MOLECULAR CHARACTERIZATION OF THE HIPPURATE HYDROLASE GENE IN HIPPURATE HYDROLASE-NEGATIVE

CAMPYLOBACTER JEJUNI ISOLATES

Tin Htwe Thin

A thesis submitted in conforrnity with the requirements for the degree of Mater of Science,

Graduate Department of Microbiology, in the University of Toronto

O Copyright by Tin Htwe Thin 1997

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Molecular Characterization of the Hippurate Hydrolase Gene in Hippurate Hydrolase-Negative Campylobacter jejuni Isolates

Degree of Mater of Science 1997 Tin Htwe Thin

Department of Microbiology University of Toronto

CampyZobacter jejuni is human entericbacterial pathogen. The present study

examined the hippurate hydrolase gene from five different hippurate hydrolase-

negative C. jejuni dinical isolates. Radiolabeled single strand confomational

polymorphic (SSCP) assay detected the polymorphism in F7 and F8 regions of the

hippurate hydrolase gene from five negative isolates compared with the respective

regions of C. jejuni TGH9011 hipO. The promoter region and the remainder coding

region revealed an identical PCR-SSCP patterns compared with the respective regions

of C. jejuni TGH9011 in five negative isolates.

The potential mutated regions were cloned and sequenced to determine the

rnolecular nature of the mutations. Most of the mutations detected were silent in

nature and they do not affect for arnino acid alteration, except for valine (Val) at

residue 250 altered to alanine (Ala). The Val-250 change was consistently present in

al1 five negative isolates. Val-250 residue may play an important role in the hippurate

hydrolase function.

Acknowledgments

My deepest thanks go to my major professor, Dr. Voo Loong (Ricky) Chan for

giving me the opportunity to participate in Campylobacter jejuni genetic studies. My

appreciation is also given to my comrnitee members Dr. A. Bognar and Dr. M.

Kregden for their advice and editorial assistance.

1 owe my sincere gratitude to my parents U Thin Tu and Daw Tin Tin Win and

my only sister, Tin 00 Thin for their permanent support through my career.

1 would like to extend rny apprteciation to my colleagues and fiiends, especialiy

to Helena Louie, for their help and encouragement.

1 aioie 01 conrenrs

Abstract Acknowledgement Table of contents List of figures List of tables List of abbreviations

Introduction

General features of the genus Campylobacter Phylogeny of Campylobacter Clinical features of Campylobacter jejuni nfect ion Virulence factors of Campylobacter jejuni Adhesins Campylobacter toxins Epidemiology of Campylobacter jejuni infection Genome size of Campylobacter jejuni Genes cloned from Campylobacter jejuni Housekeeping genes Virulence gene Antibiotics resistance gene Hippurate hydrolase of Campylobacter jejuni TGH90 1 1 Hmologous proteins belong to M40 hydrolase farnily Hippurate hydrolase-negative Campylobacter jejuni Purpose of this study

Materials and Methods

Bacterial strains Genomic DNA extraction OIigonucleotides Polymerase chah reaction Single strand conformational polymorphic(SSCP) assay and mutation detection enhancement (non denaturing) gel electrophoresis Cloning of the arnplified fragments Ligation of the DNA fragments Preparation of competent cells and transformation with

Kecombinant plasmid DNA preparation Preparation of the squencing grade plasmid DNA DNA sequencing and polyacrylarnide gel electrophoresis

Results

Detection of the hippurate hydrolase sequence heterogeneity in the hippurate hydrolase-negative C. jejuni clinical isolates Single strand conformational polymorphic analysis of naturally occuring mutant hippurate hydrolase genes Cloning of SSCP polymorphic regions and DNA sequencing Sequence comparison Predicted hydropathy profiles

Discussion

Future studies

References

Figure

Biochemical reaction of hippurate hydrolase Nucleotide sequence of C. jejuni TGH9011 hippurate hydrolase Physical map of C. jejuni TGH9011 Strategy for single Strand conformational polymorphic (SSCP) assay based on polyrnerase chain reaction PCR-SSCP analysis of F7 and F8 regions of the hippurate hydrolase gene of five different hippurate hydrolase-negative C. jejuni isolates PCR-SSCP analysis of F 1 , F2, and F3 regions of five different hippurate hydrolase-negative C. jejuni isolates Deduced amino acid sequence alignment of the mutated region of hippurate hydrolase-positive C. jejuni TGH90 1 1 and hippurate hydrolase-negative strain D835 Alignment of C. jejuni hippurate hydrolase and related proteins Comparison of the hydrophobicity profiles for arnidohydrolases of Campylobacter, Bacillus, Pseudomonas, and Synechocystis Predicted hydropathy (hydrophobicity and flexibility) profiles of C. jejuni hippurate hydrolase Comparison of the predicted secondary structure and hydrophobicity profiles of C. jejuni hippurate hydrolase and its modified sequence

Lists of tables

Table

1. Classification of the genus Campylobacter and its relatives 2 . Codon usage and molecular percentage of G+C content of C. jejuni TGH9011

hippurate hydrolase. 3 . Homologous proteins of C. jejuni hippurate hydrolase 4. Bacterial strains used in this study 5 . OIigonucleotides used in this study 6. Primer pairs used in this study 7. Summary of PCR analysis in the hippurate hydrolase gene of hippurate

hydrolase-negative C. jejuni isolates 8. Nucleotide substitutions between nucleotide 473 and 912 of the hipO gene of

C. jejuni hippurate hydrolase-negative isolates

a

P + - % ala A ~ P Arg ATP bp ' C C. CHC13 Ci CO2 CIAP CJT Ch1 CLDT cm CNW CP* CT D DNA dATP dCTP ddNTP dGTP dNTP ddTTP DMSO Dnase DTT EDTA EtBr EtOH fig g gly GTE H2

H 2 0

H2S

alpha beta positive negative percent alanine ampicillin arginine adenosine triphosphate base pair degree CeIsius Campylo bacter chloroform curie carbon dioxide calf intestinal alkaline phosphatase CampyIobucter jejuni toxin chlorarnphenicol cytolethal distension toxin centimeter catalase negative/weak counts per minute cholera toxin daltons deoxyribonucleic acid 5'-deoxyadenosine triphosphate 5'-deoxycytosine triphosphate 2',3'-dideoxynucleotide triphosphates 5'-deoxyguanine triphosphate 2'-deoxynucleotide triphophates 5'-deoxythymine triphosphate dimethy lsulphoxide deoxyribonuclease dithiothreitol ethylenediarninotetraacetic acid ethidium brornide ethanol figure gram glycine glucose, tris, EDTA buffer hydrogen water hydrogen sulfide

H 1P hr 1 A A ile IPTG K kb kDa Km L LB 1bs.lsq.in pCi Pg P 1 M Mb mg Mg MgCl2 MH min ml mM mol%G+C MOPS M r

mFWA N N-Bz- Na NaCl NaOH ng nM nt OD PAGE PCR PEG PH PI pmole PFGE phe

nlppurare nyaroiase hour Indole-3-acetic acid-arnino acid isoleucine isopropyl-P-D-thiogalactoside potassium kilobase kilodalton Michaelis constant liter Luria Bertani broth pounds per square inch microcurie microgram microliter molar megabase milligrarn magnesium magnesium chioride MuelIer Hinton minute milliliter millimolar mol percentage guanidine plus cytidine 3-[N-Morpholino]propane-sulfonic acid relative molecular mass messenger ribonucleic acid normal fi- benzoyl- sodium sodium chloride sodium hydroxide nanometer nanomolar nucleotide optical density polyacrylamide gel electrophoresis polmerase chain reaction polyethylene glycol power of hydrogen isoelectric pH picomole pulsed- field gel elctrophoresis phenylalanine resistance

KNA

RNase rRNA

SDS SSC

SPP* subspp. TBE TE TEMED tet TLC Tris R N A TS

val W

ri bonucleic acid ribonuclease ribosomal ribonucleic acid revoiution per minute sensitive sodium dodecyl sulphate sodium chloride/sodium citrate solution species species(plura1) subspecies Tris boric acid EDTA buffer Tris-EDTA N,N,N',N1-tetramethylene diamine tetracycline thin-layer chromatography tris(hydroxymethy1) benzidine transfer ribonucleic acid type strain unit microcurie ultraviolet volt valine watt weight per volume times [multiplication] times [concentration strength] 5-bromo-4-chloro-3-indoyw-D-galactopyranoside

General features of the prenus Campvlobacier

Members of the farnily Campylobacteraceae are classified as

chemoorganotrophs. They do not ferment or oxidize carbohydrates. They appear as

curved in spira-, V-, or comma-shaped or occasionally straight rods that are 0.2 to 0.9

pm wide and 0.5 to 5 pm long. They rnay occur in short or occasionally long chains.

CoIonies are usually nonpigmented and are weakIy or non-hemolytic. The cells are

Gram-negative and non-sporeforming. Motility occurs as a result of single or

occasionally multiple unsheathed flagella at one or both ends of the bacterial cells.

Campylobacters are usually microaerophilic with a respiratory metabolism. However.

some strains may grow under aerobic or anaerobic conditions. They use

menaquinones as the sole respiratory quinones. A variety of organic acids including

amino acids are used as carbon sources. Oxidation of tricarboxylic acid intermediates

and the dearnination of amino acids are the main routes through which cellular energy

is derived (Hoffman and Goodman, 1982). Optimum temperature for growth ranges

from 30' to 42'C. Biochemical tests give negative reactions in the methyl red and the

Voges-Proskauer tests and there is no production of indole. Gelatin is not liquified.

Al1 species have oxidase activity. Most species c m reduce nitrate and do not

hydrolyze hippurate (Smibert, 1 984; Penner, 1 988; Penner, 199 1 ; Ursing et al., 1 994).

Phvlogeny of Campyiobacter

Campylobacters were isolated and first named as Vibri~~fett ts due to the

- Y

Vibrio (McFadyean and Stockman, 19 1 3). Further investigations ihstrated a low

guanine plus cytosine (G+C) base composition (28 to 47 molar percentage),

microaerophilic growth requixements, nonfermentative metabolism and lack of

oxidation of carbohydrates. These Vibrio - Iike organisms were then renamed as a

new genus, Campylobacter (Sebald and Veron, 1963). Cornprehensive taxonornic

studies of Vibrio - like organisms resulted in renarning Campylobacter fetus as the

type species (with C.fetus subsp. fetus for the organisms that cause sporadic abortions

in cows and ewes and C. fetus subsp. venerealis for the organisms that are responsible

for infectious infertility), along with Campylobacter jejuni, Campylobacter coli and

Carnpylobacter sputorurn (C. sputorum subsp. sputorum, C. sputorum subsp. bubulus)

(Veron and Chatelian, 1973).

Partial 16s ribosomal ribonucleic acid sequences of Campylobacter species

were used to compare and analyze the relationships of these organisms to one another

and to other gram-negative bacteria. The genus Campylobacter was divided into three

separate ribosomal ribonucleic acid sequence homology groups. Homology group 1

contains the true Campylobacter species which are C. fetus (two subspecies), C. coli,

C. jejuni, C. laridis, C. hypointestinalis, C. concicus, C. mucosalis, C. sputorum, and

C.upsaliensis ( C N W strains). Homoiogy group II includes C. cinaedi, C. fennelliae,

C. pylori and W. succinogenes. Homology group I I I consists of C. cryaerophila and

C. nitroflgilis. These three homology groups are distantly related to the representative

of the alpha, beta, and gamma branches of the purple bacteria (Thompson et al.,

1988).

limited the genus Campylobacter to C. fetus, C. hypointestinalis, C. concisus, C.

mucosalis, C. sputorum, C. jejuni, C. coli, C. lari, and C. upsaliensis. C. cinaedi and

C. fennelliae were included in the genus Helicobacter as Helicobacter cinaedi and

Helicobacter fennelliae. Two species of the genus Carnpylobacter, C. nitrofigilis and

C. cryaerophilia were constituted as a new genus Arcobacter and were renamed as

Arcobacter nitrofigilis and Arcobacter cryaerophilus. Wolinella curva and Wolinella

recta was transferred to the genus Campylobacter as Carnpylobacter curvus and

Campylobacter rectus, respectively (Paster and Dewhirst, 1988; Vandamme et al.,

1991). Thus, Wolinella succinogenes is the only species of the genus Wolinella.

Based on the rRNA homology studies, Bacteroides gracilis and Bacteroides

ureolyticus are generically misnarned and are closely related to rnembers of the genus

Campylobacter (Vandamme et al., 1991). B. ureoZytictcs and B. graciIis are

microaerophilic and not anaerobes. Recently, B. gracilis was reclassified under the

genus CrrmpyZobacter as Campylobacter gracilis based not oniy on genotypic data, but

also on the proteolytic metabolism of respiratory quinones, protein profiles and

cellular fatty acids analysis (Vandamme et al., 1995). Presently Campylobacter hyoilei

is grouped under the genus Campylobacter and recognized as closely related to C.

jejuni and C. coli on the basis of their G+C contents, phenotypic characteristics,

hybridization with a speci'es-specific DNA probe, 16s rRNA sequence cornparison

data and DNA-DNA hybridization data (Alderton et al., 1995).

Table 1 presents the list of known species of Carnpylobacter, Helicobacter, and

Arcobacter according to the recent presentation of taxonomie position, known sources

and common diseases associated with Campylobacteria (On, 1996).

Genus Campylobacter C. coli ( Vibrio coli) C. concisus (?Pol inella curva) C. cuwus C. fetus subsp. fetus ( Vibrio fetus)

su bsp. venerealis ( Vibrio fe f us) C. gracilis C. helveticus C. hyoilei C. hyointestinalis C. jejuni subsp. jejuni ( Vibrio jejuni)

subsp. doylei C. lari C. mucosalis C. rectus C. showae C. sputorurn biovar sputoruni

biovar bubulus biovar fecalis

C. upsaliensis

Genus Arcobacter A. butderi A. cryaerophilus A. n itr0figili.s A. skirowii

Genus Bacteroides [Bacteroides] ureolyticus

Genus Helicobacter H. acirlonyx H. canis H. cinaedi H. felis H. fenrzelliae H. heilma~zrzi H. hepaticus H. muridarum H. nzustelae H. nernestrinae H. pamete~tsis H. pullorum H. pylori H. rappini

("C. butder?') (C. cryaerophila)) (C. nitrofgilis)

(C. cinaedi)

(C. fennelliae) (Gastrospirillum hominis)

(C. mustelae)

(C. pylori) (Flexispira rappini)

Gen us Wolirtella W. succinogenes

A wide spectmm of clinical features are associated with C. jejuni infections

(Skirrow and Benjamin, 1981; Allos and Blaser, 1995). The main manifestation is

diarrhea, ranging fiom watery to inflarnmatory dysentery with bloody diarrhea. Other

symptoms often present are fever, abdominal pain, nausea, headache and muscle pain

(Walker et al., 1986; Penner, 1991). Human infections with Campylobacter sp. occur

through the ingestion of contaminated food, milk and water. Generally, the most

cornmon origins of infection are uncooked poultry, beef and unpasteurized milk. The

ilhess usually occurs 2 to 5 days afier ingestion of the contarninated water or food.

The diarrhea usually continues for 2 days to a week. The organisms remain in the host

for 2 weeks to 3 months unless antibiotic therapy is taken. Early treatment with

erythromycin may reduce the length of time that infected individuals shed the bacteria

in their feces. Human feeding studies suggest that as few as 500 celis may cause

illness in some individuals. Histopathologic examination of infected colon usually

reveals diffuse inflammation of the lamina propria by neutrophils and mononuclear

cells, and injury of the surface epithelial cells. In some cases, the tissue damage

caused by C. jejuni infection resembles ulcerative colitis or Crohn's disease (Green et

al., 1984). Other abdominal complications of C. jejuni infections are gastrointestinal

hemorrhage, toxic megacolon, pseudomembranous colitis, cholecystitis, and

pancreatitis.

In addition to the gastrointestinal illness caused by C. jejuni, multi-systemic

disorders, which include septicemia, meningitis, septic abortion, Reiter's syndrome,

and reactive arthritis (Johnson et al., 1983). Most recently, Guillian-Barre syndrome

has been reported (Molnar et al., 1983; Kaldor and Speed, 1984). Guillian-Barre

etiology and pathogenesis of this illness is not completely understood. Several recent

studies (Bolton, 1995; Ho et al., 1995; Rees et al., 1995) support an earlier observation

(Kaldor, 1984) that intestinal infections with C. jejuni often precede the onset of

Guillian-Barre syndrome.

Virulence factors of Campvlobacter iejuni

Three potentially pathogenic properties have been identified for C. jejuni;

invasiveness, enterotoxin and cytotoxin production.

Adhesins

C. jejuni utilizes its adhesive ability to colonize the intestinal epithelium,

invade the intestinal mucosa and proliferate in the lamina propria and mesenteric

lymph nodes of the host. Flagella, outer membrane proteins and lipopolysaccharide

(LPS) are factors involved in bacterial adherence to epithelial cells and mucus

(McSweegan and Walker, 1986; deMe10 and Pechere, 1 990). Motility can be directed

by chemotactic factors including L-fùcose, L-aspartate, L-cystine, L-glutamate, and L-

serine, pyruvate, succinate, fumarate, citrate, malate, and oc-ketoglutarate. The C.

jejuni flagellum is able to undergo both phase and antigenic variation (Caldwell et al.,

1985; Harris et al., 1987). Both mechanisms may play a role in the ability of the

organism to evade the host immune response. The pathogen is able to revert to

flagellate forms in phase variation and can alter irnmunological specificity under

antigenic variation. Aflagellated mutants constructed by gene replacement techniques

were utilized in in vitro assays for adherence and penetration o f intestinal cells and

(Grant et al., 1993). Non invasive C. jejuni strains are also pathogenic presumably due

to the production of exotoxins that can disrupt host cells. The invasiveness of non

invasive C. jejuni strains c m be induced by coinfection with other enteroinvasive

Salmonella, Shigella, and E. coli strains (Bukholm and Kapperud, 1987).

The O side chain of LPS may be a factor contributing to the adhesive

properties for the ce11 (McSweegan and Walker, 1986). LPS has preferential binding

ability to the mucous membrane and epithelial cells.

Campvlobacters toxins

Diseases caused by C. jejuni, C. coli and C. laridis are associated with the

production of an enterotoxin or a cytotoxin (Johnson and Lior, 1986). C. jejuni

enterotoxin is a 60- to 70-kDa iron regulated heat-labile enterotoxin. It is structurally

related to cholera toxin and its 8 subunit is partially correlated with the B subunit of E.

coli heat labile enterotoxins (Ruiz-Palacios et al., 1983; Johnson and Lior, 1986;

Klipstein et al., 1986; Daikoku et al., 1990). The induction of watery diarrhea by C.

jejuni enterotoxin was postulated to be similar to cholera toxin. Like cholera toxin,

CJT elongates Chinese Hamster Ovary (CHO) cells, is detected with a GMl-based

ELISA and produces fluid accumulation in intestinal ligated loops (Fernandez et al.,

1983; Ruiz-Palacios et al., 1983; Klipstein and Engert, 1984; McCardell et al., 1984;

Walker et al., 1986; Daikoku et al., 1990).

C. jejuni strains have also been reported to produce a heat-labile protein

cytotoxin tbat is not neutralized by anti-CT, anti-Shiga, or anti-Clostridium antiserum

(Mahajan and Rodgers 1990; Guerrant et al. 1987; Johnson and Lior 1986; Walker et

i'1 -------a----, .---, A 1,

adrenal HeLa cells and the intestinal ce11 line, Int 407 (Perez-Perez et al., 1989).

Although the role of the cytotoxin in C. jejuni pathology is unknown, there is an

increase in fecal leukocytes resulting frorn epithelial ce11 damage. Another potential

toxin is a cytolethal distending toxin (CLDT) that has been identified in some C. jejuni

strains (Johnson and Lior, 1988). C. jejuni CLDT causes progressive ceIl distention

and eventually death in vitro and causes a hemorrhagic response in rat ligated

intestinal segments in vivo. The role of the C. jejuni CLDT in disease is unknown.

Recently, genes with similarity to those encoding E. coli CLDT have been isolated

from C. jejuni (Pickett et a!., 1996). A fourth potential toxin is a Shiga -1ike toxin.

Low levels of a cell-associated cytotoxic factor that is neutralized by anti-Shiga toxin

s e m have been reported from some C. jejuni strains. However, no genetic homology

has been found between the E. coli Shigu-like toxin genes and that of C. jejuni Cell-

fiee filtrates of C. jejuni have been reported to contain a heat-stable substance that

alters intestinal myoelectric activity in rabbit ileum, but no further characterization of

this factor is available (Sninsky et al., 1985).

Epidemiology of Campylobacter jejrrni infection

C. jejuni is now recognized as a common cause of diarrhea in humans. In both

the UK and USA the incidence of C. jejuni infection has been estimated to be around

1,000 per 100,000 population per year (Tauxe, 1992; Ketley, 1995). It occurs in

sporadic forrn in developed countries arnong persons living in temperate or tropical

clirnates and at a much higher incidence arnong travelers to developing countries. C.

jejuni is isolated from adults with diarrhea as ofien as Salmonella and Shigella

5------ - - 2 - - . . , - - - - - - - - - - - , - - . - , - - ------ - - -, - - . - , - - J ""- ,

1980; Young et al., 1980; Drake et al., 1981). It is the third commonest cause of

diarrhea in children of developing countries (DeMol and Bosman, 1978; Blaser et al.,

1980; Black et al., 1981 ; Ruiz-Palacios et al., l983), after enterotoxigenic E. coli, and

rotavims, and of enteritis in children of developed countries after rotavirus and

Shigella (Kapikian et al., 1976; Brunton and Heggie, 1977; Pickering et al., 1978; Pai

et al., 1979; Gurwith et al., 198 1).

Genome size of Campvlobacfer jeiuni

The genome of Campylobacter jejuni is circular and has been estimated to be

between 1.7 to 1.9 Mb by pulsed-field gel electrophoresis of digested DNA fragments

using rare cutting restriction enzymes; BssH II (g'cgcgc), Kpn 1 (ggtac'c), Nci 1

(cc'[gc]gg), Sac II (ccgc'gg), Sa1 I (g'tcgac), and Sma 1 (ccc'ggg) (Smith et al., 1988;

Chang and Taylor, 1990; Nuijten et al., 1990; Kim and Chan, 1991; Kim et al., 1992;

Taylor et al., 1991; Taylor et al., 1992). The base composition is 27 to 30 molar

percentage G+C (Owen, 1983).

Genes cloned from Can~pylobacter jeiuni

In Campylobacter jejuni, there are several chromosomal genes that have been

cloned and characterized. They play different functional roles narnely as housekeeping

genes, virulence genes or antibiotic resistance genes. Our laboratory has cloned over

30 genes from C. jejuni and completely sequenced and characterized 18 of them (Chan

et al., 1988; Kim and Chan, 1989; Chan and Bingharn, 1990; Kim and Chan, 1991;

Chan, 1994; Chan et al., 1995; Hong et al., 1995; Kim et al., 1995; Chan et al., 1997).

Housekeeping genes

Housekeeping genes are highly conserved among cross species and encode

similar functions required for the maintenance and growth of bacteria. For example,

the proA and proB genes are involved in proline biosynthesis and were isolated by

complementing proAproB mutants of E. coli (Lee et al., 1985). The proA gene

encodes gamma-glutamyl phosphate reductase which converts L-glutamate to proline.

This gene has been cloned, sequenced, and mapped on the physical map of C. jejuni

TGH9011 genome using pulse-field gel electrophoresis (PFGE) and Southern blot

hybridization (Kim et al., 1993; Louie and Chan, 1993). Furthemore, the proA gene

was expressed fiom its own promoter and the transcription start site was mapped. The

deduced arnino acid sequence of the proA gene product of C. jejuni exhibits 36.4%

and 36.0% identity to that of E. coli and Serratia marcescens respectively.

The glyA gene encoding serine hydroxymethyltransferase (SHMT) was cloned

by complementing an E. coli gEyA mutant (Chan et al., 1988). In the presence of

H4folate, SHMT catalyzes the reversible cleavage of serine to glycine and the

formation of 5,lO-CH2-H4folate which is a major contributor of CI units in ceIl

metabolism. The C. jejuni glyA gene encodes a 46 kDa protein which shows 55.6%

identity to that of E. coli. The glyA gene was mapped ont0 the C. jejuni TGH9011

chromosome (Chan and Bingham, 1991; Kim et al., 1993).

The argH gene encodes arginosuccinate lyase and was cloned by

complementing an E. coli strain deficient in ArgH activity (Hani and Chan, 1994).

argininosuccinic acid into arginine and fumarate. The C. jejuni argH gene is 1.377 kb

in size and encodes a 56 kDa protein. It was mapped ont0 the chromosome of C.

jejuni TGH9011 and lies on the Sa1 1 A, Sma 1 A, and Sac II A fragments.

The IysS encodes Iysyl-tRNA synthetase and the open reading frame overlaps 1

bp with the start codon (Met) of the glyA gene in C. jejuni (Chan and Bingham, 199 1).

The enzyme is responsible for charging the lysyl tRNA molecule with its arnino acid

moiety. The C. jejuni lysS-encoded product, LysRS shows 47.9 and 46.6% identity to

the E. coli lysS - and lysU - encoded synthetases respectively. The C. jejuni lysS gene

could complement the E. coli lysS and lysU mutations and it has been placed on C.

jejuni TGH9011 genomic map (Kim et al., 1993).

The fur ferric uptake regulatory gene is located upstream of lysS and had been

isolated by screening the genomic library with a lysS probe, characterized and mapped

ont0 the genome of C. jejuni TGH9011 (Chan et al., 1995). The C. jejuni fur gene

encodes a 18.1 kDa protein and is homologous to the E. coli fur gene. fur is

responsible for the regulation of iron-induced or repressed genes (Wooldridge et al.,

1 994).

The ileS gene encoding isoleucyl-tRNA synthetase was cloned (Hong et al.,

1995). It was completely sequenced and the deduced IleS has 91 7 arnino acids

with a molecular mass of 1 O6 kDa. Also ileS was mapped ont0 the 1360- 1 8 12 kb

region of Sma I A, Sa1 1 A and Sac II A fragments of the C. jejuni genome. The IleS is

an essential enzyme needed for the arninoacylation of isoleucyl tRNA.

The leuB, ZeuC, and leuD genes, involved in the leucine biosynthetic pathway,

were cloned by complementation of specific auxotrophs in E. coli (Labigne et al.,

The katA gene encodes catalase. The gene from C. jejuni was cloned by

functional complementation of a catalase-deficient mutant of E. coli and its nucleotide

sequence was obtained (Grant and Park, 1995). The deduced protein pxoduct of 508

amino acids with an estimated molecular mass of 59 kDa has been found to be

structurally and enzyrnatically similar to hydrogen-peroxidase from other bacterial

species.

The aroA gene encoding 5-enolpynivylshikirnate-3-phosphate (EPSP) synthase

was cloned by complementation of an E. coli auxotrophic aroA mutant. The aroA

gene has been sequenced and encodes an enzyme of 428 arnino acids which has 39%

identity to the Bacillus subtilis EPSP synthases (Wosten et al., 1996).

The cysM gene encodes O-acetylserine sulfhydrylase B which is preferentially

used for cysteine biosynthesis during anaerobic growth. It is able to utilize thiosulfate

as substrate and it has been cloned, sequenced, and expressed. The C. jejuni cysM

gene encoding a protein of 299 amino acids with a calculated molecular mass of 32

kDa was cloned by complementation of an E. coli cysteine auxotroph. The cloned C.

jejuni gene is a functional homologue of the cysM gene that codes for O-acetylserine

sulfhydrylase B in E. coli and S . îyphirnurium (Garvis et al., 1997).

Southern hybridization analysis of C. jejuni genome shows that there are 3 to 5

ribosomal RNA (rRNA) loci (Labigne-Roussel et al., 1988; Kim and Chan, 1989).

Three rRNA operons (rrn4, rrnB, and rrnC ) of C. jejuni have been isolated fiom a C.

jejuni genomic library of TGH9011 by screening with radioactively labeled C. jejuni

rRNA (Kim et al., 1993). Analysis of the structure and organization of the rRNA

genes has revealed a characteristic adjacent 16S/23S rRNA structure in the three

.,- -r"-"". "̂ ' ---- --- - - - a - - - - - - - - . - - - - - - - - - - --- -----

sites on the physical map of C. jejuni TGH9011. The isoieucine tRNA and alanine

tRNA genes were s h o w to be located in the intercistronic region between the 16s and

23s rRNA genes (Rashtchian and Shaffer, 1986; Kim et al., 1993; Kim et al., 1995).

This organization is conserved in al1 three C. jejuni rRNA operons. The rrnA rRNA

operon of C. jejuni TGH9011 was completely sequenced. The rRNAs were then

characterized by primer extension and S1 nuclease mapping analysis (Kim et al.,

1995). The comparative secondary structure analysis of the 16s and 23s rRNAs were

identified as conserved structures that are found in Proteobacteria. This finding

supports the classification of CampyIobacter in the E subdivision of the Proteobacteria

(Trust et al., 1994).

A major oxidative stress gene sodB from C. jejuni was cloned. The sodB gene

encodes an iron superoxide disrnutase (SOD) which catalyses the breakdown of

superoxide radicals to hydrogen peroxide and dioxygen which are important for

protecting the bacterial ce11 under oxidative darnage conditions (Pesci et al., 1994).

The arylsulfatase gene fiom C. jejuni has been cloned and has no sequence

similarity with other known arylsulfatase (Yao et al., 1996).

The hup gene encodes a homologue of the histone-like DNA binding protein

and the C. jejuni gene was cloned and sequenced (Konkel et al., 1994).

The C. jejuni recA gene, encoding a conserved protein involved in DNA repair

and in recombination, was cloned by PCR amplification using degenerate primers

corresponding to the conserved regions of RecA proteins found in other bacteria. The

recA gene encodes a 37 kDa protein. The terrnination codon overlaps with the

initiation codon of another open reading frarne with similarity to the E. coli enolase

a defect in generalized recombination as determined by natural transformation

fiequencies (Guerry et al., 1994).

In addition, a potential ce11 division geneflsA fiom C. jejuni was selected by

screening the C. jejuni lambda ZAP I I library with cDNA probes made by reverse

transcription of rnRNA. Although C. jejuniftsA is homologous to that of E. coli, the

structure and organization of the operon is different fiom that of E. coli The exact

fùnction of the ftsA gene product remains obscure, although together with the fstQ

gene product, it is thought to have a role in defining the site of division (Griffiths et

al., 1996).

A C. jejuni gene encoding a 29-kDa periplasmic binding protein has cloned,

sequenced, and characterized (Garvis et al., 1996). This gene has been designated as

hisJ, on the basis of complementation experiments performed with an S. fyphimurium

HisJ mutant harboring the recombinant plasmid which contains a C. jejuni

chromosomal D N A fiagment.

Furthermore, a gene conferring haemolytic activity was isolated fiom C. jejuni.

The open reading fiame encodes a protein of 36 kDa with a typical endopeptidase type

II leader sequence. The protein is modified with palmitic acid when it is processed in

E. coli, confirming it as a typical lipoprotein. The deduced gene product of 329 arnino

acids has significant homology to the group of solute binding proteins from

periplasmic-binding-protein-dependent transport systems for ferric siderophores (Park

and Richardson, 1995).

The C. jejuni tig gene encodes a 56 kDa size protein that is thought to act as a

trigger factor was cloned. It shares 31% identity to the arnino acid sequence of the

proOmpA across the cytoplasmic membrane and also serves as a potential chaperone

in ce11 division (Griffiths et al., 1995).

Virulence gene

Genetic studies have also focused on virulence deterrninants believed to play a

role in pathogenesis. Flagella play a role in internalization of C. jejuni (Grant et al.,

1993; Yao et al., 1994). ThefIaA andflrrB flagellin genes fiom C. jejuni have been

characterized (Nuitjen et al., 1990; Fisher and Nachamkin, 1991 ; Khawaja et al.,

1992). The sequence homology between the two genes in strain 8 1 1 16 is 95%. They

are expressed independently fiom two different promoters 02* and aS4 (Alm et al.,

199 1 ; Wassenaar et al., 199 1). The flagellin genes have been mapped on the C. jejuni

chromosome (Kim et al., 1993). A third flagellin (f7aC ) gene of C. jejuni has been

cloned recently (Chan et al., 1997).

Omp 18 encoding an 18-kDa outer membrane protein from C. jejuni ATCC

29428 has been isolated and expressed in E. coli. The sequence has a high degree of

similarity to the peptidoglycan-associated outer membrane lipoprotein P6 of

Haernophilus injluenzae and the peptidoglycan-associated lipoprotein PAL of E. coli

(Burnens et al., 1995; Konkel et al., 1996).

C. jejuni MapA gene encoding 24 kDa membrane-associated protein of C.

jejuni 81 1 16 has been cloned and characterized. The 18 N-terminal arnino acid

residues constitute a signal sequence characteristic of prokaryotic membrane proteins.

In a dot blot hybridization assay with a mapA probe, 120 clinical isolates of C. jejuni

were unequivocalIy discriminated fiom 126 other Campylobacters, including 34 C.

- - - - - - - - - - - - -

related Campylobocter members based on the unique differences in antigenic

structures (Stucki et al., 1995).

TheJ7hA gene was also cloned and sequenced. It is a homologue of LcrD/FlbF

family of proteins which is involved in the regulation of virulence-related proteins

(Miller et al., 1994).

Antibiotics resistance pene

Genes encoding resistance to antibiotics, like the kanarnycin resistance (Kmr)

aminoglycoside phosphotransferase gene, aphA -7, and the tetracycline resistance (Tcr)

tet(0) genes in C. jejuni have been cloned and sequenced. Resistance to antibiotics is

usually plasmid-mediated and some plasmids carry more than one antibiotic resistance

determinant (Taylor et al., 1986; Blaser et al., 1987; Tenover et al., 2989).

Hippurate hydrolase of Campvlobacter jejuni TGH9011

The C. jejuni N -benzoyl-amino-acid amidohydrolase (hippurate hydrolase)

(EC 3.5.1.32) (hipO) gene was isolated on a pBR322 recombinant clone. pHip-O was

isolated and characterized (Hani and Chan, 1995). The exact functional roles of the

hippurate hydrolase in bacterial cells have not been defined. Amidohydrolases are

able to specifically remove N -1inked amino acids or similar substitutes from larger

substrates. Similar activities have been dernonstrated in bacteria, plants, animals, and

humans. Hippuric acid serves as a sole carbon source for the growth of some

hippurate hydrolase containing microorganisms (Miyagawa et al., 1985) and hippurate

hydrolase cataIyzes the hydrolysis of hippuric acid to release benzoic acid and glycine

differentiation of C. jejuni, a cornmon cause of human enteritis, from other

Campylobacter species (Harvey, 1980; Skirrow and Benjamin, 1980).

The hipO gene of C. jejuni TGH9011 encodes a polypeptide of 383 amino

acids with a pl of 6.0. In maxicell and Western blot analysis, hippurate hydrolase

(HipO) was shown to be a 42 kDa protein (Hani and Chan, 1995). The size of HipO is

similar to proteins of Pseudornonas sp. (Karneda et al., 1968; Watabe et al., 1992;

Ishikawa et al., 1996), Bacillus sterotherrnophilus (Sakanyan et al., 1993),

Arabidopsis thaliana (Bartel and Fink, 1995), and SuIfolobus soIfataricus (Colombo,

1995).

The nucleotide sequence of hipO C. jejuni TGH9011 has been published (Hani

and Chan, 1995) and is shown in figure 2. TAA is used for its termination codon and a

AGGAGA Shine-Dalgarno sequence is located at nt-14 to -9 upstrearn of ATG the

methionine initiation codon. The hipO region is purine rich and the molecular

percentage G+C content is 33.1%. The codon usage is similar to other C. jejuni genes

except for the rare usage of UUC (phe), CTC (leu), CTG (leu), GTC (val), TCC (ser),

CCG (pro), ACC (th) , CGA (arg), and CGG (arg). Codon usage and molecuiar

percentage G+C of the hipO open reading fiame within the insert of recombinant

plasmid pHIP-O is shown in Table 2.

Open reading frarne 2 (OTCFU2) and 3(ORFU3) are upstream of 4(ORFU4), the

hipO. These three ORFs are continuous on the genome suggesting they may be

cotranscribed as an operon.

A single genomic copy of the hipO gene is present in C. jejuni TGH9011.

Southern blot hybridization of the C. jejuni genomic DNA probed with a radiolabeled

O I I

C - NHCH, Hippurate COOH Hydrolase

+ H, O +- + NH,CH,COOH

N-benzoylglycine (Hippuric acid)

Benzoic + Glycine acid

Figure 1. Biochernical reaction of hipourate hvdrolase.

Hippuric acid is formed by condensation of benzoic acid and glycine in human and

accumulates in urine. Hippuric acid is hydrolysed ta benzoic acid and glycine in

various species of microorganisms, including C. jejuni.

hippurate hydrolase. TTT (phe) 20 TTC ( p h e ) TTA ( l e u ) TTG ( l e u ) CTT ( l e u ) CTC ( l e u ) CTA ( l e u ) CTG (leu) ATT ( i l e ) ATC ( i l e ) ATA ( i l e ) ATG (met) GTT ( v a l ) GTC ( v a l ) GTA ( v a l ) GTG ( v a l ) TCT ( s e r ) TCC ( s e r ) TCA ( s e r ) TCG ( s e r ) CCT ( p r o ) CCC ( p r o ) CCA ( p r o ) CCG ( p r o ) ACT ( t h r ) ACC ( t h r ) ACA (thr) ACG ( t h r ) GCT ( a l a ) GCC ( a l a ) GCA ( a l a ) GCG ( a l a ) TAT ( tyr ) TAC ( tyr ) TAA ( e n d ) TAG ( e n d ) CAT (his) CAC (his)

(gin) W G (gin) AAT ( a s n ) AAC ( a s n )

( l y s ) AAG ( l y s GAT ( a s p l GAC ( a s p ) GAA ( glu GAG ( g l u TGT ( w s ) TGC ( w s ) TGA (end) TGG (t-1 CGT (arg) CGC (arg) CGA ( a r g ) CGG ( a r g ) AGT (ser)

AGC (ser) 6 AGA ( a r g ) 3 AGG (arg) I GGT ( g l y ) 11 GGC ( g l y 4 GGA (@Y) 1 0 G a ( q l y ) 2 T o t a l codons 383 T o t a l base pairs 1149 M o l % G+C 33.1

Figure 2. Nucleotide sequence of C. jejrr~ti TGH9Oll hippurate hvdrolase.

The deduced arnino acid sequence of hippurate hydrolase is given in single Ietter code

above the sequence for those on the "upper" strand. Termination codons are indicated

with an asterisk (Hani and Chan, 1995).

V V S W S V D K T H S F T L G F V Y I F GGTAGTATCTTGGAGTGTTGATAAAACCCATAGTTTTACTTTGGGTTTTGTTTATATTTT -81

v A L I F I S A I L A Q F V L P R R E N F

TGCTTTGATTTTTATTTCAGCGATCTTAGCACAATTTGTTTTACCTAGMGAGAAAAT -21 ORFU4 r

I Q G E K * M N L I P E I L D L Q G E TATACAAGGAGVTAGAATGAATTTmTTCCAGAAATACTAGACTTACMZGCGAAT 4 0

F E K I R H Q I H E N P E L G F D E L C TTGAAAAAATTCGTCATCAAATTCATGAAAATCCTGAGCTTGGTTTTGATGAATATGTA 100

tiprl hor2 T A K L V A Q K L K E F G Y E V Y E E I CTGCAAAATTAGTGGCGCAAAAATTAAAAGAATTAAAAGAATTTGGTTATGAGGTTTATGAGGAAATAG 160

G K T G V V G V L K K G N S D K K I G L GAAAAACAGGCGTTGTGGGGGTTTTAAAAAAGGGGAATAGCGATAAAAAAATAGGACTTC 220

R A D M D A L P L Q E C T N L P Y K S K GTGCAGATATGGATGCTTTGCCTTTGCAAGAATGCACRAATTTGCCTTATAAAAGCAAAA 280

K E N V M H A C G H D G H T T S L L L A AAGAAAATGTAATGCATGCTTGCGGTCATGATGGACATACTACTTCTTTATTGCTTGCTG 340

A K Y L A S Q N F N G T L N L Y F Q P A CAAAGTATTTAGC~GTCAGAATTTTAATGGCACTTTMTCTTTATTTTCMCCTGCTG 400

E E G L G G A K A M I E D G L F E K F D AAGAGGGTTTGGGTGGTGCTAAGGCAATGATAGAAGATGGATTGTTTGAAAAATTTGATA 460

S D Y V F G W H N M P F G S D K K F Y L GTGATTATGTTTTTGGATGGCACAATATGCCTTTTGGTAGCGATMGMTTTTATCTTA 520

K K G A M M A S S D S Y S I E V I G R G AAAAAGGTGCGATGATGGCTTCTTCGGATAGTTATAGCÀTTGÀAGTTATTGGAAGAGGTG 580

G H G S A P E K A K D P I Y A A S L L V GTCATGGAAGTGCTCCAGWGGCWGATCCTATTTATGCTGCTTCTTTACTTGTTG 640

V A L Q S I V S R N V D P Q N S A V V S TGGCTTTACMGCATAGTATCTCGCAATGTTGTTGATCCCCRAIlATTCAGCAGTTGTAAGCA 700

I G A F N A G H A F N I I P D I V T I K TAGGAGCTTTTAATGCAGGACATGCTTTTAATATCATTCCAGATATTGTMCGATT- 760

M S V R A L D N E T R K L T E E K I Y K TGAGTGTTAGAGCATTAGATAATGAAACTAGAAAGCTIMCTGMGAnAAAATTTATAAAA 820

I C K G L A Q A N D I E I K I N K N V V TTTGTMGGTCTTGCACAGGCTAATGATATAGAGATTWTCMTWTGTTGTTG 880

A P V T M N N D E A V D F A S E V A K E C A C C A G T G A C T A T G A A T A A C G A T G A A G C T G T G G A T T T T T 940

L F G E K N C E F N H R P L M A S E D F T A T T T G G C G ~ T T G T G A A T T T A A T C A T C G T C C T T T A G T G A G G T T T T G 1000

G F F C E M K K C A Y A F L E N E N D I G h T T T T T T T G C G ~ T G ~ T G T G C C T A C T T ? T T A G T G C A C A T T T 1060

4 Y A K L A L K Y L K * ATGCGAAGCTAGCTTT~TACTTAAAATAAAAACTAATCTAGAATTTCRAGCACAATT 1180

* D L I E L V I

E S K L G K I Q I E K L K L N S P L N A

~GCTCTTTATTGTTTTTTATTTGCTTTTTTTGCACTTATGGAGACTAAAATT~~~~TA 1300 L L E K N N K I Q N K A S I S L L I G R

23s rRNA probe O IGS rRNA probe

Figure 3. Phvsical map of C. jejuni TGH9011.

C. jejuni TGH90 1 1 produced a 2.1 -kb hybridizing band. DNA hybridization detected

no hipO gene sequences in C. coli, C. lari, C. upsaliensis, C. fetus, or C. sputorum or

the former member of the Campylobacter genus- Helicobacter pylori (Hani and Chan,

1995). This is correlated with the negative hippurate hydrolase activity observed in

Helicobacter, Arcobacter, and other Campylobacter species.

Substrate specificity of the C. jejuni HipO protein is highest using N -

benzoylglycine (N -B-gly), followed by N -benzoylalanine (N -Bz- ala), N -

benzoylrnethionine (N -B-met), and N -benzoylleucine (N -B-leu) respectively. No

activity is detected using N -benzoylhistidine, N -benzoylarginine, or N -

benzoylthreonine as a substrate. The hipppurate hydrolase gene was mapped by

Southern hybridization to the Sac II-A, Sa1 LA, and Sma 1-A fragments which overlap

a 450 kb region of the 1800 kb genome (Kim et al., 1993). The hipO gene was

localized to the central region of the Smu 1-A fiagrnent by mapping a hipO

::kanamycin mutant of C. jejuni (Figure 3) . The argH and ileS genes also have been

mapped to these restriction enzyme fragments (Kim et al., 1993, Hong et al., 1995).

However the proximity of these genetic markers to each other, is currently unknown.

HomoIogous proteins belong to M40 hvdrolase family

Using a BLAST search, the deduced arnino acid sequence of C. jejuni

hippurate hydrolase shows a homology score with the SuIfolobus solfataricus

carboxypeptidase (CPSA) (Colombo et al., 1995), the Arabidopsis thdania indole-3-

acetic acid -amino acid hydrolase (ILR) (Bartel and Fink, 1995; Li et al., 1996), the

Bacillus sterothermophilus N -acyl-L-arnino acid amidohydrolase (AMA)(Sakanyan et

utilizing protein C) (HYUC) (Watabe et al., 1992; Ishikawa et al.. 1996). the

Synechocystis hypothetical protein (Kaneko et al., 1996) and the Agrobacteriurn

tumefaciens cellulose synthase (CELE) (Matthysseet al., 1995) (Table 3).

The Bacillus sterothermophilus N -acyl amino acid aminoacylase enzyme

catalyses the hydrolysis of N -acyl arnino acid to yield an arnino acid and acetic acid.

The substrate specificity of this enzyme is extremely high with N -choroacetyl-L-

phenylalanine and is also high towards N -acetyl-L-ala, N -acetyl-L-tyr, N -acetyl-L-

phe, N -acetyl-L-val, N -acetyl-L-gly, N -acetyl-L-leu, IV -acetyl-L-his, N -acetyl-L-met,

and N -benzoyl-L-phe.

N -benzoylglycine amidohydrolase from Pseudomonas putida C692-3

hydrolyzes N -benzoyl amino acids with different specificity, highest with N -Bz-gly,

follow by N -Bz-L-ala and N -Bz-L-aminobutyric acid. It has no enzyme activity

towards N-acetyl-glycine, N -acetyl-L- alanine, N -carbobenzoxyglycine, or N -

carbobenzoxy-L-alanine. Amidohydrolase are able to specifically remove N -1inked

amino acids or similar substitutes fiom larger substrates.

The Pseudomonas sp. N -carbamoyl amino acid aminoacylase catalysed

reaction yields an amino acid and carbarnate. The substrate specificity of the

benzoylamino acid arnidohydrolase of Pseudomonas sp. (strain KT801) is towards L-

amino acids and not D-amino acids. N -Bz-Dl-ala is the preferred substrate, followed

by N -Bz-DL-met, N -Bz-DL-leu, N -Bz-gly, N -Bz-DL-val, N -Bz-DL- phe, N -Bz-

DL-asp, N -Bz-DL-glu, N 432-DL-trp, and N -Bz-DL-th. No activity is observed on

the substrate acetyl-DL-alanine, or phenylacetyl-DL-alanine, indicating a considerable

effect by the acyl group (Kameda et al., 1968).

activity and c m remove amino acid residues fiom benzoyloxycarbonyl derivatives

with activity towards benzyloxycarbonyl (Cbz) substrates; Cbz-glyglyphe, Cbz-arg,

Cbz-asp, and weakly with Cbz-phe and Cbz-ala. The Arabidopsis thaliana indoxyl-3-

acetic acid amidohydrolase reaction yields an arnino acid and indole-3-acetic acid.

The substrate specificity is toward indole-3-acetic acid arnino acid conjugates: IAA-

phe, IAA-leu, and weakly towards IAA-ala, IAA-gly, and IAA-ile.

Table 3. Homologous ~roteins of C. ieiuni hippurate hydrolase.

Sequences with high homolow score Homolow Score

Campylobacter jejuni hippuricase (HipO) 2.1 e-248 Sulfolobus safataricus carboxypeptidas (CpsA) 4.8e-83 Arabidopsis thaliana indoxyl-3-acetic acid amidohydrolase (Ill 1) 7.7e-72 A rabidopsis thaliana (1112) 1.8e-70 A rabidopsis thaliana (Ilr 1 ) 1.7e-62 Synechocystis sp. hypothetical protein 4.1 e-47 Bacillus stearothermophilus N-acyl-L-amino acid amidohydrolase (Amal 1.2e-44 Bacillus subtilis (YXEP) 3.4e-62 Agrobacterium tumefaciens (CelE) 7.8e-2 1 HaernophiZus infIuenae HipO homolog 7.6e- 17 Psezrdomonas sp. hydantoin utilization protein (hyuC) 0.22 Escherichia coli hemolysin B (hlyB) 0.52

An extensive study of over 556 strains of thermophilic Cumpylobacter for

species identification was reported by Totten et al., 1987. These members were

isolated either from hurnans with diarrhea or from poultry in King County,

Washington. They were analyzed by the phenotypic test, after that the classification of

the hippurate hydrolase-negative C. jejuni strains was confirmed by a quantitative

whole-ce11 DNA hybridization test. The study showed that only 1.6% (9 of 556) of the

C. jejuni strains were hippurate hydrolase negative. Thus, hippurate hydrolase-

negative C. jejuni strains represented a small percentage (1.6%) of C. jejuni strains but

a significant portion 20% (9 of 46) of hippurate hydrolase-negative strains in that

study. These C jejuni hippurate hydrolase-negative isolates were not only hippurate

hydrolase-negative in the rapid tube test but also in more sensitive assays such as gas

liquid chromatography (GLC) and thin layer chromatography (TLC) assays.

Furthermore, serotyping was also applied to these members, yet hippurate hydrolase-

negative C. jejuni isolates show a non-random association with different serotyping

systems (Totten et al., 1987). Hippurate hydrolase-negative strains fell into four

groups by the Penner serotyping system, four groups by the Lior serotyping system,

two auxotypes, and three plasmid patterns, indicating that these strains represented a

diverse group of isolates.

Hippurate hydrolase-negative isolates of C. jejuni were also examined with

respect to the presence of h i p 0 gene sequences in the genome by Southern

hybridization analysis (Hani and Chan, 1995). Four hippurate hydrolase-negative

isolates D594, D603, D941, and D 19 16 gave a positive 2.1 kb Hind I I I hybridization

band when probed with the hipO probe. One hippurate hydrolase-negative isolate,

chromatography assay (Totten et al., 1987) and showed a hybridization band in

Southern hybridization blot with the hippurate hydrolase probe (Hani and Chan, 1995).

C. jejuni and C. coli are the most comrnon thennophilic bacteria isolated fiom

humans (Ketley, 1995). C. jejuni and C. coli share many cornmon phenotypic

characteristics. Thus, very few biochernical tests can differentiate between them

(Penner and Hennessy, 1980; Penner et al., 1983; Penner, 1988; Boltan, 1984; Patton

et al., 1992). However, they are two distinct species and show only 25 to 58%

homology relative to each other by DNA hybridization (Harvey and Greenwood, 1983;

Roop et al., 1984; Morris et al., 1985). The hippurate hydrolysis (Hippuricase) assay

is the standard bicbzhemical test used to distinguish C. jejuni, C. coli, and other

Campylobacter sp. in the clinical laboratory via the detection of the product of

hydrolysis by a color reagent system. This asssay was found to correlate closely with

species differentiation in taxonomic studies. 80% of cases of Campylobacter -

mediated enteritis in hurnans are caused by C. jejuni (Ketley, 1997). C. jejuni is the

only Campylobacter species that has the hippurate hydrolase gene and is able to

hydrolyse hippurate. Recently, the hippurate hydrolase gene from C. jejuni TGH9011

strain has been cloned and characterized (Hani and Chan, 1995). Therefore, it is

important to understand the role of the hippurate hydrolase gene in C. jejuni species.

Hippurate hydrolase-negative C. jejuni strains have also been identified (Totten

et a1.,1987). It is of interest to analyse the mutant hippurate hydrolase gene in these

natural isolates. Therefore, the major objective of this study was to determine the

molecular nature of hippurate hydrolase-negative C. jejuni clinical isolates from King

County, Washington. We used a radiolabeled single strand conformational

polymorphic assay based on polymerase chain reaction (PCR-SSCP assay) to screen

1 Y A A Y A 1 - -

hydrolase-negative C. jejtrni. The S S C P assay based on polymerase chain reaction is

a sensitive and simple technique, which is capable of distinguishing single base pair

changes between single DNA strand (Orita et al., 1989; Teleni et al., 1993). It is

successfùlly used in various studies in Mycobacteriurn tuberculosis (Orita et al., 1989,

Teleni et al., 1993), Mycobacterium leprae (Heyrn et al., 1995), Hepatitis B virus

(Yusof et al., 1994), and Parvovirus B19 (Kerr et al., 1995), etc. Oligonucleotide

primers were synthesized based on the hippurate hydrolase nucleotide sequence of

hippurate hydrolase-positive C. jejuni strain TGH9011 (Hani and Chan, 1995). The

primers were used to ampliSr eleven overlapping regions of the hippurate hydrolase

gene from each of the five hippurate hydrolase-negative C. jejuni clinical isolates. The

SSCP assay was used to detect point mutations upstream and within the hippurate

hydrolase gene sequences. The regions containing the hippurate hydrolase gene of the

five diffèrent hippurate hydrolase-negative C. jejuni isolates were cloned and

sequenced in order to characterize the exact nature of the sequence heterogeneity

within the hippurate hydrolase gene sequence of C. jejuni TGH9011 strain.

Bacterial Strains

The Campylobacter jejuni serotype reference strain for 0:3 [TGH9011 (ATCC

43431) and of Lior serotype 361 was obtained fiom Dr. J. L. Penner, University of

Toronto, Canada. C. jejuni TGH9011 was used as a hippurate hydrolase-positive

controI strain. Five different hippurate hydrolase-negative clinical strains of C. jejuni

(D594, D603, D835, D977, D1713) were received fiom Dr. C. M. Patton, Center for

Disease Control and Prevention, Atlanta, Ga, USA. These hippurate hydrolase-

negative strains were isolated either fiom humans with diarrhea or fiom poultry

(Totten et al., 1987). Campylobacter species were grown routinely on Mueller-Hinton

agar plates (Merck) or in broth for 48 to 72 hours at 42'C in a carbon dioxide incubator

set for 7% CO2.

Escherichia coli (JM101) strain [A(lac-proAB ) thi strA supE44 end4 sbcB

hsdR4 F' traD36 proA+B+ Zacl A(lacZ) Ml51 was grown routinely on Luria Bertani

media aerobically at 37'C and used for making cornpetent cells.

Genomic DNA extraction

Total genomic DNA of C. jejuni TGH9011 was extracted from cells grown on

Muller-Hinton a g a plates. A plate of C. jejuni cells was washed three times in 1 ml of

saline citrate (SSC:O. 1 SM NaCl, 0.01 5M Na citrate [pH7.0]). Suspended cells were

pelleted and resuspended in 27% sucrose in 1X SSC at a concentration not higher than

10" cells per ml. The suspended cells were digested with proteinase K to a final

Table 4. Bacterial strains used in this study.

Strains Genotvpe or Characteristics References

Campylobacter jejuni

ATCC4343 1 serotype reference strain for 0:3, TGH9011 J. L. Penner

Esclierickia coli

hippurate hydrolase negative isolate C. M. Patton

hippurate hydrolase negative isolate C. M. Patton

hippurate hydrolase negative isolate C. M. Patton

hippurate hydrolase negative isolate C. M. Patton

hippurate hydrolase negative isolate C. M. Patton

hippurate hydrolase weak isolate C. M. Patton

A(1ac-pro) thi rspL supE en& sbcB hsdR F' traD36 proAB ZacP ZAM 1 5 Yanisch-Perron et al.

ATCC:Arnerican Type Culture Collection

concentration of 0.2%. The lysate was incubated at 50'C for an hour and then

extracted three times with an equal volume of 50 mM Tris-Cl, 10 mM EDTA, pH 8.0 -

saturated phenol. The aqueous phase of the phenol-extracted lysate was removed and

extensively dialyzed in Tris-EDTA (10 mM Tris [pH 8.01-lmM EDTA). DNA was

recovered by ethanol precipitation with two volumes of 95% ethanol for 30 minutes, at

-70'C. The precipitated DNA was pelleted by centrifugation, washed with 70%

ethanol and then vacuum dried. The dried pellet was resuspended in 50 pl TE (10mM

Tris-Cl pH 8.0, 1 mM EDTA pH8.O) for further use.

Olipronucleotides

To scan the hippurate hydrolase gene for the presence of mutations by

polymerase chain reaction and single strand conformational polyrnorphism analysis,

oligonucleotides were synthesized based on the C. jejuni TGH9011 hippurate

hydrolase sequence (Hani and Chan, 1995). These primers were used in polymerase

chain reactions and sequencing reactions. The location, direction, size and sequence

of primers (PC1, PC3, PC4, H l , H2, H3, H4, H5, H6, H7, H8, H9, H 1 O, H 1 1, H 12,

H13, H14, H15) designed for the polyrnerase chain reaction, single strand

conformational polymorphism analysis and sequencing reactions are s h o w in Table 5

and 6 and Figure 4. The strategy for the amplification of the various regions of the

hippurate hydrolase gene using eleven primer sets is s h o w in Figure 4.

Table 5. Uligonucleotides usecl in mis stuay.

Oligo Sequence Purpose

5'-ATCCCGGGTGGTTTCC(A/T)TTTTTCTTTAATCT(T/A)CC-3 ' Pcr 5'-ATGTCGACAAAAAAGAAAATGToATGT(T/A)ATGCATGC/TTGCGGT-3' pcr 5'-ACCCCGGGGC(T/A)GG(T/A)TTTTATGATAAATTCTACAC-3 ' Pcr 5'-TAGGATCCTCTTGGAGTGTTGATAAAACTCAT-3 ' Pcr 5 '-ATGGATCCTTGATGGCGAATTTTTTCAAATTC-3 ' Pcr 5'-GCGGATCCGAATTTGAAAAAATTCGCCATCAA-3' Pcr 5'-TTGGATCCAGCAGTACATAATTCATCAAAACC-3' Pcr 5'-AAGGATCCAGTTCCATTAAAATTTTGACTAGC-3' Pcr 5'-TAGGATCCGCAAGTCAGAATTTTAATGGCACT-3' Pcr 5'-AAGGATCCTCCATCTTCAATCATAGCTTTAGC-3' PCr 5'-TTGGATCCTGGCACAATATGCCTTTTGGTAGC-3' pcdsequencing 5'-CTGGATCCTCCAATAACTTCAATGCTATAACTATC-3' Pcr 5'-ATGGATCCGCTCCTGAAAAAGCTAAAGATCCT-3' pcr/sequencing 5'-ACGGATCCAGCAGAATTTTGAGGATCAACATT-3' pcdsequencing 5 '-CTGGATCCTTTAATGCTGGACATGCTTTTAAT-3 ' Pcr 5'-TCGGATCCAACAGCTTCATCATTATTCATAGT-3' pcdsequencing 5'-GTGGATCCTTAATGGCTAGTGAAGATTTTGGA-3' Pcr 5'-AAGGATCCAGCATAAGCACATTTTTTCATTTC-3 ' Pcr

Table 6. Primer pairs used in this study.

Primers Repions Sizes

Chromosomal DNA of Campylobacter jejuni strains was arnplified by

polyrnerase chah reaction using Taq DNA polymerase (Boehringer, Mannheim,

Germany). The amplification was carried out in a programmable thermal cycler (Gene

Arnp PCR 9600 System, Perkin-Elmer, Norwalk, CT, USA) as follows; an initial

template denaturation step at 94.C for 5-minutes followed by 25 cycles of 1-minute 30-

seconds of denaturation at 94C, 1-minute 30-seconds primer annealing at 60C, and 2-

minutes chain extension at 72'C and then a final extension step at 72'C for 10 minutes.

The reaction (50 pl) contained 50 pmol of each primer, 1 pg of genomic DNA, 1.5

mM MgC12, 160 pM each dNTP, 2.5 U Taq DNA polyrnerase and Taq buffer. Sterile

distilled water was used to adjust the total reaction volume for the negative control.

The reaction mixture was then covered with 70 pl of minera1 oil. One tenth of the

arnplified products were electrophoresed on polyacrylarnide gel (PAGE), containing

ethidium bromide in TBE buffer (0.45 M Tris-Borate, and 10 mM EDTA). PAGE

electrophoresis was performed, as described (Maniatis, 1982). Briefly, a 10%

acrylarnide gel was cast in a Biorad Mini Protein gel caster. The DNA sarnples were

separated in running buffer at 100 V for 2 hours. Aller electrophoresis, the amplified

product was exarnined under UV light. Two rnicrograms of DNA from the phage # X

174 digested with Hae III was used as the size standard. Positive control C. jejuni

genomic DNA and negative controls: water instead of DNA, are included in every gel.

For the SSCP assay, pairs of PCR primers were chosen to generate fragment sizes of

less than 400 bp to enhanced the sensitivity of the SSCP assay.

detection enhancement (non denaturing) gel electrophoresis

For the SSCP assay, double stranded DNA was denatured into single stranded

DNA and the products were separated on a mutational detection (MDE) gel under

nondenaturation conditions. The amplification reaction was performed in a 50 pl

reaction volume containing 10 pl extracted genomic DNA (approximately 1 pg), 200

pM (each) of dTTP, dGTP, and dCTP, 100 pM dATP with 0.5 pl of [alpha-32P] ATP

(5 pCi) instead of total 200 uM dATP was added to the PCR mix, 50 pmole of each

oligonucleotide primer, 5 pl 10X Taq buffer and 2.5 U of Taq DNA polymerase

(Boehringer, Mannheim, Germany). The SSCP assays were performed as described

(Teleni et al., 1993). Briefly, 5 pl of radiolabelled arnplicon was diluted in 100 pl of

the SSCP dilution solution (10 mM EDTA, O. 1% SDS). 3 pl of diluted product was

mixed with 3 pl of formamide dye (98% formamide, 0.5% bromophenol blue, 0.5%

xylene cyanol, and 20 mM EDTA), denatured at 95'C for 5-minutes, cooled on ice, and

loaded on a nondenaturing sequencing-format 0.5 X Mutation Detection Enhancement

Acrylarnide Gel (MDE gel) (Hydrolink -MDE:AT/Biochern, Malvern, PA, USA). The

MDE gel is composed of 25 ml of MDE gel, 3 ml of 10X TBE buffer, and 22 ml of

deionized water polymerized with 300 p1 10% ammonium persulfate and 30 pl

TEMED (Bio Rad, Richmond, Calif., USA). Electrophoresis was done in 90 mM Tri-

Borate (pH 8.3) - 4 mM EDTA in a sequencing gel electrophoresis apparatus (50 by 32

by 0.04 -cm gel) (Bio Rad) at room temperature at a constant power of 8 W for 20

hours. After electrophoresis, the gel was dried for an hour, exposed 3 hours to Kodak

film, and the radioactive banding patterns were inspected. Pairs of PCR primers were

Figure 4. Strategv for single strand conformational polvmorphic (SSCP) assay

based on polymerase chah reaction.

The positions of eleven sets of primer pairs used are indicated above the hippurate

hydrolase gene which are shown as box. Oligonucleotides were synthesized based on

the published sequence of hippurate hydrolase-positive C. jejuni strain TGH90 1 1

(Hani and Chan, 1995). The eleven sets of primers were used to amplify the eleven

overlapping regions which are less than 400 bp in size. Each region was overlapping

around 100 bp.

assay and at least two SSCP runs were performed for each set of sarnples to assure

reproducibility of the assay.

Cloning of the amplified fragments

1 pg PCR product was digested in 20 pl using approximately 2 U of BamHI

enzyme per microgram of DNA in BamHI reaction buffer and cloned into a pUC19

vector. Restriction enzymes were obtained fiom Boehringer Mannheim or Pharmacia

and used as recomrnended by the manufacturers.

Ligation of DNA fragments

The arnplified product, (approximately 1.0 pg), was digested with Barn HI

restriction enzyme and purified by ethanol precipitation, as described above. 0.5 pg

pUC19 vector DNA was linearized with Barn HI and dephosphorylated using calf

intestinal alkaline phosphatase (CIAP). CIAP was used at a concentration of 1U per

pg DNA for 15-minutes at 37'C. Alkaline phosphatase activity was terminated by

heating at 65'C for 10-minutes with 20 mM EDTA and 0.5% SDS. The volume was

adjusted to 240 pl and extracted with phenol in order to remove residual alkaline

phosphatase. The top phase was extracted with ether and the DNA precipitated on ice

for 30-minutes using 0.3 M sodium acetate (NaOAc) and three volume of ethanol.

The pellet was resuspended in TloEi buffer (1 0 mM Tris-HC1, pH 8.0 and 1 mM

EDTA). The vector and 100 to 500 ng of insert DNAs were then mixed together in

ligation buffer (50 mM Tris-Cl, pH 7.6, 10 mM MgC12, 1 mM rATP) in a moiar ratio

Gerrnany).

Preparation of competent celis and transformation with recombinant plasmid

Competent cells for transformation were prepared. 20 ml of Luria broth was

inoculated with 0.5 ml of an ovemight E. coli culture. The cells were then grown at

37C with aeration to early log; OD at 600 nm which was equal to 0.13 - 0.15, taking

approximately 2 hours. The cells were pelleted by centrifugation. The pellet was

resuspended in 10 ml of solution A (10 mM 3-(N-Morpholin) propane - sulfonic acid

(MOPS) pH 7.0 and 10 m M rubidium chloride). Cells were again spun down,

resuspended in 10 ml of solution B (10 mM MOPS, pH 6.5, 10 mM rubidium chloride,

and 50 mM calcium chloride) and incubated on ice for 30-minutes. Afterward, the

cells were centrifuged and the ce11 pellet was resuspended again in 1 ml of solution B.

For transformation, 0.15 - 0.20 pg of ligated DNA, approximately 10 to 20 pl

of ligation mixture was added to 200 pl of the competent cells prepared above. It was

mixed and incubated on ice for 30-minutes. The cells were then heat-shocked at 42.C

for 90-seconds. 1 ml of Luria broth was added and the mixture was incubated at 37'C

for an hour. This allowed for the expression of the antibiotic resistant genes present

on the plasmid before transformed cells were plated ont0 selective medium. 100 pl of

the transformed cells were added per plate for selection of appropriate transformants.

Recombinant plasmid DNA preparation

S,mall scale preparation of plasmid DNA from E. coli was perforrned (Maniatis

pelleted by centrifugation at 1200 rpm for 5-minutes. The bacterial ce11 pellet was

resuspended in 100 pl of GTE-lysis buffer: 50 m M glucose, 25 mM Tris-HCl pH 8.0,

10 mM EDTA, 2 mg lysozme per ml and incubated at roorn temperature for 5-

minutes. The bacterial suspension was treated with 200 pl alkaline SDS buffer (0.2N

NaOH, 1% SDS) and mixed by inverting the tubes gently several times and placed on

ice for 5-minutes. 150 pl of acetate buffer (3 M potassium acetatelacetic acid) was

added mixed by inverting and chilled on ice for 5-minutes. The samples were

centrifuged for 10-minutes at 4'C , and the supernatant was collected. Plasmid DNA,

thus extracted was concentrated by ethanol precipitation with two volumes of 95%

ethanol and incubation at -70C for 30-minutes. DNA was pelleted by centrifugation

for 20-minutes at 4C, washed with 70% ethanol and vacuum-dried. The dried pellet

was resuspended in 50 pl TloEI (1OmM Tris-Cl - 1 mM EDTA, pH 8.0) for further

use.

Preparation of sequencing grade plasrnid DNA

Bacterial cultures were grown in Luria broth containing ampicillin to ensure

the presence of the plasmid within the culture. Afier plasmid DNA was extracted, 18

pl (-1 .O pmol) of DNA was denatured for 5-minutes with 2 pl of 2 N NaOH at room

temperature. Then, the DNA was precipitated by adding 8 pl of 5M ammonium

acetate pH 7.4 and 100 pl of absolute ethanol and incubating at -70-C for 10-minutes.

The DNA collected by centrifugation was washed twice with 70% ethanol and vacuum

dried.

supernatant (after the addition of acetate buffer and centrifugation) from the

minipreparation protocol and ethanol precipitation, as mentioned above. The DNA

pellet was then resuspended in 50 pl of TE containing RNase (1 0 pg RNse per ml) and

incubated at 37.C for 30-minutes. Then 30 pl of PEG solution (20% PEG-8000,2.5 M

NaCl) was added and incubated on ice for an hou. DNA was then centrifuged,

washed in 70% ethanol, vacuum dried, and resuspended in H20.

DNA sequencing and polyacwlamide gel electrophoresis

The sequence was determined fiom subclones prepared as rnentioned above

and by using specific primers. The DNA sequence was detennined by the dideoxy

chain termination method (Sanger, 1977). It used the Sequenase sequencing kit (US

Biochemical Corp., CleveIand, OH, USA) rnodified version of T7 DNA polmerase

with [ ~ c - ~ ~ s ] dATP for labeling. For each clone, both strands of insert were cornpletely

sequenced. The denatured DNA was annealed to a specific primer and extended

products of various lengths were obtained with the use of sequenase. The DNA (3-5

pg of denatured double-stranded DNA or 1-2 pg of the single-stranded DNA per

reaction) was mixed with an appropriate primer (0.5 pmol per reaction) in 1X

Sequenase Buffer (40 mM Tris-Cl, pH 7.5, 20 mM MgC12, 50 mM NaCl), and

annealed by heating the mixture in a capped tube at 65'C for 2-minutes followed by

slowly cooling the tube to room temperature for about 30-minutes. After annealing,

the DNA was mixed with 6.5 mM DTT. 2 nM of dGTP, dCTP. dTTP, 0.5 pl of ["SI

dATP (1000 Ci per mmol), 3 U of Sequenase enzyme and labeled by incubation at

(each mix contains 50 mM NaCI, $0 uM of dNTP/8 p M one of ddATP, ddGTP,

ddCTP, or ddTTP) was added to the labeled DNA mixes in each of the four wells, and

incubated at 37'C for 5-minutes. After the termination reaction, 4 pl of the stop

solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.5% xylene

cyan01 F F ) was added to each reaction. The sample was heated to 80'C for 5-minutes

irnmediately before loading on the 6 % denaturing polyacrylamide sequencing gel.

The polyacrylamide mix for sequencing gel was prepared as 35 gm ultrapure urea, 9

ml of a 45% acrylamide stock, 350 pl of 10% ammonium persulfate and 50 pl of

TEMED per 70 ml of polyacrylarnide gel and run at 1700 Volts. After the samples

were run on the sequencing gel, the gel was soaked in a solution of 10% acetic acid,

10% methanol, transferred ont0 3MM Whatman filter paper, and dried at 80'C for 45-

minutes. The dried sequencing gel was exposed on X-ray film overnight.

Detection of hippurate hvdrolase sequence heterogeneitv in the hippurate

hydrolase-negative C. jejurzi clinical isolates

Genomic DNA of the five hippurate hydrolase-negative C. jejuni clinical

isolates was examined by polymerase chain reaction for sequence variations. The

hippurate hydrolase-positive C. jejuni TGH9011 strain was used as a control (Hani and

Chan, 1995). The PCR strategy used in this study is shown in Figure 4. The specific

sequence primers, H l , H2, H3, H4, H5, H6, H7, H8, H9, H l0 and Hl 1, with three

degenerate primers, PCl, PC3 and PC4, were used in the polyrnerase chain reaction.

The positions of the eleven primer sets used, are indicated above the hippurate

hydrolase gene which is depicted as a box in Figure 4. The degenerate primers, PC1, 3

and 4 were previously designed with the minimal number of possible degenerate

oligonucleotides required to account for al1 variations of a particular arnino acid

residue (Hani, 1997). Primers were used to ampli@ the eleven overlapping regions of

the hippurate hydrolase gene from five different hippurate hydrolase-negative C. jejuni

clinical isolates, D594, D603, D835, D 17 13, Dg77 and hippurate hydrolase-positive C.

jejuni TGH9O 1 1. Eleven primer pairs were used in polymerase chain reactions that

covered from upstream nt -259 to nt 13 17 downstream of the hippurate hydrolase

gene. The adjacent amplified products overlapped each other by about 100 bp. The

amplified products were electrophoresed in an 8% polyacrylamide gel (PAGE) with 4

X 174 Hae III fragment as markers. The results of the amplified products from eleven

overlapping fragments of the five different C. jejuni hippurate hydrolase-negative

that were identical to that of positive control C. jejzrni TGH9011. These findings

indicate that al1 tested hippurate hydrolase-negative C. jejuni isolates do not have

major sequence variations such as gross deletions or insertions at their hippurate

hydrolase gene. However, a standard gel analysis of the arnplified products would be

unable to detect minor sequence variations within the hippurate hydrolase gene of

hippurate hydrolase-negative isolates.

Single strand conformational polvmorphic analysis of the natural mutant

hippurate hydrolase Eene

The flanking regions and the coding sequence of the hippurate hydrolase loci

from hippurate hydrolase-negative C. jejzrni isolates were analyzed by 3 2 ~ labeled

single strand conformational polymorphism (SSCP) assay based on polymerase chain

reaction.

The F7 and F8 overlapping regions spanning from nucleotides 473 to 912 of

the hippurate hydrolase gene showed polymorphisms in al1 five different hippurate

hydrolase-negative C. jejuni isolates compared with those of control C. jejuni TGH

9011 (Figure 5). Other regions, F4, F5, F6, F9, F10 and F11, showed no

polymorphism in their SSCP profiles. They were indistinguishable from each other

and also similar to those of the control C, jejuni TGH9011. The polymorphic regions,

F7 and F8, may presumably account for the hippurate hydrolase-negative phenotype of

the five negative C. jejuni isolates in the hippurate hydrolysis test.

The promoter area is covered by the overlapping amplified regions F1, F2 and

F3 spanning from nucleotides -259 to 150 bp. The SSCP analysis of promoter regions

Table 7. Surnmarv of PCR analvsis in the hippurate hvdrolase gene from the five

hippurate hydrolase-ne~ative C. jejuni isolates.

Vertically presents control C. jejuni TGH9011 strain (top) along with the hippurate

hydrolase-negative C. jejuni isolates. Horizontally presents the arnplified products of

eleven overlapping regions F1 through F11. + sign represents normal and identical

arnplified product to its respective region.

Strains PCR amplified products

Figure 5. PCR-SSCP analvsis of F7 and FS regions of the hippurate hydrolase

pene of five different hippurate hydrolase-negative C. ieiuni isolates.

Figure 5(A). The sarnples anaIyzed for F7 were lane 1, C. jejuni TGH9011; lane 2,

D594; lane 3, D603; lane 4, TGH9011; lane 5, D835; lane 6 , D977; lane 7, Dl 7 13.

Figure 5(B). The sarnples analyzed for F8 were lane 1, C.jejuni TGH9011; lane 2.

D977; lane 3, D1713; lane 4, D835; lane 5, TGH9011; lane 6, D603; lane 7,

TGH90 1 1 ; lane 8, D594; lane 9, TGH90 1 1.

Mobility shifts are found in fragments F7 and F8 of the five hippurate hydrolase-

negative C. jejuni isolates when compared to the respective regions of control C. jejuni

TGH90 1 1 .

Figure 5(A).

Figure 5 (BI.

Figure 6 (A, B, and C). PCR-SSCP analvsis of FI, F2 and F3 regions of the

hippurate hydrolase gene of five different hippurate hvdrolase-negative C. iejuni

isolates.

Figure 6(A). The sarnples analyzed for FI were lane 1, C. jejuni TGH9Oll; lane 2,

D594; lane 3, D603; lane 4, TGH90I 1; lane 5, D835; lane 6, D1713; lane 7, D977;

lane 8, TGH9011.

Figure 6(B). The sarnples analyzed for F2 were lane 1, C. jejuni TGH9011; lane 2,

D594; lane 3, D603; lane 4, D835; lane 5, D1713; lane 6, TGH9011.

Figure 6(C). The sarnples analyzed for F3 were lane 1, C. jejuni TGH9011; lane 2,

D594; lane 3, D603; lane 4, TGH9011; lane 5, D835; lane 6, D1713; lane 7,

TGH9011; lane 8, D977.

The PCR-SSCP analyses of the promoter region from the five hippurate hydrolase-

negative C. jejuni isolates which were covered by the FI, F2, and F3 amplified

fragments. The assays show unaitered strand mobility compared with that of control

C. jejuni TGH90 I 1.

Figures 6 (A).

Figures 6 (BI.

Figures 6 (C).

regions of the hippurate hydrolase-negative C. jejuni isolates. They were also identical

to the respective regions of C. jejuni TGH9011 (Figure 6).

These results suggest that the promoter regions of the hippurate hydrolase-

negative isolates are unlikely to be the site of sequence variations.

Cloning of SSCP polymorphic regions and DNA sequencing

To establish the precise molecular nature in the five hippurate hydrolase-

negative isolates, the DNA coding regions which displayed the abnormal SSCP profiles

were cloned, sequenced and compared with the hippurate hydrolase sequence from C.

jejuni TGH9011. The amplified fragments of F7 and F8 regions from the five

hippurate hydrolase-negative C. jejuni isolates were cloned into pUC 1 9 vector

(Pharmacia) and sequenced by the dideoxynucleotide chain termination procedure

using T7 sequenase (USB) with specific primers.

The nucleotide sequences of the insert in pUC19 were detennined in both

directions. The sequence of each polymorphic region was almost completely

homologous with the respective region of the C. jejuni TGH90 1 1 sequence. The

respective numbers of nucleotide substitutions from the published hippurate hydrolase

C. jejzrni TGH9011 sequence for each hippurate hydrolase-negative sample are listed in

Table 8.

Most of the mutations detected were silent in nature and they do not affect the

amino acid alterations. They were TTA + TTG at nucleotide (nt) position 633, TTA

+ TTG at nt position 648, AGC + AGT at nt position 654, CCC + CCA at nt

position 678, and TCA + TCT at nucleotide position 687. Except the nt substitution

(Val) codon to alanine (Ala) at residue 250 (Figure 7). This valine at residue 250

change was common to al1 of the hippwate hydrolase-negative C. jejuni isolates.

Sequenice comparison

The hippurate hydrolase homologous sequences were obtained from a cornputer

search of the Protein Data Base GeneBank and the Swiss Protein Database. The

Clustal 1.4W program was used to compare the deduced amidohydrolase sequence of

C. jejuni TGH9011 (HipO) with those of the Bacillus sterothermophilus arninoacylase

(Ama) (Sakanyan et al., 1993), the Pseudamonas sp. amidohydrolase hydmtoin

utilizing protein C - HyuC) (Watabe et al., 1992; Ishikawa et al., 1996) and the

Synechocystis sp. hypothetical arnidohydrolase protein (Kaneko et al., 1995) of the

AMA/HIPO/HYUC M40 hydrolase family (Figure 8). The valine at residue 250 of C.

jejuni hippurate hydrolase is located as a conserved residue in these homologous

proteins from the M40 hydrolase family.

Predicted hydrophathy profiles

A hydrophobicity plot is a current method for predicting the structure of

proteins. It is a graph displaying the distribution of polar and apolar residues along

sequences. The sequences are scanned with a moving window of a given length and

computed for each position of the window, the mean hydrophobicity of al1 residues in

the window. The predicted hydropathy and flexibility profiles of the hippurate

hydrolase protein were plotted by the Kyte-Doolittle scale with a window size of 7

Table 8. Nucleotide substitutions between nucieotide 473 and 912 of the hipO genc

of C. iejuni hippurate hvdrolase-negative isolates.

HipO Sequence from C. jejrtni TGH90 1 1

Isolates

D594

D603

D835

D977

Dl713

Nucleotide # Codon

(Hani et al., 1995)

TTA TT A AGC GTA

TT A TTA AGC CCC GTA

TT A TTA AGC GTA

TTA TCA AGC GTA

TT A TT A AGC GTA

Codon Substitutions

in isolates

TTG TTG AGT GCA

TTG TTG AGT CCA GCA

TTG TTG AGT GCA

TTG TTG TCT AGT GCA

TTG TTG AGT GCA

Phenotvpe Condition

Hippurate Hydrolysis

215

TGH 901 1 positive ALQSIVSRNVDPQNSAWSIGAFNAGHAFNI 1 PDIVRTIKMSVRALDNET 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ~ 1 1 1 1 1 1 1 1

D835 negative ALQSIVSRNVDPQNSAWSIGAFNAGHAFNI 1 PDIARTIKMSVRALDNET

Hipput-ate Hydroiysis

TGH 901 1 positive KLTEEKIYKICKGLAQANDIEIKINKNW. ................... I l l l l l l l l l l l l l t l l l l 1 1 I l I I 1 I I I

. . ............... D835 neetive KLTEEKIYKICKGLAQANDIEIKINKNWA.

Figure 7. The deduced amino acid sequence alignment of the mutated region of

hippurate hvdrolase-positive C. jejuni TGH9011 and the hippurate hvdrolase-

negative strain DS35.

A single arnino acid substitution at valine to alanine at residue 250 was found in al1 the

hippurate hydrolase-negative proteins.

Figure 8. AIignment of C. jejuni hi~purate hvdrolase and related proteins.

The complete polypeptide deduced from gene hipO for N-benzoyl-arnino acid

amidohydrolase (hippurate hydrolase) was aligned using the Clustal W (1.4) program

with the complete polpeptides of related proteins. The top line gives the C. jejuni N-

benzoyl-arnino acid amidohydrolase (HipO) protein sequence, second line shows the

Bacillus stearothermophilus N-acyl amino acid arnidohydrolase (arninoacylase) (Ama)

protein sequence and the third line is the N-carbamyl-L-amino acid amidohydrolase

(DL-hydantoinase) fiom Pseudomonas sp. (hydantoin utilizing protein C) (hyzlC ) and

lastly Synechocystic hypothetical protein. Residues involved in conserved areas are

s h o w in bold face. Identical residues are marked with stars, sirnilar residues are

indicated by dots and hyphens indicate gaps introduced to maximize alignment.

Val-250 is located as an identical residue in the sequence of the

AMNHIPOMYUC M40 hydrolase family .

CLUSTAL W (1.4) Multiple Sequence Aliqnment

H ~ P O ------- MNLIPEILDLQGEFEKIRHQIHENPELGFDELCTAKLVAQKLKEFGYEVYEEI Am= ---- MTKEEIKRLVDEVKTDVIAWRRHLHAHPELSFQEEKTAQFYETLQSFGHLELSRP HyuC MKTVTISKERLRIHIEQLGEIGKTKDKGVQRLALSKEDREATLLVSEWMREAGLTVTHDH S ~ P ------- MELKNLAQTLLPRLVEIRRHLHAHPELSGQEYQTAAWAGVLSSCGLHVEEAI . . * . .. * *

HipO GKTGVVGVLKKG--NSD-KKIGLRADMDALP-------- LQECTNLPYK--SKKENVMHA Ama TKTSVMARLIGQ--QPG-RVVAIRADMDALP-------- IQEENTFEFA--SKNPGVMMA HyuC FGN-LIGRKEGE--TPSLPSVMIGSHIDSVRNGGKFDGVIGVLAGIEIVHAISEAMNHE shp GKTGVVGQLSGKGDDP--RLLAIRTDMDALP-------- IEEMVSLPFA--SRHPGVMHA

. . * . . . .*.. . * . * HipO --------c---G--- HDGHTTSLLLAAKYLASQ--NFNGTLNLYFQPAEEGLGG-AKAM Ama --------c---G--- HDGHTAMLLGTAKIFSQLRDDIRGEIRFLFQHAEELFPGGAEEM HyuC HSIEVVAFCEEEGSRFNDGLFGSRGMVGKVKPEDLQKVDDNNVTRYEALKTFGFGIDPDF shp --------c---G--- HDIHTTLGLGTAMVLSQMGHRLPGDVRFLFQPAEEIAQG-ASWM

* . * . . * HipO IEDGLFEKFDSDYVFGWHN--MPFGSDK--KFYLKKGAMMSSDSYSIEVIGRGGH-GSA Ama VQAGVMDGVD--VVIGTHL--WSPLERG--KIGIVYGPMMAAPDRFFIRIIGKGGH-GAM HyuC THQSIREIGDIKHYFEMHIEQGPYLEKNNYPIGIVSG--IAGPSWFKVRLVGEAGHAGTV shp IQDGAMKGVS--HILGVHV--FPSIPAQ--QVGIRYGALTMDDLEIFIQGESGH-GAR

* . * * . . * ** * .

HipO PEKA-KDPIYAASLLVVALQSIVSRNVDPQNSAVISIGAF~IAGH-A~I 1 PDIVTIKMSV Ama PHQT-IDAIAIGAQVVTNLQHIVSRYVDPLEPLVLSVTQFVAGT-AHNVLPGEVEIQGTV HyuC PMSLRKDPLVGAAEVIKEVETLCMN--DPNAPWGTVGRIAAFPGGSNIIPESVEFTLDI shp PHEA-IDAIWIAAQVITALQQAISRTQNPLRPMVLSLGQISGGR-APNVIADQVR~GTV

* * . * .. * . . . . . * *. . HipO RALDNETRKLTEEKIYKICKGLAQANDIEIKINKNVVAPVTMNNDEAV-EFASEVAKELF Ama RTFDETLRRTVPQWMERIVKGITEAHGASYEFRFDYGYRPVINYDEGDPRHGGNGVRAVR H ~ u C RDIELERRNKIIEKIEEKIKLVSNTRGLEYQIEKNElAAVPVKCSENLI-NSLKQSCKEL- shp RSLHPETHAQLPQWIEGIVANVCQTYGAKYEVNYRRGVPSVQNDAQLN-KLLENAVREAW

* . . . . .

HipO GEKNCEFNHRPLMASEDFGFFCEMKKCAYAFLEN----- ENDIYLHNSSYVFNDKLLARA Ama RRGSGPLETEH--GRRRFLRLFAKSARQLFLRRRGQCRKRHRLPAPPPALYD-------- H p C -EIDAPIIVSG--AGHDAMFLAEITEIGMVFVRC------ RNGISHSPKEWAEI DDILTG ohp GESALQIIPEPSLGAEDFALYLEHAPGAMFRLGTGFGDRQMNHPLHHPRFEADEAAILTG

HipO ASYYAKLALKYLK---- Ama ----------------- HyuC TKVLYESIIKHI----- shp VVTLSYAAWQYWQNIAI

surface-exposed regions (Hopp and Woods, 198 1 ).

The predicted hydrophathy profile of C. jejuni hippurate hydrolase (HIPO) is

generally simiiar to the predicted profiles of the amidohydrolase of B.

stevothermophiIus ( AMA), Pseudornonas sp. (HYUC), and Synechocystis (Figure 9).

Hydrophilic regions are located at the outside of the protein structure, and hydrophobic

regions are buried inside the protein or within other hydrophobic environment such as

membrane. Val-250 is located in the hydrophobic region of the predicted hippurate

hydrolase protein profile of C. jejuni (Figure 10). The flexibility blot is used to identify

the specific sites that are protruding or highly mobile regions on the surface, and Val-

250 is also situated in the rigid region of the protein profile (Figure 10). When alanine

is substituted in place of valine, a change in the predicted secondary structural from

beta pleated sheet (p-slieet) to alpha helix (a-helix) and also a reduction in the

hydrophobicity of the protein were observed (Figure 1 1).

CL* LO* 1 OC L O Z L 0 1

I l L

I 1 1 100. s-

O L E 00 ' S- 00 ' fr-

Hydrophi l ic i tyWindowSize= 7 Scale = Kyte-Doo l i t t le 5.00 , 1

1 . î S ............... ......................... ........................ ................ . ....................... ........................ 1 20 - *; ; ,..................*.....; .......................*+; ......*...; ; ; ................................................... ........................ ................ ........................ i ...................*.....: ...................... *..; ; ; ........................ ......................... ..... ................ ...................... ........................ i i .....*.;

...... ......... .......... ........ ............................................. .............. ......................... ....................... ........................ ............. ......................... i.. ......................... ......................... ..............*.....**.. .............*.. . ........................ ........................ ......................... ......................... 0 85 - 6 i i 6 &

......................... .......................* ................ . ........................ ........................ ......................... ................. .........*............... 0 80 - 9 6 i & &

O . 75 1 I 1 1 l 1 1

50 1 O0 150 200 250 300 350

Figure 10. Predicted hvdropathv (hvdro~hobiciw and flexibiliw) profiles of C.

jeirrni hippurate hydrolase.

The hydropathy and flexibility profiles of the HIPO protein with the Kyte-

Doolittle scale and a window size of 7. Numbers on the bottom indicate arnino acid

positions. This profiles graph the local hydrophilicity of a protein along with its amino

acid sequence. Hydrophobic regions deflect downward (negative value). In flexibility

profile of HIPO, the region deflect downward (negative value) which represents the

rigid region.

Val-250 is predicted to locate at rigid hydrophobic region of hippurate hydrolase.

Kyet & Uoolittle Hydrophobicity Protïles

Figure 11. Corn parison of the predicted secondary structure and hvdrophobicity

profiles of C. iejuni hippurate hvdrolase (Val-250) and its modified sequence

(substitution of Ala-250).

Hippurate hydrolase is a key enzyme used to identifi C. jejuni and to

differentiate C. jejuni from other species of Campylobacter. The role of hippurate

hydrolase in C. jejuni is unclear at this moment. However, kanarnycin cassette

insertion mutants of C. jejuni hippurate hydrolase have been constructed and they

appear to grow normally in Mueller-Hinton medium (Hani, 1997). Thus, the hippurate

hydrolase gene is not essential for growth under such conditions. The prevalence of

hippurate hydrolase-negative C. jejuni in poultry and humans is very rare. The

hippurate hydrolase-negative isolates which hydrolyzed low levels of hippurate could

be detected by a sensitive method, such as gas liquid chromatography (Totten et al.,

1987). Characterization of the defective hippurate hydrolase gene and the encoded

hippurate hydrolase enzyme could provide valuable information on its structure and

fünction.

In this study, we characterized five different hippurate hydrolase-negative C.

jejuni clinical isolates (D594, D603, D835, D1713, and D941) isolated fiom chicken

and humans. These strains fell into different Lior and Penner serotypes and have

different plasmid patterns. Therefore, they represent a diverse group of isolates (Totten

et al., 1987). A radiolabeled single strand conformational polymorphic (SSCP) assay

based on polymerase chain reaction was used to detect the putative mutated regions of

the hippurate hydrolase gene from five hippurate hydrolase-negative C. jejurzi isolates.

The polymorphic regions detected in the SSCP assay from each isolate were cloned and

sequenced to determine the exact nature of the mutations. We observed that valine at

hydrolase-negative clinical C. jejuni isolates.

Although valine and alanine are both nonpolar hydrophobic arnino acids and

have alkanes as side chains, stereochemical representation shows that valine has a

larger hydrocarbon side chain than alanine. The bond between the alpha-carbon and

beta-carbon of a valine residue in a polypeptide emphasizes the steric interactions

between the peptide backbone and the carbons in the two garna-positions of this arnino

acid and, by extension, the steric interactions for each of the others. One or two

substitutes might be attached to the beta-carbon, and this places one or two substituents

at either or both of the positions of the two methyl groups of valine. This does not

happen in alanine. Therefore, the change of valine to alanine may have some impact on

both the structure and fùnction of the C. jejuni hippurate hydrolase.

Sequence cornparisons of proteins between species are frequently used to

establish conserved elements which may have importance for protein function. Multiple

sequence alignment of the C. jejuni hippurate hydrolase enzyme with homologues from

M40 hydrolase family (AMA/HIPOMYUC), identified conserved sequences within the

hydrolase proteins of different species:Bacillus sterotherrnophilus, Psedornonas

species, and Synechocystis. Homologous enzymes share the cornmon feature of the

splitting action, which is similar to the enzyme that cleaves or hydrolyzes hippurate.

Significantly, Val-250 is identified as a conserved amino acid based on sequence

alignment of the AMA/HIPO/HYUC deduced amino acids. The presence of a

particular arnino acid at a conserved location in homologous proteins across different

species suggests a functional role associated with that site. This reinforces the common

identity of the protein in that farnily. Hippurate hydrolase protein from C. jejuni is

characteristic and important positions in the structure of the C. jejuni hippurate

hydrolase protein may also be characteristic and important in the other hydrolase

members. The conservation of Val-250 in al1 these homologous proteins suggests that

it may potentially be critical for hippurate hydrolase function in C. jejuni.

Particular positions in the amino acid sequence that are buried in hydrophobic

clusters are the most invariant. Moreover, the hydrophobic effect contributes favorably

to protein folding. There are usually a number of locations in the structure of a protein

where difficulties resulting fiom the packing of the backbone of the polypeptide arise.

The preference for particular amino acids rnay reflect the constraints of an intricate,

interlocking stereochemistry in the interior of protein structure. The hydropathy

profile of the C. jejuni hippurate hydrolase protein revealed that Val-250 is located in a

hydrophobic region of the C. jejuni hippurate hydrolase protein. Furthemore, the

nearest-neighbor effects are taken into account by assigning different flexibility values

to a given kind of residue depending on whether it is surrounded by "rigid" residues or

not. The flexibility profile identified that Val-250 is located in a rigid region of the

hippurate hydrolase. The difference in standard fiee energy of folding between valine

and alanine is +5 kJ mol-' (Salahuddin and Tanford, 1970). The replacement of valine

by alanine rnay result in a change of standard free energy and this will also change the

hydrophobic effect. Therefore the change could result in destabilizing the protein.

Most likely the reason is that an empty space wiil be created in the protein, unless the

native structure is rearranged to fil1 it. Therefore, the particular residue Val-250 may be

required and essential to maintain C. jejuni hippurate hydrolase structure and enzymatic

function.

iI 1 Y 1 ir - - --

alpha helix (a-helix), in place of beta pleated sheet (P-sheet) if alanine is substituted in

the place of vaiine. Although both valine and alanine have closely related aliphatic side

chains of the building blocks, alanine with a methyl group in its side chain favors the

formation of an a-helix by a higher relative frequency of 1.29. The a-helix is tightly

coiled and it tends to reside on the periphery of the protein structure. Unlike alanine

which has one carbon side chain, valine has an additional methyl goup in its side chain

and this enhances the P-pleated sheet in a relative fiequency of 1.49 (Lim, 1974;

Garnier et al., 1978). In general, a polypeptide chain is almost fully extended in the B-

pleated sheet and tends to reside in the center of crystallographic molecular models of

proteins. This indicates that the tendency of a polypeptide to adopt a regular secondary

structure depends largely on its arnino acid composition and may have some intrinsic

preferences among the amino acids for certain secondary structures. Therefore, the

conserved arnino acids have a critical role in folding or general stability. The amino

acid substitutions may be tolerated and have no deleterious effect on the protein

structure. Yet mutation of a critical structural residue might modi@ the protein and

consequently cause the catalytic ability, and the apparent conformation and stability of

mutated protein to become less efficient.

Three continuous overlapping upstream regions of the hippurate hydrolase gene

from the five hippurate hydrolase-negative C. jejuni clinical isolates revealed no

polymorphism in the SSCP assay. This indicates that there is possibly no sequence

variations in these upstream regions. A conclusive picture wouId require nucleotide

sequencing of the upstream regions of the hippurate hydrolase genes from hippurate

multiple copies of transcripts produced by these five hippurate hydrolase-negative C.

jejuni isolates (Hani, 1997). The above study did not show a defect in transcription of

the hippurate hydrolase gene in the five hippurate hydrolase-negative C. jejuni isolates.

At this stage, the fimction of hippurate hydrolase in C. jejuni is not clear. There

is no specific information available on differences in disease severity in hurnans relative

to either hippurate hydrolase-positive or -negative C. jejuni isolates. The only

distinguishing feature of C. jejuni from a closely related farnily member, C. d i , is the

presence of a hippurate hydrolase gene and its ability to hydrolyse hippurate. In

addition, C. jejuni is fiequently isolated from cases o f human diarrhea and fiom poultry.

The active sites of hippurate hydrolase in C. jejuni need to be defined. It is a

subject for future studies. Residue substitution consequently occurring o n the catalytic

component Ieads to an enzyme with dramatically reduced activity. Valine and alanine

have closely related aliphatic amino acids with the only difference in side chain

residues being carbon chain length. Mowever, this change may affect or disturb the

folding of the enzyme structure or change the amino acid size, resulting in a

nonfunctional or reduced stability of hippurate hydrolase protein. The active sites of

hippurate hydrolase need to be determined to see whether this specific residue is

essential for hippurate hydrolase function.

Analysis of the naturally mutated hipO genes from the five different C. jejuni

isolates suggests that valine at residue 250 may potentially be a key residue of hippurate

hydrolase in C. jejuni. Therefore, we can examine if this valine at residue 250 is at the

catalytic site of hippurate hydrolysis by site-directed mutagenesis and enzyme assay.

In addition, other conserved arnino acids have been identified based on a

multiple alignment of the four amino acid sequences of the M40 hydrolase farnily. We

can also examine the putative roles of other conserved residues by site-directed

mutagenesis. The enzyrnatic properties can be exarnined using a chromogenic substrate

or the hydrolysis ability by using a color reagent system.

Our lab has constmcted an isogenic hipO mutant of C. jejuni by gene

replacement with a kanamycin-insertion hippurate hydrolase mutated gene (Hani,

1997). Therefore, the correlation between the hipO gene and C. jejuni pathogenicity

can be investigated using kmamycin-inserted isogenic mutants in animal models or the

study of invasiveness with human intestinal cell lines.

In addition, there is no report on the disease severity related to particular

Cumpylobacter species and it will be interesting to reevaluate the association of

hippurate hydrolase-negative C. jejuni strains with infection.

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