Vol. 176, No. 7JOURNAL OF BACTERIOLOGY, Apr. 1994, p. 1865-18710021-9193/94/$04.00+0Copyright C 1994, American Society for Microbiology

Cloning, Characterization, and Nucleotide Sequence Analysis ofthe argH Gene from Campylobacter jejuni TGH9011

Encoding Argininosuccinate LyaseERIC KURT HANI AND VOON LOONG CHAN*

Department of Microbiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Received 11 November 1993/Accepted 19 January 1994

The complete structural gene for argininosuccinate lyase (argH) from Campylobacterjejuni TGH9011 hasbeen cloned into Escherichia coli by complementation of an E. coli argH auxotrophic mutant. The gene has beensubcloned for sequencing on a 4.1-kb DNA segment and localized by the complementing activity of deletionmutants. The complete DNA sequence of the C. jejuni argH gene was determined. The transcription start pointfor argH mRNA was determined by primer extension analysis and found to be within the coding sequence ofthe upstream gene, identified as the phosphoenolpyruvate carboxykinase gene (ppc). The argininosuccinatelyase and the phosphoenolpyruvate carboxykinase reading frames overlap by one base, the second example ofthis phenomenon in C. jejuni chromosomal genes. The enzyme has a deduced subunit molecular weight of51,831. Recombinant plasmids containing the argH gene generate a 56-kDa protein and a 43-kDa protein in E.coli maxicells. An alternate translation initiation producing a polypeptide with a deduced molecular mass of42 kDa may account for the smaller protein observed in sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. The C.jejuni argH gene shows nucleotide homology to both yeast and human argininosuccinatelyase genes, and conserved amino acid domains are evident between the corresponding proteins.

Campylobacter jejuni is a member of a diverse genus withonly distant relationships to other eubacteria (39). Our knowl-edge of the biochemistry of Campylobacter proteins and regu-lation of Campylobacter genes is rudimentary. C. jejuni utilizesamino acids and tricarboxylic acid cycle intermediates assources of energy. Amino acids can also be generated fromtricarboxylic acid cycle intermediates. Arginine and proline aresynthesized from a-ketoglutarate via glutamate. C. jejuni cangrow on a defined medium lacking arginine, demonstratingthat strain TGH9011 contains a complete arginine biosyntheticpathway. Argininosuccinase (EC 4.3.2.1) cleaves argininosuc-cinic acid into arginine and fumarate and is the last enzyme inthe arginine biosynthetic pathway.We present here the analysis of a C. jejuni gene which

complements an Escherichia coli strain deficient in ArgHactivity, as an introduction to further examinations of thearginine biosynthetic pathway in C. jejuni. The gene has beencloned and sequenced from fungal and mammalian systems(11, 25, 26, 29), and incomplete data are available from E. coli(10).Given that the C. jejuni TGH9011 genome is smaller than

that of E. coli (18-20), it is important to determine whichbiochemical pathways are present in this organism andwhether the organization and regulation of these genes iscomparable to that of bacteria with larger genomes.

MATERUILS AND METHODS

Bacterial strains and growth conditions. The C. jejunigenomic library was constructed from strain TGH9011 (ATCC43431) as described previously (7). The bacterial strains andplasmids used in this study are listed in Table 1. C. jejuni

* Corresponding author. Phone: (416) 978-6077. Fax: (416) 978-4761.

TGH9011 (ATCC 43431, serotype reference strain for 0:3)was obtained from J. L. Penner (University of Toronto,Toronto, Ontario, Canada) and was grown in Campylobacterdefined medium as described previously (38), modified byremoving arginine, and routinely in Brucella agar base supple-mented with 5% horse blood (Woodlyn Laboratories, Guelph,Ontario, Canada). E. coli JM103, E. coli C600, and E. coliW3678, obtained from B. J. Bachmann (Yale University, NewHaven, Conn.), and E. coli DR1984 were grown routinely inLB broth (10 g of tryptone, 5 g of yeast extract, and 5 g of NaClper liter); LB and M9 minimal agar medium were supple-mented with nutrients as required and, when appropriate,contained 100 ,ug of ampicillin per ml.

Materials. Ampicillin, argininosuccinic acid, arginase, argi-nine, oa-isonitrosopropeophenol (at-INPP), acrylamide, bisacyl-amide, D-cycloserine, and urea were purchased from SigmaChemical Co., St. Louis, Mo. Bacteriological growth mediumwas purchased from Difco Laboratories, Detroit, Mich. Radio-active compounds purchased consisted of [a-32P]dATP and[_y-32P]ATP (Amersham) and [ot-355]dATP (1,350 Ci/mmol;(Dupont-NEN, Mississauga, Ontario, Canada); [35S]methi-onine was purchased from ICN. Restriction enzymes and DNAmodification enzymes were purchased from Boehringer(Mannheim, Germany), Gibco-BRL (Mississauga, Ontario,Canada), and Pharmacia (Uppsala, Sweden). Moloney murineleukemia virus reverse transcriptase was purchased from Phar-macia. Sequencing was done with the Sequenase kit (UnitedStates Biochemical Corp., Cleveland, Ohio). Nick translationwas performed by using the corresponding kit from BethesdaResearch Laboratories (Mississauga, Ontario, Canada).DNA manipulation. Preparation of plasmid DNA from E.

coli was done by a rapid alkaline lysis procedure, and diges-tion with restriction endonucleases, ligation with T4 DNAligase, transformation with recombinant plasmids, and agarosegel electrophoresis were performed as described previously(24).

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TABLE 1. Strains and plasmids used in this study

Strain or plasmid Relevant characteristics Source orreference

C. jejuni TGH9011 ATCC 43431, serotype reference strain for 0:3 J. L. PennerE. coliW3678 argH B. BachmannJM101 A(lac-pro) thi rspL supE endA sbcB hsdR F' traD36 proAB lacIqZAMJ5 42JM103 A (lac-pro) thi rspL supE endA sbcB hsdR F' traD36 proAB lacIqZAM]5 42C600 F- thrl leuB6 lacYl supE44 rfbDi thi-J tonA21 Lambdadef 3DR1984 Nonmucoid form of CSR603 recAl uvrA6 phr-I 34

PlasmidspUC19 Parental cloning vector 42pPH5A 0.544-kb HindIlI fragment of C. jejuni argH subcloned into pUC19 This workpARGHI-1 4.1-kb ClaI-AccI fragment of pARGH1 inserted into AccI site of pUC19 This workpHR1 arg' deletion derivative of pARGH1-1 in the upstream region of argH This workp8R3 arg deletion derivative of pARGH1-1 removing amino-terminal region of argH This workpARGH1-2 4.1-kb ClaI-AccI fragment of pARGH1 inserted into AccI site of pUC19 in opposite This work

orientation to pARGH1-1pDTH arg+ deletion derivative of pARGH1-2 in the downstream region of argH This workpDTL arg deletion derivative of pARGH1-2 removing carboxy-terminal region of argH This workpBR322 Parental cloning vector 6pARGH- Contains incomplete argH gene isolated by homology to pPH5A insert This workpARGH1 Complements an E. coli argH mutant isolated by homology to pPH5A insert This workpARGH2 Complements an E. coli argH mutant 13pARGH3 Complements an E. coli argH mutant 13pARGH4 Complements an E. coli argH mutant 13

Construction of deletion derivatives and DNA sequencing. Aset of nested deletions of pARGH1-2 and pARGH1-1 (pUC19plus a 4.1-kb ClaI-AccI argH+ fragment; see Results) were

constructed. Plasmid pARGH1-2 was digested with KpnI togenerate a 3' overhang and with EcoRV to generate a bluntend. Plasmid pARGH1-1 was digested with KpnI and BamHI(5' overhang). The clones were then digested with exonucleaseIII and Si nuclease to generate random deletion mutantsspanning the argH gene. The conditions for the treatments withexonuclease III, S1 nuclease, and T4 DNA ligase were essen-

tially as described by Henikoff (16). The DNA sequence was

determined by the dideoxy-chain termination method de-scribed by Sanger et al. (36) for both strands.

Primer extension mapping of the transcription start site ofthe argH mRNA. RNA was isolated from late-exponential-phase cultures of E. coli W3678 containing pARGH1-1 by thehot phenol method of Aiba et al. (1). Oligonucleotide argH-1(5'-TTCTTTTAAAAGCTCATCACTTGCATCAC-3') iscomplementary to the coding strand of argH and is locatedfrom nucleotides 32 to 60 downstream from the first Metcodon of the argH gene, in a region with little similarity to theknown E. coli argH gene sequence. The oligonucleotide was

end labeled with polynucleotide kinase and mixed with 25 ,ugof total RNA prepared from E. coli W3678 containing plasmidpARGH1-1. The mixture was hybridized and extended as

described previously (9). For analysis, 2 pAl of the newlysynthesized DNA (previously suspended in 4 pul of formamideloading buffer) was loaded onto a 6% polyacrylamide sequenc-ing gel alongside dideoxy sequencing ladders of the upstreamflanking region of the argH gene generated with the identicaloligonucleotide.

Maxicell analysis. Plasmid-encoded proteins were labeled inUV-irradiated E. coli DR1984 cells as described previously(34). Cells were UV irradiated with a germicidal lamp (15 W)at a height of 50 cm. Survival was between i0-' and 10-'following 12 to 15 h of incubation with 200 pug of D-cycloserineper ml. Irradiated cells were washed two times with Hershey

salts (34, 35) and then labeled with [35S]methionine (40p.Ci/ml) for 1 h in Hershey medium. Cells were lysed by boilingfor 3 min in 50 il of 2 x Laemmli sodium dodecyl sulfate(SDS) sample buffer (LSB), and labeled proteins were sepa-rated by 0.1% SDS-13% polyacrylamide gel electrophoresis(PAGE) as described by Laemmli (22). After electrophoresis,gels were stained with Coomassie brilliant blue R-250, driedonto 3MM cellulose paper, and then exposed overnight toKodak XAR-5 film at - 70C.

Preparation of crude extracts. C. jejuni and wild-type,mutant, and transformed E. coli were grown overnight in theirrespective minimal media, supplemented with 25 ,ug of ampi-cillin per ml when appropriate. Cells were harvested at 12,000x g for 30 min, washed twice in 20 ml of 0.85% NaCl, andresuspended in 2 ml of 0.15 M potassium phosphate buffer (pH7.4). The cell suspension was passed through a French presstwice at a pressure of 16,000 lb/in2. The extract was cleared ofcell envelope by centrifugation at 20,000 x g for 40 min andstored frozen at - 20C.ASL. The argininosuccinate lyase (ASL) assay was per-

formed essentially as described previously (30, 32). Arginino-succinic acid was prepared by dissolving 4 mg of the barium saltper ml in 25 mM K2SO4 and clarifying the solution bycentrifugation. The incubation mixture contained 10 ,uMargininosuccinic acid, 50 mM potassium phosphate buffer (pH7.4), cell extract protein in the linear range of activity, and 6 Uof arginase in 0.1 ml of 0.1 M potassium phosphate buffer (pH7.4) in a final volume of 1 ml. Incubation at 38°C for 60 minwas followed by termination with 2 ml of acid mix (1:3:1concentrated H2SO4/concentrated H3PO4/H20). One millili-ter was assayed by addition of 381 ,ul of acid mix and 114 ,lI of4% ot-INPP in 95% ethanol in a final volume of 1.855 ml. Astandard curve was prepared by using 0 to 150 ,ug of urea in741 pul of acid mix and 114 ,ul of ot-INPP in a volume of 1.855ml. The assay tubes were boiled for 60 min in the dark, cooledto room temperature for 15 min, and read at an optical densityof 540 nm.

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VOL. 176, 1994 argH GENE OF C. JEJUNI 1867

E K I E K H E V N V Y L V N T G W S G G S Y G V G K R M S IGAAAAGATTGAAAAACATGAAGTTAATGTTTATCTTGTTAATACGGGTTGGAGTGGCGGAAGCTACGGTGTGGGTAAAAGAATGAGCATT

V -35K A T R T C I N A I L D G S I T K C E F E N F E V F D L A IAAGGCTACTAGGACTTGCATTAATGCTATTCTAGATGGAAGTATTACAAAATGCGAATTTGAAAATTTTGAAGTGTTTGATTTAGCTATT

-10P K A L E G V E S V L L N P I N T W L D K N A Y I A T R D KACCAAAAGCTTTAGAAGGTGTAGAAAGTGTACTTTTAAATCCTATAAATACTTGGTTAGATAAAATGCTTATATTGCAACAAGAGATAAA

L A H M F I Q N F K R Y E D V _K E _G I E_ F _S K F E T K N L RTTAGCACATATGTTTATACAAAATTTTAAACGTTATGAAGATGTCAAAGAAGGCATTGAATTTAGCAAATTTGAAACCAAAAATTTAAGG

v sS F K M* K N E M W S G R F S D A S D E L L K E F N A S L N V DTCATTTAAATGAAAAATGAAATGTGGTCAGGACGTTTTAGTGATGCAAGTGATGAGCTTTTAAAAGAATTTAATGCAAGTTTAAATGTAG

K T L F N E D I Q G S I A H A T M L E S C G I L K K E E L DATAAAACTTTGTTCAATGAAGATATACAAGGTTCTATAGCACACGCTACAATGCTTGAAAGTTGTGGTATTTTAAAAAAAGAAGAATTAG

(v)A I I K G L E Q V R S E I E Q G K F I F D I K D E D I H M A

ATGCTATTATAAAAGGTTTAGAGCAGGTTAGAAGTGAAATAGAGCAAGGTAAATTTATTTTTGATATTAAAGATGAAGATATTCATATGG(sd)

V E K R L S E I I G S E I G G R L H T A R S R N D Q V A T DCCGTAGAAAAGCGTTTAAGCGAGATTATAGGTAGTGAAATTGGTGGAAGACTTCATACTGCAAGAAGTAGAAATGATCAAGTGGCTACTG

90

180

270

360

28450

58540

88630

118720

F K L F V K K S H I E L I K L F K E L I Q T M L E H A K V H 148ATTTTAAACTTTTTGTCAAAAAATCTCATATTGAACTTATAAAGCTTTTCAAAGAATTGATTCAAACCATGCTCGAACATGCTAAAGTGC 810

K K T I M P S F T H L Q H A Q P V S F S F Y I L A Y A F M L 178ATAAAAAAACTATTATGCCAAGTTTTACACATTTACAGCACGCTCAGCCTGTGAGTTTTTCTTTTTATATTTTAGCTTATGCTTTTATGT 900

M R D I K R L Q N S L E L A D F S P L G S C A C A G T S Y R 208TAATGCGAGATATTAAGCGTTTACAAAATAGCCTAGAACTCGCAGACTTTTCGCCACTTGGATCTTGTGCATGTGCAGGAACAAGCTATA 990

T N R E L S A E I L G F K D I M L N A M D G V S D R D F A L 238GAACCAATCGTGAGTTGAGTGCTGAAATTTTAGGATTTAAAGATATTATGCTAAATGCTATGGATGGAGTGAGTGATAGGGATTTTGCTC 1080

D L L Y D I A V I F T H T S R L C E E M I L F S S S E F S F 268TTGATTTGCTTTATGATATAGCAGTTATTTTTACACACACATCAAGACTTTGCGAAGAAATGATCTTATTTTCAAGTTCGGAGTTTTCTT 1170

I T I S D S F S T G S S I M P Q K K N P D V C E L I R G K T 298TTATAACTATAAGTGATAGCTTTTCAACAGGAAGTTCTATTATGCCTCAGAAAAAAAATCCTGATGTTTGTGAGCTTATACGTGGAAAAA 1260

G R V Y G N L I S L L T I M K A L P L A Y N K D M Q E D K E 328CAGGGCGTGTTTATGGAAATTTGATTTCTCTTTTAACTATAATGAAAGCTTTACCTTTAGCCTATAATAAAGATATGCAAGAAGATAAAG 1350

G I F D S V K T A K D S L I I L N A M L K E I Q I N K E N M 358AAGGGATTTTTGATAGTGTAAAAACCGCTAAAGATAGCTTAATCATCTTAAATGCAATGCTAAAAGAAATACAAATTAATAAAGAAAATA 1440

L N A C K K G H L L A T D L A D Y L V R E K N I F F R K H I 388TGCTTAATGCTTGTAAAAAAGGTCATTTATTGGCTACCGATTTGGCAGATTATTTGGTGCGTGAAAAAAATATATTCTTTAGAAAGCACA 1530

Y S G N V V A Q A E A Q G I D I S E I K D L S K I D P V F D 418TTTATAGTGGAAATGTTGTTGCACAAGCTGAAGCACAAGGTATTGATATAAGTGAAATTAAAGATCTTTCAAAAATAGATCCTGTATTTG 1620

E K A M E L L N F E F S L N S K Q S E G S S S I A S V E K Q 448ATGAAAAAGCTATGGAACTTTTAAATTTTGAATTTTCTTTAAATTCTAAGCAAAGTGAAGGCTCAAGTTCTATTGCAAGCGTAGAAAAGC 1710

I Q I L E G F I Q N L * . 4 459AAATTCAAATTTTGGAAGGTTTTATTCAAAATTTATAAATATTGTTTAGGCTATTTTGCCTAAACATTGAATCTAAAGTGCATTACATCG 1800

FIG. 1. Complete nucleotide sequence of the C. jejuni argH gene and flanking sequences. The nucleotide sequence of the C. jejuni argH geneis shown along with the translated sequence in one-letter amino acid code. The ATG codon is indicated by an inverted solid triangle. The potentialShine-Dalgarno sequence (sd) is underlined. The ORF corresponding to the 42-kda protein is indicated by an inverted solid triangle in parenthesesalong with its corresponding Shine-Dalgarno sequence. Termination codons are indicated with asterisks. The transcription start point is indicatedby an inverted open triangle, and the putative - 10 and - 35 sites are double underlined. The two duplications upstream of the first ATG codonare indicated by solid underlining. The possible arg boxes are indicated by broken overlines. The potential transcription terminator is indicated byinverted arrows.

Nucleotide sequence accession number. The DNA sequenceshown in Fig. 1 is available in the GenBank nucleotidesequence data base under accession number M77188.

RESULTS

Cloning of the argH gene. A 0.544-kb HindIll fragmentcontaining a partial argH sequence as identified by a nucleotidedata bank search (2) was isolated incidentally from a phagelibrary and nick translated for use as a probe to screen agenomic pBR322 C. jejuni library to obtain the complete argHgene. The library was constructed as described previously (7).The argH gene was also retrieved from the C. jejuni library bycomplementation of an E. coli argH auxotrophic mutant (15).

Restriction enzyme mapping and DNA blot analysis. The C.jejuni argH gene was localized on two independent pBR322recombinants (pARGH- and pARGH1) by probing with a544-bp HindIII fragment corresponding to an internal regionof the argH gene that had been labeled with [oL-32P]dATP bynick translation. Plasmid pARGH- has a 5.0-kb insert, andplasmid pARGH1 contains a 6.0-kb insert, of C. jejuni chro-mosomal DNA. Southern blot analysis indicated that bothplasmids contain a 0.544-kb HindlIl fragment specificallyrecognized by the 0.544-kb HindlIl probe (data not shown).These results confirmed that both pARGH- and pARGH1contained C. jejuni DNA with argH-homologous sequences.There is only one copy of the argH gene in C. jejuni TGH901 1,as detected by Southern hybridization (data not shown).

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1868 HANI AND CHAN

C. jejuni ASL 1 MKNE MWSGRFSDASDELLKEFNASLNVDKTLFNEDIQGSIAHATMLESCGILKKEELDAIIKGLEQV 67yeast ASL 1 =SDGTQK-=G===-GE-=P=-MHLY===P==YK--KA=-E=-KVY--G=QKL=-=TET=-AK=HE==AE- 70human ASL 1 =AS=SGK-=G===VG=V=P--EK====-AY=-H=-EV=-===K=Y-RG==KA=-=T=A=-=Q=--==-K= 70rat ASL 1 =AS=SGK-=G===-G-V=PT-DK==-=-AY=-H=-=V=-===K=Y-RG==KA=-=T=A=-QQ=-Q==-K= 70algal ASL x PADNTKK-=G===-AK-=P=-EK==E==PF==R=-A===K xE. coli ASL 1 =A -=G===-Q=-=QRF=Q==D==RF=YG=AEQ==V==-=--KA= 44

68 RSEIEQGKFIFDIKDEDIHMAVEKRLSEIIGSEIGGRLHTARSRNDQVATDFKLFVKKSHIE LIKLFKELIQTMLEHAKVH 14871 -K=W-AD==-RHPN=====T=N=-==G=-==R==A=--==G=======V==L---C-DIVN-T=FPAL=G=-EV--K-=EGE 15271 AE=WA==T=KLNSN=====T=N=-==K=-==-T A=-===G=======V==L-=---Q-CS T=SG=LW===R==---=EAE 15071 AE=WA==I=KLYPN=====T=N=-==K=-== =AA=-===G=======V==L-=---Q-YS K=STFL=V==E-=---=EAE 150x AE=WKA=A=-INAG=====T=N=-=--=--=- -==-===G===H x

KKTIMPSFTHLQHAQPVSFSFYILAYAFMLMRDIKRLQNSLELADFSPLGSCACAGTSYRTNRELSAEILGFKDIMLNAMDGIDV-==G-====-===-R-=H--S-==TYFTE=Y===G-I=HRLNQ====-G=L==HP=GID==FL==G===NS--G=--VA-DV-F=G-====-===-R-=H--=-H=VA=T==SE==LEVRKRINVL====G=I==NPLGVD===LRAEN=GA=T==-==ACEV-F=G-====-===-R-=H--=-H=VA=T==-E==KEVQKRINVL====G=I==NPLGVD==F=CAE=N=GA=T==-==A

230234232232

x

VSDRDFALDLLYDIAVIFTHTSRLCEEMILFSSSEFSFITISDSFSTGSSIMPQKKNPDVCELIRGKTGRVYGNLISLLTIM 312-=====I--=--WG--FMN=I==FA=--=--C--==G==Q-==--=====-======A=SL==-===-===-=D=TGF=MS- 316T=-===VA-F=-WR--CM==L==-A=--==-C-K====-Q-==--=====-========SL====S=-===-=RCAG==MT- 314T=-===VA-F=-WA--CM==L==-A=--==-G-K==N=-Q-==--=====-========SL====S=-R==-=RCAG==MT- 314====- W --TV-AA---CV=L==WA=--=--==GP=G--QC==---====-========AL=== x

KALPLAYNKDMQEDKEGIFDSVKT=G==S-=D========P-==C-T==G-=S-====-=====A-=VSD==G-=S-====-=====A-=VSD=

rAKDSLIILNAMLKEIQINKENMLNACKKGHLLATDLADYLVREKNIFFRK HIYSGNV 394VEH=--=ATG--ST-T-===K=EA=LT MD-========== =G-P==ET=HI==EC 396-MSAV=Q-ATG--ST-==HQ===G-=LS PD-======Y==== =G-P==QA=EA==KA 394mMTAV=Q-ATG--ST-==H-===A-LS PD-=====Y==== =G-P==QA=EA==KA 394

LS AD-======-==== =G-P==ET=HH x

VAQAEAQGIDISE IKDLSKIDPVFDEKAMELLNFEFSLNSKQSEGSSSIASVEKQIQILEGFIQNL==T==RL=-SGI-KLT-EQYQ===SR=G-DLF=TF===Q=-ER-D-T=G--K--=L==-DN=KSE--=FM==-K=-A-NQ LS-Q-=QT=S=-=SGDV-C-WD-GH=-EQYG-L=G--R-==-W==RQ-RAL-=AQQA=VV==MK=-A-NQ LS-Q-=QT-S=-=SSDVNL-WD-SH=-EQYT-L=G--Q-===W==SQ-RAL-=M

-WD=NR=AEM-D-==G-=KR==LE=-=K-RT--AAEGQH

459463466461

x

FIG. 2. Alignment of the amino acid sequences of the various ASL proteins that have been sequenced either directly or as deduced from DNAsequence data. Identical amino acids are represented by double bars; conserved amino acids (MILV, KHR, DE, NQ, and PFY) are indicated bysingle bars. Gaps are introduced to maximize similarity.

Complementation of an E. coli argH mutant. E. coli W3678is an argH mutant with an absolute requirement for exogenousarginine. Plasmid pARGH- was able to transform E. coliW3678 to ampicillin resistance but was unable to complementthe arginine biosynthetic mutation. Plasmid pARGH1 was

capable of transforming W3678 and complementing the argHmutation. The efficiency of complementation to transforma-tion for pARGH1 was 100%, which suggested that this plasmidcontained the complete argH gene. The lack of complementa-tion by plasmid pARGH- suggested that it contained a trun-cated gene. This was confirmed by sequencing the insertdirectly in pBR322, using BamHI-specific primers (data notshown). Three additional plasmids, designated pARGH2,pARGH3, and pARGH4, were isolated from the C. jejunipBR322 library solely on the basis of complementation of theE. coli argH mutant (15).

Expression of the cloned argH gene in E. coli. E. coli C600,E. coli W3678, C. jejuni TGH9011, and transformed E. coliwere examined for ASL activity. E. coli C600, which is wild typewith respect to the argH gene, showed a specific activity of 1.1+ 0.17 ,umol of arginine per h per mg of protein. C. jejuniTGH9011 showed a specific activity of 1.3 ± 0.32 ,umol ofarginine per h per mg of protein. E. coli W3678 did not possessany significant ASL activity compared with extract-free con-

trols. The transformed strain pARGH1-1 had a specific activityof 5.7 ± 1.2 ,imol of arginine per h per mg of protein. Thisresult confirms that the protein derived from the recombinantplasmid possesses ASL activity. The C. jejuni protein is alsofunctional in the E. coli system.

Subcloning of the C. jejuni argH gene. From the comple-mentation experiments and the restriction enzyme maps ofthe recombinants, a 4.1-kb ClaI-AccI fragment of pARGH1was subcloned into pUC19, generating pARGH1-1 andpARGH1-2. Both orientations were able to complement the

ASL defect, indicating that both contained a functional argHgene and suggesting that the gene was being transcribed fromits own promoter.

Nucleotide sequencing. The ORF corresponding to theargH-complementing region was defined by examining thecomplementation ability of the various deletion mutants. The1,377-bp sequence representing the ORF of the argH gene, as

well as 540 bp of 3' and 800 bp of 5' flanking sequence DNA,was sequenced by using dideoxyribonucleotide chain termina-tion reactions. Three potential protein-coding regions (twoincomplete) were detected within the sequenced regions. TwoORFs lie on the same strand, with the phosphoenolpyruvatecarboxykinase reading frame overlapping the ASL initiationcodon by one base. The third, unidentified ORF is located on

the opposite strand and is separated from the terminationcodon of argH by 21 bp. The nucleotide sequence and thecorresponding amino acid composition of argH and the partialsequences for ppc are shown in Fig. 1.

Analysis of sequenced DNA. The coding region of the ASLgene is 1.377 kb in length and encodes a theoretical protein of459 amino acids with a calculated Mr of 51,831. The codonusage is typical of C. jejuni TGH9011 and favors codons whichare low in G+C content, typically in the third position of thecodon (8, 9, 17, 23). A Shine-Dalgarno sequence (33) is located10 bp upstream of the first ATG codon. Specific sequencescontrol the level of arginine biosynthetic gene transcripts in E.coli (14). Two regions present in the C. jejuni argH upstreamregion spanning nucleotides -65 to -48 and -43 to -26with respect to the initiation codon ATG (+ 1) show weakhomology (11 of 18 and 13 of 18 identical nucleotides) to theE. coli arg box consensus sequence (5'-[AT]ATGAATA[TA][AT]NATNCANT-3'), but particularly in the strongly con-

served bases at positions 3 to 7. Two tandem duplications occur

directly upstream of the argH initiation codon. The role of

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argH GENE OF C. JEJUNI 1869

APLASMID COMPLEMENTING

ACTIVITYpBR322pARGH-pARGH1

pARGHl-1 AX

pHR1

BKE

E Rv I

p8R3 E Rv

PUC19 AXBKE ApUC19 A EKBXA

AcGATAA

-248 G4AT

cA

FIG. 3. Mapping of the 5' end of the argH transcript, using primerextension. Lane X represents the reverse transcriptase products ofargH mRNA. Lanes G, A, T, and C represent the results of dideoxy-chain termination sequence reactions in the region encompassing thepromoter. The sequence of the 32P-end-labeled primer (5'-TTCJTrTAAAAGCTCATCACT'YGCATCAC-3') is complementary to nucle-otides 32 to 60 of argH. Bars indicate the transcription initiation siteand the upstream product.

these sequence motifs in argH regulation has not been estab-lished. Between the termination codons of argH and theunidentified ORF on the opposite strand is a short sequencewhich could form a stem-and-loop structure possibly involvedin transcription termination (Fig. 1).The C. jejuni argH gene shows 53.6% nucleotide identity to

yeast (5) and 44.3% to human (26) argH genes. The deducedamino acid residues are aligned with the complete proteinsequences of yeast (40% identity to C. jejuni ASL) (11), human(39% identity) (26), and rat (41% identity) (25) ASLs and withthe partial sequences of fungal (13) and E. coli (10) ASLs inFig. 2. The C. jejuni ASL also showed slight identity tofumarases and aspartases from a variety of sources (12 to 18%identity) (21, 27, 37, 40, 41). The upstream partially sequencedORFs showed significant identity at the amino acid level to thephosphoenolpyruvate carboxykinase (ppc) gene from a numberof organisms, as determined by the Blast algorithm of theGenBank data base search program (2).Primer extension. The 5' end of the argH mRNA was

determined via primer extension. A 28-mer synthetic oligonu-

pDTL A E

pDTH A E

pARGHl-2 A EKBX

arf3 argH PPC

B _ C4I I

a 0

CY D < m < <a 0c O- X- Q- X- X

FIG. 4. Maxicell analysis for the elucidation of the plasmid-en-coded protein corresponding to argH gene sequences. (A) Restrictionenzyme profile and phenotypic expression of ASL activity of selectedrecombinant plasmids used in maxicell analysis. Solid bars representpUC19 sequences, double bars represent pBR322 sequences, andsingle lines represent C. jejuni sequences. The transcription ofppc andargH is from right to left in the diagram. Abbreviations: A, AccI; B,BamHI; C, ClaI; E, EcoRI; K, KpnI; Rv, EcoRV; X, XbaI. (B) Maxicellproducts as analyzed by SDS-PAGE. The two protein bands producedin the recombinant pUC19 and pBR322 constructs are indicatedwith the open arrowheads. The position of the ampicillin resistanceproduct is indicated with the solid arrowhead. (C) Maxicell productsas analyzed by SDS-PAGE. The two protein bands produced inthe recombinant pUC19 deletion derivative of pARGH1-1 andpARGH1-2 are indicated by open arrowheads. The ampicillin resis-tance determinant is indicated with the solid arrowhead.

cleotide designated argH-1 complementary to argH mRNAwas chosen from an area which displayed minimal identity tothe known partial E. coli argH sequence. The size of theextended product was determined on a sequencing gel, withdideoxy sequencing reactions as size markers. As shown in Fig.3, the extended products produced were a band correspondingto the transcription start point for argH and an additionalupstream band. Transcription of the argH gene thus starts withan G residue located 248 nucleotides upstream of the initiationcodon (Fig. 1 and 3). The additional primer extension productindicates that argH may be cotranscribed with the upstreamppc gene transcript.

Maxicells. Plasmid proteins were detected through maxicell

BA A ECRvBE Rv C Rv BA A ECRv

C Rv I IXBA A ECRv

RV IXB A

;+

jXBA

IXBAIX=IXBARv

+

C

1 kb

I Is-~ p-

, i I- W.e D < a a < Icn Q- Q- a Q- Q- nQ

-200k

.116k

-97k.66k-43k <

-31 k <

4

__~~~~~~- I

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1870 HANI AND CHAN

analysis as shown in Fig. 4. Plasmid pARGH1 produces proteinbands in SDS-PAGE at 56 and 43 kDa which are not producedby plasmid pARGH- or by the pBR322 control plasmid.Plasmids pARGH1-2 and pARGH1-1, pUC19 recombinantscontaining a 4.1-kb C. jejuni insert in opposite orientationscontaining the complete argH gene, both express protein bandsat 56 and 43 kDa which are not present in the pUC19 andbackground controls (Fig. 4B). Deletion derivatives ofpARGH1-1 included plasmid pHR1, which contains argH anddownstream sequences and 300 bp of upstream sequences,and plasmid p8R3, which contains nucleotides encoding the20 amino acids of the carboxy-terminal region of ArgH andthe downstream sequences. Two deletion derivatives ofpARGH1-2 were also used. Plasmid pDTH contains approxi-mately 200 bp of sequence downstream of argH, the completeargH gene, and upstream sequences. Plasmid pDTL containsonly 200 bp of the amino-terminal region of argH and theupstream sequences (Fig. 4A). Controls included E. coliDR1984 with no plasmid and with pBR322 or pUC19.The two deletion derivates which express ArgH activity

(pDTH and pHR1) produce two protein bands at 56 and 43kDa. Since these deletion derivative lack either upstream ordownstream sequences, the polypeptides produced must there-fore come from within the coding region for argH. The twodeletion mutants which do not express ArgH activity (pDTLand p8R3) do not express either of these proteins, againindicating that both proteins come from within the codingregion of argH (Fig. 4A and C). There is no ORF apart fromthat encoding ArgH which might produce the 43-kDa protein.The smaller protein, therefore, reflects an alternate transla-tionally initiated protein or a degradation product of the56-kDa protein.

DISCUSSION

A pBR322 recombinant plasmid, pARGH1, was shown bycomplementation to contain a functional argH gene of C.jejuni. The C. jejuni argH gene is 1.377 kb in size and encodesa theoretical protein with an Mr of 51,831. The high level ofArgH activity from the recombinant plasmid pARGH1-1 indi-cates that a functional protein is being produced, as does theability of the C. jejuni plasmid product to complement the E.coli auxotrophic mutant. The size of ArgH as determined bySDS-PAGE was 56 kDa; the difference in size from thededuced Mr may represent a posttranslational modification(s)of the protein.A functional transcription start point was identified for argH

in the E. coli system and was located within the C. jejuni insertof pARGH1-1 at a position 248 bp upstream of the initiationcodon for ArgH. Significantly, the transcription start point forargH lies within an upstream ORF. This occurs within thecodon representing an amino acid 82 residues from thecarboxy-terminal end of the C. jejuni ppc gene product oramino acid 429 (of 553) as compared with the Saccharomycescerevisiae ppc gene product. An additional upstream transcrip-tional start point suggests that argH may be transcribed singlyor in concert with the ppc gene. Furthermore, the ppc ORFoverlaps one nucleotide of the initiation codon (ATG) of argH.A similar overlapping gene arrangement was also observed forthe lysS and glyA gene linkage (9), in which the two codingregions overlapped by one base pair and the promoter of glyAwas located within the coding sequence of lysS. These datatogether suggest that evolution has favored a highly condensedorganization of genes and promoter elements in C. jejuni.

C. jejuni ArgH shows considerable homology to other ASLs.There is also weaker homology to fumarases from E. coli,

Bacillus subtilis, human liver mitochondria, and S. cerevisiae, aswell as aspartases from E. coli and Pseudomonas aeruginosa,suggesting that the C. jejuni protein belongs to a family ofrelated enzymes which has developed from a single gene. Aconsistent feature of this family of enzymes is the amino acidmotif GS--M--K-N, which is also present in the C. jejunisequence from amino acid residues 278 to 287 (Fig. 2).Enzyme activity in E. coli C600 wild type and C. jejuni

TGH9011 is similar with respect to total cellular protein. In E.coli W3678, enzyme activity is not detectable, confirming thatthis strain is deficient in ASL activity. The transformed strainE. coli W3678 showed a level of enzyme activity four- tofivefold greater that wild-type E. coli. Such a results may bedue to the greater copy number of the argH gene in this cellthan in the wild-type system and may reflect a copy numberderepression of argH gene expression.Gene argH has been mapped onto the chromosome of C.

jejuni (18-20) and lies on the Sall A, SmaI A, and Sacll Afragments. Neither the genes upstream nor those downstreamof argH in C. jejuni appear to have any homology to otherarginine biosynthetic genes, and the genetic organization ofarginine biosynthetic genes in C. jejuni (15) differs from thatseen in E. coli, Salmonella typhimurium (14), B. subtilis (4, 12,28), or Neisseria gonorrhoeae (31). This unique arrangement incampylobacters prompts further analysis of the genetic ele-ments comprising this regulated system.

ACKNOWLEDGMENTS

We thank B. Bachmann for providing E. coli strains and J. L. Pennerfor providing C. jejuni TGH9011.

This work was supported by the Canadian Foundation for Ileitis andColitis and the Medical Research Council of Canada.

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