174 MALOY AND NUNN

Tl <- pur B- fad R

- hem A

trp

srlAT__~~~~~/ad D

rec A fod L

FIG. 1. Genetic linkage map of E. coli K-12 show-ing relevant markers and the episomes F10 and F125.The 25- and 90-min regions are enlarged to indicate therelative positions of markers. Adapted from the re-vised linkage map of Bachmann and Low (1).

shunt enzymes (unpublished data). However, it isconceivable that iciR mutations that suppress the pfkAmutation may be rarer than true revertants and otherindirect suppressor mutations (23). The fadR mutantswere selected as previously described (19).Transposon insertions near the aceA, aceB, and iclR

genes were constructed as previously described (11).Strains bearing transposon insertions within the aceAand aceB genes were obtained by localized mutagene-sis of a metA mutant by transduction to Met' Tcr witha PI vir phage stock grown on a pool of K-12 colonieswith random insertions of Tn)O (11). Strains bearinginsertions of TnlO were isolated after ampicillin en-richment with acetate as the sole carbon source (15).Surviving cells were plated on media containing 50mM acetate and 10 mM succinate, and small colonieswere checked for inability to grow on acetate as a solecarbon source. Such isolates were characterized ge-netically and biochemically. The TnlO insertion instrain KAT-1 eliminated isocitrate lyase activity. Inaddition, strain KAT-1 yielded Ace' recombinantswhen crossed wtih SM1021 (aceB), but not K8-Sm(aceA). Thus KAT-1 was classified as an aceA::TnlOmutant. The TnlO insertion in strain KBT-1 eliminatedboth malate synthase and isocitrate lyase activity.Strain KBT-1 yielded Ace' recombinants whencrossed with K8-Sm, but not SM1012. Thus, KBT-1 isbelieved to have a TnlO insertion in aceB which ispolar to the aceA gene.Operon fusions ofMu d (Ap lac) with the aceA gene

were constructed as described by Casadaban andCohen (3). Strain TL1 was infected with Mu d (Aplac), plated on MacConkey agar containing 25 ,ug ofampicillin per ml, and incubated at 300C. The Lac+Apr colonies were replica plated onto oleate, acetate,and succinate media. Colonies unable to grow onacetate or fatty acids as a sole carbon source, butcapable of growth on succinate, were genetically char-

acterized as aceA mutants, and the genotypes of themutants were confirmed enzymatically.To construct derivatives defective in homologous

recombination (recA), cells were infected with Pl virgrown on NK5304 (srIA::TnlO recA), and tetracyclineresistant (Tcr) transductants were selected. ThesrIA::TnlO and recA markers are about 50%o cotrans-ducible. The recA cotransductants were identified bytheir sensitivity to nitrofurantoin (2 ,ug/ml) (14). Tcsderivatives of these strains were selected on fusaricacid plates as described by Maloy and Nunn (12).

Matings were performed for 2 h at 37°C as describedby Miller (15). The mating mixtures were plated onminimal medium lacking methionine with streptomy-cin for counterselection against the donor. MutantF-prime factors were obtained by recombination inrecA+ strains as described by Miller (15). Most of themerodiploids constructed were somewhat unstablewhen grown in rich broth, giving rise to ca. S to 20%omonoploid segregants after overnight growth. Thesemerodiploids gave rise to less than 0.5% monoploidsegregants after overnight growth in minimal mediumlacking methionine. When it was difficult to obtainspontaneous monoploid segregants, merodiploidswere cured of their episome with acridine orange bythe method of Miller (15).Media and growth conditions. Bacteria were routine-

ly incubated in a Gyrotory water bath shaker (NewBrunswick Scientific Co.) at 37°C. For most geneticprocedures, cells were grown on Luria broth (15).When minimal medium was required, the bacteriawere usually grown on medium E (24). Carbon sourcesand supplements were sterilized separately and addedto the culture medium before inoculation. All organicacids were neutralized with potassium hydroxide be-fore sterilization. Acetate was added at 50 mM (finalconcentration). Fatty acids were provided at 5 mM inthe presence of Brij 58 (5 mg/ml). All other carbonsources were provided at 25 mM. When necessary,amino acid supplements were added at a final concen-tration of 0.01%. Tetracycline was added at a finalconcentration of 20 ,g/ml and ampicillin was added ata final concentration of 25 ,ug/ml. Cell growth wasmonitored at 540 nm on a Klett-Summerson colorim-eter.Enzyme asays. Crude extracts were prepared by

disrupting exponential-phase cells in a French press aspreviously described (11). Isocitrate lyase and malatesynthase were assayed as described by Maloy et al.(11). 3-Galactosidase activity was assayed as de-scribed by Miller (15). Protein was determined by amicrobiuret procedure (15), with bovine serum albu-min as a standard.

Materials. Most reagents used in this study were asdescribed (11). Nitrofurantoin, fusaric acid, chlortetra-cycline HCl, tetracycline, and ampicillin were ob-tained from Sigma Chemical Co., St. Louis, Mo.Tryptone, yeast extract, agar, and MacConkey agarwere obtained from Difco Laboratories. All otherchemicals were of reagent grade.

RESULTSFine-structure mapping of the aceA, aceB, and

icUR genes. The genetic linkage of the aceA,aceB, and iclR genes has been previously stud-ied by Brice and Kornberg (2) and Vanderwinkel

J. BACTERIOL.

GENETIC REGULATION OF THE GLYOXYLATE SHUNT

TABLE 1. Bacterial strains usedGenotype

PrototrophicfadRfadR::TniOiclR Zja::TniObaceA iclR zja::TniOmetA zja::TniOaceAl zia::TniOiclR7 zia::TniOfadRsrIA::TniO recA ilv-318 thi-l thr-3 relAmet-24 aceA3 iclR13 galK2 lacaceB6 glc-l thi-l relAl lacZ43glc-! thi-l relAl lacZ43Wlac U169 strATL1 aceA::Mu d (Ap lac)TL1 iciR aceA::Mu d (Ap lac)SMUD1 fadR::TniOSMUD2fadR::TniOaceA: :TniOaceA::TniO glcaceB::TniO glcpurB fadRpurB fadR trpF125/purB trpF1251purB fadR trpF125 fadRipurB trpF125 fadRipurB fadR trpfda-i relAl tonA22 DE32DE33 A(aceA-pgi)

argG6 metBI his-i leu-6 trp-31mtl-2 xyl-7 malAI gal-6 lacYIstr-104 tonA2 tsx-i supE44

JC1552 metB+ metA recAIMM-1 iciRMM-1 aceAMM-1 aceA iclRKFL10/MM-1KFL10/MM-3KFL10/MM-4KFL10 aceA/MM-1KFL10 aceA/MM-2KFL10 iclR/MM-3KFL10 iclR/MM-2KFL10 aceA icIRIMM-1KFL10 aceA iclR/MM-2

Source

J. Lederberg via CGSC'R. Simons et al. (19)R. Simons et al. (19)S. Maloy et al. (11)This workS. Maloy et al. (11)S. Maloy et al. (11)S. Maloy and W. Nunn (13)N. Kleckner strain (20)H. Komberg strain (23)S. Maloy et al. (11)This workG. WalkerThis workThis workThis workThis workThis workThis workThis workR. Simons et al. (20)R. Simons et al. (20)R. Simons et al. (20)R. Simons et al. (20)R. Simons et al. (20)R. Simons et al. (20)M. Jones-Mortimer strain via CGSC

J. Clark via CGSC

This workThis workThis workThis workThis workThis workThis workThis workThis workThis workThis workThis workThis work

a CGSC strains obtained from B. Bachmann, Coli Genetic Stock Center, Yale University, New Haven, Conn.b Transposon insertions are designated as previously described (13). When an insertion is not within a known

gene, it is given a three-letter symbol starting with z, and the second and third letters indicate the approximatemap location in minutes (e.g., zaf corresponds to 5 min, and zbb corresponds to 11 min).

and DeVlieghere (21). However, the work ofVinopal and Fraenkel (23) suggested that thereported linkage of the iclR gene was incorrect.Therefore, we first sought to determine the fine-structure map ofthe aceA, aceB, and iclR genes.These results are shown in Fig. 2. The aceA andaceB genes showed very close genetic linkage,in agreement with previous studies (2, 21). Inaddition, when the iciR mutation was mappedwith respect to the aceA or aceB mutations, itshowed close linkage with these genes. Howev-

er, genetic linkage between these mutations,determined independently with respect to out-side markers, suggested that the iclR gene wasabout 0.4 min away from the aceA and aceBgenes (1). Similar results were also obtained byVinopal and Fraenkel (23). These workers sug-gested that this discrepancy could be due to biasfor co-inheritance of the iclR mutation and theace+ genotype since constitutive expression ofthe glyoxylate shunt increases the growth rateon acetate plates. These studies confirmed the

Strain

K-12RS3010RS3040SM6034SM6046SM6001SM6009SM6042NK5304K8-5mSM1021SM1023TL1SMUDISMUD2SMUD3SMUD4KAT-1KAT-11KBT-1M11-i-SM2-5-SM11-iM2-1M47-1M23-1JM683

JC1552

MM-1MM-2MM-3MM-4MM-5MM-6MM-7MM-8MM-9MM-10MM-11MM-12MM-13

175VOL. 149, 1982

176 MALOY AND NUNN

QlcI..

aEl ol -I '>4

98 50_-93 |94---93- 848

* 88 - * ~ 46 '-59- 'l' ~~~33

'4 24

FIG. 2. Genetic linkage of the aceA, aceB, and iclR genes with respect to nearby markers. Values representPI cotransduction frequencies as percentages. When only a single arrowhead is shown, the direction of thearrowhead indicates the selected marker. In all other cases the values represent the average frequency ofseparate genetic crosses selecting for each marker. Not drawn exactly to scale.

gene order metA aceB aceA iclR as shown inFig. 2.

Insertion mutagenesis of the aceA and aceBgene. To characterize the genetic regulation ofthe glyoxylate shunt genes, we first needed tofind out whether these genes comprise an oper-on. We determined this by constructing transpo-son insertion mutations in the aceA. and aceBgenes. The transposon TnlO is strongly polar,preventing expression of genes downstream inthe same transcriptional unit (5). WhenaceA::TnlO insertions were constructed, theexpression of isocitrate lyase was completelyeliminated; however, malate synthase was ex-pressed in a normal fashion (Table 2). The glcgene encodes a second malate synthase activitywhich can substitute for the aceB gene product;therefore, aceB mutants were examined in a glcbackground. When aceB::TnlO insertions wereconstructed in a glc background, both malatesynthase and isocitrate lyase activities were

eliminated (Table 2). These data suggest that theaceA and aceB genes lie in an operon, and thedirection of transcription is from aceB to aceA.Mu d (Ap kw) fusions with the ace operon. The

activities of the glyoxylate shunt enzymes areelevated in both iclR and fadR mutants (Table3). However, there was no genetic evidencewhether the iciR or fadR genes regulated aceexpression at the level of transcription or atsome later step. To answer this question, weconstructed operon fusions of the aceA genewith Mu d (Ap lac). These fusions place theexpression of the lac operon under the control ofthe promoter and putative operator regions ofthe ace operon (3). Since aceA::Mu d (Ap lac)fusions lack isocitrate lyase, these strains cannotgrow on acetate as a sole carbon source. Never-theless, the malate synthase activity in strainsbearing aceA::Mu d (Ap lac) fusions was ex-pressed coordinately with the 3-galactosidaseactivity (Table 4). This finding agrees with the

TABLE 2. Polar insertion mutations in the aceA and aceB genesSp act (nmol/min per mg of protein)

Strain Relevant genotype Growth conditions Isocitrate Malate synthaselyase

K-12 aceA+ aceB+ Succinate 18 103Acetate 244 410Succinate + acetate 70 186

SM1023 aceA+ aceB+ glc Succinate 22 32Acetate 237 96Succinate + acetate 75 51

KAT-1 aceA::TnlO aceB+ Succinate <0.5 102Acetate aSuccinate + acetate <0.5 249

KAT-11 aceA::TnlO aceB+ glc Succinate <0.5 69AcetateSuccinate + acetate <0.5 198

KBT-1 aceA+ aceB::TnlO glc Succinate <0.5 0.8AcetateSuccinate + acetate <0.5 1.0

a Dashes indicate no growth on this carbon source.

J. BACTERIOL.

GENETIC REGULATION OF THE GLYOXYLATE SHUNT 177

TABLE 3. Specific activities of glyoxylate shunt enzymes in iciR and fadR mutantsSp act (nmol/min per mg of protein)

Isocitrate lyase Malate synthaseStrain Relevant genotype

Succinate Acetate Succinate + Succinate Acetate Succinate +

K-12 Prototrophic 21 244 70 103 410 186SM6034 iciR 209 320 276 267 594 330RS3040 fadR::TnlO 172 272 204 371 566 389SM6042 iciR fadR::TnlO 283 3% 386 3% 592 402

polarity of transposon insertions in the acegenes.The relative expression of 3-galactosidase in

the aceA::Mu d (Ap lac) fusion strains paralleledthe activities of isocitrate lyase and malatesynthase expressed in corresponding aceA+strains under similar growth conditions (Tables 3and 4). When the aceA::Mu d (Ap lac) fusionwas present in a fadR+ iclR+ background, theactivity of the lacZ gene product, ,B-galacto-sidase, was low on medium containing succinateas a sole carbon. source, but was induced whengrown in the presence of acetate (Table 4). TheiclR aceA::Mu d (Ap lac) strain grown on succi-nate expressed levels of ,-galactosidase equiva-lent to that of the iclR+ fadR' aceA::Mu d (Aplac) strain grown in the presence of acetate.These data indicate that induction of the aceoperon by the iclR gene occurs at the level oftranscription.When the aceA::Mu d (Ap lac) fusion was

present in afadR background, the activity of 1B-galactosidase was elevated during growth onsuccinate, but further doubled after growth inthe presence of acetate (Table 4). The 3-galacto-sidase activity expressed by the aceA::Mu d (Aplac) fusion in a fadR iclR double mutant wasconsiderably higher than that of either the fadRor iciR strain. In addition, the ,B-galactosidaseactivity of the fadR iclR aceA::Mu d (Ap lac)strain was equivalent after growth on succinate

or succinate plus acetate (Table 4). These dataare consistent with a role of thefadR gene in thetranscriptional control of the ace operon. Fur-thermore, the increased expression of the aceoperon in thefadR strain after growth on acetate(18) and the greater expression of the ace operonin the fadR iciR double mutant suggest that theiciR and fadR genes may independently affectthe expression of the ace operon.Construction of strains merodiploid for the icLR

gene. As shown in Fig. 2, the iciR gene mapsnear the ace operon at 90 min on the E. colichromosome (1) and is closely linked to themetA gene. To examine dominance betweeniclR alleles, we constructed a series of strainsmerodiploid for the genes in the 90-min region.Several F-primes previously isolated and char-acterized by Low (9) reportedly carry chromo-somal genes from this region. Three F-primefactors, KFL10, KFL11, and KFL12, wereshown to complement metA and aceA muta-tions. The F-prime KFL12 allowed only slowgrowth of a host strain bearing a metA mutationand was prone to rapid segregation. The F-primes KFL11 and KFL10 both allowed rapidgrowth of host strains and segregated less readi-ly under selective growth conditions. Since theF-prime in KFL11 was reported to be over twicethe size of that in KFL10, all further studieswere performed with KFL10. When insertedinto strain JM683, which is deleted for the region

TABLE 4. Mu d (Ap lac) operon fusions with the aceA gene

S-Galactosidase Sp act (nmol/min per mg of protein)activity (U)0 Isocitrate lyase Malate synthase

TL1 iclR+fadR+ AlacZ Succinate <1 33 125Succinate + acetate <1 151 317Acetate <1 270 584

SMUD-1 aceA::Mu d(Ap lac) iclR+ Succinate 20 <0.5 87fadR+ AlacZ Succinate + acetate 310 <0.5 368

SMUD-2 aceA::Mu d(Ap lac) iclR Succinate 300 <0.5 273fadR+ AlacZ Succinate + acetate 310 <0.5 377

SMUD-3 aceA::Mu d (Ap lac) icOR+ Succinate 200 <0.5 341fadR AlacZ Succinate + acetate 440 <0.5 429

SMUD-4 aceA::Mu d(Ap lac) iciR Succinate 650 <0.5 514fadR AlacZ Succinate + acetate 700 <0.5 582

a ,-Galactosidase activity expressed in units as defined by Miller (15).

VOL. 149, 1982

178 MALOY AND NUNN

containing the ace operon, KFL10 allowedexpression of the ace operon in an induciblemanner (data not shown). The latter resultssuggest that this episome carried the aceA+,aceB+, and iclR+ genes. The absence of theiclR+ gene in strain JM683 was later confirmedwith an F-prime bearing the genes metA+ aceA+aceB+ iciR; this strain expressed the aceA andaceB gene products constitutively. When curedof the episome, strain JM683 reverted to theAce- phenotype.The manner of construction of the merodip-

loids used in the iciR dominance tests is shownin Table 5. In all strains, the host chromosomeharbored an auxotrophic mutation in the metAgene which was complemented by the respectivewild-type allele on the F-prime. This arrange-ment facilitated strain construction and pre-vented segregation of the episome when themerodiploids were grown in the absence ofmethionine. The host strains were all recA deriv-atives of JC1552 which differed only in markersrelated to the merodiploid analysis (Table 5).The merodiploid strains constructed were all

recA and therefore defective in homologousrecombination between the F-prime and themain chromosome. To prove the diploidy of theiciR gene in these stable merodiploids, we exam-ined the phenotypes of monoploid segregantstrains obtained by acridine orange treatment.Segregants were recognized by an auxotrophicrequirement for methionine as well as an inabil-ity to transfer markers in further matings. Allsegregants were found to be phenotypicallyidentical to the monoploid host strain. In addi-tion, each merodiploid was shown to be able totransfer episomal markers in matings with ap-propriate recA recipients (data not shown). Thisevidence indicates that the merodiploids were,in fact, diploid for the iclR gene.

Expression of the ace operon in strains mero-diploid for the icR locus. In wild-type E. coli, theglyoxylate shunt enzymes are induced only dur-ing growth in the presence of acetate. In con-trast, the glyoxylate shunt enzymes are ex-pressed constitutively in iclR mutants. Thus, wesought to determine whether the iclR gene regu-lates the ace operon in a cis- or trans-dominantmanner. The data in Table 6 indicate that, when-ever at least one copy of the iclR+ gene waspresent, whether on the F-prime or the chromo-some, the expression of the aceA gene wasinducible by growth in the presence of acetate.This was true whether the iclR+ gene was cis ortrans to the aceA+ allele. Only strains with nocopies of the iclR+ allele expressed the aceAgene constitutively. This finding implies that theiclR+ allele is trans-dominant to the iclR allele.Expression of the ace operon in strains mero-

diploid for the fadR locus. The distant maplocation of the fadR gene, as well as its knownrole in the control of thefad regulon by produc-ing a negatively acting regulatory protein, sug-gested that the transcriptional control of the aceoperon by the fadR gene occurred in a trans-dominant manner. However, to confirm this, westudied the effects of mutants merodiploid forthe fadR locus on the expression of the aceoperon. The construction and characterizationoffadR merodiploids have been previously de-scribed (20). As shown in Table 7, whenever asingle copy of the fadR+ allele was present,whether on the episome or the chromosome, theglyoxylate shunt enzymes were regulated nor-mally (i.e., induced by growth on acetate). Onlywhen there were nofadR+ alleles present (M2-5-S and M23-1) was the expression of the glyoxy-late shunt elevated under noninducing growthconditions. This is consistent with a trans-domi-nant behavior of the fadR+ allele.

TABLE 5. Construction of merodiploids used for testing dominance of the iclR locus

Strain Donor Recipient Selected markers Relevant genotypeof merodiploida

MM-1 O/aceA+ icOR+MM-2 OliclRMM-3 OlaceAMM-4 OlaceA iclRMM-5 KFL10 MM-1 Met+ Strr F'aceA+ icIR+laceA+ icOR+MM-6 KFL10 MM-3 Ace+ Met+ Str+ F'aceA+ iclR+laceA icOR+MM-7 KFL10 MM-4 Ace+ Met+ Strr F'aceA+ iclR+laceA iclRMM-8 SM6009 MM-1 Met+ Strr F'aceA icIR+/aceA+ icOR+MM-9 SM6009 MM-2 Met+ Strr F'aceA iclR+laceA+ iclRMM-10 SM6034 MM-3 Ace+ Met+ Strr F'aceA+ iciRlaceA iclR+MM-11 SM6034 MM-2 Met+ Str F'aceA+ iclRlaceA+ iciRMM-12 K8-5M MM-1 Met+ Str+ F'aceA iclRlaceA+ iclR+MM-13 K8-5M MM-2 Met+ Strr F'aceA icIRlaceA+ iclR

a Genotype indicated by episomal alleles/chromosomal alleles. A zero indicates that no episome was present.

J. BACTERIOL.

GENETIC REGULATION OF THE GLYOXYLATE SHUNT 179

TABLE 6. Specific activity of isocitrate lyase in strains merodiploid for the iciR geneIsocitrate lyase activity (nmoUmin per mg of protein)

Striun Relevant genotype Succinate Succinate + Acetateacetate

MM-1 OaceA+ McR+ 21 98 316MM-2 O/aceA+ iciR 206 299 355MM-4 OlaceA iciR <0.5 <0.5 <0.5MM-S F'aceA+ iclR+IaceA+ icR+ 31 50 114MM-6 P'aceA+ iclR+/accA icIR+ 18 45 206MM-7 F'aceA+ icLR+IaccA iciR 52 79 305MM-8 F'aceA icLR+IaceA+ clR+ 3 31 209MM-9 F'aceA icR+IaceA+ iciR 19 69 276MM-10 F'aceA+ iclR/accA icIR+ 23 74 298MM-11 F'aceA+ icURlaceA+ icIR 292 326 422MM-12 F'aceA iclRaceA+ IclR 17 81 310MM-13 F'aceA icWiaceA+ icU 269 285 387

Construction of 1cU merodiploids is shown in Table 4.

DISCUSSION the ace operon in the 90-min region (Fig. 2), andthefadR gene maps at 25 min (19). The cisltrans

The genes for the glyoxylate shunt enzymes, tests for both the iciR and fadR genes indicateaceA and aceB, map at 90 min on the E. coli K- that the wild-type alleles are trans-dominant to12 chromosome (Fig. 2). Since the aceA and the mutant alleles (Tables 6 and 7). This is mostaceB genes are very closely linked genetically simply interpreted as the production of difflus-and their expression is coordinately controlled, ible, negatively acting regulatory elements orit has been proposed that they may form an repressors by both the iciR andfadR genes (4,operon (2). Polar transposon insertions in the 19). The fadR gene has been previously shownaceB gene eliminate expression ofboth the aceB to act as a repressor for thefad regulon (19). Theand aceA genes (Table 2), indicating that these fad regulon is induced during growth on long-genes are transcribed as a single, polycistronic chain fatty acids (:Cj1) (17, 19, 20). The glyoxy-message (4, 5). Thus, the aceA and aceB genes late shunt enzymes are not directly induced bycomprise an operon, and the direction of tran- acetate or acetyl-CoA (6, 11). On the basis ofscription is from the aceB gene to the accA gene. genetic studies, Kornberg (6) proposed that the

Expression of the ace operon is under the glyoxylate shunt enzymes are derepressed bytranscriptional control of two genes, iciR and low levels of pyruvate and phosphoenolpyruvatefadR (Tables 3 and 4). The iciR gene maps near that result from growth on acetate or fatty acids

TABLE 7. Specific activities of glyoxylate shunt enzymes in strains merodiploid for the fadR geneSp act (nmol/min per mg of protein)

Strain fadA genotype" Growth conditions Isocitrate lyase Malate synthaseM11-i-S 0/+ Succinate 11 42

Acetate 95 198Oleate 125 253

M2-5-S 0/- Succinate 81 111Acetate 106 315Oleate 128 320

M11-i +/+ Succinate 7 36Acetate 84 183Oleate 83 238

M12-1 +/- Succinate 12 45Acetate 64 205Oleate 121 284

M47-1 -/+ Succinate 18 52Acetate 60 210Oleate 146 277

M23-1 -/- Succinate 62 125Acetate 95 329Oleate 122 336

a fadR genotype shown as episomal alele/chromosomal allele. A zero indicates that no episome was present.

VOL. 149, 1982

180 MALOY AND NUNN

as a sole carbon source. However, more thor-ough biochemical studies have shown that thiscannot fully account for the regulation of theglyoxylate shunt enzymes (7, 10). Thus, theintracellular signal for induction of the glyoxy-late shunt during growth on acetate remains tobe elucidated.Both iclR andfadR mutants expressed elevat-

ed levels of ace enzymes during growth on thenoninducing carbon source succinate (Table 3and 4). In this case, expression of the ace operonwas even greater in iclR fadR double mutants(Tables 3 and 4). Although several models couldbe proposed from these data, the simplest inter-pretation of these results is that the iclR andfadR gene products act independently to causethe partial repression of the ace operon; actingtogether, they cause the full repression of theace operon.Although several regulatory systems have

been shown to be controlled by multiple positiveeffectors (4), no well-characterized bacterial op-erons have been shown to be controlled bymultiple repressor proteins. However, in bacte-riophage lambda, the binding of two negativelyacting regulatory proteins, cI repressor and Croprotein, to the same control region determinesthe infective pathway utilized (8). The evolutionof dual repressor control of the ace operon mayhave conferred a metabolic advantage to cellsrequired to grow on acetate or fatty acids as acarbon source. During growth on acetate, asubstantial portion of the intracellular acetyl-CoA must be fully oxidized by the completetricarboxylic acid cycle to generate metabolicenergy. On the other hand, during growth onfatty acids a considerable amount of energy isgenerated from the breakdown of fatty acids toacetyl-CoA. Therefore, less acetyl-CoA is need-ed to pass through the complete tricarboxylicacid cycle for energy production, and a greateramount of the acetyl-CoA can be shuntedthrough the glyoxylate bypass for anabolic pur-poses. A dual repressor control mechanism forthe ace operon may allow different levels ofexpression of the glyoxylate shunt enzymes un-der these conditions.

ACKNOWLEDGMENTSWe thank Graham Walker for the Mu d (Ap lac) phage and

strains used for operon fusions. Barbara Bachmann generous-ly supplied strains used for genetic studies. We are grateful toKevin Bertrand, Rowland Davis, and Paula Hennen for help-ful discussions and comments on the manuscript.

This work was supported by Public Health Service grantGM22466-1A from the National Institute of General MedicalSciences. W.D.N. is an Established Investigator of the Ameri-can Heart Association. S.R.M. was supported by TrainingGrant GM07311 from the National Institute of General Medi-cal Sciences.

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Escherichia coli K-12, edition 6. Microbiol. Rev. 44:1-56.

2. Brice, C. G., and H. L. Kornberg. 1968. Genetic control ofisocitrate lyase activity in Escherichia coli. J. Bacteriol.96:2185-2186.

3. Casadaban, M. J., and S. N. Cohen. 1979. Lactose genesfused to exogenous promoters in one step using a Mu-lacbacteriophage: In vivo probe for transcriptional controlsequences. Proc. Natl. Acad. Sci. U.S.A. 76:4530-4533.

4. Goldberger, R. F. 1979. Biological regulation and develop-ment, vol. 1, Gene expression. Plenum Press, New York.

5. Kedkner, N., J. Roth, and D. Bottebn. 1977. Geneticengineering in vivo using translocatable drug-resistanceelements. New methods in bacterial genetics. J. Mol.Biol. 116:125-159.

6. Kormberg, H. L. 1966. The role and control of the glyoxy-late cycle in Escherichia coli. Biochem. J. 99:1-11.

7. Lakshml, T. M., and R. B. Heling. 1978. Acetate metabo-lism in Escherichia coli. Can. J. Microbiol. 24:149-153.

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10. Lowry, 0. H., J. Carter, J. B. Ward, and L. Glaaer. 1971.The effect of carbon and nitrogen sources on the level ofmetabolic intermediates in Escherichia coli. J. Biol.Chem. 246:6511-6521.

11. Maloy, S. R., M. Bohlander, and W. D. Nunn. 1980.Elevated levels of glyoxylate shunt enzymes in Esche-richia coli strains constitutive for fatty acid degradation.J. Bacteriol. 143:720-725.

12. Maloy, S. R., and W. D. Nunn. 1981. Selection for loss oftetracycline resistance by Escherichia coli. J. Bacteriol.145:1110-1112.

13. Maloy, S. R., and W. D. Nunn. 1981. Role of genefadR inEscherichia coli acetate metabolism. J. Bacteriol 148:83-90.

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J. BACTERIOL.

ERRATA

Isolation and Partial Characterization of Membrane VesiclesCarrying Markers of the Membrane Adhesion SitesMARGRET H. BAYER, GREGORY P. COSTELLO, AND MANFRED E. BAYER

The Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

Volume 149, no. 2, p. 765, column 1, lines 10-17: Should read ". . . Tomita et al. (40) reported themigration of viral capsid markers from OM fractions to vesicle fractions of intermediate density;however, a redistribution of phage label may occur under similar conditions, as was reported byCrowlesmith et al. (15) for phage P22-labeled Salmonella typhimurium cells. Therefore, we....'

Genetic Regulation of the Glyoxylate Shunt in Escherichia coliK-12

STANLEY R. MALOY AND WILLIAM D. NUNN

Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92717

Volume 149, no. 1, p. 173, column 1, line 2: The statement that cis/trans analysis of iclR alleleshad not been published previously was in error. We have recently learned that dominance studies oniclR alleles had been performed before our studies (H. L. Kornberg, FEBS Symp. 19:5-18, 1969).The experimental conditions were different from ours, but the results were similar.

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