THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 28, Issue of October 5, pp. 19978-19985, 1992 Printed in U. S. A.

Characterization of the Tryptophanase Operon of Proteus vulgaris CLONING, NUCLEOTIDE SEQUENCE, AMINO ACID HOMOLOGY, AND IN VITRO SYNTHESIS OF THE LEADER PEPTIDE AND REGULATORY ANALYSIS*

(Received for publication, April 13, 1992)

Ajith V. KamathS and Charles Yanofskys From the Department of Biological Sciences, Stanford University, Stanford, California 94305-5020

The tryptophanase (tna) operon of Proteus vulgaris was cloned and characterized and found to be orga- nized similarly to the tna operon of Escherichia coli. Both operons contain two major structural genes, tnaA and tnaB, that encode tryptophanase and a tryptophan permease, respectively. tnaA of P. vulgaris is preceded by a transcribed leader region, encoding a 34-residue leader peptide, TnaC, that contains a single tryptophan residue. The tnaC coding region also has a boxA-like sequence. Regulatory studies performed in P. vulgaris, and with a plasmid carrying the P. vulgaris tna operon in E. coli, established that expression of the Proteus operon was induced by tryptophan and was subject to catabolite repression. Site-directed mutagenesis stud- ies established that translation of the tnaC coding re- gion was essential for induction. synthesis of the P. vulgaris leader peptide was demonstrated in an in vitro coupled transcription-translation system. Interest- ingly, the 5 amino acid residues of the TnaC peptide surrounding the sole tryptophan residue are identical in P. vulgaris and E. coli. We conclude that the tna operon of P. vulgaris is also regulated by tryptophan- induced transcription antitermination. Homology of tryptophanase and tryptophan permease of P. vulgaris to related proteins from other species is described.

Tryptophanase (tryptophan indole-lyase; EC 4.1.99.1.) is a catabolic enzyme that catalyzes the degradation of tryptophan to indole, pyruvate, and ammonia. It is one of most studied pyridoxal 5”phosphate (PLP)’-requiring multifunctional en- zymes that can perform cup-elimination and @-replacement reactions (1, 2). Tryptophanase (Tnase) also catalyzes the synthesis of tryptophan from indole and serine or cysteine (1-3). Tnase has been isolated from several bacterial species including Escherichia coli (3), Bacillus alvei (4), and Proteus rettgeri ( 5 ) . Tnases isolated from these sources have a similar

* This work was supported by United States Public Health Service Grant GM 09738. The costs of publication of this article were defrayed

be hereby marked “advertisement” in accordance with 18 U.S.C. in part by the payment of page charges. This article must therefore

Section 1734 solely to indicate this fact.

to the GenBankTM/EMBL Data Bank with accession number(s) The nucleotide sequence(s1 reported in thispaper has been submitted

M93277. $ Career Investigator Postdoctoral Fellow of the American Heart

Association. Career Investigator of the American Heart Association. To whom

correspondence should be addressed. Tel.: 415-725-1835; Fax: 415-

The abbreviations used are: PLP, pyridoxal 5”phosphate; Tnase, TnaA, tryptophanase; TnaB, tryptophan permease; TnaC, leader peptide coded by tnaC; kb, kilobases; bp, base pair; ACH, acid casein hydrolysate.

723-6132.

molecular weight, subunit structure, and PLP content, but they show differences in amino acid composition and anti- genic properties (1, 4). The enzyme from E. coli has been studied most extensively (1,6, 7); its amino acid sequence has been determined both by protein sequencing (8) and by de- duction from the nucleotide sequence of its encoding struc- tural gene (9). The E. coli enzyme is a tetramer composed of identical polypeptides of 52 kDa. Each subunit contains one molecule of PLP which forms a Schiffs base with the €-amino group of LysZ7O (1, 8, 9). Chemical modification studies and site-directed mutagenesis experiments have provided evidence for the role of specific cysteine (lo), arginine (ll), histidine (E?), and other residues in substrate binding and catalysis. The enzyme has been crystallized and the three-dimensional structure is being determined (13).

The tna operon of E. coli contains two major structural genes; tnaA, encoding Tnase (9), and tmB, encoding a tryp- tophan-specific permease (14). tnaA is preceded by a 319-bp transcribed leader region, tnaL, that contains a short coding region, tnaC, for a 24-residue leader peptide (9). In E. coli, expression of the tna operon is regulated transcriptionally by the combined action of catabolite repression and transcription attenuation (15, 16). A presumed CAP binding site is located immediately upstream of the tna promoter (9). Catabolite repression modulates transcription initiation at the tnu pro- moter approximately 10-fold (9). Transcription attenuation in the tna operon involves regulation of transcription termi- nation at multiple Rho-factor-dependent transcription ter- mination sites located between tnaC and tnaA (17). In the presence of L-tryptophan, or an appropriate tryptophan ana- log, transcription termination in the leader region is elimi- nated and full length tna operon transcripts are produced. Translation of the tnaC coding region is required for this tryptophan-induced transcription antitermination in the tna leader region (18, 19). Additionally, the leader peptide coding region contains a single tryptophan codon at position 12 which must be translated by tRNATv for induction to occur (18). How inducing levels of tryptophan are sensed in the induction process, and how this sensing leads to transcription antiter- mination, are not known.

Comparisons of structural and regulatory features of related operons and their polypeptide products from different species have often provided insight into the genetic and protein segments that are functionally important. To determine the conserved features of the tna operon essential to its regulation, we have cloned and characterized the tna operon of Proteus vulgaris, a bacterial species that also produces a tryptophan- inducible Tnase (20, 21), and compared these features with those of E. coli. We find that despite considerable sequence variation in the regulatory regions of the tna operons of P. vulgaris and E. coli regulation of the two operons is very similar. Particularly noteworthy is conservation of a 6-residue

19978

tna Operon of P. vulgaris 19979

segment of the leader peptide, TnaC, containing the single tryptophan residue. We demonstrated synthesis of the TnaC leader peptide, using an i n vitro system. Tnase of P. vulgaris was found to be homologous to the E. coli enzyme (9) and to the tyrosine phenol lyase of Citrobacter freundii (22). The tryptophan permease (TnaB) encoded in the tna operon of P. vulgaris is homologous to the corresponding permease of E. coli (14) and to other aromatic amino acid permeases (23,24).

MATERIALS AND METHODS

L-Tryptophan, 1-DL-methyltryptophan (lMT), PLP, dithiothrie- tol, Tricine, o-nitrophenyl-P-D-galactoside, and other chemicals were purchased from Sigma, Pharmacia LKB Biotechnology Inc., and United States Biochemicals Cop . Restriction enzymes were from New England Biolahs, Bethesda Research Labs, and IBI. ~ - [ 2 , 5 - ~ H ] Histidine (53 Ci/mmol), ~-[2,3,4,5,6-~H]phenylalanine (130 Ci/ mmol), ~-[5-~H]tryptophan (29.5 Ci/mmol), ~-[~~S]methionine (800 Ci/mmol), and [ c Y - ~ ~ S I ~ A T P (1200 Ci/mmol) were purchased from Amersham and Du Pont-New England Nuclear. S-(0-Nitropheny1)- L-cysteine was kindly supplied by Dr. R. S. Phillips of the University of Georgia.

Cloning of the tna Operon of P. vulgaris-P. vulgaris was grown overnight at 37 "C, in a liquid medium containing 1% tryptone, 0.5% yeast extract, 0.5% NaCl, and 1% glucose. The cells were harvested, washed in 50 mM Tris-HC1 buffer, pH 8.0, and chromosomal DNA was prepared according to the protocol of Nakamura et al. (25). To construct a genomic library, 40 pg of P. vulgaris DNA was digested with Sau3AI (2 units) for 45 min at 37 "C, electrophoresed on 0.9% agarose gel in 1 X TAE buffer, and fragments of approximately 4-8 kh were eluted and purified using a Gene Clean I1 kit (BIO101 Inc.). The fragments were ligated into the BamHI site of pUC13 and E. coli strain CY15073 (LE392 trpR+) was transformed with the ligation mixture and plated on LB + ampicillin (100 pg/ml) + agar plates. The recombinant cells (about 40,000 colonies) were pooled to prepare a genomic library. E. coli strain CY15077 (W3110 tnaA2 AtrpEAZ) was transformed with the genomic library, and cells were plated on a selective medium containing 0.2% ACH (acid casein hydrolysate), 0.2% succinate, 10 pg/ml indole, 10 pg/ml lMT, 1 X Vogel and Bonner minimal medium (26), 100 pg/ml ampicillin, and 1.5% agar. In this medium only recombinant cells containing the tryptophanase or tryptophan synthetase genes of P. vulgaris were capable of growth. A few colonies appeared after 2 days of incubation at 37 "C. These colonies were screened for Tnase activity by detecting indole produced in the medium, using indole reagent (27).

DNA Sequencing-Double stranded DNA sequencing of either CsC1-purified or Miniprep plasmid DNA was carried out by the dideoxy chain termination method of Sanger et al. (28) using [ o ( - ~ ~ S ] dATP. Sequencing was performed using Sequenase kit version 2.0 (United States Biochemicals Corp.). The recombinant plasmid (pAVK2) with a 2.1-kb insert was used to create nested deletions by the method of Henikoff (29). The 2.1-kb EcoRI-Hind111 fragment from this plasmid was also ligated to similarly digested pBluescript SK+ (Stratagene), and deletions were made to obtain the sequence of the opposite strand. Plasmid pAVK2 contained the entire tnaA sequence and a part of tnaB. A 1.9-kb EcoRI-AuaI fragment from pAVK4 was isolated, the ends filled in, and ligated into the EcoRV site of pBluescript SK+. These plasmids were used for creating nested deletions by treatment with exonuclease 111. Selected deletion clones were sequenced to obtain the remainder of the tnaB sequence.

Construction of a Proteus tnaA'-lacZ Translational Fusion-A tnaA'-lacZ fusion was constructed in plasmid pMLB1034. This plas- mid contains an engineered BamHI site at codon 9 of lacZ and lacks the lacZ promoter. The 5"upstream region and a part of tnaA (the first 60 nucleotides) were amplified by the polymerase chain reaction, using two synthetic oligonucleotide primers that had sequences that introduced BamHI and XbaI sites at the 5'-end and a BamHI site at the 3'-end of the amplified product. Plasmid pAVK2 was used as the template. The polymerase chain reaction product was purified on 4.0% agarose gel (NuSieve:regular, 3:1), digested with BanHI, and the resulting 426-bp fragment containing BamHI sites at both ends was ligated into plasmid p~LB1034. This plasmid (pAVK9) was transformed into E. coli strain SE5000 (MC4100 recA56) and the mixture plated on LB + ampicillin (100 pg/ml) + tryptophan (100 pg/ml) + 5-bromo-4-chloro-3-indoyl @-D-galactoside plates. Recom- binant cells harboring the plasmid (pAVK9) with the insert in the

correct orientation (in-frame fusion with lacZ) appeared blue on this plate.

Site-directed Mutagenesis of the tnaC Start Codon-A polymerase chain reaction-amplified 426-bp fragment containing BamHI restric- tion sites at both ends was ligated into plasmid pUC118 and trans- formed into E. coli strain JM107. Single stranded DNA was prepared from selected colonies using M13K07 helper phage. This DNA was used as a template to change the ATG start codon of tnaC to a stop codon (TAG), using a 21-base oligonucleotide primer (5' GATGA- G A A m C T T T C A C C C 3'), complementary to the noncoding strand and a mutagenesis kit (Amersham). Six colonies were picked for plasmid isolation. Sequencing showed that all six plasmids con- tained the desired mutation. The inserted DNA from one of the clones was completely sequenced (426 bp) and used for constructing an in-frame fusion with lacZ in plasmid pMLB1034 to give pAVK13. This recombinant plasmid was transformed into E. coli SE5000, and the resulting strain used for measuring the induction of @-galactosid- ase by tryptophan or 1MT.

Growth of P. vulgaris on a Synthetic Medium-To study the induc- tion of Tnase in P. vulgaris cells, 50-ml cultures were grown in minimal medium (26) supplemented with 1% ACH, 10 mM potassium phosphate, pH 7.0, 1 mM magnesium sulfate, and niacin (5 mg/l) (21). The medium was supplemented with tryptophan (100 pg/ml) or 1MT (20 pg/ml) and/or glucose (0.5%), as desired.

Preparation of Crude Extracts and Enzyme Assays-Wild type P. vulgaris cultures were grown to log phase, harvested, washed, and then suspended in lysis buffer (see Ref. 9 for composition of buffers). Cells were disrupted by sonication with a Heat Systems sonifier at 4 "C for 80 s. Cell debris was removed by centrifugation at 20,000 X g for 30 min, and the supernatant was assayed for Tnase activity (17) in a 1-ml assay volume, using S-(0-nitropheny1)-L-cysteine as the substrate (30). @-Galactosidase assays were performedusing sonicated extracts of E. coli strain SE5000 harboring plasmids pAVK9, pAVK13, or pPDG100. Cells were grown in 3 ml of minimal medium (26) containing 0.2% glycerol, 0.05% ACH, and 100 pg/ml ampicillin, to an A600 of 0.3--0.7. The medium was supplemented with trypto- phan (100 pg/ml) or 1MT (20 pg/ml) and/or glucose (0.5%) to study induction and catabolite repression. For @-galactosidase assays, cells from 1-ml samples were collected by centrifugation, suspended in 1- ml sonication buffer (Tris-HC1, pH 7.5, bovine serum albumin 5 mg/ ml) and sonicated for 45 s. The cell extracts were assayed for /?- galactosidase activity using o-nitrophenyl-@-&galactoside as a sub- strate, by the method of Miller (31). The enzyme activity was ex- pressed as AtJ0/30 min/ml extract/Asoo of culture.

In Vitro Tran:scrlption-Translation-E. coli strain W3110 was grown in minimal medium (26) containing 0.2% glycerol and trypto- phan (100 pg/ml). S-30 extract was prepared as previously described (32). In vitro transcription-translation reactions were carried out in the presence of all 20 amino acids including either [:''S]methionine, [3H]tryptophan, ['Hlphenylalanine, or [3H]histidine. After incuba- tion of reaction mi.xtures for 30 min at 37 "C, samples were denatured and electrophoresed in a 10% Tricine-sodium dodecyl sulfate-poly- acrylamide gel (33). The gel was fixed in 50% methanol + 10% acetic acid for 30 min, washed in water for 30-60 min and incubated with 1 M sodium salicylate as a fluor for 30 min (34). The gel was dried and exposed to Kodak XAR-5 film at -80 "C using an intensifying screen, for 1-5 days.

Primer Extension-P. vulgaris was grown to mid-log phase in ACH-medium supplemented with tryptophan (100 pg/ml). RNA was prepared from 7 ml of cells as described (35). A synthetic oligonucle- otide (mentioned above) that is complementary to the mRNA was purified on a 20% polyacrylamide gel and labeled with [-y-:32P]ATP using T4 polynucleotide kinase. The primer extension reaction was carried out with minor modifications of a described protocol (36). About lo5 cpm of labeled oligonucleotide was mixed with 10 pg of RNA in 20 pl of a. reverse transcription reaction mixture, denatured at 85 "C for 10 min and hybridized at 37 "C for 2 h. 50 units of Superscript RNase H- reverse transcriptase (GIBCO-BRL) was added, and incubation was continued for 1 h. Samples were prepared as described (36) and subjected to electrophoresis in an 8% acrylamide 7 M urea sequencing gel adjacent to a sequencing ladder generated from plasmid pAVK2 and the same primer.

Other Techniques-DNA manipulation techniques and growth me- dia are given in Sambrook et al. (35). Polymerase chain reaction reactions were carried out using an Ericomp Thermocycler and Taq polymerase (Perkins-Elmer/Cetus) according to the manufacturers' instructions. Protein estimation was by the method of Lowry et al. (37) using bovine serum albumin as a standard. Oligonucleotides were

tna Operon of P. vulgaris

GATCACGTTAATTTATAATARACACTARATAGTGTGATTATTATCC~TTTAT~TATTGTTATATTA~~CGMGCTAACTGTTATAT~CTAGTG 100 CAP binding site '-35" *- - 1 0 n

c-, SD TnaC--> TGTAAATTGTACTATTTCCTGACTA~ffiGTGARAGTATGTTCTCATCATTTAATGTATTGATMTATTAAGAGGTTTCGTCAGATTGAAGARATffiTT 200

M F S S F N V L I I L R G F V R L K K W F boxA

TAATATTGACTCTGAACTCCCTTCTTCTTTCCTAAAARATAATCCCTTTTCTATACATATCTAAATTAATTTAATAATTCTTTTCCGTTTATTTAAAAC 300 N I D S E L A F F F P K K ""><"" """, <"-

GGAGAGACTATTTTATTTAATCTTTATTTATTATTTAATTTATATT~ffiTATTATCATGGCTAAAAGAATCGTAGAACCGTTCCGTATTAAAATGGTA 400 SD TnaA-->

" ""- , <""- M A K R X V E P F R I K M V

GAhAAAATCCGAGTTCCTTCAAGAGAAGAACGTGAAGCAGCATTAAAAGAAGCAGGATATAACCCATTTTTACTACCMGCAGCGCGGTATATATTGATT 500 E K I R V P S R E E R E A A L K E A G Y N P F L L P S S A V Y I D

L L T D S G T N A M S D H Q W A A M I T G D E A Y A G S R N Y Y D L TATTGACTGACTCTGGAACTAACGCMTGAGTGATCATCAATGGGCAGCGATGATTACAGGTGATGAAGCCTATGCGGGTTCAAGAAACTATTATGATTT 6 0 0

AAAAGATAAAGCAAAAGAACTTTTTAATTATGATTATATTATTCCAGCTCACCAAGGTCGTGGCGCTGAGAATATTCTTTTTCCTGTGCTATTAAAATAT 7 0 0 K D K A K E L F N Y D Y I I P A H Q G R G A E N I L F P V L L K Y

ARACAAAAAGMGGAAAAGCGAAAAACCCAGTTTTTATTTCTAACTTCCACTTTGATACAACCGCTGCCCACGTAGAATTAAATGGTTGTARAGCAATTA 800 K Q K E G K A K N P V F I S N P H F D T T A A H V E L N G C K A I

ATATTGTTACTGAAAAAGCCTTCGATTCAGARACCTATGATGATTGGAAAGGCGATTTTGATATTAAAARATT~GAGAATATTGCACAACACGGTGC 900 N I V T E K A F D S E T Y D D W K G D F D I K K L K E N I A Q H G A

TGATAATATTGTTGCTATTGTATCAACCGTGACTTGTAATAGTGCCGGCGGCCAGCCTGTTTCTATGAGTAATTTAAAAGAAGTTTATGARATAGCGAAA 1000 D N I V A I V S T V T C N S A G G Q P V S M S N L K E V Y E I A K

CAACACGGTATTTTTGTTGTGATGGATTCAGCTCGTTTTTGTGAGAATGCTTATTTTATTAAAGCGCGTGATCCTAAATATAAAAATGCCACTATTAAAG 1100 Q H G I F V V M D S A R F C E N A Y F I K A R D P K Y K N A T I K

A A G T T A T T T T T G A T A T G T A T A R A T A T G C T G A T G C A T T A A C A A T G T C A G C G A A A A A A G A T C C A T T A T T A A 1200 E V I F D M Y K Y A D A L T M S A K K D P L L N I G G L V A I R D N

TGAAGAGATATTTACTTTAGCCAGACAACGTTGTGTACCCATGGMGGCTTTGTAACCTATGGTGGTTTAGCTGGTCGTGATATGGCTGCAATGGTACAA 1300 E E I F T L A R Q R C V P M E G F V T Y G G L A G R D M A A M V Q

GGTTTAGAAGMGGAACTGAGAAGAGTATCTGCACTATCGTATTGGTCAAGTT~GTATTTAGGTGATCGCTTACGTGAAGCGGGTATTCC~TTCAAT 1400 G L E E G T E E E Y L H Y R I G Q V K Y L G D R L R E A G I P I Q

ATCCTACTGGTGGGCATGCGGTTTTCGTTGACTGTRRAAAATTAGTTCCGCAAATTCCGGGTGATCAATTTCCTGCTCAAGCAGTTATTAATGCACTTTA 1500 Y P T G G H A V F V D C K K L V P Q I P G D Q F P A Q A V I N A L Y

TCTGGAGTCAGGTGTGCGTGCGGTAGAAATTGGTTGGTTCATTCTTATTAGGTCGTGATCCTGCAACGGGTGAAC~CATGCTGATATGGAATTTATGCGT 1600 L E S G V R A V E I G S F L L G R D P A T G E Q K H A D M E F M R

TTAACTATTGCACGCCGTGTTTATACCAATGACCATATGGATTATATTGCTGATGCATTAATTGGGTT~GAGMGTTTGCMCACTTAAAGGTCTAG 1700 L T I A R R V Y T N D H M D Y I A D A L I G L K E K F A T L K G L

AGTTCGAATATGAACCACCTGTATTAAGACACTTTACTGCAAGATTRAAACCTATCGMTMTAAGTAATMTCCACMTT~TMTGGCTCTCGTGCA 1800 E F E Y E P P V L R H F T A R L K P I E

SD ATGCGAGAGCCCTATCCTTATATATTTMffiGATACAAATAnTGG~TACTTCAGTARATARAGAACCCTCAATTATTGTAGGGGGATTTGTTCTGGG 1900

TnaB--->

M E N T S V N K E P S I I V G G F V L G

TGGTGCTATGATAGGTGCAGGTATGTTTAGCCTGCCAACAAT.~TGTCTGGTGCTTGGTTTATAAACTCACTATTTATATTGTTTATTGTTTGTTTCTTT 2000 G A M I G A G M F S L P T I M S G A W F I N S L F I L F I V C F F

ATGTTCCACTCGGGGATATATATTCTTGAATGTATTTCTAhATATGGCGCAGGAACAAATTACTTTGATATATCCAAAGAGTTATTACCGAAGTGGGCTT 2100 M F H S G I Y I L E C I S K Y G A G T N Y F D I S K E L L P K W A

GTTATATTGCCAATGCTTCATTGATCTTTGTATTATATATATTGATCTATGCTTATATCTCTGCGGCGGGTTCTATTATCTATGAnGCATCACTGTTATA 2200 C Y I A N A S L I F V L Y I L I Y A Y I S A A G S I I Y E A S L L Y

TGGTATTAATTTTAATCTGAGAGCTATATTTTTTATTTTTACGATAGCCCTTGGTGCTACMTATGGTGGGGTGGCGCTTGTGCTAGCCGTTTAACCTCA 2300 G I N F N L R A I F F I F T I A L G A T I W W G G A C A S R L T S

ATTTTCTTATTCATTAAGATAGTATTATTTATATTAGCGTTTTCGGGTTTGTTTTTTAAAGCRAAAGGTGATTTATTATTTAGTGCAACTTTTGCAGGAA 2400 I F L F I K I V L F I L A F S G L F F K A K G D L L F S A T F A G

AhAGCCAATTATATCTTTATCCTTTTATTTTTATTATCATTCCTTATGCCATTACCTCATTTGGATATCATGGTAATGTTTGTAGTCTTTATAAGCTTTA 2500 K S Q L Y L Y P F I P I I I P Y A I T S F G Y H G N V C S L Y K L Y

TAATCAAAACGAAAGAAAAGTAGTTAAGAGTTGTATCATTGGTTGCTTGTTAGCATTAGTCATCTATTTACTTTGGATGATTGGCACTATGGGTAATTTA 2 6 0 0 N Q N E R K V V K S C I I G C L L A L V I Y L L W M I G T M G N L

CCGAGAGMCAATTTATTACTATTATCCAAAAAGGCGGTAACTTAGATGCCTTTATTGATTCACTCTATACGGTATTAAATAGTAAATATATTGMGGAT 2 7 0 0 P R E Q F I T I I Q K G G N L D A F I D S L Y T V L N S K Y I E G

TTTTATTATGGTTCTCTATTAGTGCTGTTTTTTGCTCTTTTTTAGGTGTCGCGATAGGTCTTTTTGATTATATTTTAGCGTCATTAAR~TTTAAAGATAA 2 8 0 0 F L L W F S I S A V F C S F L G V A I G L F D Y I L A S L K F K D N

FIG. 1. Nucleotide sequence of the tna operon of P. vulgaris and its flanking regions. The deduced amino acid sequences of TnaC, TnaA, and TnaB are presented below the nucleotide sequence in the single letter code. The putative CAP binding site, promoter "-35," and "-10" regions, Shine-Dalgarno ( S D ) sequence and boxA-like sequence are given in bold letters. -, potential transcription start site as determined by primer extension analysis. The codon for the critical tryptophan residue in the TnaC peptide is also given in bold letters. Arrows mark the inverted repeat sequences present in the transcribed leader region.

tna Operon of P. vulgaris 19981

TARAACAGGGCGATTG~TCAGGTGTGCTTTGTTTTACACCTCCATTGCTATTATGTCTATT’rTTCCCTMTGGTTTCTTMTTGCTATTGCGTATGCA 2900 K T G R L K S G V L C F T P P L L L C L F F P N G F L I A I A Y A

GGCACTGCTGCTTGTGTATGGGCCATTATTTGTCCGGCAGTAATGGCACTG~GCACGACAGMGTTTCCTMCTCAGGCTTT~GTATGGGGTGGTA 3 0 0 0 G T A A C V W A I I C P A V M A L K A R Q K F P N S G F K V W G G

~GCTGATTTATGCAGTTATTGCTTTTGGTGTTGTTGGTATTATTTGTCAATCGTGGCGCMTTT~TTTACTGCCTATTTATCGTT~TAAG~CT 3100 K K L I Y A V I A F G V V G I I C Q S W R N L I Y C L F I V K

CARRTCAGGGAAAATAAAACCAGAGTAGCACCATTGAATATACGTTAT 3148

FIG. 1-continued

1 5 10 15 30 25 30 35 40 45 50 55 60

cftplse

61 65 70 75 80 8 5 90 95 100 105 110 115 120

121 125 130 135 140 145 150 155 160 165 170 175 180

181 185 190 195 200 205 210 215 220 225 230 235 240

241 245 250 255 260 265 270 215 280 285 290 295 300

301 305 310 315 320 325 330 33s 34 0 34 5 350 355 360

361 365 31 0 315 380 385 390 395 400 405 410 415 420

421 425 433 435 440 445 450 455 4 60 465 470

cftplse Y Q H K E D

FIG. 2. Alignment of the amino acid sequences of Tnases of P. vulgaris (pvtnase) and E. coli (ectnase) and tyrosine phenol lyase of C. freundii (cftplse). Residues common to two or three of the polypeptides are enclosed in boxes. An asterisk marks the lysine residue that forms the internal aldimine bond with PLP in these enzymes.

synthesized on an Applied Biosystems Synthesizer. The MacVector program (IBI) was used to compile and analyze nucleotide sequences. Amino acid alignments were performed using the TULLAl program of Subbaiah and Harrison (38).

RESULTS

Cloning and Sequencing of the tna Operon of P. vulgaris- A genomic library of P. vulgaris DNA was constructed in pUC13, and the plasmid mixture was transformed into tryp- tophan-requiring mutant strain CY15077 (W3110 tnaA2 AtrpEA2) of E . coli, selecting for growth on indole plus 1MT. Growth on this medium presumably would occur only if the introduced plasmid directed synthesis of the enzymes Tnase or tryptophan synthetase. A few positive clones were obtained. Two of these, pAVK2 and pAVK4, had 2.1 and approximately 6-kb inserts, respectively. The 2.1-kbp insert of pAVK2 was

entirely sequenced on both strands, using nested deletions. The insert contained tnaA and part of tnaB. Restriction digest analyses of pAVK4 showed that it contained all of tnaA and tnaB. Digestion of pAVK4 with EcoRI and AvaI gave a 1.9- kb fragment that had the remainder of tnaB. A total of about 90 clones containing overlapping segments were sequenced to obtain the entire tna operon sequence on both strands.

The sequence of the tna operon of P. vulgaris is presented in Fig. 1. The tnaA structural gene is preceded by a 341-bp region that contains an open reading frame analogous to tnaC of E. coli that could encode a 34-residue polypeptide (Fig. 1). This short coding region was preceded by a well defined Shine- Dalgarno sequence and putative “-35” and “-10” promoter elements. The transcription start site was identified by primer extension analysis. A potential CAP binding site resembling the consensus CAP sequence (39) was present in the presumed

19982 tna Operon of P. vulgaris

1 5 1 0 1 5 2 0 2 5 3 0 P 40 45 50 55 60

pvtnab ectnab emtr

ectnab pvtnab

eantr

ectnab pvtnab

eant r

pvtnab ectnab eantr

pvtnab

eCmt1 ectnab

pvtnab ectnab emtr

pvtnab ectnab emtr

I I1

61 65 70 75 80 85 90 , 95 100 1 0 5 110 115 120

- I l l

121 1 2 5 130 1 3 5 1 4 0 1 4 5 1 5 0 1 5 5 1 6 0 1 6 5 110 1 7 5 180

I V 1 8 1 1 8 5 1 9 0 1 9 5 2 0 0 2 0 5 2 1 0 2 1 5 2 2 0 2 2 5 230 235 240

V

VI 241 245 250 255 260 265 270 215 280 285 290 295 300

V I 1 VIII 301 305 310 315 320 325 330 335 340 345 350 355 3 60

361 365 370 375 380 385 3 90 395 400 405 410 415

FIG. 3. Alignment of the amino acid sequences of the tryptophan permeases of P. vulgaris (putnab), and E. coli (ectnab) and the Mtr Dermease (ecmtr) of E. coli. Residues common to two or three of the polypeptides are enclosed in boxes. The putative hydrophobic domains are indicated by u&rlining.

promoter region. The predicted tnaC leader peptide of P. vulgaris contains a single tryptophan residue at position 20. A single tryptophan residue is present at position 12 of the E. coli tnaC leader peptide (18). The tnaC peptide of P. oulgaris is unusually hydrophobic in nature, containing multiple phen- ylalanine, valine, isoleucine, and leucine residues.

Predicted Amino Acid Sequence of P. vulgaris Tnase-The tnaA structural gene is 1401 bp in length and encodes a 467- residue polypeptide. The codon bias of the t n d open reading frame is similar to that of tnaA of E. coli. The predicted molecular mass of the encoded polypeptide is 52,485 Da, which compares favorably with the estimated molecular weight for the polypeptide from this species (40). The calculated isoelec- tric point of the polypeptide is 5.9.

Comparison of the amino acid sequences of the Tnases of P. vulgaris and E. coli (9) indicate that they are 52% identical and 69% similar (Fig. 2). There are many segments that are highly conserved, e.g. 15 of the 16 residues from 224 to 239 are identical, and 12 of the 14 residues near the carboxyl- terminal end of the polypeptide are identical. The amino acid residues around Lys2'j6, the residue that is covalently bound to PLP, are completely conserved. Tnase is also homologous to the functionally related PLP-dependent enzyme, tyrosine phenol lyase, of Citrobacter freundii. The structural gene for this enzyme has been cloned and sequenced (22). The nucleo- tide sequence of this gene was determined independently by P. Gollnick (State University of New York at Buffalo).' The predicted amino acid sequence of the tyrosine phenol lyase

P. Gollnick, personal communication.

polypeptide is compared with the sequences of the two Tnases in Fig. 2.

Predicted Amino Acid Sequence of TnaB, the Tryptophan Permease-P. vulgaris tnaA is followed by a gene that is homologous to tnaB of E. coli (Fig. 1). In the Proteus operon, approximately 80 bp separate tnaA from tnaB (Fig. 1); the tnaB coding region is preceded by a Shine-Dalgarno sequence. The tnaB structural gene codes for a 417-residue polypeptide that has a predicted mass of 46,283 Da. The polypeptide is highly hydrophobic; approximately 43% of the residues are hydrophobic, 3.3% of the residues are acidic, and 8% are basic. The calculated PI is 9.7. The high content of hydrophobic residues suggests that the TnaB polypeptide, like its homol- ogous polypeptides, is membrane-bound and has multiple membrane-spanning domains (14). The amino acid sequence of the P. vulgaris TnaB permease is compared with that of E. coli (14) and the high affinity tryptophan-specific transporter of E. coli, Mtr (23), in Fig. 3. TnaB of P. vulgaris is 42% identical to TnaB and Mtr of E. coli; 30% of the residues are conserved in all three proteins. Most of the conserved residues are in the presumed membrane-spanning domains of TnaB of E. coli (Fig. 3) .

Detection of the Leader Peptide of P. vulgaris-tnaC of E. coli encodes a 24-residue leader peptide that has a single tryptophan residue that is crucial to regulation of transcrip- tion of the operon by attenuation (18). Analyses with an E. coli tnaC'-lacZ translational fusion demonstrated that the tnaC coding region was translated in E. coli (17). It was also shown that translation of the leader peptide coding region

tna Operon of P. vulgaris 19983

was essential for tryptophan induction (18, 19). The leader region of the tna operon of P. vulgaris contains a short coding region analogous to tnaC of E. coli that encodes a 34-residue peptide. To demonstrate synthesis of the leader peptide (TnaC) of P. uulgaris, we employed an in vitro transcription- translation system from E. coli and a template containing the P. vulgaris tna operon leader region. To prepare the template,

1 2 3 4 5 6 7 8

46.5-

29.7-

19.1-

15.1-

6.2-

3.0-

+ TnaA

+ B-Lac

+ TnaC

FIG. 4. Detection of synthesis of the TnaC peptide in a cou- pled in vitro transcription-translation system. The reactions were carried out according to the method of Zubay et al. (32), in a final volume of 20 p1 and incubated for 30 min a t 37 "C. Reactions were terminated by adding 30 p1 of 2 X loading buffer (125 mM Tris- HC1 buffer, pH 6.8,4% sodium dodecyl sulfate, 20% glycerol, 10% 8- mercaptoethanol, and 0.01% bromphenol blue) and were incubated at 37 "C for 30-60 min. Samples were clarified by centrifugation and 5-20-p1 aliquots were loaded onto the gel, electrophoresed (33), and processed as described in the text. Plasmids were purified on CsCI- gradients twice and 2-3 pg was used as templates. Polypeptide bands in lanes I, 2, 3, 7, and 8 were labeled with ["S]methionine (3 pCi); those in lanes 4-6 were labeled with ['Hltryptophan, ["Hlphenylala- nine, and ["Hlhistidine (20 pCi each), respectively. The templates used were, pUC118 (lane I); pUC118 containing the 426-bp tnaC- tnaA' fragment (pAVKll), (lane 2); self ligated 426-bp RarnHI mol- ecule (approximately 0.3-0.5 pg) (lanes 3-6); pUC118 containing the 426-bp tnaC-tnaA' fragment in which the ATG start codon of tnaC was replaced by a stop (TAG) codon (lane 7) and pAVK2 (lane 8). See text for construction of the plasmids which were used as tem- plates. To obtain a clear band, 40 p1 of the sample was loaded in lane 4, and this overloading slightly retarded the mobility. Prestained protein standards (GIBCO-BRL) were used as markers, and their apparent molecular weights are given. The positions of the TnaC peptide, Tnase, and p-lactamase are marked with arrows. P-Lac, p- lactamase.

an appropriate 426-bp DNA fragment containing BamHI sites a t both ends was ligated into pUC118. The resulting plasmid, pAVKl1 was used as the template in the reaction. In addition, large amounts of this 426-bp fragment were purified and self- ligated to form small circular molecules which were also used as templates. By employing the latter construct, we could increase the molar concentration of the template in the in vitro reaction mixture, and in addition, the smaller template presumably would provide a simpler pattern of translation products. With pAVKll as template (Fig. 4, lane 2), a labeled peptide band appeared that migrated at a rate corresponding to a polypeptide of about 4.2 kDa. Synthesis of this peptide was more evident when the self-ligated BamHI fragment was used as template (Fig. 4, lane 3 ) . The P. vulgaris TnaC peptide is predicted to contain 1 tryptophan, 7 phenylalanine, but no histidine residues. I n vitro synthesis of the leader peptide was performed in the presence of "H-labeled tryptophan, phenyl- alanine, or histidine. As expected, the peptide contained tryp- tophan (lane 4 ) , phenylalanine (lane 5), but no histidine (lane 6). Additional proof that the low molecular weight band was the leader peptide was provided by constructing a plasmid, derived from pAVK11, in which the initiator ATG codon of tnaC was replaced by TAG. This change eliminated synthesis of the presumed leader peptide band (lane 7). A labeled 53- kDa band corresponding to Tnase was also observed when pAVK2 was the template (lane 8). The stability of the TnaC peptide was not investigated.

Induction of tna Operon Expression by Tryptophan and IMT-To determine if the tna operon of P. vulgaris was regulated in the same manner as the tna operon of E. coli, namely by catabolite repression and tryptophan-mediated transcription antitermination, appropriate in vivo regulatory studies were performed. To examine regulation of the P. vulgaris tna operon, a culture was grown in a medium con- taining 1% ACH as carbon source, with appropriate supple- ments, and Tnase activity was assayed in crude cell extracts. I t can be seen in Table I that Tnase-specific activity increased approximately 7-8-fold when the culture medium was supple- mented with tryptophan or 1MT. Addition of glucose to the medium appreciably decreased Tnase specific activity, and induction was not detectable. For comparison E. coli W3110 was also grown in the same medium, and induction of synthe- sis of Tnase by tryptophan or 1MT was determined (Table I). Addition of either inducer increased Tnase production 20- 30-fold, while glucose, in the presence or absence of inducer, substantially reduced the Tnase level.

To determine if the Proteus tna operon could be regulated in E. coli, a tnaA'-lacZ translational fusion was constructed in plasmid pMLB1034. The derived plasmid (pAVK9) was introduced into E. coli SE5000, and induction experiments were carried out (Table 11). p-Galactosidase activity increased 3-6-fold when tryptophan or 1MT was added. Glucose re-

TABLE I Induction of Tnase synthesis in P. vulgaris and E. coli by tryptophan or 1 M T

P. vulgaris E. coli W3110

Growth medium" Specific activity Induction Specific activity Induction

-Glut' +Gluc -GIuc +Gluc - G ~ c +Gluc -GIuc +Gluc ~ ~ ~~

unitsfmg' -fold unitsfmg' -fold ACH medium 0.040 0.009 1.0 1.0 0.163 0.046 1 .o 1.0 ACH medium + Trp 0.286 0.010 7.2 1.1 3.564 0.123 21.8 2.8 ACH medium + 1MT 0.306 0.013 7.9 1.4 4.845 0.110 28.8 2.4

Medium composition and concentrations of inducers are given under "Materials and Methods." Micromoles of o-nitrothiophenol formed/min/mg protein. Trp, tryptophan; lMT, 1 methyltryptophan; Gluc, glucose.

19984 tna Operon of P. vulgaris

TABLE I1 Induction of &galactosidase s.ynthesis by tryptophan or 1MT using plasmids containing tnaA '-lacZ fusions of P. vulgaris and E. coli

Plasmid in E. coli SE 5000

pAVKSb pPDG100' ~AvK13~ Growth medium" @-Galactosidase

activity' Induction ratio 8-Galactosidase activitf Induction ratio @Galactosidase

activitf

ACH medium 13.8 6.9 1.0 1.0 437.0 11.6 1.0 1.0 6.6 5.8 ACH medium + Trp 37.0 12.8 2.1 1.9 2977.0 33.0 6.8 2.8 5.4 5.7 ACH medium + 1MT 79.0 18.5 5.7 2.7 3653.0 62.0 8.4 5.3 6.2 6.0

"Medium composition and concentrations of inducers are given under "Materials and Methods." The plasmids used in this study are

*Plasmid pAVK9 contains the Proteus tnaA'-lac2 translational fusion in vector pMLB1034. stable under all the growth conditions used. Plasmid copy number was not determined.

Plasmid pPDGlOO contains the E. coli tnaA'-lacZ translational fusion in vector pRS552. Plasmid pAVK13 has a construction similar to pAVK9, except that the ATG start codon of tnaC was changed to a TAG stop codon. Activity is expressed as A120/30 min/ml extract/Asm of culture.

'Trp, tryptophan; lMT, 1 methyltryptophan; Gluc, glucose.

TABLE I11 Comparison of regulatory features of tna operons of P. vulgaris and E. coli

The residues that are identical in the leader peptides are in bold letters.

Organism CAP binding site Leader peptide sequence bonA sequence Distance between tnaC and tmA

1 34 P. vulgaris AATTTATACTATTGTTATATT MFSSFNVLIILRGFVRLKKWFNIDSELAFFFPKK CGCCTTCTT 115 bp

E. coli ATTGTGATTCGATTCACATTT MNILHICV TSKWFNIDNKIVDHRP CGCCCTTGA 220 bp 1 24

pressed enzyme synthesis as it did in P. vulgaris cells. Values obtained with a tnaA'-lacZ fusion plasmid (pPDG100) carry- ing a tnu operon segment from E. coli, are given for compari- son (Table 11). The @-galactosidase activity obtained for pPDGlOO under both noninducing and inducing conditions was significantly higher than that for the corresponding P. vulgaris construct, pAVK9. When the ATG initiator codon of P. vulgaris tnaC was replaced by a TAG stop codon (plasmid pAVK13), the basal level of @-galactosidase activity decreased, and the fusion operon was not inducible (Table 11). While glucose repressed enzyme synthesis in pAVK9, the repression was not significant in the case of pAVK13. The explanation for this observation is not known.

DISCUSSION

Comparison of the Features of the tna Operons of E. coli and P. vulgaris"Severa1 bacterial species have the capacity to utilize tryptophan as a source of carbon or nitrogen. The mechanism of tryptophan utilization invariably involves pro- duction of the enzyme Tnase, which degrades tryptophan to indole, pyruvate, and ammonia. The pyruvate and ammonia can be used as a carbon and nitrogen source, respectively, while the toxic indole is generally secreted. Tnase also can catalyze what is essentially the reverse reaction, synthesis of tryptophan from indole and serine or cysteine (1-3). This property has been exploited in various mutant selection schemes in E. coli since mutants that lack tryptophan synthe- tase can use their Tnase to synthesize tryptophan from indole. However, for rapid growth on indole an inducer of Tnase production, such as a tryptophan analog, must be provided. The Tnase-specifying tna operon of E. coli also encodes a tryptophan permease. Transport studies have shown that this permease has a low affinity and high capacity for the amino acid (41), as would be expected of a transporter associated with a catabolic function.

In this report we described the cloning, sequencing, and initial regulatory analysis of the tna operon of P. vulgaris.

Our principal objective was to determine the structural fea- tures of this operon that are conserved in E. coli and P. vulgaris, on the assumption that these features would reveal important regulatory elements and likely regulatory events. We found that the tna operon of P. vulgaris, like that of E. coli, contains two genes, tnuA and tnaB, encoding Tnase and a tryptophan permease, respectively. The promoter region of the tna operon of each organism contains a binding site for the CAMP-dependent CRP protein (Fig. 1 and Table 111). In addition, as in the tna operon of E. coli, the tna operon leader region of P. vulgaris contains a coding region for a short leader peptide, designated TnaC. This peptide is 24 residues in length in E. coli and 34 residues in length in P. vulgaris. The coding regions for both leader peptides contain a single tryp- tophan codon; studies with E. coli have demonstrated that translation of this codon by tRNATv is essential for trypto- phan-induced transcription antitermination in the leader re- gion of the operon (18). Interestingly, the 5 amino acids (Lys- Trp-Phe-Asn-Ile-Asp) surrounding the single tryptophan res- idue are conserved (Table 111); also conserved are 17 of the 18 nucleotides specifying these amino acids. Few of the other leader peptide residues are conserved (Table 111). There are 7 phenylalanine residues in TnaC of P. vulgaris but only 1 in TnaC of E. coli; the significance of this, if any, is not known.

We used a coupled transcription-translation system and differential labeling to demonstrate synthesis of the P. uul- garis TnaC peptide. Synthesis of E. coli TnaC is not demon- strable in a comparable ~ y s t e m . ~ However, a translational fusion was used to demonstrate that the E. coli tnaC peptide coding region was translated (17). The ability to detect the TnaC peptide of P. vulgaris in vitro will permit an analysis of the effects of inducing conditions on synthesis of the leader peptide.

The TnaC coding region is followed by a 115-bp segment that precedes the tnaA start codon. This region, presumably

A. V. Kamath, unpublished observation.

tna Operon of P. vulgaris 19985

containing one or more Rho-dependent transcription termi- nation sites, is half the length of the corresponding region of the E. coli tna operon, which is 220 bp. This region has an unusually high A+T nucleotide content, 80%, and contains three indirect repeats. The sequence is unrelated to the se- quence of the corresponding region of the E. coli tna operon. As in E. coli, the TnaC coding region of P. vulgaris has a boxA-like sequence (42) near its end (Table 111 and Fig. 1). Studies with E. coli have established the importance of this boxA sequence in attenuation control, e.g. mutations that alter the boxA sequence generally result in constitutive expression (17).

Regulation of the tna Operon in P. vulgaris-Expression of the tna operon of P. vulgaris was shown to be subject to catabolite repression and induction by tryptophan (Table I). When the P. vulgaris tna operon was present on a plasmid in E. coli, induction was only 2-3-fold, whereas in the strain with the comparable plasmid containing the E. coli tna operon induction was 6-8-fold (Table 11). Note that expression with the E. coli plasmid is elevated under all conditions tested. The common features of the leader regions of the tnu operons of the two organisms suggest that similar regulatory mechanisms are employed in both organisms to sense tryptophan and prevent Rho-mediated transcription termination (43). Fur- thermore, as our findings indicate, the cell components of E. coli can regulate the P. vulgaris operon. As in E. coli (18, 19), translation of tnaC of P. vulgaris was essential for tryptophan induction (Table 11).

Polypeptide Homology-The Tnase of E. coli and the func- tionally related enzyme, tyrosine ammonia lyase, of C. freundii are 42% identical at the amino acid level (22). The P. vulgaris Tnase appears to be more closely related to the C. freundii tyrosine ammonia lyase since there are 50% amino acid iden- tities. As in the Tnase of E. coli, the P. vulgaris enzyme has two classes of -SH groups (44). Gollnick and Phillips (10) recently demonstrated that CysZg8 of the E. coli enzyme is located in the active site; a cysteine residue is present at the same position in the Proteus enzyme (Fig. 2). In many decar- boxylases, aminotransferases and other PLP-requiring en- zymes, serine, and aspartate residues are conserved a t posi- tions 2 and 8, respectively, preceding the PLP-lysine residue (22,45). These 2 residues are conserved in both Tnases. The Tnases of E. coli and P. vulgaris have 52% amino acid iden- tities. Comparison of the three polypeptides reveals that 33% of the residues are identical (Fig. 2), indicating that they probably evolved from a common ancestral polypeptide.

The tryptophan permeases of E. coli and P. vulgaris have 42% amino acid identities. Both are homologous to the tryp- tophan-specific permease, Mtr (23) (Fig. 3), and are members of an aromatic permease family (14) that includes the tyrosine transporter, TyrP (24), which is 29% identical to the trypto- phan permease of P. vulgaris. The proposed membrane-span- ning domains of these polypeptides (14) are highly conserved.

Acknowledgments-We are grateful to Dr. Paul Gollnick for the generous gift of plasmid pPDG100. We thank Kurt Gish, Mark Eshoo, Carl Yamashiro, Paul Babitzke, and Frank Lauter for critical reading of the manuscript.

REFERENCES

1. Snell, E. E. (1975) Adu. Enzymol. 42 , 287-333 2. Miles, E. W. (1986) in Vitamins B6: Pyridoxal Phosphate (Dolphin, D.,

Poulson, R., and Avramoric, O., eds) Part B, pp. 253-310. John Wiley &

3. Newton, W. A., and Snell, E. E. (1964) Proc. Natl. Acud. Sei. U. S. A. 5 1 , Sons, New York

4. Hoch, J. A., Simpson, F. J., and DeMoss, R. D. (1966) Biochemistry 5 , 382-389

5. Yoshida, H., Kumagai, H., and Yamada, H. (1974) FEBS Lett. 48,56-59 2229-2237

6. Phillips, R. S., Bender, S. L., Brzovic, P., and Dunn, M. F. (1990) Biochem-

7. Metzler, C. M., Vishwanath, R., and Metzler. D. E. (19911 J. Biol. Chem. istry 29,8608-8614

266.9274-9281

13

14.

15.

16. 17 18

20 19

21.

22.

23. 24. 25.

27. 26.

28.

29. 30. 31.

32.

33. 34. 35.

36.

37.

38. 39. 40.

41.

42.

43.

44.

45.

"_, ". . ."_

8. Kagamiyama, H., Matsubara, H., and Snell, E. E. (1972) J. Biol. Chem.

9. Deeley, M. C., and Yanofsky, C. (1981) J. Bacteriol. 147,787-796 247,1576-1586

10. Phillips, R. S., and Gollnick, P. D. (1989) J. Biol. Chem. 264,10627-10632 11. Kazarinoff, M. N., and Snell, E. E. (1977) J. Biol. Chem. 2 5 2 , 7598-7602 12. Nihira, T., Toyora, T., and Fukui, S. (1979) Eur. J . Biochem. 101 , 341-

747 Tawata, Y., Tani, S., Sato, M., Katsube, Y., and Tokusbige, M. (1991)

Sarsero, J. P., Wookey, P. J., Gollnick, P. D., Yanofsky, C., and Pittard, A.

Bilezikian, J., Kaempfer, R., and Magasanik, B. (1967) J. Mol. Biol. 2 7 ,

"-,

FEBS Lett. 284,270-272

J. (1991) J. Bacteriol. 173,3231-3234

m-mc Botsford, J. L., and DeMoss, R. D. (1971) J. Bacteriol. 105 , 303-312 Stewart, V., and Yanofsky, C. (1985) J. Bacteriol. 164 , 731-740 Gollnick, P., and Yanofsky, C. (1990) J. Bacteriol. 172 , 3100-3107 Stewart, V., and Yanofsky, C. (1986) J . Bacteriol. 167,383-386

Hoffman, P. S., and Falkinham, J. O., I11 (1981) J. Bacteriol. 148 , 736- Demoss, R. D., and Moser, K. (1969) J. Bacteriol. 98, 167-171

Iwamori, S., Yoshino, S., Ishitawa, K., and Makaguchi, N. (1991) J. Ferm.

Heatwole, V. M., and Somerville, R. L. (1991) J. Bacteriol. 173 , 108-115 Wookey, P. J., and Pittard, A. J. (1991) J. Bacteriol. 170,4946-4949 Nakamura, K., Pirtle, R. M., and Inouye, M. (1979) J. Bacteriol. 137,595-

Vogel, H. J., and Bonner, D. M. (1956) J. Biol. Chem. 218.97-106 Smith, 0. H., and Yanofsky, C. (1962) Methods Enzymol. 5,794-806 Sanger, F., Nickleu, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci.

Henikoff, S. (1987) Methods Enzymol. 125 , 157-165 Suelter, C. H., Wang, J., and Snell, E. E. (1976) FEES Lett. 6 6 , 230-232 Miller, J. (1972) Experiments in Molecular Genetm, Cold Spring Harbor

Laboratory, Cold Spring Harbor, NY Zubay, G., Chambers, D. A,, and Cheong, L. C. (1970) in The Lactose

Operon (Beckwith, J. R., and Zisper, D. Z., eds) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

I" ""

738

Bioeng. 72, 147-151

604

U. S. A. 7 4 , 5463-5467

Schagger, H., and van Jagow, G. (1987) And. Biochem. 166,368-379 Chamberlain, J. P. (1979) Anal. Biochem. 9 8 , 132-135 Babitzke, P., and Kushner, S. R. (1991) Proc. Natl. Acud. Sei. U. S. A. 88,

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A 1-5

Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NV

Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J.

Simpson, R. B. (1980) Nucleic Acids Res. 8,759-766 Subbaiah, S., and Harrison, S. C. (1989) J. Mol. Biol. 209 , 539-548

Sirnard, C., Mardini, A,, and Bordeleau, L. M. (1975) Can. J . Microbiol. 21 ,

Biol. Chem. 193 , 265-275

828-833 Yanofsky, C., Horn, V., and Gollnick, P. D. (1991) J . Bacteriol. 173 , 6009-

~~~ ~~~

cnl7 Friedman, D. I. (1988) in The Bacteriophages (Calender, R., ed) Vol. 2, pp.

Stewart, V., Landick, R., and Yanofsky, C. (1986) J. Bacteriol. 166 , 217- 263-319, Plenum Publishing Corp., New York ""

Simard, C., Mardini, A., and Bordeleau, L. M. (1975) Can. J . Microbiol. 21 ,

Vaaler, G. V., and Snell, E. E. (1989) Biochemistry 2 8 , 7306-7313

Z Z J

841-845