Vol. 171, No. 10JOURNAL OF BACTERIOLOGY, OCt. 1989, p. 5567-55710021-9193/89/105567-05$02.00/0Copyright © 1989, American Society for Microbiology

(S)-3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase, a Productof the mva Operon of Pseudomonas mevalonii, Is Regulated

at the Transcriptional LeveltYULI WANG, MICHAEL J. BEACH,t AND VICTOR W. RODWELL*

Department ofBiochemistry, Purdue University, West Lafayette, Indiana 47907

Received 7 February 1989/Accepted 17 July 1989

We have cloned and sequenced a 505-base-pair (bp) segment of DNA situated upstream of mvaA, thestructural gene for (S)-3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.88) of Pseudomonasmevalonii. The DNA segment that we characterized includes the promoter region for the mva operon. NucleaseSl mapping and primer extension analysis showed that mvaA is the promoter-proximal gene of the mva operon.Transcription initiates at -56 bp relative to the first A (+1) of the translation start site. Transcription in vivowas induced by mevalonate. Structural features of the mva promoter region include an 80-bp A+T-rich region,and -12, -24 consensus sequences that resemble sequences of r54 promoters in enteric organisms. The relativeamplitudes of catalytic activity, enzyme protein, and mvaA mRNA are consistent with a model of regulation ofthis operon at the transcriptional level.

Pseudomonads are well known for their diverse metabolicactivities. Unlike enteric bacteria such as Escherichia coli,whose natural habitat provides high concentrations ofreadily assimilated growth substrates, soil bacteria such asPseudomonas spp. occupy ecological niches characterizedby low concentrations of poor growth substrates (26). Whilepseudomonads have evolved well-controlled systems to en-sure their survival under these conditions, our understandingof the molecular events that govern the expression andregulation of Pseudomonas genes lags far behind that for E.coli.

Catabolism of mevalonate by Pseudomonas mevalonii, anorganism originally isolated from soil by enrichment culturein media having mevalonate as the sole carbon source (12),involves internalization of the carbon source by an inducible(R)-mevalonate transport system (12) and conversion to(S)-3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) byan inducible HMG-CoA reductase (EC 1.1.1.88) (12, 13).This HMG-CoA reductase has been purified and character-ized (13). The corresponding gene, mvaA, has been cloned,sequenced, and overexpressed in E. coli (2). It is nowpossible to investigate the regulation of mevalonate catabo-lism at the DNA level in P. mevalonii. This study has thepotential to provide not only new knowledge concerning theregulation of mevalonate metabolism but also fresh insightinto the mechanisms ofgene regulation in Pseudomonas spp.

(The data in this report was derived in part from the Ph.D.thesis material of Yuli Wang.)

MATERIALS AND METHODS

Materials. Materials from commercial sources included4-chloronaphthol, PIPES [piperazine-N,N'-bis(2-ethanesul-fonic acid)], polyethylene glycol 8000, yeast RNA (SigmaChemical Co.); [a-32P]dATP, [a-35S]dATP, and [_y-32P]ATP(Amersham Corp.); (R,S)-mevalonolactone, Klenow frag-

* Corresponding author.t Journal paper 11928 from the Purdue University Agricultural

Experiment Station.t Present address: Hepatitis Branch, Centers for Disease Control,

Atlanta, GA 30333.

ment of DNA polymerase I and T4 polynucleotide kinase(U.S. Biochemical Corp.); restriction endonucleases (NewEngland BioLabs, Inc.); S1 nuclease and avian myeloblas-tosis virus reverse transcriptase (Boehringer Mannheim Bio-chemicals); horseradish peroxidase conjugated to rabbitanti-chicken immunoglobulin G (IgG) (ICN Pharmaceuti-cals, Inc.); and RNasin (Promega Biotec). Additional chem-icals were purchased from previously cited sources (2, 13).

Oligonucleotide probes. Probe 1, the 20-mer oligonucleo-tide 5'-ATGTGGTCCAGGCGCGCGGC-3' complementaryto the mRNA that codes for amino acids 16 to 22 ofHMG-CoA reductase (2), was synthesized in the laboratoryof Jack Dixon, Department of Biochemistry, Purdue Univer-sity, on an Applied Biosystems model 308 nucleotide syn-thesizer utilizing phosphoramidate chemistry. This 20-merwas used as a primer for synthesis of probe 2. After endlabeling with [y-32P]ATP using T4 polynucleotide kinase(28), the same oligonucleotide was utilized for primer exten-sion analysis. Probe 2, a 570-base-pair (bp) antisense oligo-nucleotide that spanned base pairs -505 to +65, was uni-formly labeled with [a-32P]dATP as described below undernuclease S1 mapping. Probe 3, an 840-bp SalI-PstI fragmentof mvaA (2, submitted) (Fig. 1) labeled with [a-32P]dATPusing an Amersham nick translation kit, was used to screenthe lambda EMBL4 library.

Bacteria and growth conditions. E. coli JM101 and JM103(28) were grown as previously described (2). P. mevalonii (2)was grown in ionic medium (12) plus either 25 mM ammo-nium mevalonate or 17.5 mM D-glucose plus 25 mM NH4Cl.

Cell extracts. Cells from 25-ml cultures of P. mevaloniigrown, with shaking at 30°C, to early stationary phase onmevalonate or glucose were harvested by centrifugation andwashed once in ionic medium. Subsequent operations wereconducted at about 0°C. The washed cells were suspended in1 ml of a mixture of 2 mM dithiothreitol, 1 mM EDTA, 1 mMphenylmethylsulfonyl fluoride, and 10% glycerol in 10 mMpotassium phosphate (pH 7.2), ruptured in a French pressurecell, and centrifuged (10,000 x g, 30 min; 4°C). The resultingsupernatant liquid was used as the cell extract.Plasmid and bacteriophage. A P. mevalonii genomic li-

brary was constructed utilizing lambda EMBL4 as previ-

5567

5568 WANG ET AL.

O.lkb

Pst I

-505

Kpn I

Probe 2 (570bp) AProbe 1(20bp)

SaIl Pst I

Probe 3 (840bp)

SEQUENCING STRATEGY

FIG. 1. Positional relationships of the 1,849-bp PstI fragmentand probes 1, 2, and 3, and the strategy employed for sequencing themva promoter region. Portions of the PstI fragment are the 505 bpupstream of mvaA (EO), the 1,287 bp of mvaA (Ell), and the 58 bpdownstream ofmvaA ( M ). Nested deletions of lengths suitable forsequencing (arrows) were prepared by using an IBI Cyclone kit.

ously described (2). The phage derivative M13mp1965 wasconstructed by cloning the 1,849-bp PstI fragment fromlambda HMGR-5 (Fig. 1) into M13mpl9 (38) and deletingfrom the 3' end up to +440 bp (relative to the translationstart site) using an IBI Cyclone kit according to the direc-tions of the manufacturer (2). pHMGR-2, a derivative ofpUC19 (38), contained the 840-bp SalI-PstI fragment ofmvaA (2).DNA and RNA manipulation. Transformation of single-

stranded M13mp1965 and template preparation were con-ducted according to the IBI Cyclone kit manual. The pro-cedures for restriction endonuclease digestion, ligation,transformation, DNA isolation, and gel electrophoresis havebeen previously described (25). RNA was isolated by hotphenol extraction (11) of early-log-phase cultures of P.mevalonii.

Isolation of the region upstream of mvaA. The P. mevaloniigenomic library was screened as previously described (2)using probe 3, an 840-bp Sall-HindIll fragment of mvaA(Fig. 1). The resulting 14-kilobase clone, lambda HMGR-5,contained a 0.5-kilobase PstI-KpnI fragment that includedsequences upstream of mvaA. The nucleotide sequence ofboth strands was determined by the dideoxy-chain termina-tion method (34). Figure 1 illustrates the sequencing strat-egy.IgY directed against HMG-CoA reductase. HMG-CoA re-

ductase, Blue Dextran Fraction (13), was first subjected tosodium dodecyl sulfate-polyacrylamide gel electrophoresisto remove a trace impurity. HMG-CoA reductase protein,visualized with Coomassie brilliant blue, was electroelutedwith an IBI unidirectional electroeluter, desalted with anAmicon Centricon 30 microconcentrator, and used as anti-gen to prepare immune chicken IgY (35). After three bi-weekly injections of about 30 ,ug of protein, IgY wasextracted from egg yolk and partially purified by successiveprecipitation with polyethylene glycol 8000 and ammoniumsulfate (35).

Nuclease Si mapping. Probe 2 (Fig. 1), a 570-bp, 32p_labeled, single-stranded DNA from -505 to +65 (relative tothe mvaA translation start site), was prepared using 2.4 ng ofprobe 1 as primer and 1 jxg of M13mp1965 as template.Conditions for annealing and extension catalyzed by DNApolymerase I (Klenow fragment) were identical to thoseemployed for the DNA sequencing reaction (1), except thatdideoxynucleoside triphosphates were omitted. The reaction

CTGCA GCCAGGCCAC CGCGGCAAAA CCTTCACCCA -471

-470 ACGGCGCCAG GGGTTCGCGA ACGAACTGCT GCCGCATCAT CCAGATGGTG -421

-420 TCCATGGCCC GGGTGTACGG CGGTGTTTCA TCCAGCTCGG CGGCCTGCAG -371

-370 CCTGCGCACC GACAAAAAAC CCCTGCCCGT GGCGCGCCTC CACCAAGCCC -321

-320 TTGAGAGGCG AGAATCTCGT ACGCGGTGAT CAGCCGTATT TTTCGAGTGT -271

-270 CCGTGCAAAC GGCATGTACT CGCGTAACGA CGGCAGGCGG TCGCCAACCT -221

-220 TCAGCACGCC TGTGCGGATC TGCTGCTCGA CCGCATCGGC GACTTGCTGG -171

-170 GCAAGGGTGA CACGAGGCAT CGGCTGGCTC CTGTGCTGTC ATGGGTACAG -121

-120 TCTGGGTACT GlTGGGCAAA ACTGTACCTT CAATCAAGr& CACAGTGGCT---> RNA

-70 r&ACA1TTAG CACCACGCCG GCGACGCGCT CAAGCCGCCG CCCAACAACA*

-20 ATGACGCCMALgGCCCTC ATG AGC CTC GATMet Ser Leu Asp

-71

-21

+12

FIG. 2. Nucleotide sequence and selected structural features ofthe 517-bp fragment of DNA which contains the mva promoter. Thesequences of both strands were determined by the dideoxy-chaintermination method (34) (Fig. 1). The transcription initiation site (*)was determined by primer extension analysis (described in text).The sequence presented contains the transcription initiation site, the-12 and -24 promoter consensus sequences (underlined), a puta-tive ribosome-binding site (doubly underlined), and the first fouramino acids of mvaA. The AUG codon was previously shown to bethe translation initiation site for HMG-CoA reductase (2). TheA+T-rich region, shown in italics, extends from base pair -60through -140, relative to the translation start site (+1).

mixture was digested with HindIII and subjected to electro-phoresis on a 3.5% polyacrylamide-8 M urea gel. A 32p_labeled DNA fragment of approximately 570 bases wasisolated and suspended in TE buffer (25).

Nuclease Si mapping was conducted as described by Berkand Sharp (2a) with some modifications. For hybridization,0.1 to 20 Fig of RNA from cells grown on glucose ormevalonate was mixed with excess labeled probe and incu-bated at 53°C for 3 h. After treatment with 175 U of Sinuclease, the mixture was precipitated with ethanol, sus-pended in sequencing gel loading buffer (1), and applied to asequencing gel.Primer extension. Primer extension by avian myeloblasto-

sis virus reverse transcriptase was conducted according toCalzone et al. (6). Approximately 0.6 to 6 pug of RNA frommevalonate-grown cells or 1.3 to 13 ,ug of RNA fromglucose-grown cells and 5 x 105 cpm of probe 1 were used.Hybridization was conducted at 53°C. After primer exten-sion, the sample was phenol extracted, precipitated, sus-pended in sequencing gel loading buffer (1), and applied to asequencing gel.

RESULTS

Nucleotide sequence of the DNA upstream of the mvaoperon. The nucleotide sequence of 505 bp upstream ofmvaA (Fig. 2) was determined by the dideoxynucleotidemethod of Sanger et al. (34). The GC content of thisupstream region (62%) was similar to but slightly less thanthat of mvaA (65%). However, the region encompassed byresidues - 140 to -60, which lie immediately upstream of thetranscription initiation site (see below), had a GC content ofabout 50%, which is significantly below that of mvaA, theupstream region as a whole, or Pseudomonas spp. in gen-eral.

Identification of the transcriptional start site. The transcrip-tion initiation site was initially localized by nuclease S1

J. BACTERIOL.

REGULATION OF THE mvaA OPERON 5569

B M G M

r--TT6I. T

TIA11

Gi_ A

r

A*

rb

'1 f-

L e_

1j-4

iA~~~MIXA

GLU

...~~~~~~1-g

AV'

IS]a

.

GA.

.6. g

GLU1.3ug

FIG. 3. Identification of the start point of mva mRNA by Simapping and by primer extension analysis. (A) Si mapping. Lane 1,RNA extracted from P. mevalonii grown on mevalonate as carbonsource was hybridized to 32P-labeled probe 2, digested with nucleaseSi, and subjected to electrophoresis on an 8% polyacrylamidesequencing gel. Lanes A, C, G, and T are dideoxy sequencingreactions for which the probe 1 oligonucleotide was used as primer.(B) Primer extension analysis. RNA from cells grown either onmevalonate or on glucose was hybridized to probe 1. The primerwas then extended using avian myeloblastosis virus reverse tran-scriptase. Lanes marked MVA contained 6 or 0.6 jig of RNA frommevalonate-grown cells. Lanes marked GLU contained 13 or 1.3 jigof RNA from glucose-grown cells. GLU, Glucose; MVA, meval-onate.

mapping (Fig. 3, left). Primer extension analysis was thenemployed for precise localization (Fig. 3, right).HMG-CoA reductase levels in induced cells, measured

immunochemically, parallel induced levels of HMG-CoA re-ductase catalytic activity. The activity of HMG-CoA reduc-tase, the product of mvaA, rises several hundredfold whencells are grown on mevalonate or when mevalonate is addedto cells growing on glucose or other neutral sources ofcarbon (12, 13). Although increases in activity of bacterialenzymes upon induction are generally attributed to in-creased levels of specific mRNAs, the activity of mammalianHMG-CoA reductase (33) and of certain bacterial enzymes(8, 9, 22, 23) can also be modulated by posttranscriptionalprocesses such as covalent phosphorylation and regulationat the translational level. We therefore asked whether induc-tion of HMG-CoA reductase activity was accompanied by acomparable increase in the quantity of HMG-CoA reductaseprotein. Chicken egg antibody raised against HMG-CoAreductase was used as the first reagent in the semiquantita-tive analysis of the sodium dodecyl sulfate-polyacrylamidegel electrophoresis-resolved proteins of crude extracts ofcells grown on mevalonate or glucose (Fig. 4). HMG-CoAreductase protein was readily detectable in mevalonate-grown cells but undetectable in glucose-grown cells. Fromestimates of total protein (3) and of HMG-CoA reductaseprotein by scanning densitometry, we calculated that HMG-

G Snr

-----~97.4

1!P= 66.2

....

VA. .--31.0

-21.5

14.4

FIG. 4. Detection of HMG-CoA reductase protein by immuno-blotting. Crude extract protein, 20 jig, from P. mevalonii growneither on mevalonate (M) or on glucose (G) was applied to twohalves of a sodium dodecyl sulfate-polyacrylamide gel electropho-resis gel (12% separating gel; 4% stacking gel). Lane S containedmolecular weight standards. After electrophoresis, one half (rightpanel) was stained with Coomassie brilliant blue. The other half (leftpanel) was transferred to a nitrocellulose filter for immunoblotting,which was conducted essentially as described by Towbin et al. (37)and Burnette (5). Approximately 27 ,ug of chicken anti-HMG-CoAreductase IgY per ml was used as the first antibody and a 1:3,000dilution of horseradish peroxidase-conjugated anti-chicken IgG wasused as the second antibody.

CoA reductase constituted 2 to 4% of the total protein ofmevalonate-grown cells.Mevalonate-induced cells contain high levels of mvaA-

specific RNA. Northern (RNA) blot hybridization (data notshown) and densitometric scanning after nuclease Si map-ping (Fig. 5) were employed to detect and quantify mvaA-specific mRNA, respectively. While as little as 0.1 ,ug ofRNA from induced cells gave detectable bands (Fig. 5, lane2), no bands were detected using 200 times more RNA fromuninduced cells (Fig. 5, lane 1). This large induction ofmvaA-specific mRNA, taken together with the immunoblot-ting data, suggests that elevations in HMG-CoA reductaseactivity reflect primarily, and possibly exclusively, increasedlevels of mva mRNA.

DISCUSSION

Nuclease Si mapping and primer extension analysis dem-onstrated that mvaA is the promoter-proximal gene in themva operon. (In addition to the 1.3-kilobase gene mvaA,mva contains the 0.9-kilobase gene mvaB, which encodesHMG-CoA lyase [D. H. Anderson and V. W. Rodwell,submitted for publication].) Transcription initiated at -56 bprelative to the translation start site. mvaA RNA was detectedin RNA from mevalonate-grown but not from glucose-growncells. The putative mva promoter exhibits strong sequencehomology with the recognition sequence GG-[10 bp]-GC (4,10, 20, 21) for the core of the a"1 factor encoded by ntrA (14,27) (Fig. 6). Other Pseudomonas promoters which exhibitthis sequence similarity include xylS (17), xylCAB (16),CPG2 (29), and PAK (19). Apart from the -56 transcriptionstart site, three other bands at -21 to -23 relative to thetranslation start site also were detected in mevalonate-growncells. These may be degradation products of mva mRNA. Ifso, degradation seems sequence specific, since although the

A

VOL. 171, 1989

wl.

i" I

5570 WANG ET AL.

20.49 0.1z9 1.049 5.049GL.. MVA MVA MVA

FIG. 5. Growth on mevalonate induces production of RNAspecific for mvaA. The indicated quantities of RNA, isolated by hotphenol extraction (11) of early-log-phase cultures grown on glucose(GLU) or on mevalonate (MVA), were hybridized to probe 2,digested with S1 nuclease, electrophoresed on an 8% polyacryl-amide-8 M urea gel, and subjected to autoradiography.

amount of these short mRNAs varies from preparation topreparation, the position remains constant at -21 to -23 bprelative to the translation start site. However, we cannotexclude the possibility that these may represent true tran-scription initiation sites. Dual promoters for a single genecan facilitate the differential regulation of gene expression(15, 30, 31, 32, 36), and the coordinate regulation of over-lapping promoters has been reported (26).

Relatively A+T-rich regions similar to that found imme-diately upstream of the promoter have been reported forseveral highly expressed genes (7, 18, 24, 36). Furthermore,it has been suggested that these A+T-rich sequences mayfacilitate RNA polymerase binding (7), reduce the kineticbarrier for formation of the open complex (24), or both. Therelatively A+T-rich region located -60 to -140 bp upstreamofmvaA thus may have significance for regulation of the mvaoperon.The observed increase in immunoreactive protein in me-

valonate-grown cells is consistent with the elevated HMG-CoA reductase levels we have observed. As estimated fromenzyme purification data (13), approximately 4% of the totalcell protein is HMG-CoA reductase. The over-200-fold in-duction of mvaA-specific mRNA also supports the conclu-sion that induction of HMG-CoA reductase is achievedprimarily, if not exclusively, at the transcriptional level.

ACKNOWLEDGMENTS

This work was funded by Research Grant GM 33457 from theNational Institutes of Health. M.J.B. was supported by Public

Consensus

mva

-24 -12

C C A

T G G A (S bp) T T G CT T T

G G G C A C (5 bp) C T G C A

FIG. 6. Comparison of the mva promoter sequence to the -24and -12 consensus sequence proposed by Dixon (10).

Health Service training grant GM 07211 from the National Institutesof Health.We thank our colleagues Doug Anderson, Dan Ebbole, Jack

Green, Tuajuanda Jordan-Starck, David Kuhn, Janice Morand,David Scher, Ronald Somerville, Edwin Umbarger, and HowardZalkin for many helpful suggestions.

LITERATURE CITED

1. Amersham Corporation. 1983. M13 cloning and sequencinghandbook. Amersham Corporation, Arlington Heights, Ill.

2. Beach, M. J., and V. W. Rodwell. 1989. Cloning, sequencing,and overexpression of mvaA, which encodes Pseudomonasmevalonii 3-hydroxy-3-methylglutaryl coenzyme A reductase.J. Bacteriol. 171:2994-3001.

2a.Berk, A. J., and P. A. Sharp. 1977. Sizing and mapping of earlyadenovirus mRNA by gel electrophoresis of S1 endonuclease-digested hybrids. Cell 12:721-732.

3. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

4. Buck, M. 1986. Deletion analysis of the Klebsiella pneumoniaenitrogenase promoter: importance of spacing between con-served sequences around - 12 and -24 for activation by the nifAand ntrC (glnG) products. J. Bacteriol. 166:545-551.

5. Burnette, W. N. 1981. "Western blotting": electrophoretictransfer of proteins from sodium dodecyl sulfate-polyacryla-mide gels to unmodified nitrocellulose and radiographic detec-tion with antibody and radioiodinated protein A. Anal. Bio-chem. 112:195-203.

6. Calzone, F. J., R. J. Britten, and E. H. Davidson. 1987. Mappingof gene transcripts by nuclease protection assays and cDNAprimer extension. Methods Enzymol. 152:611-632.

7. Cole, S. T., E. Bremer, I. Hindennach, and U. Henning. 1982.Characterization of the promoters for the ompA gene whichencodes a major outer membrane protein of Escherichia coli.Mol. Gen. Genet. 188:472-479.

8. Cortay, J. C., C. Rieul, B. Duclos, and A. J. Cozzone. 1986.Characterization of the phosphoproteins of Escherichia colicells by electrophoretic analysis. Eur. J. Biochem. 159:227-237.

9. Cozzone, A. J. 1988. Protein phosphorylation in prokaryotes.Annu. Rev. Microbiol. 42:97-125.

10. Dixon, R. 1986. The xylABC promoter from Pseudomonasputida TOL plasmid is activated by nitrogen regulatory genes inEscherichia coli. Mol. Gen. Genet. 203:129-136.

11. Ebina, Y., and A. Nakazawa. 1983. Cyclic AMP-dependentinitiation and rho-dependent termination of colicin El genetranscription. J. Biol. Chem. 258:7072-7078.

12. Gill, J. F., Jr., M. J. Beach, and V. W. Rodwell. 1984. Transportof mevalonate by Pseudomonas sp. strain M. J. Bacteriol.160:294-298.

13. Gill, J. F., Jr., M. J. Beach, and V. W. Rodwell. 1985. Meval-onate utilization in Pseudomonas sp. M. Purification and char-acterization of an inducible 3-hydroxy-3-methylglutaryl coen-zyme A reductase. J. Biol. Chem. 260:9393-9398.

14. Hirschman, J. P., K. Wong, K. Sei, J. Keener, and S. Kustu.1985. Products of nitrogen regulatory genes ntrA and ntrC ofenteric bacteria activate gInA transcription in vitro: evidencethat the ntrA product is a cr factor. Proc. Natl. Acad. Sci. USA82:7525-7529.

15. Hu, L., and A. T. Phillips. 1988. Organization and multipleregulation of histidine utilization genes in Pseudomonas putida.J. Bacteriol. 170:4272-4279.

16. Inouye, S., Y. Ebina, A. Nakazawa, and T. Nakazawa. 1984.Nucleotide sequence surrounding transcription initiation site ofxylABC operon on TOL plasmid of Pseudomonas putida. Proc.Natl. Acad. Sci. USA 81:1688-1691.

17. Inouye, S., A. Nakazawa, and T. Nakazawa. 1986. Nucleotidesequence of the regulatory gene xylS on the Pseudomonasputida TOL plasmid and identification of the protein product.Gene 44:235-242.

18. Jaurin, B., and S. N. Cohen. 1985. Streptomyces contain Esch-erichia coli-type A+T-rich promoters having novel structural

J. BACTERIOL.

REGULATION OF THE mvaA OPERON 5571

features. Gene 39:191-201.19. Johnson, K., M. L. Parker, and S. Lory. 1986. Nucleotide

sequence and transcription initiation site of two Pseudomonasaeruginosa pilin genes. J. Biol. Chem. 261:15703-15708.

20. Kennelly, P. J., and V. W. Rodweil. 1985. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by reversiblephosphorylation-dephosphorylation. J. Lipid Res. 26:903-914.

21. Khan, H., M. Buck, and R. Dixon. 1986. Deletion loop muta-genesis of the nifL promoter from Klebsiella pneumoniae: roleof the -26 to -12 region in promoter function. Gene 45:281-288.

22. Klumpp, D. J., D. W. Plank, L. J. Bowdin, C. S. Stueland, T.Chung, and D. C. LaPorte. 1988. Nucleotide sequence of aceK,the gene encoding isocitrate dehydrogenase kinase/phos-phatase. J. Bacteriol. 170:2763-2769.

23. LaPorte, D. C., and D. E. Koshland, Jr. 1983. Phosphorylationof isocitrate dehydrogenase as a demonstration of enhancedsensitivity in covalent regulation. Nature (London) 305:286-290.

24. Lin, E., and D. B. Wilson. 1988. Transcription of the celE genein Thermomonospora fusca. J. Bacteriol. 170:3838-3842.

25. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

26. Mermod, N., P. R. Lehrbach, W. Reineke, and K. N. Timmis.1984. Transcription of the TOL plasmid toluate catabolic path-way operon of Pseudomonas putida is determined by a pair ofco-ordinately and positively regulated overlapping promoters.EMBO J. 3:2461-2466.

27. Merrick, M. J., and Gibbins, J. R. 1985. The nucleotide se-quence of the nitrogen-regulation gene ntrA of Klebsiella pneu-moniae and comparison with conserved features in bacterialRNA polymerase sigma factors. Nucleic Acids Res. 13:7607-7620.

28. Messing, J. 1983. New M13 vectors for cloning. Methods

Enzymol. 101:20-78.29. Minton, N. P., and L. E. Clarke. 1985. Identification of the

promoter of the Pseudomonas gene coding for carboxypeptid-ase G2. J. Mol. Appl. Genet. 3:26-35.

30. Musso, R. E., R. DiLavro, S. Adhya, and B. de Crombrugghe.1977. Dual control for transcription of the galactose operon bycyclic AMP and its receptor protein at two interspersed promot-ers. Cell 12:847-854.

31. Nesin, M., J. R. Lupski, and G. N. Godson. 1988. Role of the 5'upstream sequence and tandem promoters in regulation of therpsU-dnaG-rpoD macromolecular synthesis operon. J. Bacte-riol. 170:5759-5764.

32. Reitzer, L. J., and B. Magasanik. 1985. Expression of glnA inEscherichia coli is regulated at tandem promoters. Proc. Natl.Acad. Sci. USA 82:1979-1983.

33. Rodwell, V. W., J. L. Nordstrom, and J. J. Mitschelen. 1976.Regulation of HMG-CoA reductase. Adv. Lipid Res. 14:1-74.

34. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci.USA 74:5463-5467.

35. Song, C.-S., J.-H. Yu, D. H. Bai, P. Y. Hester, and K.-H. Kim.1985. Antibodies to the a-subunit of insulin receptor from eggsof immunized hens. J. Immunol. 135:3354-3359.

36. Stephens, R. S., E. A. Wager, and U. Edman. 1988. Develop-mental regulation of tandem promoters for the major outermembrane protein gene of Chlamydia trachomatis. J. Bacteriol.170:744-750.

37. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

38. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. ImprovedM13 phage cloning vectors and host strains: nucleotide se-quences of the M13mpl8 and pUC19 vectors. Gene 33:103-119.

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