A gene complementation strategy in cloning an oxidative stress response

gene of Campylobucter jejuni.

Ashley Edward Roy Soosay

A thesis submitted in confonnity with the requirements for the Degree of Master of Science

in the Giaduate Department of Molecdar & Medical Genetics

University of Toronto

Q Copyright by Ashley E. R. Soosay (1998)

National Library Bibliothèque nationale du Canada

Acquisitions and AccpMtions et Bibliographie Services services bibliographiques

395 Wellington Street 395. nie Wdington OîtawaON K 1 A W OttawaON K1AON4 canada Canada

The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Lïbrary of Canada to Bibliothèque nationale du Canada de reproduce, loan, distriibute or seil reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de

reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantie1s may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.

ABSTRACT

In order to clone oxidative stress response gene of C~mpylobacter jejwu' TGH9ûI 1,

an endonuclease IV (nfo) mutant s w i n of Escherichia wli AB 1 157, RPC5ûû that has

increased sensitivity to ten-butyl hdyroperoxide (Tl3HP) was tramfomied with a pBR322

genomic DNA library of C. jejiuu TGH9011. A pBR322 combinant clone, pBRC 1 that

conrains a 4.1 kb fiagrnent of the C. jejMi genome was shown to confer increased

resistance for RPCSOO against TBHP. The 4.1-kb fragment has been completely sequenced

and contains five coxnplete open reading hunes (ORFs) and two partial ORFs. Using

deletion mutant analysis I was able to ident* a 6Wbp ORF. that is responsible for the

&tance to TBHP, desig~ated ORF3. ORF3 shows homology to a thioredoxin-like

protein of Drosophiülo melanogaster. The tmsription start-site of ORF3 (d) was

identifieci. Fur-Box-like sequences were identifjied at the ribosome-binding-site (rbs),

within the coding region and dowmtream of ibe gene. A catabolite activator protein (CAP)-

Wce-binding sequences are present at the promoter region and r the rbs. Two OxyR-like-

binding sequences are present upstnam of t d gene. ïhe rr*C gene has been mapped to

the SalI-A, SacII-A and S d - A fiagrnent on the physical genomic map of C. jejwu'

TGEW) 1 1 . Southem blot hybridization anaiysis identified a mC homolog in C. lari and C.

coli, and also if low stringency conditions were use& in C. upsalienFis. trxC an oxidative

stress response gene of C. jejwii, may be regulated by CAP, Fur and also rnight be part of

the OxyR regdon. The pçesence of a nxC homolog in three other carnpylobacters suggest it

may be an important gene.

My h d e l t gratitude to Dr. Ricky Chan for king a constant motivator during my

graduate study. His helpN M o n and constructive advise pertainjng to my research

project is much appteckted. The two years of aaaing at his lab has been a resourceful

experience and will be neasured

Without the help and guidance of my labmares it would not have been possible for

me to master the molecular biology techniques. Firstiy 1 wish to convey my suicere thanks

to Helena Louie and Shahnaz Al-Rashid For these two ladies have been my source of

reference in the lab. I am also greaîly grateful to David Ng and Thin for helping me

whenever possible. Shahnaz, David and Thin. thank you very much for also king my

bestfriend. Many thanks to Angela and Jennifer. for helping me in my routine lab work and

also king my niend. 1 rhank Eric Hani for clearing my doubts at t h e s and also for the late

discussions. F d y a note of gratitude to Billy Bourke, who made it possible for me to

grasp the PFGE technique.

My life in Toronto would not have been compkte without my suitemates. Yoahan

Kong, Hector Pons. Manwinder Singh and not forgetting, Gillian Patton. You guys are

what they call true friends. Your friendship will always be treasured.

AU work and no play makes "anyones7* life a miserable one. Well for this 1 thank

Penny Chan for being my constant squash game parmer during my stay at Toronto. I

would also iike to thank Dr. Chan's family for taking me into their home when 1 h t

anived in Toronto.

Finaily, 1 would like to thank my supe~sory cornmitte, Dr. Andrew Becker and

Dr. Andrew Bognar for their constructive advise in writing of my thesis.

ABSTRACT ACKNOWLEDGEMENT TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS

i i i iii v i i viii x i

INTRODUCTION

Chernical nature of oxygen

Reactivity of reactive oxygen-derived species (ROS)

Superoxide

Hydrogen peroxide

ïiydroxyl radical

Iron-oxygen complex

Sources of reactive oxygen-derived species (ROSI

Superoxide

Hydrogen peroxide

Hydroxyl radicals

ROS from macrophage

Cellular defence mechanisms

The primary defence

Superoxide dismutase (Sod)

Catalase (Kat)

The thioredoxin (Tm) & glutaredoxin (Ga) system

The secondary defence

Proteia damage

DNA damage

Mutagenicity

DN A repair enzymes

Oxidative stress response

Superoxide stress response

Superoxide stress proteins

SoxRS regulon

Role of iron in regalation of sodA expression

Role of superoxide stress proteins

Peroxide stress response

Peroxide stress proteins

OxyR regdon

Eiow is an oxidative stress signal transduced to OxyR

protein?

Role of peroxide stress proteins

PURPOSE OF STUDY

METHODS & MATERIALS

Bacterial strains, plasmids and oligonucleotides

Media and general growth conditions

tert.-butyl hydroperoxide (TBBP)-mediated oxidative stress assay

Preparation of comptent cells and Transformation

Transformation of C. jejuni TG89011 recombinant pBR322

genomic DNA library into RPCSOO

Plasmid DNA isolation and A DNA extraction

Preparation of recombinant plasmid DNA for generating a

deletion mutant

Preparation of sequencing grade plasmid DNA

Dideoxynucleotide sequencing procedure

Polymerase c h a h reaction (PCR) amplification of genomic DNA and

piasmid DNA

End-labelling of oligonucleotides

Nick translation

Southern transfer

Screening of a pBluescript II SIC(+) library of C. jejuni (Colony Blot) 48

Screenhg of a S-Gemll Ubrary of C. jejrrni (Piaque lift)

Hybridization and Autoradiography

Pulsed field gel electrophoresis (PFGE)

RNA extraction

Primer extension analysis

RESULTS

TBHP-mediated oxidative stress assay

Clonhg an oxidative stress response gene of C. jejuni TGH9011

Snbcloning of pBSPCl and construction of pBPEORFl

Screening of a pBluescript II SIC(+) library & a A-Gemll übrary

of C. jejuni TG-O1 1

Mapping complementation region of pBSPCl and pBSAG4-LI

in RPCSOO agains t TBEP-mediated oxidative stress asssay

DNA sequencing of pBSPC1

Nucleotide sequence of ORF3 (trxC )

Deduced amino acid (aa) sequence of i rxC and alignment

Mapping of the lrrC promoter

Locating the trxC on the C. jejuni TGH9011 physical genomic map

Campylobacter species genomic DNA bybridizption

DISCUSSION

Oxidative stress via TBEIP

TxxC a regenerative protein during oxidative stress

The promoter region of C. jejuni trxC

Locating C. jejuni frxC on the physical genomic map and Southern

analysis of the gene in Campylobacter genus

DNA sequence of the pBSPCl insert

Gent-arrangement coiistrvatioa

FUTURE STUDIES

REFERENCES

List of Tables

Table 1. Bacterial strains used in this study.

Table 2. Plasmid vectors used in this study. 38

Table 3. Recombinant plasmid vectors used in this study. 39

Table 4. Oligonucleotides used in this study. 4 0

Table 5. Amino acid profile of TrxC and various other Trx €rom different prokaryotes. 93

vii

List of Figures*

F i . 1.

Fig. 2.

An ideaiïzed condition of an idectious pathogen.

Complete reduction of molecular oxygen to water, and the generation of reactive oxygen-derived species (ROS).

Fig. 3.

Fig. 4.

Fenton reaction and Haber- Weiss cycle.

Oxygen-dependent antirnierobial systems in phagocytic vacuoles.

Fig. 5. The tetravalent reduction of oxygen to water and the target site of superoxide dismutase (Sod) and cataiase (Kat).

Fig. 6.

Fig. 7.

The two-step reaction of superoxide dismutase (Sod).

Thioredoxin oxidoreductase reaction and the role as a hydrogen donor for the reductive enzyme.

Fige 8.

Fig. 9.

Glutathione disulfide transhydrogenase reaction.

Glutathione reaction with hydroperoxyl radical and hydroperoxide.

Fig. 10.

Fig. 11.

Fig. 12.

Degradation pathway of abnormal proteins.

DNA base excision repair pathway.

9% of suMval against tes-butyl hydroperoxide (TBBP) of AB1157, with its isogenic mutant strains, RPCSOO (nfo'), BW9091 (xth') and WC501 (xth-nfo').

Fig. 13. % of sumvai against teri.-butyl hydroperoxide (TBHP) of RPCSOO harbouring pBRC1, pBRC2, pBRC7 and appropriate negative and positive controls.

Fig. 14.

Fig. 15.

Fig, 16a.

Subcloning of pBSPC1.

Subcloning of pBPEORF1.

Primary screening of a pBluescript II SK(+) library of C. jejuni TGHJOll.

Fig. 16b. Secondary screening of a pBluescript II SK(+) Iibrary of C. jejuni TGH9011.

Fig. 16c. Secondary screening of a pBluescript II SK(+) library of C. jejuni TGH9011.

Fig. 17.

Fig 18.

Fig. 19.

Fig. 20%

Fig. 20b.

Fig. 21.

Fig. 22.

Fig. 23.

Fig. 24.

Fig. 25.

Fig. 26.

Fig. 27.

Fig. 28.

Fig. 29.

Fig. 30.

Restriction enzyme digested plasmid DNA from pBSPC1, pBSDCPA1 and pBSAGCL1.

ORF1, ORF2 and ORE3 and the relative positioning of Sol1 and Xbal sites.

The five complete open reading &ames (ORFs) and two partial ORFs.

Southern hybridization analysis of restriction enzyme digested plasmid DNA from pBSPC1, pBSDC9-Al and pBSAGQL1.

Tertiary screening of a LGemll library of C. jejuni TGH9011.

Relative positioning and detailed restriction enzyme map of pBSPC1, pBSDC9-Aland pBSAGCL1.

Relative positioning and detailed restriction enzyme map of pBSPCl and the k6 phage clone.

tee-butyl hydroperoxide (TBBP)-mediated oxidative stress assay on the deletion mutant of pBSPCl and pBSAG4-LI.

Nucleotide and deduced amino acid sequence of the trxC gene (ORF3) of C. jejuni TGE9û11 with its 5' and 3' Elanking regions.

CAP-1 and CAP-II of the trxC gene of C. jejuni: aiignrnent with the CAP binding consensus sequence.

FBS-1, FBS-II, FBS-III and FBS-N of the trxC gene of C. jejunf: aügnment with the Fur box consensus sequence.

OxyR-like-binding site 1 of the trxC gene of C jejuni: location and alignment with OxyR binding conseosus sequence.

OxyR-like-binding site II of the trxC gene of C. jejuni: location and aügnment 6 t h OxyR binding consensus sequence.

Amino acid aiignment of TrxC of C. jejuni and various thioredoxin proteins from prokaryotes.

Primer extension mapping of the tmC promoter.

Fig. 31. The curent physical genomic map of C. jejuni TGH9û11 showing the location of the trrC gene. 98

Fig. 32. Pulsed field gel electrophoresis analysis of C. jejuni TGEW)ll genomic DNA. 100

Fig. 33. Campylobacter species genomic DNA hybridization. 102

Fig. 34. Gene-arrangementconservatiorr of ORFl and ORF2 in C. jejuni as compared to E. coli and 61. pyion' 112

ABBREVIATIONS

GENERAL

aa AMV-RT AP-site ATCC CAP CAP-BCS cw FBS FBCS Fig . Fur His HMS kan LB Met nts O W s ) PAPS PCR PFGE rbs ROS RT SH TBHPItBuOH

ATP CaCl, CHCI, c- AMP DNA dATP dH,O m DEPC EDTA EtOH HC1 MOPS Mg Cl* NaCi NaOH NaOAc PEG Crp2 dATP

amino acid avian myeloblastosis vLus reverse mmcriptase apuRnidapyrUnidinic site amencan type culture collection catabolite activator protein cataboiite activator protein-like-binduig coosensus sequence cyclic adenosine monophosphate receptor protein fur box-îibbinding consensus sequence fur box consemus sequence figure femc uptake regulator protein histidine hexose monophosphate shunt kanamycin Luria-Bertani methionine nucleotides open reading frame(s) 3-phosphoadenosine-5'-phosphosulfate polymerase chain reaction pulsed field gel eiectmphoresis ribosome binding site reative oxygen-derived species rom tempetature thiol rem-bu ty 1 h ydroperoxide

CHEMICALS

adenosine triphosphate calcium chloride chlorofonn cyclic adenosine monophosphate deoxyribonucleic acid deox yadenosine triphosphate distilled water dithiothreitol diethyl pyrocarbonate ethylenediaminetetra acetic acid ethanol hydrochloric acid 3m-morphohoJpropane-saonic acid magnesium chloride natrium (sodium) chloride natrium (sodium) hydroxide nauium (sodium) acetate polyethylene glycol deoxyadenosine 5'- triphosphate tetra(ûiethylammonium) [@]

YP" RbC1, SDS au dATP

ihf kat lon lys merR nfo nth O ~ Y R porA proA rec P H sod soi soxR nx trxB xrh zwf

adenosine 5'- triphosphate tetra(triethy1ammonium) [yp] rubidium c hloride sodium (naaium) dodecyl suifate

deoxyadenosine 5'-[a-thio] triphosphate [asD]

GREEK SYMBOLS

lambda

MNEMONICS FOR GENES

all@hydroperoxide aerobic pathways cona01 caseinoiytic pmtease (proteme Ti) cyclic adenosine monophosphate DNA-binding protein, starvation anaeiobic respiratory femc uptake regdator glutathione reductase glutaredoXia glutathione hydroperoxidase integration host factor catalaSe long fom (protease La) lysine merciiry response endonuclease N endonuclease III oxygen response DNA polymerase 1 proline recorn bination RNA polymerase su peroxide dismutase superoxide induci ble superoxide response thioredoxin thioredoxin reductase exonuclease III gIucose-6-phosphate dehydrogenase

UNITS

w mL mM Ml % pmol

degree (temperature) in celcius counts per minute rnolar micro gram micro iiter micro molar miltlitcr mi7imola.r nanometer pe=ntage picomoiar rotation per minute enzyme unit (according to manufachirer's definition)

INTRODUCTION

Cmnpylobacter jejmi, a tumeber of the family Campylobacteriaiceae, ïs a Gram-

negative, spiral-shaped mimaemphilic bacterim. It is a major cause of enteric disease

worldwide. Acute symptoms of infection include dysentery. fever and abdominal pain.

Sequelae can include colitis (Blaser 1986), reactive arthntis (Johnson et al. 1983; Lang et

ai. 1980) and Guillain-Barre syndrome (Mishu et ai. 1993; Gnienwald et al. 1991; Rhodes

and Tattersfield 1982).

C. jejmi is a mimaerophilic organism, thus is Milnerable to the normal level of

oxygen in the air. This constraint is due to the nature of oxygen metabohm (Cadenas

1989; D& 1978). where every organism that thrives in an ambic environment is bound

to encounter the intermediate reactiw oxygen-denved species (ROS), superoxide (Cadenas

1989; Duke 1978). hydrogen peroxide (Imlay et al. 1991) and hydroxyl radical (Imlay a

al. 1991). So the rnimaemphilic naaire of an organism is correlated to its high

susceptibility to low levels of ROS. ûxidative stress occus in a ceil when ROS are not

adequately removed or are made additionally by exogenous sources.

Fig. 1 is a description in an i d e a h x i condition of an infectious pathogen. C. jejw*

a s a pathogen will encounter oxidative stxess at two stages (indicated by * in Fig. 1). first

during transmission of the live bacteriun from one host to the other, and the next stage is

while resisting the initial host immune respoose.

Survivd in an aerobic or an oxidative stress environment requires the presence of

cellular defense mechanisms which can protect the celk against ROS. The cellular defense

mechanisns include superoxide dismutase (Fridovich 1989; Fm et al. 1986), catalase

(Grant and Park 1995; Heimberger and Eisenstark 1988). iron storage proteins such as

f e m ~ (Wai et al. 1996; Wai et al. 1995) and pmtein degrading enzymes such as the ATP-

dependent proteases. 'The defence also includes repair mechanisms such as the protein

repairing enzymes of the thioredoxin and giutatredoxin systems (Fernando et al. 1992;

Holxngren 1989), and DNA repair enzymes (Cunningham et al. 1986).

An infectious pathogen

*Transmission- Establishment in host

~Triggering host immune response

Seeking new host 4- Survival in host by counter measures

Effects on the host

Vinilence factors

Fig. 1: An ideahxi condition of an infectious pathogen. (*) denotes places where the

infectùig organism encounters oxidative stress.

The cellular defence mechanisms could increase the rate of successfiil transmission

of C. jejuni, since a recent study has suggesred the possibility of C. jejwi survival in the

environment afnr adaptation to aembic metaboiism (Jones et al. 1993; VerceIlone et al.

1990). These &fence mechanisms also serve a purpose to neutraiize the effects of

macrophages. which use hydrogen peroxide and hydroxyl radical to eliminate bacteria in

host systems (see ROS from Macrophages. pp 8).

Chernicd nature of oxygen

What makes the usage of oxygen as a terminal electron acceptor a M y encounter?

Molecular oxygen is inert with most compounds except radicals. This is due to spin

restriction of the elecûons in the molecuiar orbital (Farr and Kogoma 1991). Molecular

oxygen has an even nimiber of elecaons. The molecular orbital has two unpaired electrons

which have the same spin quantum number. In order for a molecule to react with another

molecule it must have an antiparalle1 spin in its molecular orbital. Since oxygen has a

p d e l spin electmn in its molecular orbitai (Farr and Kogoma 1991; Cadenas 1989). it is

unable to oxidize another molecule with ease by accepting a pair of electrons. Due to

thermodynamic rasons, oxygen is a poor acceptor of a single electron. Molecular oxygen

has a way to overcome this problem by interacting with another paramagnetic center.

Transition metais such as iron and copper are good catalysts for singleelectron reduction of

molecular oxygea.

Molecular oxygen's reactivity increases upon acœptance of one. two or three

electrons. A complete reduction of a molecule of oxygen to water requires four electrons

(Duke, 1978). The intemediates foxmed in the hcomplete reduction pathway are

superoxide (4'). hydrogen peroxide (H202) and hydroxyl radical (OH). Under acidic

conditions. 0; can be protonated to fom hydroperoxyl radid (HOO). An aerobic life-

style will genemte these intermediates in a living system. Fig. 2 depicts the o v e d four-

electron reduction of molecular oxygen to water.

Fig. 2: Complete reduction of molecuiar oxygen to water. The reactive oxygen-derived

intemediates are shown in bold text O;. superoxide; &O,, hydrogen peroxide; HOO..

hydroperoxyl radical; and 08; hydroxyl radical,

Fe2+ + H,O, + H+ + ~ e % + HO* + H20 (Fenton Reaction)

HO- + H,O, + HOO' + H20

HO0 + ~ e ~ + -, Oz+ ~ e ' + + H+

Fe Overall reaction : 2 H 2 4 ---+ O,+ 2H20

Fig. 3: Fenton reaction and Haber & Weiss cycle. HO', hydroxyl radical; and HO0 *,

hydroperoxyl radical.

Reactivity of reactive oxygen-derived species (ROS)

Soperoxide

Superoxide is able to oxidize thiols, ascorbate, tocopherol and catecholamines

(Fridovich 1989). Proteins which have (Fe-S), clusters are highly sensitive to attack by O;

(Farr and Kogoma 1991). Unlike these reactions the spontaaeous dismutation of Oi to

&O2 and 4 is a more important conhbutor to the oxidative stress in a ceiL The laaer

~eai~tion has the ability io reduce transition metals and metal complexes. Superoxide oui

also be protonated in an acidic environment to produce hydroperoxyl radical, Due to the

n e u ~ o n of the aegative charge in O,', hydroperoxyl is more d v e comparai to

superoxide.

Hydrogen peroxide

The univalent reduction of %O2 was postulated by Fenton and by Haber & Weiss

to explain the iron-&pendent decomposition of ho2 at andic pH (Imlay et al. 1991). Fig.

3 depicts iron's role as a redox otalyst in the Fenton reaction.

Metal chelators can block the Fenton reaction (see Fig. 3) by occupying the metal

coordination site. Hydrogen peroxide reacts with reduced iron to generate hydroxyl radical.

Since superoxide has the ability to reduce ~ e * to Fe2+ and its dismutation produces %O,, it

is likely that when the intraceUular concennation of 0; increases, the concentrations of

H202 and OH- will also rise. From the Haber & Weiss cycle one may conclude that

hydroperoxyl radicals c m signifcantly contribute to oxidative stress. The hydroxyl radical

and hydroperoxyl radicai are show in bold text in Fig. 3.

Hydroxyl radical

The reactions of hydroxyl radcals are numerous because hydroxyl radiais are

highly reactive in a celL Hydroxyl radicals react at diffusion-limited rates with most

biomolecules. This high d v i t y is conaibuteci by its very high standard eiecM>de

potentiai, +23V (the standard eiectrode potential of 4 is +0.8V). Hydroxyl radicds can

ofidize almost anything exœpt ozone but due to its high reactivity, the average diffunon

distance of this m o l d e is only a few nanometers (m) (Singh and Singh 1982). So its

effect on any biomolecules will depend highly upon the location of its formation.

Iron-oxygen complex

"Perferryl" radical (2Fe2+OJ can be formed by incorporation of superoxide with

Fer( F m and Kogoma 1991). Although this cornplex is not thennodynarnically capable of

undergohg oxidative reactions with biomolecules, it will undergo a series of reactions to

form ferryl radical (2Fe2+0 )' (Fm and Kogoma 1991). The later complex is rich in

electrow, has radical characteristics and is not spin restricted in its reacàon. This reactive

complex is proposed as one of the major iniriating species of Lipid pemxidation and

possibly DNA damage (Fm and Kogoma 199 1).

Sources of reactive oxygenaerived species (ROS)

Superoxide

Superoxide a n be generated by two ways. via enymatic and nonenzymatic

reactions. The enzymatic reactions include several membrane-associated respiratory-chain

enzymes which d u c e oxygen through the temvalent reduction to f o m water. It has been

established that in Eschenchia coli the autoxidation of NADH dehydrogenase, sucçinate

dehydrogenase and D-lactate dehydrogenase are major sources of superoxide formation

(Imlay et al. 1991). Glutathione reductase. a cyu>solic enzyme is capable of generating

superoxide by using NADH as an electron source (Imiay et al. 1991). in marnmaüan

tissues, cytochrome P-450 is an important source of active oxygen species. For example.

in certain mammalian tissue like liver, cytochrome P-450 cornpises 4 % of the miai ceIl

protein (Estabrook and Peterson 1990).

The autoxidation of ceUular componenu such as ubiquinois. catechols. thiois and

flavins conaibute to the nonenzymatic production of superoxides. Eiectrophilic quinone

compounds are readily reduced to semiquinones. which in tum wiU reduce oxygen to

superoxides and generate oxidized quinones. A nanirally occuring electrophüic quinone is

ubiquinone and exogenous sources are plumbagin and menadione. The oxidued quinone

now will act as a redox-cycling agent A very effective redox-cycling agent is paraquat

(methyl viologen), which is a dipyridyl. Transition met& in their reduœd state, both in

free and cornplexed forms, axe capable of donating a single electron to oxygen to form

superoxide (Fm and Kogoma 199 1).

Hydrogen peroxide

The second intermediate ROS, hydrogen peroxide, is produced either

spontaneously or by superoxide dismutase (Sod)-catalyzed dismutation of superoxide.

There is a h evidence stating that hydrogen peroxide rnight be a photoproduct of near-UV

irradiation. Oxidases such as Damino acid oxidase can also catalyz the dismutation of

oxygen to form hydrogen peroxide (Eisenstark 1989).

Hydroxyl radical

Hydroxyl radicals are fonned in many ways. An absorption of a photon at 365 nm

by tryptophan residues will generate hydroxyl radicals. Radiolysis of water wiU also

produce this type of ROS (Fan and Kogoma 1991). A ~ i g ~ c a n t contributor to the

formation of hydroxyl radical is the Fenton reactioa When the steady-state concentration of

superoxides increase in proportion to the steady-state concentration of hydrogen peroxides,

hydroxyl radicals will rise.

ROS from macrophages

Macrophages derived h m bone marrow promonocytes are long-iived cells with

mugh-surfaceci endoplaanic xeticulum and rnitochondna Macrophages are the defence of

the host system against foreign particles such as bacteria, v i n s and protozoa, which have

the ability to invade host ce&. Macrophages neutralue invacihg pathogens by ingesting the

foreign matter (phagocyctosis) and later killing the mimorgganisms by two dinerent

rnethods. oxygen-dependent mechanisms and oxygen-independent mechanisms.

During phagocyctosis there is a Sgnificant increase in the activity of the hexose

monophosphate shunt (IIMS) or pentose phosphate pathway. Through this pathway

NADPH is provided to the phagocytes. Pksma membrane NADPH oxidase or a

cytochrome oxidase is activated during phagocytosis and this produces a burst of oxygen

consumption. Powerful microbicidal agents in the form of ROS are formed through the

oxygen coasumption. A potent system comprised of peroxides. myloperoxidases and

halide ions are generated within the phagocytes, and are able to kill both bacteria and

Wuses. Fig. 4 shows some of the reactions of the oxygen-dependent mechanisms.

Glucose + NADP r pentose phosphate + NADPH

NADPH oxidase NADP + 0;

Fig. 4: Oxygendependent antirniaobiai systems in phagocytic vacuoles. Micmbiocidal

O S ) s@es are in bold ietters. O;, superoxide anion; ' 0,. singlet (activared) oxygen;

OH. hydmxyl free radical; H,O,, hydrogen peroxide and HMS. hexose monophosphate

shunt.

Cellular defence mechanisms

Active speQes of oxygen nahxaüy occur in ambic-life styles and they can also be

made to arise by various intraceiluiar and extracellular sources, these are fraught with

danger for the cell. Ceils are equiped with &face rnechanisns that overcome the damage

by oxidative stress. These mechanisms are grouped inta Rimary defence and Secondary

defence.

The primary defence

Enzymatic components, the primary defence are u t i h d by the cellular defence to

resist damaging effects of oxidative stms. The primary kfence is a preventive mechanism

utilized by the cell to protect major macromolecules from king destroyed

The enzyrnatic components directly scavenge the reactive species and detoufy or

d u c e the toxicity of reactive species by producing nonenzymatic antioxidants (Farr and

Kogoma 199 1). Superoxide dismutase (Sod), c a W . glutathione synthetase and

glutathione reductase comprise the enymatic defense against oxygen-denved xeactive

species. Sod dismutates superoxide to hydrogen peroxide whereas catalase

dispropationates hydrogen peroxide to water and oxygen.

Alkyhydroperoxide reductase provides additional defense by reducing various

organic hydroperoxides. Some prokaryotes have NADH-dependent or glutathione

peroxidase but some like E. coli and S. ryphimurUun do not ( F m and Kogoma 1991).

In E. coü the two Sod proteins are encoded by sodA and sod.3. Whiie katE and

kanj code for catalase. Glutathione synthetase is encoded by gshAB and -y glutathione

reductase is encoded by gor. Fig. 5 depicts the univalent pathway and the neuvaliPng

effect of the antioxidant enzymes. Aliqhydroperoxide reductase is encoded by ohpC and

ahpF.

Fig. 5: The teuavalent reduction of oxygen to water and the target site of superoxide

dismutase (Sod) and catalase (Kat). The reactive oxygen-derived intermedi- are shown

in bold text 0;. supemxide; &O2, hydrogm peroxide; and OH-. hydroxyl radical.

Fig. 6: The two-step reaction of superoxide dismutase (Sod).

Soperoxide dismutase

There are thRe types of Sod based on its metal ligand incorporation. they are

CuZoSod, FeSod and MnSod. These metals fanlitate electmn transport. FeSod is primarily

found in prokaryotes. MnSod is found in prokaryotes and eukaryotes. CuZnSod is

generally not found in bacteria with the exception of Photobacter lewgnathi FeSod is

expresseci in both anaerobiosis and aembiosis, whereas MnSod is present ody under

aerobic conditions The reaction catalyzed by Sod û a two-step reaction (see Fig. 6) (Fm

and Kogoma 1991). Sod converts Oi to -0, (Takeda and Avila 1986).

The steady-state concentration of 0; in a wüd-type aerobically growing E. c d c d

is about lu9 to IO-'' M. In a sodAsodB murant which lacks Sod activity, the dculated

steady-state concentration of O,' is about 5 x 104 M (Hantke 1988). So we cm conclude

that the presence of Sod in E coli, reduces the steady-state concentration of superoxide by

up to three orders of magnitude. The significance of this enzyme is established.

The product of 0; dismutation is H,O, (Touati et al. 1988), which is a reactive

species by itself and it is a substrate for catalase. A question arises here. whether Sod acts

as a primary defence against oxygen-derived mtive species? It would be a ~ ~ g as a

primary defence if:

0; is more toxic than

&OZ can be eliminated h m the ceil more rapidly than 0;

%O2 can be eliminated by many routes compared to O;.

Catalase

There is no evidence saying that the relative toxicity of &O2 is less compared to O;

or vice versa It is dficult t draw a conclusion about the toxicity of &O2 and 0; in vivo.

On the other hand there is evidence that there is an element that can remove H202 with

remarkable rapidiq. It is catalase which does this remarkable job by a disproportionation

reaction. The airnover number for a t y p i d catalase is about 109 molecules of %O2

disproportionated per active site per second at 1 M H202 (Fm and Kogoma 1991). The

reasons for this remarkable rapidity in catalase reaaion toward %O2 are: - the elecmn source for the disproportionaàon reacbon lies within the &O2

molecule. This makes the reaction independent of an exogenous reducing agent - the reaction is exothermic.

the reaction does not require ATP. So even in an energy depleted ceil catalase

provides protection against hydrogen peroxide.

The two catalases in E. coli are found in Werent cellular locations. HP1 is found

in the periplasm whereas HPII is found in the cytoplasm (Heimberger and Eisenstadt

1988). Only HP1 is activami by the peroxide smss response and is a part of the OxyRS

regdon. HPII is induced by stravation and stationary phase and is regulated by VOS. ?he

ciifferenbal in location could be explained by looking at the sources of H,O, which may

Vary during starvation-dependent and starvation-independent oxidative stress.

The facts presented above indicate that the presence of superoxide dismutase does

serve the purpose of the initial primary defence against superoxide. The dismutation

reaction of Sod with superoxide generating &O2, paves the way for an efficient elimination

of MO by disproportionation reaction of catalase.

The thioredoxin & glntaredoxin system

lbioredoxin is a powemil protein dïsulfïde oxidoreductase (see Fig. 7) (Gleason

and Holmgren 1988). NADPH, the flavoprotein thioredoxin reductase (d) and

thioredoxin (axA) are coliectively known as the thioredoxin system (Hoimgren 1989).

Glutaredoxin exhibits glutatbione disuifide transhydrogenase activity (see Fig. 8)

(Gleason and Holmgren 1988). NADPH, the fiavoprotein glutatbione reductase (gor),

glutathione (gshB) and glutaredoxin (gn) comprise the glutaredoxin system (Holmgren

1989).

Thioredoxin contains an active-site (CGPC) with a redox-active disulfide

(Fernando et al. 1992; Gleason and Holmgren 1988; Holmgren 1985). Glutaredoxin has a

si& active-site but it is different in amino acid sequence (CPTC). Trx and Grx hurtion

in elecaon aansfer via a reversible oxidation of two vicinal protein-thiol (SH) groups to a

disuIfide bridge.

Thioredoxin participates in a cyclic oxidoreduction system (see Fig. 7), where

reducing power is king transferred fmm NADPH to a s@c oxidant The reduced fom

of thioredoxh (Tm-[Sm,) acts as a hydrogen donor for the reductive enzymes

nbonucleotide reductase, methionine sulfoxide reductase and 3-phosphoadenosine-5'-

phosphosuifate (PAPS) reductase (Gleason and Hohgren 1988). These enzymes rntalyze

irreversibie reactions where the SH-group on the enzyme are oxidized to disulfide,

thioredoxin subsequently reduces the enzyme to its reduced state. Cells require

nbonucleotide for DNA synthesis, sulfate reduction to generate reduced sulfur as in

cysteine (Kredich 1996). and reduction of methionine sulfoxide either to fonn the free

amino acid or for repair of oxidatively damaged side chains in proteins.

Bacteriophage T4 coded thioredoxin is more sirnilar to E. coü glutaredoxin (32 96

amino acid identity) than to E. coli thioredoxin (Gleason and Holmgren 1988). T4 phage

thioredoxin serves as a Specinc hydrogen donor for T4 nbonucleotide reductase. Although

Fig. 7: Thioredoxin oxidoreductase reaction and the rde as a hydrogen donor for the

reductive enzyme. A 0 is a substrate. A is the reduced enzyme, Trx-(S H), is reduced

thioredoxin and Tnt-S, oxidized thioredoxin.

gIuruodoxin 2GSH + X-S-S-X GSSG + 2XSH

glutathione reductase

NADP+

Fig. 8: Glutathione disuifide tmshydrogenase reaction. GSH is reduced glutathione.

GSSG is oxidized glutathione, XSH is reduced substrate and X-S-S-X is oxidized

substmte.

it has w si-cant amino acid homology to E. culi thioredoxin, T4 phage thioredoxin is a

good subsaate for E culi. thioredoxin reductase (Glewn and Holmgren 1988). T4 phage

thioredoxh has an active site sequence, CVTC which is similar to glutaredoxin. Like

glutaredoxh activity, T4 phage thioredoxin catalyzes GSH-dependent ribonucleoti&

reduction. E. CO& giutaredoxin displays good activity with T4 phage ribonucleotide

reductase and is a more efficient hydrogen donor for ribonucleotide reductase than

thioredoxin. Its concentration in the cell is highly variable but is les than that of

thioredoxin (Gleason and Holmgrem 1988).

Genetic analysis revealed that both thioredoxin and glutaredoxin are not essential

for viability (Gleason and H o l m p n 198 8). A double mutant of tm and g n . A4 10 was

obtained and is able to grow in rich medium but quires cysteine in minimai medium. It

has been established now that Trx and Grx are essential for sulfate reduction but not

required for DNA synthesis. A Gnr mutant generated through mutagenesis in a ml3 mutant

background, grew in rich medium with added glutathione or cysteine but displayed long

filamentous morphology (Kren et al. 1988). This phenotype is characteristic of impaireci

ce11 division (Clark 1968). On the other hand it has been shown that thioredoxin and not

glutaredoxin. is the essential subunit for TI phage DNA polymerase. To make maaers

more complicated, discovery of an anaerobic ribonucleotide reductase system in E. coü

strongly implies that the deoxyrïbonucleotide metabolism is more complicated than what

was thought originaüy (Hantke 1988).

Peroxidase can also destroy &O2 but it is dependent on the presence of NADH or

NADPH as an electron source (Farr and Kogoma 199 1). If a œii lacks the reducing power

then the role of peroxidase is limitai Glutathione is an important antioxidant and is

synthesized by glutathione synthetase (Meister and Anderson 1983). In E. coli the steady-

state concentration of glutathione is high (Loewen 1979). Glutathione has the ability to

react with %O,, O; or HOO and the product of this reaction is a stable glutathione radical

(GS3.

Upon formation of a stable glutathione radical the next step will be the dimerimion

of the glutathione radicalS. Glutathione reductase will M e r an electron from NADPH to

the dimer and form the reduced glutathione. (nie of the most important functions of GSH

is to reduce disulfide bridges caused by oxidative stress in proteins Fig. 9 depicts the

reaction of glutathione with hydroperoxyl radical (Gleason and Holmgren 1988).

Alkylhydroperoxide reductase enco&d by ahp has the ability to reduce organic

hydroperoxide in vitro such as cumene hydroperoxide and zen-butyl hydroperoxide

(Jacobsen et al. 1989).

The secondary defence

'Ihere is great deal of evidence showing that oxygen radicals cause damage to

macromoiecules in vivo and in vitro (Altman et al. 1994). Damage to mammolecules can

be classified into two categories, one that leads to ceil death and the other one which does

not Oxidative damage that has an &ect on the c d membrane or DNA may have a more

deleterious effect on the celI than damage targetiag protein or RNA. Nevertheles such

damage to protein and RNA will at the very least deplete the ceU of its energy and

resources, since the damaged molecules have to be replaced.

The secondary defence's d e , unlike the primary defence is to repair damage to the

cell. In secondary defence the cell resowes are ualized to repair damage made by

oxidative stress upon major macrornolecules. The secondary defence does not detoxify

ROS. instead it rectifies the byproducts of the ceaction between the DNA rnolecule or

protein molecules and ROS.

Protein damage

Protein damage during oxi&tive stress is common (Saran and Bors 1990). The

damage can be a direct assault to the native protein or an indirect damage. Direct damage to

GSH + HOOo- GS- + H,O,

GS* + GS- -- GSSG

GSSG + NADPH + H+ + 2GSH + NADF

Fig. 9: Glutathione miction with hydroperoxyl radical (HOO) and hydroperoxide

m2w

si& chains in polypeptides cm be &letenous. Oxidation of si& chah groups in sdfhydryl

proteins can cause the hee dimensional smicnire of proteins to be altered Structural

proteins would lose their integrity while enzymatic proteins would lose their ability as

cataiysts.

Indirect damage is a result of the damage to the DNA molecule. A mutation

occuring on the DNA molecule rnay mult in an inactive protein. This deficient protein can

be the result of a nonsense mutation or missense mutation. During translation a non-sense

mutation would be aanslated into a tnincated protein, it will be inactive in performing its

function. If the missense mutation occureci in an active-site or other important sites of the

protein, the function of the protan will be altered.

Damaged protein may be either repaired or degraded to its fundamental units. The

repair functions can be carried out by the thiol-disulnde oxidoreductase enzyme system.

This includes the thioredoxin and glutaredoxin systems. The degradation of damaged

proteins are undertaken by ATP-dependent endoproteases, endoproteases and peptidases.

Fig. 10 illustrates the pathway for degradation of abnormal proteins.

Rotease La (Lon) and protease Ti (Clp) are two of the many ATP-dependent

endoproteases. Lon is encoded by lon, while Clp is composed of two distinct subunits

ClpA and ClpP. ClpA is the product of clpA while ClpP is encoded by clpP. The Lon gene

is not essential for viability un&r nomal conditions. Mutants lacking Lon have a lower rate

of degradation of abnormal proteins. The residual proteolysis in ion mutants is ttributed to

the presence of Clp. A Clp mutant is viable but unable to degrade casein. Under conditions

where abnomal proteins persis~ the avaihbility of Lon and Clp might be of importance to

the viability of the ceil. This is because, abnormal proteins tend to form inhacellular

aggregates, these aggregam if not degraded, retard the fluidity of the cytoplasm.

Abnormal proteins (Intracellular aggregates)

ATP-dependen t endoprotease / polypeptides (MW A 5 0 )

endopro \ tease

peptides -

I pep tidases

amino acid

Fig. 10: Degradation pathway of abnormai proteins.

DNA damage

When cells are exposed to an agent causing oxidative stress there is evidence

indicating that the DNA moiecule is being damaged. The damage is either targeted to the

bases or to the sugar moieties. Damage to the bases will produce either irregular bases or

apurinidapyrknydinic siw (AP-Site), whereas attack on sugar moieties always leaves a

break in the phosphate backbone.

There are at least three categories of DNA damage, they are as follows:

oxygen derived reactive species

immediate intermediate organic radicals

termination products of the radicals

Hydroxyl radicals can attack sugar moieties and produce strand breaks with 3'-

phosphate or 3'-phosphoglycolate termini. Base modifications axe also common such as

formation of hydroxymethyluracil, which is a product of hydroxylation of thymine. are

common lesions in oxidative stress. Thymine can also be oxidatively degraded to produce

thymine glycol or a urea residue (Fm and Kogoma 1991). On the other hand, addition

reactions of hydroxyl radical to guanosine can generate 8-hydroxydeoxyguanosine (Farr

a d Kogoma 1991). Formation of thymine glycol has been shown to block DNA

replication. Removal of this irregular base contributes to premutagenic lesions (HalliweIl

and Aruoma 199 1).

Intermediate organic radicals which are formed during Lipid peroxidation can react

with DNA. Bulky adducts or decomposition of purines are the end products that leave a

lesion on the DNA (Vaca et al. 1988). Site-s-c cleavage of double-stranded DNA

adjacent to guanidylate residues are lesions that occur when a DNA molecule is incubated in

the presence of linoleic acid hydro peroxide (Vaca et al. 1988).

F d y , the stable termination product of oxygen derived reactive species such as 4-

hydroxyallcenals. epoxides and aldehydes can react directly with DNA by allqhting bases

(Far and Kogoma 1991) or forming intrasûand or interstrand crosslinks (Summerfield and

Tappel 1983).

What is the signincance of this damage to the DNA? A s m d break or other lesion

that impedes the replication proces will contribute to lethality. A base modification will not

cause lethality but that does not mean it is not a serious problem for the cell. Base

modification will contribute to a mutagenic effect on the ce1

Mutagenicity

To understand the potential and speQficity of H202 mutagenicity, a reversion assay

using histidine requirement as a m a r k was undertaken in S. typhimurium . When oxyR

mutants were grown in nomal oxygen tension, they showed a His' + His' reversion

frequency 1 1-fold higher than isogenic wild-type cells (Storz et al. 1987).

The mutation rate in ceils void of Sod activity during aerobic growth was rneasured

by a fluctuation test that masures the rate of rifampicin-sensitive to rifampicui-resistance-

The double mutant of sodAsodB showed 40-fold increa~e in the rate of aerobic

spontaneous mutation toward rifampicin resistance ( F m et al. 1986). These two tesuIts

indicate the magnitude of mutagenicity in cells that lack the necessary defence against

oxidative stress-

DNA repair enzymes

Thymine glycol and its immediate decomposition product are readily removed by an

enzyme hown as endonuclease III (Nth) which has N-glycosylase activity (Demple and

Luui 1982). The gene coding for this enzyme is nrh (Cunningham and Weiss 1985).

Mutants of nth are not sensitive to H,O, or y-radiation. This suggests that thymine glycol

is a premutagenic lesion but not a lethal lesion.

recA mutants are hypersensitive to exposure to H , 4 . This shows that the RecA

protein which plays a role in the recombinant repair pathway is crucial to ce11 survival,

upon peroxide stns (Imlay and Linn 1987).

xrhA encodes for exonuclease III (Zzumi et al. 1992) and xrhA mutants anz

hypersensitive to exposure to H,O,. This suggests that sugar firagmentation m u t be

occuring at the 3' end of the strand breaks- The sugar Sagrnent seem to act as a blocking

agent. This speculation was confirmed when &O2-nicked DNA was found unable to serve

as primer for DNA synthesis unless exonuclease III is present (Cunningham et al. 1986).

Exonuclease III cleaves 5' of apurinidapyrimidinic (AP) sites to m e in the DNA base

excision repair pathway.

Endonuclease IV the nfo gene product (Saporito and Cunningham 1988; and

Cunningham et al. 1986) removes the 3'-blocking group and cleaves 5' of AP site

(Cunningham et al. 1986). Over expression of this enzyme in a ndrA mutant seem to relieve

the sensitivity of the mutant to H20,. Exonuclease (A)BC are encoded by uvrA, uvr% and

UV< respectively. The ixnponance of this set of enzymes is in the repair of UV-damaged

DNA. A combination of wAnfoxzM mutant was not possible but any combination of two

mutants were viabIe.niis is an indication of the functional rerlundancy of exonuclease

(A)BC, Nfo and Xth (Fm and Kogoma 1991). In support of this notion Exonuclease

(A)BC was shown to be capable of removhg thymine glycol and AP-sites. Keeping this in

mind the investigators were able rn construct a txiple mutant by relieving the c d of the AP

site. This was done by using a m g mutant (Fm and Kogoma 199 1). ung codes for uracil

N-glycosylase which removes uracil from DNA whereby generating AP-sites.

The requhement of DNA polymerase 1 activity upon H202 damage was

dernonstrated when polA mutants were found to be hypersensitive to killing by H202

(Ananthaswamy and Linn 1977). Later the d e of DNA polymerase III was also found to

be essentiai in DNA repair synthesis upon &O7 s t m s (Hagensee et al., 1987).

A simple DNA repair mechanism is the base excision repair pathway . in which the

apurinidapyrimidinic site (AP-site) is repaireci by AP endonuclease IV (Nfo). The AP-site

is created by the &ect of endonuclease III which has DNA glycosylase activity i.e it

removes thymine glycol from DNA. Nfo has the ability to nick 5' to the AP-site leaving a

normal 3'-OH and an irreguIar s'-PO,. The action of DNA deoxyribophosphodiesterase

wiU then cleave the irregular site to fonn a normal s'-PO,. On completion of this. DNA

polymerase by using the 3'-OH wiU synthesize the missing portion and halIy. to complete

the repair mechanism. DNA ligase ligates the newly synthesized base(s) to the normal 5'-

PO, end Fig. 11 depicis the base excision repair pathway.

1 DNA glycusylase

5' $ Free base excised 3'

5' AP endonuclease t

DNA deoxyribophosphodies terase

DNA polymerase + DNA Ligase t

Fig. 11. DNA base excision repair pathway. Open boxes denote nomal bases while filled

box is an irregular base. Upon excising the irregular base the empty space within the DNA

strand indicates AP-site. The top most diagram depicts DNA double stranded while ail

other drawings are depicting single stranded DNA (highlighàng the suand that has the

irregular base).

Oxidative stress response

Naairal oxidative stress conditions occur when the concentration of active oxygen

species rise to a level that overwhelm the basal level of the scavenging capacity of a c d .

The responses to this oxidative stress can be grouped into two stress responses, superoxide

stress respoose and peroxide stress response.

Superoxide stress response

Under elevated level of the superoxide radical anion, bacteria respond by

stimulating a response wbich is known as superoxide stress response. As we know that an

elevated level of MnSOD will increase the level of H202 production, so in order to leam

more about 0;-stress stimulated proteins sodAsod3 double mutants were utilized to

rninimi.IP. any secondary induction by the H,O, response. This experiment done in E. cok'

indicated that 30 Soi (superoxide inducible) proteins were induced by the 0; stress

conditions. Six of these proteins were identified, 2 were associated with HP1 cataiase, 2

proteins of alkylhydroperoxide reducuise (Ahp), heat shock protein GroEL and

endonuclease IV (Wallnip and Kogoma 1989). These superoxide stimulon protans are

known to be regulated by the product of two loci. soxR and soxS (Greenberg et al. 1990).

Inactivating either of these two loci render the œll noninducible to the six superoxide

stimulon pmteins.

In a different experiment, investigators found that GroES, two other heat shock

proteins. MnSOD, endonuclease N and glucose-6-phosphate dehydrogenase were

induced, when induction of proteins were compared using wild type cek exposed to O,'

and &O2 (Greenberg et al. 1990).

Presisatment of ce& with a non-lethal dose of plumbagin. a superoxide radical

anion generator, enhances the survival rate of those cek upon exposure to a lethal

challenge dose (Fm et al., 1986). This indicates that the superoxide stress response

includes an increased DNA repair capacity. Evidence that supports this notion is the fmding

that the ievel of endonuchse IV is greatly in tells upon m e n t with

superonde radical anion generators. Endonucfease N is encoded by the nfo gene

(Cunningham et al. 1986) which is a member of the SoxRS regdon (Chan and Weiss

1987).

Superoxide stress proteins

MnSOD was the k t enzyme which was known to be iaduced by 0; genecating

agents. Then endonuclease IV. which is a minor apwinidapyrimidinic (AP)-endonuclease

was identified. Glucose-6-phosphate dehydrogenase and NAD(P)Hdehydrogenase

(diaphorase) were found to be induced by paraquat and menadione.

A signiscant level of induction of HP1 catalase was observed in Sod void cells,

when challenged with menadione (Gieenberg and Demple 1989). This suggests that

production of H,O, is possible even in the absence of Sod. However the possibility of the

redox-cycling agent generating H202 directly or even the notion that some of the oxygen

response (OxyR) regdon gens may Fosses a cis-acting control element that is sensitive to

an O,'-mediated inducing signal. cannot be d e d out (Fm and Kogoma 1991).

SoxRS regulon

The superoxide radical response (soxR) locus was established when it was

discovered that the induction of a soi::lacZ hision protein was completely independent of

the reguiatory loci, oxyR. p H and recA (Kogoma et al. 1988). The soxR locus was

defineci when the SoxR(Con) mutant was isolated, which rendered the expression of

several 02'-inducible genes to be constitutive in E. c d . Two mutations were isolated,

soxRI and soxZU. The genes tbat were coastitutively expresseci in these mutants were nfo.

zwf, sodA, soi- 17. soi-19 and soi-28. nfo codes for a DNA =pair enzyme endonuclease

IV. zwf codes for giucose-6-phosphate dehydrogenase. sodA codes for superoxide

dismutase that requires rnanganese as a CO-factor and the soi loci code for superoxide

inducible proteins.

Reguiation of tbe soxR locus appears to be primarily at the traoscriptional level

usaneva and Weiss. 1990). Inducibility by paraquat at the soxR gene is stopped by

creating a large deletion in the su& gene. nK inducibility is retained upon introduction of

a plasmid carrying the portion of the deletion (Greenberg et al. 1990). So so*R codes for a

tram-acting positive factor which is required to activate the soxRS regdon genes.

DNA sequence determination of soxR and soxS predicted the synthesis of two

polypeptides* 17-kDa (SoxEt) and 13-kDa (SoxS) respectively. nie two genes. soxR and

soxS are tmrmibed divergeatly (Tsaneva and Weiss 1990) and are essential for

inducibility of the SoxRS regulon gens (Wu and Weiss 1991). The soxR promoier Lies

within the soxS gene (Wu and Weiss 1991). SoxS is related to the arabinose (AraC) family

of proteins ( W u and Weiss 1991). that function as positive aanscriptional regulators and

SoxR protein shares region of homology to the rnercury response (MerR) protein

(AmabileCuevas and h p l e 1991). MerR is involved in activating genes that d e t o w

rnercury. SoxR contains four cysteine clusters near the carboxy ~rminus suggesting the

possible role of these residues as a redox-cycle center. The purifieci SoxS binds tightly and

specifically upstxeam of the -35 elements of target promoters (SoxRS regulon genes) and

d t s RNA polymerase to these promoters in vitro, in the absences of SoxR or paraquat.

This indicates that SoxS can switch on the SoxRS =guion on its own (Fawcett and Wolf

1994; Li and Demple 1994).

Role of iron in replation of sodA expression

The product of the fur (femc uptake regdatory gene) requires iron a bind to the

Ûon box, in order to repress the expression of die iron repuiated genes. The iron box is a

19-bp consensus sequence that is found at the regdatory region of genes that are involved

in iron uptake (Bagg and Neilands 1987). Subsequently a stretch of sequence that is

homologous to the iron box was found at the promoter region of sodA flakeda and Avila

1986). Derepression of a d- lac2 operon fusion in aerobically and anaerobicaiiy

growing ficr mutants demonstrated the role of Fur protein in reguiating the sodA gene

(Neiderhoffer et al 1990). It was shown that Fur negarively regdates sodA in a classsical

iron-dependent repression but the possibility that other cellular factors may be involved in

its total reguiation cannot be d e d out The synthesis of SodA is aiso controled negatively

by Arc (aerobic pathways coatrol regdatory system). FNR (anaerobic respiratory control)

and MF (mtegration host factor). while the products of SoxRS and SoxQ acts positively

(Compan and Touati 1993).

By analyzing mutants that are capable of hi& expression of SodA in anaembic

conditions another level of reguiation was discovered. In order for the high expression in

anaerobic conditions, a mutation at mc and fur were required. any single mutation only

showed partiai expression (Walkup and Kogoma 1989). The arc regdatory genes

negatively conml expression of genes involved in aerobiosis (Iuchi et al. 1989). In a

double mutant of fur and mc MnSOD is elevated when chailenged with paraquat,

sugges~g that the SoxRS is independent of the Fur and Arc systems. Overproduction of

MnSOD represses the tranmiption of sodA gene. suggesting autoregdation (T'ouati 1988).

Role of the superoxide stress proteins

The function of Sod is to reduce the level of superoxide radicals. ï h e product of

nfo repairs oxidatively damaged DNA sites such as AP-sites.

The role of glucose-6-phosphate dehydrogenase in the response to oxidative stress

is to produce NADPH. NADPH is an electron source for thioredoxin reductase and

glutathione reductase. Tfiese two proteins are required as a primary defence during

oxidative stress. They are utilized as cellular reductants. The presence of NADPH does not

reduce ~ e " whemu NADH does. NADH is capable of reducing ~ e " rn ~ e ~ ' . ~ e ~ + wiii

react with H,O, to generate OH'. another source of oxidative stress. So in produchg

NADPH the cell would have the necessary reducing power but without the risk of

generating hydroxyl radicals via the Fenton reaction. This is another indirect role which is

undertaken by glucose-6-phosphate dehydrogenase, in reducing the levels of OH'.

The function of NADH-dependent diaphorase activities during oxidative stress has

not been clearly elucidated but them are at Least three possible roles which this reductant

could play in defending the cell against oxidative damage. The first is to reduce the level of

NADH in order to prevent the formation of hydroxyl radical via the Fenton reaction. The

second function would be to reduce cellular respiration thereby reducing the electrun flow

through the electron transport chain, which is a source of superoxide radical. The final

possible role would be to & t o w redox-active quinones to hydroquinones by a two-

electron reduction. Redox-active quinones can go on to be a redoxcycling agent, thus

generating oxidative stress.

Peroxide stress response

An increased flux of H24 and other organic peroxides induce production of 30

proteins over their n o d level of production. This response has been coined as the

peroxide stress response (Morgan et al. 1986). There are at least nine proteins in S.

typhimuriwn and eight proteins in El coli, which are part of the peroxide stress response,

known to be positively regulated by oxyR locus. The genes encoding these proteins

constitute the OxyR regulon.

Concomitantly with the relief of the peroxide stimulus, bacteria acquire resistance to

peroxide stress (Farr and Kogoma 199 1). The conclusion from this observation is that at

least in part the increased resistance to peroxide stress would be due to increased DNA

repair capacity. Evidence shows that DNA repair ability is not a part of the OxyR regulon.

The resistance to peroxide stress upon initial exposure would then be a response which is

part of a broader peroxide stimulon (Fan and Kogoma 1991).

Peroxide stress proteins

In S. ryphmriwn the peroxik-mediated stress response is cornprised of 9

proteins. which have been fomd to be regulated by the oxyR locus. Out of this nine, two

are heat shock proteins. two are elecmmorphs of HP1 catalase (kzffi), and two are

subunits of Ahp. Inferes~gly MoSOD and glutbathione reductase activity also seemed to

be elevated moderately.

In E. coli, WO proteins of HP1 catalase. two proteins of ahp operon, MnSOD and

gm flao 1997) were identifieci as part of OxyR regulon. Although initially MnSOD was

found to be regulated by oxyR locus OranBogelen et aL 1987). a sodA-Lac2 operon fusion

study indicated that there was no significant elevation of the SodA protein upon treatrnent

with H,02 (Touati 1988).

The OxyR regulon

The ûxyR reguion positively reguiates the peroxide stimulon proteins, this

conclusion was derived upon isolation of a oxyN mutant in S. ryphirnuriwn. In this

mutant, the nine proteins were found to be constitutively expressed. The deletion of thk

oxyR locus renders the nine proteins uninducible. It was inferred then, that the oxyR

product is essential as a positive factor for the activation of the OxyR regulon gens

(Christman et al. 1985). OxyR protein also negatively regdates its own expression (Stoa

et al. 1990). and belongs to the LysR (lysine) f d y of DNA-binding proteins (Christman

et al. 1985).

Evidence from OxyR-binding sites extending into the -35-0'' indicates that OxyR

protein interacts with RNA Polymerase to activate transcription. Regions upstream of kaG,

M F operon and oxyR were identEed by using purifid OxyR protein in a footprinting

experiment but sequence analysis at the OxyR-binding sites revealed poor conservation of

nucleotides flaaagiia et al. 1989).

Reoent study by Toledano et al. has elucidated the presence of an OxyR-Wre

binding sequenœ upsaeam of oxyR regdon genes They showed that oxidized OxyR

recognizes a motif comprised of four ATAG nucleotides element spaced at 10 bp intervals

and contacts these elements in four adjacent major grooves on one face of the DNA helix.

Oxidized OxyR binding at this site. enables OxyR to activate aanscription of katG. &pC,

dps. gor and grx. While the reduced OxyR bhds at two pairs of adjacent major grooves

separated by one helical tum. The oxyR gene is repressed by the binding of both oxidized

and reduced OxyR floledano et ai. 1994).

How is an oxidative stress signal transduced to OxyR protein?

Induction of the OxyR regulon did not show an increase in the amount of OxyR

expression. Evidence supporthg this notion is, that upon exposure of cek to hydrogen

peroxide the transcription of oxyR-fucZ operon fusion did not change (Vadogelen et ai.

1987) nor did the rate of OxyR protein synthesis (Storz et al. 1990). The possibility of

direct activation by oxidative stress upon OxyR protein was then postulated.

OxyR protein prepared in the absence of oxygen was found to be inactive as a

aansrription activator but it cm be readily converted to its active form by exposing to air

(Ston et ai. 1990). Removal of reductants such as dithiothreitoi from the purification

bufTers permits the ~ f o m a t i o n from an inactive to an active fom of the protein but this

phenornenon can be prevented by adding catalase. These results indicate that upon the

influx of hydrogen peroxide, OxyR protein is king oxidized, enabhg the protein to act as

transcriptional activator of the OxyR regulon genes

There are six cysteine residues in OxyR protein (Christman et al. 1985). Although

these are good candidates for a redox-active center, conversion of five of the six residues to

a serine residues did not effect the activation of the protein. In contrast, conversion to

serine from cysteine at position 199 inactivates the protein ( S t o n et al. 1990).

Evidenœ from footprinting experiment with the oxidized and reduced form of

OxyR prorein at the regions upsûeam of kaffi gene. ohpCF o p n and uxyR gene suggest

a distinct confoxmational change is raking place upon oxidation and reduction. because the

footprinting pattern seems to be clearfy different (Storz et al. 1990). The conformational

change seem to alter the interaction of the protein with RNA polymerase. leading to

activation of transcription The OxyR mutant protein (C199S). when prepared in the

presence of oxygen produces an identical footprinting poteni as a reduced form of the

wild-type protein (Storz et al. 1990). This observation highlights the importance of the

cysteine residue at position 199 on OxyR

By altering DNA contacts (conformational change) in response to enviromenta1

signals. OxyR represes its own expression. under both oxininng and reducing conditions.

Whiie in response to oxidative saess OxyR activates tfatlscfiption of kaffi. ahpC. gor, dps

and gn.

Role of peroxide stress response

The role of HPI catalase is to reduœ the cellular concentration of H,O,. The

presence of reductants such as glutathione reductase will ensure the reducing environment

to directly react with H202 to detoxify the oxygen-derived reactive species. Similarly the

role of Ahp is l ik glutathione reductase reduchg the offending peroxides. including

organic peroxides. Gnt exhibits glutathione disuEde transhydrogenase activity ( s e pp 14).

GroEL functions as a chapemne protein that maintains prefolded proteins in the

unfolded state and facilitates their aanspon through the inner membrane. Apart from this

bct ion it &O refolds d e n a d proteins. During oxidative stress there is an increased

level of misfolded proteins. These might be due to the damaged nascent polypeptides. by

direct oxidation of amino acids or iridirectly fiom either rnistranscribed or mistranslateci

genes. The increased production of this GroEL protein would then be of importance to the

cell during oxidative stress. It is known rhat membrane damage does occur diiring oxidative

stress, so expression of GroEL would be to compensate the damaged membrane export

apparatus.

The heat shock protein DnaK makes up at least 1 % of the total cellular protein t

37°C and approximateiy 4 % upon temperature upshifl in E. culi. The most important d e

of this protein is as a chaperone protein. DnaK has a similar role as GroEL

The role of NADH-dependent diaphorase activities are as in the role of superoxide

stress response (see pp 13).

PURPOSE OF THIS STUDY

BW9091 and RPCSOO are congenic mutants of E. coli AB1157 (wild type)

defective in the a h and nfo gene, respectively, and are sensitive to rem-butyl

hydroperoxide (TBW). BW9û91 is less sensitive than RPC500 to TBHP-rnediated

oxidative stress (Cunningham et al. 1986). xrh encodes the major AP endonuclease

enyme, exonuclease III (Weiss 1976; and Yajko and Weiss 1975), while nfo codes for the

endonuclease IV (Cunningham et al. 1986). Both enzymes are involved in the base

excision DNA repair pathway. A double mutant of AB1157, RPCSOl(xrh- nfu-) is

extremely sensitive to oxidizing agents (Cunningham et al. 1986). im plicating the profound

importance of a functional DNA repair ability during oxidative stress.

The C. jejunifur gene has k e n cloned, sequenad and codes for the femc uptake

regdatory protein (Chan et al. 1995). Its fiuiction is in reguiating iron-regulated genes.

pUH5C 10 is a recombinant pUC 19 vector bearing the C. jejwu' fur gene. Over expression

of this recombinant plasmid DNA in AB 1157 and BW9091, rendes the cells more fesistant

to H,O, and TBHP (unpublished data; Chan 1996). Increased resistance to both these

oxidizing agent was nulliiied in the RPCSOO suain, although canying pUHSC10.

irnplicating possible activation of the nfo gene or endonuclease IV byfir.

SodA is the mangrnese containing superoxide dismutase enzyme (NeiderhofTer et

al. 1990). A recent study has impiicated (Cu. Zn)Sod (homolog of the SodC of E. coli) in

the pathogenicity of Salmonella typhimurium (Farxant et al. 1997). A Fur-box-like

sequence has been i&nîîfied upstream of sodA of 6 coli (Neiderhoffer et al. 1990). This

implicates Fur in repuiating a major oxidative stress respome gene. The presence of the

Fur-box-like-sequence has also been noted at the promoter region of the E. coü nfo gene.

The intricate mechanism(s) by which Fur regdates the oxidative stress response genes have

not been characterized,

C. jejwi, a rnimaerophilic organism is a major causative agent of diarrhea-

ûrganisrns that thrive or are able to survive at normal levels of oxygen are at the mexcy of

ROS. Cellular defence against ROS is needed so tbat oxygen can be u9lized as a terminal

electron accepter. A reoent study has implicated the emergence of an aerotolaant variant of

C. jejwu' (Jones et aL 1993; Verceilone et al. 1990). It is thus important to elucidate the

mechanism(s) that C. jejunï utilizes for a successfui oxygen metabolking ability.

We propose to clone the oxidative stress response genes of C. jejwu' in order to

gain a better understanding of the mechanïsms of oxidative stress response in this

microaero philic bac terium.

E. coli RPCSOO, an nfo mutant with increased sensitivity to TBHP was used as

host in a TBHP-mediated oxidative stress screening strategy. This strategy shouid lead to

the cloning of the C. jejwu' nfo gene and other oxidative stress response genes.

A recombinant plasmid (pBRC1) of the pBR322 genomic DNA library of C. jejuni

TGH9011 was isolateci through a complementation smtegy. The ability of pBRCl to

increase resistance of RPCSOO to TBW, is attributed to an open reading frame encoding a

deduced protein which has an active site distinctly similar to another oxidative stress

response gene. known as n* codes for the thioredoxin protein. Recent studies have

implicated Trx as a protein thar reduces oxidatively damaged proteiw (Fernando et al.

1992). The direct role of Trx in DNA repair has b e n noted by Hirota et al. (1997).

METHODS & MATERIALS

Bacterial strains, piasmids and oügonncleotides

C. jejimi strain TGH9011 (ATCC 43431) was used in the construction of a

genomic DNA Iibraty (Chan et aL 1988). The bactaial stmins, plasmids and

oligonucleotides used in this study are listed in Table 1-4.

Media and gewral growth conditions

C. jejwii strain TGEW)lI was grown in Muller Hinton agar or broth (BDH,

Darmstadt, Gemany) at 37'C with 5 1 CO,. Ail E. c d strains were grown in Luria-

Bertani (LB) medium on plates or in broth (1 % Trytoae, 0 5 46 Yeast Ex= and 1 %

NaCI ) at 37OC. Strains harbouring plasnid constnicts were maintaineci on LB medium

plates containing ampicilin (SIGMA Chemicals, SL Louis, USA) at a final concentration of

100 pg/mL. For plasnid analysis, E. CO& strains harbouring appropriate plasmid

consaucts were grown ovemight in the presence of 50 p g / d of ampicilin.

ter#.-butyl hydroperoxide (TBHP)-mediated oxidative stress assay

E coli strains (AB 1 157, BW9109, RPCSûû or ReC501) with or without plasmid

were grown ovemight either in the presence or absence of ampicilin selection. The

ovemight saturateci cultures were then diluted at 1 : 10 and 1 5 (mutant strains) in LB broth,

glucose was added to a final concentration of 0.1 %. The diluted culaires were grown to

iate-log-phase. The late-log-phase cultures were then challenged with TBHP (SIGMA, St.

Louis, USA) by a pour plate method. In a tube containing 2 rnL top agar (0.6 96). sining in

a 4S°C waterbath. various concentration of TBHP (50-200 pdplate) were added and an

appropriate volume (5û-200 pL) of ce& diluied in M9 Sala (Sambrook et al. 1989) were

Table 1: Bacterial strains used in this study

Cumpylobacter jejuni - ATCC33559 type strain for species - ATCC43431 serotype reference strain for 0:3 (TGH90 1 1)

Campylobacter coli - ATCC33560 type s~ain for species

Campylubacter lari - ATCC35221 type slrain for species

Campylobacter sputorurn - ATCC33562 type strain for species

Campylobacter upsaliensis - ATCC43954 type strain for species

hcherichia coli - JM101 A(lac-pro) thi rspL supE endA sbcB hsdR

F' (naD36 proAB lacIq ZNM 151

FargE2 thr- 1 feuB6 proA2 his-4 thi- 1 lacY 1 galK2 rpsL supE44 ara- 1 4 xyl- 1 5 m l - 1 tsx-33

- RPCSOO As AB 1 157 plus njo- 1 ::kan

- RPCSOl As AB 1 1 57 plus nfo- l ::kan A(xth-pncA)W

ATCC J.L. Penner

I.L. Penner

Cunningham et al. 1986

Milcarek

Cunningham et al. 1986

Cunningham et al. 1986

Brock

Table 2: Plasmid vectors used in this study

pBR322 436 1 bp Ampr, Te(, low copy cloning vector

pBluescript II SK(+) 296 1 bp Ampr, high copy cloning vector lacZa, multiple cloning site

- Bolivar et al.

Table 3: Recombinant plasmid vectors used in this study

pBRC 1 gBRC2 and pBRC7

pBSPC1, pBSPC2 and pBSPC7

pBSAG4-LI and pBSDC9-Al

pBSPC 1-2J

pBSPC 1 -2B

C. jejuni TGH90 1 1 genomic DNA library clone complementing TBHP-oxidative stress assay in E. coli RPC5ûû

A Sa1 1-Pst I subclone of pBRC1, pBRC2 and pBRC7 into pBluescript SK(+)

C. jejuni TGH90 1 1 genomic DNA library clone obtained through Southem hybridization screening

A deletion mutant derived from pBSPC 1

A deletion mutant derived from pBSPCl

A deletion mutant derived from pBAGCL1

A deletion mutant derived from pBAG4-LI

A deletion mutant derived from pBAGQL1

A deletion mutant derived from pBAG4-LI

A deletion mutant derived from pBAG4-L 1

A deletion mutant derived from pBAG4-L 1

A deletion mutant derived from pBAOCL1

A deletion mutant derived from pBAG4-L l

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

Table 4: Oligonucleotide sequences used in this study

TCC CCC GûG AAT GAA AAA AAT TTC AGC CC

TCC CCC GCXi ?TA TIT TCC T i ' GTA G

CTï A T ï AAA AAT AGG GCT G

AAC 'IüC AGC AAA ATA TGG AGC AAC T T . C

ATG CGT CCG GCG TAG A

GAA AAG CTC ATA CAG GAT TC

GCT CTA GAC ATC TAA AAC AAC AGA TAT TGT AGG

* restriction enzyme linkers included # oligonucleotide complementa~ to pBR322 vector sequence ai nucleotides 38 1 thmugh 396.

mixe& The mixture was immediateiy poured onto a LB medium plate. These plates were

incubated at 37OC und the maximum nurnber of colonies appeared (2-3 days). A graph

depicting the sumival rate against various TBHP concentrations was ploued

Preparation of competent celis and Transformation

An ovemight culture of an E. coü strairi grown in LB bmth was diluwl5X and

incubated for 2 hours at 37OC with shaking. Cek are harvested by centrifugation at 3 0

rpm. These ceils were then resuspended gently in Solution A (10 rnM MOPS [SIGMA

Chemicals, ST Louis, USA], pH 7.0. 10 mM RbClJ and spun down immedimly. 'Ihe

supematant was decanied and Solution B (1 0 mM MOPS pH 6.5. 10 mM RKL, (SIGMA

Chemicals, ST Louis, USA). 50 mM CaClJ was added. The cells were once agah

muspended gently and the mixture was placed on ice for 30 minutes. Finally the cells were

spun down at 3000 rpm, supematant was decanted and the pellet was resuspended in 1-2

rnL of Solution B. The competent ceh were stored ovemight at 4OC. For transfomation.

100 p.L to 150 pL of competent ceils were mixeci with 1-20 pL of a DNA solution. The

mixture was placed on ice for 30 minutes and then hat shocked at 4Z°C for 90 seconds.

Upon heat shock, 1 mL of LB broth was added to the mixture and grown for 1 hour a

37OC. 10 pL to 100 pL of transfonned c e k were then plated on seleetion plates. These

plates were incubated at 37OC for 8-12 hours.

Transformation of C. jejuni TGH90 11 recombinant pBR322 genomic D NA

library into RPC500

E. coli RPC500 was made competent by the RbCYCaCI, method. as desaibed

eariier and 3 pL of C. jejuni TGH9011 recombinant pBR322 genomic DNA library was

aansformed into i t The aansformed ce& were challenged with TBHP chernical on an

ampicilin selection plate. The TBHP concentration used was 200 pg /plate. Afkr 2 days of

growth the cells were pooled and a secondary challenge of TBHP-rnediated oxidative stress

was carried out.

Plasmid DNA isolation and k phage DNA extraction

For the analysis of plasmid. E. coli saains harbouring appropriate plasmid

construct were grown ovemight in the presence of 50 pg/rnL of ampicilin Plasmid DNA

was isolated using the alkaline lysis method (Sambrook et al. 1989). Plasmid DNA

preparation was aeated with a noal concentration of 20 mg/mL RNAase A (Sigma

Chemicals, St Louis, USA) at 37OC for at least 30 minutes. The f e ~ d ~ g Plasmid DNA

solution is then stored at -20°C mil further use. Infection of )c phage was c d e d out by

growing E coli LW92 ovemight in LB broth, in the presence of glucose (SIGMA

Chernicals. St Louis, USA) and MgCI, (BDH Inc., Toronto. Canada) at a final

concentration 0.2 % and 20 mM respectively. The ovemight LW92 culture was then

diluted 1: 10 in LB broth and maltose was added to a nnal concentration of 0.4 96. 200 pL

of a 2 hour cultureci LE392 ceils were then mked with 200 pL absorbtion buffer (10 mM

MgCJ, 10 mM CaC4 BDH Inc.. Toronto, Canada]) this mixture was then idkcted with

appropriate dilution of  phage particles and either grown in broth medium to harvest h

DNA (Grossberger, 1987) or plated on LB medium plates by pour plate method to tiût the

phage. h phage lysates with 2 % Chloroform is stored as stock h particles.

Preparation of recombinant plasmid DNA for generating deletion mutants

Deletion mutants were geaerated ushg exonuclease III and S1 nuclease (Henikoff

1984). A San (Boehringer Manheim) -Pst1 (Gibco. BRL) W e n t h m the original

recombinant pBR322 genomic DNA construct were p d e d using the Geneclean Kit (BI0

101 hc.) These fkagments were ligated to pBluescript II SK (+) (Stratagene) phmid

which was digested with S d and PstL The multing consaict was then subjected to San

digestion foilowed by a KpnI @oehruiger Manheim) digesoon to produce a S'overhang

and a 3'overhang respectively. The plasmid DNA was percipitated using 0.3 M sodium

acetate (NaOAc) and 100 96 ethanol at -70°C overnight The DNA pellet was washed with

70 % ethanol and dried under vacuum. The DNA pellet was resuspended gently in a 1X

exonuclease III b a e r and incubated at 37'C for at least 5 minutes prior to subjecting it to

a time limitai 200 U of exonuclease III (Pharmacia) digest The digestion was carried out

at 37OC . At the appropriate times an aliquot fmm the exonuclease III digest tube was added

to a fresh 1.5 mL polypropylene tube containhg an equal volume of prewamied 2X S 1

nuclease b a e r with 200 U S 1 nuclease (Phannacia) at 37OC. nie S 1 nuclease digest was

carried out for 5 minutes and then an equal volume of phenol (BDH Chemicals, Toronto.

Canada) is added to stop the digestion. The tube was shaken vigorously to mix the

emulsion and spun 5 minutes at 9000 rpm, at room temperature to separate the aqueous and

phenolic phase. The aqueous phase was then transfered to a fresh 1.5 mL tube and an equal

volume of chlorofom (CHC13) (Caledon. Georgetown. Canada) is added and the resulting

emulsion was once again shaken vigorously and then spun for 5 minutes at 9000 rpm, at

room temperature. The aqueous phase was then msfered to a fresh 1.5 mL tube and then

sodium accetate was added to a bal concentration of 0.3 M and 2 volumes of 100 %

ethanol was added. The solution was placed at -70°C for 30 minutes to overnight. Then the

percipitated DNA was peUted by centrifugation at 13000 rpm for 15 minutes at 4'C. The

DNA pellet was washed with 70 46 ethanol and dried under vacuum. The dried DNA pekt

was dissolved in 20 Cu, 1X NTB (1 50 mM Tris-HC1 pH 7.5, 30 mM MgC4, 3 mM DTT

and 150 pdmL bovine s e m albumin) baer . 100 U of DNA pdymearse I large Eragment

(IUenow) (Gibco, BRL) was added and incubateci at room temperaaire for 5 minutes. The

four deoxyribonucleotides (dATP, dGTP, dCTP and d m ) (Pharmacia) were then ad&d

to a h a 1 concentration of 0.25 m M each and incubated further at room temperature for 10

minutes. Einally ATP (SIGMA Chemicals, St Louis. USA) was added to a final

concentration of 10 mM followed by 30 U of T4 DNA ligase (Bœhringer Manheim). The

ligation mixture was incubated at 15OC oveniighr 'The ovemight ligation mixture was then

tmnsformed into competent E. coü Ml01 cek. Transformed cek were plated on

ampicilin selection plates. Transformants were then subjected to phsrnid DNA anatysis.

Preparation of sequencing grade plasmid DNA

The method desaibed here was modified h m Grossberger. 1987 and a protocol

published by Applied Biosystems. Inc. (1991). An aliquot of plasmid DNA solution (10

pg) was mixed with ammonium accetate (BDH Inc., Toronto, Canada) to a final

concentration of 1.875 M and then 3 volumes of 100 96 ethanol was added. The mixture

was placed at -70°C for 30 minutes to ovemight. ïhe percipitate was then spun at 4OC, at

13Wû rpm for 15 minutes. The DNA pellet was washed with 70 % ethanol and then dried

under vacuum. The dxied DNA pellet was resuspended in 32 pL of dH30 and NaCl was

added to a fmal concentmtion of 0.8 M. An equal volume of 13 Q polyethyelene glycol

(PEG) (SIGMA Chemicals, ST Louis, USA) was added and the solution was incubated on

ice for at l es t 1 hou. After incubation the solution was œntrifùged at 4OC for 20 minutes

at 13000 rpm. The supernatant was aspyated carehilly and the DNA pellet was washed

with 70 % ethanol and dried under vacuum. The resulting DNA pellet was resuspended

gently in ~ 0 . The DNA was now ready for denaturation and sequencing.

Dideoxynucleotide sequencing procedure

Sequencing was performed with the Sequenase kit h m United States Biochemical

using the dideoxy chah-tennination method (Sanger et al. 1977). The PEG percipitated

DNA was denanued by adding fresh NaOH to a final concentartion of 0.2 M and

incubated at 37OC for 10 minutes. The denanired DNA was precipitated with ammonium

acetate and ethano1 as desaibed earkr in the "Preparation of sequencing grade plasmid

DNA". Tbe resulting pellet was resuspended in 7 pL %O. 3-10 pmol of primer (1 &)

and 2 pL of 10X sequenase reaction bufKer (USB) was added. To anneal the primer to the

template the tube was innibated at 6S°C for 2 minutes and then placed in a plastic container

containing 500 mL of water preheated to 65OC. The container was left at room temperature

(approximately 30 minutes) to permit slow cooling and annealing. The tubes were placed

on ice und labelhg reactions were carrieci out In the labelling raction dithiothreitol (Dm was added to a finai concentration of 10 mM- 2 p L of SX premade labelling nucledde mix

(USB), 5 U of Sequenase enzyme (USB) and 10 pCi of US" dATP (ICN, Biochemicals),

were added. This mixm was incuba& at room temperature for 5 minutes. An aliquot of

3.5 pL of the labelling reation rnix was added to 2.5 p.L of dideoxy termination mix (USB)

prewarmed at 37OC. The reaction mix was incubated at 37OC for 5 minutes and the

extension reaction was stopped by adding 4 pL of gel loading buffer (USB). An aliquot of

2-3 pi, of the reaction product was fractionated on a 5 % or a 6 % polyacrylamide

sequencing gel with a voltage of 1900 Volts. Oroe the sequencing plaies are separated.

with the gel still adhering to one of the glass plates. a 10 % methanol and 10 96 acetic acïd

solution was slowly poured ont0 the gel- This k e s the gel and removes the urea. which

otherwise prevents the gel h m dqmg complete!y. The h e d acrylaïnide gel was transfered

onto a blotring paper (3MM) and dried un&r vacuum at 80°C for 90 to 120 minutes. The

dried gel was exposed to an X-ray film ovemight. The nucleotide sequences were read off

nom the autoradiogaph. Sequence data were managed by using the MacDNASIS software.

Polymerase chain reaction (PCR) amplification of genomic DNA and

plasmid DNA

Amplification of DNA using PCR was usually c d out in a 50 p L miction

volume. In a 1X PCR resu:tion buffer (Boehringer Manheim) void of MgC1, the final

concenhation of al1 reaction constituents were as follow: apppropriate set of primers were

used at 1.0-5.0 pmol, four deoxyribonucleotides at 0.2 mM, various concentration of

MgCI, at 1.0-3.0 mM and 2.5 U Taq polymerase (Boehringer Manheim). Minerai oil

(ACP, Chernicals Inc.) was added at 2X the reaction volume. The amount of genomic

DNA used in the PCR reaction is 28 ng while the concentration of plasmid DNA was at

5.0-10.0 ng. The amplification reaction was always initiated with a denamration at 95OC

for 90 seconds followed by annealing temperature suitable for the primers useci, for another

90 seconds and f d y extension of the annealed primers was carrieci out always at 72OC

for an appropriate tirne. The cycle usually was repeated 30 to 35 tirnes. Upon completion of

the cycles a fmal 10 minutes extension was canied out at 72OC. 5 5% to 10 96 of the PCR

product was resolved in an appropriate concentration of agarose gel. The remaining PCR

product was separated from the mineral oil and stored at -20°C untii further use.

End-labelling of oligonucleotides

Oligonucleotide were end-labelled as described in Sambrook et al. (1989). In a 20

IL reaction volume containing IX kinase b&er (NEB. USA), 0.1-0.3 w of

oligonucleotide was added with 10 pCi ofy ATP (ICN Biochemicals) and 10 U of T4

polynucleotide kinase (NEB, USA). The mixture was incubated at 37°C for 30 minutes.

The reaction was stopped by adding 1 pL of O J M FDTA pH 8.0. Pnor to separahon of

the labelleci product from the unlabelied y ATP by G-50 column purification, 79 pL of

STE buffer (10 rnM Tris pH 8.0.100 mM NaC4, 1 mM FDTA pH 8.0) was added to the

21 pL of the reaction mix. 1 pL of the purïfied labelleci product was mixed with 10 mL of

scintillation fluid (Bechan Instrument Inc. Ca USA) and the radioactivity was determined

using the Beckman LS3801 Liquid Scintillation System.

Nick translation

Double stranded DNA was labelled d g the nick translation method. In a 50 pL

reaction volume, 5 of solution Al (each of dGTP, oITP and dCïP at 0.2 mM and void

of dATP, 500 m M Tris-HC1 pH 7.0.50 m M MgC4, 100 mM 2-mercaptoethanol and 100

pg/mL of nuclease-free bovine senun albumin). 10 pCi of ap3* dATP and 0.4 U of

enzyme mixture @NA poiyrnerase 1 and DNAase i ) (Gibco, BRL). nie labelling reaction

was done at 15OC for 1 hour and stopped by adding 1 pL of 0.5 M EDTA pH 8.0. 49 p.L

of STE buffer (as described above) was added to the reaction prior to purifying through rhe

G-50 column. 1 pL of the purified labelled product is mixed with 10 mL of scintillation

Buid and the radioactivity was determineci.

Southern Transfer

AU high prwrntage agame gels were bloOed using the vacuum blot. w h e m low

percentage agamse gels weE blotted using the conventional capiUary uansfer. DNA

digested with restriction enzymes and fractionated on a 0.75 Sb- 1.2 96 agarose ge1,were

stahed with 250 pg/rnL of ethidium bromi& and destained in water for at least 30 minutes.

The destained gel was photographed using the FotoDyne camera under UV light. The gel

was b10üed onto a GeneScreen Plus (DUPONT) membrane according to the LKB 2016

VacuGene Vacuum Blotting System (Phmacia) ïnsmtction manual Before air drying the

membrane, it was washed in a 2X SSC (20X SSC: 3 M sodium chloride and 0.3 M

sodium citrate) solution for at least 10 minutes.

For 0.4 96-0.8 % agamse gel the capilary uansfer method was used. The restriction

enzyme digested DNA was fractionated overnight at a low voltage using a prestained 0.4 %

gel. The gel was photographed as above without destaining. Then the gel was treated using

the salt m f e r protocol as deScnbed in the GeneScreen & GeneScreen Plus hybridization

a s f e r membranes transfer and detection protocols manual. The aeated gel was transfered

by capillary transfer (Sambrook et al. 1989) ont0 a GeneScreen Plus membrane.

Screening of a pBIuescript II SK(+) iibrary of C. jsjuni (Colony Blot)

A genomic iibrary of C. jejuni constructed using 4 to 9-kb S a 3 A fragments

inserted into the BarnHI site of pBluescript II SIC(+) was used in the screening. Nine

hundred and sixty individual clones harbou~g the pBluescript II SIC(+) recombinant

plasmid DNA were grown in 10 different %-well microtim plates. Using the BIOMEK

1 0 (Beckman Instrument Inc. Ca. USA) apparatus these 960 individual clones were

blotted onto a nitrocellulose backed membrane (soaked in LB broth with ampicilin). placed

on a microtitre plate lids (containing LB medium aga with ampicilin). ïhe ceils on the

fdters were then grown at 37OC for approximately 8-10 hours (growth was rnonitored so as

to avoid overlapping colonies). Once SuffiCient growth was visible, the cells were lysed for

10 minutes by placing the membrane on a 3MM Whaîmm blotting paper sahirated with 10

9b SDS. The membrane was then uaosferred to a 3MM Whaanan bloüing paper sahirated

with 0.5 M NaOW 1.5 M NaCl for 5 minutes to denature DNA To neutralize the

denaturation reaction the membrane was tramferreci to a 3MM Whafman blothg papa

sahuated with 0.5 M Tris (pH 8.0) for 5 minutes. This process was repeated. The

neutfalized membrane was then immersed in 2X SSW. 1 % SDS solution for 5 rninutes

with gentle agitation. F d y the membrane was washed in 2X SSC for 5 minutes. 'Ihe

washed membrane was air dried and the appropria column matching the 96 microtitre

wells were marked. The dried membrane was baked for 2 hours at 80°C under vacuum.

This membrane was hybridizied in 10 mL hybridization solution. with 3.5 ng of nick

translated 0.9-kb probe (PCR product of oligonucleotide #1219 and p2Ja with pBSPC 1 as

template, see Fig. 18), which had a radioactivity count of 1.0 x 106 cpm.

Secondary screening was Carried out using ovemight grown colonies iifM ont0 a

ColonylPlaque sneen hybribt ion tramfer membrane (Dupont). The blotting procedure is

as follows, the plates harbouring the appropriate colonies h m the primary sc~eening were

cWed at 4OC for at least 1 hour pnor to placing a Colony/Plaque Screen hybndization

transfer membrane for lifting. The membrane was placed on the plates for 3 minutes and

then it was carefuily lifted. The lifted membrane was placed for 2 minutes on a pool of 0.75

mL of 0.5 N NaOH dispenseci onto a saran wrap which was placed on a clean blotting

paper. After 2 minutes the membrane was dned on a clean bloning paper. The above

procedure was repeated. The denaairation process was stopped by placing the membrane

on 0.75 mL of 1 M Tris-HC1 pH 7.5 for 2 minutes. 'Ihe membrane was dned on a clean

b l o b g paper and this procedure was repeated. FmaIly the membrane was air dried. The

resulting membrane was hybrïdkied in a 5 mL hybridization solution, with 3.5 ng of 0.9-

kb probe which had a radioac tivity count of 1 .O x 106 cpm.

Plasmid DNA was harvested h m each positive secondary screening clone and the

DNA was digested with various restriction enzymes. The digested product were

fractionated in a 1.2 % agarose gel (see Fig. 17) under 70 Volts for 3 1R hours. The g4

was then W e r r e d by the vacuum blot method ont0 a GeneScreen Plus membrane. This

membrane was used in a rertïary screening. The hybridization was done in 5 mL

hybridization solution. with 50 ng of end labeled p C l b (see Fig. 18) probe which had

5.37 x ld cpm of radioactivity.

Saeening of a LGemll iibrary of C. jejuni (Plaque iift)

From the h-Geml 1 Iibrary of C. jejwi which has a titre of approxhately 6.8 x 10'

pWmL. 3.0 x 10' pfu were used to infect LE392 and plateci on a 150 x 15 mm petri dish.

An overnight growth displayed well isolated plaques. The plates were sealed with lab

parafilm (Amencan National Cm, WI, USA) and placed at 4OC for at least 1 hour prior to

placing a membrane for üfüng plaques. Once the plates were well chilled and ready for

plaque lifüng, a 137 mm hybridization transfer membrane disc (DUPONT) was placed on

the 150 x 15 mm petri dish for 3 minutes. Using the holes on the d i x , marks were

punched on the plates with sterile pasteur pipettes. After 3 minutes the membrane was lifted

carefully using a sterile twhmr. Onto a saran wrap stretched on a clean blotting paper, 4

mL of 0.5 N NaOH was dispenseci (0.75 mL for a 82 mm hybridization transfer membrane

disc). the plaque lifted membrane was placed on the solution and soaked for 2 minutes with

the plaques facing upward. Then the membrane was uansferred ont0 a clean blotting paper

to dry. The process was repeated. To neuaaiize the denaturation process. the dried

membrane was placed ont0 4 mL of 1 M Tris-HCl (pH 7.5) for 2 minutes (0.75 rnL for a

82 mm hybridization transfer membrane disc). The membrane was dned on a dean blotàng

paper and the neuaalization process was repeated The air dried membrane was h y b ~ ~

with 0.9-kb probe (0.5 ng per mL of hybridization solution), which had a r a d i d v i t y

count of 1.45 x 16 cpm per m . of hybridization solution.

Positive plaques from the primary screening were picked and p w n and plated for

secondary meening. An 82 mm hybridization aander membrane disc was used to lift

plaques off a 100 x 15 mm petri dish. The membrane was processed as above. Six

individual membranes were made with apprOpnate negative controls and these membranes

were hybridized in a 10 mL hybndization solution. with 5 ng of 0.9-kb probe having a

radioactivity count of 1.45 x 106 cpm.

From the secondary ~creening, well isolated positive plaques were picked and

phage lysates were prepared. 2 pL of these lysates were bloüed onto GeneScleen Plus

membrane. The membrane was treated as an ordinary plaque iift membrane. in this tertiary

screening, a 5 mL hybndization solution camed 5 ng of 0.9-kb probe. which had

radioactivity count of 1.45 x 106 cpm.

Hybridization and Autoradiography

Hybridization was either çarried out with deionizied fonnamide (prepared according

to the Bio-Rad instruction manuai) or water as the main component in the hybridization

solution. Hybridization was always done in two stages. nie îkst was the prehybridization

where the membrane was equlibrated with pre-hybridization solution. The pre-

hybridization solution with deionizied fornamide. contained 50 % deionizied fonnamide. 10

% Dextxan Sulphate, 1 M NaCl and 1 % SDS. The pre-hybridiztation solution without

deionizied formamide contained 10 % Dextran Sulphate, 1 M NaCl and 1 % SDS. The pre-

hybridization was camïed out at the hybridization temperature for at least 1-2 hours. Afkr

this the probe was mixed with the pre-hybndization solution. Double stranded DIVA probe

was denatured by boiling for 10 minutes with 100 pL of 5 mg/rnL of sahon sperm DNA

and then chilling the boiled mixture on ice for 15 minutes. Once chilled a quick spin was

done to collect the condensaiion. The denaaired DNA probe was then mixed with the pre-

hybridizaîîon solution. Oligonucieotide probes, were mixed with 100 @ of 5 mghnL of

salmon sperm DNA which was denatured and chilled as described eariier. The probe

mixture was then mùred with the pre-hybridization solution. When hybridizing with

membrane harbouring digested plasmid DNA, salmon spemi DNA was excluded.

Pulsed field gel electrophoresis (PFGE)

PFGE inserts were prepared according to the method of Smith et al. (1987) with

minor modification (Maslow et al. 1993). t i o r to restriction enzyme digestion the insert,

with the intact C. jejimi genomic DNA. was washed in TE (10 mM Tris-HC1 pH 7.5. 1

mM EDTA pH 8.0) for at least 2 hours. The digestion was later carrieci out in a 0.3 mL

volume with 1X concentration of the appropriate b a e r for SmaI. Sali or SacIL n e

respective enzyme was in excess of 40 U to 50 U in each digestion. Restriction enzyme

digests of S m 1 were incubated ovemight at 25OC and the rest were also incubated

ovemight but at 37OC.

Upon completion of digestion, each of the huer& was removed and 113 of the insert

was carefully planted into a 1.0 % agame gel well and electmphoresised in a 0.5X TBE

ninnuig b&er at 14OC with 10 Vlcm. The electrophonsis was carried out ushg a contour-

clamped hornogenous-electric field (CHEF) apparatus with programmable, autonomously

controlled digitdanalogue converters at each electrode (PACE system) (LKB 2015

Pulsaphor gel elecaophoresis unit and pulse time controller-Phamiacia LKB

Biotechnology, Uppsala. Sweden). The gel was subjected to 4 running phases, with the

starting and the ending puIse as 5. 10,25 and 45 seconds in each phase. Each phase lasted

for 5 hours.

The C. jejwi chromosomal DNA fragments n a c t i o d on the gel were stained

with etùidium bromide (Boehringer Manheim) and photographeci. The gel was then

transferred by the vacuum blot meuiod onto a GeneScreen Plus membrane. The membrane

was hybridized in a 10 mL hybridhuion solution, with 44 ng of nick translated ORE3

probe (PCR product of oligonucleati& f-orf3 and r-orf3 with pBSPCl as template. see

Fig. 18), which had 3.66 x 1 O6 cprn of radioactivity. For reprobing th: membrane was

then stripped by treating in 0.1X SSC with 1 % SDS at 65OC for 15 minutes.Using a

Geiger cuunter the tadioactivity erniüed nom the membrane was monitored and if necessary

the stripping procedure was prolonged. The membrane was then exposed to X-ray nùn to

ensure a successfuI stripping procedure. 'The stripped membrane was hybndued with a

new probe. This second hybridization was carried out in a 10 mL hybridization solution,

with 50 ng of nick translated WSalI probe 6 6 was obtained through ~cfeening of h-

Gem l l library of C. jejwii) which had radioactivity count of 5.1 x 106 cpm.

RNA extraction

An ovemight culture of E. coü sPain M l 0 1 harbouring the recombinant plasmid

pBSPC 1 was diluted 10X in LB and grown for 2 hours at 37OC, till the ceh were at mid

log growth. The cells were then spun at 5 0 rpm for 7 minutes in a 50 mL polypropylene

tube. The supernatant was discardeci and the pellet was subjected to two diferent ways of

RNA extraction, the hot phenol method (Aiba et al. 198 1) and the TNol method (Gibco.

BK). A brief account of the hot phenol method is as follows; the pellet was resuspended

in 1 mL of 0.02 M NaOAc pH 5.5,0.5 % SDS and 1 mM EDTA pH 8.0. 0.5 mL of the

resuspended peilet was transfered to two different 1.5 mL polypropylene tubes and an

equal amount of hot phenol (equilibrated with 0.02 M NaOAc) was added. The phenol

extraction was carrieci out 3X for 5 miautes in a 60°C waterbath. The resuiting aqueous

phase was precipitated with 0.3 M NaOAc pH 5.5 and 3 volumes of EtOH at -70°C for 30

minutes. The precipitate was spun at 4OC. 13000 rpm for 30 minutes. The RNA pellet was

washed with 1 mL of 70 % ElOH and the pellet was air dried, resuspended in 50 pL of

diethyl pyrocarbonate (DEPC) treated -0.

The Trizol method is as follows, the cell pellet was resuspended using vigorous

pipetting action in 1 mL of Trizol solution. The resuspended pellet was incubated at RT for

5 minutes. 200 pL, of CHCl, was then added and the emulsion was vigorously mixed and

incubated at RT for 3 minutes. The mixture was then sep- by centrifugation at 4OC.

13000 rpm for 30 minutes. The colourIess aqueous phase was then transfered to a fresh

1.5 mL polypropylene tube and equal amount of isopropyl alcohol (BDH Inc.. Toronto,

Canada) was added. This mixture was incubated for 10 minutes at RT. The phpitate was

spun at 4OC, 12000 rpm for 10 minutes. The supematant was discarded and the RNA pellet

was washed with 1 mL of 70 96 EtOH. the mixture was vortexed bnefly and then spun at

4'C. 7500 rpm for 15 minutes. The supematant was discarded, the RNA pellet was air

dned and resuspended in 50 pL of DEPC treated 30.

RNA purity and concentration was cietennineci by measuring absorbante of samples

at 260 nm and 280 nm. The RNA was either directiy used in the foilowing expriment or

stored at -70°C with 3 volumes of EtOH. AU solutions in dkct contact with RNA during

extraction were prepared using DEPC aeated *O and the fresh chernicals used were

reserved for RNA work only.

Primer extension anaiysis

The oligo deoxyribonucleotide primer, p2Fa (see Fig. 24) was end-labelleci as

described previously. 50 pg of isolated total RNA and ld cpm of end-IabeUed p-2Fa was

precipitated with 0.3 M NaOAc pH 5.2 and 2.5 volumes of EtOH at -70°C for 30 minutes.

The resulting pellet was lesuspended in 30 pL of 1X aqueous hybridization solution (3X

aqueous hybridization solution coosists of 3 M NaCl, 0.5 M Hepes pH 7.5 and 1 mM

EDTA pH 8.0). The mixture was incubated at 8S°C for 10 minutes and then was transfered

to 30°C and incubated overnight The annealed primer was then precipitated with 170

of 0.3 M NaOAc and 500 pL of EtOH at -70°C for 30 minutes. The precipitate was spun at

4OC, 12000 rpm for 30 minutes. The pellet was washed with 75 46 EtOW 25 46 0.1 M

NaOAc and air dned. The dried pellet was resuspenki in 25 jL of primer extension mix

(0.5 m M of each of dGTP, dATP. dTTP and d m , 34 mM Tris-HC1 pH 8.3. 50 mM

NaCl. 5 mM MgCl,. 5 rnM MT and 40 U RNAguard [Pharmacial). 40 U of avian

myeloblastosis virus reverse transcriptase (AMV-RT) (Pharmacia) was added and the mix

was incubated for 90 minutes at 42OC. 1 pL of 0.5 M EDTA and 1 p.L of 1 m m

pancreatic nbonuclease A was added and incubared at 37OC for 30 minutes. 100 pL of 2.5

M ammonium aceiate was added and extracted with 125 pL of phenol/CHCI,. nie aqueous

phase was then transfered to fresh 1.5 rnL polypropylene cube and an equal volume of

EtOH was added and precipitated at -70°C for 30 minutes. The praipitate was spun at

4OC. 12000 rpm for 30 minutes. The pellet was washed with 70 9b EtOH and was vacuum

dried. The dried pellet was resuspended in 6 pL of TE (10 mM Tris-HCL. 1 mM EDTA)

and 4 p.L, formamide gel loading buffer (USB). The samples were boiled for 5 minutes and

irnmediately chilled on ice before loajing onto a 5 % polyacrylamide sequencing gel with a

sequencing ladder of the pBSPCl obtained with p2Fa primer. The gel was then pxepared

as M b e d eadier in the dideoxynucleotide sequencing procedure.

RESULTS

TBE?P-mediated oxidative stress assay.

AB1 157. wild type E. coli strain shows considerable &stance to 200 pg/p& of

TBHP. Isogenic mutants of AB 1157, RPCSûû (nfo-1:W) and BW9091 (A-1) are

sensitive to TBHP (Cunningham et aL 1986; see Fig. 12). RPC500 (nfo-l:-) is more

sensitive to the oxininng chernical compared to BW9091 (xth'). RPC501 (xrhnfo-) a

double mutant of AB1 157 is extremely sensitive to TBHP (see Fig. 12) as has been

observeci by Cunningham et ai. (1986).

Cloning an oxidative stress response gene of C. jejuni TGH9011

In order to clone oxidative stress response genes of C. jejuni TGH901. RPCSOO

an nfo mutant with increased sensitivity to TBHP was used as the host in a TBHP-

mediated oxidative stress screening strategy. 'Ihree different profiles of pBR322

recombinant plasmid DNA were obtained h m clones that showed resistance to TBHP at

200 pg/plaîe upon transformation of a pBR.322 genomic DNA library of C. j e +

TGH9û11 into RPCSOO. The three recombinant pIasmids are designated pBRC1, pBRC2

and pBRC7 and the respective RPCSOO transfomesi ce&, Cl, C2 and C7 showed

increased resistance to TBHP. when compared to RPCSûû with or without pBR322. C l

showed the highest survival rate compared to C2 and C7 (see Rg. 13).

Subcloning of pBSPCl and construction of pBPEORF1

A San-Pst1 fragment from pBRCl was subcloned into pBluescript II SIC(+)

digested with the same enzymes, to fom pBSPC1. The subcloning procedure is depicted

in Fig. 14. pBSPCl was used to characteriz the C. jejwu' genomic DNA insert.

pBPEORFl was consmicted with a PCR product generated by using primers p C l a and p

Zia with pBSPC1 as the template. The PCR product was digested with Pst1 and ligated

Fig 12: TBHP-media& oxidative stress assay of AB1157. with its isogenic mutant

strains, RPCSOO (nfo'). BW9091 (A-) and RPCS01 (nhnfo-). The graph depicts the

survival rate of the wild type cell and its isogenic mutants against ten.-butyl hydroperoxide.

% of Survival against TBHP (uglplate)

O 50 IO0 150 200 250

TBHP (ug/plate)

Fig. 13: TBHP-mediated oxidative stress assay of RPCSOO harbouring pBRC1. pBRC2.

pBRC7 and appropriate negative and positive controls.

% of Survival against TBHP

% of Survival

O 50 1 O0 150 200 250

TBHP (ug/plate)

Fig. 14: Subclonïng of pBSPC1. A SaNPstI fragment from pBRCl was subcloned into a

pBluescript II SIC(+) which was digested with SaNPstI.

SaA/M digest

Fragment cantaining the C. jejuni insert DNA was purified by gel purification

DNA

multiple cloning site

I

SaWPsti digest

Vector DNA was purified by gel purification

C. jejuni DNA - Ligated by using T4 DNA ligase

I

DNA

22 DNA 7

into pBluescript II SK(+) digesteci with EcoRV-Psrl The construction of pBPEORFl is

depicted in Fig. 15. pBPEORFl was used in the TBHP-mediated oxidative stress assay.

Screening of a pBluescript II SK(+) library and a k-Gemll iibrary of C.

jejuni TGHMl1.

In order to obtain pBluescript recombinant clones that have inserts overlapping that

of pBSPC1, a 0.9-kb K R product obtained using primers p-2Ja and #1219 and pBSPC 1

as the template (see Fig. 18), was labeled by nick translation and used as a probe to screen

a membrane bearing 960 isolated clones of the pBluescript II SIC(+) library of C. jej*

TGHW 1 1. The primary sneening identifiexi eight potential clones but a secondq

screening n m w e d the potential clones to only two (see Fig. 16% 16b, and Mc). Plasmid

DNAs from the two clones designami pBSAG4-LI (see Fig. 16c) and pBSDC9-Al (see

Fig. 16c) were prepared. To i&nW cornmon C. jejwi DNA fragments in pBSAG4-L1,

pBSDC9-A 1 and pBSPC1, these plasmids were subjected to restriction enzyme analysis. A

single and double digest with XbaI and Sac1 were performed on these plasmids. The

restriction hgments were fractionated in a 1.2 % agarose gel (see Fig. 17). Southem

hybridization was used to identify DNA fragments in pBSAG4-LI and pBSDC9-AI that

are common to the ORF2 region on pBSPC 1 (see Fig. 18). The DNA blot was h y b r i b d

with p-C l b, an end-labeled oligonucleotide complementary to 20 nucleo tides of the non-

coding strand of ORF2 ( s e Fig. 18). Southem hybridization result (see Fig. 20a) and

restriction enzyme digest profile (see Fig. 17) revealed two distinct clones. pBSDC9-Al

has an insert approximately 4.5-kb, but has an extended portion, u p s m of ORE?

compared to pBSPCl (see Fig. 21). While pBSAG4-LI has a bigger insert (approximately

Il-kb) with the largest portion extendhg downsrream of ORF3 (see Fig. 21). Deletion

mutants generated from pBSAG4-LI were used in the TBHP-mediated oxidative stress

assay and were also used to obtain the complete sequence of ORF2.

Fig 15: Subcloning of pBPEOWI. A PCR product of prime= pCla and p-2Ja with

pBSPC1 as the template was digested with Pst1 and ligated into a pBl-pt II SIC(+)

which was digested with PstYEcoRV.

ORFl ORF5

XbaI XbaI XbaI Sac1 Nhe 1 Sali , 1 1 1 1 l i K p n l I 1

PCR product

Primers

. digested with Pst1 digested with PstVEcoRV

ligated by using T4 DNA ligase + pBPE0R.F 1

Fig. 16a: Rimary screening of pBluescript SK(+) iibrary of C. jejruu' TGH9û11. Ten

different set of 96-well plate bearing 96 isolated clones of tbe library were compacted onto

one piece of membrane. For example in lane ES each background hybridued dots represent

clone E5 fiom 10 diffenent (A-J) 96weM plate. EG4, BG4, AG4, DC9, DB9. AClO,

GHIO, HHlO, M10. JH10, BC5 and EE7 were selected for secondary screening.

Fig. 16b. & Mc: Pure cultures were made from EG4 (C). BG4 0). AG4 (LJ. DC9

(A), DE59 (E), AClO (B), GHlO (F), HHlO (G), Ml0 (H). JHlO (I), BCS (J) and EE7

(K), where the alphabets in bracket correspond to Fig. 16b. and Mc. The numerals within

the figure represent different isolated colony from the pure cu1tw-e. Five isolated colonies

from A @C9) and four from L (AG4) were found as positive clones. Plasmici from clone

Al and clone L 1 were designated as pBSDCC9-Al and pBSAG4Ll. respectively.

Fig. 17: Analysis of restriction enzyme digested plasmid DNA h m pBSPC1. pBSDC9-

Al and pBSAG4-LI. Lanes X repment XbaI digesf laaes S represent Sad digest and

lanes XIS represent a double digest of Xbd and SacL Lanes h represent =dm marker

while L represent 100-bp m e r .

Open reading Frames ( )

O-: hyphothetical protein ORFS: clpA OW3: trxC

transcriptional direction - pBR322 vector DNA

C. j e j u n i DNA

Fig. 18: Depicts ORF1. 0RFJ2 and ORF3 that was generated by using MacDNASIS

software. The dative positionhg of Sa& XbaI sites and the location of the relevant

primers used in this study are also shown within the figure. Vertical h e withh the figure

indicates a stop codon while an open inverteci triangle represents a methione site.

File : pBSPCI-4083M Mode : Normal Range : mit : ATG Term : TAA TAG TGA

Open reading £rame 1: hypothetical protein

hypothetical protein

Fig. 19: The five complete open reading b e s (ORFs) and two paràal ORFs generated

by using MacDNASIS software. Vertical line withui the figure indicates a stop codon while

an open invemd triangle represents a methione site.

Fig. 20a: Southern hybxidization analysis of restriction enzyme digested plasmid DNA

from pBSPC1, pBSDC9-Al and pBSAG4-LI, with end-labeled oligonucleotide p-C l b as

the probe. Lanes X represent XbaI digest, l m S represent Sac1 digest and lanes XIS

represent a double digest of XbaI and Sad.

Fig. 20b: Te* ~creening of L G e m l l library of C. jejuni TGH9011. Sixteen phage

lysates were prepared fiom sixteen well isolateci plaques fiom a secondary ~creening and

blotted onto a Genescreen membrane. Ten out of the Sxteen phage clones were positive

when hybridized with a Nick d a t e c i PCR product of primers p-2Ja and #1219 with

pBSPCl as the template. XbaI restriction enzyme profde of the ten phage clones revealed a

singuiar pattern.

pBSPC1 pBSDC9-A1 pBSAG441

x w s s X X/s S X ws S

Fig. 21: Relative positioning and detailed restriction enzyme map of pBSPC1, pBSDC9-

Al and pBSAG4-L1. Thin horizontal lines represent vector DNA while open boxes denote

C. jejuni DNA. Closed head w w s denote open reading frame (ORF) and the direction of

transcription. The ORF designations within the figure correspond to Fig. 19. Vertical short

bars indicate restriction enzyme sites.

ORFl ORF5

XbaI XbaI XbaI SacI NheI Sul 1

I I l 1 I l IP"'

I HincII

XbaI XbaI XbaI Sa11

I , I I 1 , ;KpnI

XbaI XbaI XbaI Sac1 NheI EcoRI EcoRV NheI Pst1 Kpn 1 I I

I I I I I I I I I sa1 1 4 1

I

HincII

The 0.9-kb fragment was also used for saeening the LGem 1 1 iibxary of C. jejwi

TGH9011. Ten phage clones were identified as positive clones in a te- screening (see

Fig. 20b). Rirified phage DIVA h m these ten clones showed a singuiar profie when

digested with XbaI. The restriction map of the insert obtaioed h m one of these phage

clones designated A6 is shown in Fig. 22. The h6 phage DNA insert was used as a probe.

in a Southern analysis of fractionated C' jejwii TGH9011 genomic DNA by pulsed field gel

electrophoresis.

Mapping complementation region of pBSPCl and pBSAG4-LI in RPCSOO

against TBHP-mediated oxidative stress assay.

Deletion mutants geaerated h m pBSPCl and pBSAG4-L1 plus a subclone

pBPEOE2Fl were used to locate the specific region of the insert that is required to confer

increased resistance to TBHP by uansformed cells of RPCSOO. The evidence that

irnplicates ORF3 in the increased &tance of RPCSOO is as foiiow: (i) pBSPC1-2J

(denved nom pBSPCl), having 33 amino acids deleted into the carboxy terminal of ORE3.

is unable to render the incleased resistance; (ii) pBPEORFl (a subclone), having complete

OW1, with 0-1-kb of its ups- sequence is unable CO render increased resistance to

RPCSOO; (iü) pBSL1-60.10 (derived from pBSAG4-LI). having a deletion that stops 33-

bp upsaeam of the -35 region of ORF3, with intact ORF3, is able to render i n c r d

mistance; and (iv) pBSL 1-f.l'l(denved from pBSAG4-L 1 ), having a deletion extending

past ORF3 and h d ~ g IO-bp away from the stop codon of ORF3 is unable to confer

increased resistance. The results of the detail complementation expriment are depicted in

Fig. 23.

Fig. 22: Relative positionhg and detailed restriction enzyme map of pBSPC 1 and the h5

phage clone. L and R represent left ami and right ami of the h vector. Vertical short bars

indicaie restriction enzyme sites.

Sa11 XbaI XbaI XbaI Sac1 KpnI

I 4 1

XbaI XbaI Xbai XbaI Sac1 Sa11

1 .O-kB

L - left arrn of the lambda-Gem 1 1 vector

R - right arm of the lambda-Gem 1 1 vector

Fig. 23: ten.-butyl hydroperoxide (TBHP)-mediated oxidative stress assay on the

deletion mutant of pBSPCl and pBSAGCL1. The thick horizontal h e and open boxes

represents C. jejmi DNA insert while thùi horizontal lines indicate vector DNA. The

verticd short bars indicate restriction enzyme sites. The various deletion mutants. indicated

by the appropriate recombinant plasmid consmict, are named in the figure itseIf. Deletion is

from the S d site of pBSPCl and pBSAG4-LI. Double head closed arrows indicaie

complete open reading frames (ORF) while the single closed head arrow indicates an

incomplete ORE The numerals represent the ORF as in Fig. 19. (+). denotes increased

resistance for RPCSOO against TBHP. While (-), indicates no signifïcant resistance

O bserved relative to negative control.

DNA sequenung of pBSPC1.

The complete DNA iosert of pBSPC 1 has k e n sequenced using the dideoxy chah

tamination method. The 4.1-kb fragment had 5 complete potential open reading h n e s

(ORFs) and two partial ORFs. They were designated ORE, ORFI. ORF3.ORF4. ORFS.

ORF6 and ORF7. respectively frorn the San site (see Fig. 18; and 19). Using the NCBI

Basic local alignment search tool (Blast) (Altschul et al. 1990). ORFl naoslared product is

highly homologous to an uncharacterizci region on the E. cofi genome. ORF2 protein

shows high homology to the N-terminal of CipA of E. coli, while the ORE3 protein is

rnoderately homologous to a thioredoxinCrn<)-like protein of Dmsophiün rnehgaster.

ORF4 protein shows high homology to a 18.3-kDa protein known as the srnail protein A

(smpA) in E. coli. ORFS aanslated product is rnoderateIy homologous ro a hypothetical

protein in El coli. ORF6 protein is highly homologous to tRNA pseudouridine 55 synthase

encoded by truB of E. COÜ- Fdy the partial ORF7 translated product is highly

homologous to the C-terminai end of DNA helicase II encoded by uvrD of E. coli.

The organization of the ORF within the 4.1-kb fragment of pBSPC1 is extrexnely

cornpack with no intergenic region between the coding sequences, as has been observed in

other regions of the C. jejwu' genome (Chan and Bingham 1992; and Hani and Chan

1994). This iikely reflects the smail size of the C. jejurù genome (Kim and Chan 199 1 ).

The potential initiation codon of ORF2 overlaps the ORFl by one base (data not shown).

while the h t Met codon of ORFl overlaps one base with the stop codon of ORF3 (see

Fig. 24). The predicted initiation codon of O R B is immediately &ter a putative stop codon

of ORF4 (see Fig. 24). An intergenic region of 17-bp sepatates the putative stop codon of

ORF5 and the putative initiaEon codon of OW4. The biggest intergenic region amongst

these ORF is between ORF5 and ORF6 where the putative stop codon of ORF6 is

separated from the first Met codon of ORF5 by 93-bp. There is no intergenic region

between ORF6 and ORF7. The potential initiation codon of ORF6 overlaps the ORF7 by

one base, distinctly Smilar to the region between ORF2 and ORFl (data not shown).

Fig. 24: Nucleotide and deduceci amino acid sequence of the mC (ORF3) of C. j e w

TGIW)11 with its 5' and 3' flanking regions. The proposed Shine-Dalgamo site is denoted

by horizontal double broken lines, while the -10 and -35 hexamers are indiçated by

horizontal Iiple broken lines. The putative mmcriptionai stan site is indicated by a

horizontal arrow. Pmposed transcriptionai tenninator sequences are indicated with a closed

head arrow below the nucleotide sequences. The carabolite activator protein (CAP)-like

binding consensus sequences (CAP4 and CAP-LI). are denoted by an overline. The

proposed Fur-box-like binding consensus sequences (FBS-1. FBS-II, FBS-III and FBS-

IV) are &noted by an undexline below the nucleotide sequences. The highly conserved

cysteine redox-active residues are denoted by an underline below the deduced amino acid

sequences. The stop codon of the ORE3 is Uidicated by an asterisk below the nucleotide

sequence. Potential start codon of an open reading frame downstream of ORF3 is indicated

by small boldfaced underlined letters.

CAP-II - FBS-I

- FBS-II

. . ~ ~ G C C C ~ A C i c A ~ C T T A A T G C m C T C C A A G G A A G A A ~ T G m A A G A G I S A L F L I S L A F F L N A C S K E E E I Q N D F M F E E

T A T C A C A A A G ( 3 A G A T A A A A T A ( 3 T C T T A G T C T T A A A T A G E T M W ~ M G C ~ C ~ M T A A G M C A G A T M N ~ ~ ~ G M f f i A Y H K G D K I V L N S V N G G S K T L I R T D K G F V V E G

G A G G A A G G A A A A G ~ T M T G ~ A T T T T T T T G G ~ A C ~ C A C C C C A T G T A A A G A A G A A G C ~ A G A T C T T A G C A A A C ~ G I V V I E E G K V L M F D F F G T F T P K E E A L D L S K L W K

. . MTAATTCTAGCAAATTTATCATTATAGGACTTACACAmAAGATG'I"rAGCGATGAAACAGTTAAAAAATTCGCGGAmATTATGGT N N S S K F I I I G L T H F E D V S D E T V K K F A D D Y G

. e

C C ~ A A A G T T C T I T T A A A A A A T G G A A T T T A T C C P F K V V L K N G I Y Q K I S D Y W N N N T P T N F Y L G K

ATTCCMCAGAACTCATGCAAGAAGATTTAAATAAAATCTACAAAGGAAAATAarncCAAAAACCCAAACTCTAGAGCAAACAAAACTTA I P T E L M Q E D L N K I Y K G K *

FBS-IV

Nucleotide sequence of ORF3 (trxC ).

A region of pBSPCl col~e~ponding to ORF3 (W) was completely sequenced on

both strands using overlapping deietion clones of pBSPC 1. A detailed restriction m . of

pBSPC 1 is depicted in Fig. 2 1. The DNA sequence of the fr;rC of jejuni TGH90 1 1 is

shown in Fig. 24. An ORF siamng with an ATG codon at nucleotide 82 and ending at

nucleotide 68 1 with a TAA stop codon was identifid on this DNA sequence. Upst~am of

this ORF was a potentid -10 hexamer consensus of a 8' promoters at nucleotides (nts) 35

through 40 and a potential -35 hexamer consensus sequence at nucleotides 14 through 19

with a spacing of 15 nts. A potential ribosome-binding site (rbs) of AGGAAA was found

four bases ups- of the initiation codon at nts 72 through 77. At nts 789 through 8 14 a

stem loop structure is located. It would be expected that this region of dyad symmetry with

a hair pin structure. 105 nts 3' to the stop codon, is a potential transcriptional terminator.

At nts 6 through 21 (CAP-1) and 53 through 67 (CAP-II), tsvo sequences

resembling the consensus catabolite activator protein (CAP)-like-binding sequences (Jae-

Hoon et aL 1997) (see Fig. 24; and 25) can be seen at the 5'-flanking region of the d.

These putative CAP sequences suggest potential influence of the level of c-AMP and the

involvement of the c-AMP receptor protein gene (crp) in regula~g the expression of r d .

CAP-1 completely overlaps the -35 region while CAP-II is located 4 bases upstream of the

rbs. CAP-like-binding consensus sequences have also been 1-d at the upstream region

of C. jejmifur (Chan et al. 1995) and E. coü m (Jae-Hoon et al. 1997).

Four Fur-box-like binding consensus sequenoes (FBS) (Chan et al. 1995; and

International Union of Biochernistry, 1986) have been identifid within the 833-bp region

(see Fig. 24; and 26). FBS-1 (nts 78 thmugh 96) is located irnmediately downstream of die

rbs. FB S-II (nts 1 0 through 1 18) and FBS-III (nts 150 through 168) are located inside the

ORF. while FBS-IV (nts 775 through 794) is located 90 bases downstream of the ORF3

stop codon. A FBS has been located within the coding region of C. jejunifur gene and also

identined at the upstream region of jejunifur gene (Chan et al. 1995) and E. coli sodA

CAP TGT GAn ATn MT C h Hl10 T/15

1 CGT GAA ACT =A AAA 6 f 1 II TAT GA- AAA =A CAA 7 12

CAP represents CAP Binding Consensus Sequence (CAP-BCS) (Jae- Hoon et al 1997), where the upper-case boldfaced alphabets are highly conserved bases. Lowercase alphabet represent Intemationai Union of Biochemistry (WB) arnbiguity code.

HI10 10 highly conserved bases on the CAP-BCS

T/15 15 bases of the CAP-BCS

Roman numerals - represent the two CAP-Wre-binding consensus sequences identified at the 5' region of the mC gene of C. jejuni

- upper-case boldfaced alphabets are identicai to the highîy conserved bases on the CAP-BCS.

- underiined upper-case alphabets are identicai match based on the IUB ambiguity code for CAP-BCS.

Fig. 25: CAP4 and CAP-II of the trXC gene of C. jejwii alignrnent with the CAP binding

consensus squence.

c-FBS bAT wAT kAd wwk y ATT hdy H/8 Tl19

1 ATA -AT GAA AAA A ATT I C A 6 11 II TAT D T I A A TAA G TTT AGC 6 15 III GAA ATT CAA AAT G ATT =A 6 14 IV - TTT LGT TAP AAA 1 TTT TGA 5 14

c-FBS represents the Fur Box Consensus Sequence @CS), where lower- case alphabets are W B ambiguity code and uppercase boldfaced alphabets are highly C O W N ~ ~ bases.

Hf8 8 highly conserveci bases on the FBCS.

Tl19 19 bases of the FBCS.

Roman numerals - represent the four Fur-Box-like binding (FBS-1, FBS-II, FBS-III and FBS-IV) sequences identifieci at the gene of C. jejuni

- upper-case boldfaced alphabets reptesent identical match to the highly conserved bases on the FBCS.

- underlined uppercase alphabets represent identical match based on the IUB ambiguity code for the FBCS.

Fig. 26: FBS-1, FBS-II, FBS-III and FBS-IV of the OZC gene of C. jejwi alignment

with the Fur box consensus sequence.

(Neiderhoffer et al. 1990). sodA is a major oxidative stress response gene. The existence

of the four FBS in C. jejwii tmC suggests the involvement of the fur product in regulating

this gene.

Two OxyR-Iike-bindiiig sequences (Toledano et al. 1994) have been identified

within the 5' flanlong region of rrrC (see Fig. 27; and 28). OxyR-like-binding site 1 is

found overlapping with the -35 regioe While OxyR-He-binding site II is located 93 bases

upstream of the -35 region.

OxyR-like-binding sites have been located upstream of oxyR, hG, cd@

(Christman et al. 1989; Tartaglia et ai., 1989, 1992). grx flao 1997)' 4 s (Altuvia et al.,

1994) and the Mu phage m m gene (Bolker and Kahmann 1989). These respective genes

are part of the OxyR regulon.

Deduced amino acid (aa) sequence of t n C and nlignment.

The deduced aa sequence of the C. jejuni TGEW) I 1 rr*C product is shown in Fig.

24. It is initiated by Met and consists of 2ûû aa (23-kDa and a p l of 4.9). The TrxC of C.

jejwu' was compared and aligned with 4 other Tm sequences in the database using the

CLUSTAL V (Higgins et ai. 1992) program. The N-terminal region is quite variable while

the C-terminal half shows consenmi aa sequence but with 4 gaps. The highiy conserved

motif is situaîed near the redox-active cysteine residues at c~~ and (see Fig. 29). These

redox-active cysteine residues are known to be at the active site of the thioredoxin molecule

(Hirota et ai. 1997; Fernando et al. 1992; and Gleason and Holmgren 1988). The

conservation irnplicates an important d e of these cysteine residues for the hinction of

thioredoxin.

In the complementation experiment, pBSPCI-21 fails to show an increase in the

resistance for RPC500 against TBHP, when 33 amino acids were deleted fiom the carboxy

terminal end. This is consistent with the k t that the carboxy terminal end is an important

feature of the thioredoxin molecule (Hirota et al. 1997).

tac -35 -10 +

5' t 1 AGGAAA - ATG

O x v R consensus bindincr si te

ATAGN~~CTATNNNNNNNATAGN-TNNNAN - CTAT

upper-case boldfaced alphabets represent highly consemed bases. 0 upper-case underlined alphabets represent degenerate bases.

the alphabet N represents NB ambiguity code

0 Vertical line indicates perfect match (10 bases 1 20 bases).

Fig. 27.OxyR-like-binding site 1 of rr*C gene of C. jejuni depicting the location and the

alignment with the OxyR consensus binding site.

OxvR consensus bindinn site

ATAGN-TNNN-ANC TATNNNNNNNATAGN-WC TAT

0 upper-case boldfaced alphabets represent highly conserved bases. 0 upper-case underlined alphabets represent degenerate bases. 0 the alphabet N represents IUB ambiguity code

ATAGNm-gNCTATNNNNNNNATAONm-m- CTAT *OxyR I I I I l i l 1 1 1 1 I I I I I A T ~ ~ T A ~ G G T ~ G T A A G T A T A G M G G A T ~ T A ~ A T OxyR-like binding site

Vertical line indicates perfect match (16 bases / 20 bases). a * - modined OxyR consensus binding site.

Fig. 28. OxyR-like-binding site II of rr*C gene of C. jejuni depicting the location and the

alignment with the OxyR consensus binding site.

je juni co l i f esoxi dans i n f l uenzae subtilis

VLMFDFFGTFCTPCKEEALDLSKLWK AILVDFWAEWCGPCKMIMILDEIRD

Upper-case boldfaced letters indicate the redox-active

cysteine residues s i t e s (Trx family active-site), where ( " 1

denotes an identical match, while . indicates a conserved

residue .

Fig. 29: Amino acid alignment of TrxC of C. jejwzi and various thioredoxin proteins from

prokaryotes.

The Grx and Tm molecules do not share àigh homology in aa sequence (Gleason

and Holmgren 1988) (the redox-active cysteine sites are conserveci) but it is believed that

the three dimensionai structures of the two rnolecuie are simiiar. The three dimensional

structure of thioredoxin moiecuie shows two f%strands lying dongside each other, their

interaction is presumed to stabilize the structure and maintain the disuüide loop (active-site)

(Gleason and Holmgren 1988). The alignrnent data show that the region encoding the two

pstrands are rdativeiy c o l l ~ e ~ e d in the C- jejwii T e

The aa composition of Trx proteins and tbat of the C. jejuni Tm indicate minimum

usage of histidine (see Table 5). Stadman (1986) has shown that histidine in glutamate

dehydrogenase is prone to oxidation during oxidative stress. While Fernando et al. (1992)

has shown that endothelid œUs aeated with 12 mM of H202 for 5 minutes (E. c d Trx

[Spector et al. 19881 has a limit of 8 rnM of H20,). resulted in a marked b a s e in

glyceraldehyde-3-phosphate dehydrogenase activity but no effect on the activity of

thioredoxin. The resistance to H202 might be due to the structure and the composition of

the Tm,

Mapping of the trxC promoter.

Analysis of the 5'-flanking region of the DNA sequence of mC revealed several

potential promoters. 'Zhe in vivo site of transcriptional initiation was identifid by primer

extension analysis. A synthetic oligonucleoti& 17 nucleotides long ( p-2Fa) ,

complementary to nts 97 thmugh 1 12 (see Fig. 24) was end-labeled, annealed to total RNA

extracted from a s d bearing pBSPCl (to enrich for a*C aanscripts) and extended with

AMV-RT. This oligo was also used in a dideoxy DNA sequencing reaction with plasmid

DNA from svain bearing pBSPC 1. The extension product ended at T45 and with a minor

site at T46 (see Fig. 30). Since this is the sequence of the template strand, transcription

Table 5: The amino acid content (Md $6) of TrxC and various thioredoxin proteins from prokaryotes.

C. jej Cmnpylobacter jejmi T, fer ThiobacilluF feroxiclans E coli Escherichia coii H. inf Haemophilus injlunzae B. sub BaciUus subtilii M. tub Mycobmerium tubercuhsis

P Y ~ Heiicobacter pylon' SL aer Streptomyces aureofaciens St. coe Strepsomyces coeiicolor

*** Stop codon # Total number of iimino acids

Minimum usage of Histidine residue in the thioredoxin prorein is depicted in boldface while Cysteine content i s in italic and boldfaced (al nurnenc values represent Mol 46).

Fig. 30: 'Ibe autoradiogram of a 5 % poiyacryiamide gel used to analyze the primer

extension mapping of the t d promoter. The sequencing ladder of the tempiate s m d was

genmted by dideoxy sequencing of pBSPC1. Laue 1, primer extension product of the

Trizol method and Lane 2. is the primer extension product of hot phenol method. The

horizontal arrow indiates the major start 4ie (T45) while one base below (T46) is the

minor stan site. The sequences are of the ternplate strand, so A45 and A46 &notes the

major and minor start sites, respectively. The DNA sequeoces are consistent with the

cornplementary sequence in Fig. 24.

initiates at A45, with a minor start site at A46. Thû major start site is consistent with the

location of the predicted prornoter region,

Loeating the trxC on the C. jejuni TGH9011 physical genome map.

Pulsed field gel electmphoresis was carxied out using genomic DNA of C. jejwu

TGH9011 digested with Sa& SmaI and SacIL The Southem analysis with ORF3. obtained

by PCR amplification using oligonucleotides f-orf3 and r-orf'3, as probes, showed tbat

ORF3 is locared on the Sd-A fragment, SmaI-A fiagrnent and S d - A fragment of the

TGH9û11 physical genomic map (see Fig. 31). Southem analysis with insen fmm h6 as

probe (approximately 16-kb) revealed a 4milar hybridization pattem. The Southem

hybridization results are shown on Fig. 32.

Campylobacter species genomic DNA hybridization.

Genomic DNA h m five different ATCC type saaùis of Curnpylobacrer spp. (C.

jejwi; C. coli; C. lori; C. sputorwn; and C. upsdiensis) and one serotype reference suain

for 0:3. C. jejwi TGH9û11 was digested with BamHI, XbaI and Xho 1. The digested

genomic DNA was fractionated on a 0.7 % agarose gel. A membrane bearing these

genomic DNA profdes was hybndized with ORF3, a nick translated PCR product of

oligonucleotides f-orf3 and r-orf3 (approximately 0.7-kb).

Two ciifterient hybridization conditions were used. a high suingency, 50 96

fornamide hybridization reveded the presence of rrirC in the species type strain of C.

jejuni, C. coli, and C. lari The hybridization patterns of C. coü and C. lan' were similar.

The hybridization pattern of TGH90 1 1 was different compared to the C. jejuni type strain.

C. jejuni TGH90 1 1 genomic DNA produces a 1.7-kb fragment when digested with XbaI

which hybridized with the probe. The 1.7-kb fragment is consistent with the expected size

based on the nucleotide sequenœ.

Hybridization using 30 46 formamide showed the presence of PXC in al1 of the type

svains for species tested except C. sputonrm The type SM of C. Imi and C. jejwu'

showed Smilar hybridization intensity. The Southem hybridization results are shown in

Fig. 33.

Fig. 31: The current physical genomic map of C. jejwu' TGH9011 showing the location

of the axC gene. Initiai mapping work showed the pcesence of 5 Sali sites but recmt

discovery of an extra San hgment, Sd-E, is inclucied in this map.

23s rRNA probe 0 16s rRNA probe

Fig. 32: Puîsed field gel elecmphore9s analysis of C. jejwù TGEW 1 1 genomic DNA

digested with San (Sa), SmaI (Sm) and SacII (Sc). The horizontai closed head arrows

indicate pamal digest products of SacII. b e l (a) shows genomic DNA fractionated in a

1.0 % agarose gel; panel (b) shows Southern analysis with a 0.7-kB ORF3 specitic probe;

while panel (c) shows the same membrane upon stripping. probeci with a 16-kB k6 insert.

The lanes L indicate lambda marker. The hybridinag fragments on panels (a) and (b)

correspond to the SalI-A fragment, SmaI-A fiagrnent and SocII-A fkagment on the C. j e j d

TGH9011 physical genomic map (see Fig. 3 1).

Fig. 33. Campylobacter species genomic DNA hybridization. Genomic DNA from five

différent ATCC type snains of Ccunpylobacrer spp (C. jejwi [CJ], C. coü [CC], C. krn'

[CL]. C. upsaliencis [Cu, and C. spu~orwlt [CS] ) and one serotype refemce stxain for

0:3, C. jejuni TGH9011 was digested with BamHI [BI, XbaI [Xb] and XhoI ml. nie

digested genomic DNA was fiactionated on a 0.7 % agarose gel. The Southem analysis

was camîed out with a 0.7-kB ORF3 probe. Panel (a) and (b) are results of 50 5%

formamide hybridization while panel (c) is of 30 % formamide hybrkiization.

ci CC cc CU Cs BXbXh BXbXh BXbXh BXbXh BXbXh

DISCUSSION

Oxidative stress via TBHP

Nfo accounts for l e s than 10 46 of the AP endonucleolytic activity in crude tell

extracts of E. coü (Ljungquist et ai. 1976). The major contributor to the AP endonucleolytic

activity is the product of xrh gene (Weiss 1976; and Ya$o and Weiss 1975), exonuclease

IIi cepresenting about 85 %. Both these enzymes catalyze the cleavage of a phosphodiester

bond 5' to the AP site. R e e l y BW9091 (nh3 is l e s sensitive to TBHP and

bleomycin compared to RPCSOO (nfo-) but the nfo mutant is less susceptible to H202

compared to BW9û91. It seems lesions introduced by TBHP and bleomycin are recognized

predominantly by endonuclease IV (Cunningham et al., 1986).

Studies with iron ion chelators and Sod revealed the possible involvement of iron

ions and superoxide (O;) in the toxicity of TBHP (Nakae et aL 1990; Masaki et ai. 1989;

and Nicotera 1988). O; can d u c e Felm] to F e W (Cadenas 1989; and Dtke 1978).

TBHP in the presence of reduced iron has the ability to generate ter?.-butoxyl radical (te*

bu0) (Nakae et aL 1990; Masaki et al. 1989) in a similar fashion as the formation of OH.,

when H,02 reacts with reduced F e m , which is widely hown as the Fenton reaction

(Imlay et al. 1991). The proposed mechanism for DNA damage by TBHP is a metai ion-

site specifïc lesion ( A h a n et ai. 1994). During oxidative stress by TBHP, metal ions are

released h m their storage sites and these intracellular free ions (Kon and copper) may bind

to DNA The increased level of DNA-bound memi ions make DNA a likely target for site

specific damage by O; and H24. through the generation of OH-.

Nevertheles, during oxidative stress DNA is not the only macromolecule that bears

injury, protein oxidation is also common. Stadman (1986) has shown that oxidation of

amino acid residues in proteins occua predominantly at metal binding sites. So during

oxidative stress proteins bound with metal ions are also a lïkely targefs for site specific

damage by 0; and &O2, through the generation of OH-.

TrxC a regenerative protein during oxidative stress

RPCSûû when transformed with a recombinant plasmid (pBRC1) canying C- j e j d

TGEW)11 genomic DNA displays increased resistance to TBHP. The product of the genes

within the insert of pBRCI is directly responsible for the ability of RPCMO to withstand

oxidative stress generated by TBHP. The generation of unidirectional deletions of the insert

have made it possible to locate the specinc region that is responsible for the phenotype of

the transformed RPCSOO. This region contains an ORF. that codes for a thioredoxin-like

protein and thus is designateci as RIC, denoting thioredoxin of C. jejwii.

Thioredoxin is a small ubiquitous protein with two redox-active cysteine residues

which act as its active Sie (Fernando et al. 1992; Gleason and Holmgren 1988; and

Holmgren 1985). It participates in various redox reactioas via reversible oxidation of its

active-site disulfide bond. The redox-active cysteioe residues are oxidized to fom a

disulfide bridge in the oxidized state but are reduced to a dithiol by thioredoxin ductase

and NADPH. Reduced thioredoxin functions as a hydrogen donor for the reduction of

protein disulfdes So the thioredoxin system is composed of thioredoxin, thioredoxin

reductase and NADPH.

The sequence around the redox-active disulfide bond of thioredoxins (thioredoxin

family active-site) is well conserved. Bactenophage T4 codes for a thioredoxin but its

prirnaiy saucture is not homologous to the bacterial. plant or vertebrate thioredoxins.

Protein disdphide isomerase (PDI). found in eukaryotic ceils contains a domain

that is evolutionarily related to thioredoxin. PD1 is an endoplasmic reticulum enzyme that

catalyzes the tearrangement of disulphide bond in various proteinS.

The thioredoxin domain is also found in some bacterial periplasmic proteins that act

a s thiokdisulphide interchange proteins. In E. coli some of these periplasmic pro teins are

encoded by &bA, dsbc, &bD and drbE.

The thioredoxin system has b e n proposed as a regenerative machinery for proteins

inactivated by oxidative stress (Fernando et al. 1992). It has been shown that E. coIi

thioredoxin added exogenously exerts a regenerative effect on oxidative damages in E. c d

and in epithelial ceih (Spector et aL 1988).

The &duceci amino acid sequence of the C. jejuni TrxC. has dyad-cysteine &dues

at c'~ and Ca' (see Fig. 24). When aügned with other known thioredoxins. a cooserved

motif can be identified between residues 70 through 92 (see Fig. 29), indicating the

importance of this redox-active cysteine site. AU known thioredoxins have an approximate

molecular mass within the range of 10 to 12-kDa (Fernando et al. 1992). C. jejuni TrxC is

encoded by a 600-bp OFlF, predicted to produce a polypeptide of 200 amino acid residues

(see Fig. 24) with a molecular mass of 23-kDa 'Ihe large size protein of TnrC might

suggest another functional role for the proteia The amino acid profile of TrxC and

thioredoxin from various other prokaryotes shows mimmum usage of histidine residue (se-

Table 5). Histidine residues are pmne to oxidation during oxidative stress due to their high

affiity for metal binding. The restricted number of Histidine residues in TrxC suggesu

that during oxidative stress this protein may be mistance to oxidation.

C. jejuni TrxC with the redox-active site domain presumably is able to repair

oxidized proteins that are formed during oxidative stress in RPCSOO under the influence of

TBHP.

The promoter region of C. jejuni trxC

Two CAP-like binding consensus sequences have been identified at the promotei

region of C. jejuni nxC (see Fig 24; and 25). CAP-1 completely overlaps the -35 region

(typical of a class II CAP-dependent promoiers) while CAP-II is located 4 bases upstream

of the rbs.

In the presence of cyclic AMP (c-AMP), CAP binds to CAP-binding consensus

sequences and either upregulates or downregulates (Kawamukai et al. 1985; and Mon and

Aiba 1985) the gene located immediately downstream of this sequence. The cellular

concentration of c-AMP is inversely related to the concentration of glucose. Glucose lowers

the intmcellular concentration of c-AMP by mhibiting adenylate cyclase (Hassan and

Fridovich 1977), tbrough the phosphoenolpynivate-dependent glucose transport system

(Isbmka et al. 1993). Adenylate cyclase is encodexi by cya gene. Exogenous c-AMP

completely elllninates catabolite repression in CRP-overproducing ce&. while it does not

hùly reverse the effect of glucose on ~galactosidase expression in wild-type celis

@hiaika et al. 1993). It is id& then that glucose not only inhibits die expression of

Cya but also CRP (Ishiaika et ai. 1993). On the other hand the traascnption of np is both

negatively and positively autoreguiated by the different concentration of CRP a d o r c-

AMP in E. coli (Hanamura and Aiba 1992).

A recent study has implied that the E. coli nx gene is negatively reguiated by c-

AMP. ?he m gene expression increases when the comtrat ion of c-AMP is low due to

the presence of glucose (Jae-Hoon et al. 1997). E. coli EX has a class 1 CAP-dependent

promoter. Ln the presence of glucose as the carbon source El coü will be actively growing

by deriving energy h m the Embden-Meyerhof pathway, while utiliang the aicarboxylic

cycle mainly for the purpose of biosynthesis. The notion mat c-AMP negatively regulates

trr is in accord with the knowledge that actively growing cells wiU require

deoxyribonucleotides and Trx is known to be a cofactor in the synthesis of

deox yribonucleo tides.

Transcription activation at class II CAP-dependent promoters has been proposed

(Busby and Ebright 1997). A class II CAPdependent promoter is pcesent upstream of the

C. jejmi R*C but a second CAP binding site is &O present downsueam of the class II

CAP-dependent promoter (4-bp upstream of rbs) (see Fig. 24; and 25). it is then proposed

that binding of CAP to these sites in the presenœ of c-AMP might downregulate the

expression of TrxC. It will also be interesthg to elucidate whether oxidative stress has any

effect on the expression of the cya and c p genes directly.

Fur-Box-like consensus sequences have also k e n located on the C. jejuni nxC (see

Fig 24; and 26). unlike the CAP-binding consensus sequences, one of these sites is located

UnmediateIy downstrem of the rbs and two are within the ORF. A FBS (FBS-IV) is aiso

found downstream of M. FBS-IV is a c W y located within ORFl (see Fig. 24). FBS are

f o n d at the promoter region of many iron-regulated genes but the location of FBS within

the ORF is yet to be understood.

Under the influence of hydrogen peroxide. vanous oxidative stress response gene

products are elevated such as OxyS (Alaivia et al. 1997), KatG. Dps .Ger. Grx and Ahp.

These proteins are part of the OxyR regulon (Tolechno et al 1994; and Tao 1997),.

Analysis of 5' flanking region of g e n s coding for those proteins have identified a binding

site for OxyR protein. ï h e reduced and oxidized OxyR negatively regulates its own

expression. The oxidized fom acts as a aanscriptiod activator at the promoter region of

ûxyR regulon genes. The presence of OxyR-like-binding site I and II (see Fig. 27; and 28)

upstream of rr*C suggest that expression of TmC may be under the influence of OxyR,

thus tnrC might be part of the OxyR repuion.

Locating C. jejuni tnC on the physical genomic map and Southern analysis

of the gene in the genus Campyfobaeter

The unique C. jejlmi rr*C has been located in the San-A. SmI-A and SacII-A

fragments on the C. jejwu' TGZW) I 1 physical genomic map (see Fig. 3 1). A homologue

of thioredoxin reductase (mB) is present in C. jejuni TGH9û11. It is located upsueam of

the proA gene of C. jejuni In the physical genomic map of C. jejuni TGH90 1 1. proA is

located on the SUD€. SacII-E and SmaI-C fragments (see Fig. 31). This location seems to

be directly opposite to the tnrC of C. jejuni Thioredoxin reductase is part of the

thioredoxin system dong with NADPH and thioredoxin.

The organization of the nx and axB in most bacwia are physicaliy separated, with

the exœption of Mycobacterium leprae (Wieles et al. 1997). Streptomyces c h l i g e m s

(Cohen et al. 1993). Huernophilu inpuenuie and Heücobacterpylon'. Ln 'M. leprue the ax

and trxB are expressed as a single hybrid protein where TrxB at its N-terminus and Trx at

its C-terminus. This protein has been proposed to play a key role in one of the mechanisms

by which lepue resist oxidative stress within the human mononuclear phagocytes

(Wiiles et al. 1997). Whiie in Sr. chdigenrs although the Trx system is organized in a

clustal formation. it is separaied by 33 nts with the rrxB u p s m of tm.

There seem to be more than one copy of the thioredoxin gene present in H.

inpiremae and H. pybri. In H. infuenzae. there is one region on the genome where

RxA(hi1159) and Rxg(hi1158) are present in tandem. Away fkom this region at two

different sites. there are genes that show homology to a thioredoxin gene, they were

designated -(hi1 115) and nxM(hiûû84) of H. uipuenz~e-

Unlike If. intuenme. H. pybn has two copies of both ou\ and mB. The

organktion is such that at one site the RxA(hp0824) and RxB(hp0825) are in tandem and

the homolog of these genes are present physically separateci elsewhere on the H. pylori

genome. designated t r a p 1458) and d ( h p 1 164). The alphanumeric value within the

parentheses (gene identification number) is refening to the location of the respective genes

on their respective genomes. These musuai organizations suggest the importance of this

thioredoxin family of gene in various redox-potential reactions in the celL

A Southem hybndization has indicated that the unique nzC of C. jejwu' is a single

copy gene and a homologous gene is present in C. coli. C. (mi and C. upsaliensis but

surprisingly not in C. sputonun (se. Fig. 33). The C. sputonun homolog if presenb only

has a weak homology with the C. jejuni t d . The C. hi homolog seems to be more

closely related to the C. jejuni tnC compared to the tested four type strains.

DNA sequence of the pBSPC1 insert

Five complete OMS and two partial ORFs have been identified on the pBRCl

insert (se Fig. 19). ORFl translated product shows homology to an uncharacterized

region at the 19.9 minutes locus of the E coli genome. Downstrearn of this O W is ORE!

predicted D produce a protein with high homology to the C-temiinal of ClpA (Gottesman et

al. 1990) of E. coli. The product of c lpk is a subunit of an ATP-dependent protease. In E.

coii this protein and ClpP (Maurizi et aL 1990) maks an active ATP-dependent protease Ti

(Clp). It has been proposed that this pro- is involved in ATPaependent degradation of

abnormal proteins (Goldberg 1992).

ORF3 ( t d ) translated product shows moderaie homology to a thioredoxin protein

of D. rnelanogaster. ORF4 is located upsûeam of OEW. ORF4 is encoded by 450-bp of

DNA and shows high homology to smpA. a locus located at 59.1 minutes on the E. cok'

genome. Upstream of ORF4 is ORF5 which codes for a protein of 236 aa. that shows

good homology to a hypothetical protein in E. coli. This hypothetical protein gene is

situated at 27.1 minutes on the E coli genome. ORF6 is located upsûam of ORF5. ORF6

codes for a protein of 315 aa that has high homology to tRNA pseudouridine 55 (W55)

synthase. This enzyme is encoded by the m B locus located at 7 1.3 minutes on the E. coü

genome. The product of mrB is specific for the conversion of uridine (U) 55 to ~ 5 5 in the

mSUwG loop in most tEWAs (Nurse 1995). Fmally upstream of ORF6 is ORFI which

codes for a protein with high homology to the N-terminai of m D of E. coli, situateci at

86.1 minutes on its genome. UvrD is single-stranded DNA-dependent ATPase. It unwinds

double stranded DNA with a 3' to 5' polarity with respect to the strand of DNA on which

the enzyme initially binds (George et al. 1994).

The seven ORF in the pBSPCl insert are organized in a highly compact manner

(see Fig. 19). Intergenic space is either eradicated or miaimkd similar to thefir-lysS-glyA

(Chan et al. 1988) region and the argH locus (Hani and Chan 1994). C. jejwu' can grow in

chemically defmed medium and thus have most of the biosynthetic pathways that are found

in E. coli. Therefore genes and operons in C. jejuni genome of 1.8 Mbp needs to be

compactly organized cornpared to the E. coli genome of 4.7 Mbp.

Gene-arrangement conservation

This study uncovers a gene-arrangement thaî is coaserved in C. jejuni, E. coü and

H. pyiori. ORFl codes for a polypeptide that is highly homologous to that of ORF106 at

19.9 minutes of the E coli genome and a hypothetical protein coùed by HP0032 of the H.

pylori genome. These three homologous genes, ORFl of C. jejwi, ORF106 of E. coli and

HP0032 of H. pylori are a i l in tandem with the clpA gene (see Fig. 32). cl'A of E. coi5

encodes for ClpA ( G o m a n et al. 1990) which is a subunit of a ATP-dependent protase

ri that has been implicated in the protein degradation pathway (Goldberg 1992). The

significance of this conserved gene-anangement will only be appreciated when the function

of ORFl of jejuni. ORF106 of E. c d and HP0032 of H. pylori are defmed. The only

other conserved gene arrangement reported for these three bacteria is the 23s rRNA-SS

rRNA cluster (Tomb et al. 1997; and Berlyn et al 1996).

This stuciy shows that the E. coli nfo mutant (rcPcs00) can be of general use to

clone genes of C. jejwi and other bacteria that are involved in the oxidative stress

response. The rrxC gene clones will greatly facilitate future studies on the role of oxidative

genes of C. jejwu' in infections of various animal models.

1 Gene Arrangement Conservation 1

- ORFl cZpA (ORF2)

1 C. jejuni 1

Fig. 34: Gene mangement conservation of ORFl and ORF2 in C. jejuni as compared to E. coli and H. pylori.

FUTURE STUDIES

C jejuni, king a microaerophüic orgaaism, is susceptible to ROS. therefore

presence of genes that plausibly rentier resistance to oxidative damage is essential It has

ken shown that the nx gene of E. coü is not a requisite for viabiiity. Generating an

isogenic mutant of C. jejuni TGH9ûI 1 lacking the functional mC by kanamycin cassette

insertion into the RXC ORF would eIucidate the phy901ogical role of this protein. To seek

further knowledge of the in vivo function of the nAC product, oligonucleotide site-directed

mutagenesis study of the dyad cysteine active-site should be undeaaken.

The pTeSence of a class II CAP-like binding sequence at the -35 region should be

investigated by reporter gene assay. Construction of a R*C-lac translational fusion gene in

which the ~galactosidase expression is directed by the mmcriptional signals of the RxC

gene, should reveai the regulatory fiinction of this putative CAP binding site.

Iron bas ken linked with oxidative s t n s through the Fenton reaction and FBS has

been identified within the nxC gene of C. jejwù. The in vivo role of Fur in regulating this

gene is also an area that should be elucidated. Fhtly Fur-binding assay should be

undertaken and followed by a reporter gene assay.

The TrxC have been identifiecl in other campylobacters, indicating that it might be a

house keeping gene. It has also been linked to oxidative stress in this study. so it should be

intemting to seek its physiological d e üz vivo.

Aiba, H., S. Adhya, and B. de Crombrngghe. 1981. Evidence for two

fiuictional gal promoters in intact EÎcherichia coli cek. J. BioL Chem. 256: 1 1905-

l l 9 l O .

Altman, S. A., T. H. Zastawny, L. Randers, Z. Lin, J. A. Lumpkin,

J. Remacle, M. Dizdaroglu, and G. Rao. 1994. tefi-butyl hydmperoxide-

rnediated DNA base damage in cultureci cells. Mutat. Res. 306:35-44.

Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J .

Lipman. 1990. Basic local alignment search tool. J. Mol- Biol. 215:403-4 10

AltuMa, S., D. Weinstein-Fischer, A. Zhang, Lm Postow, and G. A .

Ston . 1997. Small. stable RNA induœd by oxidative stress: d e as a pleiotmpic

replator and antixnutator. CeL 90(1):43-53.

Altuvia, S., M. Almiron, G. Huisman, R KoIter, and G. Storz.

1994. The clpr promoter is activated by OxyR during growth and by IHF and sigma

S in stationary phase. Mol. Micro. 13(2):265-72.

Amabile-Cuevas, C. F., and B. Demple. 1991. MoIecular characterization

of the SoxRS g e n s of Eschenchia coli: two genes control superoxide stress

regulon. Nucleic Acids Res. 19(6): 4479-4484.

Ananthaswamy, H. N., and A. Eisenstark. 1977. Repair of hydrogen

peroxide-induœd single-strand breaks in Escherichia coü deoxyribonucleic acid. J .

Bacteriol. 130(1): 187-9 1.

Applied Biosystems, Inc. 1991. Highquality templaie DNA for Taq cycle

sequencing using DyeDeoxyTM Teminaton: An improved preparation procedure.

Bagg, A., and J. B. Neilands. 1987. Molecular mechanisrn of regdation of

siderphore-mediated iron assimilation. Microbiol. Rev. Sl:5W-5 18.

Berlyn, M. K. B., K. B. Low, and K. E. Rudd. 1996. Biosynthesis of

cysteine in &chenchia wlî and SalmoneUa ryphmurium. In ficheri& coü and

SalmoneUa iyphimurium: Cellular and molecular biology . (Neidhard~ F. C.. chief

ed.) Vol 2:pp1715-1902. ASM Press. Washington, D. C.

Blaser, M. J. 1986. Baclerial gastrointestinal infections. Gastetoenterol. Ana

3:3 17-340.

Bolker, M., and R Kabmann. 1989. The Escherich coli regdatory protein

OxyR discriminates between methylated and unmethylated States of the phage Mu

mom promoter. EMB O Journal. 8(8):2403- 10.

Busby, S., and R H. Ebright. 1997. Transcription activation at Class II

CAP-dependent promoters. Mol. Mimbiol. 23(5):853-859.

Cadenas, E. 1989. Biochemistry of oxygen toxicity. Annu. Rev. Biochem.

S8:79- 1 10,

Chan, E., and B. Weiss. 1987. Endonuclease N of Ercherichuz coli is

induced by paraquat. Pmc. Nad. Acad. Sci. USA. 84:3189-3193.

Chan, V. L. 1996. Unpubished data.

Chan, V. L., Hm Louie, and H. Lm Bingham. 1995. Cloning and

transaiption regulation of the femc uptake regdatory gene of Ccuttpylo~ter

jejuni. TGH9û 1 1. Gene. l'14:695-70 1.

Chan, V. L., and 8. L. Bingham. 1992. Lysyl-tRNA synthetase gene of

Cumpylobacter jejwri. J. Bacteriol. 16425-3 1.

Chan, V. L., H. Bingham, A. Kibue, P. R V. Nayudu, and J. L.

Penoer. 1988. Cloning and expression of the Campylobacter jejwu' glyA gene in

Escherichia coli, Gene. 73: 185- 19 1.

Christman, M. F., G. Ston, and B. N. Ames. 1989. OxyR, a positive

reguiator of hydrogen peroxide-inducible genes in Eschenchtcr colï and Salmonefh

ryphimriwn, is homologous to a family of bacterial regulatory proteins. Proc.

Natl. Acad. Sci. U S A 86(10):34û4-8.

Christman, M. F., RWa Morgan, Fa S. Jacobson and B. N. Ames.

1985. Positive control of a regdon for defences agaiast oxidative stress and some

heat shock proteins in SaZmneiia typhimrium Cell. 41:753-762.

Clark, D. J. 1968. Reguiation of deoxyribonucleic acid replication and cell

division in Escherichia coli B Ir. J. Bacteriol. 96: 12 1 4 1224.

Cohen, G., M. Yanlro, M. Mislovati, A. Argaman, R Schreiber, Y .

Av-Gay, and Y. Aharonowitz. 1993. Thioredoxin-Thioredoxin miuctase

system of Streptomyces clmligerus: sequenœ, expression, and organization of the

genes. J. Bacteriol. 175(16):5 159-5 167.

Compan, I., and D. Touati. 1993. Interaction of six global aanscription

regulators in expression of manganese superoxide dismutase in Eschenchia coli K-

12. J, Bacteriol. 175(6): 1687-96.

Cunningham, R P., S. M. Saporito, S. G. Spitzer, and B. Weiss.

1986. Endonuclease N (nfo) mutant of Escherichia coli. J. Bacteriol. 168: 1 120-

1127.

Cunningham, R P., and B. Weiss. 1985. Endonuclease III (nth ) mutant of

Escherichia cofi. Proc. Nati. Acad. Sci. USA. 82:474-478.

Demple, B., and S. Linn. 1982. 5.6-Saturated thymine lesions in DNA:

production by UV light and hydrogen peroxide. Nucleic Acids. Res. 10:378 1 -

3789.

Duke, J. B. 1978. The biology of oxygen radical. Science. 2û1:875-880.

Eisenstark, A. 1989. Bacteriai genes involved in response to near-ultraviolet

radiation. Adv. Genet. 26:99- 147.

Estabrwk, R., and J.A. Peterson. 1990. CytochmeP-450 and oxidative

stress. Free Radic. Biol. M e d 9(sappl 1): 161.

Farr, S. B., and T. Kogoma. 1991. ûxidative stress response in Escherichia

coli and Salmonella typhimurïum. Microbiol. Rev. 55(4):56 1-585.

Farr, S. B., R D'Ad, and D. Touati. 1986. Oxygen-dependent

mutagenesis in fidienchia coü laclong superoxide dismutase. Proc. Natl. Acad.

Sci. USA 83:8268-8272.

Farrant, J. Lm, A. Sansone, J. R Canvin, M. J. Pallen, P. R .

Langford, T. S. Wallis, G. Dougan, and J. S. Kroll. 1997. Bacterial

copper- and zinccofactored superoxide dismutase contributes to the pathogenesis

of systemic salmonellosis. Mol. Microbiol. 25(4):785-796.

Fawcett, W. P., and R. E. Wolf Jr. 1994 hirification of a MalE-SoxS

fusion protein and identification of the connol sites of Escherichia coli superoxide-

inducible genes. Mol. Microbiol. 14(4):669-679.

Fernando, M. R., H. Nanri, S. Yoshitake, K. Nagata-Kuno, and S .

Minakami. 1992. Thioredoxin regenerates proteins inactivaied by oxidative stress

in endotheliai celis. Eur. J. Biochern. 209(3):917-922.

Fridovich, 1. 1989. Superoxide dismutase. J. Biol. Chem. 264:776 1-7764.

George, J. W., R M. Brosh, Jr., and S. W. Matson. 1994. A dominant

negative a l l e of the Ercherichh coli uvrD gene encoding DNA Heiicase II. A

biochernicai and genetic characaization. J. Mol. BioL 235424-435.

Gleason, F. K., and A. Holmgren. 1988. Thioredoxin and related proteins

in prokaryotes. FEMS Microbiol. Rev. 54:27 1-298.

Goldberg, A. L. 1992. The mechaniSm and hinctions of ATP-dependent

proteases in bacterial and animal celis. Eur. J. Biochem. 2û3(1-2):9-23.

Gottesman, S., C. Sqaires, E. Pickersky, M. Carrington, M. Hobbs,

J. S. Mattick, B. Dalrymple, H. Kuramitsu, T. Shiroza, T. Foster,

W. P. Clark, B. Ross, and M. R Maurizi. 1990. Conservation of the

regulatory subunit for the Clp ATP-dependent protease in prokaryotes and

eukaryotes. Proc. W. Acad. Sci. USA 87:3513-35 17.

Grant, K. A., and S. F. Park. 1995. Molecular characterization of katA from

CMiPylobacrer jejmi and generation of a catahedefiecient mutant of

C~mpybbacter jejmi by interspecinc dieiic exchange. Microbiol. 141: 1369- 1376.

Grossberger, D. 1987. Minipreps of DNA nom bacteriophage iambdê Nucleic

Acids Res. 15(16):6737.

Gruenwald, R, A. H. Ropper, H. Lior, J. Chan, R Lee, and V. S .

Molinaro. 1991. Serological evidenœ of ~ b b u c t e r je jdcoi i enteritis in

patients with Guillain-Barre syndrome. Arch. Neurol. 48: 1080-1082.

Greenberg, J. T., P. Monach, J. EI. Chou, P. D. Josephy and B .

Demple. 1990. Positive control of a global antioxidant defense activated by

superoxide- generating agents in Escheridria coli. Proc. Nad. Acad. Sci. 87:6 18 1 - 6185.

Greenberg, J. T., and B. Dernple. 1989. A global response induced in

Escherichin coli by redox-cycling agents in oxyR mutants. J. Bacteriol. 171:3933-

3939.

Hagensee, M.E., S. K. Bryan, and R. E. Moses. 1987. DNA polyrnerase

ïII requirement for repair of DNA damage caused by methyi rnethanesuifonate and

hy drogen peroxide. J. Bacteriol. l69(10):4608- 13.

Halliwell, B., and 0. 1. Aruoma. 1991. DNA damage by oxygen-denved

species. Its mechanism and measment in mammalian system. Fed. European

Biochemical Soc. 281(1-2): 9- 19.

Hanamura, A., and H. Aiba. 1992. A new aspect of tmsaiptional control of

the ficherichia CO& cxp gene: positive autoreguiation. Mol. Mimbiol. 6(17):2489-

97.

Hani, E. K., and V. L. Chan. 1994. Cloning, characterization and nucIeotide

sequence of nnalysis of the mgH gene from Campylobacrer jejwu' TGH9011

encoding argininosuccinate lyase. J. Bacteriol. 176: 1865- 187 1.

Eantke, K. 1988. Cbaracterization of an iron sensitive mudl mutant in E. coli

lacking the ribonucleotide reductase subunit B2. Arch. MicrobioL 149:344-349.

Hassan, H. M., and Fddovich, 1. 1977. Regulation of Superoxide

Dismutase Synthesis in EFchen'chia coi2 Glucose e f f e c ~ J. BacterioL 132(2):505-

5 10.

Eeimberger, A., and A. Eisenstark. 1988. Cornpartmentakation of

catalases in Estherichia d i . Biochem. Biophys. Res. Commun. 154:392-397.

Renikoff, S. 1984. Unidirectional digestion with exonuclease m -tes targeted

breakpoint for DNA sequencing. Gene. 28:35 1-359.

Holmgren, A. 1989. Thioredoxin and glutaredoxin systems. J. Biol. Chem.

264(24): 13963- 13966.

Holrngren, A. 1985. Thioredoxin. Annu. Rev. Biochem. 54:237-27 1.

mggins, D. G., A. J. Bleasby, and R Fuchs. 1992. Clustal V: Irnproved

software for multiple sequence alignment Comput AppL Biosci. 8: 18% 19 1.

Hirota, K., M. Matsui, S. Iwata, K. Mori, and J. Yodoi. 1997. AP-1

transcriptional activity is regulated by a direct association benveen thiredoxin and

Ref- 1. Proc. Natl. Acad. Sci. USA. 94:3633-3638.

Imlay, J. A., S. H. Chin, and S. Linn. 199 1. Toxic DNA damage by

hydrogen peroxide through the fenton reaction in vivo and Vi vitro. Science

240: 640-642,

M a y , J. A., and S. Linn. 1987. Mutagenesis and stress responses induced in

Escherichia coli by hydrogen peroxide. J. BacterioL 169(7):2%7-76.

International Union of Biochemistry. 1986. Nomenclature for incompletely

specifïed bases in nucleic acid sequence. J. Biol. Chem. 261:13-17

Ishizuka, H., A. Hanamura, T. Kunimura, and H. Aiba. 1993. A

lowered concentration ofcAMP receptor protein caused by glucose is an important

detemiinant for catabolite repression in ES- . . coli, Mol. Microbiol-

10(2):341-350.

Iuchi, S., D. C. Cameron, a d E. C. C. Lin. 1989. A second global

reguiaior gene (arcB) rnedizting repression of enzymes in aerobic pathways of

Escherichia coli. J. Bacteriol. 171:868-873.

Izumi, T., K. Ishizaki, M. kenaga, and S. Yonei. 1992. A mutant

Endonuclease TV of fichenchia coli loses the abiiity to repair lethal DNA damage

induced by hydrogen peroxide but not that induced by methyl methanesulfonate. J .

Bacteriol. 174:77 11-77 16.

Jacobsen, F. S., R. W. Morgan, M. F. Christman, and B. N. Ames.

1989. An alkyhydroperoxide reductase from SalmoneIla typhimurizun involved in

the defence of DNA damage by oxidative damage. J. Biol. Chem. 264: 1488- 1496.

Jae-Hoon, S., M. A. Nampng, L. Chang-Jin and J. A. Fuchs. 1997.

Expression of the Escherichia coli thioredoxin gene is negatively regulated by cyclic

AMP. Biochem. Biophy. Res. Comm. 233564-567.

Johnson, K., M. Ostensen, A. C. S. Melby, and K. Melby. 1983.

HLAB27-negative arthritis relaced to C~tpylobacter jejuni enteritis in three

children and two addts. Acta. Med. Scand. 214:165-168.

Jones, D. M., E. M. Sutciiffe, R Rios, A. J. Fox and A. Curry.

1993. Camp):lobacter jej* adapts to aerobic metabohm in the environment I . Med. Micribiol. 38: 145- 150.

Kawamukai, M, J. Kishimoto, R Utsud, M. Himeno, T. Kornano,

and H Aiba. 1985. Negative regdation of adenylare cyclase gene (cya)

expression by cyclic AMP-cyclic AMP receptor protein in fichenchia coli: studies

with cyu-lac protein and operon fusion plasmids. J. Bacterio1. 164(2):872-877

Kim, N. W., and V. Lm Chan. 1992. Genoinïc characterimion of

Cùmpylobacrer jejwU by field inversion gel elecmphoresis. Curr. Mimbiol.

22: 123-127.

Kogoma, T., S. B. F m , K.M. Joyce and D.O. Natvig. 1988. Isolation

of gene hision ( soi::kZ) inducible by oxidative stress in Escherichia coü Roc.

Nad. Acad. Sci. 85:4799-4803.

Kredich, N. M. 1996. Biosynthesis of cysteine in &chenchia coli and

Salmonella ryphimurium. In Escherichia coli and Salnonelïa ryphimurium: CeUular

and molecular biology. (Neidhardt, P. C., chief ed.) Vol 1:ppS 14-528. ASM

Press. Washington. D. C.

Kren, B., D. Parsell, J. A. Fuchs. 1988. Isolation and characterization of an

Escherichia coli K- 12 mutant defiecient in giutaredolan. J. Bacteriol. l70:308-3 15.

Leung, F. Y-K., G. O. Littlejohn, and Cm Bombardier. 1980. Reiter's

syndrome after Campylobacter jejuni entetitis Arth. Rheum. 2394-950.

Loewen, P. C. 1979. Levels of glutathione in Eschen'chia coli. C a J .

Biochem. 154:787-792.

Ljungquist, S., T. Lindhal, and P. Howard-Flanders. 1976. MethyI

rnethanesulfonate-sensitive mutant of Escherichia coli deficient in an endonuclease

specficfor purinic sites in deoxyribonucleic acid. J. Bacteriol. 126646-653.

Li, Z., and B. Demple. 1994. SoxS, an activator of superoxide stress genes in

Eschenchia colt'. -cation and interaction with DNA. J. Biol. Chem.

269(28): 1837 1-1 8377.

77. Maslow, Je N., A. M. Slotsky, and R Dm Arbeit. 1993. Application of

pulsed-field gel electrophoresis to moleculai epidemiology. In: Persing D. H., T.

F. Smith, F. C. Tenover. and T. J. White (ed). Diagnostic Mo1ecuia.r

Mimbiology: principles and Applications. American Society for Microbiology,

Washington D. C. pp563-572.

78. Masaki, N., M. E. Kyle, and J. L. Farber. 1989. $en.-butyl

hydroperoxide kills hepatocytes by peroxinin'ng membrane lipids. Arch Biochem.

Biopyhs. 269:390-399.

79. MauriP, M. R, W. P. Clark, Y. Katayama, S. Rudikoff, J .

Dumphry, B. Bowers, and S. Gottesman. 1990. Sequence and structure

of ClpP, the proteolytic component of the, Amdependent Clp protease of

Escherichia coli. J. Biol. Chem. 265: 12536- 12545.

80. Meister, A., and M. E. Anderson. 1983. Glutathione. Annu. Rev.

Biochem. 52:7 1 1-760.

81. Mishu, B., A. A. Ilyas, C. Lm Koski, F. Vriesendorp, S. D. Cook,

F. A. Mithen, and M. J. Blaser. 1993. Serological evideace of previous

Campylohter jejwu' infection in patients with the Guillain-Barre syndrome. Ann.

Intern. Med. 118:947-958.

82. Morgan R W., M. F. Christman, F. S. Jacobsen, G. Ston and B.

N. Ames. 1986. Hydrogen peroxide-inducible protein in Salmonella typhimurium

overlap with kat shock and other stress proteins. Proc. Natl. Acad. Sci. 83:8059-

8063.

83. Mori K., and He Aiba. 1985. Evidence for negative control of cya mmsaiption

by CAMP and CAMP receptor protein in intact Erdierichia coli ceIls. J. Biol. Chem.

260(27): 14838- 14843,

Nakae, D. He, H. Yoshiji, T. Amaooma, T. Kinugasa, J. L. Farber,

and Y. Konishi. 1990. Endocytosis-independent uptake of iiposome

encapsuiated suproXide dismutase prevents the killing of cultured hepatocytes by

tem- butyl h ydroperoxide. Arch. Biochem. Biopyhs. 279:3 15-3 1 9.

Neiderhoffer, E. C., C. M. Naranjo, K. L. Bradley, and J. A. Fee.

1990. Conml of EFdierichia coü superoxide dismutase (sodA and sods) genes by

the femc uptake regulation mr) locus. J. Bacteriol. 172: 1930- 1938.

Nicotera, P., D. McConkey, S. -A. Svensson, G. Bellomo, and S .

Orrenius. 1988. Correlation between cytosolic ca2+ concentration and cytotoxicity

in hepatocytes exposed to oxidative stress. Toxicology. 5235563.

Nurse, K., J. Wrzesinski, A. Bakin, B. G. lane, and JO Ofengand.

1995. Purification, cloning, and properties of the tRNA psi 55 synthase fiom

Eschekhia coli. RNA. l(1): 102-1 12.

Rhodes, K. M., and A. E. Tattersfield. 1982. Guillain-Barre syndrome

associated with Campylobacter infection. Br. Med. J. 285:173-174.

Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular Cloning.

A Laboratory Manual. 2nd ed. Cold Spring Harbour Laboratory Press. Cold

Spring Harbour, W.

Sanger, F., S. Nicklen, and A. Coulson. 1977. DNA sequencing with

chah-terminating inhibitors. Proc. Nad. Acad. Sci. USA. 74:5463-5467.

Saporito, S. M., R. P. Cunningham. 1988. Nucleotide sequence of the nfo

gene of ficherichia coli. K12. J . Bacteriol. 170: 5 141-5 145.

Saran, M., and W. Bors. 1990. Radical reactions in vivo-an overview. Radiat.

Environ. Biophys. 29:249-262.

Singh, A., and 8. Singh. 1982. Time scale and naaire of radiation biological

damage. Prog. Biophys. Mol. Biol. 39:69-107.

Smith, Cm Lm, Je G. Econome, A. Schutt, S. Kko, and C. R Cantor.

1987. A physical map of the E. coli K12 genome. Science. 236: l448- 1453.

Spector, A, G. -2. Yan, R. -R C. Huang, M. J. McDermott, P. R.

C. Gascogne, and V. Pigiet. 1988. The effect of &O2 upon thioredoxin-

enriched lens epithelid ce&. J. Biol. Chem. 2634984-4990.

Ston, G., Lm A. Tartagüa and B. N. Ames. 1990. Transcriptional

regulator of oxidative stress-inducible genes: direct activation of oxidation. Science.

248: 189- 194.

Ston, Ge, M. Christman, 8. Sies and B.N. Ames. 1987. Spontaneous

mutagenesis and oxidative damage to DNA in Salmonelia yphimurium. Froc. Nati.

Acad. Sci. 84:88 17-8921,

Stadman, E. R. 1986. Oxidation of proteins by mued-function oxidation

systems: implication in protein turnover, ageing and neutrophil function. Trends

Biochem. Sci, 11: 1 1 - 12.

Summerfeild, Fm W., and A. L. Tappel. 1983. Eletennination by

fluorescence quenching of the environment of DNA crosslinks made by

malondialdehyde. Biochim. Biophys. Acta. 740: 1 85- 189.

Takeda, Y., and 8. Avila. 1986. Structure and gene expression of the

Escheri& coli Mn-superoxide dismutase gene. Nucleic Acids Res. 14:4577-

4589.

Tao, K. 1997. oxyR-Dependent induction of Escherichia coli grx gene expression

by peroxide stress. J. Bactenol. l79(18):5967-5970.

Tartaglia, L. A., Ce J. Gimeno, Cm Ston and B. N. Ames. 1992.

Multidegenerate DNA recognition by the OxyR transcriptional regulator. J. Bi01

Tartaglia L. A., G. Ston and B. N. Ames. 1989. Identification and

molecular analysis of OxyR-reguiated promoters important for bactexhl adaptation

to oxidative stress. J, Mol. BioL 210:709-7 19,

Toledano, M. B., 1. Kulük, Fe Trinb, P. T. Baird, Te D. Schneider,

and G. Storz. 1994. Redox-dependent shift of OnyR-DNA contaas dong an

extendeci DNA-binding site: a mechanism for differentiai promoter selectioa CeU.

78(5) :897-9O9.

Tomb, J-F., O. White, A. R Kerlavage, et al. 1997. The complete

genome sequence of the gastnc pathogen Helicobacrer pylori. Nature. 388539-

547.

Touati, D. 1988. Transcriptional and postmmaiptional ieguIation of rnanganase

superoxide dismutase biosynthesis in fichenchia coü. studied with operon and

protein fusion. J. Bacteriol. l7O:Z 1 1-2520.

Tsaneva, I.R., and B. Weiss. 1990. SOXR~ a locus governing a superoxide

response regulon in Escherichia coii. K12. J. Bacteriol WZ:4 197-4205.

Vaca, C. E., J. Wilhelm, and M. Harms-Ringdahl. 1988. Interaction of

lipid peroxidation products with DNA: a review. Mutat. Res. 195: 137- 149.

VanBogelen, R A., P. M. Kelley and F. Ce Neidhardt. 1987.

Differential induction of heat shock, SOS and oxidative stress regulons and

accumulation of nucleotides in Escherichia d i . J. Bac terioL 169:26-32.

Vercellone, P. A., R M. Srnibert, and N. R Krieg. 1990. Catalase

activity in Ccunpybbacter jejuni: mmparison of a wild-type strain with aerotolerant

variant. Can. J, Microbiol, 36:#9-45 1.

Wai, S. N., K. Nakayama, K. Umene, T. Moriya, and K. Amako.

1996. Consvuchon of a fem~-defiecient mutant of Campybbacter jejwti:

contribution of femtin to iron storage and protection against oxidative stress. Mol.

Microbiol. 20(6): 1 127- 1 134.

112. Wai, S. N., T. Takata, A. Takade, N. Hamasaki, and K. Amako.

1995. Pinincation and characterization of ferritin f h n Campybbaccter jejwii. Arch.

Microbiol. 164: 1-6.

113. Walkup, L. K. B. and T. Kogoma. 1989. Escherichia coli proteins inducible

by oxidative stress mediated by the superoxide radicai. J. BacterioL 171:1476-

1484.

114. Wieles, B., T. He M. Ottenhoff, T. Mo Steenwijk, K. L. M. C.

Franken, R. R P. de Vries, and J. A. M. Langermans. 1997. Increased

i n m u l a r survival of Mycobuctenm srnegmdF containhg the Mycobacteriwn

leprae Thioredoxin-'Ihioredoxin reductase gene. Infect Immun. 65(7):2537-2% 1.

115. Weiss, B. 1976. Endonuclease II of Escherichia coli is exonuclease III. J. Biol.

Chem. 251: 1896-1901.

116. Wu, J., and B. Weiss. 1991. Two divergentiytninscnbed genessoxR and

soxS. control a superoxide response regulon in Escherichia coü. J. Bacteriol.

173:3488-349 1.

117. Yajko, D. M., and B. Weiss. 1975. Mutations simultaneousiy a f f e c ~ g

endonuclease II and exonuclease III in Escherichia coli. Proc. Natl. Acad. Sci.

USA. 72:688-692.

l MAGE EVALUATION TEST TARGET (QA-3)