Mol Gen Genet (1993) 240:29-35 © Springer-Verlag 1993

Cloning and characterization of the gamma-glutamyl phosphate reductase gene of Campylobacterjejuni H. Louie, V.L. Chan Department of Microbiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8 Received: 22 September 1992 / Accepted: 8 February 1993

Abstract. The gamma-glutamyl phosphate reductase gene, proA, of Campylobacter jejuni was isolated from a recombinant pBR322 clone. A HindIII fragment of the insert containing the gene was subcloned into pUC19 and sequenced in both orientations. The deduced amino acid sequence of gamma-glutamyl phosphate reductase (EC 1.2.1.41) of C.jejuni exhibits 36.4% identity to that of Escherichia coli and 36.0% identity to Serratia marcescens. Two highly conserved regions in the amino acid sequence were identified from the alignment of the three available gamma-glutamyl phosphate reductase gene sequences. The gene was expressed from its own promoter and the transcription start site was mapped. The proline biosynthetic genes of C. jejuni are not located tandemly and thus differ in this respect from those of E. coli and S. marcescens, where gamma-glutamyl phosphate reductase and gamma-glutamyl kinase (proB) are located in a single operon. Key words: Campylobacter jejuni - proA - Proline - Sequencing

The conversion of L-glutamate to proline in Escherichia coIi is achieved in a four-step reaction catalysed by gamma-glutamyl kinase (EC 2.7.2.11), gamma-glutamyl phosphate reductase (EC 1.2.1.41), and 1-pyrroline-5carboxylate reductase (EC 1.5.1.2) which are encoded by proB, proA, and proC, respectively. In E. coli and Serratia marcescens, the proB gene is located directly upstream of the proA gene and they appear to share a common promoter (Deutch et al. 1984; Omari et al. 1991). Similarly, the proB and proA genes of Salmonella typhimurium have been reported to form a single operon with proB located proximally to the promoter (Mahan and Csonka 1983). In C.jejuni, Lee et al. (1985) demonstrated that a cloned 4.8 kb fragment of C. jejuni DNA was able to complement both proA and proB mutants of E. coli, and proposed that the C. jejuni proA and proB genes are also disposed in tandem. In this communication, we report the isolation and mapping of the C.jejuni proA gene and its complete coding sequence along with the 5' and 3' flanking sequences. This flanking region exhibits no homology with the E. eoli proB gene. Materials and methods

Introduction

Campylobacter jejuni is a gram-negative spiral microaerophilic bacterium, which is commonly associated with secretory type diarrhoea and enteritis (Penner 1988). Genetic studies of this organism are greatly hampered by the lack of genetic markers. Fifty-eight per cent of naturally isolated C. jejuni are auxotrophs requiring at least one supplementing amino acid (Tenover et al. 1985); Met- and Pro- are the two most common phenotypes and are potentially useful genetic markers. However neither the mutations nor the wild-type alleles have been physically mapped in the organism. Communicated by W. Goebel Correspondence to: V.L. Chan

Bacterial strains and plasmids. The C. jejuni genomic library was constructed from strain TGH9011 (ATCC 43431) (Chan et al. 1988). The bacterial strains and plasmids used in this study are listed in Table 1. C. jejuni was grown on chemically defined media (Tenover et al. 1985) in the presence or absence of proline at 37 ° C with 5% CO2. E. coli strains were grown in Luria medium at 37 ° C on plates or in broth cultures. For complementation tests, the E. coli strains were grown on minimal medium (Davis and Mingioli 1950) with 0.36% glucose as a source of carbon, and supplemented with required vitamins and amino acids (Novick and Maas 1961). For the analysis of plasmid-encoded proteins, overnight cultures in Luria broth were used to inoculate into minimal medium supplemented by 0.2% glucose and 1% casamino acids. Ampicillin and chloramphenicol were added when

30 Table 1, Bacterial strains and plasmids Strains or plasmids

Genotype or relevant characteristic

Source or reference

Campylobacter jejuni ATCC 43431

Serotype0:3

J.L. Penner

A (lac-proAB) thi strA supE44 endA sbcB hsdR4 F'traD36 proA +B+ laeI A (lacZ)M15) Nonmucoid form of CSR603 reeA1 uvrA6 phr-1 F- (X-) leu thr thi lac tsx strA proA

This laboratory

4.6 kb plasmid with the Amp r gene as selectable marker pBR322 recombinant clone containing 4.5 kb C. jejuni insert 2.69 kb plasmid with amino-terminal fragment of lacZ gene product and Amp' gene as selectable marker pUC19 containing 2.3 kb HindIII fragment of pBHL-8 pUC19 containing 2.3 kb HindIII fragment of pBHL-8 in reverse orientation pUC19 containing 2.2 kb claI fragment of pBHL-8 4.24 kb plasmid with Cm ~ and Tetr genes as selectable markers pACYC containing 2.3 kb HindIII fragment of pBHL-8

Bolivar et al. 1977 This work Messing 1983

Escherichia coli:

JM101 DR1984 X680 Plasmids: pBR322 pBHL-8 pUC19 pUH23 pUH23r pCC22 pACYC184 pACH23

Sancar et al. 1979 Hayzer and Leisinger 1980

This work This work This work Chang and Cohen 1978 This work

necessary to the media at final concentrations of 100 gg/ml and 20 gg/ml, respectively. Transformation of bacteria with plasmids was by the CaC12-RbCI procedure (Maniatis et al. 1982).

SDS-polyacrylamide gel. Standard low mol. wt. protein markers from Bio-Rad Laboratories were run alongside and the gel was stained and exposed to Kodak XAR-5 film.

Construction of deletion derivatives and D N A sequencing. Plasmid DNA was isolated by the alkaline lysis method (Maniatis et al. 1982). Nested deletions of C. jejuni DNA inserted in pUC19 were constructed using ExoIII and S1 nucleases (Henikoff 1984). DNA was sequenced by the dideoxy chain-termination method of Sanger et al. (1977) using the Sequenase kit from United States Biochemicals. The nucleotide sequence data given in Fig. 2 are available in the EMBL, GenBank, and DDBJ nucleotide sequence data bases under accession number M74579.

Primer extension analys& f o r localization o f the transcription start site. RNA was extracted from C. jejuni and from E. coli JM101 harbouring pBHL-8 and pUH23 by

the hot phenol method (Aiba et al. 1981). Oligonucleotide 5'-GTTTTTGAGAATTTTTCTTAATATTTTCAAGC-3', designated as P1 and located at nucleotides (nt) 154-184, was end-labelled and hybridized to the RNA extracts. The extension products generated with reverse transcriptase were fractionated on an 11% polyacrylamide gel along with the DNA sequence ladder generated from the same primer (Maniatis et al. 1982).

Southern analysis. DNA probes were labelled using

[a-3ZP]dATP and the Nick Translation Reagent kit from Bethesda Research Laboratories Life Technologies. After restriction enzyme digestion and electrophoresis, the target DNA was transferred to GeneScreen (NEN Research Products) by the method of Southern (1975). Filters were hybridized at 37 ° C in 50% deionized formamide, 1% sodium dodecyl sulphate (SDS), 1 M NaC1, and 10% dextran sulphate with the denatured probe at a final concentration of 1 x 105 cpm/ml and denatured salmon sperm DNA at 100 gg/ml, washed with 2 x SSC (1 x SSC is 0.15 M NaC1, 15 mM sodium citrate) with increase in stringency achieved by increase of temperature, followed by exposure to Kodak XAR-5 film. Analysis o f plasmid-encoded proteins.

E. coli strain

DR1984 was transformed with pBR322, pBHL-8, pACYC184 and pACH23. Maxicell preparations of the resulting strains were analysed for plasmid-encoded proteins as described (Chan and Bingham 1992). Briefly, the UV-irradiated cells were labelled with [asS]methionine, lysed and the protein extracts electrophoresed on an

C I

HHHSpplC SspI ~

i I

L7

(

r

H HH I

"IF"

-I proA pBHL-8

sppi

Sspl

HI

proA pUH23 C I

H H H SppI C "~'11 I I

VproA

j

j

lkb

pCC22

Fig. 1. Restriction enzyme map of pBHL-8, pUH23 and pCC22. The thick lines represent vector sequences and the thin lines represent Campylobacter DNA insert. The location of the proA gene is denoted by the horizontal line. The completeproA gene is located in the 2.3 kb HindIII fragmentwith approximately1.5 kb of insert DNA between the putative promoter of proA and the tetracycline promoter of pBR322 in pBHL-8. The direction of transcription of the vector and the putative proA promoters are indicated by the arrows. Restriction site abbreviations: C. ClaI; H, HindIII

pShl-Bh 10

20

30

40

50

60

70

80

90

100

110

120

AAG~TTGG~TTCAACTGc&cAGccTATCccTGTTGGACCTGcAc~TACTAcAATTAAATcTATTTTTTTCATTTATATTTACcTTT~AATTCTAAATTTTTTTGTATAATTTATcAAGTT

I----~130

140

150

160

170

180

190

200

210

220

230

240

TTTTGTAAATAAAGGATTAAAAATGCGAAAT T T GCTT GAAAATATTAAGAAAAATTCTCAAAAACTACTTAACT TAACGCCCAAAGATAAAGAAAAAAT TATTCTAAAATTAGCTCAAAT M

250

R

260

N

k

L

270

E

N

I

280

K

K

N

S

290

Q

K

L

L

300

N

L

3 I0

T

P

K

D

320

K

E

K

330

I

I

L

340

K: L

A

Q

350

I

360

T T TAAGAGAAAAT T T CAAAAT TAT C C TAGAAG CAAATAAAAAAGATA T GG CAAA T T T CACAAAAAGCGGCAAGAT GAAA GA TAG G C T T T T G T TAGA T GAAAAA CGTA T T T TAGC TCT T T G L

R E

N

F

370

K

I

I

L

380

E A

N K

390

K D M A

400

N

F

4 I0

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420

G K

M K D

430

R

L

440

L

L

D

450

E

K

R

460

I

L

A

L

470

C

480

C GAAGGCCTAGAAAAAATCGCTTACATCGAAGATCCTATAGGCAAAATTTCTAAAGGCTGGAAAAATTATG•GGGTTTAAACATACAAAAAATAAGCATTCCTTTAGGACTCATTTGTGT E

G

L

E

K

490

I

A

Y

I

500

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D

P

5 I0

I

G

K

520

I

$

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G W K

530

N

540

Y

A

550

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A

R

610

P

S

L

S

620

A

E

I

A

630

A

L

640

M

I

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S

650

S

N

A

C

660

L

N

I

Q

560

TATTTACGAAGCAAGGCCTAGTCT TAGCGCTGAAATCGCAGCTTTAATGATAAAAAGTTCCAATGCTTGTGTAT I

G

V

K

I

S

570

I

P

L

580

G

L

I

C

590

V

600

TTAAAGGCGGAAGTGAAGCAAAATTTACAAATGAGGCTATATTTAC F

670

K

G

G

S

680

E

A

K

690

F

T

N

700

E

A

I

F

710

T

720

TCTTGTTAATAAAGTACTTAAGGAATTTGATTTG•AAGATTGTTTTGCTATGTTTA••CAAAGAGATGAAATCTTGCAAATTCTAGCCTTTGATGATTTAATCGATGTGATCATACCTCG L

V

N K

V

730

L

K

E

F

740

D

L

Q D

750

C

F A

760

M F

T

770

Q R D

780

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I

L

790

Q

I

L

A

800

F D

D

810

L

I

D V

820

I

I

P

830

R

840

C GGAAGTTCAAATATGATACAAGAAATT GCAAACAATACCAAAATTCCTCTCATTAAGCAAAATAAAGGCTTGTGTCATG~TTTTGTAGATCAAAG1~G~TAACTTAGATATGG~TTTAAA G S

S

N M

850

I

Q E

I

860

A

N N

870

T

K

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880

P

L

I

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890

Q N K

900

G L

C H A

910

F

V

920

D Q $

930

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940

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950

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960

AATAATCCTT~T GCAAAGTGTCAAAGAGTAAGTGTTTGTAACGCTTTAGAAACGCTTTTAATCCATGAAAAAAT TGCTAAAAATTTTATAJ~GTCTTTTAATACCTGAATT TGA/blb~TT I

I

L

N

A

970

K

C

Q

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980

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S

V

990

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I000

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1060

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1080

TAAGGTAAAAATTCACGCCCATGAAAATACTTTGGCTTATTTTAACAACTCAAATTTAGAAATTTTTAAAGCAAATGAAAATACCTTTGATACAGAATGGCTTGATTTTGCTTTAAGTGT K

V

K

!

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1090

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1100

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1110

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1120

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1130

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1140

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N

1150

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1160

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1170

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L

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1180

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A

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1190

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V

1200

AAAAT TAG TAAAA GA T T G C GA T GAA G C T A TA GA G CA TA T CAA TAAA CA CA G C T CC T T G CA T T C T GAAA C CA T TA T C T CAAA T GA T G C T T CAAA TA T C G C TAAA T T T CAAC G C C T TA TAAA K

L

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1220

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1230

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1280

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1300

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1310

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1320

•TCATCTTGTATTTATGCTAATGCTTCAA•G•GTTTTAGCGATGGAGGAGAATTTGGCTTTGGTGGAGAAGTTGGAATTT•AACCAGCAAATTGCATGCAAGAGGTCCTATGGGGATTGA S

S

C

!

Y

1330

A

N

A

1340

S

T

R

F

S

1350

D

G

1360

G

E

1370

F

G

F

1380

G

G

E

1390

V

G

I

1400

S

T

S

K

14 I0

L

H

A

1420

R

G

P

M

G

1430

I

E

1440

A GATAT T TGCACT TATAAATA TA TAA T C AAT G GGGAAG GACAAA T T C GAGAGTG_G_AATAAAATAG.___~TACTAAAGCAA CACT T TAAAGAAAT T GC TAAAT TAAAT T CAAGCGAACAAGT T D

I

C

1450

T

Y

K

1460

Y

I

I

N

1470

G

E

G

1480

Q

I

R

1490

E

1500

1510

1520

1530

1540

1550

1560

TGCA~TGCCIITTTTTGCGGCTTTAGGCGTAGGTATAGITTTAGGGCTTAGTGTATITTTTGG~'TI~CTATGGACTCATTGCCATGATAGGAGCTTTGTCTTT~ITATATGTTc

1570

1580

1590

1600

1610

1620

1630

1640

1650

1660

1670

1680

C~`A~ACTCCTTTATAT~ATAGAATGGCTGTTGTGATGTGCTGTTCTTTTGGCATAGTTTCTAGTTTTTTCTTAGGAATTTTAACT~ATTTTTTGC~TGCAATTTTTGC~TTCATC~TA

Fig, 2. The nucleotide sequence and deduced amino acid sequence ofproA. The putative promoter and the ribosomal binding site are denoted by horizontal lines above the sequences. The stop codons are underlined and the amino acid sequence ofproA is given below the nucleotide sequence. The region hybridizing to the oligonucleotide probe used in the initial screening is boxed. The transcription

start sites are marked with a bent arrow. The DNA fragment was sequenced in forward and reverse directions by dideoxy chaintermination methods (Sanger et al. 1977). A total of 700 nucleotides upstream and 700 nucleotides downstream of the proA encoding sequence were sequenced but only part of these flanking sequences are shown

32 Results

Nucleotide sequence of proA The C. jejuni T G H 9 0 1 1

r e c o m b i n a n t p B R 3 2 2 library

(Chan et al. 1988) was screened with a mixed oligonucleotide probe for the enterotoxin gene of C. jejuni. A false positive recombinant pBR322 clone, pBHL-8, was shown to contain a gene that displayed homology to the gamma-glutamyl phosphate reductase gene of E. coli in a region directly upstream of the site homologous to the oligonucleotide P1. The 2.3 kb HindlII C. jejuni D N A fragment containing the putative proA gene and the overlapping ClaI fragment from pBHL-8 (Fig. 1) were subcloned into pUC19 forming pUH23 and pCC22, respectively, then sequenced in the forward and reverse directions. A 1680 nt sequence consisting of the putative proA gene along with the predicted 411 amino acids are shown in Fig. 2. The molecular weight of the putative proA protein is 46 kDa. The codon usage resembles that of the 91yA (Chan and Bingham 1991), lysS (Chan and Bingham 1992) andfla (Nuijten et al. 1990; Khawaja et al. 1992) genes of C. jejuni, and is typical for a gene in a genome with low G + C content. Synonymous codons with A or T in the third base are preferentially used. The percentage of A + T at the third base position of all the

codons is 77%, which clearly correlates with the high A + T content (69%) of the C. jejuni genome. A 31 mer oligonucleotide, P1, complementary to the proA m R N A transcript was used to locate the transcription start site. Two equally strong transcription start sites were located in the sequence, separated by 3 bp, at nucleotides 123 and 127 (Fig. 3). Located five nucteotides upstream of the Met start codon (ATG) is the sequence AAGGAT, which matches five of the six nucleotides of a Shine-Dalgarno (Gold 1988) sequence (Fig. 2). At nt 107-112, the sequence TAATTT, conserved in three of the six nucleotides of the Pribnow box, occurs. Located 2 bp upstream of this Pribnow box the sequence TATAAT is found. However the mapping of the transcriptional start sites at nucleotides 123 and 127 indicates that the sequence at nt 107-112 is more likely to be the Pribnow box. Upstream of this putative - 10 region at nt 86-90 is the sequence TTCAAT which matches the - 3 5 consensus sequence (TTGACA) in three of the six nucleotides (Hawley and McClure 1983). There are also several repeats and inverted repeat sequences in the up-

1

MLEQMGIAAKQASYKLAQLS ...... K ...... WQ--V--RNLLENIK-NSQ. --LN-T

SREKNRVLEKIADELEAQSE TAK--Q--SVM--R---N-PKD-EKIIL-L-QI-RENFK

I ILNANAQDVADARANGLSE A--L--E--M-Q---T-M----E--I~K-M-NFTKS-. K.

AMLDRLALTPARLKGIADDV -L .... L ...... AA--N-.-K---L-DEK-ILALCEGL

RQVCNLADPVGQVIDG. GVL . . . . R - N . . . . H-L--. N L EKIAYIE--I-KISK-WKNY

A.--NIQKIS~---L-C---

116

---~SLSAEI-A-MI-SS--

VILRGGKETCRTNAATV~VI ......... HN--Q---K-CVFK--S-AKF--E-IFTLV

160 156

QDALKSCGLPAGAVQAIDNP -Q--EQ ..... A ...... SNKV--EFD-QD. CFAMF. TQ

DRALVSEMLRMDKYIDMLIP ..... N-L--L-R-V ..... .-DEILQI-AF-DLI-VI--

200

RGGAGLHKLCREQSTIPVIT

193

--SSNMIQEIANNTK--L-K

GGIGVCHIYVDESVEIAEAL ....... T---AD-DFDK-QNK-L--AF--Q-ANLDM--

240

KVIVNAKTQRPSTCNTVETL T--E---I .... A--SL---I-L---C--V-V--AL---

LVNKNIADSFLPALSKQMAE ---RS--AE ...... AK--A -IHEK--KN-ISL-IPEFEK

SGVTLHADAAALAQLQAGPA A ...... AEN--PL--G--FK-KI--HENT--YFNNSNL

KVVAVKAEEYDDEFLSLDLN T--P-N--D .... W ...... EIFKANENTF-T-W-DFA-S

313

VKINSDLDDAIAHIREHGTQ -LL-D-I-Q--D---T---N --LVK-C-E--E--NK-SSL

HSDAILTRDMRNAQRFV... ........ SLSS-EH--RAV --ET-ISN-AS-IAK-QRLI

3s7

;;~;V~N~ST~F~DGGQFG

353

NSSCIYI~- .....

390

EALTTYKWIGIGDYTIRA D ......... Y--DLV-S -DIC---Y-IN-EGQ--E

1 41

12

3 G A

T

40

C

81 77

T

>

C A A A A A* A C A T* T

233 280 273 320

393

Fig. 3. Primer extension mapping of the transcription start site on the proA mRNA. Lanes G, A, T, C are the chain-terminated products showing the complementary sequence of the transcription start site area, Lane 1 shows C.jejuni mRNA extension, lanes 2 and 3 show the mRNA extension products of DR1984 containing pBHL-8, and pUH23, respectively. The nucleotides corresponding to the transcription start sites are indicated by the asterisks

S---E--

F-G--GI--S

........

~I

Fig. 4. Amino acid sequence comparison ofproA gene product of C.jejuni, Escherichia coli and Serratia marcescens. The top sequence in the comparison is the proA gene product of E. coli, followed by S. marcescens and C.jejuni at the bottom. Identical amino acids are designated by dashes,missing amino acids are indicated by dots, and the highly conserved amino acid regions common to all three bacteria are boxed

33

kDa

1

2

3

4

5

kb

66 ProA 43 31

21 lO 8.4

Fig. 5. Maxicell analysis of plasmid encoded protein. Lanes 1-4: DR1984 containing pACH23, pACYC, pBHL-8, and pBR322, respectively;lane 5, bacterial strain DR 1984. The molecularweight markers are indicated by the arrowheads at the left-hand side stream region of this putative proA (data not shown). These sequences may be involved in the regulation of this gene but their exact role remains to be established. The nucleotide sequence of the putative C. jejuni proA gene exhibits 48.6% identity to the proA gene of E. coli and 45.5% identity to the proA gene of S. marcescens, while the translated amino acid sequence exhibits 36.4% identity to the corresponding sequence of E. coli and 36.0% identity to S. marcescens (Fig. 4).

Maxicell analysis A 2.3 kb HindIII C.jejuni insert from pBHL-8 containing the putative proA gene was subcloned into plasmid pACYC 184 in order to determine whether the gene could be expressed in E. coIi. Plasmids pBHL-8, pBR322, pACH23, and pACYC 184 were transformed into E. coli strain DR1984. As shown in Fig. 5 (lanes 1 and 2) plasmid pACH23 produced a 46 kDa polypeptide, which was not synthesized from the parental vector pACYC184. Plasmid pBHL-8 also encoded two extra polypeptides, of 40 kDa and 46 kDa, when compared to the pBR322 plasmid (see lanes 3 and 4). Since the 2.3 kb HindlII fragment was common to both pACH23 and pBHL-8, this suggests that the 46 kDa protein is the proA gene product of C. jejuni. The 27 kDa protein in pACYC184 and pACH23 is the chloramphenicol acetyltransferase. The 28 kDa and 31 kDa proteins in pBR322 and pBHL-8 are beta-lactamase and its precursor. The 37 kDa protein common in pACYC184 and pACH23 is the tetracycline resistance determinant (Tet). The 40 kDa protein seen in pBHL-8 and not pACH23 may be a Tet-C.jejuni fusion protein.

2.3 2.2

Fig. 6. Southern blot hybridization of C. jejuni chromosomal digest using as probe a radiolabelled 1.0 kb SppISspI DNA fragment of the proA sequence. Lanes 1-3: ClaI, HindIII, and EcoRV digested genomic C. jejuni DNA respectively. The Southern transfer of the gel was exposed overnight at -70 ° C after washing with 2 x SSC for 30 rain at 60° C

(Fig. 6) in order to determine the copy number of this gene in C. jejuni. This probe hybridized to two ClaI fragments, of 10 kb and a 2.2 kb, a single 8.4 kb EcoRV band, and a 2.3 kb HindIII band. These findings indicate the presence of a single copy gene in C. jejuni.

Complementation analysis A complementation test was performed by transforming X680, an E. coli proA mutant, with plasmids containing the 2.3 kb HindIII fragment and with the corresponding controls. These plasmids included pBHE-8, pACH23, pUH23, and pUH23r. Plasmids containing the 2.3 kb HindIII fragment were all capable of complementing the proA E. coli mutant, X680. DNA preparations of all Pro ÷ Amp + transformants contained the 2.3 kb HindIII fragment. However plasmid DNA isolated from those transformants on ampicillin-containing plates revealed size heterogeneity and all of the plasmid DNAs were smaller than the corresponding plasmid vectors transformed. The frequency of Amp + transformants on plates with proline was also 10-fold higher than the transformation frequency on Amp plates without proline. These results suggest that sequences located within the 2.3 kb HindIII fragment may be involved in the destabilization of plasmid DNA. Instability in E. coli of cosmid recombinant clones of C. jejuni DNA carrying leucine biosynthesis genes has been reported (Labigne et al. 1992).

Southern hybridization Discussion

A portion of the putative proA gene, a 1.0 kb SppI-SspI fragment containing the internal ClaI site, was labelled and hybridized to digested genomic Campylobacter DNA

In this study, the C. jejuni gene encoding gammaglutamyl phosphate reductase (proA), an enzyme in-

34 volved in proline biosynthesis was sequenced and characterized. There is 36.4% and 36.0% amino acid identity between the gene product of C. jejuni and the proA gene products of E. coli and S. marcescens, respectively; moreover, the ability of the C. jejuniproA gene to complement proA E. coli mutants suggests the presence of a similar proline biosynthesis pathway. The protein sequence alignment of all three proA gene products revealed two highly conserved regions (Fig. 4.). Although gamma-glutamyl phosphate reductase is N A D P + dependent, neither of these regions contains the typical Gly-X-Gly-X-X-Ala-XX-X-Ala NADP-binding domain (Scrutton et al. 1990). These highly conserved regions may therefore be responsible for substrate binding. The proBA mutation in E. coli can be complemented by a single cloned D N A fragment from C. jejuni (Lee et al. 1985). These findings suggested that the proB gene of C.jejuni should be in the vicinity ofproA. In E. coli and S. marcescens, proA and proB genes had been found to be contiguous and their coding sequences were within 15 or 12 bp of each other (Deutch et al. 1984; Omori et al. 1991). In contrast in C.jejuni, proB could not be detected by sequence homology within 700 bp upstream or downstream ofproA. These results indicate that the proA and proB genes in C. jejuni are not disposed in tandem and are probably in separate operons located at least 700 bp apart. The proA gene also includes its own promoter containing putative - 3 5 and - 1 0 regions, which are capable of directing expression the gene in an E. coliproA mutant. The transcription start site of this gene has also been located at positions 123 and 127, 16-20 nucleotides upstream of the initial Met residue. The plasmids pBHL-8, and its subclones pUH23, pUH23r and pACH23 containing the 2.3 kb HindIII fragment were all capable o f complementing the E. coli proA mutant, strain X680. Since the transcriptional start sites were mapped on the m R N A extension products of pBHL-8 and pUH23, this confirms the presence of a functioning promoter adjacent to the proA coding sequence of C. jejuni. The discrepancy observed between the complementation and transformation frequencies suggest the possibility that sequences located close to proA are involved in the destabilization of the plasmid DNA. This effect may partly account for the high frequency of P r o - auxotrophs seen in natural isolates of C. jejuni. However the precise region of the D N A that is responsible for the rearrangement o f the plasmid still remains to be elucidated. The C.jejuniproA has also been shown to be a single copy gene. Southern blot hybridization with the labelled proA gene fractionated on pulsedfield agarose gels of digested C. jejuni genomic D N A show that proA maps in the 220 kb SaII (C), the 300 kb SmaI (C), and the 185 kb SacII (E) genomic fragments (Kim et al. 1992). Further studies are likely to include the identification of the precise region within the 2.3 kb HindIII fragment that is responsible for D N A rearrangement and the isolation and characterization of the proB and proC genes of C. jejuni. Such studies would further our understanding of the proline biosynthesis pathway in C. jejuni and also provide the tools with which to analyse the molecular

basis of the high frequency of proline auxotrophic C. jejuni isolates.

Acknowledgements. We thank Dr. T. Leisinger for providing the E. coli strain, Mrs. Hermine Bingham for her technical assistance and invaluable advice and Mr. Eric Hani for his help in the preparation of the manuscript. This work was supported by the Medical Research Council of Canada and the Canadian Foundation for Ileitis and Colitis.

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