Vol. 171, No. 4

Bacillus subtilis pur Operon Expression and RegulationtDANIEL J. EBBOLE AND HOWARD ZALKIN*

Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907

Received 9 November 1988/Accepted 24 January 1989

The Bacillus subtilis pur operon is a 12-gene cluster, purEKB-purC(orf)QLF-purMNH(J)-purD, organized ingroups of overlapping coding units separated by intercistronic gaps. Translational fusions of Escherichia colilacZ were constructed to purE, purC, and purM, the first gene of each group. Analyses of gene fusionsintegrated into the chromosomal pur operon exclude the possibility of internal promoters in intercistronicregions and support the view that transcription is from the single fr43 promoter at the 5' end of the operon.Enzyme and mRNA measurements indicate that transcriptional regulation occurs solely at the 5' end of theoperon. The relative levels of 0-galactosidase from purE-lacZ, purC-lacZ, and purM-lacZ were determinedunder repressing and nonrepressing conditions. These results indicate that expression ofpurC-lacZ was 3.0- to6.8-fold higher than purE-lacZ because of enhanced translational efficiency. The enhanced translationalefficiency ofpurC-lacZ was accompanied by a partial escape from regulation by purines. This anomalous effecton purC-lacZ was the only suggestion for posttranscriptional regulation.

A cluster of 12 contiguous genes, designated the pur

operon, encodes all of the enzymes required for de novosynthesis of IMP in Bacillus subtilis (1). The operon consistsof three clusters of overlapping genes separated by intercis-tronic spaces: purEKB-purC(orf)QLF-purMNH(J)-purD. Inthis gene cluster, orf is an unidentified open reading frame of84 codons and purH(J) is the designation for a gene encodinga bifunctional enzyme that catalyzes steps 9 and 10 in thepathway. The purBC and purFM gaps are 73 and 101 basepairs (bp), respectively. Gene purD is separated frompurH(J) by 15 bp. A single c43-dependent promoter has beenidentified which initiates transcription 242 nucleotides up-stream of purE, the first structural gene. Enzyme activitiescorresponding to genes purB, purD, purF, and purH(J) aredecreased in cells grown with added purines or purinenucleosides (8, 10), indicative of gene regulation. We havereported nuclease Si mapping experiments of the mRNA 5'end which provide evidence for dual regulation of transcrip-tion (1). Transcription initiation is repressed upon addition ofadenine or adenosine to the growth medium. Transcription isindependently regulated by a termination-antiterminationmechanism in the 242-nucleotide untranslated mRNA leaderregion. Addition of guanine or guanosine to cells promotestranscription termination. For simplicity, we arbitrarily referto the regulatory molecules as the purine bases, recognizing,however, that the active coregulators may be purines, purine5' nucleosides, 5' nucleotides, or metabolites of these mol-ecules.Thus far, the only known cis-acting regulatory sites are in

the vicinity of the promoter and the leader region at the 5'end of the operon. Deletion ofDNA between positions -193and -64, relative to the start of transcription, abolishesrepression by adenine and thus identifies a likely region forinteraction of a repressor protein (D. J. Ebbole and H.Zalkin, J. Biol. Chem., in press). A candidate repressor

protein has been partially purified and shown to interact withpur operon DNA between positions -145 and -29. The sitefor guanine-mediated transcription attenuation follows aterminatorlike secondary structure at position 151 to 198 (1).

* Corresponding author.t Journal paper no. 11,823 from the Purdue University Agricul-

tural Experiment Station.

Attenuated pur operon mRNA having 3' ends at nucleotides200 to 205 accumulates to high levels in guanine-grown cells(2) and thus provides strong evidence for the independentregulation of transcription initiation and attenuation.We now report results of studies to examine three aspects

of gene expression and regulation. We have investigated (i)the translational yield of several enzymes from the operon,(ii) whether all adenine and guanine-mediated transcriptionalregulation occurs in the promoter-leader region of the op-eron, and (iii) whether there is translational regulation inaddition to transcriptional control. Fusions of Escherichiacoli lacZ to three genes in the operon have been constructedto facilitate measurements of enzyme levels. Levels of puroperon mRNA have been determined under repressing andnonrepressing conditions and have been compared withlevels of P-galactosidase to evaluate gene regulation.

MATERIALS AND METHODS

Strains and plasmids. E. coli plasmid constructions andpreparations were made in strain DH5Sa (Bethesda ResearchLaboratories). Plasmid pCATZ1 was used to construct lacZfusions for integration into the B. subtilis chromosome instrain DE1 (1). Plasmid pCATZ1, formerly designated pCZ1(Ebbole and Zalkin, in press), contains the chloramphenicolacetyltransferase gene from pDE194 (1) cloned into theBamHI and HindIII polylinker sites of pSP64 (PromegaBiotec). The BamHI 'lacZ cartridge from pMC1871 (11) wasthen added to produce pCATZ1. This arrangement providesa unique SmaI site for introduction ofpur gene fragments forconstruction of lacZ fusions. For the purE-lacZ fusion, a

HincII fragment was isolated from pDE264, a pUC19 deriv-ative containing pur operon DNA from positions -1000 to+303 (1) cloned into the pUC19 HindIII and SphI polylinkersites. The HinclI fragment contains pur operon DNA frompositions -31 to +303 plus 3 codons derived from theSphI-to-HincII polylinker sequence of pUC19. The resultingplasmid, pEZ, contains the first 20 codons of purE and 3codons of pUC19 polylinker fused to codon 8 of lacZ. ThepurC-lacZ fusion plasmid was constructed by cloning theHinclI (nucleotide 2200) to BglII (nucleotide 3278) fragmentfrom pDE55 (1) into pCATZ1. The BglII end was filled inwith the Klenow fragment of DNA polymerase I. Thisproduced plasmid pCZ, having a fusion of codon 17 ofpurC

2136

JOURNAL OF BACTERIOLOGY, Apr. 1989, p. 2136-21410021-9193/89/042136-06$02.00/0Copyright X) 1989, American Society for Microbiology

B. SUBTILIS pur OPERON EXPRESSION AND REGULATION

p-

-31 puE

I, +303

pEZ

'\I, acZCAToc

P+-31 +303

purE purK

TransformSelect CmR

P+ p_-31 +303 -31 +303i6 _1_11

purE-locZ CAT purEFIG. 1. Integration of purE-lacZ into the pur operon. Plasmid

pEZ contains pur operon nucleotides -31 to + 303. This fusion joinscodon 20 ofpurE and 3 codons of polylinker sequence with the 'lacZgene from pMC1871. Transformation of B. subtilis with circularplasmid DNA results in chloramphenicol resistance by integration ofthe plasmid by homologous recombination in the pur operon. Theintegration results in duplication of identical sequences (-31 to+303). The strain is Ade- because of integration of the inactivepromoter P-. The -35 promoter element is represented by a smallbox centered at nucleotide -31. P+, Intact -35 element.

with codon 8 of lacZ. The purM-lacZ fusion was constructedby cloning the NruI (nucleotide 7720)-to-HincII (nucleotide8629) fragment from pDE312 (1) into the SmaI site ofpCATZ1. Plasmid pMZ thus contains a fusion of codon 10 ofpurM to codon 8 of lacZ. The fusion plasmids were used totransform B. subtilis DE1 to chloramphenicol resistance toproduce strains DElEZ, DElCZ, and DElMZ. In thesestrain designations, the last two letters refer to the pur geneand lacZ. Integration of the plasmid into the pur operon wasverified by screening for purine auxotrophy. Construction ofthe purE-lacZ strain is illustrated in Fig. 1. The purE-lacZfusion strain had a specific requirement for adenine sincepurB, which is required in synthesis of AMP from IMP, wasnot expressed. The purine requirement of strains DElCZand DElMZ could be satisfied by xanthine, adenine, orguanine.Enzyme assays. For assay of P-galactosidase, overnight

cultures were grown in minimal medium (1) containing 5 jigof chloramphenicol per ml and 100 jig of xanthine per ml or100 jig of the indicated repressing purine base(s) per ml.Cultures of strain DElEZ contained 10 jig of adenine per mlas well. These cultures were used to prepare log-phase cellsin the same medium. Log-phase cells were in turn used toinoculate the final cultures. For purine starvation, 1 ml oflog-phase cells from a culture grown with 100 jig of xanthineper ml (for DE1CZ and DElMZ) or 10 jig of adenine per mlplus 100 jig of xanthine per ml (DElEZ) was centrifuged andthen suspended in 5 ml of minimal medium with 5 jig ofchloramphenicol per ml and shaken for 3 to 4 h at 37°C. Toexamine the effect of purines on gene regulation, the mediumcontained 100 jig of either xanthine, adenine, guanine, or a

combination of adenine and guanine per ml, and cells weregrown for 3 to 4 h at 37°C. Whole-cell assays were performedas described by Miller (7) with chloroform-sodium dodecylsulfate-permeabilized cells.

In another experiment (see Table 2), P-galactosidase was

assayed in cell extracts. Cells were grown in 50 ml ofminimal medium with S ,ug of chloramphenicol per ml and100 ,ug of xanthine per ml (DElCZ, DElMZ) or 10 ,ug ofadenine per ml and 100 jig of xanthine per ml (DElEZ) for 4h. Cells were then harvested by centrifugation, suspended in100 ml of minimal medium with 5 jig of chloramphenicol perml, and grown for 3 h at 37°C. Cells were harvested bycentrifugation, suspended in 3 ml of 0.1 M potassium phos-phate (pH 7.5), and broken by two passes through a Frenchpress at 20,000 lb/in2. A supernatant fraction was obtainedby centrifugation at 16,000 x g for 15 min. Glycerol wasadded to 20% (vol/vol), and extracts were stored at -70°C.Protein concentration was measured by the Lowry assay (4).

Extracts for assay of glutamine phosphoribosylpyrophos-phate amidotransferase (amidotransferase) were preparedfrom cells grown in 500 ml of minimal medium. For strainDE1, the medium contained either no purines, 200 jig ofadenine, or 200 ,ug of guanine per ml. For strain DElMZ, themedium contained 5 ,ug of chloramphenicol per ml and either7 ,ug of adenine per ml, 100 ,ug of xanthine per ml, 200 jig ofadenine per ml, 200 ,ug of guanine per ml, or 200 ,ig each ofadenine and guanine per ml. The cultures were harvested atmid-log phase (-70 Klett units with a no. 66 filter) exceptstrain DElMZ with 7 ,ug of adenine per ml, for whichincubation was continued for 1.5 h after the adenine wasexhausted. The point of adenine exhaustion was defined asthe time at which the growth rate slowed. The cell turbidityat the time of harvest was 35 Klett units. This protocol was

necessary to obtain derepression and avoid enzyme inacti-vation and degradation (13). Cells were suspended in bufferB (50 mM Tris hydrochloride [pH 8.0], 10 mM MgCl2, 0.1mM EDTA, 2 mM dithiothreitol, 0.5 mM phenylmethylsul-fonyl fluoride) and broken as described above. Glycerol wasadded to 20% (vol/vol), and extracts were frozen at -70°C.Protein concentration was determined by the biuret method(4). Amidotransferase was assayed by the glutamate dehy-drogenase method (6). The minimum detectable level ofenzyme activity was 0.2 nmol/min per mg.

Preparation of RNA. Strain DE1 was grown in 100 ml ofminimal medium or minimal medium supplemented witheither 100 ,ug of xanthine per ml, 200 jig of adenine per ml,200 jig of guanine per ml, or 200 jig of both adenine andguanine per ml. For strains DE1CZ and DElMZ, cells weregrown with xanthine as described above. At mid-log phase(70 Klett units with a no. 66 filter), cells were harvestedrapidly by filtration of 10-ml aliquots onto 2.4-cm glass fiberfilters. Filters were dropped into 125-ml flasks containing 3.5ml of phenol, 3.5 ml of chloroform, and 7 ml of RNA buffer(0.25 M NaCl, 5 mM EDTA, 63 mM Tris hydrochloride [pH7.5], 0.1% sodium dodecyl sulfate) preheated to 75°C. Filterswere swirled frequently for 10 min at 75°C, and then eachsample was poured into a 30-ml Corex tube, leaving thefilters behind. After centrifugation at 16,000 x g for 15 min,the aqueous layer was transferred to another centrifuge tubeand RNA was precipitated with 3 volumes of ethanol. Aftercentrifugation, the pellets were suspended in 100 jil ofDNase I buffer (50 mM sodium acetate [pH 6.5], 10 mMMgCl2, 2 mM CaC12), and 100 U of RNasin (Promega Biotec)and 10 jig of RNase-free DNase I (Worthington Diagnostics)were added. DNase I digestion was for 30 min at 37°C. Thereaction was stopped by extracting with an equal volume ofphenol. The aqueous layer was extracted with ether, sodiumacetate was added to a final concentration of 0.3 M, and theRNA was precipitated with 2.5 volumes of ethanol. Thepellets were washed with 75% ethanol and then dried undervacuum. The RNA was suspended in 0.1 ml of diethylpyro-

VOL. 171, 1989 2137

2138 EBBOLE AND ZALKIN

TABLE 1. ,-Galactosidase levels in lacZ fusion strains grown with limiting or excess purinesValue for cells"

StrainStarved Xan Ade Gua Ade + Gua

DE1EZ 260 + 38 130 ± 58 (2.0) 9.2 ± 5.1 (28) 5.8 ± 1.3 (45) 2.6 ± 1.0 (100)DE1CZ 1,440 ± 304 1,470 ± 238 (1.0) 100 ± 40 (14) 70 ± 28 (21) 12 ± 6.8 (120)DE1MZ 700 ± 107 310 ± 37 (2.3) 20 ± 5.9 (35) 13 ± 2.8 (54) 4.0 ± 1.8 (175)

a The values are expressed as Miller units plus or minus standard deviation from 3 to 17 independent experiments. Repression ratios relative to the values forstarved cells are given in parentheses. Cells were grown as described in Materials and Methods.

carbonate-treated TE (10 mM Tris hydrochloride [pH 7.5], 1mM EDTA). The A260-to-A280 ratio was -2.1, and the yieldwas approximately 200 jig from a 100-ml culture.mRNA measurements. RNA was quantitated by hybridiza-

tion with DNA probes with a slot blot apparatus. For eachRNA sample, at least three different quantities ofRNA wereused in the range of 1 to 8 jig per slot. RNA samples in 50 ,ulofTE were prepared for denaturation by adding 30 ,ul of 20xSSC (1 x SSC is 0.15 M sodium chloride plus 0.015 M sodiumcitrate) and 20 ,ul of 37% formaldehyde. After being heated at60°C for 15 min, samples were applied to the nitrocellulosemembrane. Hybridizations and washing of the nitrocellulosefilter were performed as previously described (14). Quanti-tation was by liquid scintillation counting of uniformlyexcised portions of the hybridized membrane. There was

essentially no nonspecific hybridization to E. coli RNA incontrol reactions.

Hybridization probes. Probes for purE, purC, purM, andlacZ were prepared by random primed synthesis (3) withisolated DNA fragments as templates. The templates usedwere as follows: purE, 1,141-bp HindIll fragment frompDE55 (1); purC, 1,164-bp SspI fragment from pDE51 (1);purM, 1,067-bp EcoRI-to-MluI fragment from pDE71 (1);lacZ, -3,000-bp BamHI fragment from pMC1871 (11). Eachlabeling reaction used 50 ng of DNA and 50 jiCi (-3,000Ci/mmol) of [a-32P]dCTP. Probes for purE, purC, and purMhad specific activities of 2.0 x 108 to 2.2 x 108 cpm/jg. ThelacZ probe had a specific activity of 2.7 x 109 cpm/ jig.

Quantitative Western blots (immunoblots). Cell extracts(100 jig) were electrophoresed on a 10% polyacrylamide gelcontaining sodium dodecyl sulfate and electroblotted tonitrocellulose (12). After transfer, the nitrocellulose was

rinsed in TST (10 mM Tris [pH 8.0], 150 mM NaCl, 0.05%Tween 20) and then blocked with 2% nonfat dry milk in TSTfor 30 min. The nitrocellulose was then treated with a 1:1,000dilution of anti-,-galactosidase antibodies (Organon Tek-nika) for 60 min at room temperature. The nitrocellulose waswashed three times for 5 min each in TST, then added to 50ml of TST containing 2% nonfat dry milk and 50 ,uCi of125I-protein A (ICN Pharmaceuticals Inc.) for 2 h at roomtemperature. The nitrocellulose was then washed three timesfor 5 min each in TST before incubation with alkalinephosphatase-conjugated anti-rabbit antibodies (PromegaBiotec) at a 1:7,500 dilution for 30 min. The blots were

washed three times in TST before final visualization of the,-galactosidase bands with Nitro Blue Tetrazolium and5-bromo-4-chloro-3-indolyl phosphate. The bands were ex-cised from the nitrocellulose, and the radioactivity was

quantitated by liquid scintillation counting. Purified E. coli3-galactosidase (Bethesda Research Laboratories, Inc.) was

used as a control for enzyme amount and enzyme activity.Stability of fusion proteins. Cultures (5 ml) were grown in

minimal medium with 5 jig of chloramphenicol per ml plus100 jig of xanthine per ml (DE1CZ, DElMZ), or 10 jig ofadenine per ml (DElEZ). To start the experiment, cultures

were diluted in identical medium containing 200 jg ofchloramphenicol per ml to an A6,0 of 0.1. Growth of thestrains was blocked by 20 ,ug of chloramphenicol per ml.Whole-cell assays of 3-galactosidase were performed at 0, 1,2, and 4 h.

RESULTS

Translational lacZ fusions. The 12 genes of the B. subtilispur operon are organized in groups of overlapping codingunits separated by intercistronic gaps: purEKB-purC(orf)QLF-purMNH(J)-purD. Fusions of purE, purC, and purM(genes 1, 4, and 9 in the operon) with E. coli lacZ were madein order to facilitate measurements of expression and regu-lation. Plasmid fusions were integrated into the chromo-somal pur operon as diagrammed in Fig. 1. Plasmid integra-tion disrupts the chromosomal pur operon and results in apurine growth requirement. The three lacZ fusion strainswere grown under conditions of purine limitation to dere-press the pur operon or purine excess to obtain repression,and cells were assayed for ,3-galactosidase. The results ofthese assays (Table 1) provide information on patterns ofgene expression and regulation.Gene expression. Maximal levels of ,B-galactosidase were

obtained from purine-starved cells (Table 1). The activityratio in purine-starved cells was 1.0:5.5:2.7 for fusions topurE, purC, and purM, respectively. With xanthine as alimiting purine source, purE-lacZ and purM-lacZ, but notpurC-lacZ, were partially repressed compared with thelevels in purine-starved cells. For this growth condition, theratio of ,B-galactosidase activity was 1.0:11:2.4 for fusions topurE, purC, and purM, respectively. We have consideredthree possibilities to account for the different P-galactosidaseactivities obtained from the gene fusions. The enzyme activ-ities may reflect different rates of enzyme synthesis, differentrates of enzyme degradation, or different specific activitiesof the three fusion enzymes. Enzyme stability and specificactivity were determined to distinguish between these pos-sibilities.The stability of each of the enzymes was measured in cells

at 37°C over a period of 4 h following inhibition of proteinsynthesis with chloramphenicol. The ,B-galactosidase activ-ity was constant for all three lacZ fusion strains over the 4-hperiod. Therefore, the lower 3-galactosidase activities ofpurE-lacZ and purM-lacZ fusions compared with that ofpurC-lacZ cannot be due to enzyme lability.The specific activity of each fusion protein was deter-

mined in extracts of strains DElEZ, DElCZ, and DElMZand is summarized in Table 2. The amount of 3-galactosidasein each extract was determined by quantitative Westernblotting. By using this information, the specific activity ofeach enzyme was calculated and is listed in Table 2. Thecalculated specific activity of 3-galactosidase from purC-lacZ was approximately the same as that of purified, native,-galactosidase. The specific activities of ,-galactosidase

J. BACTERIOL.

B. SUBTILIS pur OPERON EXPRESSION AND REGULATION

TABLE 2. Calculated specific activities of 3-galactosidasefusion enzymes

Strain Activity in Protein in Calculated CorrectionStrain extracta extractb sp act' factor

DElEZ 320 1.9 168,000 0.60DElCZ 2,150 7.7 279,000 1.00DElMZ 1,480 6.1 243,000 0.87

a Means of five measurements for a single extract from purine-starved cells.The units are nanomoles per minutes per milligram of extract protein.

b 13-Galactosidase protein in micrograms per milligram of extract proteinwas determined by qu?intitative Western blotting.

Calculated P-galactosidase specific activity in nanomoles per minute permilligram of pure enzyme. The specific activity of purified 3-galactosidase was271,000.

from purE-lacZ and purM-lacZ were 40 and 13% lower,respectively, than that of the enzyme from purC-lacZ.Correction factors are given in Table 2 to convert the,-galactosidase activities in cell extracts to relative enzymelevels.By using the correction factors for 3-galactosidase fusion

enzymes given in Table 2, the calculated stoichiometry forexpression of purE-lacZ, purC-1acZ, and purM-lacZ is 1.0:3.3:1.9 in cells derepressed by starvation for purines and1.0:6.8:1.6 in derepressed cells grown with Xanthine. Thesedifferent levels of pur operon gene expression could reflectmRNA levels, rates of translation of a single polycistronicmRNA, or a combination of both factors. We show belowthat the observed ratios for expression of genes within theoperon are likely determined by differential rates of transla-tion.Gene regulation. The data in Table 1 show that P-galac-

tosidase levels from purine-starved and xanthine-grown cellswere repressed by adenine and guanine. Repression ratiosfor each strain, calculated relative to the maximal level ofexpression in purine-starved cells, are also given in Table 1.Repression of purE-lacZ and purM-lacZ by adenine was-32-fold and by guanine was -50-fold. Repression by ade-nine plus guanine was 100-fold or more. Repression ofpurC-lacZ by a single purine was less than half that ofpurE-lacZ and purM-lacZ.We have attempted tQ determine whether there is authen-

tic differential gene regulation in the operon by individualpurines or whether repression values for purC-lacZ are

adversely affected by the lacZ fusion. Fusions of lacZ topurE, purC, and purM were initially chosen to monitorexpression and regulation of the three multigene clusters inthe operon. In the simplest case, we might imagine that eachcluster of overlapping genes is coregulated, if not the entireoperon. We have therefore determined the regulation of theamidotransferase product of purF, the distal gene in thepurC cluster. The data summarized in Table 3 show that thepurF-encoded amidotransferase and P-galactosidase frompurM-lacZ are coregulated by purines. We must thereforeconsider the possibility that regulation of purC and purF isdiscoordinate or that fusion of lacZ to purC adversely affectsregulation of this gene by single purines. Evidence givenbelow favors the interpretation of a low-level translationalmodulation of purC.Measurements of mRNA. Levels of lacZ mRNA were

determined in an attempt to distinguish whether expressionof P-galactosidase is determined entirely by the amount ofmRNA or whether there is posttranscriptional regulation.Levels of lacZ mRNA were measured by probing RNA blotswith a labeled lacZ probe. RNA was isolated from cellsderepressed by growth with xanthine. Isolation ofRNA from

TABLE 3. Coregulation of purF and purM-lacZaCoregulation

GrowthB-Galactosidase Amidotransferase

Starved 1,320 79Xan 380 (3.5) 34 (2.3)Ade 58 (23) 5 (16)Gua 29 (46) 2 (40)Ade + Gua 3.0 (440) <0.2 (>395)

"Strain DEIMZ was grown under derepressing or repressing conditions,and purM-lacZ-encoded P-galactosidase and purF-encoded amidotransferasewere assayed in extracts. Enzyme activities are in nanomoles per minute permilligram. Repression ratios are given in parentheses.

purine-starved cells was unsatisfactory because of lowyields. The values for expression of purC-lacZ and purM-lacZ were 1,380 and 345 Miller units, respectively. Thesevalues correspond to those in Table 1. These cells containedsimilar amounts of purC-lacZ and purM-IacZ mRNA. Thevalues were 25,200 and 20,100 cpm/,ug for purC-lacZ andpurM-lacZ mRNA, respectively. Thus, the fourfold-higherlevel of 3-galactosidase in strain DE1CZ than in DElMZ wasnot due to a difference in mRNA level but rather could beattributed to a posttranscriptional effect, such as the abilityof the RNA to be translated.RNA was also isolated from wild-type cells for mRNA

measurements from the intact pur operon. Hybridizationprobes of approximately the same length (-1,000 bp) werelabeled to similar specific activities so that levels of purE,purC, and purM mRNA could be directly compared. Thedata summarized in Table 4 show that in most cases therewas approximately 1.5-fold more purEmRNA than purC andpurM mRNA, levels ofpurC and purM mRNA were similar,and there was three- to fourfold repression by adenine orguanine. In cells repressed by adenine plus guanine, RNAlevels were near the background level and are therefore notincluded. Repression by xanthine was approximately 1.5- to3-fold.Measurements of amidotransferase activity indicated that

basal operon expression in the prototrophic strain DE1 wasapproximately 25% of the expression in pur-lacZ strainsstarved for purines. Repression of amidotransferase activityby purines was approximately fourfold, comparable to therepression of mRNA shown in Table 4.

DISCUSSION

We have determined enzyme and mRNA levels fromnonrepressed and repressed cells as a first step to character-ize the expression and regulation of the B. subtilis puroperon. Fusions of lacZ were made to genes 1, 4, and 9

TABLE 4. Steady-state levels of mRNA from the intactpur operona

Steady-state level with purine supplemeptProbe

None Xan Ade Gua

purE 470 147 (3.2) 116 (4.0) 107 (4.4)purC 266 163 (1.6) 88 (3.0) 64 (4.2)purM 274 197 (1.4) 84 (3.3) 62 (4.4)

a The prototrophic strain was grown and RNA was isolated as described inMaterials and Methods. Values obtained by hybridization are expressed ascounts per minute per microgram of total RNA. These values are from a singledetermination. The numbers in parentheses are repression ratios relative tobasal expression (no added purines).

VOL. 171, 1989 2139

2140 EBBOLE AND ZALKIN

-31 +303 -31 +303

DElEZ I__I_-_-_-_-_-_-_--purE-IacZ CAT purE

2220 3278 2220 3278

DEIC4 -------

purB purC-IocZ CAT purB purC

7720 8630 7720 8630

DEIlMZ . 77773'purF purM-IacZ CAT purF pwrM

FIG. 2. Structure of the B. subtilis pur operon in lacZ fusionstrains. DElEZ, DElCZ, and DElMZ contain the intact pur operonup to the site of the lacZ fusion. Following plasmid sequences(dashed line), a duplicated region of B. subtilig pur operon DNAprecedes an intact copy of the gene to which lacZ was fused, as wellas the remaining genes of the operon. CAT, Chloramphenicolacetyltransferase gene. The genes are not drawn to scale.

(purE, purC, and purM) to facilitate measurements of en-

zyme activity. The 5'-proximal gene purE is best situated toevaluate operon expression and regulation from the pro-moter. Genes purC and purM follow the two major intercis-tronic gaps in the operon. The intercistronic gaps are poten-tial sites for any internal regulation that might occur. Thepertinent features of pur operon expression and regulationare discussed below.The pur operon promoter. The pur operon is over 13 kbp in

length, and all significant transcription appears to be initiatedfrom the c43-dependent promoter at the 5' end of theoperon; internal promoters have not been detected. We haveeliminated the possibility of internal promoters from threespecific regions of the operon as a result of the integration oflacZ fusions. Integration of each lacZ fusion results inoperon disruption and duplication of the plasmid-encodedpur gene sequences (Fig. 1). In tfrain DElCZ, purEKB aretranscribed from the pur promoter (Fig. 2). Following theintegrated lacZ and plasmid sequences, nucleotides 2220 to3228 are duplicated, followed by intact purC(orf)QLFMNH(J)D. Likewise for DElMZ, purEKBC(orf)QLF precedespurM-lacZ while nucleotides 7720 to 8600 and purMNH(J)Dfollow purM-lacZ (Fig. 2). The three lacZ fusions are purineauxotrophs, meaning that promoters must be absent fromthe following duplicated regions, numbered (1) as followsfrom the start of transcription: -31 and the start of purE at243, 2220 and the start ofpurC at 3228, and 7720 and the startof purM at 8600. The latter two segments contain thepurB-purC and purF-purM intercistronic gaps, respectively.In addition, we have not detected transcription from internalpromoters by measurement of mRNA levels. The data inTable 4 show that the level of distal purM mRNA is similarto the level ofpurC mRNA and is less than the level ofpurEmRNA. Furthermore, there is comparable regulation ofpurE and purM mRNA by adenine and by guanine, indicat-ing that if there were internal transcription initiation, itwould have to be subject to control, similar to that at the 5'end of the operon. By comppter analysis, we could not findinternal sequences similar to those involved in adenine andguanine control at the 5' end of the operon. For thesereasons, internal promoters are excluded from the intercis-tronic gap regions and are unlikely in other positions.

Sites of regulation. One of the objectives of this work wasto determine whether all regulation is exerted at the 5' end ofthe operon or whether there is additional purine-mediatedregulation at sites within the operon. Data in Table 4 showcomparable three- to fourfold repression of basal levels of

purE mRNA and purM mRNA by adenine or guanine.Likewise, data in Table 1 show comparable repression byadenine and guanine of ,-galactosidase from purE-lacZ andpurM-lacZ. On the basis of these results, there do not appearto be internal sites between the first gene, purE, and thepurM gene for purine-mediated transcriptional regulation.

Other experiments, however, sugge§t the possibility oflow-level internal regulation of purC by individual purines.Data in Table 1 indicate that P-galactosidase from purC-lacZwas partially jRsensitive to repression by xanthine, adenine,or guanine while purC mRNA was coregulated with purEand purM mRNA (Table 4). There are at least two possibleexplanations: (i) low-level translational control of the purCenzyme and (ii) an unknown adverse effect of the lacZ fusionthat desensitizes purC-lacZ to regulation. Although it isdifficult to distinguish between these two possibilities, inte-gration of purC-IacZ into the pur operon is not expected toinfluence transcriptional regulation at the 5' end of theoperon. Whatever the basis for the partial escape of purC-lacZ from repression by individual purines, this effect is nottransmitted to purF, the distal gene in the purC cluster. Datain Table 3 show that purF and purM-lacZ are coordinatelyregulated by purines, indicating that purF is not partiallyinsensitive to repression control.

Stoichiometry for enzyme synthesis and translational effi-ciency. The corrected ratio for synthesis of P-galactosidasefrom purE-lacZ, purC-lacZ, and purM-lacZ is 1.0:3.3:1.9 inpurine-starved cells and 1.0:6;8:1.6 in xanthine-grown cells.On the basis of similar levels of lacZ mRNA in strainsDElCZ and DE1MZ, these ratios for ,B-galactosidase mustreflect different translational efficiencies. The higher trans-lation efficiency of purC-lacZ relative to purE-lacZ andpurM-lacZ cannot be explained by the nucleotide sequencesof the respective translation initiation regions. All threegenes have comparably good ribosome-binding sites (1). Inaddition, purC has two feature$ usually thought to decreasetranslation initiation: a GUG initiation codon instead ofAUG (9) and a potential secondary structure of AG = -23kcal/mol (- -96, 200 J/mol) that could sequester the ribo-some-binding site. Enhanced translation of purC is possiblydue to a 5'-flanking sequence in the purB-purC gap. Trans-lational enhancemnent of an internal coding sequence in apolycistronic mRNA has been described for the E. coli atpoperon (5). Enhancement of atpE is due to a sequence thatextends more than 20 nucleotides upstream of the ribosome-binding site. The involvement of a cis-acting site to enhancetranslation of purC could provide a means for purine-mediated regulation at this site.

ACKNOWLEDGMENTS

We thank Jerry Grandoni and Robert L. Switzer (University ofIllinois) for helpful discussions.

This work was supported by U.S. Public Health Service grant GM24658 from the National Institutes of Health.

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