DNA Sequences and Characterization of Four Early Genes of the

Vol. 172, No. 2

DNA Sequences and Characterization of Four Early Genes of theTryptophan Pathway in Pseudomonas aeruginosatDAVID W. ESSAR,t* LEE EBERLY, CHUN-YA HAN, AND IRVING P. CRAWFORD§

Microbiology Department, University of Iowa, Iowa City, Iowa 52242

Received 22 August 1989/Accepted 23 October 1989

Two pairs of related but easily distinguishable genes for the two subunits of anthranilate synthase have beenidentified in Pseudomonas aeruginosa. These were cloned, sequenced, inactivated in vitro by insertion of an

antibiotic resistance cassette, and returned to the P. aeruginosa chromosome, replacing the wild-type gene.

Gene replacement implicated only one of the pairs in tryptophan biosynthesis. This report describes the cloningand sequencing of the tryptophan-related gene pair, designated trpE and trpG, and presents experimentsimplicating their gene products in tryptophan production. DNA sequence analysis as well as growth andenzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon thatalso includes trpD and trpC. Complementation of Trp auxotrophs by R-prime plasmids (T. Shinomiya, S.Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983) has shown that a large cluster of pyocin R2genes is flanked at one end by trpE and the other end by trpDC; the physical map that was obtained shows thedistance between trpE and trpDC to be about 25 kilobases. Our restriction map of the trpE and trpGDC regionsagrees with data presented by Shinomiya et al.

In 1978, R. W. Hedges obtained an anthranilate synthase-containing R-prime plasmid by mating Pseudomonas aerug-

inosa PAC174 (R68.44) with Escherichia coli W3110 tnaAtrpE5. We subsequently subcloned the anthranilate syn-

thase-encoding segment of this plasmid, determining thatboth the large (a)- and small (13)-subunit genes were present,and we reported the sequence of the latter along with a

portion of the former (7, 9). It is clear now that these twogenes do not give rise to the enzyme catalyzing the firstreaction of the tryptophan synthetic pathway but in factencode a second anthranilate synthase in P. aeruginosa.This resolves a paradox found in the initial report (7),namely, that the amin acid sequence deduced for the P.aeruginosa 13-subunit gene product differed considerablyfrom the Pseudomonas putida amino acid sequence obtainedearlier from the purified enzyme subunit (20). In this report,we describe the cloning and sequencing of the genes for a

second anthranilate synthase in P. aeruginosa, along withexperiments implicating these gene products in the synthesisof tryptophan. An accompanying report (11) details thecloning and sequencing of the homologous anthranilatesynthase gene pair from P. putida. In both organisms, thegenes are designated trpE (encoding the a subunit, or

component I) and trpG (encoding the subunit, componentII, or the glutamine amidotransferase subunit), in accordwith current usage (4, 5). Subsequently, we will presentevidence for the role of the first anthranilate synthase genescloned (7, 9) in secondary metabolism; we propose torename them phnA and phnB for phenazine synthesis (lla).

* Corresponding author.t This paper is dedicated to the memory of Robert W. Hedges.

Without his contributions from the start and later encouragement,this work would not have been accomplished.

t Present address: Root Disease and Biological Control ResearchUnit, Agricultural Research Service, U.S. Department of Agricul-ture, Washington State University, Pullman, WA 99164-6430.

§ Deceased 8 October 1989.

We began these studies by insertionally inactivating thefirst 1-subunit gene cloned (7) and recombining the in-activated gene into the chromosome of P. aeruginosa,showing that this did not result in a growth requirementfor tryptophan. Low-stringency southern hybridization withthis gene as a probe identified a second, cross-reacting13-subunit gene on the P. aeruginosa chromosome. Once thatgene had been cloned and shown by sequence analysis to beclosely related to the gene for the P. putida subunit, weused it to identify and clone the P. putida trpG gene. Boththese new 13-subunit genes proved to be at the 5' end of a

three-gene operon, trpGDC. We made use of the knownclose linkage of trpE to trpDC in P. putida (15) and per-formed a short chromosomal walk in that organism to findtrpE, which was recognized by its similarity in sequence toother anthranilate synthase a-subunit genes. Again employ-ing low-stringency interspecific DNA hybridization, we thenidentified and cloned the homologous P. aeruginosa trpEgene. The normal chromosomal trpE and trpG genes werereplaced with insertionally inactivated versions in both spe-cies, thereby confirming their participation in the tryptophanpathway.(Some of these experiments were presented at the 88th

Annual Meeting of the American Society for Microbiology,1988.)

MATERIALS AND METHODS

Bacterial strains and plasmids. The bacterial strains andplasmids used in this work are listed in Table 1.Media and antibiotics. E. coli and P. aeruginosa were

grown at 37°C unless specified. Complete medium was LB(27), and minimal medium was Vogel-Bonner medium E(27). Modified M9 minimal medium (28) was used in pheno-typic analysis of trpG mutants. Antibiotic concentrationswere as follows: for E. coli, 100 jig of ampicillin per ml,25 ,ug of chloramphenicol per ml, 50 ,ug of kanamycin perml, 25 ,ug of tetracycline per ml, 15 ,ug of mercuric chloride

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TABLE 1. Bacterial strains and plasmids

Strain or plasmid Genotype or phenotype' Source or reference

Pseudomonas aeruginosaPAO1PA04290PADE ElPADE E2PADE GiPADE G2

Escherichia coliJM109

S17-1

Wild typeaphA argF leu-10trpE Tcr exconjugant of PAO1 with pDE1533trpE Tcr exconjugant of PA04290 with pDE1533trpG Kmr exconjugant of PA04290 with pDE1410trpGDC Hgr exconjugant of PAO1 with pDE1574

recAl endAl gyrA96 thi hsdRJ7 supE44 rel A- A(lac-proAB)(F' traD36 proAB lacIqZ,&MJ5)

[RP4-2 (Tc::Mu) (Km::Tn7) Tra (IncP)] pro hsdR recA Tp' Smr

PlasmidspDE-KmpDE-TcpDE-HgpDE-HgXColEl::TnS-132pBR322pBR322::TnSpDG106pUC9pUC18pUC18-XhoIpUC18-BglIIpUCl9pSUP205pIA14p1391p1395p1472p1450p1460pDE1406pDE1408pDE1409pDE1410pDE1525pDE1528pDE1530pDE1533pDE1574

Bacteriophage (X b221 c1857)

Ampr KmrAmpr TcrAmpr HgrAmpr HgrTcrAmpr TcrAmpr Kmr

Hgr KmrAmprAmprAmprAmprAmprCmr Tcr MobAmpr P. aeruginosa phnBAmpr P. aeruginosa trpDAmpr P. aeruginosa trpGAmpr P. aeruginosa trpCAmpr P. putida trpEAmpr P. aeruginosa trpEAmpr P. aeruginosa trpGAmpr P. aeruginosa trpG-XhoIAmpr Kmr P. aeruginosa trpG-KmCmr Kmr Mob P. aeruginosa trpG-KmAmpr P. aeruginosa trpEAmpr P. aeruginosa trpE-BglllCmr Mob P. aeruginosa trpE-BglIllCmr Tcr Mob P. aeruginosa trpE-TcCmr Hgr Mob P. aeruginosa trpG-Hg

X b221 rex::TnS c1857

This studyThis studyThis studyThis studyM. Vasil (1)2This studyB. D. Gambill (13)3738This studyThis study3A. Puhler (34)7, 9This studyThis studyThis study11This studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis study

G. Stauffer (36)a Abbreviations: Amp, ampicillin; Cm, chloramphenicol; Hg, mercuric chloride; Km, kanamycin; Sm, streptomycin; Tc, tetracycline; Tp, trimethoprim.

per ml, and 100 ,ug of cefazolin per ml; for P. aeruginosaPA04290, 250 ,ug of kanamycin per ml; for P. aeruginosaPA01, 15 jxg of mercuric chloride per ml; and for P.aeruginosa PA04290 and PAO1, 100 ,ug of tetracycline perml. Solid media contained 1.5% agar (Difco Laboratories,Detroit, Mich.).

Transformation and conjugation. E. coli was transformedby a minor modification of the Mandel and Higa procedure(23). Bacterial matings were performed as described bySimon et al. (34). Donor and recipient cells were grown inliquid culture to the exponential phase (P. aeruginosa at42°C and E. coli at 37°C), mixed (1:1), and pelleted bycentrifugation. The mating mixture was carefully suspendedin 200 ,lI of LB and spread onto a nitrocellulose filter (13-mmdiameter, 0.45-,um pore size) on a prewarmed LB agar plate.Matings were incubated for 16 to 20 h at 37°C, and then thecells were suspended and diluted in LB and spread ontoselective media.

Cell extracts and enzyme assays. Preparation of cell ex-tracts by sonication and assays for enzyme activity andprotein concentration were as described previously (9).DNA isolation. High-molecular-weight genomic DNA was

prepared as described previously (30). Large amounts of E.coli plasmid DNA were isolated by alkaline lysis and purifiedin CsCl-ethidium bromide gradients (24). Small amounts ofrecombinant plasmid DNA were isolated by the alkalinelysis method (24). Small amounts of genomic DNA for use inSouthern hybridization analysis were isolated from P. aerug-inosa by a Sarkosyl-pronase lysis procedure (10) followed byphenol extraction and ethanol precipitation.

Southern hybridization. Restriction fragments to be usedas probes were electroeluted from 5% polyacrylamide gels.After ethanol precipitation, the DNA fragments were 32plabeled by nick translation (29). Restriction endonucleasedigests of P. aeruginosa DNA were separated on horizontal0.8% agarose gels. After electrophoresis, DNA was dena-

B. HollowayH. MatsumotoThis studyThis studyThis studyThis study

38

A. Puhler (34)

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PSEUDOMONAS AERUGINOSA trpE AND trpGDC 855

Z O<>q , tured and neutralized as described previously (35) and thenw> 5 t j i transferred to Gene Screen Plus hybridization transfer mem-

Q fr branes (Dupont, NEN Research Products, Boston, Mass.)._ I ,, ~ Hybridizations were done as described previously (35). Final

Z ,* I high-stringency washes in triplicate were in 0.5x SSC (lxI j t . s SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1%

° 3 B o- | 1 l z X ~~~~~~sodium dodecyl sulfate at 65°C for 15 min. Low-stringencywashes were done in duplicate with 5x SSC-0.1% sodiumdodecyl sulfate at room temperature. Autoradiography was

tcn o I I I I I-Z I performed at -70°C with Kodak XAR-5 film and CronexLightning-Plus intensifying screens.

I) n X i DNA sequencing. Fragments were usually labeled with 32pc£ c2, _ - ,by 3' fill-in labeling with the large fragment ofE. coli DNA

-qn = O t r o " polymerase I and the appropriate (x labeled radioactiveddeoxynucleotide triphosphate (26). Occasionally, the 5' ends

0 (> 5 oIwere labeled with T4 polynucleotide kinase and [y- P]ATP¢;30° and then reduced by restriction enzyme cleavage to smaller

°Q < cL ,rfragments labeled at one end (26). Labeled fragments elec-. p, = O t , I troeluted from polyacrylamide gels were sequenced by the

T. ,j, r l l l l Maxam and Gilbert procedure (26); reaction mixtures wereI x 0 | | g | 4 l l resolved by 8% urea-polyacrylamide gel electrophoresis by

(O ,.Othe method of Sanger and Coulson (31), except that the gelsa-oW Z contained 25% formamide to minimize compressions. Se-

n, ~ ~ Iquence data were analyzed with the aid of the PCS computerI. > program (21).

I = X - . Construction of BglII-Tcr cassette. A 2.7-kilobase (kb) TcrC - I cartridge containing the tetAR region from TnlO was isolated-^ O' W from TnS-132 by digesting ColEl::TnS-132 (1) with BglII.>=O X | rJ This cartridge was inserted into pUC18-BglII (pUC18 with a

0 I BglII adaptor inserted into the EcoRI site) to create a0x l_ | high-copy-number, nontransposable source of the BglII-TCr

4 5 5 *i;_t cassette; this plasmid was designated pDE-Tc.I o 5 B _ @ Construction ofXIoKr cassette. Isolation of pBR322::

r I ea _ TnS was accomplished essentially as described by Stauffer etI+ I n t , " al. (36). E. coli cells carrying pBR322 were transfected with

r CL p OAX::Tn5 bacteriophage and spread on LB plates containingkanamycin and ampicillin. Kmr Ampr cells were harvested

CDUQ- from the plates, and plasmid DNA was prepared by alkaline

t c X I l l l lysis (24). This DNA was used to transform E. coli JM109cells with selection for Kmr pmpr; one such transformant

_-0I was designated pBR322::TnS. pBR322::Tn5 was digestedtz I with XhoI, and the 2.5-kb Kr fragment was inserted into

CDp,O z the XhoI site of pUC18-XhoI (pUC18 with an XhoI adaptor0 o rI inserted into the EcoRI site); this plasmid, a high-copy-5:~ce g. 1number, nontransposable source of the AhoI-KMr cassette,

_ I. 0 was designated pDE-Km.I n o n o o = Construction ofXoI-Hgr cassette. A 5.2-kb Hgr cartridgeI 9 4 containing the mer operon of IncFII plasmid NR1 was

tZ cB isolated from pDG106 (13) by digestion with BamHI andPstI. This cartridge was inserted into pUC18-XhoI digested

-!t I _* , with BamHI and PstI to create pDE-Hg. pDE-Hg wasQ & x +! n+ ^ > 1 1; subsequently digested with HindIII and treated with DNA

I I X | . X _ ,,, polymerase I Klenow fragment and the appropriate deoxy-ribonucleotide triphosphates, and an XhoI linker was in-

U, =n l lserted; this plasmid, a high-copy-number source of the0 t| = XhtoI-Hg' cassette, was designated pDE-HgX.Io I = || s , | " Enzymes and chemicals. All enzymatic reactions were

=On t 1 t Jr ffi o . performed essentially as described by Maniatis et al. (24)=° I or as recommended by the supplier (New England BioLabs,BO:¢ I Inc., Beverly, Mass.). Restriction endonucleases, DNA

> > t IT + I X polymerase I, DNA polymerase I Klenow fragment, T4= g J |.IDNA ligase, and T4 polynucleotide kinase were purchasedCD <°. l l _"cosine were purchased from Sigma Chemical Co. (St. Louis,

Mo.). Radiolabeled deoxynucleotide triphosphates were ob-'r:~° OrQ tained from Amersham Corp. (Arlington Heights, Ill.). Link-

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856 ESSAR ET AL.

30 60 90CTCAGCAGCATCATCACCTCCCTCATCMGGCATTCGCCCGTTGGCGCTGGCGCGCCTGACTTCTTCTTCGCCGGCCTGGCCGGTCACGT

120 150 180CTCCCTGCGTACCCCGTTTTCCACAGGCATAATGCCGACGATCCACTCCCCCTTCCTCCGCGAGCGCGCCGGGAAACGGGTGTGGCGAAA

210 240 270GCCGGGTGATGCGATTCGCATAGGAATGAAATCAAGAGGTTACAGCCAGCATGCTGCTGATGATCGATAACTACGACTCCTTCACCTACA

N L L M I D N Y D S F T Y

300 330 360ACCTGGTGCAGTACTTCGGCGAGCTCAAGGCCGAGGTCMGGTGGTGCGTAACGACGMCTGAGCGTGGMCAGATCGAGGCCCTGGCGCN L V Q Y F G E L K A E V K V V R N D E L S V E Q I E A L A

390 420 450CCGAGCGCATCGTCCTTTCCCCCGGTCCCTGCACCCCCAACGAGGCCGGCGTCTCGCTGGCCGTTATCGAGCGCTTCGCCGGCAAGCTGCP E R I V L S P G P C T P N E A G V S L A V I E R F A G K L

480 510 540CGCTGCTCGGTGTCTGCCTTGGCCATCAGAGCATCGGCCAGGCCTTCGGCGGCGMGTGGTGCGGGCGCGGCAGGTGATGCACGGCMGAP L L G V C L G H Q S I G Q A F G G E V V R A R Q V M H G K

570 600 630CCAGCCCGATCCACCACAAGGACCTCGGCGTGTTCGCCGGCCTGGCCAATCCCCTGACGGTGACGCGCTACCACTCGCTGGTGGTGMGCT S P I H H K D L G V F A G L A N P L T V T R Y H S L V V K

660 690 720GAGAGAGCCTGCCGGAGTGCCTGGAGGTCACCGCCTGGACCCAACATGCCGACGGATCGCTCGACGAGATCATGGGCGTACGCCACAAGAR E S L P E C L E V T A W T Q H A D G S L D E I M G V RH R

750 780 810CCCTGAATGTCGAGGGCGTGCAGTTCCATCCCGAGTCCGTCCTCACCGAGCAGGGCCACGAGTTGCTGGCCMCTTCCTCCGCCAGCAGGT L N V E G V Q F H P E S V L T E Q G H E L L A N F L R Q Q

840 870 900GCGGCGTGCGTGGGGAGGGTAACTGAGATGGATATCAAGGGAGCCCTCAATCGCATCGTCAACCAGCTCGACCTGACCACCGAGGAMTGG G V R G E G N M D I K G A L N R I V N Q L D L T T E E H

930 960 990CAGGCGGTCATGCGCCAGATCATGACCGGGCAGTGCACCGACGCGCAGATCGGCGCCTTCCTGATGGGCATGCGGATGMGAGCGAAACCQ A V M R Q I H T G Q C T D A Q I G A F L M G M R n K S E T

1020 1050 1080ATCGACGAGATCGTCGGCGCGGTGGCAGTGATGCGCGAACTGGCCGACGGCGTGCAGTTGCCTACGCTGAAGCATGTGGTCGACGTGGTCI D E I V G A V A V M R E L A D G V Q L P T L K H V V D V V

1110 1140 1170GGCACCGGCGGCGATGGCGCGAACATCTTCAACGTGTCCTCGGCGGCGTCCTTCGTGGTCGCCGCCGCTGGCGGCAAGGTCGCCAAACACG T G G D G A N I F N V S S A A S F V V A A A G GK V A K H

1200 1230 1260GGTAACCGCGCGGTCTCCGGCMGAGCGGCAGCOCCGACTTGCTGGMGCCGCCGGCATCTACCTGGAGCTGACCTCCGAACAGGTGGCGG N R A V S G K S G S A D L L E A A G I Y L E L T S E Q V A

1290 1320 1350CGTTGCATCGACACCGTCGGCGTCGGGTTCATGTTCGCCCAGGTCCACCACMGGCGATGMGTACGCCGCCGGTCCGCGCCGCGAGCTGR C I D T V G V G F M F A Q V H H K A H K Y A A G P R R E L

1380 1410 1440GGCTTGCGGACTCTGTTCMCATGCTTGGCCCACTGACCMCCCGGCGGGAGTCAGGCACCAGGTGGTCGGGGTGTTCACCCAGGMCTGG L R T L F N M L G P L T N P A G V R H Q V V G V F T Q E L

1470 1500 1530TGCMGCCGCTGGCTGMGTGCTCMGCGTCTCGGCAGCGAGCATGTGCTGGTGGTGCATTCGCGCGACGGGCTGGACGAGTTCAGTCTGC K P L A E V L K R L G S E H V L V V H S R D G L D E F S L

1560 1590 1620GCCGCGGCGACCCACATTGCCGAGTTGAAGGACGGCGAGGTACGCGAGTACGMGTGCGTCCCGAGGACTTCGGGATCMGAGCCAGACCA A A T H I A E L K D G E V R E Y E V R P E D F G I K S Q T

1650 1680 1710CTGATGGGGCTGGAGGTCGACAGTCCGCAGGCCTCGCTGGAACTGATCCGCGACGCTTTGGGGCGGCGCAAGACCGAGGCTGGGCAGAAGL M G L E V D S P Q A S L E L I RD A L G R R K T E A G Q K

1740 1770 1800GCCGCCGAGCTGATCGTGATGAATGCCGGCCCGGCACTGTACGCTGCCGATCTGGCGACCAGCCTGCACGAGGGCATTCAACTGGCCCACA A E L I V M N A G P A L Y A A D L A T S L H E G I Q L A H

1830 1860 1890GATGCCCTGCACACCGGGCTGGCACGGGAGMGATGGACGAACTGGTGGCCTTCACCGCCGTTTACAGAGAGGAGAACGCACAGTGAGTGD A L H T G L A R E K M D E L V A F T A V Y R E E N A Q H:; S

1920 1950 1980TGCCGACGGTTCTGCAGMGATCCTCGCCCGCAAGGCCGAGGAGGTCGCCQAGCGCCGTGCGCGCGTCAACCTGGCAGAGGTCGAGCGGCV P T V L Q K I L A R K A E E V A E R R A R V N L A E V E R

2010 2040 2070TGGCGCGTAGCGCCGATGCGCCGCGCGGCTTCGCCAATGCCCTGCTGGAGCGGGCCMGCGCAAGGAGCCGGCAGTGATCGCCGAGATCAL A R S A D A P R G F A N A L L E R A K R K E P A V I A E I

2100 2130 2160AGAAGGCATCGCCGAGCAAGGGCGTGCTGCGCGAACACTTCGTCCCGGCGGAGATCGCCCGCAGCTACGAGGCGGGTGGCGCGGCGTGCCK K A S P S K G V L R E H F V P A E I A R S Y E A G C A A C

2190 2220 2250TGTCGGTGCTCACCGACGTGGACTTCTTCCAGGGCGCCGATGCCTATCTGMGGMGCGCGGGCCGCCTGTGCGCTGCCGGTGATCCGCAL S V L T D V D F F Q G A D A Y L K E A R A A C A L P V I R

2280 2310 2340AGGACTTCATGATCGATCCGTACCAGATCGTCGAGGCGCGGGCGATCGGTGCCGACTGCATCCTGCTGATCGTCTCGGCGCTGGACGACGK D F M I D P Y Q I V E A R A I G A D C I L L I V S A L D D

2370 2400 2430TGCTGATGGCCGAACTGGCGGCGACTGCCMGTCGGTCGGTCTCGACGTACTGGTCGMGTGCATGACGGCACCGAGCTGGMCGTGCACV L M A E L A A T A K S V G L D V L V E V H D G T E L E R A

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2460 2490 2520TGAAGACCCTGGACACGCCGTTGGTGGGCATCAACAACCGCAACCTGCACACCTTCGAGGTGAGCCTGGAAACCACCCTCGACCTGTTGCL K T L D T P L V G I N N R N L H T F E V S L E T T L D L L

2550 2580 2610CGGAAATTCCCCGCGACCGCCTGGTGGTCACCGAGAGCGGTATTCTCAACCGGGCCGACGTGGAGCTGATGGMGTCAGCGAGGTCTACGP E I P R D R L V V T E S G I L N R A D V E L M E V S E V Y

2640 2670 2700CCTTCCTGGTTGGCGAGGCGTTCATGCGGGCCGACGATCCTGGCCTCGAGCTGMGCGCCTGTTCTTCCAGGAGCGTGGTGCTGTGGTGCA F L V G E A F M R A D D P G L E L K R L F F Q E R G A V V

2730 2760 2790TGGGCGCCGATCCTGACTGATCCGCGCTGTCGAACCCCATGAAAAAGGCCGGTTGCGACCGGCCTTTTTCATGGGTGCTGTTCAGCGGGTL G A D P D*

2807GCCGAAGACCACCATGG

FIG. 2. DNA sequence and deduced amino acid sequence of P. aeruginosa trpGDC operon and flanking DNA. Start and stop codons andShine-Dalgarno sequences are underlined, as is an inverted repeat at the end of the operon.

ers and adaptors were obtained from the University of IowaRecombinant DNA Core Facility or New England Bio-Labs.

RESULTS

Cloning P. aeruginosa thpG, trpD, and tipC. Lack of a

requirement for tryptophan in recombinants bearing an

insertionally inactivated form of the first P-subunit gene

cloned (7, 9) led us to search for the real P. aeruginosa trpGgene. We purified and used as a probe a 896-base-pair (bp)BamHI-KpnI fragment containing P. aeruginosa phnB (7)obtained from plasmid pIAl4 and labeled with 32P by nicktranslation. This probe was hybridized to P. aeruginosaPAG1 fragments presumably containing trpG.We first cloned a weakly hybridizing 2.1-kb ClaI fragment

from P. aeruginosa after electroelution from agarose andligation to pUC19 DNA that had been digested with AccI anddephosphorylated. The ligation mixture was transformedinto E. coli JM109, and the desired plasmid, designatedp1391, was identified by colony hybridization (24). WhenDNA sequencing showed the insert in p1391 to lack a portionof the trpG gene at the 5' end, a second, overlappingchromosomal fragment was isolated from P. aeruginosachromosomal DNA by using the EcoRV-PstI fragment at the5' end of the p1391 insert as a probe. This identified a 3.0-kbSalI fragment which was isolated and cloned into SalI-digested pUC19 in the manner described for p1391. Thisplasmid was designated p1395; its DNA sequence confirmedthat it contained the P. aeruginosa trpG gene and more than2 kb of DNA ahead of the gene.DNA sequencing of p1395 and p1391 disclosed another

open reading frame immediately downstream from trpG.

When the nucleotide sequence of this open reading framewas compared with other known trp genes, we found 60%identity with the trpD gene of Acinetobacter calcoaceticusand weaker but still significant homology to trpD genes fromother species (6). From earlier studies, it was known thattrpD and trpC are tightly linked in P. aeruginosa (17);therefore, we suspected that trpC might also be present on

p1391. The complete DNA sequence of the p1391 insertconfirmed that it contained the entire P. aeruginosa trpDgene preceded by the 3' end of trpG and followed by the 5'end of trpC.Because p1391 lacks a large portion of the trpC gene, a

third overlapping chromosomal fragment was isolated fromP. aeruginosa chromosomal DNA by using the 660-bpNarI-SacII fragment at the 3' end of the p1391 insert as a

probe. This identified a 3.0-kb Sall fragment; this fragmentwas isolated and cloned into Sall-digested pUC19 in themanner previously described for p1391 and p1395. Thisplasmid was designated p1472; its DNA sequence confirmedthat it contained the 3' end of the trpD gene, the entire P.aeruginosa trpC gene, and almost 2 kb of DNA beyond theend of trpC.The sequencing strategy for the relevant portions of

p1391, p1395, and p1472 is shown in Fig. 1. All restrictionsites were bridged, and complementary sequences were

obtained from both strands.Sequence of P. aeruginosa trpGDC. Figure 2 presents the

DNA sequence of the trpG, trpD, and trpC genes of P.aeruginosa as well as the deduced amino acid sequences ofthe three gene products. All three coding sequences haveconventional ribosome-binding sequences (32) 8 to 11 bpahead of the suggested ATG or GTG start codon. All threeutilize the TGA stop codon. At the trpGD interface a single

TABLE 2. Growth characteristics of P. aeruginosa PA01, PA04290, and their derived mutants

Growth on: PAO1 PA04290 PADE G1 PADE G2 PADE El PADE E2

M9 medium with ammonium content ofa:1 mM + + - - - -50 mM + + +

Vogel-Bonner medium supplemented witha:None + + +AAb + + + _ + +

TRPb + + + + + +

a Cultures of PA04290 and its mutants PADE G1 and PADE E2 were supplemented with arginine (1 ,ug/ml) and leucine (10 ,ug/ml).b AA, Anthranilate; TRP, L-tryptophan.

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AA

CL ~~~~~~~~~~~~~~.0h.I -

2~~~~~~~~~~~~~~~~~~~~~~~~~r

(,U

- 4,~~~~~~~~~~~~~~~~~~~~~~~~-

I~~~~~~~~~~

t~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~t

TABLE 3. Enzyme assays of P. aeruginosa PA01, PA04290,and their derived mutants

P. aeruginosa Enzymatic activity' with: Phosphoribosylcelexrc transferasecell extract Ammonia Glutamine activity

PA01 0.014 0.058 0.400PA04290 0.050 0.046 0.270PADE Gl 1.27 0 2.45PADE G2 0.55 0 0PADE El 0 0 1.07PADE E2 0 0 5.69PADE E1 + PADE G2 NTb 0.069 NTPADE E2 + PADE G1 NT 0.37 NT

' Enzymatic activities were measured with cell extracts prepared fromtryptophan-starved cultures (3). Extracts were assayed fluorometrically foranthranilate synthesis with ammonia or glutamine as the nitrogen source. Thesecond enzyme of the pathway, phosphoribosyl transferase, was also assayedas a control. Enzymatic activities are shown in units per milligram of protein.One unit is defined as the production or utilization of 1 nmol of product orsubstrate per min at 21°C.

b NT, Not tested.

G separates the stop and start codons, whereas at the trpDCinterface the genes overlap slightly, with the last two basesof the trpC start codon forming the first two bases of the trpDstop codon. The designated stop and start codons wereassigned based on alignment with sequences of homologousproteins (or domains of proteins) performing the same reac-tions in other bacteria and fungi (6).The deduced amino acid sequence of the P. aeruginosa

trpG gene showed 83% identity with the amino acid se-quence of the small subunit of P. putida anthranilate syn-thase (20), with no gaps required for alignment. In contrast,it showed only 42% identity with the P. aeruginosa phnBgene product reported earlier (7), and three gaps had to beinserted for alignment of those two proteins. Comparison ofthe trpD and trpC sequences of Fig. 2 with those ofP. putidaand other more distantly related organisms is deferred to theaccompanying report (11).

Cloning P. aeruginosa IrpE. From transduction studies, theP. putida trpE gene was known to be very closely linked totrpDC (15); in contrast, in P. aeruginosa they are separatedby at least 25 kb (33). A chromosomal walk in P. putidalocated the trpE gene 2.2 kb upstream from the trpGDCcluster (11). The P. putida trpE gene was cloned andsequenced; it was found to encode a protein similar to otheranthranilate synthase large subunits (11). We subsequentlypurified a 770-bp XhoI-BstEII fragment located near the 5'end of P. putida trpE, using it as a probe to identify P.aeruginosa trpE. The probe, obtained from plasmid p1450(11), hybridized readily under high stringency with P. aerug-inosa PAQ1 chromosomal DNA that had been digested witha variety of restriction enzymes.We cloned a 3.8-kb tbpE-containing HindIII-BglII frag-

ment from the P. aeruginosa chromosome by electroelutionfrom agarose and ligation into HindIII-BamHI-digestedpUC19. The ligation mixture was transformed into E. coliJM109, and the desired plasmid, termed p1460, was identi-fied by colony hybridization (24). The DNA sequence ofp1460 confirmed that its insert contained the entire codingregion of P. aeruginosa trpE.

Relevant portions of p1460 were sequenced by the strat-egy shown in Fig. 3. All restriction sites were bridged,and complementary sequences were obtained from bothstrands.

Sequence of P. aeruginosa trpE. Figure 4 shows the se-

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30 60 90CTGAGATAGTGTTGCCGGACGACTCCCTTTCCCCCTCCGACCAGCGAGATCAAGCCGTGGCGGTCAGCAMCTCTGGATGAAGGTCATCA

120 150 180AGGCCCTGGCGCGTTGGCGCTGGCGCGCCTGACATCATCCTCCGCCGGATAGCCGGCGCGCGTTTGCACCCTGTTGACCGTTCTCTCCTC

210 240 270CGATGAGGCTGATCATGMTCGCGAAGAATTCCTGCGGCTGGCCGCCGATGGCTACAACCGCATCCCGCTGTCCTTCGAGACCCTTGCCG

M N R E E F L R L A A D G Y N R I P L S F E T L A

300 330 360ACTTCGACACGCCGCTGTCGATCTACCTGAAGCTGGCCGACGCGCCGAACTCCTACCTGCTGGAGTCGGTGCAGGGCGGCGAGAAATGGGD F D T P L S I Y L K L A D A P N S Y L L E S V Q G G E K W

390 420 450GGCGCTATTCGATCATCGGCCTGCCGTGCCGCACGGTGCTGCGGGTCTACGACCATCAAGTGCGGATCAGCATCGATGGCGTGGAAACCGG R Y S I I G L P C R T V L R V Y D H Q V R I S I D G V E T

480 510 540AGCGCTTCGATTGCGCCGACCCGTTGGCTTTCGTCGAGGAGTTCAAGGCGCGCTACCAGGTGCCCACCGTGCCCGGCTTGCCACGTTTCGE R F D C A D P L A F V E E F K A R Y Q V P T V P G L P R F

570 600 630ATGGCGGCCTGGTCGGCTACTTCGGTTACGACTGCGTGCGCTACGTGGAAAAACGCCTGGCCACCTGTCCGAACCCGGACCCGCTGGGCAD G G L V G Y F G Y D C V R Y V E K R L A T C P N P D P L G

660 690 720ACCCGGATATCCTGTTGATGGTGTCCGATGCCGTAGTGGTATTCGACAACCTGGCCGGGAAGATCCACGCCATCGTCCTCGCCGATCCCTN P D I L L M V S D A V V V F D N L A G K I H A I V L A D P

750 780 810CCGAGGAAAATGCCTACGAGCGCGGCCAGGCACGTCTGGAGGAGCTGCTGGAGCGTCTACGCCAGCCGATCACCCCGCGTCGCGGCCTCGS E E N A Y E R G Q A R L E E L L E R L R Q P I T P R R G L

840 870 900ACCTCGAGGCGGCCCAGGGTCGTGAGCCGGCGTTTCGTGCCAGCTTCACCCGCGAGGACTATGAAAACGCGGTAGGAAGGATCAAGGACTD L E A A Q G R E P A F R A S F T R E D Y E N A V G R I K D

930 960 990ACATCCTGGCCGGCGACTGCATGCAGGTGGTGCCGTCGCAGCGCATGTCCATCGAATTCAAGGCGGCGCCCATCGACCTGTACCGCGCGCY I L A G D C M Q V V P S Q R M S I E F K A A P I D L Y R A

1020 1050 1080TGCGTTGTTTCAATCCGACGCCCTACATGTACTTCTTCAACTTCGGCGACTTCCATGTCGTGGGCAGCTCGCCGGAGGTGCTGGTACGGGL R C F N P T P Y M Y F F N F G D F H V V G S S P E V L V R

1110 1140 1170TCGAGGATGGCCTGGTGACGGTGCGCCCGATCGCCGGTACCCGTCCGCGCGGGATCAACGAAGAGGCCGACCTGGCACTGGAGCAGGATCV E D G L V T V R P I A G T R P R G I N E E A D L A L E Q D

1200 1230 1260TGCTGTCGGACGCCAAGGAGATCGCCGAGCACCTGATGCTGATCGACCTGGGGCGCAACGACGTGGGGCGGGTGTCCGATATCGGCGCGGL L S D A K E I A E H L M L I D L G R N D V G R V S D I G A

1290 1320 1350TGAAGGTCACCGAAAAAATGGTGATCGAACGTTACTCCAACGTCATGCACATCGTGTCCAACGTCACCGGGCMTTGCGCGAGGGGCTCAV K V T E K M V I E R Y S N V M H I V S N V T G Q L R E G L

1380 1410 1440GCGCGATGGACGCGCTGCGGGCGATTCTGCCGGCGGGCACTCTATCCGGCGCGCCGAAGATCCGCGCCATGGAGATCATCGACGAGCTGGS A M D A L R A I L P A G T L S G A P K I R A M E I I D E L

1470 1500 1530AGCCGGTCAAGCGTGGAGTCTACGGCGGCGCGGTCGGCTACCTGGCATGGMCGGCMCATGGACACCGCCATTGCCATCCGCACCGCGGE P V K R G V Y G G A V G Y L A W N G N M D T A I A I R T A

1560 1590 1620TGATCAAGAACGGTGAACTCCACGTGCAGGCCGGCGGCGGTATCGTTGCCGACTCGGTGCCCGCGCTGGAGTGGAAGAAACCATCAACAV I K N G E L H V Q A G G G I V A D S V P A L E W E E T I N

1650 1680 1710AGCGCCGGGCGATGTTCCGCGCCGTGGCGCTGGCCGAGCAGAGCGTCGAATAAGACGGCGCGAGGCGGAAGAAAAGAAGGCGGATGGCGTK R R A M F R A V A L A E Q S V E *

1740 1770 1800TTGCGTCATCCGCCTTTTTTGTGCCCGGCGTGGACCCACGCATTGGCTTCCGCCCTACGCGGCACCGCTTTCGTAGGGCGMTATCGCGG

1830 1860 1890CGCGATATCCGCCAGGGAGCAGGACGCCGAACCCGCCTCAAGGTGAAGTGATCGATATGTCCGGTTGGCGGATGACGCCTTCGGCTTACT

1920 1950 1980TGCCCACGCAGAAGGCGCTTTCGTAGGGGGGACATCACCACGCGATATCCGCCAGGCGGAGCATGGTGTCGCTCCCTACGATGCTGGCM

1995CGGCGGGCCGCTCAG

FIG. 4. DNA sequence and deduced amino acid sequence of P. aeruginosa trpE and flanking DNA. Start and stop codons andShine-Dalgarno sequences are underlined, as is an inverted repeat at the end of the gene.

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A 10 20E.c.TrpE M Q T Q K P T L E L LT C E G A Y R D N P T A L FB.l.TrpE M S T N P H V F S L D V R Y H E D A S A L FP.a.TrpE M N R E E F L R L A A D G Y N R IP L S F E T L A D F D T P LB.s.TrpE M N F Q S N I S A F L E D S L S H H T IP I V E T F T V D T L T P IS.c.TrpE* K Q L Q Q Q N D D S S I N M Y P V Y A Y L P S L D L T P H V A Y L K LE.c.PabB MKTLSPAVITLLWRQDAAEF

40 50C G D R P A T L L L E SA D I D S K D D L K S L LG T T A D D A A L L E S A D I T T E N G I S S L AL A D A P N S Y L L E S V Q G G E K W G R Y S I IK L D R E I T YL LE S K D D T S T W S R Y S F IN P D R K E S FL LE S- A K T N N E D R Y S F IL S H L P W A ML L H S G Y A D H P Y S R F D I V

60 70L V D S A L R I T A L G D T V T I Q A LV L K S S V R I T C T G N T V V T Q P LG L P C R - - - - - - - - - - - - - - -

G L N P - - - - - - - - - - - - - - - -

G I S P - - - - - - - - - - - - - - - -

V A E P - - - - - - - - - - - - - - - -

80 90 100 110 120S G N G E A L L A L L D N A L P A G V E S E Q S P N C R V L R F P P V S P L L D E D A R LT D S G R A V V A R L T Q Q L G Q Y N T A E N - - - - - - T F S F P A S D A V D E R E R L- - - - - - - - - - - - - - - - - - - - - T V L R V Y D H Q V R I S I D G V E T E R F D C- - - - - - - - - - - - - - - - - - - - - F L T I K E E Q G R F S A A D Q D S K S L Y T G- - - - - - - - - - - - - - - - - - - - - R K T I K T G P T E G I E T D - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - I C T L T T F G K E T V V S E S E K R T T T T D

130 140 150 160- -C S L S V F D A F R L L Q N L L N V P K E E R E A M F F S G L F S Y L V A G F D L

T A P S T I E V L R K L Q F - - E S G Y S D A S L P L L M G G F A F D F L E T F E T LA D P L A F V E E F K A R Y Q V P - T V P G L P R F D G G L V G Y F G Y D C V R Y V E K RN E L K E V L N W M N T T Y K I K T P E L G I P - F V G G A V G Y L S YDH I P L I E P S- - P L E I L E K E M S T F K V A E N V P G L P K L S G G A I G Y I S Y DC V R Y F E P KD - P L Q V L Q Q V L D R A D I R P T H N E D L P F Q G G A L G L F G Y IL G R R F E S L

170 180 190 200 210P Q L S A E N N C - - - P D F C F Y L A E T L M V I D H Q K K S T R I Q A S L F A P N E EP A V E E S V N T Y - - P D Y Q F V L A E I V L D I N H Q D Q T A K L T G V S N A P G E LL A T C P N P D P L G N P D I L L M V S D A V V V F D N L A G K I H A I V L A D P S E E NV P S H T K E T D M - E K C M L F V C R T L I A Y D H E T K N V H F I Q Y A R L T G E ET R R P L K D V L R -L P E A Y L M L C D T I I A F D N V F Q R F Q I I H N I N T N E T SP E I A E Q D I V L P D M A V G I Y D W A L I V D H Q R H T V S L L S H N D V N A R R

220 230 240 250E - - - - - - - - - - - - - - K Q R L T A R L N E L R Q Q L T E A A P P L P V V S V P H ME - - - - - - - - - - - - - A E L N K L S L L I D A A L P A T E H A Y Q T T P H D G D T LA - - - - - - - - - Y E R G Q A R L E E L L E R L R Q P I T P R R G L D L E A A Q G R E PT K N E K M D V F H Q N H L E L Q N L I E K M M D Q K N I K E L F L S A D S Y K T P S F EL E E G Y Q A - - - - - - - A A Q I I T D I V S K L D R R F L A N T I P E Q P P I K P N QA - - - - - - - - - - - - - - - - - - - - - - - - - - - W L E S Q Q F S P Q E D F T L T S

FIG. 5. (A) Alignment of TrpE proteins of E. coli (E.c.), Brevibacterium lactofermentum (B.l.), P. aeruginosa (P.a.), Bacillus subtilis(B.s.), and S. cerevisiae (S.c.) and PabB protein of E. coli. Residues identical in all sequences are boxed. Hyphens indicate gaps introducedto increase similarity. (B) Alignment of TrpG proteins of E. coli, B. lactofermentum, P. aeruginosa, A. calcoaceticus (A.c.), B. subtilis, andS. cerevisiae and PabA protein of E. coli. Residues identical in all sequences are boxed. Hyphens indicate gaps introduced to increasesimilarity.

quence of P. aeruginosa trpE along with some flankingDNA. The deduced amino acid sequence is shown as well.The presumed ATG start codon is six bases downstreamfrom a potential ribosome-binding site (GAGG) and is con-

sistent with alignment of the deduced protein sequence withsequences of 22 other anthranilate synthases. An open

reading frame of 492 codons is followed by a TAA stopcodon. Downstream 23 bp from this stop codon is a potentialstem-loop structure preceded by a string of As and followedby a string of Ts; this structure resembles bidirectional,rho-independent terminators found near the ends of manytranscripts in bacteria (12).

It is a characteristic of moderately expressed genes inbacteria whose DNA has a high G+C content that a highproportion of the codons end in G or C. Sequencing errors

resulting in frameshifted regions can often be recognized byan examination of codon usage. The distribution of bases inthe TrpE open reading frame is as follows: first codonposition = 66.9% G+C; second codon position = 40.8%G+C; third codon position = 85.4% G+C; total base com-

position = 64.4% G+C. There are no stretches with abnor-mally low G+C content in the third codon position. Thecodon usage is very similar to that seen for the P. aeruginosa

TrpG, TrpD, and TrpC proteins (analysis not shown) and thetryptophan synthase subunits (16).Comparison of TrpE and TrpG sequences with those of

other bacteria. Little difficulty was encountered in aligningthe amino acid sequences deduced for P. aeruginosa anth-ranilate synthase with other published sequences throughoutTrpG and in the C-terminal half of TrpE. In fact, as shown inFig. 5, the alignment also accommodates a eucaryotic en-

zyme, Saccharomyces cerevisiae anthranilate synthase, andthe paralogous p-aminobenzoate synthase from E. coli (14,19). Amino acid identities occur at 38 positions in thealignment of seven glutamine amidotransferase subunits(Fig. SB) and at 50 positions beyond position 270 in thealignment of six a subunits (Fig. SA). The amino-terminalhalves of the a subunits are much harder to align. Figure 5Acontains an inordinate number of gaps in this region, some ofthem quite large, and even so shows identity at only sixpositions. This alignment was extracted from a more com-

prehensive one presented elsewhere consisting of 17 large-subunit sequences (6). Some of the sequences presentin the larger collection but omitted from Fig. SA, such as

Salmonella typhimurium TrpE, closely resemble others, andsome are still unpublished. Even when all are present,

30- - H Q L- A H L GS I Y LK- Q H I E- A Q L N- Y F S R

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260 270R C E C N Q S D E E F G G V V R L L Q KR V V A D I P D A Q F R T Q I N E L K EA F R A S F T R E D Y E N A V G R I K DT V S S N Y E K S A F M A D V E K I K SL L N R M W A R K V T K I T S P T L K KD W Q S N M T R E Q Y G E K F R Q V Q E

ANyyHy

IRIyILIKIKLH

280A E I F Q V VN G D I Y Q V VA G D C M Q V VA G D I F Q G VK GD I I Q G VS G D C Y Q V N

290 300P S R R F S L P C P S P L AP A R T F T A P C P D A F AP S Q R M S I E F K A A P IL S Q K F E V P I K A D A FP S Q R V A R P S R Y I L SL A Q R F H A T Y S G D E W

310 320----A Y Y V K K SNP S Y F F M Q D N D

- - - - A Y L Q L R A T N P S P Y M F Y I R G L N ED - - - L Y R A L R C F N P T P Y M Y F F N F G D -E - - - L Y R V LR I V N P S P Y M Y Y M K L L D -I F T D I Y R H R T I N P S P Y L F Y I D C L D -Q - - - A F L Q LN Q A N R A P F S A F L R L E Q -

350 360T S R Q I E I Y P I A G T R P G R R A DA N R E L Q L Y P I A G T R P R G L N P D- - G L V T V R P I A G T R P R G I - - -- - G H L E I H P I A G T R K R G A - - -- K N R V I T H P I A G T V K R G A - - -- - S E I Q T R P I K G T L P R L P---

GG

SS

370L DI N- N- D- A- D

400 410H L ML V D L A R N D L A RI C T P G S R Y V A DD T M L V D L A R N D L A R V S V P A S R R V A DH L M L I D L G R N D V G R V S D I G A V K V T EH Y M L V D L A R N D I G R V A E Y G S V S V P EH V M L V D L A R N D I N R I C D P L T T S V D KN L I V DLM I G R V A V A G S V K V P E

RDEKTp

LLKFLL

G

DEEAEQ

TLMTLF

R

330- F TS Y E- F H- R E- F Q- G A

L D SL D IA D LE D EE D DE D S

KQVKTV

420V DV DI EIIV

vQE

RRARAK

RRRSKp

LFLFVVIVIIIL

INLLGQ

yyyFFF

EEEKAA

SSSSSp

340G A S P E S S L K Y D AG A S P E S N L K F T AG S S P E V L V R V E DG S S P E R L I H V Q DG A S P E L L C K S D SS L S ER F I L C D N

380L EL DQ DV ED QV K

MMLLLL

390R T D H K E L S ER T D A K E I A DL S D A K E I A EM K D E K E K A ER G S L K D R A EA N S A K D R A E

4aYRIVIH L VISIRR|V|M|H|L V|S|RN|V|M|H| I V|S|NH|V|M|H| I I |S|VH|V|Q|H|L V|S|QAVHHLVWT

VVVVVI

VTTTsT

GAGGGA

ETQRvQ

440 450 460 470 480L R H D L D A L H A Y R A C M N MG T L S G A P K V R A MIQ L I A E A G R R nG s Y G G|L|D P E L D A L D A Y R A C M N M|G|T L T|G A P K|L|R A M|E L L R G V|E|K R R|R|G S Y G|G|LIR E G L S A M D A L R A I L PALG|T LSL G A PKIIIR A M|E I I D E LIEIP V KIR|G V Y GIG|L|K K G V H P V D A L M S A F P A|G|T L T|G A P K|I|R A M|Q L L Q E L|E|P T P|R|E T Y G|G|L|R P E K T R F D A F R T N F P A G|T V S|G A P K V|R A M|E L I A E L|E|G E R|R|G V Y A|GL P E Q L H A S D L L R A A F P GGS I TIG A P KVR A E I I D E LEP Q RRN A W CG

490 510 520A V G Y F T A H- D L T C V I R S A L V E N I A T V QAGA V LDSV P Q S EA V G Y L R G NG- D MD|N C|I|VIRIS A F V Q DIGIV A A V QIA GIAIGVVRIVRD SIN P Q SIEIA V G Y L A W NG- NMDD T A|IIAIRIT A V I K NIGIE L H V QA GIGIGIIVIAID SIV P A L IEIC I A Y I G F DG- N I|D|S C I T IIRITM S V K NIGIV A S I QA GIAIGIIVIAID SIV P E A IEIA V G H W S Y D|G|K T M|D|N C|I|A L|RIT M v Y K DIGII L T L QIA GIGIGIIIVYI SI E Y DIES I G Y L S F C G- N M D T S IT I RT L T A I N GQ I F C SIA G GG I AD SQ E E AS

530 540A DIET R N K A R A V L R A I A T A H HA D E T L H K A Y A V L N A I A L A A GW E E T I N K R R A HF R A V A L A E QY E E S C N KA G A L L K T I H I A E DM L E T M N N D G Q S Q Y Y C A S R R IY QWET F D K V N R I L K Q L E K

550 560A Q E T FS T L E V I RS V EH F H S K E D K A D E Q I S T I V RV G R Y R R I S L K R A F S V F F P L D D I F I V

* S.c. TrpE starts: H T A S I K I Q P D I D S L . . and ends . . F E

FIG. 5-Continued

however, the discrepancies in the two halves of TrpE andPabB remain striking, and aligning the N-terminal halves ofthe proteins poses problems. Nevertheless, Fig. 5 indicatesthat the P. aeruginosa sequences conform to the group aswell as any.

Construction of pDE1410 and pDE1574 and insertion intothe P. aeruginosa chromosome. Plasmid pDE1410 was con-structed as follows (Fig. 6). p1395 containing the cloned P.aeruginosa PA01 trpG gene was digested with BamHI, andthe purified 2.0-kb BamHI fragment was inserted into theBamHI site of pUC9 to create pDE1406. pDE1406 wassubsequently digested with Sacl, and a SacI-XhoI adaptor(CCTCGAGGAGCT) was inserted to yield pDE1408.pDE1408 was then digested with XhoI, and the XhoI-Kmrcassette from pDE-Km was inserted into the trpG gene,resulting in the formation of the Ampr Kmr plasmidpDE1409. pDE1409 was digested with BamHI, and thepurified 4.5-kb BamHI-trpG-Kmr fragment was inserted into

the BamHI site of pSUP205, yielding the Cmr Kmr Mobplasmid pDE1410.

E. coli S17-1(pDE1410) was mated with PA04290. Themating mixture was plated on LB agar containing cefazolinand kanamycin to select for P. aeruginosa TrpG- Kmrexconjugants. (Cefazolin was present to select against the E.coli donor strain.)

P. aeruginosa PA01 proved to be very resistant to kana-mycin; therefore, we could not use pDE1410 to construct aPAO1 trpG mutant. We subsequently constructed a mercuryresistance cassette and used it to replace the kanamycincassette of pDE1410. The Kmr cassette has an internalpromoter which allows expression of the downstream genestrpD and trpC; however, the Hgr cassette lacks a down-stream-directed promoter, and expression of trpD and trpCare also affected. Figure 6 details the construction of plasmidpDE1574. pDE1410 was digested with XhoI, and the XhoI-Hgr cassette from pDE-HgX was inserted into the trpG gene,

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0 20 30M A D I L L L D N I D S F T Y NL A D Q L R S N G H N V V I Y R N H IM T H V V L I D N H D S F V Y N L V D A F A V A G Y K C T V F R N T V

N L L M I D N D S F T Y N L V Q Y F G E L K A E V K V V R N D EM L L M I D N Y D S F T Y N I V Q Y F G E L N Q D V K V V R N D QM I L M I D N Y D S F T Y N L V Q Y L G E L G E E L V V K R N D S

. . N K H V V L I D N Y D S F T W N V Y E Y L C Q E G A K V S V Y R N D AM I L L I D N Y D S F T W N L Y Q Y F C E L G A D V L V KRND A

40P A Q T L I E R L A TP V E T I L A A N P DL S V E Q I E A L A PV T L E D I E R W Q PI T I D E I E E L S PI T V P E I A A L N PL T L A D I D A L K P

oI I GI CL GH Q A IL L G I C L G Y Q A LL L C V C L G H Q S IL L G V C L G H Q A II F G V C L G H Q S IV F G I C n G Q QC MIL AVLGLH A M

NLEKDDQ

VIGGAFA

S

RyFTK

EEQQQDQ

30N P

AyAAvvA

V LIILLLI

NCVVNLV

Y G G YH G G KF G G EF G G NF G G DF G G EF G G K

LLLVII

60 70 g0SPGPGVP--SEA GCNPELLTRLRK LPS|P G P|G Y|P|- A D AGN M M A L I E R T L|G|Q I PSIP G PIC T|P|- N E A|G|V S L A V I E R F A|G|K L|P|G|P G P|C S|P|- T E A|G|I S I P A I H H F A|G|R I|PS|P G P|C S|P|- D E AIGII S L E A I K H F AIGIK IPS|P G P|G H|P|K T D S |G |I S R D C I R Y F T|G |K I |P|S|P G P|C T P- D E A G I S L D V I R H Y A G R L P

100V GV Ev vI Iv vV Av v

QAPcRARARAYARA

GEG -RQKTERGEAK

110 120I L H G K A S S I E H D G Q A - M F A G LP V H G T T D N M I L T D A G - V Q S P VV M H G K T S P I H H K D L G - V F A G LV M H G R L S D M Y H T D K G - I F S N LL M H G K T S D I E H D G K T - I F E G LI V H G K T S P I S H D N C G - I F R N VV NHG K T S P I T H N G E G - V F R G L

P

140

E V P

VRHDARTTVRHKVKHKI RHNVRH KI RHR

G R KLvLFLIL

PPTSVAT

vIvAAvv

150

A Y H S LlV G S N -

GIR Y H S LIG C V V -

T|R Y H S L|V V K R ET|R Y H S L|V I E Q ETIR Y H S LII V K P ET|R Y H S L|A G T E STRY H S L|V V E P D

160 170- I F A G L T I N A H F- A P D G I E S L G T CS L P E C L E V T A W TS L P E C L E V T C W TT L P S C F T V T A Q TS L P S C L K V T A S TS L P A C F D V T A W S

190 20A D R V C GF Q F H P E SI L T T Q G A R LD G K A I G L Q F H P E S V L S P T G P I IT L N V E G V Q F H P E S V L T E Q G H E LT L P V E G V Q F H P E S I L S Q H1G H Q ID L P I E G V Q F H P E S I

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220 230A Q H K L E P A N T L Q P.L L ANQ G G V R G E G NY AY R K E V I AS G G T W E E N K S S P S.

FIG. S-Continued

replacing the XhoI-Kmr cassette and resulting in the forma-tion of the Cmr Hgr Mob plasmid pDE1574.

E. coli S17-1(pDE1574) was mated with PAO1. The matingmixture was plated on LB agar containing cefazolin andmercuric chloride to select for P. aeruginosa TrpGDC- Hgrexconjugants.To demonstrate directly the replacement of trpG on the P.

aeruginosa chromosome with the Kmr or Hgr insertionallyinactivated gene, we subjected DNA from the parentalstrains PAO1 and PA04290 and the two derivative strains,the TrpG- Kmr mutant, PADE Gl, and the TrpGDC- Hgrmutant, PADE G2, to restriction and hybridization analysis.Chromosomal, p1395, pDE1410, and pDE1574 fragmentsgenerated by cleavage with EcoRV were separated on a0.8% agarose gel and transferred to nylon membranes. Themembranes were hybridized with a 32P-labeled 1.0-kbEcoRV fragment from p1395, a 32P-labeled 750-bp XhoI-HindHI fragment from pDE-Km, or a 32P-labeled 1.2-kbPstI-ScaI fragment from pDG106. The p1395-derived proberecognized a 1.0-kb EcoRV fragment from p1395, PA04290,and PAQ1 DNA (Fig. 7A, lanes 1, 2, and 3). This same proberecognized a 3.5-kb EcoRV fragment from PADE Gl and

pDE1410 DNA (Fig. 7A, lanes 4 and 5), indicating loss of thewild-type trpG and replacement with the Kmi, insertionallyinactivated trpG. The probe also recognized a 6.2-kb EcoRVfragment from PADE G2 and pDE1574 DNA (Fig. 7A, lanes6 and 7), indicating replacement of wild-type trpG with theHgr, insertionally inactivated trpG. The pDE-Km-derivedprobe failed to hybridize with any of the EcoRV fragmentsfrom p1395, PA04290, PA01, PADE G2, and pDE1574DNA (Fig. 7B, lanes 1, 2, 3, 6, and 7). The probe did,however, recognize the same 3.5-kb EcoRV fragments fromPADE Gl and pDE1410 DNA as the p1395-derived probe(Fig. 7B, lanes 4 and 5), demonstrating that the KmT cassettewas indeed inserted into the mutant trpG. The pDG106-derived probe failed to hybridize with any of the EcoRVfragments from p1395, PA04290, PA01, PADE Gl, andpDE1410 (Fig. 7C, lanes 1 to 5) but did recognize the same6.2-kb EcoRV fragments from PADE G2 and pDE1574DNAas the p1395-derived probe (Fig. 7C, lanes 6 and 7). Thehybridization results indicate that the Hgr cassette wasindeed inserted into trpG.

Construction of pDE1533 and isrtion into the P. aerugi-nosa chromosome. Plasmid pDE1533 was constructed as

BE.B.P.A.B.S.E.

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FIG. 6. Construction of pDE1410 and pDE1574. The followingletters are used to denote different restriction enzyme sites on themaps: B, BamHI; E, EcoRI; H, Hindlll; P, PstI; RV, EcoRV; S,Sall; Sa, Sacl; Sc, ScaI; and X, XhoI.

follows (Fig. 8). p1460 (pUC19 containing the P. aeruginosaPAO1 trpE gene in a HindIII-BglII fragment) was digestedwith HindIll and SmaI, and the resulting 3.7-kb fragmentwas inserted into Hindlll- and Smal-digested pUC18-XhoIto create pDE1525. pDE1525 was digested with EcoRI toremove a 747-bp fragment internal to trpE, and an EcoRI-BglII adaptor (AATTAGATCT) was inserted to yieldpDE1528. pDE1528 was subsequently digested with Hindllland SmaI, and the purified 3.0-kb fragment carrying themutant trpE was inserted into HindIll- and EcoRV-digestedpSUP205 to form pDE1530. pDE1530 was digested withBglII, and the 2.7-kb BglII Tcr cassette from pDE-Tc was

inserted to yield the Cmr Tcr Mob plasmid pDE1533.E. coli S17-1(pDE1533) was mated with PAO1 and

PA04290. The mating mixtures were plated on LB agar

containing cefazolin and tetracycline to select for P. aerug-inosa TrpE- Tcr exconjugants.Replacement of the trpE gene on the P. aeruginosa

chromosome with the Tcr mutant trpE gene was demon-strated directly by subjecting DNA from the parental strainsPAO1 and PA04290 and the two derivative TrpE- Tcrstrains, PADE El and PADE E2, to restriction and hybrid-ization analysis. Chromosomal, p1460, and pDE1533 frag-ments generated by cleavage with KpnI were electro-phoresed in 0.8% agarose and transferred to nylonmembranes. The membranes were hybridized with the 32p_labeled 868-bp Hindlll-EcoRI fragment from pDE-Tc or the32P-labeled 1.1-kb KpnI-EcoRI fragment from p1460. Thep1460-derived probe recognized a 2.0-kb KpnI fragmentfrom p1460, PAO1, and PA04290 DNA (Fig. 9A, lanes 1 to3). The same probe recognized a 3.9-kb KpnI fragment fromPADE El, PADE E2, and pDE1533 DNA (Fig. 9A, lanes 4to 6), indicating the loss of wild-type trpE and its replace-ment with the Tcr mutant trpE. The pDE-Tc-derived probefailed to hybridize with either of the KpnI fragments fromp1460, PAO1, or PA04290 DNA (Fig. 9B, lanes 1 to 3). Thisprobe recognized the same 3.9-kb KpnI fragment fromPADE El, PADE E2, and pDE1533 DNA as the p1460-derived probe (Fig. 9B, lanes 4, 5, and 6), demonstrating thatthe Tcr cassette was indeed inserted into trpE.

Phenotypic characterization of PADE Gi, PADE G2, PADEEl, and PADE E2. Even in the absence of the ,B subunit, theao subunit of anthranilate synthase can synthesize anthra-nilate directly from chorismate and high concentrations ofammonia. TrpE mutants require either anthranilate or tryp-tophan for growth on minimal media. The P subunit ofanthranilate synthase provides glutamine amidotransferaseactivity, allowing glutamine at moderate concentrations tosubstitute for ammonia in the above reaction. Paluh et al.(28) showed that the a subunit alone suffices for growth inminimal medium if the ammonium content is high but thatgrowth in low ammonium concentrations requires the pres-ence of both subunits. Vogel-Bonner minimal medium hastoo high an ammonium content to select against TrpGmutants, so we used a reformulated M9 minimal medium (28)with the ammonium content adjusted to allow their detec-tion. TrpG mutants show a definite requirement for anthra-nilate or tryptophan in low-ammonium medium (1 mMNH4+) that disappears when the ammonium content israised to 50 mM.The results of growth of P. aeruginosa PAO1, PA04290,

PADE Gl, PADE G2, PADE El, and PADE E2 on definedmedia are shown in Table 2. Growth tests were done both inliquid culture and on plates; identical results were obtained.The TrpG mutant, PADE Gl, failed to grow on M9 low-ammonium medium but grew in 50 mM ammonia. TheTrpGDC mutant, PADE G2, and the two TrpE mutants,PADE El and PADE E2, were unable to grow on either M9medium. PADE Gl was indistinguishable from wild type onVogel-Bonner minimal medium. PADE G2, PADE El, andPADE E2 all required supplementation for growth on Vogel-Bonner minimal medium; PADE G2 required tryptophan,while PADE El and PADE E2 required either anthranilateor tryptophan for growth.

Anthranilate synthase (with either ammonia or glutamineas the nitrogen source) and TrpD-derived phosphoribosyltransferase enzymatic activities were measured in extractsfrom tryptophan-starved cultures (3). The results (Table 3)were consistent with the growth requirements observed.

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FIG. 7. Hybridization of EcoRV-digested p1395, pDE1410, pDE1574, PA04290, PAO1, PADE Gl, and PADE G2 DNA with 32P-labeled1.0-kb EcoRV fragment from p1395 (A), 32P-labeled 750-bp XhoI-HindIII fragment from pDE-Km (B), and 32P-labeled 1.2-kb PstI-ScaIfragment from pDG106 (C). Lanes: 1, p1395; 2, PA04290; 3, PAO1; 4, PADE Gl; 5, pDE1410; 6, PADE G2; and 7, pDE1574.

DISCUSSIONSeveral lines of argument lead to the conclusion that the

anthranilate synthase a- and P-subunit genes reported here,not the ones described earlier (7, 9), are the source of thetryptophan biosynthetic activity in P. aeruginosa. Shi-nomiya et al. (33) obtained from P. aeruginosa PAO1 a largeseries of R-prime plasmids bearing the pyocin R2 genecluster and some neighboring DNA. By complementation ofTrp auxotrophs, they determined that this pyocin genecluster is flanked by trpE at one end and the trpDC pair at theother. Their physical map shows the distance between trpEand trpDC to be about 25 kb, including a pyocin-encodingsegment 13 kb in length. Their restriction map of this regionindicates the presence of two fairly closely spaced EcoRIsites within trpE and a 5'-HindIII-EcoRI-BamHI-3' groupingin the vicinity of trpD. The former can be found in thesequence of Fig. 4 at positions 207 and 954; the latter is notpresent in the sequence of Fig. 2, but we have found aBamHI site about 860 bp ahead of the first base in thatsequence (data not shown), suggesting that the other twosites occur upstream from there. No similar groupings arepresent in the phnAB sequence reported earlier from strainPAC174 (7) or ones obtained more recently for the samegene pair in strain PA01 (lla). Extending the sequencedownstream from trpE somewhat (data not shown), weobserved two open reading frames that Shinomiya and hiscolleagues have identified as prtN and prtR, regulatory locifor the pyocin R2 structural loci (T. Shinomiya, personalcommunication). Thus, our sequence results appear to beentirely compatible with the known topography of the region(33).

It is clear now that the trpDC region includes trpG as thefirst gene in a three-gene operon. Our insertional inactivationresults indicate that this trpG gene is the sole source of the Psubunit for the tryptophan-related anthranilate synthase,since insertion of a Kmr cassette having an internal promoter

(strain PADE Gl) gave cells requiring tryptophan only in amedium with low ammonium ion content the phenotypeexpected in a strain defective in only the 1B subunit. Enzymeassays in extracts confirmed the inability of the PADE Glenzyme to use glutamine as an amide-group source. Resultswith a strain containing an Hgr cassette in the same locationin the trpG gene, with this insertion lacing a downstream-directed promoter, showed a more severely defective phe-notype in which the TrpD and TrpC activities were alsomissing. This confirms the structural inference that trpGDCrepresents a single transcriptional unit, probably exhibitingcoordinate regulation and coupled transcription. All ourgenetic results are in agreement with earlier data by Calhounet al. (3) on the regulation of the enzymes of the tryptophanpathway in P. aeruginosa auxotrophs.The existence in both P. putida (25) and P. aeruginosa (3)

of regulatory mutants that constitutively overproduce TrpE,TrpG, TrpD, and TrpC suggests that these four gene prod-ucts derived from two transcriptional units share a commonregulatory element formally analogous to the trpR repressorin E. coli. Suggestive sequence features compatible with thisare described and discussed in the accompanying report (11).The strongest evidence that trpGDC functions as a singleoperon is the inactivational result discussed above, but thevirtual absence of intercistronic spaces, with the obligatoryinclusion of the Shine-Dalgarno sequences in codons nearthe end of the preceding cistron, is quite characteristic ofoperon structure in this organism. A very similar juxtaposi-tion of trpB and trpA is seen in the genes of the tryptophansynthase operons of P. aeruginosa (16) and P. putida (8).Similarly, codon usage favoring codons ending in G or C isseen for all the trp genes sequenced in these pseudomonadsand is presumably characteristic of moderately expressedPseudomonas genes in general.Why should P. aeruginosa be unique among bacteria

studied to date in possessing two pairs of genes for anthra-

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VOL. 172, 1990 PSEUDOMONAS AERUGINOSA trpE AND trpGDC 865

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nilate synthase? Clearly these genes are not the result of arecent duplication. Their sequences are much less alike thanthe homologous trpE and trpG sequences from P. putida (o1)or even A. calcoaceticus (18). The phnA and phnll readingframes overlap by 23 bp, whereas the trpE and trpG genesare on different transcriptional units separated by 25 kb ofDNA of unrelated function. It seems obvious that the genesfor these two anthranilate synthases duplicated and began todiverge in sequence much earlier in evolution than the timeof speciation of the two pseudomonads under study. Analy-sis of the relatedness of the two P. aeruginosa anthranilatesynthases to homologous enzymes of distantly related or-ganisms will be deferred until the entire phnA sequence isavailable (lla). From the finding that mutations in phnA andphnB result in decreased synthesis of pyocyanin, a phena-zine pigment produced in the stationary phase of growth(22), we hypothesize that trpE and trpG function primarily in

A 1 2 3 4 5 6 B 1 2 3 4 5 6

3.9 kb -.

2.Okb- ---

FIG. 9. Hybridization of KpnI-digested p1460, pDE1533, PAO1,PA04290, PADE El, and PADE E2 DNA with 32P-labeled 1.1-kbKpnI-EcoRI fragment from p1460 (A) and 32P-labeled 868-bpHindIII-EcoRI fragment from pDE-Tc (B). Lanes: 1, p1460; 2,PAO1; 3, PA04290; 4, PADE El; 5, PADE E2; and 6, pDE1533.

the exponential growth phase while phnA and phnB normallyprovide anthranilate only for secondary metabolism. Thishypothesis is currently being tested.

ACKNOWLEDGMENTS

We thank B. Holloway, H. Matsumoto, M. Vasil, B. D. Gambill,and G. V. Stauffer for strains and plasmids used and T. Shinomiyafor providing unpublished data.

This work was supported by grant DMB 8606653 from theNational Science Foundation.

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DNA Sequences and Characterization of Four Early Genes of the