating phage DNA, then one might expect mostof the T4 DNA polymerase to become bound tointermediates in host DNA breakdown. If nu-clear disruption is the process normally respon-sible for separating these two pools, one might,superficially at least, expect the nuclear disrup-tion-deficient mutants to grow less rapidly thanwild type. The fallacy of this reasoning is thatT4 DNA polymerase is probably synthesized inexcess under optimal growth conditions (130).Moreover, the synthesis of DNA polymerase is"self-regulated," that is, when the amount offunctional polymerase is limiting, increased syn-thesis of polymerase occurs (109). Thus, underoptimal growth conditions, if DNA polymeraseactivity were to become limiting because manyof the polymerase molecules became bound tofragments of host DNA, this would trigger in-creased synthesis of polymerase to compensatefor the loss of activity.The possibility that nuclear disruption serves

a compartmentalization role is supported by thecomparison of thin-section electron micrographsof cells infected with endonuclease 11-deficientmutants and cells infected with DNA synthesis-deficient, endonuclease II-deficient double mu-tants (132). When host DNA degradation isblocked (endonuclease II) but phage DNA syn-thesis is allowed to occur normally, two distinctpools ofDNA are apparent (Fig. 5). When phageDNA synthesis and host DNA degradation areboth blocked, all of the DNA is located at theperiphery of the cell (Fig. 3b). This indicatesthat the largely centrally located (electron-densemembrane associations are also observed) poolsin cells infected with endonuclease II-deficientphage are the pools of replicating phage DNA.A conclusion as to whether or not the role ofnuclear disruption is indeed to separate the hostDNA degradation pathway from the phageDNA biosynthetic pathway with respect to theT4 DNA polymerase must await further evi-dence.

Recently, a phage-induced protein that is ab-sent in cells infected with ndd mutants has beenobserved by polyacrylamide gel electrophoresis(J. F. Koerner, S. K. Thies, and D. P. Snustad,unpublished data). It has a molecular weight of15,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This is con-sistent with the coding capacity ofthe ndd (D2b)gene determined by physical mapping (18). Thusthis protein may be the product of the ndd gene.Pulse-labeling experiments show that the nddprotein is synthesized in large quantities from


shortly after infection (consistent with earlierevidence that it is a pre-early gene product) (123,133) until about 12 min after infection. Veryrecent experiments have demonstrated that thisprotein is associated with a sedimentable frac-tion which may be derived from the cell enve-lope. It is the most prominent protein observedwhen the sedimentable fraction of cells labeled3 to 6 min after infection is subjected to electro-phoresis on urea-acetic acid polyacrylamide gels.Further experiments will be required to deter-mine whether it is associated with the plasmamembrane and whether it binds to DNA.

In summary, nuclear disruption is responsiblefor the early (=4 min postinfection) shutoff ofhost DNA synthesis by wild-type T4 phage(132). In its absence, however, host DNA syn-thesis (at least net incorporation of [3H]thymi-dine) is shut off 10 min after infection (132).There is no evidence that nuclear disruption isinvolved in the shutoff of host RNA and proteinsynthesis. The latter are shut off with normal(T4D+-induced) kinetics in the absence of nu-clear disruption (131, 132).

Unfolding of the Host NucleoidThe chromosomes of bacteria such as E. coli,

when isolated very gently in solutions of highionic strength, exist as compact structures con-ferring relatively little viscosity to solutions (146,173). When these isolated structures are exam-ined by electron microscopy, they are found tohave approximately the same dimensions as thecell nucleoid in vivo (101). These folded genomesor nucleoids have been shown to consist of nu-merous (12 to 80) negatively supercoiled loopsor "domains," whose structural integrity is de-pendent on RNA and protein (16, 17, 100, 173-175). If E. coli cells are lysed at 0 to 10°C, thefolded chromosomes remain attached to the cellmembrane and sediment at 3,000-4,OOOS,whereas if the cells are lysed at 25°C or if thelysates are treated with 1% sodium lauroyl sar-cosinate (Sarkosyl), the folded chromosomes ap-pear to be released from the membrane andsediment at 1,300-2,200S (26, 101, 110, 174). The"membrane-free" folded genomes consist ofDNA, RNA, and a small amount of protein,primarily core RNA polymerase. Ryder andSmith (111) found the sedimentation velocity ofmembrane-associated folded genomes to be af-fected in some unknown way by the activity ofthe E. coli dnaC gene product just prior to theinitiation of chromosome replication.Treatment of cells with rifampin causes the

nucleoids of E. coli to unfold, at least as isolatedusing the standard procedures in 1 M sodium


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i

FIG. 5. Apparent compartmentalization of host and viral DNA. Thin-section electron micrograph of E.coli B/5 cells fixed 20 min after infection with endonuclease II-deficient T4 phage (nd28x6 [gene denA].Magnification marker: I um. Compare this photograph with Fig. 3b, in which viral DNA synthesis did notoccur. (From Fig. 9, reference 132, reproduced with permission.)

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chloride (26, 100, 101). Dworsky (25) has pre-sented evidence for two distinct states of thefolded genomes relative to their stability underdifferent conditions of isolation. He has demon-strated a "less stable" state (which unfolds in 1M NaCl under the commonly used lysis condi-tions), which exists after rifampin treatment.These results suggest that transcription is re-

quired to maintain the "stable" folded genomestate.Tutas et al. (155) discovered that the nu-

cleoids of E. coli are rapidly unfolded (within 5min) after infection with wild-type T4 or thehostDNA degradation-deficient mutant nd28x6(gene denA, endonuclease 11-deficient). This T4-induced unfolding of the host genome does notoccur after infection with heavily ultravioletlight-irradiated phage or in the presence ofchloramphenicol (155). Snustad et al. (134) dem-onstrated that the T4 gene responsible for nu-

clear disruption does not control the phage-in-duced unfolding of the folded genomes of E. coli,since the host chromosomes are unfolded within5 min after infection with nuclear disruption-deficient, host DNA degradation-deficient mul-tiple mutants. More recent studies (135) showthat the nucleoids of E. coli cells are rapidlyunfolded at about 3 min after infection withwild-type T4 bacteriophage or with nuclear dis-ruption-deficient, host DNA degradation-defi-cient multiple mutants of phage T4. Unfoldingdoes not occur after infection with T4 phageghosts. Experiments using chloramphenicol toinhibit protein synthesis indicate that the T4-induced unfolding of the E. coli chromosomes isdependent on the presence of one or more pro-teins synthesized 2 to 3 min after infection.Snustad et al. (135) also identified and par-

tially characterized a mutant of T4 which isdefective in the phage-induced unfolding of thehost genome. Mass screening of clones of a mu-tagenized culture of phage for such mutants wasmade possible by development of a simple assaycalled the "pour test." Suspensions of infectedcells were lysed under conditions which preserve

folded chromosomes. The folded or unfoldedstate of the DNA was judged by the obviouslyhigher viscosity of the latter cultures when theywere poured.The mutant, termed untf (for "unfoldase de-

ficient"), maps near gene 63 between genes 31and 63 and identifies a new gene. The foldedgenomes of E. coli cells remain essentially intact(2,000-3,000S) at 5 min after infection with un-

foldase-deficient, nuclear disruption-deficient,host DNA degradation-deficient T4 phage. Nu-clear disruption occurs normally after infectionwith unfoldase-deficient, host DNA degrada-tion-deficient, but nuclear disruption-proficient


(ndd') phage. The host chromosomes remainpartially folded (1,200-1,800S) at 5 min afterinfection with the unfoldase single mutantunf39x5 or an unfoldase-deficient, host DNAdegradation-deficient, but nuclear disruption-proficient strain. The unfoldase mutation doesnot affect the rate of host DNA degradation, nordoes its presence in nuclear disruption-deficient,host DNA degradation-deficient multiple mu-tants alter the shutoff of host DNA or proteinsynthesis.The two most obvious explanations of the T4-

induced unfolding of the host chromosomes are(i) that the phage genome codes for an unfoldaseprotein, such as a specific ribonuclease, which isdefective in the unf mutant or (ii) that unfold-ing is an indirect effect of the inhibition of hosttranscription. As was mentioned above, thechromosomes of cells in which transcription hasbeen inhibited by rifampin unfold, suggestingthat continual RNA synthesis is necessary tomaintain folded genomes. This leads one to pre-dict that host chromosomes would unfold afterinfection with T4 ghosts, since these DNA-freephage coats prepared by osmotic shock areknown to inhibit host RNA synthesis (22). How-ever, the host nucleoids do not unfold afterinfection with T4 ghosts. The most likely expla-nation of the rifampin and ghost effects is thatwhether or not unfolding will occur as a resultof inhibiting transcription depends on the pre-cise mechanism of inhibition. Rifampin preventsthe initiation of new transcripts, but allows thecompletion and release of transcripts alreadyinitiated at the time of treatment. Rifampintreatment would be expected, therefore, to leadto genomes free of nascent RNA. Ghost infec-tion, on the other hand, may lead to a generalinhibition of energy metabolism (32). In thiscase, the formation of all RNA transcripts mightbe arrested regardless of their stage of synthesis,resulting in genomes that are transcriptionallyinactive but associated with their normal con-tent of nascent RNA molecules. In the nascentRNA-free form, the genomes may unfold; in thenascent RNA-containing forn, they may remainfolded. On the basis of this evidence, the mostobvious explanation of the unf- mutant is thatit is defective in the shutoff of host transcription.

Sirotkin et al. (128), in fact, claim that unf-mutants which they isolated as mutants thatallow late transcription on cytosine-containingDNA (akc mutants [137]) do not shut off hosttranscription. However, these results have beencriticized by Tigges et al. (152). Their geneticdata are consistent with the conclusion of Sir-otkin et al. that alc and unf are probably thesame gene. Their DNA-RNA hybridization data,however, are inconsistent with the claim that


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the unflalc mutants do not shut off transcrip-tion. A major reservation in accepting the resultsof Sirotkin et al. is their observation that 30% ofthe RNA synthesized between 14 and 16 minafter infection with T4 wild type is E. coli RNA.Many studies (66, 67, 80, 96) have failed to

detect significant amounts of E. coli-specificRNA synthesis beyond about 5 min after infec-tion. In all these studies, pulse-labeled RNA washybridized to both E. coli DNA and T4 DNA.Sirotkin et al., on the other hand, only carriedout hybridization of their pulse-labeled RNAsamples to E. coli DNA. Thus they may haveoverestimated the amount of E. coli-specificRNA synthesis occurring at late times after in-fection with both wild-type T4 and all.The shutoff of host transcription after infec-

tion with unf/alc mutants has been reexaminedby P. Dennis (personal communication) usingDNA of various lambda transducing phages ashybridization probes. His experiments were doneusing lambda chromosomes carrying (i) the lacz gene, (ii) genes for 15 ribosomal proteins plusthe a RNA polymerase subunit, (iii) genes forthe ,B and P' RNA polymerase subunits, and (iv)ilv genes (isoleucine-valine genes). RNA pulse-labeled with [3H]uracil between 9 and 10 minafter infection with a wild-type T4 phage, analcl strain (137), or an isogenic alc+ strain washybridized to the above-mentioned probes andto T4 DNA. Dennis concluded that althoughalcl may be somewhat slower in turning off hosttranscription, virtually no host transcription oc-curs after 9 min postinfection. A very low butdetectable amount of transcription of the ribo-somal protein genes was observed after alclinfection. However, higher proportions ofsurviv-ing cells were observed in experiments exhibitinghigher amounts of ribosomal protein gene tran-scription. No differences were observed in tran-scription levels of the E. coli lac, ilv, rpoB, orrpoC (lactose, isoleucine-valine, RNA polymer-ase ,B and ,') genes between 9 and 10 min afterinfection with alc- and alc+ phage.

Nevertheless, it does seem likely that someunflaic mutants are altered in the shutoff of thesynthesis of at least some species of host RNA.R. Buckland and A. Travers (A. Travers, per-sonal communication) have shown that cells in-fected with the original unf- mutant (135) syn-thesize several times as much ribosomal RNA(rRNA) between 5 and 10 min after infection ascells infected with wild-type T4. Moreover,Buckland and Travers (personal communica-tion) have shown that the RNA polymeraseisolated from unf39X5-infected cells is more sen-sitive to inhibition of rRNA synthesis by gua-nosine 5'-diphosphate 3'-diphosphate (154) than

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RNA polymerase isolated from T4+-infectedcells. Travers (154) has presented evidence thatthe E. coli RNA polymerase holoenzyme canexist in at least two different conformations, onewith a high affinity for rRNA promoters and onewith a low affinity, and that guanosine 5'-di-phosphate 3'-diphosphate stabilizes the enzymein the low affimity state. Baralle and Travers (4)showed that T4 infection has the same effect onRNA polymerase with respect to rRNA synthe-sis as guanosine 5'-diphosphate 3'-diphosphate.The addition of guanosine 5'-diphosphate 3'-di-phosphate does not decrease the fraction ofrRNA synthesized by the enzyme from T4-in-fected cells, in contrast to its effect on the en-zyme from uninfected cells. Baralle and Travers(4) proposed that one of the four T4 proteinsnoncovalently attached to the E. coli RNA po-lymerase (143) stabilizes the enzyme in therRNA low affinity conformation. Sirotkin et al.(128) reported that RNA polymerase from alc-infected cells is missing one of these proteins,the 15,000-dalton polypeptide. However, whenRNA polymerase from alc-infected cells is con-centrated by antibody precipitation and exam-ined on polyacrylamide gels, the 15,000-daltonpolypeptide is present (C. G. Goff, personal com-munication). Furthermore, it now appears thata mutation which somehow affects the structureof the 15,000-dalton polypeptide maps in a dif-ferent region of the genome (near rII) than unflalc (Goff, personal communication). Thus, thefactors involved in shutoff of rRNA remain un-certain. Of particular significance is the fact thata few unflalc mutants have no detectable effecton the kinetics of shutoff (152).The effect of the unflalc mutations on the

shutoff of host mRNA synthesis is less clear.However, the results of Tigges et al. (152) andP. Dennis (personal communication) requirethat most mRNA synthesis be shut off by 8 to10 min after infection with unf- mutants.

R. Buckland and A. Travers (A. Travers, per-sonal communication) observed that the RNApolymerase from unf39X5-infected cells fails tobind the restriction fragment ofphage )80 PSUIII+carrying the suljl+ transfer RNA (tRNA) pro-moter and some p80 promoter fragments, likethe RNA polymerase from T4+-infected cellsand unlike the RNA polymerase from uninfectedE. coli cells. Nomura et al. (96) have shown thatthe shutoff of host stable RNA responds differ-ently from the shutoff of mRNA to chloram-phenicol and varying multiplicity of T4 infec-tion. The putative role of the unflalc gene in theshutoff of host mRNA synthesis clearly needsfurther study before any conclusions can bedrawn.


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T-EVEN BACT}ERIOPHAGE INFECTION 213

Ribonucleic Acid PolymeraseModifications

T4 phage transcription is known to be de-pendent on the E. coli DNA-dependent RNApolymerase (43). The host RNA polymerase ismodified in several ways after infection, however(39, 124, 153). In particular, four T4-inducedpolypeptides become tightly associated (nonco-valently) with the E. coli RNA polymerase afterinfection (143). Two of these polypeptides (theproducts of genes 33 and 55) are required for thesynthesis of T4 late RNA (51, 105). The othertwo, molecular weights 15,000 and 10,000, areprime candidates for roles in host RNA synthesisshutoff. As mentioned in the preceding section,Sirotkin et al. (128) have concluded that the15,000-dalton protein is the product of the unflalc gene (a conclusion presently being reexam-ined in other laboratories). Stevens and Rhoton(145) and Stevens (144) have presented evidencethat the 10,000-dalton polypeptide, still unde-fined genetically, may be involved in the inacti-vation of the RNA polymerase sigma subunit. Ifso, it should play a key role in the shutoff of hostRNA synthesis.

In addition to the new polypeptides bound tothe E. coli RNA polymerase, adenosine diphos-phate (ADP)-ribose moieties are added to thea-subunits of RNA polymerase after T4 infec-tion (37). Two genes control the ADP-ribosyla-tion of the a-subunits (53). One, termed alt foralteration, codes for a protein that is carried inthe virion and is injected into the host cell withthe phage DNA (38, 53, 108). Alteration doesnot require the expression of the infecting phagegenome (37). It results in the ADP-ribosylationof only a fraction of the host RNA polymerasemolecules. The a-subunits show the highest de-gree of ADP-ribosylation, but the other subunitsare also altered to a lesser degree (37, 108). Thesecond gene, termed mod for modification, re-quires phage-specific protein synthesis after in-fection and results in the ADP-ribosylation ofall RNA polymerase a-subunits (37, 52, 53).Clearly, alt and mod would appear to be idealcandidates for roles in the genetic control of hostshutoff.

Single mutants deficient in alteration andmodification, respectively, have been shown toshut off host RNA and protein synthesis nor-mally (53). Likewise, the alt -mod- double mu-tant, which fails to induce any ADP-ribosylationof RNA polymerase after infection, shuts offsynthesis of E. coli rRNA with the same rapidkinetics as wild-type T4 (C. G. Goff, personalcommunication). That does not mean that altand mod are not involved in host shutoff, or

even that they are not capable of shutting offhost RNA synthesis by themselves. It merelyindicates that they are not solely responsible forhost shutoff. It is very possible that obligatepathogens such as the T4 virus have evolvedredundant mechanisms for the shutoff of theexpression of the host genome. This would max-imize their control of the metabolic machineryof the host cell (i.e., minimize the frequency atwhich the host could mutate to a "T4-shutoff-resistant" state). Thus, to obtain a T4 strain thatdoes not shut off the host, one may well have toconstruct a complex multiple mutant carryingalt, mod, unf, ndd, and possibly other mutations.

It is instructive to compare these mechanismswith those of other phages. In Ti coliphageinfections, host mRNA synthesis continues afterinfection, but f)-galactosidase cannot be induced(87). In T7, the earliest phage genes to be ex-pressed are transcribed by unmodified hostRNA polymerase (7, 58, 125). Among the earlygene products induced by this phage are a novelRNA polymerase comprised of a single 110,000-dalton peptide (9, 93) and a protein kinase thatprobably catalyzes inhibition of host RNA po-lymerase (98, 180). Late transcription is cata-lyzed by the phage-induced RNA polymerase. Alarge virulent phage of B. subtilis, SPOl, alsocatalyzes early gene transcription with unmodi-fied host RNA polymerase. One of the proteinscoded by an early transcript replaces the hostsigma factor, and the modified host polymerasenow transcribes only phage middle genes. Twoproducts of these genes further modify the hostpolymerase for transcription of late genes. Thusthe host core polymerase remains activethroughout infection, and shutoff of host tran-scription accompanies its modifications for tran-scription of phage genes (31, 34).

Ribosome and Transfer Ribonucleic AcidModifications

Smith and Haselkorn (129) first reported thatnew, T4-induced proteins become associatedwith the host ribosomes after infection. At leastone of the proteins is synthesized early after T4infection. Hsu and Weiss (56) first demonstratedthat the T4-induced alteration of the host ribo-somes results in changes in translational tem-plate specificity-namely, restriction of transla-tion of host RNAs while allowing normal trans-lation of T4 RNAs. These observations weresubsequently verified and extended in severallaboratories (20, 81, 103, 104, 120, 126, 127, 141).Studies by Dube and Rudland (20) and Steitz etal. (141) indicate that ribosomes from T4-in-fected cells have altered mRNA binding prop-erties. These altered properties were shown to

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be dependent on the initiation factor fractionsof the infected cells. Lee-Huang and Ochoa (81)have proposed on the basis of their work that E.coli contains two F3 initiation factors, at leastone of which is inactivated after T4 infection. Amore extensive study by Rahmsdorf et al. (104)has shown that T4 infection results in the addi-tion of at least three, and probably four, newproteins to the ribosomes. These polypeptidesare present on ribosomes after high-salt (1 MNH4Cl) washes and thus do not include theinitiation factors such as F3 mentioned above.They were separable from the ribosomal pro-teins from uninfected cells by two-dimensionalpolyacrylamide gel electrophoresis.

Extensive biochemical data are thus availabledocumenting the T4-induced modifications ofthe host ribosomes. Unfortunately, however, noinformation as to the genetic control of thesemodifications is available. Clearly, definitive in-formation as to the putative role(s) of theseribosomal modifications in host translation shut-off (see the following section) can most easily beobtained by isolating and studying mutants de-ficient in the ability to induce each of the mod-ifications.

Modifications of the tRNA pools of E. colicells after T-even phage infection are also welldocumented. These include structural modifi-cations of host tRNA's (61, 62, 147, 148, 169) andthe synthesis of eight new tRNA's specified bythe phage genome (15, 88, 121, 170, 171). Kano-Sueoka and Sueoka (62, 148) have proposed thatthese tRNA changes may explain the arrest ofhost protein synthesis. Specifically, they pro-posed that "phage T2 infection induces a specificribonuclease that cleaves leucyl-tRNA, at thesite of the translation of E. coli mRNA on ribo-somes; this, in turn, leads to the cessation of hostprotein synthesis." Whereas the modification ofthe host leucyl-tRNA, described by Kano-Sueoka and Sueoka (62, 148), may be involvedin the altered pattern of protein synthesis ob-served after T2 phage infection, it seems some-what unlikely to be totally responsible for thevery rapid, complete shutoff of host protein syn-thesis, since only about one-half of the leucyl-tRNA is inactivated. The eight tRNA's specifiedby the T-even phage genomes (15, 88, 121, 171)are clearly not required for the shutoff of hostprotein synthesis because deletion mutants ofphage T4 lacking all the phage-specific tRNAgenes are known to turn off host protein synthe-sis with apparently normal kinetics (170).

MECHANISM OF SHUTOFFThe once popular proposal that phage inhibit

macromolecular synthesis (DNA, RNA, and

protein synthesis) of the host cell by the samemechanisms as phage ghosts (DNA-free phagecoats prepared by osmotic shock) no longerseems tenable. The results of French and Simi-novitch (32) suggested that the shutoff of mac-romolecular synthesis in ghost-infected cellsmight be the result of the inhibition of energymetabolism. An inhibition of energy metabolismcould not occur during a productive phage infec-tion, of course. Lehman and Herriott (82), how-ever, observed the same rates of respiration inuninfected, phage-infected, and ghost-infectedcells. Further studies on the effects of ghostinfection on host energy metabolism seem nec-essary before any definite conclusions can bedrawn (21).The differences between the effects of ghosts

and intact phage on host metabolism have beenreviewed in detail by Duckworth (21). The majordifferences between ghost-infected cells andphage-infected cells are (i) that host macromol-ecules continue to be synthesized for at least 3min after phage infection (46, 55, 66, 80, 122),whereas the attachment of ghosts appears tocause an almost immediate inhibition of all mac-romolecular synthesis (22, 32, 48, 82), and (ii)the complete inhibition of macromolecular syn-thesis requires protein synthesis after phage in-fection but not after ghost attachment (10, 23,40, 95, 96).

After a very detailed discussion of the effectsof ghost infection and phage infection on hostmetabolism, Duckworth (21) proposed a veryinteresting and simple mechanism to explain theearly events following phage and ghost infec-tions."The hypothesis that I believe best fits all the

known facts regarding the earliest events inphage and ghost infection is the following: thatattachment of the phage protein coat to the cellwall causes events which result in allostericchanges in some membrane components andlead to functional detachment of the host DNAand protein-synthesizing systems, and also causeloss ofother membrane-associated functions; theinjection of the phage DNA or internal protein(or both) and its attachment to the cell mem-brane allows the membrane to retain its func-tionality, but in a slightly altered state thatallows the production of phage macromoleculesin lieu of host DNA and RNA." (21, reprintedwith permission)

It is clearly difficult to prove or disprove theexistence of "functional detachment" of the hostDNA from the cell membrane; however, we nowknow that the host DNA retains its physicalattachment to the cell membrane after ghostinfection (136). In cells infected with nuclear

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disruption-deficient and host DNA degradation-deficient T4 phage, most of the host DNA isreleased from the membrane (134). In all ofthese cases, host protein synthesis is shut off (22,135). Thus, the mere existence of attachment ordetachment of host DNA from the cell mem-brane would not appear to explain the inhibitionof host cell metabolism. Duckworth's (21) pro-posal of a functional detachment of the hostDNA from the membrane cannot, of course, beruled out by these results.

Also the results of Takeishi and Kaji (149)indicate that the ribosomes of cells infected withT4 ghosts in the presence of 10 mM Mg2e areactive, whereas those from cells infected in thepresence of 1 mM Mg2e are inactive. Moreover,they reported that, in the presence of 10 mMMg2+, adenosine triphosphate, guanosine tri-phosphate, phosphoenolpyruvate, and pyruvatekinase, T4 ghost-infected cells synthesized ,B-ga-lactosidase after induction with isopropyl thio-galactoside (150). These results support the ideathat the rapid cessation of protein synthesisobserved after T4 ghost infection is caused, atleast in part, by a selective leakage of certaincritical small molecules such as nucleotides frominfected cells (24, 150).Whatever the effects of ghost and phage in-

fection on host metabolism, the requirement ofprotein synthesis prior to the inhibition of hostmacromolecular synthesis after phage infectionsuggests that the expression of one or morephage genes is involved in the shutoff process orprocesses after phage infection. The identifica-tion and elucidation of the mode of action of thisgene or genes are prerequisite to understandingphage-induced shutoff of host metabolism.The results of Snustad et al. (132) suggest that

nuclear disruption is responsible for the early 4-min shutoff of synthesis of host DNA after T4phage infection. No information is available,however, as to the genetic control of the shutoffof host DNA synthesis occurring at about 10min after infection with nuclear disruption-de-ficient mutants.Phage T4 clearly exerts genetic control over

the expression of the host chromosome at boththe transcriptional level (1, 80, 95, 96) and thetranslational level (45, 66, 67). In the unf mu-tants (135), where host RNA synthesis shutoffoften is slow (152), host protein synthesis is shutoff at the normal time (135). Snyder and hiscolleagues report that translation is shut off afterinfection with akc mutants, under conditionswhere they claim RNA synthesis is not shut off(128). Thus, there is agreement that T4 infectionbrings about the arrest of host mRNA transla-tion.


Most of the attention given to the mechanismof the T4-induced shutoff of host transcriptionhas focused on the numerous modifications ofRNA polymerase that occur after viral infection(discussed earlier). As mentioned, mutants defi-cient in alteration and modification, respec-tively, shut off host transcription like wild-typeT4.

Clearly, the unflalc gene plays some role inhost RNA synthesis shutoff. This may be a keycomponent of transcription shutoff (if the con-clusion of Sirotkin et al. [128] is correct), or itmay be just one component of a more complexshutoff mechanism (if the data of Tigges et al.[152] and P. Dennis [personal communication]are indicative of the true situation).Mailhammer et al. (85) observed a reduction

of-E. coli f,-galactosidase synthesis in a coupledin vitro protein-synthesizing system when RNApolymerase from T4-infected cells was used. Onthe basis of reconstruction experiments, theyconcluded that "modification of the a-subunit ofthe RNA polymerase is sufficient for inhibitionof host transcription." Their results thus indi-cate a role for the ADP-ribosylation of the a-subunit of RNA polymerase in the shutoff ofhost mRNA synthesis. They observed a T4DNA concentration-dependent inhibition of E.coli transcription in their in vitro system andsuggested that Horvitz's experiments (53) mayhave been done using too high a phage multi-plicity to see the true effects of alteration andmodification on shutoff of host-specific tran-scription.

Stevens and Rhoton (145) and Stevens (144)have evidence indicating that the 10,000-daltonpolypeptide found associated with RNA poly-merase after T4 infection inactivates or altersthe sigma subunit. Assuming that sigma is re-quired for in vivo protein synthesis, this 10,000-dalton protein would be expected to inhibitRNAsynthesis.Thus, present evidence implicates the ADP-

ribosylation of the a-subunit of RNA polymer-ase, the 10,000-dalton and possibly the 15,000-dalton RNA polymerase-associated proteins,and the product(s) of the unflalc gene(s) in theT4-induced shutoff of host transcription.Results from our laboratory (132-135) prompt

us to suggest that the effects of alteration, ormodification, ofthe addition of the 10,000-daltonpolypeptide to RNA polymerase, and of othergene products that might be involved in hostshutoff, might not be detectable in the presenceof nuclear disruption (ndd+) and/or unfolding(unf+) of the host nucleoid, both of which occurwithin 3 to 4 min after infection and both ofwhich may be sufficient to shut off or at least


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interfere with host transcription. It should nowbe possible to construct multiple mutants defi-cient in several ofthese processes to more clearlyobserve the consequences of expression of indi-vidual genes.

CONCLUSIONSIn summary, a multitude of biochemical

changes and physiological events which occurshortly after T-even phage infection could, inprinciple, contribute to shutoff of host macro-molecular synthesis. Thus enzymatic degrada-tion of the host DNA is clearly capable of ter-minating replication and perhaps does so in thecase of infection with T5. However, after infec-tion with T-even phages, DNA synthesis is ac-tually terminated early in infection before de-tectable DNA degradation commences, andknown T4 phage mutants deficient in inducingDNA degradation are without demonstrable ef-fect on shutoff of host DNA, RNA, or proteinsynthesis.Thus, two hypotheses can be suggested for

the roles of the systems discussed in this review:(i) that many of the genes that have been pro-posed to be involved in shutoff are actuallyinvolved in other functions of the phage and (ii)that many of the proposed shutoff genes providemultiple mechanisms for shutoff, of which dif-ferent ones become most important under dif-ferent conditions. Although no firm conclusionscan be drawn, it is instructive to summarize whatis known about the roles of these systems underthe laboratory conditions in which they havebeen studied.The alterations to nucleotide metabolism are

directed to synthesis of viral DNA containinghydroxymethylcytosine and excluding cytosine.Host DNA degradation provides about 20% ofthe nucleotide precursors for viral DNA underlaboratory conditions of infection. None of thesesystems have been demonstrated to be involvedin shutoff.Nuclear disruption, which was originally ob-

served as a dramatic morphological rearrange-ment of the host nucleoid, has now been dem-onstrated to be essential for the prompt shutoffof host DNA synthesis at 4 min after infection.In the absence of nuclear disruption, host DNAsynthesis is shut off at 10 min after infection.Also of interest is the fact that nuclear disruptionis required for a productive T4 infection of E.coli strain CT447 and that this mechanism mayinvolve a bacterial plasmid. Thus the gene fornuclear disruption may have roles both in shut-off and in other functions, i.e., restriction of T4phage growth by plasmids.Another early event, unfolding of the host

nucleoid, occurs simultaneously with shutoff ofRNA and protein synthesis. Many, but not all,phage mutants defective in unfolding show aslight delay in shutoff of host transcription butnot replication or translation. It seems likelythat another event is primarily responsible forshutoff of transcription in the strains and underthe conditions that have been studied, but thetemporal expression of this event is somewhatmodified by unfolding. A reasonable approachto future investigations is to seek the primaryevent leading to shutoff of transcription underlaboratory conditions by unfoldase-defective(unflaic) mutants. When this event has beenidentified, a comparison of its role with that ofunflaic under other conditions can be ap-proached more systematically.

Prime candidates for roles in the shutoff oftranscription are changes of the host RNA po-lymerase. The ADP-ribosylation of the RNApolymerase by the products of genes alt andmod does not appear to affect shutoff underlaboratory conditions. This leaves the 15,000-dalton and 10,000-dalton phage-induced poly-peptides associated with RNA polymerase asprime candidates for transcriptional shutoff. Al-though implication of these by default is clearlyan unsatisfactory state of affairs, it does providean attractive model for possible experiments.The most direct approach seems to be to seekphage mutants deficient in these functions, pref-erably using a parental strain with defectivegenes for alt, mod, unf/alc, and ndd.Two possible sites for translational shutoff are

phage-induced changes of the tRNA pool and ofthe bacterial ribosomes. The known phage-in-duced tRNA's have been excluded from a rolein shutoff under laboratory conditions of infec-tion. Although it has been suggested that nu-cleolytic damage to host tRNA is responsible forshutoff, the extent of the known damage seemsto exclude this as a primary mechanism. Thus,again by default, the phage-induced proteinsassociated with the ribosomes are prime candi-dates for a role in translational shutoff. Theseproteins are still undefined genetically, so seek-ing mutants, particularly using parental strainsthat are multiply defective in other nonessentialearly functions, is of high priority.Although infection with phage ghosts causes

a prompt shutoff of host macromolecular syn-thesis, available evidence suggests that this oc-curs by a different mechanism from shutoff in-duced by intact viruses. The most reasonablemechanism for shutoff by ghosts is loss of criticalmetabolites through the damaged cell envelope.Since there is evidence that leakage from ghost-infected cells is qualitatively and quantitatively

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different from phage-infected cells, it is unlikelythat ghost-induced leakage plays an importantrole in phage infection.

Besides those experiments which may finallydisclose the primary mechanisms for transcrip-tional and translational shutoff, other directionsfor future research are evident from this review.All the systems discussed have been incom-pletely studied, particularly at the biochemicallevel. The pathway of host DNA degradation isincomplete, particularly the terminal stages andother steps thought to use exonucleases. Themechanisms by which the ndd gene productattaches host DNA to the cell membrane andterminates host DNA replication are of greatinterest but unknown. The identity and bio-chemical role or roles of the unflalc gene prod-uct are unknown. Finally, the physiological rolesof all the so-called nonessential functions thathave been disclosed by this research deserveinvestigation. The restriction of nuclear disrup-tion-deficient virus by E. coli CT447 provides amodel for one experimental approach to theseroles. It is likely that other strains of E. coliisolated from nature will restrict other viral mu-tants. Alternatively, growing standard strainsunder conditions unlike "laboratory conditions"but reflecting those encountered in nature maydisclose essential roles for these genes.

ACKNOWLEDGMENTSResearch by the authors of this review was sup-

ported by Public Health Service grants AI-04479, AI-07946, and GM-25417 from the National Institutes ofHealth and by National Science Foundation grantPCM76-01841.

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