structure and regulation of the carab operon in pseudomonas

JOURNAL OF BACrERIOLOGY, Apr. 1994, p. 2532-2542 Vol. 176, No. 90021-9193/94/$04.00+0Copyright © 1994, American Society for Microbiology

Structure and Regulation of the carAB Operon in Pseudomonasaeruginosa and Pseudomonas stutzeri: No Untranslated

Region ExistsDONG-HYEON KWON, CHUNG-DAR LU, DEBORAH A. WALTHALL, TIMOTHY M. BROWN,

JOHN E. HOUGHTON, AND AHMED T. ABDELAL*Biology Department, Georgia State University, Atlanta, Georgia 30303

Received 17 September 1993/Accepted 13 January 1994

The carAB operons from Pseudomonas aeruginosa PAO1 and Pseudomonas stutzeri JM300 were characterizedby Southern and DNA sequence analyses. The results show that the previously reported sequence for carA(S. C. Wong and A. T. Abdelal, J. Bacteriol. 172:630-642, 1990) is derived from P. stutzeri and not P. aeruginosa,as originally reported. Therefore, the amino-terminal sequence of the purified carA product is identical to thatderived from the nucleotide sequence in both organisms, P. stutzeri having four additional amino acids. Theresults also show that while carA and carB are contiguous in P. stutzeri, as is the case in other bacteria, theyare surprisingly separated by an open reading frame (ORF) of 216 amino acids in P. aeruginosa. S1 nucleasemapping experiments with RNA extracted under a variety of growth conditions, as well as experiments usingdifferent lacZ fusions, indicate that the carA-ORF-carB operon of P. aeruginosa is transcribed from a singlepromoter. Moreover, these experiments demonstrate that expression of this single transcript is controlled byboth arginine and pyrimidines and that variation in arginine levels specifically modulates transcriptionalinitiation, while pyrimidine regulation is exerted subsequent to transcriptional initiation. Modification of arho-independent terminator-like structure, which is present upstream of carA in P. aeruginosa, removed alltranscriptional sensitivity of a carA::lacZ fusion to pyrimidines. This result, when coupled with the finding thattranslation of an 18-amino-acid leader polypeptide (associated with this putative rho-independent terminator),is inversely proportional to pyrimidine concentration in the cell, strongly suggests that regulation of carA bypyrimidines is mediated through an attenuation-type mechanism in P. aeruginosa.

Carbamoylphosphate synthetase (CPSase) catalyzes the syn-thesis of carbamoylphosphate, a common precursor of arginineand pyrimidines (16). In enteric bacteria, carbamoylphosphateis synthesized by a single enzyme consisting of two unequalsubunits. The small subunit (encoded by carA) functions as aglutamine amidotransferase; the large subunit (encoded bycarB) carries all other catalytic functions (16). In these organ-isms, the two genes are organized in one operon which istranscribed from tandem promoters independently controlledby pyrimidines and arginine (14, 28, 32, 33, 37).We have extended our studies on carbamoylphosphate

metabolism to Pseudomonas aeruginosa in view of the ques-tions posed by the presence in this organism of a catabolicenzyme (carbamate kinase) that degrades carbamoylphosphatevia the arginine deiminase pathway (1). We have previouslyreported the characterization of CPSase (3) and carbamatekinase (1) of P. aeruginosa and shown how this organism avoidsthe operation of a futile cycle involving carbamoylphosphatethrough the regulation of synthesis and activity of the twoenzymes. These studies also showed that while CPSase from P.aeruginosa is similar in subunit structure to the enteric CP-Sases, it differs in regulation of its activity and expression (3).Here we report the characterization of carAB clones from P.aeruginosa and Pseudomonas stutzeri. The results demonstratethat while carA and carB are contiguous in P. stutzeri, as theyare in other bacteria (16, 38), they are surprisingly separated bya 648-bp open reading frame (ORF) in P. aeruginosa. Contraryto our earlier report (47) of the presence of an untranslated

* Corresponding author. Mailing address: Biology Department,Georgia State University, Atlanta, GA 30303. Phone: (404) 651-1410.Fax: (404) 651-1542.

region within carA in P. aeruginosa, this paper presents evi-dence that the amino-terminal amino acid sequence of thesmall subunit is in fact identical to the derived sequence forcarA of this organism. This paper also reports studies onregulation of carA expression in P. aeruginosa. The resultsshow that carAB is transcribed from a single promoter in P.aeruginosa and that this transcript is controlled by both py-rimidines and arginine. Evidence is presented to support thehypothesis that arginine control of carA is exerted at the levelof transcriptional initiation, while pyrimidine regulation isexerted subsequent to transcriptional initiation, possiblythrough an attenuation mechanism (29).

MATERIALS AND METHODS

Bacterial strains, plasmids, media, and growth conditions.The bacterial strains and plasmids used are listed in Table 1.The Luria-Bertani (LB) enriched medium (36) was used forstrain construction with the following supplements as required:ampicillin at 50 ,ug/ml (Escherichia coli); carbenicillin at 500,g/ml (P. aeruginosa); and 5-bromo-4-chloro-3-indolyl-13-D-galactoside (X-Gal) at 0.03%. For experiments on regulationof CPSase synthesis in P. aeruginosa, strains were grown at37°C in the citrate minimal medium described by Stalon et al.(42).Enzyme assays. Logarithmically growing cultures were har-

vested by centrifugation at an optical density of 0.5 at 600 nm.Cells were washed, suspended in 0.1 M potassium phosphate(pH 7.6) containing 0.5 mM EDTA, and disrupted by passagethrough an Aminco French pressure cell at 8,000 lb/in2.CPSase activity was assayed by incorporation of ['4C]bicarbon-ate into carbamoylphosphate as previously described (26).

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TABLE 1. Strains, vectors, and plasmids

Straind

vector, Genotype or description (reference)

StrainsE. coli

DH5os F - +80dlac AM15 l(lacZYA-argF) U169 deoR recA] endAl hsdRJ 7(rK MK) supE44 X thi-I Bethesda ResearchgyrA96 relA] Laboratories

Jef8 AcarB8 rK mK thr thi; (the carB deletion encompasses 75% of the gene) N. Glansdorff (15)NM554 recA13 araD139 A(ara leu)7679 A(lac) 17A galU galK hsrR rpsI(Strr) mcrA mcrB Stratagene

P. aeruginosaPAOI Wild type D. HaasPA0303 argB18 D. Haas (25)PA0483 pyrE70 D. Haas (25)

P. stutzeri JM300 Wild type J. L. Ingraham (10)Plasmids

Cloning vectorspUC118 bla, lacZ J. Messing (45)pMC1403 bla, lacZYA translational fusion vector M. Casadaban (11)pQF50 bla, lacZ promoter fusion vector, broad-host-range plasmid M. A. Farinha (18)SuperCosl bla, kan, cosmid vector Stratagene

P. aeruginosapCOS1 carAB cosmid clone This studypCOS7 carAB cosmid clone This studypCOS8 carAB cosmid clone This studypKAl carAB of P. aeruginosa; the 6.1-kb HindIll-EcoRI fragment from pCOS8 into pUC118 This studypKA9 carA::lacZ promoter fusion in pQF50; insert is the HindIII-KpnI fragment (nt 1-782 of Fig. 2) This studypKA20 carA::lacZ promoter fusion in pQF50; insert (nt 1-447 of Fig. 2) was isolated after HindIII- This study

NruI digestionpKA24 Identical to pKA9 except for a 10-bp deletion (nt 530-539 of Fig. 2) This studypKA26 carA::lacZ promoter fusion in pQF50; insert (nt 241-486 of Fig. 2) was amplified by PCR This studypKA31 carA::lacZ translational fusion; insert (nt 1-565 of Fig. 2) was amplified by PCR This studypKA34 SalI-EcoRI (nt 1533-2483) fragment fused to pQF50 This studypMO 010749 Cosmid which complements car-9 of P. aeruginosa B. Holloway (39)pMO 011344 Cosmid which complements car-9 of P. aeruginosa B. Holloway (39)pMO 012147 Cosmid which complements car-9 of P. aeruginosa B. Holloway (39)

P. stutzeripHC79-4 carAB cosmid clone of P. stutzeri which complements E. coli Jef8 J. L. Ingraham

CPSase assays were done in duplicates that varied within 2%.,B-Galactosidase activity was determined by the method ofMiller (36). Protein concentration was determined by themethod of Bradford (9), using bovine serum albumin as thestandard.The relative levels of plasmid DNA under different growth

conditions were determined by quantitative isolation of plas-mid DNA, using the alkaline lysis method (6), digestion withBamHI, and densitometric measurement of the linearizedDNA bands in photographic negatives of ethidium bromide-stained agarose gels as described by Roland et al. (40).HindIII-digested A phage DNA was used as the molecularweight standard.Gene cloning and sequencing. A cosmid vector with two X

cos signals (Supercos 1; Stratagene Cloning Systems, SanDiego, Calif.) was used for preparation of a genomic library ofP. aeruginosa PAO1 essentially as described by Evans et al.(17). The vector was linearized with XbaI and dephosphory-lated with calf intestinal alkaline phosphatase. The vector wasthen digested with BamHI, and the vector arms were ligatedwith chromosomal DNA from P. aeruginosa that had beenpartially digested with Sau3A. The ligation mixture was pack-aged into E. coli NM554 by using an in vitro X packagingsystem (Gigapak; Stratagene). Cosmid DNA was extracted,transformed into E. coli Jef8 (AcarB8), and plated on minimalmedium that selected for ampicillin resistance and prototrophyfor arginine and pyrimidines. Subclones were obtained bycomplete digestion of the recombinant cosmid with Hindlll

and EcoRI ligation with pUCI 18 cut by the same enzymes,transformation into strain Jef8, and selection for arginine andpyrimidine prototrophy. DNA sequences were determined bythe dideoxynucleotide chain termination method of Sanger etal. (41) with synthetic oligonucleotide primers and double-stranded plasmid templates (35).

Southern analysis. Cloned DNA was completely digestedwith appropriate restriction endonucleases, and the resultingDNA fragments were separated by agarose gel electrophoresis.The desired DNA fragments were eluted and labeled withdigoxigenin-1 1-dUTP (Genius System; Boehringer Mann-heim), using the random-primed method (19). The labeledDNA was hybridized for 16 h at 65°C to a Southern blot ofdigested genomic DNA on a nylon membrane. The hybridswere detected by enzyme-linked immunoassay using anti-digoxigenin-alkalinephosphataseconjugate (BoehringerMann-heim) and subsequent enzyme-catalyzed color reaction with5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazo-lium salt as described by the manufacturer. The sizes of DNAfragments were determined by comparison with DNA stan-dards.

Construction of carA::lacZ fusions. The DNA fragment ofinterest was isolated by agarose gel electrophoresis aftersuitable restriction enzyme digestion. The DNA fragment wastreated with the Klenow fragment to fill recessed ends and thenligated into the SmaI site of pQF50 for transcriptional (pro-moter) fusions or into the SmaI site of pMC1403 for transla-tional fusions. The resulting plasmids were transformed into E.

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2534 KWON ET AL.

coli DH5cx and selected by plating on LB medium containingampicillin and X-Gal. Insert orientation was confirmed byDNA sequencing. To study the regulation of promoter expres-sion in P. aeruginosa, promoter fusions were directly trans-formed by the method of Berry and Kropinski (5). Fortranslational fusions carried on pMC1403, the EcoRI-SalIfragment carrying the carA::lacZ cassette was used to replacethe lacZ on the broad-host-range vector pQF50 before trans-formation into P. aeruginosa strains.

Generation of deletion by PCR. The recombinant PCRprocedure described by Higuchi (27) was used to generate adeletion in the leader sequence of carA in P. aeruginosa. Tworeactions were carried out to generate PCR products thatoverlapped between nucleotides (nt) 510 and 559. The primerscomplementary to the overlapping region created the desireddeletion between nt 530 and 539.

Si nuclease mapping. Total cellular RNA was extractedfrom exponentially growing cells as previously described (33).Si nuclease mapping using an end-labeled single-strandedprobe was carried out as described by Greene and Struhl (21),with one modification. To overcome problems associated withthe significant secondary structures found in P. aeruginosa,extension of the labeled oligonucleotide for preparation of thesingle-stranded probe was carried out with Taq DNA poly-merase at 75°C. Experiments were carried out quantitatively topermit comparison of levels of transcripts under differentgrowth conditions. The relative levels of transcripts weredetermined by scanning autoradiographs with a MolecularDynamics personal laser densitometer.

Purification of CPSase and determination of amino-termi-nal amino acid sequence. CPSases were purified from P.aeruginosa and P. stutzeri by chromatography on MonoQanion-exchange, phenyl-Superose, and Superose 6 columns(Pharmacia) as previously described (47). The two subunits ofCPSase were separated by application onto a sodium dodecylsulfate (SDS)-7.5% polyacrylamide gel. The polypeptides werethen transferred by electroblotting to polyvinylidene difluoridemembranes as described by Matsudaira (34). The amino-terminal sequences were determined at the Molecular Genet-ics Facility of the University of Georgia at Athens and at theWistar Protein Microchemistry Laboratory in Philadelphia.

Purification of protein fusions. Cell extract of the P. aerugi-nosa culture carrying a carA::lacZ translational fusion waspassed through a P-galactosidase immunoaffinity column (Pro-tosorb lacZ; Promega). The fusion protein was eluted with 0.1M sodium carbonate (pH 10.8), and the eluent was concen-trated by an Amicon Centricon 10 microconcentrator. Thesmall subunit/,B-galactosidase fusion was further separatedfrom other polypeptides by SDS-polyacrylamide gel electro-phoresis and electroblotted, and its amino-terminal sequencewas determined as described above.

Nucleotide sequence accession numbers. The sequences ofcarA from P. aeruginosa and P. stutzeri have been deposited inthe GenBank data base under accession numbers U04992 andU04993.

RESULTS

Identity of the derived sequence for carA of P. aeruginosawith the amino-terminal amino acid sequence. We previouslyconcluded that codons 5 to 8 of carA of P. aeruginosa were nottranslated because these four codons in the sequence derivedfrom carA in pSW2 had no corresponding amino acids in CarAfrom a plasmid-free strain of P. aeruginosa (47). To explain theapparent lack of colinearity, we hypothesized ribosomal hop-ping during translation, but experiments designed to test this

hypothesis led us to question our conclusion. Specifically, acarA::lacZ translational fusion carrying most of carA frompSW2 was constructed and introduced into P. aeruginosa. Wepurified the fusion protein and determined its amino-terminalamino acid sequence as described in Materials and Methods.The amino-terminal amino acid sequence was identical to thederived sequence; i.e., codons 5 to 8 were translated. Also,replacing codon 7 with a UAG stop codon caused translationaltermination. Since these results are inconsistent with ribo-somal hopping, we questioned whether the carAB in pSW2 wasan authentic P. aeruginosa operon.To address this question, a number of independent clones of

the carAB operon from P. aeruginosa were characterized.Three cosmid clones of P. aeruginosa (pCOS1, pCOS7, andpCOS8) which complement the carB deletion of E. coli Jef8were constructed as described in Materials and Methods.Three additional cosmid clones (pMO 010749, pMO 011344,and pMO 012147) which complement the car-9 mutation of P.aeruginosa (39) were generously provided by B. W. Holloway(Monash University). The carAB operon from one of thecosmids (pCOS8) was then cloned into pUC118, and theresulting plasmid was designated pKAL. The chromosomalinsert in pKAl was labeled and used to probe the restrictedgenomic DNA of P. aeruginosa as described in Materials andMethods. The results (Fig. la) show the same hybridizationbands for pKAl and chromosomal DNA from P. aeruginosa.Southern hybridization using the previously characterized plas-mid, pSW2 (47), and genomic DNA from P. stutzeri show thatthe hybridization bands are indeed similar (Fig. lb). Identicalhybridization bands were obtained (data not shown) withpSW2 and cosmid pHC79-4 carrying carAB from P. stutzeri(generously provided by J. L. Ingraham, University of Califor-nia, Davis). Additional Southern analysis using pSW2 as aprobe and genomic DNA from P. aeruginosa (not shown)confirmed that the chromosomal insert on this plasmid was notderived from this organism.The 5'-terminal sequence for carA was determined by

plasmid sequencing in pKAl and in each of the six availablecosmids carrying carAB of P. aeruginosa. The derived amino-terminal sequence (Fig. 2) in each case is identical to theamino-terminal amino acid sequence for the small subunitwhich has been previously reported (47) and also confirmed inthis work.

Nucleotide sequence of carA and flanking regions in P.aeruginosa. The nucleotide sequence of carA and its flankingregions was determined by plasmid sequencing of pKA1, usingsynthetic oligonucleotides. The sequence of the nontranscribedstrand is shown in Fig. 2. The sequence contains an ORFextending from a TTG triplet at position 588 to a TAG tripletat position 1722. This ORF has a coding capacity for 378 aminoacids, yielding a polypeptide with a molecular mass of 40,800Da, which is in good agreement with the value of 44,000 Dapreviously reported (3) for the small subunit of CPSase fromthis organism. Twenty-two residues of the amino-terminalsequence were determined as described in Materials andMethods and found to be identical to the derived sequenceshown in Fig. 2.

Analysis of the nucleotide sequence upstream of carArevealed that it encodes a homolog of dapB from E. coli (8).The derived amino acid sequence from nucleotide 1 (thesubcloning site) to 402 showed 82.8% identity to dapB of E.coli. The distance between the stop codon of dapB and the startcodon of carA of P. aeruginosa is 186 bases. The dapB gene isalso found 455 bp upstream of the corresponding codon forcarA of E. coli (7).

Analysis of the nucleotide sequence downstream of carA

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1 2 3 4 5 (b)(kbp)

23.1--9.4----6.64.4-2.3 -

2.0

0.6

1 2 3 4 5 6 7

HindIII EeoRI EcoRIda1.35 kbp I 1.1 kbpOR

dapB' carA ORF

EcoRI EcoRI EcoRI

I I_35 kbP

car carA

Pseudomonas aeruginosa (pKA1) Pseudomonas stutzeri (pSW2)FIG. 1. (a) Southern hybridization of cloned and genomic DNA that encompass carAB from P. aeruginosa PAO1. Chromosomal DNA from

P. aeruginosa was digested with Hindlll and EcoRI (lane 1) and HindlIl, EcoRI, and PstI (lane 2), electrophoresed in a 1% agarose gel, andhybridized to probe DNA. Lanes 4 and 5 contain similarly cut DNA from plasmid pKA1, which was likewise hybridized to probe DNA. The DNAprobe was derived from pKAI plasmid DNA, which had been restricted with EcoRI and HindlIl, separated from vector DNA, and labeled asdescribed in Materials and Methods. Molecular weights of the various fragments were determined by using A phage DNA which had been digestedwith HindlIl (lane 3), the mobility of which is referenced on the left. Also shown below the gel is a schematic diagram and relevant restriction mapof cloned DNA encoded within plasmid pKAL. (b) Southern hybridization of cloned and genomic DNA that encompass carAB from P. stutzeriJM300. Chromosomal DNA from P. stutzeri was digested with EcoRI and BamHI (lane 1), EcoRI, BamHI, and PstI (lane 2), and EcoRI, BamHI,and SmaI (lane 3), electrophoresed in a 1% agarose gel, and hybridized to probe DNA. Lanes 5 to 7 show plasmid pSW2 (47) cut in a similarmanner and likewise hybridized to probe DNA. Molecular weights of the various fragments were determined by using k phage DNA, digested withHindlIl (lane 4), the mobility of which is referenced on the left side. Probe DNA was derived from a combination of the 1.8-kbp EcoRI-EcoRIand 7.0-kbp EcoRI-BamHI DNA fragments of pSW2 which had been separated from vector DNA and labeled (as previously described). Alsoshown below the gel is a schematic diagram and relevant restriction map of cloned DNA encoded within plasmid pSW2 (47).

revealed that carA and carB are separated by a 682-bp regionthat contains an ORF with a coding capacity for a 23-kDapolypeptide. This ORF is preceded by a putative ribosomebinding site (GGAG) complementary to the 3' end of the 16SrRNA of P. aeruginosa, 3'-AUUCCUCU (20). An extensiveGenBank library search did not reveal any significant homol-ogy between the derived amino acid sequence for this ORFand known protein sequences. We have confirmed the pres-ence of the ORF found in pKA1 in five of the available cosmidclones (Table 1) by DNA sequencing and restriction map

analysis (data not shown).Following this ORF is carB, as evidenced by the identity of

residues 2 to 16 of the derived sequence to the first 15 residuesof the amino-terminal amino acid sequence that was deter-mined for the large subunit of CPSase as described in Mate-rials and Methods. The terminal methionine residue was

absent from the mature polypeptide. The translational startsite for carB is an ATG triplet which is preceded by a putativeribosome binding site (GAGG).

Nucleotide sequence of carA and flanking regions in P.stutzeri. The nucleotide sequence of carA and its flankingregions from P. stutzeri was determined by plasmid DNAsequencing of pHC79-4 (kindly provided by J. L. Ingraham).This is a cosmid clone of P. stutzeri wherein an EcoRI partialdigest of the P. stutzeri chromosomal DNA had been inserted

into the cosmid vector pHC79. This clone has a chromosomalinsert of approximately 26 kbp. A comparison of this newlydefined sequence (GenBank accession no. U04993) with thatpreviously reported for carA from pSW2 (47) by employing thesame oligonucleotide primers showed that, with few excep-tions, the two sequences are identical. The differences involveG-N inversions at eight locations of high GC content within thestructural gene. None of these changes affect the regions ofinterest in this work, i.e., the 5'-terminal region of carA (47);the 300-bp upstream region containing all the control ele-ments. Moreover, additional sequence comparisons of carAfrom both pHC79 and pSW2 on the same gel showed thatthese two sequences are indeed identical. These results sup-port our above conclusion that pSW2, and hence the previouslyreported sequence of carA, was likely derived from P. stutzeni.The amino-terminal amino acid sequences for the small and

large subunits for CPSase from P. stutzeri were determined as

described in Materials and Methods. The first 22 residues ofthe small subunit were identical to the derived sequence forcarA of this organism. In the case of the large subunit, the first22 residues were identical to residues 2 to 23 of the derivedsequence of carB, indicating the removal of the terminalmethionine. In contrast to results for P. aeruginosa (this work)and E. coli (7), analysis of the upstream flanking region showedthe absence of DNA sequence homologous to the dapB gene.

(a)(kbp)

23.19.4 -

6.64.42.32.0

0.6

::..... ....

...,,......,:Mf:

BamHI

carBt-i

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120AAGCTTCTGGATACCGCGGCACGGGTGTTGGGCGACGAAGTGGATATCGAGATCATCGAGGCCCATCACCGGCACAAGGTCGATGCACCGTCCGGCACCGCCTTGCGCATGGGCGAGGTGK L L D T A A R V L G D E V D I E I E A H H R H K V D A P S G T A L R N G E V

240GTAGCCCAGGCGCTCGGCCGGGACCTCCAGGAAGTGGCGGTCTACGGACGCGAGGGGCAGACCGGCGCGCGGGCGCGGGAAACCATCGGCTTCGCCACCGTGCGCGCCGGCGACGTGGTGV A Q A L G R D L a E V A V Y G R E G Q T G A R A R E T I G F A T V R A G D V V

360GGCGACCACACCGTGCTGTTCGCCGCCGAGGGCGAGCGCGTGGAGATCACCCACAAGGCTTCCAGCCGCATGACCTTCGCCCGTGGCGCGGTGCGTGCGGCGCTGTGGCTGGAGGGCAAGG D H T V L F A A E G E R V E I T H K A S S R M T F A R G A V R A A L U L E G K

-35 -10 *GAGAACGGCCTGTACGACATGCAGGATGTGCTCGGCCTACGCTGAGGCGCTGCCGCACGTCGTCTTATTGGTAGGACGGAATGTCGCGATTCTGTMMCTACAGCTTTAGTGTGTCCACTE N G L Y D N E D V L G L R ***

> <---------------599AAAAGCAGCGCAGCATGAATCGAAAAAAAGCGGGATGACTCTTCACGGTGTCATCCCGCTTTTTTACACCTGCGCGACCAGTCAGGCTTGATTTAC6E&AfiGTCTTC TTGACTAAGCCA

S.D. N T K P719

GCCATACTTGCACTTGCCGACGGCAGCATTTTTCGCGGTGAAGCCATCGGTGCCGACGGCCAGACCGTTGGCGAGGTGGTGTTCAACACCGCCATGACCGGCTACCAGGAAATCCTTACCA I L A L A D G S I F R G E A I G A D G Q T V G E V V F N T A N T G Y Q E I

GATCCTTCCTACGCCCAGCAGATCGTCACCCTGACCTACCCGCACATCGGCAATACCGGTACCACCCCGGAAGACGCCGAGGCCAACCGTGTCTGGGCCGCCGGCCTGATCATTCID P S Y A Q Q I V T L T Y P H I G N T G T T P E D A E A N R V W A A G L I I

CTGCCGCTGATCGCCAGCAACTGGCGCAGCAAGCAGTCGCTGCCTGACTACCTGAAAGCCAACGGCACCGTCGCCATCGCCGGCATCGATACCCGTCGCCTGACTCGCATCCTCCIL P L I A S N U R S K Q S L P D Y L K A N G T V A I A G I D T R R L T R I L

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

GCCGAGCGCTACGAGTGGCGCTCCAGCGTCTGGAACCTGGAAAGCGACAGTCACCCGGAGATCCCGGCCGGTGAACTGCCGTATCACGTGGTGGCCTACGACTACGGCGTCAAGC1A E R Y E W R S S V U N L E S D S H P E I P A G E L P Y H V V A Y D Y G V K

ATCCTGCGCATGCTGGTGGCGCGCGGCTGCCGCCTGACCGTGGTGCCGGCGCAGACACCGGCCAGCGAAGTGCTGGCGCTGAACCCTGACGGCATCTTCCTTTCCAACGGCCCCGII L R M L V A R G C R L T V V P A Q T P A S E V L A L N P D G I F L S N G P I

CCCGACCGTGCGACTACGCGATCCAGGCGATCCGCGAATTCCTCGATACCGAGATCCCGGTGTTCGGCATCTGCCTCGGCCATCAGTTGCTGGCCCTGGCCTCTGGCGCCAGA(P E P C D Y A I Q A I R E F L D T E I P V F G I C L G H Q L L A L A S G A KF

AAGATGGGCCACGGCCACCACGGCGCCAACCACCCGGTGCAGGACCTGGACAGCGGCGTGGATGATCACCAGCCAGAACCACGGTTTCGCGGTCGACGAGAGCACCCTGCCGK M G H G H H G A N H P V Q D L D S G V V M I T S Q N H G F A V D E S T L P 1

CTGCGAGCCACTCACAAGTCGCTGTTCGACGGTACCCTGCAGGGTATCGAGCGCACCGACAAGGTCGCTTTCAGCTTCCAGGGCCACCCCGAGGCGAGCCCCGGTCCGCATGACGIL R A T H K S L F D G T L Q G I E R T D K V A F S F Q G H P E A S P G P H DR

CCGCTGTTCGACCGCTTCATCTCGGCt;ATGGCCGAGCGTCGCTGAGGAGCAGAGCC ATGCCGACCCTGGGGATAACCGATTTCTGGACCTACGTGCTGGGTGTGGTTTTCGTGAIP L F D R F I S AA A E R R *** S.D.F P T L G I T D FA T Y V L G V V F V I

TTGCCCGGACCGAACTCGTTGTTCGTCCTCGCGACTTCGGCCCAGCGTGGCGTGGCGACCGGCTACCGGGCCGCCTGCGGCGTGTTCCTCGGCGACGCGGTGCTGATGCTGCTGTCL P G P N S L F V L A T S A Q R G V A T G Y R A A C G V F L G D A V L M4 L L '

CTTGGCGTGGCGTCCCTGCTGAAGGCCGAGCCGATGCTGTTCATCGGCCTGAAGTACCTCGGTGCGGCCTACCTGTTCTATCTCGGCGTCGGCATGCTGCGGGGCGCCTGGCGCMjL G V A S L L K A E P M L F I G L K Y L G A A Y L F Y L G V G N L R G A W R k

CGCAMTCCCGAAGCGACGGCGGGGCAGGCCGAACAGGTGGATGTCCACCAGCGGTTCCGCCAGGCGCTGCTGCTCAGCCTGTCGAATCCCAAGGCGATCCTGTTCTTCATCTCCTIR N P E A T A G Q A E Q V D V H Q R F R Q A L L L S L S N P K A I L F F I S F

ATCCAGTTCGTCGATCCCGGCTATGCCTATCCGGGGCTGTCGTTCCTGGTGCTGGCGGTGATCCTGGAACTGGTCAGCGCCCTTTACCTGAGCTTCCTGATTTTCACCGGTGTGClI Q F V D P G Y A Y P G L S F L V L A V I L E L V S A L Y L S F L I F T G V A

GCGGCCTGGTTCCGCCGGCGGCAACGGTTGGCTGCCGGCGCCACTTCGGGCGTCGGCGCCCTGTTCGTCGGCTTTGGTGTGAAGCTGGCCACCGCAACCCTGTCCTGA TTATTtA A U F R R R Q R L A A G A T S G V G A L F V G F G V K L A T A T L S ***

L T839

:GCGACR D959

:GCGAGR E1079

kCCACCT T1199

:TGAACL N1319

IGCGACG D1439

iCCCTGT L1559

ACAACD N1679TCGCCV A1798

,TCCTGI L1918CGGCCS A2038AGCTGK L2158TCTTCF F2278GGCTGR L2396GCAAC

S.D. N P K R T D I K S I L I L G A G P I V I G 0 A C E F

FIG. 2. Nucleotide sequence of the carA gene and flanking regions from P. aeruginosa PAO1. The amino acid sequences deduced for thecarboxy-terminal region of dapB, carA, ORF, and the amino-terminal region of carB are shown below the DNA sequence. Also shown are therelevant Shine-Dalgarno (S.D.) sequences. The transcriptional initiation site for the carA-ORF-carB transcript is indicated with an asterisk abovethe nucleotide. The putative - 10 and - 35 regions of the promoter are underlined and labeled accordingly. Converging arrows define a regionof dyad symmetry which lies upstream of the carA structural gene and may form a stable stem-loop structure within the transcribed RNA.

Furthermore, as is the case in E. coli, as previously reported(47), the carA and carB genes from P. stutzeri are contiguous.

Regulation of CPSase synthesis in P. aeruginosa. We havepreviously shown (3) that while exogenous arginine or uracildid not significantly affect CPSase activity in P. aeruginosa, the

enzyme was derepressed under conditions of limitation ofarginine or pyrimidines. To assess the effect of limitationconditions on CPSase levels in balanced exponentially growingcultures, we searched for compounds that could serve as poorsources of arginine or pyrimidines for auxotrophic derivatives


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TABLE 2. Regulation of CPSase synthesis in auxotrophicderivatives of P. aeruginosa

CPSase

Strain Relevant Growth conditiona Sp actgenotype (U/mg of Fold de-protein repression[SE])"

PA0483 pyrE Excess pyrimidines 4.3 (0.07) 1(1 mM uracil)

Limiting pyrimidines 47.4 (0.21) 10.8(0.1 mM cytosine)

PA0303 argB Excess arginine (10 mM 1.0 (0.14) 1arginine)

Limiting arginine (1 mM 2.8 (0.01) 2.8arginyl-glutamate)

a Concentrations of poor sources of arginine (arginyl-glutamate) and pyrimi-dines (cytosine) were selected such that auxotrophic derivatives grew withdoubling times of 180 min. This rate is one-third of that (60 min) obtained underexcess conditions of arginine or pyrimidines. Exponentially growing cultureswere harvested at optical densities of 0.5 at 600 nm, and CPSase was assayedin duplicate as described in Materials and Methods.

h One unit of CPSase activity catalyzes the formation of 1 nmol of car-bamoylphosphate per min.

of P. aeruginosa. Preliminary experiments showed that thedipeptides arginyl-glutamate (1 mM) and arginyl-valine (1mM) are poor sources of arginine such that they permit anarginine auxotroph to grow only at one-third of the specificrate obtained in the presence of excess arginine (10 mM). Aninvestigation of pyrimidine utilization by an auxotroph showedthat UMP (10 mM), CMP (1 mM), or TMP (1 mM) did notpermit growth in citrate minimal medium. However, cytosine(0.1 mM) permitted logarithmic growth at one-third of thespecific rate obtained with excess pyrimidines (1 mM uracil).Given these results, 1 mM arginyl-glutamate and 0.1 mMcytosine were selected to provide logarithmic growth underlimiting conditions for arginine and pyrimidines, respectively.Table 2 shows CPSase levels determined under conditions oflimitation and excess of arginine or pyrimidines. The results(Table 2) show that limitation for arginine and pyrimidinesresults in 2.8- and 10.8-fold derepression of CPSase, respec-tively.Mapping of transcriptional initiation and regulation of

transcript level in P. aeruginosa. The 5' terminus of carAmRNA was determined by Si mapping using an end-labeledsingle-stranded probe with a sequence complementary to nt 1to 692 of Fig. 2. The results (Fig. 3) show four consecutivebands corresponding to positions 475 to 478 of the sequence.Multiple bands are usually considered the result of nibbling byS1 nuclease (12). Accordingly, the most distal site, correspond-ing to position 478, is the most likely transcription site for carA,particularly since it is preceded by -10 and -35 sequencesthat are homologous to the sigma 70 consensus promoter of E.coli. Similar results were obtained with P. stutzeni (data notshown).To determine the levels of this transcript under different

physiological conditions, quantitative S1 experiments werecarried out. Densitometric measurement of the carA transcriptin the subsequent autoradiographs (Fig. 3) showed that argi-nine and pyrimidine limitation result in elevating the level ofthis transcript by two- and ninefold, respectively. The elevatedtranscript levels under conditions of either arginine or pyrim-idine limitation are in reasonable agreement with the elevatedlevels of CPSase under the same conditions. Thus, regulationby both arginine and pyrimidine in P. aeruginosa is mediated bycontrol of the level of a single major transcript.

A C G T l 2... ... ... .. _ _. ...ww wa:7Slt we e w... ......

,,-,f ,, B--a-

........ , .. ....... w;

"N, ........ __

.. .rwr sws

. - w q

_ _

AW_ _ ?"

t *-W swe

*_ _ " sssew* :.s. aw_:.: :f.::.ws

%:_* r._t ....._

-

_ _.,,,__ 4." :\>

i.......W:,

4ibq <

:*, .._

bV

3 4

I -+1

il

N.;............. ........ ...j

FIG. 3. Si nuclease mapping of the 5' end of carA transcript fromP. aeruginosa. A single-stranded DNA fragment (nt 1 to 692; Fig. 2)which has sequence complementary to the mRNA was labeled at the5' end with 3 P as described in Materials and Methods. After hybrid-ization to cellular RNA, the hybridization mixture was treated with SInuclease and analyzed on a 6% sequencing gel. The dideoxy sequenceladder (lanes A, C, G, and T) was derived by using the same primerthat was used to generate the probe for the Si nuclease mapping. Lane1, PA0483 grown in the presence of 0.1 mM cytosine (pyrimidinelimitation); lane 2, PA0483 grown in the presence of 1 mM uracil(excess pyrimidines); lane 3, PA0303 grown in the presence of 1 mMarginyl-glutamate (arginine limitation); lane 4, PA0303 grown in thepresence of 10 mM arginine (excess arginine). Also shown is the 5'terminus of the mRNA, defined as + 1, corresponding to the T residueat nt 467 (Fig. 2).

To address the possibility of an additional carA promoterupstream from that defined by the Sl mapping experiments,the lacZ gene was fused to this region (pKA20; Fig. 4). NoP-galactosidase activity was detected in P. aeruginosa transfor-mants of this plasmid construct. Similarly, the possibility ofadditional promoters preceding the ORF between carA andcarB or preceding carB was assessed by construction of aSalI-EcoRI fragment (960 bp) carrying the 3' terminus of carA,the ORF, and the 5' terminus of carB (pKA34). Transformantsof P. aeruginosa PA0483 carrying this fusion did not possess,B-galactosidase activity.

Regulation of carA::lacZ fusions in P. aeruginosa. A numberof carA::lacZ transcriptional fusions were constructed intopQF50 as shown in Fig. 4 and introduced into P. aeruginosaPAO1 and its auxotrophic derivatives. pKA9 contains the782-bp HindIII-KpnI fragment (nt 1 to 782; Fig. 2) thatincludes the first 194 bp of the structural gene encoding thesmall subunit of CPSase and the flanking upstream sequence.Measurements of ,B-galactosidase levels in P. aeruginosa strainscarrying this fusion (Table 3) showed that the enzyme levelsare elevated 2.1- and 9.1-fold under conditions of limitation ofarginine and pyrimidine, respectively. These results are inreasonable accordance with those obtained from measure-ments of CPSase and transcript levels.

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HindIll

KpnVlSmaI11 1~~~~~~~~~~~~~~~~~~~~~~~~~~~~

pKA9I

Small--

dapB'Hindlll

+1 carA'

pKA20

lacZ reporter gene

pKA24

lacZ reporter gene

KpnI/SmaI

carA'

+1

--lSmal pKA26

1 lacZ reporter gene

9 pKA31

___________________771 L _dapB' +1 lacZ reporter gene

(in-frame translational fusion)

FIG. 4. Schematic representation of various promoter fusions between the carA promoter of P. aeruginosa and a promoterless lacZ gene withinthe expression vector pQF50 (18) (Table 1). Depicted are restriction sites that either were used to form the different gene fusions or serve to orientthe cloned fragments with respect to the defined +1 of the carA-ORF-carB transcript and pKA1 (Fig. la). --/SmaI (in the pKA26 construct)denotes a ligation junction between a 247-bp PCR-generated DNA fragment and the SmaI site that lies within the multiple cloning site of thepQF50 vector. The partial ORFs of the cloned genes are depicted as variously shaded boxes (dapB' [striped box] and carA' [shaded box]). Alsoshown, when present, are the stem-loop structure and the proposed carA leader polypeptide (thick black line) that lie 5' proximal to the carAstructural gene. The lacZ gene from the original pQF50 expression vector is depicted as an unfilled box in all transcriptional gene fusions and a

filled box in the carA leader::lacZ translational gene fusion within pKA31.

Analysis of the sequence of the transcript upstream of thetranslational initiation site for carA revealed the presence of a

potential leader peptide consisting of 18 amino acids (Fig. 5).This ORF is preceded by a putative ribosome binding site withonly three bases (AAG) that are complementary to the 3' endof the 16S rRNA of P. aeruginosa (20). This potential leaderpeptide overlaps a potential stem-loop structure (AG = - 21.1kcal [ca. -88.3 kJ]/mol) that precedes six uridine residues,thus possessing features that characterize rho-independentterminators (48). To test the possibility that this structure isinvolved in regulation, a transcriptional fusion (pKA26) carry-ing only part of the leader sequence upstream of the potential

stem-loop structure was constructed. f-Galactosidase mea-surements with this fusion (Table 3) showed the absence ofpyrimidine control. Further, pyrimidine control was signifi-cantly reduced in pKA24, in which 10 bp was deleted from theright arm of the potential stem. Thus, the potential stem-loopstructure is essential for full pyrimidine control of carA. BothpKA24 and pKA26 retained arginine control (Table 3), indi-cating that this control is mediated by an independent mech-anism.To test for the potential leader peptide, a DNA fragment (nt

1 to 565; Fig. 2) was generated by PCR using oligonucleotideprimers specifically designed for this purpose. One of these

TABLE 3. Regulation of carA expression in lacZ fusions

(3-Galactosidase Fl-Galactosidase activity (SE)

Fusion Plasmid ~~~~~activity' (SE) Fold _______________FoldFusion PlasmiddeersineepsioExcess Limiting derepression Excess Limiting derepressionarginine arginine pyrimidines pyrimidines

TranscriptionalpKA9 47 (5) 99 (6) 2.1 69 (2) 628 (8) 9.1pKA26 125 (9) 313 (1) 2.5 203 (6) 228 (14) 1.1pKA24 202 (3) 566 (44) 2.8 322 (7) 988 (8) 3.1

TranslationalpKA31 13 (1) 235 (3) 18.1

"Auxotrophic derivatives of P. aeruginosa were grown under different growth conditions as described in Table 2. Exponentially growing cultures were harvested atan optical density of 0.5 at 600 nm. (3-Galactosidase activities were determined, and the specific activities (Miller units [361) were normalized relative to the copynumbers determined as described in Materials and Methods. Values shown are means of two independent cultures in each case.

HindIlldapB'

NruVSmaI

dapB'

lacZ reporter gene

Hindrl

dapB'L.-

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P. aerugrinosa

P. atutzeri

TGTGGCTGG-AGGGCAAGGAGAACGGCC-TGTACGAC-----ATGCAGGATG-TGCTCGGCCTACGCTGA----GGCGCTGCCGCACGTCGTCTTATTGGTGGACCGGAATGTCGCGAIII 11 1111 11 1111 1 I 11 IIIIIIIII IIIIII 11 11 111 11 111111 III 11 11TGTCACTCGCAGGAGATG-AGAATGATAATTGTCAACCAGTCATGCAGAATGCTGCTCGCGCTTTTTAGATCGTTGCGGTGGGATA--TCGTCT------ GGATAGAAAGGCCGGCT

+1

fMetThrLeuHlsGlyValIleProLeuPheTyrThr CysAlaThrSerGlnAla***

111111111 IIIIIIIIIIIIIIIII 111111 1111111 IIIIIIIIIIIIIIII 111111 1111111111111111111 11111TTCTGTATAGTGTTCCGTTT,AGTGTGTCCACTAAAGTT-GCGCAGAAATGAATCAAACAAAAAAGCGGGATGAC-CTTCAC-ACGTCATCCCGCTTTTTTACAATCTGCGTCTGCTC -

fMetThr PheThr

+1

III 111111

-CAGCCTTGATCTACd lC-TCTTGACTAAG-ro AlaLeuIleTyrGlySerSer LeuAsp***

fMetThrLys->

FIG. 5. Alignment of the nucleotide sequences of P. aeruginosa and P. stutzeni carA promoter regions. Identical nucleotides are labeled withshort vertical bars between two adjacent sequences. The convergent arrows indicate the positions of the proposed rho-independent terminators.The deduced amino acid sequences of the two different putative leader sequences are presented below the corresponding DNA sequences. Thetranscriptional initiation site is labeled +1 and marked with an arrow. The Shine-Dalgarno sequences for carA are boxed and precede theamino-terminal sequences and nucleotide sequences of the respective carA structural genes. Dashed lines indicate gaps that have been introducedinto the DNA sequence alignment to maximize sequence homology.

primers encoded a SmaI restriction site, which permitted anin-frame fusion of the promoterless reporter gene of pMC1403with amino acid 17 of the leader peptide. This fusion possessedP-galactosidase activity and was fully regulated by the avail-ability of pyrimidines (Table 3).

DISCUSSION

We previously reported that codons 5 to 8 of carA from P.aeruginosa were not translated because the four amino acidscorresponding to these codons were absent from the proteinsequence (47). Further, the sequence of the 5' terminus forcarA was confirmed by direct sequencing of chromosomalDNA and cDNA after amplification by PCR (47). In thepresent work, additional experiments with the previously re-

ported clone (47) raised concerns about whether it carried theauthentic carAB of P. aeruginosa. Determination of the DNAsequence for the 5' terminus of carA in six independent clonesof P. aeruginosa, reported here, established that the derivedamino acid sequence is identical to the protein sequence.Southern analysis showed that the previously reported clone ofcarAB (47) was not derived from P. aeruginosa PAO1. Rather,it was most likely derived from P. stutzeri, a conclusionsupported by the finding that the sequence reported here forcarA from P. stutzeri is identical to that derived from thepreviously reported clone (47). An erroneously labeled ormisidentified stock culture is the most likely basis for theearlier report.Comparisons among the derived amino acid sequences for

carA among P. aeruginosa, P. stutzeri, and E. coli (Fig. 6) showsignificant homology throughout the structural genes. How-ever, while the amino-terminal sequence for the small subunitof P. aeruginosa (carA) shows homology with those from E. coli(Fig. 6) and Salmonella typhimurium (not shown) (28), thisterminus in P. stutzeri surprisingly has four additional aminoacids. These residues apparently resulted from a 12-basetandem duplication, unique to carA from P. stutzeri. Thederived sequence for carA of P. aeruginosa shows identities of87 and 68% with those from P. stutzeri and E. coli, respectively.The sequence identity between the two pseudomonads and E.coli is more pronounced in the glutamine amidotransferasedomain (residues 204 to 380; Fig. 6) (46). The function of theless conserved amino-terminal portion of the small subunit hasnot been well established. However, studies with deletionmutants of E. coli (23) led to the conclusion that one of thefunctions of the amino-terminal third of the small subunit is tostabilize the complex with the large subunit. Consequently,

some of the variations among the different sequences of thesmall subunits may relate to the specific variations in subunitinteractions among the CPSase holoenzymes from each spe-cies. In this regard, it is of interest that CPSase from P.aeruginosa (3) does not undergo self-association into dimericand tetrameric forms as do CPSases from enteric bacteria (2,16).

Despite the excellent conservation of amino acid sequenceand function that is apparent between CPSase from P. aerugi-nosa, P. stutzeri, and the enteric bacteria, the carA gene from P.aeruginosa, along with flanking sequences of DNA, differssignificantly from the analogous sequences in E. coli and S.typhimurium in three major aspects. The first and most obviousdifference can be seen in the genetic arrangement of the carAand carB structural genes. The carA and carB genes from E.coli and S. typhimurium are characteristically found to becontiguous (16, 28), as is the case in P. stutzeri (this work). Suchgenetic association between the genes encoding the small andlarge subunits of CPSase is even more dramatically empha-sized in Bacillus subtilis, in which the reading frames of thecorresponding pyrimidine specific genes, pyrAA and pyrAB,actually overlap by 16 nt (38). This is not the case for P.aeruginosa PAO1, in which carA and carB are surprisinglyseparated by a 682-bp sequence that encompasses an ORF of216 amino acids. An extensive search in the GenBank libraryfailed to reveal any significant homology of this ORF with anyknown sequence, including the ORFs associated with thepyrimidine cluster of B. subtilis (38). Interestingly, analysis ofDNA encoding the ORF reported here revealed that its use ofcodons is consistent with that of a highly expressed gene in

pseudomonads (22), as is the GC content at the third baseposition (87%), which closely matches that of defined Pseudo-monas structural genes (4).The second major difference between P. aeruginosa and the

enteric bacteria is that the carAB operons of L. coli and S.typhimurium have been shown to utilize tandem promoters thatare differentially and independently regulated by pyrimidinesand arginine (28, 37). This is not the case for the carA-ORF-carB operon from P. aeruginosa. The S1 nuclease mappingexperiments reported here, using RNA extracted under differ-ent growth conditions, indicate that this operon is transcribedfrom a single promoter in P. aeruginosa and that the level ofthis transcript is controlled by both arginine and pyrimidines.Moreover, the results reported here on expression of variouscarA::lacZ fusions (Table 3) indicate that variation in argininelevels specifically modulates transcriptional initiation, while

fMetThrLys->TTCTGTAAACTA-CAGCTTTAGTGTGTCCACTAAAAGCAGCGCAG-CATGAATCG--AAAAAAAGCGGGATGACTCTTCACGGTGTCATCCCGCTTTTTTACAC-CTGCGCGACCAGTCAGGCTTGATTTAC..CTTCTTGACTAAG-

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2540 KWON ETAL.J.BTEIL

10 20

carA S. coil: L:Jo~]IX. S L V E

carA P. aeruglnoara:IL:KAG S' G

carA P. atuz.ri: T T P AI sl

30 40

VV... T ..............S..V..T .........D

.V....M.G....L...

50 60RQIVTLTY.HIHG

QQOVTLTYPHOGH:::T:4::N::T GT

K QGI VVTl;T::Y:::PHGT

90 100 110 1020 130 140

Y:::::L::::K: R H N I D N P D A A L .Al:iL: E

1.A D` T:" L:':X:'A G L'.. T R::j 1.::T,: A: .G: R K:.Ll.'E

V P, E.,A :,G 'N O', ..G ."C .'I "Li'A .G. A: 0::'A:T :: ::':E K A: ..L :E

150 160 170 iso 190 200 210 220

fl S F,1C. I D I riv.::GG L P E A K K E D :F,:::::L k:.G. ic

W, 1, -::X::VG.LA::A:xX.:: R s v W N: L.E sD:::SH G E:..: ':'F.'Y::.H:V .V:.A X'.D..Y R: W::VG.:V:i KLN ij:: .:T :P:F L's, -X 1: .:L R

L A :R-GCL..' .i.nsi.. .R: y L WW.L. E TD S:. It S Y:::H.4::WA T:,U Y-:::t.. V. KYIN 1:.L R.:WI`Wv P A

270

350 360 370 380

L I E Q Y K T A K (382)

R*(378)

V V (380)

FIG. 6. Alignment of the amino acid sequences of carA from E. coli, P. aeruginosa, and P. stutzeri. Shaded boxes denote identical residues in

two or more neig-hboring- sequences. Shaded circles indicate identical residues in any two nonadjacent sequences. Dashed lines indicate gaps that

have been introduced into the sequence alignment to maximize sequence homology. The double-shaded regions represent three regions of

similarity between carA products and defined glutamine amidotransferases (46). The numbers in parentheses indicate the total amino residues

found in each of the deduced amino acid sequences.

pyrimidine regulation is exerted subsequent to transcriptionalinitiation.

The third major difference between carA from P. aeruginosa

and the enterobacteriaceae is the presence in the carA leader

in P. aeruginosa of a region (Fig. 5) that possesses the

characteristics of a rho-independent terminator, namely, a

GC-rich hairpin with reasonable stability (AIG = -21.1 kcal/

mol) that precedes a run of six uridine residues (48). Experi-ments with a series of carA::lacZ transcriptional fusions sup-

port the hypothesis that the region containing the potentialattenuator is essential for full pyrimidine control. One such

fusion which precedes this region (pKA26; Fig. 4) was found to

express dramatically higher levels of enzyme, but at the

expense of any sensitivity to pyrimidine variations in the cell

(Table 3). Further, partial removal of the stem-loop structure

by a 10-base deletion (pKA24; Fig. 4) enhanced expression of

a carA::lacZ fusion, along with a 68% reduction in sensitivity to

fluctuations in pyrimidine concentrations (Table 3).The carA leader of P. aeruginosa also contains an ORF with

a coding capacity for an 18-amino-acid leader peptide that

terminates 16 bases upstream of carA and would be subject to

translational initiation from a weak ribosome binding site. This

ribosome binding site lies within the first stem of the proposed

rho-independent terminator, and any translation initiatingfrom it would likely be conditional upon its availability as a

function of mRNA secondary structure. If the RNA poly-merase were to be stalled at the continuous run of uridine

residues (because of a low concentration of pyrimidines in the

cell), translational initiation of the leader peptide would

remove the possibility of stem-loop formation. The in vivo

synthesis of the leader polypeptide from P. aeruginosa was

verified by the pyrimidine-sensitive expression of P3-galactosi-

dase from a plasmid-borne translational fusion of this se-

quence with lacZ (pKA31; Table 3 and Fig. 4).

Similar regulatory features are also found within the up-

stream region of carA from P. stutzeri (Fig. 5). Curiously, while

the length of the putative attenuating leader polypeptide

sequence is slightly greater in P. stutzeri (25 amino acids) as a

result of a couple of single-base insertions and deletions, the

position of the terminator loop relative to carA is similar to

that found in P. aeruginosa. Further, the sequence of the

stem-loop, the weak ribosome binding site, and the position of

the initiation codon within the stem-loop sequence are identi-

cal (Fig. 5).While all of the data reported here for carA of P. aeruginosa

are consistent with pyrimidine regulation by an attenuation

mechanism, the details of such a mechanism can be expected

to differ significantly from the well-characterized UTP-sensi-

tive attenuation of pyrBI of enteric bacteria (29, 30, 44).

Notably absent from the upstream regzulatory sequences in P.

aeruginosa is a transcriptional pause site, which is considered to

be critical for optimal regulation of the pyrBI operon in E. co/i

(29, 30, 44). Furthermore, the relative positions of the ribo-

some binding site and initiation codon of the leader peptideare also different. Indeed, it is possible that the proximity of the

ribosome binding site to the rho-independent terminator of the

carA transcript in P. aeruginosa obviates the requirement for a

transcriptional pause site. Moreover, if pyrimidine control of

carA in P. aeruginosa actually occurs through an attenuation-

type mechanism, this would be in marked contrast to that

observed for carAB in enteric bacteria, where the sequence

features that characterize attenuation mechanisms are absent

(16) and where evidence indicates the involvement of one or

70

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340

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more transacting elements in control of transcriptional initia-tion (13, 28, 31).

Pyrimidine-insensitive expression of a carA::lacZ fusion(pKA26; Table 3), which is still mediated by arginine availabil-ity in the cell, indicates that regulation of transcriptionalinitiation for this system in P. aeruginosa is exclusive to argininecontrol. However, sequences homologous to the ARG box,which characterizes operators for arg and car genes in entericbacteria (14, 32, 43), are absent in P. aeruginosa. It will be ofinterest, therefore, to determine whether the arginine controlof carA in P. aeruginosa occurs by a similar repressor-typemechanism. Of significance in this regard is the identification(24, 25) of a regulatory element that is involved in theregulation of argF (a gene involved in the arginine biosyntheticpathway) as well as certain genes of the major aerobic pathwayfor arginine catabolism in P. aeruginosa. It should be noted thatP. aeruginosa has a pumber of arginine catabolic pathways thatdo not occur in enteric bacteria; these pathways enable P.aeruginosa to utilize arginine as a source of carbon, nitrogen,and energy (24, 25), in contrast to enteric bacteria, whichutilize arginine only as a source of nitrogen (16). Accordingly,while arginine control of gene expression in enteric bacteria ismediated solely by ArgR, optimal metabolism of arginine bythe extensive network of pathways in P. aeruginosa (24) is likelyto be more complex and may employ additional regulatoryelements.

ACKNOWLEDGMENTS

We are very grateful to M. Casadaban, M. Farinha, D. Haas, B. W.Holloway, and J. L. Ingraham for gifts of strains and plasmids. Wethank J. L. Ingraham, J. Neuhard, and P. C. Tai for careful review ofthe manuscript.

This work was supported in part by research grant GM47926 fromthe National Institute of General Medical Sciences.

ADDENDUM IN PROOF

Tuohy et al. (J. Bacteriol. 176: p. 265-267) recently reportedstudies with a plasmid carrying carA (47), provided by ourlaboratory, and which is now believed to be derived from P.stutzeri (see above). Their determination of the N-terminalamino acid sequences for carA::lacZ translational fusionsshowed continuous translation, as also reported in this work.

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15. Crabeel, M., D. Charlier, G. Weyens, A. Feller, A. Pierard, and N.Glansdorff. 1980. Use of gene cloning to determine polarity of anoperon: genes carAB of Escherichia coli. J. Bacteriol. 143:921-925.

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