Roles of Genes 44, 50, and 51 in Regulating Gene Expression and Host Takeover during Infection of Bacillus subtilis by Bacteriophage SPO1

We show that the products of SPO1 genes 44, 50, and 51 are requiredfor the normal transition from early to middle gene expressionduring infection of Bacillus subtilis by bacteriophage SPO1;that they are also required for control of the shutoff of hostDNA, RNA, and protein synthesis; and that their effects on hostshutoff could be accounted for by their effects on the regulationof gene expression. These three gene products had four distinguishableeffects in regulating SPO1 gene expression: (i) gp44-50-51 actedto restrain expression of all SPO1 genes tested, (ii) gp44 and/orgp50-51 caused additional specific repression of immediate-earlygenes, (iii) gp44 and/or gp50-51 stimulated expression of middlegenes, and (iv) gp44 and/or gp50-51 stimulated expression ofsome delayed-early genes. Shutoff of immediate-early gene expressionalso required the activity of gp28, the middle-gene-specificsigma factor. Shutoff of host RNA and protein synthesis wasaccelerated by either the 44^- single mutant or the 50^-51^- doublemutant and more so by the 44^-50^-51^- triple mutant. Shutoff ofhost DNA synthesis was accelerated by the mutants early in infectionbut delayed by the 44^-50^-51^- triple mutant at later times. Althoughgp50 is a very small protein, consisting almost entirely ofan apparent membrane-spanning domain, it contributed significantlyto each activity tested. We identify SPO1 genes 41 to 51 and53 to 60 as immediate-early genes; genes 27, 28, and 37 to 40as delayed-early genes; and gene 52 as a middle gene.

INTRODUCTION

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Introduction

During infection of Bacillus subtilis by bacteriophage SPO1,there is a complex program of gene actions. Early genes aretranscribed by the unmodified host RNA polymerase (RNAP), frompromoters recognized by B. subtilis ${sigma}$ A. Early gene 28 specifiesa new sigma factor, gp28, which substitutes for ${sigma}$ A and directstranscription of the middle genes. Middle genes 33 and 34 specifya new sigma factor and accessory protein, which direct transcriptionof the late genes (4, 5, 10, 18). Each of the major groups ofgenes can be divided into subsets showing different regulatorypatterns, which cannot be accounted for by the known regulatorymechanisms. For the most part, middle genes specify the phageDNA replication machinery, while the late genes specify thestructural and morphogenetic proteins (16).

The early genes appear to be involved primarily in host takeover,in which the host's biosynthetic machinery is subverted to thepurposes of the infecting bacteriophage (17). Host mRNA, protein,and DNA syntheses are shut off rapidly and are replaced by synthesisof the corresponding phage-specific macromolecules (5, 9, 12,15).

SPO1 has 26 known early genes, arranged in 13 operons, whosetranscription is directed by early promoters P_E1 to P_E13, eachrecognized by the host RNAP with ${sigma}$ A (1, 13, 16, 17). (Two otherearly promoters are known [1], but the downstream genes havenot been identified.) Operons 1 to 12 form a continuous clusterof 24 genes, genes 37 to 60, which we have called the "hosttakeover module," located within an 11.5-kb segment that constitutesmost of the SPO1 terminal redundancy (17) (accession numberAF031901). In operon 13, genes 28 and 27 are transcribed fromP_E13, and gene 27 is also transcribed from a middle promoterthat lies between 28 and 27 (2).

Our initial studies of the roles of individual genes in hosttakeover have focused on genes 44, 50, and 51. When gene 44was expressed in uninfected cells of either B. subtilis or Escherichiacoli, it caused the shutoff of bacterial RNA synthesis and celldeath (20, 21). DNA and protein synthesis were also shut off,presumably as indirect results of the effect on RNA synthesis.An E. coli mutation substituting valine for glutamate at position1272 of the B subunit of the RNAP provided resistance to thelethal effects of gene 44 (21; A. Sampath, unpublished results).Genes 51 and 50 constitute a two-gene operon which, when expressedin uninfected cells, also caused the shutoff of RNA, DNA, andprotein synthesis and cell death (our unpublished results).The products of both gene 44 and gene 51 include acidic-hydrophobicdomains similar to the domain in E. coli ${sigma}$ 54 that is requiredfor binding to RNAP (19), and one segment of gp51 shows substantialsimilarity to the product of SPO1 gene 27, which is requiredfor normal transcription of late genes (7, 17). Thus, it seemedlikely that the roles of gp44 and gp51 in infection were tointeract with the host RNAP, presumably for the purpose of changingits specificity from host to phage transcription, and that oneconsequence of this interaction with either gp44 or gp50-51was inactivation of the RNAP with respect to transcription ofhost genes.

To test that hypothesis, we inactivated each of genes 44, 50,and 51 by mutation and tested the effects of these mutationson host shutoff during infection. (Gene 50 was included becausethe last 8 nucleotides of gene 51 overlap the first 8 nucleotidesof gene 50, suggesting the possibility of translational couplingand joint activity. Its product includes only 23 amino acidsand consists almost entirely of an apparent membrane-spanningdomain, with charged amino acids at each end.) We show that,contrary to our expectations, the mutations accelerated shutoff,suggesting that the role of these gene products in host shutoffmay be regulatory, rather than directly causative. In confirmationof that suggestion, we show that the mutations do affect theexpression of all known SPO1 early genes and of all of the middlegenes tested and that these effects on gene expression are sufficientto account for the observed effects on host shutoff.

In the course of assaying their expression, we have been ableto identify each of the known early genes as immediate-earlygenes, delayed-early genes, or, in one case, actually a middlegene.

MATERIALS AND METHODS

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Materials and Methods

Growth conditions. Bacterial strains, growth media, bacterial growth, and phageinfection were as described previously (20). Routine propagationof phage cultures was at 37°C in veal-yeast extract (VY)medium on CB313, a nonsense-suppressor strain. Experiments testingthe effects of the mutations were done at 30°C on CB10,the nonsuppressing strain, in the medium designated for eachtype of experiment. The generation time and the latent periodwere about twice as great at 30°C as at 37°C, providinggreater resolution of events occurring at different times andalso, serendipitously, increasing observed differences betweenmutants and wild type.

SPO1 strains. The formation of the gene 44 mutation was described previously(20), where it was called e3m3. For genes 50 and 51, clonedgenes were mutagenized using the Stratagene QuikChange kit.In each case, a nonsense codon was substituted for the firstlysine codon, which was codon 3 for each of the three genes.Each mutagenized plasmid was allowed to recombine with superinfectingSPO1. Recombinant progeny were identified by plaque-lift hybridization,using the Amersham ECL 3'-oligolabeling and detection kit, andallowed to segregate pure mutant strains as described previously(20). Multiple mutants were produced by successive recombinationwith the appropriate mutagenized plasmids, and all genotypeswere confirmed by DNA sequencing. The mutants were propagatedon a suppressor strain, CB313, which inserts lysine at nonsensecodons. The first 8 nucleotides of gene 50 overlap the last8 nucleotides of gene 51, and the gene 50 mutation destroysthe termination codon of gene 51, apparently permitting translationof gene 51 to run on for about 30 more amino acids, beyond itsnormal 151. Thus, definitive conclusions about the role of gp50could not be made from single mutants in which only gene 50was mutated. The gene 28 mutant was derived from mutant F21,initially described by Okubo et al. (11), by five successivebackcrosses to wild-type SPO1. We have previously called itsus28-1 (3), and our lab bookkeeping lists it as F21 (5).

RNA preparation. Cultures in VY medium were harvested at the indicated timesafter infection, as described previously (20). The RNA was purifiedusing the Qiagen RNeasy Mini kit, following the manufacturer'sprotocol.

Dot blots. PCR products carrying a particular gene or genes were denaturedby incubating them for 15 min at 37°C in 0.2 M NaOH. Tennanograms of DNA in 2 µl was spotted onto positively chargednylon membranes, Amersham Hybond N⁺, and bound by UV cross-linking.Each 11- by 5-cm membrane held an array of about 23 spots. ThePCR fragments included all, or nearly all, of the specifiedgene or genes, except that the one for gene 31 included onlythe first exon.

Hybridization. For each RNA preparation, a 1.0-µg aliquot was labeledwith alkaline phosphatase, using the Amersham AlkPhos directlabeling and detection kit. The entire labeled preparation washybridized to one membrane carrying a dot blot array, with useof the hybridization buffer defined in the kit. Hybridizationwas at 60°C for 2 h in a Robbins Model 400 hybridizationincubator rotating at about 4 rpm. Wash buffers were as definedin the kit, with the primary wash at 65°C and the secondarywash at room temperature. Detection of hybridized RNA used theAmersham CDP Star chemiluminescent detection reagent, followingprocedures from the kit. The alkaline phosphatase catalyzesdecomposition of dioxetane with emission of light, which isdetected on film, with the use of Hyperfilm ECL from Amersham.All hybridizations were done under the same conditions, withoutattempting to identify optimal conditions for each gene, andwe have not made independent measurements of the concentrationsof the various transcripts or of their intrinsic hybridizationefficiencies. Thus, gene-to-gene differences in the absoluteamount of signal detected could be due to differences in intrinsichybridization or labeling efficiency. Conclusions were drawnonly from comparisons between signal intensities for the samegene under different conditions.

Densitometry. The intensities of the dot blot signals were measured with aMolecular Dynamics computing densitometer. Positions at whichno spot was visible on the film gave intensity values rangingfrom -20 to +20. Some spots that were faintly visible gave intensityreadings less than 20, which are reported as measured, withthe recognition that they represent only approximations of theactual value. Spots not visible on film are reported as 0. Intensityvalues as high as 1,700 were within the linear dose-responserange. Where undiluted samples gave values beyond the linearrange, we measured hybridization by diluted samples and correctedfor the dilution factor and the experiment-to-experiment differencein hybridization efficiency (see details for Table 1 in nextsection).

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TABLE 1. Effect of triple mutant on stability of specific RNAs^a

Measurements of RNA stability, as reported in Table 1. Rifampin was obtained from Sigma and was used at a final concentrationof 4 µg/ml, which is four times the concentration of rifamycinthat was sufficient to prevent SPO1 RNA synthesis (6). Pilotexperiments showed that that concentration was sufficient toprevent synthesis of SPO1 RNA detectable by our assay system.In cultures parallel to those analyzed in Fig. 1, rifampin wasadded at 15 min after infection, and the RNA was extracted 10min later. Hybridization and detection of this RNA were performedas described above. For two delayed-early and seven immediate-earlygenes (ranging from the lowest to the highest signal intensities),Table 1 shows the intensity at 15 min, the intensity remainingafter 10 min of further incubation with rifampin, and the proportionof the activity present at 15 min that survived the 10-min incubationwith rifampin (the ratio of the first two numbers). For genes44, 45-46, 56-58, and 59-60, hybridization to the undilutedRNA from the triple mutant at 15 min gave a signal beyond thelinear range. Therefore, the data for those genes from the triplemutant were taken from spots made by hybridization to 1:4 dilutionsof the 15-min RNA preparations. Since those values were comparedwith the values for the same genes after 10 more min of incubationwith rifampin, the latter values for the same four genes werealso taken from 1:4 dilutions. Each value thus obtained wasmultiplied by the dilution factor and by a normalization factor,which corrects for the experiment-to-experiment difference inhybridization efficiency. The normalization factor was the ratioof the average signal given by hybridization of undiluted RNAsin the original experiment to the average signal given by thesame undiluted RNAs in a control hybridization done simultaneouslywith the diluted samples, making use only of signals that werein the linear range. Since both the dilution factor and thenormalization factor were the same for the two sets of samples,they did not affect the calculation of the fraction surviving.These measurements of stability are imprecise, because of thelow signal-to-noise ratio at low RNA concentrations (particularlysignificant for wild type, where most starting values were alreadylow) and because the activity that survived rifampin treatmentincluded RNA that was synthesized after the rifampin was addedbut before it could completely stop RNA synthesis. Thus, thecalculated ratios probably overestimate the proportion of RNAexisting at 15 min that survived until 25 min, so the triplemutant RNA probably was even less stable than indicated by thecalculated ratios.

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FIG. 1. Gene-specific expression patterns. Cultures infected with wild-type (wt) SPO1, mutant 28^-, or triple mutant 44^-50^-51^- were harvested at 5, 15, or 25 min (x axis) after infection at 30°C. RNA was prepared, labeled, hybridized to dot blots, and quantitated by densitometry as described in Materials and Methods. The densitometric values for each gene (y axis) are plotted as a function of time after infection. In each graph, the number in the upper left corner indicates the gene whose activity is plotted. Where an operon of more than one gene was assayed, the graph is labeled with the number of the first gene in the operon. This applies to operons 37-39, 45-46, 48-49, 51-50, 55-53, 58-56, and 60-59. Note that the scales are individualized for each gene and that the maximum levels of activity vary by as much as 20-fold. To a first approximation, the immediate-early genes have been arranged from top to bottom in order of increasing activity. Gene 27 could have been displayed with either the delayed-early or the middle genes..

Pulse-labeling. Rates of synthesis of host and phage RNA or protein were measuredin C4 medium as described previously (20), by pulse-labelingfor 5 min with [5-³H]uridine or [4,5-³H]leucine, precipitatingwith trichloroacetic acid, and counting the precipitate. SinceSPO1 DNA contains hmUra in place of thymine, host DNA synthesiscould be measured independently by pulsing with [methyl-³H]thymidine.Phage DNA synthesis was measured by pulsing with [2,8-³H]adenineand measuring alkali-stable, trichloroacetic acid-precipitablecounts. That measures host DNA synthesis as well, but most hostDNA synthesis has been shut off by the time that phage replicationbegins, even in mutant infections, so incorporation of labelafter 12 min is overwhelmingly into phage DNA. The time givenfor each pulse is the time at which the pulse began.

RESULTS

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Results

Identification of immediate-early, delayed-early, and middle genes. Expression of specific genes was measured by dot blot hybridizationand densitometry, as described in Materials and Methods. Figure1 shows the expression patterns of all of the known early genesand several representative middle genes, in the wild type orwhen altered by either the 28^- mutation or the 44^-50^-51^- triplemutant. The 28^- mutation inactivates the middle-gene-specificsigma factor, permitting identification of middle gene activity.Note that different genes are expressed on different scales,as the absolute level of expression varies widely from geneto gene.

Genes 31 and TF1 were previously known to be middle genes (8,14). The others are now identified as immediate-early (genes41 to 51 and 53 to 60), delayed-early (genes 27, 28, and 37to 40), or middle (gene 52), on the basis of time of expressionin wild-type or 28^- infection and of gp28 dependence of theirexpression. Gene 27 had characteristics of both delayed-earlyand middle genes. Genes 48 and 49 also showed substantial gp28-dependentexpression at late times (data not shown).

Roles of genes 44, 50, and 51 in regulating early and middle gene expression. The top three rows of Fig. 1 show that expression of every immediate-earlygene was dramatically increased by the triple mutant, in comparisonwith its expression in wild-type infection. In contrast, thetriple mutant decreased expression of the middle genes, slightlyfor TF1 and substantially for genes 31 and 52. Thus, the mutationof genes 44, 50, and 51 inhibited the transition from immediate-earlyto middle gene expression, suggesting that those gene productsare required for normal progress of that transition. (The delayed-earlygenes are more complicated and will be discussed with Fig. 2.)

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FIG. 2. Differential effects of mutations on immediate-early, delayed-early, and middle transcription. Cultures infected with wild-type (WT) SPO1, mutant 44^-, double mutant 50^-51^-, or triple mutant 44^-50^-51^- were harvested 15 min after infection at 30°C. RNA was prepared, labeled, hybridized to dot blots, and detected on film as described in Materials and Methods. The films were scanned using the UMAX Vista Scan program with a UMAX Astra 1220S scanner. A 25- by 25-pixel square surrounding each spot was copied and pasted into the grid displayed. The numbers at the top represent the gene or genes present in that spot on the dot blot. All of the middle and delayed-early genes assayed are presented, along with three representative immediate-early operons. The gene categories are indicated across the top. Gene 27 falls under both middle and delayed-early, since it is expressed from both an early and a middle promoter.

Since the dot blot hybridizations measure the total amount ofspecific RNAs present at a particular time, rather than therate of synthesis, it was possible that the increased quantitiesof specific RNAs obtained from cells infected by the triplemutant were due to increased stability rather than to increasedsynthesis. We tested this by using rifampin to inhibit new RNAsynthesis and observing the persistence of RNA already present.In cultures parallel to those analyzed for Fig. 1, rifampinwas added at 15 min after infection, and the RNA was extractedat 25 min. Table 1 shows that, except for gene 44 RNA, the RNAsmade by the triple mutant were not substantially more stablethan those made by the wild type or the 28^- mutant.

Figure 2 shows the effects of the separate components of thetriple mutant, the single mutant 44^- and the double mutant 50^-51^-.It shows that the products of genes 44, 50, and 51 had fourdistinguishable effects, one requiring all of gp44-50-51 andthe others requiring either gp44 and/or gp50-51.

Effect 1. gp44-50-51 acted to repress every gene's expression, and thefull effect of this repression required the participation ofboth gp44 and gp50-51. This can be seen by comparing the datain the wild-type row with those in the next two rows. The expressionof every gene was increased when either gene 44 or genes 50and 51 were inactivated by mutation.

Effect 2. Either gp44 or gp50-51, even in the absence of the other one,caused residual repression of immediate-early genes. This canbe seen by comparing the two middle rows with the bottom row,looking specifically at the immediate-early genes. Althoughthe repressive activity of effect 1 had been inactivated byeither the 44^- or 50^-51^- mutation, those strains retained substantialrepressive activity, which was lost when the remaining wild-typegene(s) was inactivated to form the triple mutant.

Effect 3. Either gp44 or gp50-51 stimulated middle gene activity. Thiscan be seen by comparing the two middle rows with the bottomrow, looking specifically at the middle genes. When the repressiveactivity of effect 1 had been inactivated by either the 44^-or the 50^-51^- mutation, those strains had active middle geneexpression, as seen in the two middle rows. Much of this expressiondepended upon the remaining active gp44 (in the 50^-51^- mutant)or gp50-51 (in the 44^- mutant), since it was lost when thosegenes were inactivated to form the triple mutant.

Effect 4. Either gp44 or gp50-51 stimulated the expression of certaindelayed-early genes. This can be seen by comparing the two middlerows with the bottom row, looking specifically at the delayed-earlygenes. When the repressive activity of effect 1 had been inactivatedby either the 44^- or the 50^-51^- mutation, those strains hadactive delayed-early gene expression, as seen in the two middlerows. For genes 27 and 28, much of this expression dependedupon the remaining active gp44 (in the 50^-51^- mutant) or gp50-51(in the 44^- mutant), since it was lost when those genes wereinactivated to form the triple mutant. For genes 37 to 40, thesame activities appear to be present, but the effect of gp50-51was so slight that it could not be documented conclusively.

In effects 2 and 3, both gp44 and gp50-51 had activities thatrepressed immediate-early gene expression and stimulated middlegene expression, thus facilitating the transition from immediate-earlyto middle gene expression.

Roles of genes 44, 50, and 51 in shutoff of host-specific biosyntheses. (i) Shutoff of host gene expression. The triple mutant caused host RNA synthesis to be shut off muchmore rapidly and completely than in wild-type infection. Figure3 shows the rate of RNA synthesis as a function of time afterinfection. These assays measure the sum of host and phage RNAsynthesis, so the decrease in total RNA synthesis by 12 minafter infection provides a minimum estimate of the extent towhich host RNA synthesis had been shut off by that time. By12 min after infection, the rate of RNA synthesis in triplemutant infection was decreased to less than 10% of the ratein wild-type infection and to less than 5% of the rate in uninfectedcells.

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FIG. 3. Shutoff of host RNA synthesis. Rates of RNA synthesis were measured by pulse-labeling with [5-³H]uridine as described in Materials and Methods, as a function of time after infection by the indicated strains.

Each of the three mutations contributed to this effect. 44^-and 50^-51^- each shut off host RNA synthesis more completelythan wild type did but less completely than the triple mutantdid. Figure 4 shows that, while each of the 50^- and 51^- mutationshad little effect by itself, each was necessary for the fulleffect of the triple mutant, 44^-50^-51^-, and the double mutant,50^-51^-.

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FIG. 4. Distinguishable effects of genes 50 and 51. Rates of RNA synthesis were measured by pulse-labeling with [5-³H]uridine as described in Materials and Methods, as a function of time after infection by the indicated strains.

Similar results were seen when shutoff of host protein synthesiswas measured by pulse-labeling with [³H]leucine or by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis of proteinspulse-labeled with ³⁵S-labeled amino acids (data not shown).Thus, gp44-50-51 restrained the normal shutoff of host geneexpression, and each of genes 44, 50, and 51 contributed significantlyto that activity.

(ii) Shutoff of host DNA synthesis. Figure 5 shows that gp44-50-51 also affected the shutoff ofhost DNA synthesis. The 44^- mutant caused hyperfast shutoff.The triple mutant caused hyperfast shutoff early in infectionbut delayed the completion of the shutoff that occurred laterin infection. Thus, there appear to be at least two componentsto the shutoff, an early component which is restrained by gp44-50-51and a later component which requires the activity of eithergp44 or gp50-51.

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FIG. 5. Shutoff of host DNA synthesis. Rates of host DNA synthesis were measured by pulse-labeling with [methyl-³H]thymidine as described in Materials and Methods, as a function of time after infection by the indicated strains.

It is possible that the phage-induced decrease in total RNAsynthesis could indirectly increase the pool size of deoxyribonucleosidetriphosphates and thereby cause a spurious decrease in measuredrates of DNA synthesis. However, such an effect could not accountfor the actual differences observed. Such an effect would causethe measured rate of host DNA synthesis to be decreased morein the 44^-50^-51^- infection than in the 44^- infection, whereasthe opposite is true. Such an effect would cause the measuredrate of host DNA synthesis to be decreased more in 44^-50^-51^-than in wild type at all times during infection, whereas theobserved difference is in one direction at 5 min and in theopposite direction at 12 and 25 min. The amount of the differencebetween wild type and the triple mutant caused by any such effectmust be substantially less than twofold, since even wild-typeinfection decreases total RNA synthesis by more than 50%; thus,such an effect could not account for the eightfold differencein phage DNA synthesis between wild type and the triple mutant,which is seen below in Fig. 6.

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FIG. 6. Phage DNA synthesis. Rates of phage DNA synthesis were measured by pulse-labeling with [2,8-³H]adenine as described in Materials and Methods, as a function of time after infection by the indicated strains.

Roles of genes 44, 50, and 51 in phage DNA synthesis and growth. Figure 6 shows that phage DNA synthesis was substantially deficientin infection by the triple mutant. Presumably this was due tothe deficiency in expression of the middle genes, which specifymost of the enzymes required for phage DNA synthesis. The 44^-and 50^-51^- mutations alone caused slight inhibitory or stimulatoryeffects, respectively. Presumably because of the delay in middlegene expression and DNA synthesis, the triple mutation causeda modest extension of the latent period and a modest decreasein burst size (Fig. 7). Plating efficiency and plaque size ona Sup^- lawn were less than half of those of wild-type phage,and the plaque size ranged down to barely visible. Neither 44^-nor 50^-51^- had any consistently observable effect on phage growth.

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FIG. 7. Single-step growth curves. Cultures of CB10, growing in VY medium at 30°C, were infected with SPO1 wild type (WT) or 44^-50^-51^- at a multiplicity of infection of about 0.1. After 10 min the cultures were diluted 1:2 into VY medium containing anti-SPO1 antibody. After being shaken for 10 more min, the cultures were diluted 1:2 x 10⁴ into fresh VY medium. They continued to be shaken at 30°C and were assayed for PFU at 25-min intervals. The value plotted for each time point is the ratio of the PFU at that time point to the PFU for the same culture at 25 min.

DISCUSSION

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Discussion

Delayed-early genes. Since genes 27, 28, and 37 to 40 were expressed primarily atmiddle time but independently of gp28, we are calling them delayed-earlygenes. There is nothing known about the early promoters fromwhich these genes are transcribed that would account for sucha delay in their expression, given the high level ultimatelyachieved (1, 2, 13, 17). Thus, there must be a heretofore-unrecognizedactivity that stimulates transcription of these two early operonsat middle times, independently of gp28 and without similarlyaffecting transcription of the immediate-early genes. That activitycould be provided by gp44 (with or without gp50-51), since itstimulates expression of the delayed-early genes. If so, thedelay in expression of the delayed-early genes could be thetime required for synthesis of sufficient quantities of gp44.

Effects of the products of genes 44, 50, and 51 on regulation of SPO1 gene expression. Table 2 summarizes the effects of wild-type SPO1 and of mutants44^-, 50^-51^-, and 44^-50^-51^- on each of the processes studiedduring SPO1 infection. Pluses and minuses show the directionand relative amount of the effect of each SPO1 strain on eachof the processes listed. The first three columns of Table 2summarize the data in Fig. 2 and show that these gene productshad four distinguishable effects on expression of SPO1 genes.

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TABLE 2. Summary of effects of SPO1 strains^a

Effect 1. In comparison with wild type, either the 44^- mutation or the50^-51^- double mutant caused increased expression of all genestested, including immediate-early, delayed-early, and middlegenes. Therefore, gp44-50-51 repressed expression of all ofthe SPO1 genes tested, and the full effect of this repressionrequired the activity of both gp44 and gp50-51.

Effect 2. In comparison with 44^- or 50^-51^-, the triple mutant 44^-50^-51^-caused a further increase in expression of immediate-early genes.Therefore, there must be residual repressive activity presentin the 44^- or 50^-51^- strains, which is no longer there in 44^-50^-51^-and which therefore must be caused by gp50-51 in the 44^- strainand by gp44 in the 50^-51^- strain.

Effect 3. In comparison with 44^- or 50^-51^-, the triple mutant caused adecrease in expression of middle genes. Therefore, there mustbe an activity present in the 44^- or 50^-51^- strains which stimulatesmiddle gene expression. Since that activity is no longer therein 44^-50^-51^-, it must be caused by gp50-51 in the 44^- strainand by gp44 in the 50^-51^- strain.

Effect 4. In comparison with 44^- or 50^-51^-, the triple mutant caused adecrease in expression of delayed-early genes 27 and 28. Therefore,there must be an activity present in the 44^- or 50^-51^- strainswhich stimulates delayed-early gene expression. Since that activityis no longer there in 44^-50^-51^-, it must be caused by gp50-51in the 44^- strain and by gp44 in the 50^-51^- strain. With respectto delayed-early genes 37 to 40, such an activity can be seenfor gp44 in the 50^-51^- strain but not conclusively for gp50-51in the 44^- strain.

Note that, in the wild type, both activities 1 and 3 or 1 and4 would be active and would cause effects in opposite directions.To varying extents they would cancel each other out, sometimesobscuring the difference between wild type (which has both activities)and the triple mutant (which has neither).

Thus, the four activities are (i) repression of all SPO1 genes,requiring the activity of both gp44 and gp50-51; (ii) residualrepression of immediate-early genes by either gp44 or gp50-51,even in the absence of the other; (iii) stimulation of expressionof middle genes by either gp44 or gp50-51; and (iv) stimulationof expression of certain delayed-early genes by either gp44or gp50-51.

Effects of the products of genes 44, 50, and 51 on host shutoff. These gene products also played significant roles in the shutoffof host DNA, RNA, and protein synthesis. gp44-50-51 restrainedthe rate of shutoff of host gene expression, and each of genes44, 50, and 51 contributed significantly to that activity. gp44-50-51restrained the early component of the shutoff of host DNA synthesis,while a later component required the activity of gp44 and/orgp50-51. These effects might have been accomplished by additionaldirect activities of gp44-50-51. However, we prefer the simplerexplanation that the effects on host shutoff resulted from thealready-documented effects of gp44-50-51 on phage gene expression.Table 2 summarizes the effects of the mutations on host shutoffand suggests a plausible model. Note that the increase fromstrain to strain in the expression of immediate-early genesparallels the increase in the efficiency with which host RNAsynthesis is shut off. This can be explained if one or moreof the immediate-early genes, regulated by gp44-50-51, specifiesa product that causes shutoff of host RNA synthesis. The overexpressionof such genes, caused by the triple mutant, could cause theaccelerated shutoff of host RNA synthesis that was observedin triple mutant infection. The intermediate levels of overexpressioncaused by mutants 44^- and 50^-51^- could account for their alsocausing intermediate levels of accelerated shutoff of host RNAsynthesis. Similarly, the deficient shutoff of host DNA synthesisby the triple mutant at middle times could be explained if thatshutoff required one or more of the middle gene products whoseexpression is deficient in the triple mutant.

ACKNOWLEDGMENTS

The research reported here was supported by grants 003604-0020-1999and 003604-0035-2001 from the Texas Advanced Technology Program.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251. Phone: (713) 348-4926. Fax: (713) 348-5154. E-mail: crs{at}bioc.rice.edu.

Present address: Novartis Institute for Tropical Diseases, Singapore117528.

REFERENCES

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