Isolation and Characterization of Salmonella typhimurium Glyoxylate Shunt Mutantst REBECCA B. WILSON AND STANLEY R. MALOY * Department of Microbiology , University of Illinois , Urbana , Illinois 61801 Growth of SalmoneUa typhimurium on acetate as a sole carbon source requires expression of the glyoxylate shunt ; however , the genes for the glyoxylate shunt enzymes have not been previously identified in S. typhimurium .
In this study , we isolated transposon insertions in the genes for the two unique enzymes of this pathway , aceA ( isocitrate lyase ) and aceB ( malate synthase ) .
The aceA and aceB genes were located at 89.5 min on the S. typhimurium genetic map .
Genetic linkage to nearby loci indicated that the relative gene order is purDJ metA aceB aceA .
Transposon insertions in aceB were polar on aceA , suggesting that the genes form an operon transcribed from aceB to aceA .
Transcriptional regulation of the aceBA operon was studied by constructing mini-Mu d ( lac Kan ) operon fusions .
Analysis of these fusions indicated that expression of the aceBA operon is regulated at the level of transcription ; the aceBA genes were induced when acetate was present and repressing carbon sources were absent .
Although glucose represses expression of the aceBA operon , repression does not seem to be mediated solely by cyclic AMP-cyclic-AMP-receptor-protein complex .
Mutants with altered regulation of the aceBA operon were isolated .
The glyoxylate shunt is an anapleurotic pathway that allows the net accumulation of four carbon compounds during-growth on two carbon substrates such as acetate or acetyl coenzyme A generated by fatty acid degradation .
This accumulation is accomplished by bypassing the two C02-evolving steps of the tricarboxylic acid cycle ( 5 , 13 ) .
In Escherichia coli , the two unique enzymes of the glyoxylate shunt , isocitrate lyase ( aceA ) and malate synthase ( aceB ) , are induced during-growth on acetate or fatty-acids .
In E. coli , the aceA and aceB genes are in an operon that is transcriptionally regulated by two repressors encoded by the iclR and fadR genes ( 20 ) .
In addition to regulating the aceBA operon , the fadR repressor also controls expression of the genes for fatty acid degradation ; expression of the fad genes is induced by long-chain-fatty-acids ( > C12 ) ( reviewed in reference 24 ) .
Although the glyoxylate shunt in E. coli has been studied for many years , the mechanism of genetic regulation remains unclear .
Genetic studies suggest that acetate or acetyl coenzyme A is not the inducer of the glyoxylate shunt ( 14 ) .
Other potential regulatory mechanisms have been proposed based on the physiology of growth on acetate .
When cells grown on another carbon source are transferred to media with acetate as the sole carbon source , dicarboxylic acids from the tricarboxylic acid cycle are depleted for biosynthetic uses .
This depletion has at least two consequences : there is inadequate oxaloacetate to combine with acetyl coenzyme A to run the tricarboxylic acid cycle , and there is a consequential drop in the intracellular concentration of other intermediates which must be derived from the tricarboxylic acid cycle .
Decreasing the concentration of an intermediate which also functions as a corepressor of the glyoxylate shunt could allow induction of the glyoxylate shunt enzymes .
Kornberg ( 14 ) suggested that phosphoenolpyruvate may be t This paper is dedicated to the memory of our late friend and colleague William Nunn .
the corepressor which interacts with the iclR gene product to regulate expression of the glyoxylate shunt .
However , later studies showed that phosphoenolpyruvate levels do not decrease significantly during-growth on acetate and hence can not account for the induction of the glyoxylate shunt enzymes ( 15 , 18 ) .
Thus , the actual corepressor or inducer that interacts with the iclR gene product is not yet known .
In some organisms , expression of the glyoxylate shunt is related to pathogenicity ( 23 ; J. Paznokas , personal communication ) .
Therefore , we have begun to characterize the glyoxylate shunt from the closely related bacterium , Salmo-nella typhimurium .
Expression of the glyoxylate shunt in S. typhimurium has not been previously reported .
In this paper , we describe the isolation and characterization of aceA and aceB mutations in S. typhimurium and the initial isolation of aceBA regulatory mutants .
MATERIALS AND METHODS Bacterial strains .
The genotypes of the strains used in this study are shown in Table 1 .
All S. typhimurium strains were derived from LT2 .
Nutrient broth ( 0.8 % ; Difco Laboratories , Detroit , Mich. ) with 0.5 % NaCl added was used for rich-medium .
The minimal-medium used was NCE ( 2 ) .
Carbon sources were added to minimal-medium at the following final concentrations : 0.4 % potassium acetate , 0.6 % sodium succinate , and 5 mM potassium oleate .
When oleate was added to media , the detergent Brij 58 was also added at 0.5 % .
Amino acid supplements were added at the concentration listed in Davis et al. ( 8 ) .
Antibiotics were added at the following final concentrations : kanamycin sulfate , 125 , ug/ml in minimal-medium or 50 , ug/ml in rich-medium tetracycline hydrochloride , 10 , ug/ml in minimal-medium or 20 pug/ml in rich-medium ; sodium ampicillin , 15 , ug/ml in minimal me-dium or 30 , ug/ml in rich-medium ; and streptomycin sulfate , 2 mg/ml in minimal-medium with 1 % nutrient broth added .
5-Bromo-4-chloro-3-indolyl-p-D-galactoside ( X-gal ) was dissolved in N,N-dimethylformamide at a concentration of 20 mg/ml and then added to media to yield a final concentration of 20 , g/ml .
3 ' , 5 ' - Cyclic AMP ( cAMP ) was added to media at a final concentration of 5 mM .
The carbon sources , antibiotics , and supplements were all obtained from Sigma Chemical Co. , St. Louis , Mo. .
Solid medium contained 1.5 % Bacto-Agar ( Difco ) .
The high-frequency generalized transducing bacteriophage P22 HT105/1 int-201 was used for all transductions ( 26 ) .
Phage lysates were prepared as described by Davis et al. ( 8 ) .
Transductions were performed by infecting cells with phage at a multiplicity of about 1 PFU per cell .
Usually phage and bacteria were mixed directly on selective medium .
When Kanr and Strr colonies were selected , phage and bacteria were spread on nonselective medium , incubated for 4 to 5 h at 37 °C , and then replica plated onto a medium containing the antibiotic .
Transductions were purified , and phage-free clones were isolated by streaking them nonselectively on green indicator plates ( 6 ) .
The phage-free colonies were then checked for P22 sensitivity by cross-streaking them against P22 H5 ( a clear-plaque mutant ) ( 8 ) .
Conjugational matings were performed directly on selective plates as described previously ( 22 ) .
Donor strains containing temperature-sensitive F-prime plasmids were grown at 300C .
Hfr strains were formed by recombination between the lac homology on F'ts 114 lac + and the lac region on the chromosomal Mu d fusion as described by Maloy and Roth ( 21 ) .
A simplified nomenclature suggested by K. Hughes and J. Roth ( in press ) is used to describe the Mu d operon fusion vectors used in this study .
Mu dl ( Amp lac ) is the original operon fusion vector constructed by Casadaban and Cohen ( 4 ) .
It carries all essential Mu transposition functions , and expression of these functions is controlled by a temperature-sensitive repressor ( cts ) .
This vector is simply designated Mu dl .
Mu d11734 is a derivative of Mu dl which has been deleted for transposition functions and carries Kanr instead of Ampr ( 5 ) .
Mu d11734 is designated Mu dJ .
Mu dl-8 is a derivative of Mu dl that has amber mutations in the Mu transposition functions ( 12 ) .
Mu dl-8 is designated Mu dA .
Isolation of aceBA mutants .
Stable ace : : Mu dJ fusions were isolated in two ways .
Random Mu dJ insertions were obtained by transitory trans-acting transposition ( Hughes and Roth , in press ) .
LT2 was transduced to Kanr with phage grown on strain TT10289 .
This strain carries two Mu d insertions in the his operon , Mu dJ ( Kan ' ) and Mu dl ( Amp ' ) .
Because of size limitations , P22 can not package both Mu d phage into a single transducing particle .
Mu dJ does not carry the Mu transposition functions , but Mu dl does .
When a transducing fragment carries the entire Mu dJ genome and the portion of the Mu dl genome that encodes transposase , the transposase gene can be transiently expressed to provide transposase which can act in trans on the Mu dJ genome , allowing Mu dJ to transpose to new sites in the chromosome .
The linear transducing fragment is rapidly degraded by cellular nucleases , eliminating transposase expression .
Thus , once the Mu dJ is integrated into the chromosome , it is stable .
Homologous recombination of the Mu dJ transducing fragment into the chromosome yields His-Kanr colonies , while transposition usually yields His ' Kanr prog-eny .
To eliminate inheritance of the Mu dJ by homologous recombination , transductants were selected on minimal me-dium containing kanamycin sulfate .
The fHis + Kanr transductants were replicated onto X-gal , acetate , oleate , succinate , and sodium ampicillin plates .
Nomenclature for Mu d insertions is described in Materials and Methods .
b Obtained from K. Sanderson at the Salmonella Genetic Stock Center ( SGSC ) , Department of Biology , University of Calgary , Calgary , Alberta , Canada .
c Obtained from B. Bachmann at the E. coli Genetic Stock Center ( CGSC ) , Yale University , New Haven , Conn. .
Suc + Amps Kanr colonies were picked as potential aceBA : : Mu dJ fusions .
The fusions were then mapped with respect to metA , and enzymatic assays were performed to confirm the genotypes of the mutants .
We also isolated aceBA : : Mu dJ insertions by localized mutagenesis ( 8 , 11 ) .
To accomplish this , strain TR5670 ( metA ) transduced with P22 phage stock was a grown on a pool of colonies with random Mu dJ insertions , and Met + His + Kanr transductants were selected on minimal-medium containing kanamycin sulfate .
About 13 % of these insertions had the phenotype expected for aceBA mutants ( Ace-OleSuc + Amps ) .
TnJQ insertions in and near the aceBA genes were also isolated by localized mutagenesis .
Strain TR5670 ( metA ) was transduced to Met + Tetr with phage grown on a pool of random TnlO insertions in LT2 .
The transductants were screened and characterized as described above .
Mutant derivatives of the ace : : Mu dJ fusions that were defective for cya or crp were constructed by transduction of the fusions with phage grown on PP1002 ( cya : : TnlO ) or PP1037 ( crp : : TnlO ) .
Transductants were selected on MacConkey ribose glycerol plates ( 1 ) that contained tetracycline hydrochloride .
White Tetr colonies were chosen or as cya crp mutapts .
Although the aceBA : : Mu dJ fusions were X-gal + , the level of expression of the lac genes was insufficient to allow-growth on lactose as a sole carbon source ( Lac - ) .
Regulatory mutants were isolated by selecting for increased expressions of the lac operon from aceBA : : Mu dJ operon fusions .
Samples ( 0.1 ml ) of Qvermnght cultures of ace : : Mu dJ fusion strains were spread on minimal lactose plates , and a few crystals of nitrosoguanidine were placed in the center of the plate .
Lac + colonies that arose were restreaked on minimal lactose plates and tested for constitutive 3-galactosidase expression .
F ' 110 carries the region of the E. coli K-12 chromosome between polA and malB ( 17 ) , including the metA and aceBA + genes ( 20 ) .
To transfer F ' 110 to S. typhimurium , the E. coli donor JC15533/KFL10 ( F ' 110 ) was mated with the S. typhimurium recipient TR5878 ( r-m + metA metE ) , with selection for MetA + and counterselection against the auxotrophic mutations in the donor .
Vitamin B12 was included in the medium to suppress the metE mutation in TR5878 ( 13 ) .
The resulting exconjugants ( TR5877/F ' 110 ) were transduced to Tetr with phage P22 grown on TR627 , yielding TR5877/F ' 110 zzf : : TnJO .
F ' 110 zzf : - TnJO was then mated into other S. typhimurium strains by selection for Tetr and counterselection against the auxotrophic mutations in the donor .
Cells were grown to mid-log phase ( 100 to 120 Klett units ) in 100 ml of minimal-medium , and crude extracts were prepared in a French press ( 19 ) .
Specific activities of isocitrate lyase and malate synthase were determined by using spectrophotometric assays previously described ( 19 ) .
Isocitrate lyase activity was measured by determining the rate of glyoxylate formation , and malate synthase activity was measured by determining the rate of hydrolysis of acetyl coenzyme A. Protein concentrations of the crude extracts were determined by a modified Bradford assay ( Bio-Rad Laboratories , Richmond , Calif. ) with bovine serum albumin as a standard .
, - Galactosidase activity was measured as described by Miller ( 22 ) by using the chloroform-sodium dodecyl sulfate permeabilization procedure .
P-galactosidase activity was expressed as nanomoles per minute per optical density unit at 650 nm .
cQ ) * < Q ) E. I a I 16 % ( 31/200 ) 62 % ( 124/200 ) 74 % ( 1631/2200 ) 25 % ( 49/196 ) 63 % ( 190/300 ) 81 % ( 2207/2510 ) 1 % ( 4/452 ) 5 % ( 22/468 ) 23 % ( 183/803 ) 43 % ( 217/500 ) l1 % ( 0/315 ) FIG. 1 .
Genetic linkage of aceBA mutations with nearby loci .
P22 cotransduction frequencies are shown as percentages .
The number of transductants is shown in parentheses .
The direction of the arrowhead indicates the selected marker .
The distances between markers are not drawn exactly to scale .
RESULTS Isolation of aceBA mutants .
Mutants defective in the glyoxylate shunt enzymes should be unable to use acetate ( Ace - ) or fatty-acids ( Fad - ) as a sole carbon source but should grow on succinate ( Suc + ) .
Screening of random pools of TnlO and Mu dJ transposon insertions yielded a large number of Ace-insertion mutants , but less than 20 % of the Ace-Suc + mutants had the phenotype expected for aceBA mutants .
Some Ace-Suc + mutants were able to grow on fatty-acids as expected for ack ( acetate kinase ) or pta ( phosphotransacetylase ) mutants ( 16 ) .
Other Ace-Suc ' mutants were able to grown on acetate or fatty-acids if supplemental levels of isoleucine were provided ( 7 ) .
In contrast , after localized mutagenesis of the metA region , all of the Ace-Suc + mutants obtained had the phenotype expected for aceBA mutants .
Potential aceBA mutants with the Ace-Fad-Suc + phenotype were further characterized .
Genetic mapping of aceBA mutations .
The aceBA genes had not been previously mapped in S. typhimurium .
Knowing that the aceBA genes are cotransducible with metA at 90 min on the E. coli genetic map ( 3 , 20 ) and given the close similarity of the genetic maps of E. coli and S. typhimurium ( 25 ) , we tested the linkage of the Ace-mutations to the metA locus .
All the potential aceBA mutations were approximately 80o cotransducible with metA by P22 transduction .
Two factor crosses between these mutants and nearby loci indicated that the gene order is purJD metA ace ( Fig. 1 ) .
These results agree with the fine-structure genetic map of this region from E. coli ( 3 , 20 ) .
Specific activity of the glyoxylate shunt enzymes .
Expression of the glyoxylate shunt enzymes was induced in S. typhimurium LT2 during-growth on acetate ( Table 2 ) .
When succinate and acetate were provided , isocitrate lyase and malate synthase expression were partially repressed , and very low levels of activity were expressed during-growth-on-succinate alone ( Table 2 ) .
Although the pattern of induction was similar , expression of both isocitrate lyase and malate synthase was about fourfold lower in S. typhimurium LT2 than in E. coli K-12 ( Table 2 ) .
When transferred to S FIG. 1 .
Genetic linkage of aceBA mutations with nearby loci .
P22 cotransduction frequencies are shown as percentages .
The number of transductants is shown in parentheses .
The direction of the arrowhead indicates the selected marker .
The distances between markers are not drawn exactly to scale .
LT2 ace + 6 19 MS229 aceB : : TnlO 2 3 MS227 aceA : : TnlO 3 3 MS1311 aceB : : Mu dJ 2 4 MS1309 aceA : : Mu dJ 2 3 MS1361C aceB : : Mu dJ/F ' ace + c 7 18 MS1360C aceA : : Mu dJ/F ' ace + c 5 22 K412 ace + 16 49 a All activities are expressed as nanomoles minute-1 milligram of protein - ' .
b , No growth on acetate as a sole carbon source .
c F'ace + is an E. coli F ' factor ( F ' 110 ) that carries the region of the E. coli K-12 chromosomne between approximately 84 and 91 min .
This F ' is known to complement the metA aceBA genes of E. coli ( 18 ) .
40 43 112-123-490 239 typhimurium on an F-prime plasmid , the E. coli aceBA genes complemented the S. typhimurium aceBA mutants ( Table 2 ) , and the activities of isocitrate lyase and malate synthase expressed were similar to the activities of the S. typhimu-rium enzymes .
Although lower activities of glyoxylate shunt enzymes were expressed in S. typhimurium than in E. coli , both organisms grew at about the same rate on acetate as a sole carbon source ( approximately 110-min doubling time ) .
We obtained two classes of Ace-insertion mutants .
One class had very low levels of isocitrate lyase activity ( aceA ) but retained normral levels of malate synthase activity ( Table 2 ) .
The other class had very low levels of both isocitrate lyase activity and malate sypthase activity ( aceB ; Table 2 ) .
A low , uninducible residual level of malate synthase activity was observed in all of the mutants isolated .
In E. coli , residual malate synthase activity in aceB insertion mutants is due to a second malate synthase activity encoded by the glc gene ( 27 ) .
However , no glc mutants of S. typhimurium have been isolated .
This is the result expected if transposon insertions in the aceB gene were polar on expression of the aceA gene .
This result suggests that in S. typhimurium , as in E. coli , the aceA and aceB genes form an operon that is transcribed from aceB to aceA .
Regulation of operon ftsons .
Operon fusions between the aceB and aceA genes and the lac operon were constructed to study the transcriptional control of the aceBA operon .
Insertions of Mu dJ in the ace genes in the correct orientation form stable operon fusions which place the transcription of the lac operon under control of the aceBA promoteroperator sites .
Hence , transcription of the aceBA operon can be studied by measuring the expression of,3-galactosidase .
Catabolite-repression of the aceBA operon ( - Galactosidase octivity '' in : Glucose plus acetate plus cAMP BL TA Strain Genotype Glucose plus TABLE 3 .
Expression of , B-galactosidase from aceBA operon fusions Glucose acetate , B-Galactosidase activitya in : Succinate Strain Genotype Succinate plus Acetate acetate MS1311 aceB : : Mu dJ 22 110 MS1309 aceA : : Mu dJ 20 49 MS1393 aceB : : Mu dJ aceA : : TnlO 17 104 MS1398 aceB : : TnlO aceA : : Mu dJ < 1 < 1 MS1361 aceB : : Mu dJ/F ` ace + 16 76 MS1360 aceA : : Mu dJ/F ` ace + 10 50 a Expressed as nanomoles minute - ' milligram of protein - ' .
b - , No growth on acetate as the sole carbon source .
Expression of,-galactosidase in aceB : : Mu dJ and aceA : : Mu dJ operon fusions was induced when cells were grown in the presence of acetate plus succinate rather than with succinate only ( Table 3 ) .
The level of induction of , B-galactosidase was comparable with the level of induction of isocitrate lyase and malate synthase .
This level of induction indicates that , as in E. coli , expression of the glyoxylate shunt in S. typhimurium is regulated at the level of transcription .
To determine whether the lack of isocitrate lyase and malate synthase activities in the aceB insertions was due to polarity , we examined the polarity of Th1O insertions on aceB and aceA operon fusions .
Different ace : : Mu dJ mutants were transduced to Tetr with phage P22 grown on ace : : TnlO mutants .
Approximately 1 % of the transductants inherited both markers .
An aceA : : TnlO insertion bad no effect on expression of an aceB : : Mu d -RCB- operon fussion ( Table 3 ) , but an aceB : : TnlO insertion prevented expression of 3-galactosidase from an aceA : : Mu dJ operon fusion ( Table 3 ) .
This polarity is further evidence that the aceB and aceA genes form an operon as suggested above .
CaaboUte repression of the aceBA operon .
The glyoxylate shunt enzymes were strongly repressed during-growth-on-glucose .
However , the glyoxylate shunt was also repressed by metabolic intermediates such aF succinate that do not cause strong catabolite-repression .
Therefore , we wanted to determine whether the ace operon is controlled by cAMP ¬ MS1309 aceA : : Mu dJ 9 8 6 MS1380 aceA : : Mu dJ cya 3 7 3 MS1381 aceA : : Mu dJ crp 4 6 3 MS1514 putA : : Mu dA 7 NDb 85 MS1519 putA : : Mu dA cya 6 ND 134c MS1517 putA : : Mu dA crp 5 ND 5c aStrains were grown to mid-log phase in the indicated medium .
Glucose was added at 0.2 % , acetate was added at 0,4 % , and cAMP was added at 5 mM .
b ND , Not determined .
c Data for the putA : : Mu dA mutants were determined after growth-on-glucose plus cAMP without acetate ( 10 ) cAMP-receptor-protein complex .
Isogenic aceA : : Mu dJ strains were constructed with cya : : TnJO or crp : : TnlO mutations , and,-galactosidase expression was measured in these mutants .
The addition of cAMP to the growth medium had no effect on the induction of,B-galactosidase in the aceA : : Mu dJ cya , or aceA : : Mu dJ crp mutants ( Table 4 ) .
In contrast , expression of the put operon which is known to be regulated by cAMP-cAMP-receptor-protein complex is fully activated by cAMP under these conditions ; putA : : Mu dA and putA : : Mu dA cya mutants are activated by cAMP , but putA : : Mu dA crp mutants can not be activated by cAMP ( Table 4 ) .
These results suggest that the glucose repression of the aceBA operon was not solely due to cAMP .
Isolation of regulatory mutants .
Mutants with altered expression of isocitrate lyase and malate synthase are difficult to isolate .
However , availability of stable ace : : Mu dJ operon fusions allowed the use of lac selections to isolate ace regulatory mutants .
The level of lac enzymes expressed from the aceB operon fusions was not high enough to allow-growth on lactose as a sole carbon source .
Thus , it was possible to isolate mutants that overexpress lac from the aceBA : : Mu dJ operon fusions by selecting for growth on lactose minimal-medium .
Mutants that express the aceBA operon constitutively were isolated after mutagenesis with nitrosoguanidine .
Two classes of constitutive mutants were obtained : mutants tightly linked to aceBA ( > 99 % cotransducible ) and mutants that were approximately 70 % cotransducible with aceBA .
The constitutive mutations that were 70 % o linked to aceBA were only about 36 % linked to metA , indicating that these mutations map on the opposite side of aceBA ( Fig. 1 ) .
No constitutive mutants were isolated that mapped outside of the 90-min region of the S. typhimu-rium chromosome .
The mutants that are very tightly linked to the aceBA operon are potential operator mutations .
The iclR gene in E. coli maps in a position comparable to that of the constitutive mutants that are 70 % o linked to the S. typhimurium aceBA operon , suggesting that this class of mutation may be iclR defects .
Direction of transcription of the aceBA operon .
The direction of transcription of the aceBA operon was determined by using Mu dJ-mediated Hfr formation .
F'ts 114 lac + TnJO was mated into aceB : : Mu dJ and aceA : : Mu dJ insertion mutants selecting for Tetr at 30 °C .
Hfr strains generated by recombination between the lac genes on the F ' plasmid and the Mu dJ fusion were selected by requiring Tetr at 42 °C .
These Hfr strains were then mated with recipient strains carrying auxotrophic mutations that map on either side of the aceBA region .
The frequency of repair of the auxotrophic markers indicates the direction of transcription of the original aceA : : Mu dJ fusion ( 21 ) .
All X-gal + aceA : : Mu dJ mutants tested transferred the thr gene ( O min ) at about 100 times the frequency of the pyrE gene ( 79 min ) .
This indicates that the aceBA operon is transcribed in a clockwise orientation on the chromosome ( 25 ) .
Catabolite-repression of the aceBA operon ( - Galactosidase octivity '' in : Glucose plus acetate plus cAMP BL TA Strain Genotype Glucose plus TABLE 3 .
Expression of , B-galactosidase from aceBA operon fusions Glucose acetate , B-Galactosidase activitya in : Succinate Strain Genotype b 116 85 DISCUSSION The organization and regulation of the glyoxylate shunt in S. typhimurium LT2 was determined .
The genes for the glyoxylate shunt enzymes isocitrate lyase ( aceA ) and malate synthase ( aceB ) mapped at 89.5 min on the S. typhimurium genetic map ( Fig. 1 ) .
The aceB and aceA genes were tightly linked by cotransduction , and their expression was coordinately controlled ( Tables 2 and 3 ) .
Polar insertion mutations in the aceB gene eliminated expression of the aceA gene ( Tables 2 and 3 ) , indicating that the ace genes lie in an operon that is transcribed from aceB to aceA .
Transcription of the aceBA operon was induced during-growth on acetate ( Table 3 ) , but induction of the aceBA operon was repressed by other carbon sources ( Table 4 ) .
The organization of the aceBA operon in S. typhimurium is similar to that in E. coli , but the aceBA operon was induced about fourfold greater in E. coli ( 20 ) than in S. typhimurium .
Transcription of the aceBA operon in E. coli is regulated in trans by the iclR gene product and the fadR gene product ( 20 ) .
The primary role of the fadR gene product is the regulation of fatty acid degradation ( 24 ) .
Long-chain fatty-acids inactivate the fadR repressor , resulting in about a 10-fold induction of the fad regulon during-growth on long-chain-fatty-acids .
Regulation of the aceBA operon by the iclR and fadR repressors ' may allow the rapid induction of the glyoxylate shunt enzymes by either carbon source in E. coli ( 20 ) .
S. typhimurium LT2 can also grow on long-chain-fatty-acids as a sole carbon source ( 9 ) .
However , growth studies indicate that the fad genes in S. typhimurium LT2 are expressed at a low constitutive level ( S. Maloy , unpublished results ) .
The inability to fully induce the fad genes may explain the lower expression of the aceBA genes in S. typhimurium compared with gene expression in E. coli .
This inability may also explain why no fadR mutants were obtained in S. typhimurium that resulted in constitutive expression of the aceBA genes .
The reason the aceBA operon is regulated differently in these two closely related bacteria is not clear .
However , the ability to isolate and characterize a large number of aceBA regulatory mutants may clarify the molecular mechanism of regulation of the glyoxylate shunt in S. typhimurium .
John Roth , Ken Sanderson , Barbara Bachmann , and Don Hahn generously provided some of the strains used in this study .
This work was supported by Public Health Service grant GM34715 from the National Institutes of Health to S. Maloy .
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