NssR, a member of the Crp-Fnr superfamily from Campylobacter jejuni, regulates a nitrosative...

Molecular Microbiology (2005)

57

(3) 735ndash750 doi101111j1365-2958200504723x

copy 2005 Blackwell Publishing Ltd

Blackwell Science LtdOxford UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd 2005

2005

57

3735750

Original Article

NssR a nitrosative stress-responding regulator in C jejuniK T Elvers

et al

Accepted 2 May 2005 For correspondence E-mail sparksurreyacuk Tel (

+

44) 1483 689 024 Fax (

+

44) 1483 300 374

dagger

These authors contributed equally to this work

NssR a member of the Crp-Fnr superfamily from

Campylobacter jejuni

regulates a nitrosative stress-responsive regulon that includes both a single-domain and a truncated haemoglobin

Karen T Elvers

1dagger

Sue M Turner

2dagger

Laura M Wainwright

3

Gemma Marsden

4

Jason Hinds

4

Jeffrey A Cole

2

Robert K Poole

3

Charles W Penn

2

and Simon F Park

1

1

School of Biomedical and Molecular Sciences University of Surrey Guildford GU2 7XH UK

2

School of Biosciences University of Birmingham Birmingham B15 2TT UK

3

Department of Molecular Biology and Biotechnology The University of Sheffield Sheffield S10 2TN UK

4

Bacterial Microarray Group Department of Cellular and Molecular Medicine St Georgersquos Hospital Medical School Cranmer Terrace London SW17 0RE UK

Summary

Consistent with its role as a nitric oxide (NO)-detoxi-fying globin in


Cgb (

Campylo-bacter

globin) expression is strongly and specificallyinduced following exposure to nitrosative stress sug-gesting a previously unrecognized capacity for NO-related stress sensing in this food-borne pathogen Inthis study Fur and PerR have been eliminated asmajor regulators of

cgb

and NssR (Cj0466) a memberof the Crp-Fnr superfamily has been identified as themajor positive regulatory factor that controls nitrosa-tive stress-responsive expression of this geneAccordingly disruption of

nssR

resulted in the aboli-tion of inducible

cgb

expression which was restoredby a complementing chromosomal insertion of thewild-type gene with its indigenous promoter at a sec-ond location The NssR-deficient mutant was moresensitive to NO-related stress than a

cgb

mutant andthis phenotype most likely arises from the failure ofthese cells to induce other NO-responsive compo-nents in addition to Cgb Indeed analysis of globalgene expression by microarray and confirmatoryreal-time polymerase chain reaction (PCR) in the wildtype and

nssR

mutant not only confirmed the depen-

dence of inducible

cgb

expression on NssR but alsorevealed for the first time a novel NssR-dependentnitrosative stress-responsive regulon This regulon ofat least four genes includes Cj0465c a truncatedglobin Consistent with NssR being a Crp-Fnr super-family member an Fnr-like binding sequence (TTAAC-N

4

-GTTAA) was found upstream of each gene atlocations

----

405 to

----

425 relative to the centre of thebinding sites and the transcription start point Site-directed mutagenesis confirmed that this

cis

-actingmotif mediates the nitrosative stress-inducibleexpression of

cgb

Introduction

At elevated levels nitric oxide (NO) reacts readily withnumerous cellular targets predominantly thiols and metalcentres (Poole and Hughes 2000) As these targetsinclude haem centres and Fe-S clusters NO is a potentinhibitor of for example terminal oxidases (Stevanin

et al

2000) aconitases (Gardner

et al

1997) and certaintranscription regulators (Cruz-Ramos

et al

2002)Accordingly NO and its congeners can exert strong bac-tericidal activity NO can also react with superoxide at anear diffusion limited rate to generate the highly damagingoxidizing agent peroxynitrite (Pfeiffer

et al

1997) Bacte-ria encounter NO from a variety of sources sometimes asa consequence of denitrification during which it is gener-ated as an intermediate in the reduction pathway fromnitrate to dinitrogen (Kwiatkowski and Shapleigh 1996Watmough

et al

1999) and also because it is a promi-nent agent in macrophage killing (Nozaki

et al

1997Stevanin

et al

2002) In both macrophages and entero-cytes NO is generated through the action of inducible NOsynthases (iNOS) (Salzman

et al

1998 Witthoft

et al

1998 Weinberg 1999 Alderton

et al

2001) NO is bothlipophilic and freely diffusible so production from thesecells can result in the death or inhibition of both intracel-lular and extracellular pathogenic bacteria (Webb

et al

2001)

Bacteria have evolved a number of activities that detox-ify NO and its redox products (reviewed in Poole 2005)and are accordingly able to modulate gene expression in

736

K T Elvers

et al


Molecular Microbiology

57

735ndash750

response to NO and reactive nitrogen species The NorRprotein of

Escherichia coli

which belongs to the family oftwo-component response regulators and which regulatesthe expression of flavorubredoxin in response to nitrosa-tive stress (Hutchings

et al

2002 Gardner

et al

2003)appears to be a dedicated sensor of reactive nitrogenspecies (Mukhopadhyay

et al

2004) NnrR and Nnrmembers of the Crp-Fnr superfamily have evolved prima-rily to regulate the NO-responsive induction of nitritereductase and NO reductase in the denitrifying bacteria

Rhodobacter sphaeroides

and

Paracoccus denitrificans

(van Spanning

et al

1995 1999 Tosques

et al

1996Kwiatkowski

et al

1997) Most recently prokaryotic pro-teins which possess the H-NOX domain of the eukaryoticNO-responsive soluble guanylate cyclases have beenimplicated as NO sensors (Nioche

et al

2004 Boon andMarletta 2005) In addition a number of other regulatorswhich play prominent roles in other stress responses havealso been implicated in NO-signalling For example SoxRand OxyR are sensors for superoxide and hydrogen per-oxide respectively but as NO interacts with the C199residue in OxyR (Kim

et al

2002) and also directly withthe 2Fe-2S centres of SoxR (Ding and Demple 2000)these regulators have been implicated in NO signallingSimilarly while the primary role of Fur is iron sensing NOalso reacts with the Fe

2

+

cofactor (DrsquoAutreaux

et al

2002)and consequently a role in

Salmonella

for Fur in NOdetection has been suggested (Crawford and Goldberg1998) The Fur and PerR regulons in

Bacillus subtilis

respond primarily to iron limitation and peroxide stressrespectively (Bsat

et al

1998) but the ability of NO tonitrosylate the Fe

2

+

centres in these metalloregulators alsoexplains its ability to induce the expression of componentsof the Fur and PerR regulons in this organism (Moore

et al

2004) Finally in

E coli

the global anaerobic regu-lator Fnr (Green

et al

2001) plays a role in the NO-responsive regulation of

hmp

(Cruz-Ramos

et al

2002)


is a Gram-negative microaero-philic enteric pathogen that is recognized as the predom-inant agent of bacterial gastrointestinal disease worldwide(Friedman

et al

2000)

C jejuni

encounters elevated lev-els of nitrosative stress during infection as NO synthesisis markedly increased in patients with infective gastroen-teritis (Forte

et al

1999) and particularly so following

Campylobacter

infection (Enocksson

et al

2004) Wehave demonstrated previously that the single domainglobin Cgb performs a major NO scavenging and detox-ification function in this pathogen (Elvers

et al

2004)Consistent with the protective role of Cgb against NO-related stress

cgb

expression is minimal in standard lab-oratory media but strongly and specifically induced fol-lowing exposure to nitrosative stress This study hasfocused on elucidating the identity of the NO-sensing reg-ulator for

cgb

expression and here Cj0466 a member of

the Crp-Fnr superfamily of transcriptional regulatorswas identified as a regulator of the nitrosative stress-responsive expression of Cgb Furthermore Cj0466 (des-ignated NssR) was shown to regulate the expression of anitrosative stress-responsive regulon of which Cgb is acomponent and which also includes among other pro-teins a truncated haemoglobin

Results

Cj0466 but not Fur or PerR mediates the expression of Cgb in response to reactive nitrogen species

Analysis of the

C jejuni

genome sequence (Parkhill

et al

2000) reveals a number of potential regulators which byanalogy with other bacteria could sense NO and mediatethe NO-responsive expression of Cgb in

C jejuni

(Elvers

et al

2004) These include Fur (van Vliet

et al

1998)PerR which is related to Fur and also a Fe

2

+

-containingmetalloregulator (van Vliet

et al

1999) and Cj0466 amember of the superfamily of Crp-Fnr transcription regu-lators (Korner

et al

2003) The gene encoding Cj0466was disrupted in

C jejuni

NCTC 11168 by insertion of akanamycin resistance cassette to generate CJNSSR1 Todetermine whether this protein or other potential regula-tors played a role in modulating

cgb

expression mutantswere tested for inducible expression of this globin in com-parison to the parental strain NCTC 11168 using immu-noblotting as described previously (Elvers

et al

2004) Inthe wild type and the

fur

mutant AV17 (van Vliet

et al

1998) the expression profile of Cgb was identical Expres-sion was not detectable in the absence of nitrosativestress but was strongly induced 25 h after the addition of01 mM GSNO (Fig 1) While Cgb expression was clearlyinducible by GSNO in the

perR


et al

1999) the level of induced expression was slightlylower than that detected in the wild type In contrastinducible expression of Cgb was almost totally abolishedin the Cj0466 mutant suggesting a prominent role for thisregulator in NO-responsive

cgb

expression As a result ofthese studies Cj0466 was designated as NssR (Nitrosa-tive stress sensing Regulator)

Phenotypic analysis of the

nssR

mutant CJNSSR1

Diffusion assays on solid medium were used to screenthe sensitivity of CJNSSR1 to a range of stress-inducingagents in comparison to the wild type a

cgb

mutant(CJCGB01) a

fur

mutant (AV17) and a

perR

mutant(AV63) As expected CJNSSR1 was sensitive to GSNOwith a significantly greater zone of inhibition comparedwith the wild type (Table 1) As the

cgb

mutant exhibiteda similar zone of killing (Elvers

et al

2004) the sensitivityof CJNSSR1 to GSNO is likely to be at least in part aconsequence of its failure to induce Cgb expression

NssR a nitrosative stress-responding regulator in

C jejuni 737



57

735ndash750

NssR is unlikely to contribute to resistance to peroxidesas CJNSSR1 had sensitivities to

tert

-butyl hydroperoxideand hydrogen peroxide that were similar to those of wild-type cells However CJNSSR1 did display an increasedsensitivity to methyl viologen which is typically attributedto increased superoxide production in

Campylobacter

(Purdy

et al

1999) The

perR

mutant was more resistantto organic hydroperoxides and hydrogen peroxide thanthe wild type This is in accordance with previous reportsand results from the derepression of AhpC and catalaseexpression (van Vliet

et al

1999) Notably the

fur

mutantwas also significantly more sensitive to GSNO than wild-type cells which has not been reported previously

The effect of the

nssR

mutation was probed further by

exposing the wild type CJCGB01 (Cgb

ndash

) and CJNSSR1to various concentrations of GSNO and assessing viabilityThe

nssR

mutant was markedly more sensitive to GSNOthan either the wild type or

cgb

mutant (Fig 2) For exam-ple at 04 mM GSNO there was little to no change in theviability of either the wild-type strain or the strain lackingCgb whereas there was a 100-fold decrease in the viabilityof the strain lacking NssR At 2 mM GSNO all strainsappeared to be equally sensitive and here the levels oftoxic nitrosative stress mediators present may haveexceeded the capacity of the detoxification mechanisms

Microarray analysis

The NssR mutant was more sensitive to killing by GSNOthan the strain lacking only Cgb (Fig 2) and consequentlythere must be other targets of NssR that are important inthe cellular response nitrosative stress Microarray exper-iments were thus conducted in order to define the extentof the regulon influenced by NssR and thus identify othergenes involved in the nitrosative stress response through-out the genome Preliminary growth experiments and real-time polymerase chain reaction (PCR) were used to deter-mine the growth conditions that gave sufficient biomassto obtain concentrated RNA to ensure cells were in thesame growth phase and to define a level of GSNO thatupregulated

cgb

expression but did not significantly affectthe growth rate Thus differences observed from themicroarray results would result from the

nssR mutation ortreatment applied and not from growth-related effectsThese growth experiments showed that before treatmentboth the wild type and CJNSSR1 had identical growthrates Previous experiments have shown that induction ofCgb occurs maximally 2 h after exposure to GSNO (Elverset al 2004) Exposure to 200 mM GSNO at an opticaldensity at 600 nm (OD600) of 02ndash03 for 2 h did not affectsubsequent growth rates for both cell types (data notshown) but after this period sufficient RNA was obtainedfor microarray analysis and cgb expression in wild-type

Fig 1 Cj0466 (NssR) mediates the inducible expression of Cgb in response to reactive nitrogen species GSNO (01 mM) was added to growing cultures of wild-type C jejuni AV17 (Furndash) AV63 (PerRndash) CJNSSR1 (Cj0466ndash) and CJNSSRC (Cj0466-complemented CJNSSR1) The expression of Cgb was detected at the times spec-ified using anti-Cgb antibody Each lane of the 10 SDS gels was loaded with 45 mg of total protein

Wild type

AV17 (Fur-)

AV63 (PerR-)

CJNSSR1 (Cj0466-)

Time (h) after addition of GSNO

0 25 45

C jejuni strainmutant

CJNSSRC

Wild type

AV17 (Fur-)

AV63 (PerR-)

CJNSSR1 (Cj0466-)


0 25 45


CJNSSRC

Table 1 Resistance and sensitivity of C jejuni strains to agents of oxidative and nitrosative stress

Stress inducer

Diameter of disk inhibition zone (mm)a

Initial concentrationon disk

NCTC 11168(n = 6)

AV17 (Furndash)(n = 4)

AV63 (PerRndash)(n = 4)

CJNSSR1 (Cj0466ndash)(n = 6)

S-nitrosoglutathione 100 mM 112 plusmn 03 160 plusmn 06(P = 00007)

118 plusmn 08(P = 051)

147 plusmn 04(P = 89 yen 10-5)

Tert-butyl hydroperoxide 05 (vv) 450 plusmn 09 gt 850 plusmn 00(P = 85 yen 10-8)

348 plusmn 08(P = 00003)

422 plusmn 48(P = 059)

Methyl viologen 3 (wv) 353 plusmn 08 343 plusmn 19(P = 001)

323 plusmn 08(P = 002)

397 plusmn 06(P = 0001)

Hydrogen peroxide 3 (vv) 417 plusmn 08 425 plusmn 03(P = 037)

250 plusmn 00(P = 48 yen 10-6)

385 plusmn 35(P = 041)

a Values are expressed as mean diameter of zone of killing plusmn SE of the mean Data shown are from four or six replicatesSignificant P-values where P lt 005 P-values are a result of t-test assuming equal variance (two-tailed) comparing wild type with each mutant

738 K T Elvers et al

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 57 735ndash750

cells was maximal as assessed by real-time reverse tran-scription polymerase chain reaction (RT-PCR) (data notshown)

Two type I microarray experiments where test cDNA iscompared with a reference cDNA were used to examinethe nitrosative stress response of NCTC 11168 and toidentify the genes controlled by NssR The first experimentidentified the nitrosative stress response of C jejuni bycomparing the transcriptome of NCTC 11168 grown in thepresence of 200 mM GSNO to that of an untreated cultureof NCTC 11168 Here of the genes that passed the initialfilters (flags and error see Experimental procedures)1340 of 1553 were non-changing including most riboso-mal proteins and metabolic enzymes (Tables S1 and S2)This corresponds with the lack of effect of GSNO treat-ment on growth rate Of the 231 significantly changinggenes 60 passed the false discovery rate correction (with

a maximum significance cut-off at 005) Eight of thesegenes were over twofold upregulated (Table 2 column 2)suggesting they might be involved in the nitrosative stressresponse These include cgb the product of which isknown to protect the bacteria from nitrosative stressCj0465c which encodes the truncated globin and sixgenes with no known function Fourteen genes were overtwofold downregulated (Table S2) and of these onlyCj0564 and Cj0635 were over fourfold downregulated

The second experiment compared the transcriptomesof both NCTC 11168 and CJNSSR1 when treated withGSNO to identify which of the genes induced by nitrosa-tive stress in the first experiment were under the controlof NssR Here NCTC 11168 DNA was labelled with Cy3and CJNSSR1 was labelled with Cy5 Thus genesexpressed at a higher level in CJNSSR1 than in the wildtype were designated upregulated while downregulated

0 05 1 15 2

GSNO (mM)

cfu

ml-1

108

107

106

105

104

103

Fig 2 Effects of GSNO on the viability of C jejuni Growing cultures of C jejuni wild type (filled triangles) CJCGB01 (open squares) and CJNSSR1 mutant (filled circles) were exposed to different concentrations of GSNO for 1 h and viability was assessed by plating onto MH agar The results are a mean of two independent experiments Bars indicate one standard error of the mean

Table 2 Genes as assessed by microarray showing at least a twofold induction by 200 mM GSNO in the wild type and the effect of an nssRmutation

Gene

Fold increase of geneexpression in 11168 inresponse to 200 mM GSNO Function

Expression level in GSNO-treated CJNSSR1 compared with GSNO-treated 11168

Cj1586 152 (P = 0029) Single-domain globin (Cgb) 47-fold decrease but failed SD filterCj0465c 114 (P = 0011) Truncated globin 88-fold decrease (P = 0003)Cj0761 74 (P = 0008) Unknown 70-fold decrease (P = 0049)Cj0830 43 (P = 0007) Probable integral membrane protein 28-fold decrease (P = 0022)Cj1582c 31 (P = 0029) Probable peptide ABC transport system

permease protein25-fold increase (P = 0001)

Cj0851c 22 (P = 0008) Probable integral membrane protein 37-fold increase (P = 002)Cj0313 20 (P = 0008) Probable integral membrane protein 14-fold increase but failed FDR correctionCj0430 20 (P = 0031) Probable integral membrane protein 21-fold increase but failed FDR correction

SD standard deviation FDR false discovery rateP-values quoted correspond to the value after FDR correctionExpression in CJNSSR1 is low or below detectable limits leading to a standard deviation greater than 1Significant P-values

NssR a nitrosative stress-responding regulator in C jejuni 739


genes are those expressed at a lower level in CJNSSR1Again the majority of the genes were non-changing (75)but there were more changes than in the first experimentwhich could result from the reduced ability of the mutantto counter the stress caused by NO exposure Of the 512changing genes 178 passed the false discovery rate cor-rection Fifty-one genes were upregulated over twofold inthe mutant including Cj0851c and Cj0430 (Table 2 alsosee Table S3) suggesting that the changes in thesegenes are an NssR-independent response Genes cgbCj0465c Cj0830 and Cj0761 were among the 31 thatwere at least twofold downregulated in the mutant(Table S4) This change is opposite to that seen in thesegenes in the wild type following exposure to GSNO whichstrongly indicates that they are regulated by NssR

Confirmation of microarray results by real-time PCR

Real-time PCR was used to verify independently theresults of the microarray experiment and the predictedNssR regulon (Fig 3) All genes which showed at least atwofold upregulation in response to exposure to GSNOwere selected for this analysis The DNA gyrase gene(gyrA) has previously been used in C jejuni as a controlin experiments to quantify transcript levels by real-timePCR (Wosten et al 2004) In this study its expressionwas shown by real-time PCR and confirmed in themicroarray analysis not to be affected by GSNO treat-ment The expression levels of the significantly upregu-lated genes and two apparently non-changing genes flhFand flgE2 were tested by real-time PCR

The expression of cgb cannot be quantified as a foldchange because in untreated cells of NCTC 11168 andCJNSSR1 the signal was below the limits of detectionHowever after treatment with GSNO a signal was

detected in NCTC 11168 but not in CJNSSR1 indicatingit is upregulated in response to GSNO and dependent onNssR

The real-time PCR experiment (Fig 3) compared theexpression level of the wild type in response to GSNOcompared to an untreated control (open bars) and themutant treated with GSNO compared to the wild typetreated with GSNO (filled bars) Genes upregulated in thewild type respond to GSNO Relative downregulation inthe mutant implies a dependence on NssR No change inexpression level implies the gene is regulated identicallyin both strains in response to GSNO and relative upreg-ulation implies that the gene is expressed at a higher levelby the mutant probably as part of an additional stressdefence mechanism due to the lack of the NssR-controlled regulon

Cj0465c Cj0761 Cj0830 and Cj0430 were all found tobe significantly upregulated in three independent culturesof NCTC 11168 treated with GSNO compared with cellsof NCTC 11168 not treated (Fig 3) The relative expres-sion of Cj0465c Cj0761 and Cj0830 was significantlydownregulated in the CJNSSR1 mutant compared withcells of NCTC 11168 when both were treated with GSNOconfirming that their expression is dependent on NssR Intreated cells of NCTC 11168 Cj1582c Cj0851c andCj0313 were upregulated but not over twofold It is likelythat for marginally changing genes such as these thecompetitive hybridization that is the basis of microarraytechnology is more discriminatory than real-time PCR Asin the microarray experiment the expression of Cj0851cand Cj0430 was stimulated by GSNO but independentlyof NssR The upregulation of Cj0851c in the mutant mayhelp to compensate for the lack of the NssR-dependentnitrosative stress responses flhF and flgE2 were non-changing in both microarray experiments and real-time

001

01

1

10

100

Cj0465c Cj0761 Cj0830 Cj1582c Cj0851c Cj0313 Cj0430 flhF flgE2

Cha

nge

inge

neex

pres

sion

Relative expression level in 11168 + GSNO compared to 11168

Relative expression level in CJNSSR1 + GSNO compared to 11168 + GSNO

001

01

1

10

100


Cha

nge

inge

neex

pres

sion


Relative expression level in CJNSSR1 + GSNO compared to 11168

Fig 3 Real-time PCR assays of Cj0465c Cj0761 Cj0830 Cj1582c Cj0851c Cj0313 Cj0430 flhF and flgE2 expression in wild-type and CJNSSR1 cells cDNA synthesized using random hexamer primers was amplified by real-time PCR using gene-specific primers and SYBR Green The level of gene expression in response to 200 mM GSNO was quantified rel-ative to the internal control gyrA using the 2ndashDDCT method The expression level in NCTC 11168 exposed to GSNO was compared with untreated cells of NCTC 11168 (open bars) The gene expression level for CJNSSR1 exposed to GSNO was compared with GSNO-treated NCTC 11168 (filled bars) The relative change in gene expression compared with the reference sample is shown The error bars rep-resent the range determined using the 2ndashDDCT with DDCT + s and DDCT - s where s = the standard deviation of the value for DDCT These results represent one data set the results were reproducible using different RNA extracts from independent cultures



PCR which confirmed that the expression of neither genechanged in response to GSNO either in NCTC 11168 orin the CJNSSR1 mutant

Complementation of CJNSSR1

To confirm that the phenotype of CJNSSR1 solely resultedfrom disruption of the nssR gene the strain was comple-mented by insertion of a single copy of the Cj0466 genewith its indigenous promoter into the chromosome to gen-erate strain CJNSSRC Restoration of GSNO resistancein strain CJNSSRC was tested by disk diffusion assayCJNSSRC had equivalent resistance to the wild typewith diameters of growth inhibition of 95 plusmn 050 mm(mean plusmn standard error of the mean) and 90 plusmn 052 mmrespectively indicating that its ability to resist GSNO hadbeen restored As observed previously the mutant washypersensitive to GSNO with a diameter of growth inhibi-tion of 232 plusmn 054 mm Furthermore GSNO-inducibleCgb expression as monitored by immunoblotting wasfully restored in the complemented strain (Fig 1) Real-time PCR was used to confirm that strain CJNSSRC wasrestored in its ability to upregulate other members of theNssR-dependent regulon RNA was extracted from thewild type CJNSSR1 and CJNSSRC and reverse-tran-scribed for quantitative PCR as previously described Therelative gene expression of Cj0465c Cj0830 and Cj0761in the GSNO-treated cultures was compared with thatdetected in the untreated cultures (Fig 4) After 2 hgrowth in the presence of GSNO compared with theuntreated control the mutant showed relative decreasesin expression of Cj0465c Cj0830 and Cj0761 Both thewild type and complemented strain significantly upregu-lated to essentially identical levels Cj0465c Cj0830 andCj0761 the genes identified as part of the NssR regulonThe complemented strain also showed unquantifiableupregulation of cgb expression Consequently the possi-bility that transcriptional and phenotypic responses of

CJNSSR1 result from polar effects in gene expressioncaused by insertion of the kanamycin cassette can beexcluded

Transcription at the nssR promoter and NssR-dependent promoters

In view of the clear evidence obtained for regulation byNssR of the genes indicated above the existence ofactive promoters upstream of these genes was investi-gated The 5cent end of the mRNA for NssR and the genesof the NssR regulon including cgb Cj0465c Cj0830 andCj0761 were mapped using 5cent RACE which resulted in asingle PCR fragment around 200ndash300 bp for all targetgenes The sequence of this fragment revealed the startof transcription for each gene (Fig 5) The transcriptionalstart for cgb was also confirmed using reverse ligation-mediated PCR (see Experimental procedures) In Cjejuni the promoter consensus sequence for s70 isunusual compared with other Gram-negative bacteria inthat the region upstream of the -10 TATA box does notcontain a conserved -35 motif (Petersen et al 2003)Consensus s70 -10 sequences but not -35 sequenceswere identified for nssR cgb Cj0465c Cj0830 andCj0761 (Fig 5) However an Fnr-like binding sequencewas discovered upstream of each of these genes Theconsensus for this inverted repeat based on the fourNssR-dependent genes is TTAAC-N4-GTTAA (Fig 5)which is similar to the recognition sequences proposedfor Nnr an NO-sensing regulator from Paracoccus(TTAAC-N4-GTCAA) (Saunders et al 2000) and PrfA(TTAACA-N2-TGTTAA) the regulator of virulence geneexpression in Listeria monocytogenes (Korner et al2003) For cgb Cj0465c Cj0830 and Cj0761 the motifwas centred at -415 -425 -405 and -415 res-pectively from the start point of transcription (Fig 5)which is consistent with the architecture of a class II FNR-dependent promoter (Guest et al 1996) The region

001

01

1

10

100

11168

CJNSSR1

CJNSSRC

Cha

nge

inge

neex

pres

sion

Cj0761Cj0830Cj0465c

001

01

1

10

100

11168

CJNSSR1

CJNSSRC

Cha

nge

inge

neex

pres

sion

Cj0761Cj0830Cj0465c

Fig 4 Real-time PCR assays of Cj0761 Cj0830 and Cj0465c expression in wild-type CJNSSR1 CJNSSRC cells The expression of Cj0761 (dark bars) Cj0830 (grey bars) and Cj0465c (open bars) in response to 200 mM GSNO was quantified relative to the untreated sample using gyrA as the internal control gene and the 2ndashDDCT method as described for Fig 3



upstream of the nssR gene did not contain a putativeNssR recognition sequence in the equivalent locationHowever nssR is adjacent to Cj0465c but is divergentlytranscribed and as a consequence the putative NssRrecognition sequence for Cj0465c lies between the tran-scriptional start point for nssR and the ATG start codon(Fig 5) When the lsquoPattern Searchrsquo tool on CampyDB(httpcampybhamacuk) was used to search for addi-tional putative NssR binding regions an additional con-sensus sequence was found upstream of Cj0580c aHemN-like protein (Fig 5) However from the microarrayexperiments transcription of this gene was not elevatedin the presence of GSNO and it is therefore not likely tobe part of the NssR regulon

To confirm the function of the suggested NssR bindingsite the sequence upstream of cgb (TTAACacaaGTCAA)was altered to CTAACacaaGTCAG a change that basedon studies on Nnr (Hutchings and Spiro 2000) was pre-dicted to prevent recognition by NssR The wild type andaltered promoter sequences were introduced intopMW10 a plasmid for generating transcriptional fusionsto lacZ in C jejuni (Wosten et al 1998) The constructswere then introduced into C jejuni 480 (NCTC 12744) andlacZ expression was measured in the presence andabsence of 005 mM GSNO C jejuni 480 was used as asurrogate in these experiments as this stain accepts shut-tle vectors whereas NCTC 11168 does not (Wassenaaret al 1993) In cells containing pKE117 (wild-type cgbndashlacZ fusion) LacZ expression was induced 23-fold follow-ing exposure to GSNO whereas inducible promoter activ-ity was abolished in cells containing pKE120 (altered cgbndashlacZ fusion) (Fig 6) Inducible cgb promoter activity istherefore completely dependent on the integrity on theputative NssR binding site

Regulation of expression of Cj0465c the truncated globin

To confirm the regulation of Cj0465c and to identify otherstresses that could stimulate expression of Cj0465clevels of this protein were assessed by immunoblottingfollowing exposure to various stresses (Fig 7) Cj0465cexpression clearly occurred at low levels in the absenceof GSNO and although not induced by methyl viologenhydrogen peroxide organic peroxides or the solvent(DMSO) was stimulated markedly by exposure to GSNOand S-nitroso-N-acetylpenicillamine (SNAP) (a nitrosatingagent) While unstimulated levels of Cj0465c were thesame in both the wild type and CJNSSR1 nitrosativestress-responsive expression was abolished in the NssR-deficient mutant

Discussion

The aim of this study was to identify regulatory proteinswhich mediate the nitrosative stress-responsive expres-sion of Cgb one of two members of the microbial globinfamily of proteins (Wu et al 2003) in C jejuni (Elverset al 2004) The nitrosative stress-responsive expressionof Cgb was abolished in a Cj0466-deficient backgroundbut not in Fur or PerR mutants indicating that this proteinis a major positive regulatory factor for controlling cgbexpression Consequently Cj0466 was designated asNssR While Cgb expression in the PerR mutant wasclearly induced upon exposure to GSNO final levels ofCgb were lower than in the wild type However it seemsunlikely that the peroxide sensor PerR plays any signifi-cant role in Cgb regulation as PerR generally functions asa repressor (van Vliet et al 1999) and in the absence ofnitrosative stress Cgb expression is not elevated in the

Fig 5 Transcription start sites of promoters upstream of cgb Cj0465c Cj0830 Cj0761 and nssR The transcript start sites as determined by 5cent RACE are marked in bold with an arrow indicating direction of transcription Proposed -10 sequences are underlined Putative NssR binding sites are boxed and nucleotides identical to the consensus TTAAC-N4-GTTAA shown in negative print while TG residues characteristic of extended -10 promoters are in grey boxes A putative NssR binding sequence upstream of Cj0580c is also shown

Cgb AATTTTAACACAAGTCAATTTTTTTCTCCTTTTTAAGATATAAAATATCTCTTTTACAACAAAAAGGAGAAACTATG

Cj0465c ATTCTTAACTTATGTTAAATTTAATTTATCTTATTTTTGCTATATTAACGCCATAAAATTAACATTTAAGAAAGGCTTATATG

Cj0830 AAAATTAACTAAAGTGAATTCAAAAAATGAAAAAAGTGTTATAATATAGCAAATTCCGAAGTTTAATAAGGAGGGAAAAATG

Cj0761 TAAATTAACACAAGTTTATAATTTATATTTTGGCTTTTGCTATAGTTTTGTAAAACTAAAAAAGGAGATTTTGTG

Cj0466 CAAATTTCATATAAGCCTTTCTTAAATGTTAATTTTATGGGTTAATATAGCAAAAATAAGATAAATTAAATTTAACATAAGTTAAG-17bp-ATG

Cj0580c AATTTTAACCTAAATCAAATCAAATTTTTATTAAAATACATTAAAAAATTTAATAAAATATTTTAGGGTTTTTATG



perR mutant The reduced level of Cgb expression is thusmost likely indirect and may arise from changes in thereactivity or metabolism of GSNO due to its interactionwith derepressed proteins such as KatA and AhpC whichare highly expressed in the perR mutant but not the wildtype (van Vliet et al 1999)

A mutant deficient in NssR was even more sensitive toGSNO than a Cgb-deficient mutant suggesting that NssRupregulates other genes that are involved in tolerance tonitrosative stress (see below) In comparison to wild-typecells CJNSSR1 also displayed a modest increase in sen-sitivity to methyl viologen a superoxide-generating agentThe reason for this is not yet clear but it might suggest anadditional role for the NssR regulon or that superoxidecan interact with NO to form the highly bactericidal agent

peroxynitrite Interestingly the fur mutant also displayedincreased sensitivity to GSNO although this regulator wasnot found to contribute to inducible Cgb expression Sim-ilarly E coli and B subtilis fur mutants are hypersensitiveto nitrosative stress (Moore et al 2004 Mukhopadhyayet al 2004) In the former permanent derepression ofiron assimilation systems in fur mutants generates oxida-tive stress (Touati et al 1995) and consequently the sen-sitivity of C jejuni AV17 (Furndash) to GSNO may reflect theadditive effects of nitrosative stress and endogenous oxi-dative stress in this strain

The global role of NssR in the response of C jejuni toreactive nitrogen species revealed by microarray analysisand confirmed by real-time PCR and complementationstudies clearly defines NssR-dependent nitrosative

0

20

40

60

80

100

120

140

160

180

pMW10 pKE117 pKE120

Reporter construct

b-ga

lact

osid

ase

activ

ity (

Mil

ler

unit

s)Fig 6 b-Galactosidase activities of cells of C jejuni containing variants of the promoter probe vector pMW10 grown in the absence (white bars) or presence of GSNO for 2 h (black bars) pKE120 is identical to pKE117 except for two point mutations which result in an altered NssR binding site Values are the means plusmn the stan-dard error of three separate experiments

100 mM 1 h

100 mM 2 h

200 mM 1h

200 mM 2 h

1 2 3 4 5 6 7 8

Wild type

200 mM 2 h CJNSSR1

Fig 7 Cj0465c expression as assessed by Western blotting Wild-type and CJNSSR1 cells were grown to mid-exponential phase and incu-bated for 1 or 2 h in the absence of stress (lane 1) or in the presence of DMSO (solvent lane 2) GSNO (lane 3) SNAP (lane 4) methyl viol-ogen (lane 5) H2O2 (lane 6) cumene hydrop-eroxide (lane 7) and tert-butyl hydroperoxide (lane 8) as indicated The expression of Cj0465c was detected using the anti-Cj0465c antibody



stress-responsive regulon comprising at least four genesGiven its role in NO scavenging (Elvers et al 2004) thefact that Cgb is a member of this regulon is not unex-pected However the truncated globin (Cj0465c) is alsopart of this regulon and this is of interest because trun-cated globins (Pesce et al 2000 Wittenberg et al 2002)also play a role in NO detoxification in mycobacteriawhere they act as NO dioxygenases (Ouellet et al 2002Pathania et al 2002) It is also apparent that whileCj0465c (and most likely Cj0761 and Cj0830) expressionis strongly stimulated by NO stress it is expressed at lowlevels in a NssR-independent manner in the absence ofthis stress Thus its regulation shows distinct differencescompared with Cgb the expression of which is minimal instandard laboratory media (Elvers et al 2004) Theremaining NssR-dependent genes Cj0761 and Cj0830at present have no predicted function (Parkhill et al2000) In addition the expression of Cj0851c and Cj0430was clearly induced by nitrosative stress in a NssR-independent manner It is possible that these representa NssR-independent NO-responsive network Howeverother than Fur and NssR the genome does not encodepreviously characterized NO-responsive regulators(Parkhill et al 2000) and as these genes do not appearto be part of the Fur regulon (Holmes et al 2005) theidentity of potential regulators for these genes is obscureAlso because the nssR mutant is deficient in theprimary defence against nitrosative stress it is possiblethat the induction of Cj0851c and Cj0430 is simply aconsequence of increased levels of general stress due theinability of these cells to detoxify GSNO-derived nitrogencompounds

While nitrosative stress clearly induces changes ingene expression in C jejuni in terms of the numbers ofgenes involved the response seems to be limited in com-parison to other bacteria In B subtilis for example whilenitrosative stress induces flavohaemoglobin expressionmembers of the Fur PerR and sB general stress regulonsare also induced (Moore et al 2004) In Pseudomonasaeruginosa some 30 genes including those encoding aputative NO-detoxifying flavohaemoglobin a putative NOreductase and antioxidative functions are upregulated bymore than threefold by GSNO treatments (Firoved et al2004) Similarly in E coli some 34 genes are induced bymore than fivefold after treatment with 1 mM GSNO(Mukhopadhyay et al 2004) although the number ofgenes directly regulated by NO-related stresses is likelyto be much lower as the conditions described by Mukho-padhyay et al (2004) favour induction of genes respon-sive to iron limitation or disrupted methionine biosynthesiscaused by homocysteine S-nitrosation (Flatley et al2005) The response of E coli to NO also depends onoxygen tension as only 10 of the genes induced byexposure to NO under anaerobic conditions are also

induced following exposure in aerobic conditions (Justinoet al 2004) The capacity of C jejuni to regulate NO-responsive gene expression under conditions of differentoxygen tension was not investigated in this current studyHowever the effect of oxygen is likely to be less significantthan in E coli as C jejuni tolerates only a limited variationin the concentration of this gas and cannot grow either inaerobic or strictly anaerobic conditions

The promoter regions of genes regulated by membersof the Crp-Fnr superfamily contain distinctive recognitionmotifs (Korner et al 2003) For example FNR proteinsrecognize a DNA target consisting of an inverted repeat(TTGAT-N4-ATCAA Eiglmeier et al 1989) In this contextthe consensus sequence TTAAC-N4-GTTAA foundupstream of all NssR-dependent genes in this study ispredicted to be the NssR-binding motif This conclusion issupported by the finding that alteration of the motif abol-ishes nitrosative stress-inducible Cgb expression The-10 promoter regions of all genes except that of cgb alsofeature a TG motif at positions -14 and -15 similar to thelsquoextended -10rsquo motif found in numerous E coli promoters(Mitchell et al 2003) This feature is characteristic of pro-moters with deviation from the consensus sequence in the-35 region and its absence from the cgb promoter mightcorrelate with the observed tighter regulation of the latterIn addition because nssR and Cj0465c are divergentlytranscribed the putative NssR-binding sequence is poten-tially able to influence the transcription of both genes butin the case of nssR is located between the transcriptionstart point and the start point for translation Thus it ispossible that NssR represses its own expression

NssR is the sole representative of the Crp-Fnr super-family of DNA-binding proteins in C jejuni NCTC 11168(Parkhill et al 2000) and based on its phylogenetic rela-tionship with other members NssR is a member of branchE of the Crp-Fnr superfamily (Korner et al 2003) Whileother members of this superfamily such as members ofthe Dnr and Nnr branch have been implicated in NOsensing in denitrifying bacteria (van Spanning et al 1995Tosques et al 1996 Kwiatkowski et al 1997) this is thefirst time a function has been attributed to a member ofbranch E of the Crp-Fnr superfamily While NssR is clearlycentral to the nitrosative stress response of C jejuni it ispossible that NssR may not be the actual NO sensor Thisis because certain members of the Crp-Fnr family do notinteract directly with a signal molecule and instead aseparate system acts as the sensor which operates toincrease the intracellular concentration of the regulator (inthis case NssR) The FixK proteins which resemble Fnrbut lack the sensory module operate in this way Activa-tion of fixK expression by the oxygen-responsive FixLJsystem increases expression of FixK to a level that per-mits DNA recognition and transcription regulation of targetgenes (Fischer 1994) However from the microarray anal-



ysis it is clear that NssR expression is not upregulated inresponse to nitrosative stress and thus nssR expressioncannot be responding to a cognate sensor It is alsoconceivable that NssR acts indirectly by modulatingthe expression of a second regulator which interactsdirectly with the upstream regions of the nitrosative stress-responsive genes Again the results of the microarrayexperiments eliminate this possibility as transcriptionalregulators that responded to nitrosative stress or thatwere dependent on NssR for expression were not identi-fied Consequently it is most likely that NssR is the actualsensor for NO and that it acts directly on the regulatorysequences

In nearly all Dnr family proteins the motif for DNA bind-ing is conserved as an E-SR motif Similarly a distinguish-ing feature of NnrR family proteins is the substitution of ahistidine residue for glutamate in the E-SR sequence (Kor-ner et al 2003) This region appears to be partially con-served in NssR which has the motif Q-SR in the putativerecognition helix Activation of gene expression by NO isonly now becoming fully understood but NO is known toreact with a variety of metal-containing proteins Accord-ingly as Fnr contains a 4Fe-4S cluster this has beenimplicated in regulating NO-responsive hmp expression inE coli (Cruz-Ramos et al 2002) Intriguingly while NssRis a member of the same superfamily it does not possessthe cysteine signature for binding the 4Fe-4S cluster andlike members of the NnrDnr group of NO-sensing regu-lators which also lack the same domain (Korner et al2003) the mechanism of NO sensing is not known

While this study has clearly shown that C jejuni exhibitsa response to nitrosative stress and that NssR is a keyregulator of this the source of NO in vivo is not yetrecognized Cgb-deficient mutants are not attenuated forsurvival in enterocytes and thus Cgb does not appear toplay a role in survival in this environment (Elvers et al2004) Nevertheless this protein and other members ofthe NssR regulon may be important for intracellular sur-vival in other cell types and during the colonization ofother hosts For example C jejuni is able to colonizepoultry asymptomatically and it has been shown recentlyto stimulate this host in a proinflammatory manner Sig-nificantly infection of chick kidney cells (a model for avianepithelial cells) and avian macrophages results in anincrease in iNOS-derived NO levels (Smith et al 2005)Thus a role for the NssR regulon during the interaction ofC jejuni with this host can be envisaged

In conclusion the data presented here demonstrate forthe first time that NssR a member of the Fnr-Crp super-family controls the expression of a previously unknownnitrosative stress-responsive regulon in C jejuni whichincludes both a single-domain globin (Cgb) and a sepa-rate truncated globin This system is likely to be a key partof the defence of C jejuni against the toxic activities of

NO and its congeners during its interaction with a numberof different hosts

Experimental procedures

Bacterial strains plasmids and growth conditions

The bacterial strains and plasmids used in this study arelisted in Table 3 C jejuni NCTC 11168 and E coli DH5awere obtained from the National Collection of Type Cultures(Colindale London) and Life Technologies respectively Cjejuni AV17 (furndash) and C jejuni AV63 (perRndash) both derivativesof NCTC 11168 and also C jejuni 480 were kindly donatedby J Ketley University of Leicester

Campylobacters were grown routinely at 37infinC in Mueller-Hinton (MH Oxoid) broth with shaking or on MH agar undermicroaerobic conditions generated using the CampyGen(Oxoid) gas generating kit or a MACS 500 variable atmo-sphere incubator (Don Whitley Scientific) For cloning exper-iments E coli DH5a was grown at 37infinC in LuriandashBertani (LB)broth or on agar When antibiotic selection was necessarythe growth media were supplemented with ampicillin(100 mg ml-1) kanamycin (50 mg ml-1) or chloramphenicol(25 mg ml-1)

Construction of a C jejuni Cj0466 (NssR)-deficient mutant

The region containing the Cj0466 gene and flankingsequences was amplified by PCR using oligonucleotide prim-ers NssR1 5cent-CACCTTCGCCTAAAAGCATGCCTG-3cent andNssR2 5cent-GGATCCCAAGCGGTGGAATTTGCTCTAAATG-3centusing the PCR conditions described previously (Elvers andPark 2002) The resulting 1167 bp PCR fragment was clonedinto the TA cloning vector pBAD-TOPO (Invitrogen) to formplasmid pKE77 Inverse PCR (Wren et al 1993) was thenused to introduce a small deletion of 134 bp in the Cj0466open reading frame and a unique BglII restriction site usingoligonucleotide primers NssR3 5cent-gaacaAGATCTTTAGCCTCTTCGCCTTCATAAA-3cent and NssR4 5cent-gaacaAGATCTCCTGCTAATGCTGTTTTTGAAGAG-3cent and the PCR protocolsdescribed previously (Elvers et al 2004) The resulting plas-mid pKE78 was digested with BglII and ligated to the1480 bp kanamycin resistance gene cassette excised byBamHI digestion from pJMK30 (Baillon et al 1999) resultingin the suicide plasmid pKE79 In this construct the antibioticresistance cassette was orientated with the same transcrip-tional polarity as the Cj0466 gene Electroporation was usedto introduce pKE79 into C jejuni NCTC 11168 The homolo-gous recombination that resulted in a double cross-overevent was verified by PCR and Southern hybridization (datanot shown) The resulting mutant was designated CJNSSR1

Construction of an NssR-complemented strain

The Cj0466 gene including its promoter region was amplifiedby PCR from C jejuni strain NCTC 11168 using oligonucle-otide primers Cj0466FP3 5cent-CGCGGATCCCCATGAGTTTTGCTATGC-3cent containing a BamHI restriction site andCj0466RP3 5cent-CGGGGTACCCAAAATCCATTCTCTACC-3centcontaining a KpnI site The resulting 1 kb PCR fragment was



digested with BamHI and KpnI before ligation into the Cj0752sequence cloned in pGEMCWH01 In plasmid pGEMCWH01a multiple cloning site has been inserted into Cj0752 a genethat in its chromosomal location is an isolated pseudogeneencoding a probable IS element transposase (C Holmes andPA Lund unpublished results) The chloramphenicol cas-sette from pAV35 (a gift from A van Vliet) was then insertedinto the KpnI site to create construct pSTCJ4 Thus the sui-cide plasmid pSTCJ4 contains a wild-type copy of Cj0466and downstream of it the chloramphenicol resistance select-able marker flanked by contiguous sequences from pseudo-gene Cj0752 For homologous recombination pSTCJ4 wasintroduced into CJNSSR1 by electroporation Transformantswere selected using chloramphenicol (10 mg ml-1 on the firstselection plate and 25 mg ml-1 for subsequent subcultures)The occurrence of a double cross-over event which resultedin excision of the plasmid backbone was confirmed by PCRThe resulting strain was designated CJNSSRC

Western blot detection of Cgb and Cj0465c

Anti-Cgb and -Cj0465c antibodies were prepared asdescribed by Elvers et al (2004) To assess Cgb expressioncultures of C jejuni NCTC 11168 C jejuni AV17 C jejuniAV63 CJNSSR1 and CJNSSRC were grown in 100 ml of MHbroth microaerobically at 37infinC and 15 ml of samples weretaken at time 0 after which 01 mM GSNO was added Fur-ther 15 ml of samples were taken after 25 and 45 h Tomonitor Cj0465c expression cells were grown to mid-expo-nential phase and then exposed to 100 or 200 mM GSNOSNAP methyl viologen hydrogen peroxide cumene hydrop-eroxide or butyl hydroperoxide for 1 or 2 h Controls were

performed using DMSO (solvent for cumene and tert-butylhydroperoxide) and with cultures to which no additions weremade Sample preparation and Western blotting was thencarried out as described previously (Elvers et al 2004)

Sensitivity of C jejuni to GSNO methyl viologen tert-butyl hydroperoxide and hydrogen peroxide

The sensitivity of C jejuni NCTC 11168 C jejuni AV17 Cjejuni AV63 CJCGB01 CJNSSR1 and CJNSSRC to a rangeof stress-inducing agents including tert-butyl hydroperoxide(05 vv) methyl viologen (3 wv) hydrogen peroxide (3vv) and S-nitrosoglutathione (100 mM) was initially assayedin a plate diffusion assay using the method described byBaillon et al (1999) For viability assays cells were exposedto 02 04 08 16 and 2 mM GSNO and viability wasassessed as described previously (Elvers et al 2004)

Growth and RNA extraction for microarrays

Overnight cultures were diluted 11 with MH broth and grownshaking at 37infinC for 3 h in a microaerobic atmosphere Thesecultures were used to inoculate fresh broth at an OD600 of008 Cultures were then grown to an OD600 between 02 and03 before treatment with 200 mM GSNO where appropriateCultures were grown for an additional 2 h to an OD600 ofapproximately 035ndash045 before 5 ml of culture was mixedwith 10 ml of RNAprotectTM bacterial reagent (Qiagen Craw-ley UK) before centrifugation at 6000 g for 15 min Total RNAwas extracted using an RNeasy kit (Qiagen) according to themanufacturerrsquos instructions with the additional on-column

Table 3 Bacterial strains and plasmids used in this study

Strain or plasmid Characteristicsa Source or reference

StrainsC jejuni

NCTC 11168 Parental strain National Collection of Type Cultures480 (NCTC 12744) Shuttle plasmid accepting strain National Collection of Type CulturesCJCGB01 11168 cgbKanr Elvers et al (2004)CJNSSR1 11168 nssRKanr This studyCJNSSRC CJNSSR1 with mutation complemented by insertion of nssR Cmr into Cj0752 This studyAV17 11168 furKanr van Vliet et al (1998)AV63 11168 perRKanr van Vliet et al (1999)

E coli DH5a Fndash f80dLacZ DM15 Life Technologies

PlasmidspJMK30 C coli Kanr cassette in pUC19 Baillon et al (1999)pAV35 C coli Cmr cassette in pBluescript Van Vliet et al (1998)pBAD-TOPO Cloning vector Apr InvitrogenpGEM-T Easy Cloning vector Apr PromegapKE77 1160 bp PCR product containing nssR in pBAD-TOPO This studypKE78 pKE77 with a unique PCR generated BglII site in nssR This studypKE79 Kanr cassette cloned into BglII site in pKE78 This studypGEMCWH01 Cj0752 with an internal cloning site in pGEMT-Vector C Holmes and PA Lund

(unpublished results)pSTCJ4 nssR with downstream Cmr cassette in pGEMCWH01 This studypMW10 lacZ transcriptional fusion vector Kanr Wosten et al (1998)pKE116 cgb promoter fragment in pGEM-T This studypKE117 cgb promoter fragment in pMW10 This studypKE119 pKE116 with mutated cgb promoter fragment This studypKE120 Mutated cgb promoter fragment in pMW10 This study

a Apr ampicillin resistance Kanr kanamycin resistance Cmr chloramphenicol resistance



DNase I digestion step to remove genomic DNA contamina-tion The concentration of RNA was determined on an Agilent2100 Bioanalyser

Labelling and hybridization to DNA microarrays

cDNA was synthesized from 15 mg of RNA and indirectlylabelled using the CyScribe Post-Labelling Kit (AmershamBiosciences) according to the manufacturerrsquos protocol Wholegenome C jejuni microarrays comprised PCR ampliconsprinted on Ultragaps (Corning) glass slides (BacterialMicroarray Group St Georgersquos Hospital Medical School Lon-don UK) (Jones et al 2004) Slides were incubated in a pre-hybridization solution of 5yen SSC 01 BSA and 01 SDSfor at least 4 h before the slides were washed in water andisopropanol and dried by centrifugation at 1500 rpm for5 min Cy3- and Cy5-labelled cDNA samples (30 pmoles)were vacuum dried and hybridized to a microarray slide in afinal volume of 30 ml in 30 formamide 5yen SSC 01 SDSsalmon sperm DNA (01 mg ml-1) and 1yen Denhardtrsquos solutionat 42infinC for 16 h in the dark Slides were washed for 2 min in2yen SSC01 SDS buffer 2 min in 01yen SSC01 SDSbuffer and 2 yen 2 min in 01yen SSC buffer The slides were driedby centrifugation at 1500 rpm for 5 min The hybridizedslides were scanned with a confocal laser scanner (AxonGenePix 4000A) to obtain the highest intensity withoutsaturation

Microarray analysis

Microarray images were analysed using GenePix Pro 3(Axon Instruments) and resulting data with GeneSpring 7(Silicon Genetics) Data were normalized using intensity-dependent Lowess with 40 of the data used to calculatethe Lowess fit to eliminate Cy dye incorporation bias Signalsbelow 001 were taken as 001 Three biological replicateswere used in each experiment and stringent criteria appliedfor analysis Data were filtered on flags (three out of threepresent or marginal) and error (one standard deviation) toremove bad spots absent spots and spots where the geneexpression level was inconsistent between replicates Theywere then filtered on expression level to remove non-chang-ing genes those between 0667- and 1334-fold changeChanging genes were then filtered on confidence applyingthe Benjamini and Hochberg (1995) false discovery rate algo-rithm with a maximum significance cut-off at 005 to eliminatethe chance of false-positives Data were further analysed inGeneSpring using volcano plots and Microsoft Excel Onlygenes that changed by twofold were considered biologicallysignificant

RNA extraction and cDNA synthesis for real-time PCR

Cultures were grown according to the protocol described forthe microarray experiments After 2 h of treatment withGSNO where required 2 ml of culture was mixed with 4 mlof RNAprotecTM bacterial reagent RNA was extracted usingRNeasyreg (Qiagen) with an additional 1 h off-column DNasedigestion step at 37infinC and the additional RNA clean-up stepto minimize genomic DNA contamination Total RNA was

converted to cDNA using random hexanucleotide primersand Superscript II Reverse Transcriptase (Invitrogen) The RTreaction was performed as follows 500 ng of total RNA with300 ng of random hexanucleotide primers (Invitrogen) and500 mM dNTPs in a 20 ml reaction mix were heated to 65infinCfor 5 min before cooling on ice First-strand buffer DTT and40 units of RNasinreg Ribonuclease Inhibitor (Promega) wereadded and the reaction was incubated at 25infinC for 2 minSuperscript II (200 units) was added to RT+ reactions andRNase-free water added to RT- reactions before incubationat 25infinC for 10 min 42infinC for 50 min and 70infinC for 15 min

Relative quantification of gene expression using SYBR Green real-time PCR

Specific primers for real-time PCR were designed usingPrimer Express software (Applied Biosystems Table 4)cDNA (3 ml) was used as a template for real-time PCR Thepositive control was the supernatant extracted after centrifu-gation of whole cells of NCTC 11168 boiled for 10 min Anoptical 96-well microtitre plate (Applied Biosystems) wasused with a 25 ml reaction volume consisting of 2yen RainbowSYBR Green Master Mix (Bioline) 50 nM of gene-specificprimers and the template An ABI Prism 7000 SequenceDetector (Applied Biosystems) was programmed for an initialstep of 10 min at 95infinC followed by 40 cycles of 15 s at 95infinCand 1 min at 60infinC SYBR Green technology detects double-stranded DNA (dsDNA) so melting curves were used to mon-itor specificity To ensure validity of relative quantificationusing the DDCT method the amplification of the target andreference genes must be approximately equal (Livak andSchmittgen 2001) The supernatants from boiled whole-cellsuspensions of C jejuni NCTC 11168 at approximately5 yen 108 cells ml-1 were diluted 10-fold to 11000 and real-timePCR was used to determine the average crossing threshold(CT) for each target gene and for the control gene gyrA Thelog10 of template dilution was plotted against DCT (differencein crossing threshold target-reference) A gradient of closeto zero was calculated for all the target genes indicating thatthe DDCT calculation could be used (not shown) Geneexpression level was determined relative to a reference groupusing the 2ndashDDCT method and gyrA as the internal control gene(Livak 1997 Livak and Schmittgen 2001) Signal intensityfrom genomic DNA contamination was monitored usingRTndashcDNA reactions as the template for real-time PCR

RNA extraction 5cent rapid amplification of cDNA ends (RACE) and reverse ligation-mediated PCR

NCTC 11168 was grown in the presence of GSNO (005 mM)for 25 h and total RNA extracted using TRIzolreg reagent(Invitrogen) as described by the manufacturer

Rapid amplification of cDNA ends (RACE) was used tomap the 5cent ends of the mRNA for Cj1586 (cgb) Cj0465c(trHb) Cj0466 (nssR) Cj0830 and Cj0761 using the 5cent RACESystem (Invitrogen) Briefly for each target gene two gene-specific primers that hybridized to target sites between 80and 371 bp downstream of the ATG start were acquired(Table 4) For first-strand cDNA synthesis 2 mg of total RNAwas combined with gene-specific primer A (GSA 25 pmol)



and 1 ml of SuperScriptTM II RT After cDNA synthesis RNAtemplate was removed by treating with an RNase mix andthe first-strand product was purified An oligo-dC tail wasadded to the 3cent end by terminal deoxynucleotide transferasecreating the abridged anchor primer binding site PCR wasperformed on the dC-tailed cDNA using gene-specific primerB (GSB) and the abridged anchor primer using the conditionsas described by the manufacturer The 5cent RACE productswere purified using QIAquick enzymegel extraction kit(Qiagen) and directly sequenced on an automated capillarysequencer

The 5cent end of mRNA for Cgb was also mapped using analternative procedure derived from Bertrand et al (1993) andSzymkowiak et al (2003) RNA was extracted using hot phe-nol (Aiba et al 1981) and 1 mg treated with DNA-freeTM

DNase and inactivation reagent (Ambion) for 30 min at 37infinCThe DNase-free RNA was circularized by incubating 200 ngof RNA with 20 U of T4 RNA ligase (New England Biolabs)and RNase OUT (40 m Invitrogen) for 1 h at 37infinC The T4RNA ligase was inactivated by heating at 65infinC for 15 min andthe ligated products were precipitated by addition of 1 mlsodium acetate (pH 6) and 30 ml of ethanol The pellet wasrinsed with 70 ethanol and resuspended in 12 ml of waterThe gene-specific primer HMP7 5cent-GCAACAAGATCTAAGCTTTTGGTTGTTCTCCTGAAATT-3cent (2 pmol) was annealedto the RNA template at 65infinC for 5 min in a mixture containing50 mM of each dNTP To initiate first-strand cDNA synthesis

a mixture containing SuperScript III RT and buffer (200 mInvitrogen) 40 U of RNase OUT and 5 mM DTT was addedto the denatured ligated RNA and incubation carried out at65infinC for 1 h Using HMP7 (above) and HMP8 5cent-GCAACAAGATCTGCAATGGCGATTTTAATGGCGGCT-3cent (final concen-tration of 10 mM) PCR was applied to amplify the 5cent-3centjunction generated by the RNA ligase This consisted of 35cycles of 45 s at 94infinC 1 min at 55infinC and 1 min at 72infinCFollowing PCR amplification the products were cloned intopCR21 (Invitrogen) The nucleotide sequence of the insertsof five plasmids was identical and revealed the 5cent and 3cent endof the cgb mRNA

Characterization and mutagenesis of the putative cgb NssR-binding motif

The promoter region of cgb was amplified by PCR using theoligonucleotide primers mut3 5cent-GGATCCAAATTTGCCATAGTCCCTCTTTT-3cent and mut2 5cent-AGATCTTGTTCTTTTGTCATAGTTTCTCCTT-3cent containing unique BamHI and BglII sitesrespectively The resulting 187 bp fragment was cloned intopGEMreg-T Easy vector (Promega) to generate pKE116 TheQuikChangeregII Site-Directed Mutagenesis Kit (Sratagene)was used to incorporate two single-point mutations into thecgb promoter region of pKE116 using the complementaryoligonucleotide primers mut4 5cent-GAGCATAATTCTAACACAA

Primer name and usea Sequence (5cent-3cent)

RACEGSA Cgb TTGGTTGTTCTCCTGAAATTTGTGSA 0466a CAGCATTAGCAGGATAATTTGSA 0761a CATTTGCCTCTAGGCTTATCAGSA 0830a ATCGCGATACAAAGCACAAAGSA 0465c1 TGCCCATTATAATCACCTTCGGSB CgbR2 AACTCATTGGTTAAATCCTCTCCAGSB 0466b AAGCAGGCATTTCTGCTATAAGSB 0761b GGAGCTTTATGCTCTTTCATCAGSB 0830b TGCCAAAGCACCTCCACTTAGSB 0465c2 CATCGCTTGTTCCAATAGCA

Real-time PCR forwardCGB TTGGAAAATATGAGAAGCTTTGTTGAFLHF TGATACCTCCTGAATTTGCAAGTATTFLGE2 CCCTGTGGATATAGGTCCTATGTATAATCGYRA ATGCTCTTTGCAGTAACCAAAAAACj0313 TTAAAGATACAAGATTTCTCCGAGCTTCj0430 TGCCGGGTACTGTTGCAACj0465c TGCTATTGGAACAAGCGATGAACj0761 GCATAAAGCTCCTTTTGCTATACATGCj0830 TGCTACTGGAGTTAAAATAATGCCTTTCj0851c GCTTATGTAATATTGGTTGCTGGATTTTCj1582c GGGCTGGGTTTAGGATTTGAA

Real-time PCR reverseCGB TGGCTTCATCAGGATTTAAAAGATTFLHF ATAGTTGCTTTCATAATCGCTTCTAGATGFLGE2 AGTTGGTCTTGTAGTTGCATTAGCAGYRA GGCCGATTTCACGCACTTTACj0313 CTAGCAACCCCTTCTATAATCTTTGGCj0430 GTAAGCACATAATATAGCAAGGGTTAAAAACj0465c GCCTGCCCAAAAATTTCCTATCj0761 TTTCCTTTTCCACTTCAAACCAACj0830 GGTTTAAAATGAAAACCTGCCAAACj0851c AGCAAGGATGAAATTCCACAAATATACj1582c CCAAGCGCCTATAAAAACTGCTT

a GSA gene-specific primer A GSB gene-specific primer B

Table 4 Gene-specific primers for real-timePCR and 5cent RACE



GTCAGTTTTTTTCTCC-3cent and mut5 5cent-GGAGAAAAAAAGTGACTTGTGTTACAATTATGCTC-3cent The PCR reaction con-tained 50 ng of plasmid 02 mM of each primer and 25 U ofPfuUltratrade High-Fidelity DNA polymerase and used the fol-lowing conditions 95infinC for 30 s then 12 cycles of 95infinC for30 s 55infinC for 1 min and 68infinC for 35 min The DNA wasdigested with 10 U of DpnI for 1 h at 37infinC to removed super-coiled parental dsDNA and introduced into E coli XL-1 Bluecells Plasmids containing the altered sequence were identi-fied by DNA sequencing and one containing the mutatedNssR binding site designated pKE119 The wild type andaltered cgb promoter regions were excised from pKE116 andpKE119 using BamHI and BglII and the fragments ligated intothe corresponding sites of the lacZ reporter plasmid pMW10(Wosten et al 1998) to generate plasmids pKE117 andpKE120 respectively

Plasmids were introduced into C jejuni by electroporationand transformants selected and maintained using kanamycin(50 mg ml-1) Overnight lawns of the transformants were har-vested in broth and the OD600 was adjusted to 05 Aliquots(2 ml) of these suspensions were inoculated into 10 ml ofbroth and incubated microaerobically for 5 h at 37infinC at100 rpm When required GSNO was added to a final con-centration of 005 mM and cells were incubated for a further2 h (final OD600 approximately 02) Cultures were then cen-trifuged at 3500 rpm for 20 min and resuspended in 1 ml ofZ buffer b-Galactosidase activity was then measured in01 ml aliquots of these suspensions using o-nitrophenyl-b-D-galactopyranoside as described previously (Wosten et al1998)

Bioinformatic analysis

lsquoPattern Searchrsquo on CampyDB (httpcampybhamacuk)which uses the fuzznuc part of the EMBOSS software suitewas used to search the genome for putative NssR bindingsites by defining the number of mismatches permitted fromthe input consensus sequence The consensus sequence ofTTAAC-N4-GTTAA was used with up to three mismatches toidentify putative binding sites in regions up to 150 bpupstream of predicted translation start sites

Acknowledgements

This work was supported by BBSRC grants D18368 D18084and D14520 to SFP RKP and CWP and by a BBSRCcommittee studentship to LMW We thank Dr Simon Smith(University of Sheffield Antibody Resource Centre) for prep-aration of antibody Also we acknowledge Chris Holmes andPete Lund (University of Birmingham) and Julian Ketley (Uni-versity of Leicester) for the kind gifts of pGEMCWH01 andpMW10

References

Aiba H Adhya S and de Crombrugghe B (1981) Evi-dence for two functional gal promoters in intact Escherichiacoli cells J Biol Chem 256 11905ndash11910

Alderton WK Cooper CE and Knowles RG (2001)

Nitric oxide synthases structure function and inhibitionBiochem J 357 593ndash615

Baillon ML van Vliet AH Ketley JM Constantinidou Cand Penn CW (1999) An iron-regulated alkyl hydroper-oxide reductase (AhpC) confers aerotolerance and oxida-tive stress resistance to the microaerophilic pathogenCampylobacter jejuni J Bacteriol 181 4798ndash4804

Benjamini Y and Hochberg Y (1995) Controlling the falsediscovery rate a practical and powerful approach to multi-ple testing J Roy Stat Soc B 57 289ndash300

Bertrand E Fromont-Racine M Pictet R and Grange T(1993) Visualization of the interaction of a regulatory pro-tein with RNA in vivo Proc Natl Acad Sci USA 90 3496ndash3500

Boon EM and Marletta MA (2005) Ligand specificity ofH-NOX domains from sGC to bacterial NO sensors JInorg Biochem 99 892ndash902

Bsat N Herbig A Casillas-Martinez L Setlow P andHelmann JD (1998) Bacillus subtilis contains multiple Furhomologues identification of the iron uptake (Fur) andperoxide regulon (PerR) repressors Mol Microbiol 29189ndash198

Crawford MJ and Goldberg DE (1998) Regulation of theSalmonella typhimurium flavohemoglobin gene A newpathway for bacterial gene expression in response to nitricoxide J Biol Chem 273 34028ndash34032

Cruz-Ramos H Crack J Wu G Hughes MN Scott CThomson AJ et al (2002) NO sensing by FNR regula-tion of the Escherichia coli NO-detoxifying flavohaemoglo-bin Hmp EMBO J 21 3235ndash3244

DrsquoAutreaux B Touati D Bersch B Latour JM andMichaud-Soret I (2002) Direct inhibition by nitric oxide ofthe transcriptional ferric uptake regulation protein vianitrosylation of the iron Proc Natl Acad Sci USA 9916619ndash16624

Ding H and Demple B (2000) Direct nitric oxide signaltransduction via nitrosylation of iron-sulfur centers in theSoxR transcription activator Proc Natl Acad Sci USA 975146ndash5150

Eiglmeier K Honore N Iuchi S Lin EC and Cole ST(1989) Molecular genetic analysis of FNR-dependent pro-moters Mol Microbiol 13 869ndash878

Elvers KT and Park SF (2002) Quorum sensing inCampylobacter jejuni detection of a luxS encoded signal-ing molecule Microbiology 148 1475ndash1481

Elvers KT Wu G Gilberthorpe NJ Poole RK andPark SF (2004) Role of an inducible single-domainhemoglobin in mediating resistance to nitric oxide and nit-rosative stress in Campylobacter jejuni and Campylobactercoli J Bacteriol 186 5332ndash5341

Enocksson A Lundberg J Weitzberg E Norrby-TeglundA and Svenungsson B (2004) Rectal nitric oxide gas andstool cytokine levels during the course of infectious gastro-enteritis Clin Diagn Lab Immunol 11 250ndash254

Firoved AM Wood SR Ornatowski W Deretic V andTimmins GS (2004) Microarray analysis and functionalcharacterization of the nitrosative stress response in non-mucoid and mucoid Pseudomonas aeruginosa J Bacteriol186 4046ndash4050

Fischer HM (1994) Genetic regulation of nitrogen fixationin rhizobia Microbiol Rev 58 352ndash386



Flatley J Barrett J Pullan ST Hughes MN Green Jand Poole RK (2005) Transcriptional responses ofEscherichia coli to S-nitrosoglutathione under definedchemostat conditions reveal major changes in methioninebiosynthesis J Biol Chem 280 10065ndash10072

Forte P Dykhuizen RS Milne E McKenzie A SmithCC and Benjamin N (1999) Nitric oxide synthesis inpatients with infective gastroenteritis Gut 45 355ndash361

Friedman CR Neimann J Wegener HC and TauxeRV (2000) Epidemiology of Campylobacter jejuni infec-tions in the United States and other industrialized nationsIn Campylobacter 2nd edn Nachamkin I and BlaserMJ (eds) Washington DC American Society for Micro-biology Press pp 121ndash138

Gardner PR Costantino G Szabo C and Salzman AL(1997) Nitric oxide sensitivity of the aconitases J BiolChem 272 25071ndash25076

Gardner AM Gessner CR and Gardner PR (2003)Regulation of the nitric oxide reduction operon (norRVW)in Escherichia coli Role of NorR and sigma54 in the nitricoxide stress response J Biol Chem 278 10081ndash10086

Green J Scott C and Guest JR (2001) Functional ver-satility in the CRP-FNR superfamily of transcription factorsFNR and FLP Adv Microb Physiol 44 1ndash34

Guest JR Green J Irvine AS and Spiro S (1996) TheFNR modulon and FNR-regulated gene expression InRegulation of Gene Expression in Escherichia coli LinECC and Lynch AS (eds) Texas R G Landes amp CoAustin pp 317ndash342

Holmes K Mulholland F Pearson BM Pin C McNi-choll-Kennedy J Ketley JM and Wells JM (2005)Campylobacter jejuni gene expression in response toiron limitation and the role of Fur Microbiology 151243ndash257

Hutchings MI and Spiro S (2000) The nitric oxide regu-lated nor promoter of Paracoccus denitrificans Microbiol-ogy 246 2635ndash2641

Hutchings MI Mandhana N and Spiro S (2002) TheNorR protein of Escherichia coli activates expression of theflavorubredoxin gene norV in response to reactive nitrogenspecies J Bacteriol 184 4640ndash4643

Jones MA Marston KL Woodall CA Maskell DJLinton D Karlyshev AV et al (2004) Adaptation ofCampylobacter jejuni NCTC 11168 to high-level coloniza-tion of the avian gastrointestinal tract Infect Immun 723769ndash3776

Justino MC Vicente JB Teixeira M and Saraiva LM(2004) New genes implicated in the protection of anaero-bically grown Escherichia coli against nitric oxide J BiolChem 280 2636ndash2643

Kim SO Merchant K Nudelman R Beyer WF JrKeng T DeAngelo J et al (2002) OxyR a molecularcode for redox-related signaling Cell 109 383ndash396

Korner H Sofia HJ and Zumft WG (2003) Phylogeny ofthe bacterial superfamily of Crp-Fnr transcription regula-tors exploiting the metabolic spectrum by controlling alter-native gene programs FEMS Microbiol Rev 27 559ndash592

Kwiatkowski AV and Shapleigh JP (1996) Requirementof nitric oxide for induction of genes whose products areinvolved in nitric oxide metabolism in Rhodobactersphaeroides 243 J Biol Chem 271 24382ndash24388

Kwiatkowski AV Laratta WP Toffanin A and ShapleighJP (1997) Analysis of the role of the nnrR gene productin the response of Rhodobacter sphaeroides 241 to exog-enous nitric oxide J Bacteriol 179 5618ndash5620

Livak KJ (1997) ABI Prism 7700 Sequence Detection Sys-tem User Bulletin No 2 PE Applied Biosystems AB web-site bulletin reference 4303859B 777802-002 httpdocsappliedbiosystenscompediodocs04303859pdf

Livak KJ and Schmittgen TD (2001) Analysis of relativegene expression data using real-time quantitative PCR andthe 2(-Delta Delta C(T)) method Methods 25 402ndash408

Mitchell JE Zheng D Busby SJ and Minchin SD(2003) Identification and analysis of lsquoextended -10rsquo promot-ers in Escherichia coli Nucleic Acids Res 16 4689ndash4695

Moore CM Nakano MM Wang T Ye RW and Hel-mann JD (2004) Response of Bacillus subtilis to nitricoxide and the nitrosating agent sodium nitroprusside JBacteriol 186 4655ndash4664

Mukhopadhyay P Zheng M Bedzyk LA LaRossa RAand Storz G (2004) Prominent roles of the NorR and Furregulators in the Escherichia coli transcriptional responseto reactive nitrogen species Proc Natl Acad Sci USA 101745ndash750

Nioche P Berka V Vipond J Minton N Tsai AL andRaman CS (2004) Femtomolar sensitivity of a NO sensorfrom Clostridium botulinum Science 2004 1550ndash1553

Nozaki Y Hasegawa Y Ichiyama S Nakashima I andShimokata K (1997) Mechanism of nitric oxide-dependentkilling of Mycobacterium bovis BCG in human alveolarmacrophages Infect Immun 65 3644ndash3647

Ouellet H Ouellet Y Richard C Labarre M WittenbergB Wittenberg J and Guertin M (2002) Truncated hemo-globin HbN protects Mycobacterium bovis from nitric oxideProc Natl Acad Sci USA 99 5902ndash5907

Parkhill J Wren BW Mungall K Ketley JM ChurcherC Basham D et al (2000) The genome sequence of thefoodborne pathogen Campylobacter jejuni reveals hyper-variable sequences Nature 403 665ndash668

Pathania R Navani NK Gardner AM Gardner PRand Dikshit KL (2002) Nitric oxide scavenging and detox-ification by the Mycobacterium tuberculosis haemoglobinHbN in Escherichia coli Mol Microbiol 45 1303ndash1314

Pesce A Couture M Dewilde S Guertin M YamauchiK Ascenzi P et al (2000) A novel two-over-two alpha-helical sandwich fold is characteristic of the truncatedhemoglobin family EMBO J 19 2424ndash2434

Petersen L Larsen TS Ussery DW On SL andKrogh A (2003) RpoD promoters in Campylobacter jejuniexhibit a strong periodic signal instead of a -35 box J MolBiol 326 1361ndash1372

Pfeiffer S Gorren AC Schmidt K Werner ER HansertB Bohle DS and Mayer B (1997) Metabolic fate ofperoxynitrite in aqueous solution Reaction with nitric oxideand pH-dependent decomposition to nitrite and oxygen ina 21 stoichiometry J Biol Chem 272 3465ndash3470

Poole RK (2005) Nitric oxide and nitrosative stress toler-ance in bacteria Biochem Soc Trans 33 176ndash180

Poole RK and Hughes MN (2000) New functions for theancient globin family bacterial responses to nitric oxideand nitrosative stress Mol Microbiol 36 775ndash783

Purdy D Cawthraw S Dickinson JH Newell DG and



Park SF (1999) Generation of a superoxide dismutase(SOD)-deficient mutant of Campylobacter coli evidence forthe significance of SOD in Campylobacter survival andcolonization Appl Environ Microbiol 65 2540ndash2546

Salzman AL Eaves-Pyles T Linn SC Denenberg AGand Szabo C (1998) Bacterial induction of inducible nitricoxide synthase in cultured human intestinal epithelial cellsGastroenterology 114 93ndash102

Saunders NF Ferguson SJ and Baker SC (2000) Tran-scriptional analysis of the nirS gene encoding cytochromecd1 nitrite reductase of Paracoccus pantotrophus LMD9263 Microbiology 146 509ndash516

Smith CK Kaiser P Rothwell L Humphrey T BarrowPA and Jones MA (2005) Campylobacter-inducedcytokine responses in avian cells Infect Immun 73 2094ndash2100

van Spanning RJ De Boer AP Reijnders WN SpiroS Westerhoff HV Stouthamer AH and Van der OostJ (1995) Nitrite and nitric oxide reduction in Paracoccusdenitrificans is under the control of NNR a regulatory pro-tein that belongs to the FNR family of transcriptional acti-vators FEBS Lett 360 151ndash154

van Spanning RJ Houben E Reijnders WN Spiro SWesterhoff HV and Saunders N (1999) Nitric oxide isa signal for NNR-mediated transcription activation in Para-coccus denitrificans J Bacteriol 181 4129ndash4132

Stevanin TM Ioannidis N Mills CE Kim SO HughesMN and Poole RK (2000) Flavohemoglobin Hmpaffords inducible protection for Escherichia coli respirationcatalyzed by cytochromes bocent or bd from nitric oxideJ Biol Chem 275 35868ndash35875

Stevanin TM Poole RK Demoncheaux EA and ReadRC (2002) Flavohemoglobin Hmp protects Salmonellaenterica serovar Typhimurium from nitric oxide-relatedkilling by human macrophages Infect Immun 70 4399ndash4405

Szymkowiak C Kwan W-S Su Q Toner TJ ShawAR and Youil R (2003) Rapid method for the character-ization of 3cent and 5cent UTRs of influenza viruses J VirolMethods 107 15ndash20

Tosques IE Shi J and Shapleigh JP (1996) Cloning andcharacterization of nnrR whose product is required for theexpression of proteins involved in nitric oxide metabolismin Rhodobacter sphaeroides 243 J Bacteriol 178 4958ndash4964

Touati D Jacques M Tardat B Bouchard L andDespied S (1995) Lethal oxidative damage and mutagen-esis are generated by iron in delta fur mutants of Escher-ichia coli protective role of superoxide dismutase JBacteriol 177 2305ndash2314

van Vliet AHM Wooldridge KG and Ketley JM (1998)Iron-responsive gene regulation in a Campylobacter jejunifur mutant J Bacteriol 180 5291ndash5298

van Vliet AHM Baillon MH Penn CW and Ketley JM(1999) Campylobacter jejuni contains two Fur homologscharacterization of iron-responsive regulation of peroxide

stress defense genes by the PerR repressor J Bacteriol181 6371ndash6376

Wassenaar TM Fry BN and van der Zeijst BA (1993)Genetic manipulation of Campylobacter evaluation of nat-ural transformation and electro-transformation Gene 132131ndash135

Watmough NJ Butland G Cheesman MR Moir JWRichardson DJ and Spiro S (1999) Nitric oxide in bac-teria synthesis and consumption Biochim Biophys Acta1411 456ndash474

Webb JL Harvey MW Holden DW and Evans TJ(2001) Macrophage nitric oxide synthase associates withcortical actin but is not recruited to phagosomes InfectImmun 69 6391ndash6400

Weinberg JB (1999) Human mononuclear phagocyte nitricoxide production and inducible nitric oxide synthaseexpression In Nitric Oxide and Infection Fang FC (ed)New York Academic Press pp 95ndash150

Wittenberg JB Bolognesi M Wittenberg BA and Guer-tin M (2002) Truncated hemoglobins a new family ofhemoglobins widely distributed in bacteria unicellulareukaryotes and plants J Biol Chem 277 871ndash874

Witthoft T Eckmann L Kim JM and Kagnoff MF(1998) Enteroinvasive bacteria directly activate expressionof iNOS and NO production in human colon epithelial cellsAm J Physiol 275 G564ndashG571

Wosten MM Boeve M Koot MG van Nuene AC andvan der Zeijst BA (1998) Identification of Campylobacterjejuni promoter sequences J Bacteriol 180 594ndash599

Wosten MM Wagenaar JA and van Putten JP (2004)The FlgSFlgR two-component signal transduction systemregulates the fla regulon in Campylobacter jejuni J BiolChem 279 16214ndash16222

Wren BW Henderson J and Ketley JM (1993) A PCR-based strategy for the rapid construction of defined bacte-rial deletion mutants Biotechniques 16 994ndash996

Wu G Wainwright LM and Poole RK (2003) Microbialglobins Adv Microbial Physiol 47 257ndash300

Supplementary material

The following supplementary material is available for thisarticle onlineTable S1 FDR corrected genes over twofold upregulated in11168 treated with GSNO (experiment 1 11168 versus11168 GSNO treated)Table S2 FDR corrected genes over twofold downregulatedin GSNO-treated 11168 (experiment 1 11168 versus 11168GSNO treated)Table S3 FDR corrected genes over twofold upregulated inCJNSSR1 (experiment 2 11168 treated with GSNO versusCJNSSR1 treated with GSNO)Table S4 FDR corrected genes over twofold downregulatedin CJNSSR1 (experiment 2 11168 treated with GSNO versusCJNSSR1 treated with GSNO)

736

K T Elvers

et al



57

735ndash750

response to NO and reactive nitrogen species The NorRprotein of

Escherichia coli

which belongs to the family oftwo-component response regulators and which regulatesthe expression of flavorubredoxin in response to nitrosa-tive stress (Hutchings

et al

2002 Gardner

et al

2003)appears to be a dedicated sensor of reactive nitrogenspecies (Mukhopadhyay

et al

2004) NnrR and Nnrmembers of the Crp-Fnr superfamily have evolved prima-rily to regulate the NO-responsive induction of nitritereductase and NO reductase in the denitrifying bacteria

Rhodobacter sphaeroides

and

Paracoccus denitrificans

(van Spanning

et al

1995 1999 Tosques

et al

1996Kwiatkowski

et al

1997) Most recently prokaryotic pro-teins which possess the H-NOX domain of the eukaryoticNO-responsive soluble guanylate cyclases have beenimplicated as NO sensors (Nioche

et al

2004 Boon andMarletta 2005) In addition a number of other regulatorswhich play prominent roles in other stress responses havealso been implicated in NO-signalling For example SoxRand OxyR are sensors for superoxide and hydrogen per-oxide respectively but as NO interacts with the C199residue in OxyR (Kim

et al

2002) and also directly withthe 2Fe-2S centres of SoxR (Ding and Demple 2000)these regulators have been implicated in NO signallingSimilarly while the primary role of Fur is iron sensing NOalso reacts with the Fe

2

+

cofactor (DrsquoAutreaux

et al

2002)and consequently a role in

Salmonella

for Fur in NOdetection has been suggested (Crawford and Goldberg1998) The Fur and PerR regulons in

Bacillus subtilis

respond primarily to iron limitation and peroxide stressrespectively (Bsat

et al

1998) but the ability of NO tonitrosylate the Fe

2

+

centres in these metalloregulators alsoexplains its ability to induce the expression of componentsof the Fur and PerR regulons in this organism (Moore

et al

2004) Finally in

E coli

the global anaerobic regu-lator Fnr (Green

et al

2001) plays a role in the NO-responsive regulation of

hmp

(Cruz-Ramos

et al

2002)


is a Gram-negative microaero-philic enteric pathogen that is recognized as the predom-inant agent of bacterial gastrointestinal disease worldwide(Friedman

et al

2000)

C jejuni

encounters elevated lev-els of nitrosative stress during infection as NO synthesisis markedly increased in patients with infective gastroen-teritis (Forte

et al

1999) and particularly so following

Campylobacter

infection (Enocksson

et al

2004) Wehave demonstrated previously that the single domainglobin Cgb performs a major NO scavenging and detox-ification function in this pathogen (Elvers

et al

2004)Consistent with the protective role of Cgb against NO-related stress

cgb

expression is minimal in standard lab-oratory media but strongly and specifically induced fol-lowing exposure to nitrosative stress This study hasfocused on elucidating the identity of the NO-sensing reg-ulator for

cgb

expression and here Cj0466 a member of

the Crp-Fnr superfamily of transcriptional regulatorswas identified as a regulator of the nitrosative stress-responsive expression of Cgb Furthermore Cj0466 (des-ignated NssR) was shown to regulate the expression of anitrosative stress-responsive regulon of which Cgb is acomponent and which also includes among other pro-teins a truncated haemoglobin

Results

Cj0466 but not Fur or PerR mediates the expression of Cgb in response to reactive nitrogen species

Analysis of the

C jejuni

genome sequence (Parkhill

et al

2000) reveals a number of potential regulators which byanalogy with other bacteria could sense NO and mediatethe NO-responsive expression of Cgb in

C jejuni

(Elvers

et al

2004) These include Fur (van Vliet

et al

1998)PerR which is related to Fur and also a Fe

2

+

-containingmetalloregulator (van Vliet

et al

1999) and Cj0466 amember of the superfamily of Crp-Fnr transcription regu-lators (Korner

et al

2003) The gene encoding Cj0466was disrupted in

C jejuni

NCTC 11168 by insertion of akanamycin resistance cassette to generate CJNSSR1 Todetermine whether this protein or other potential regula-tors played a role in modulating

cgb

expression mutantswere tested for inducible expression of this globin in com-parison to the parental strain NCTC 11168 using immu-noblotting as described previously (Elvers

et al

2004) Inthe wild type and the

fur


et al

1998) the expression profile of Cgb was identical Expres-sion was not detectable in the absence of nitrosativestress but was strongly induced 25 h after the addition of01 mM GSNO (Fig 1) While Cgb expression was clearlyinducible by GSNO in the

perR


et al

1999) the level of induced expression was slightlylower than that detected in the wild type In contrastinducible expression of Cgb was almost totally abolishedin the Cj0466 mutant suggesting a prominent role for thisregulator in NO-responsive

cgb

expression As a result ofthese studies Cj0466 was designated as NssR (Nitrosa-tive stress sensing Regulator)

Phenotypic analysis of the

nssR

mutant CJNSSR1

Diffusion assays on solid medium were used to screenthe sensitivity of CJNSSR1 to a range of stress-inducingagents in comparison to the wild type a

cgb

mutant(CJCGB01) a

fur

mutant (AV17) and a

perR

mutant(AV63) As expected CJNSSR1 was sensitive to GSNOwith a significantly greater zone of inhibition comparedwith the wild type (Table 1) As the

cgb

mutant exhibiteda similar zone of killing (Elvers

et al

2004) the sensitivityof CJNSSR1 to GSNO is likely to be at least in part aconsequence of its failure to induce Cgb expression


C jejuni 737



57

735ndash750


tert


Campylobacter

(Purdy

et al

1999) The

perR


et al

1999) Notably the

fur


The effect of the

nssR



ndash


nssR


cgb


Microarray analysis


cgb




Wild type

AV17 (Fur-)

AV63 (PerR-)

CJNSSR1 (Cj0466-)


0 25 45


CJNSSRC

Wild type

AV17 (Fur-)

AV63 (PerR-)

CJNSSR1 (Cj0466-)


0 25 45


CJNSSRC


Stress inducer



NCTC 11168(n = 6)





118 plusmn 08(P = 051)

147 plusmn 04(P = 89 yen 10-5)


348 plusmn 08(P = 00003)

422 plusmn 48(P = 059)


323 plusmn 08(P = 002)

397 plusmn 06(P = 0001)


250 plusmn 00(P = 48 yen 10-6)

385 plusmn 35(P = 041)








0 05 1 15 2

GSNO (mM)

cfu

ml-1

108

107

106

105

104

103



Gene
















001

01

1

10

100


Cha

nge

inge

neex

pres

sion



001

01

1

10

100


Cha

nge

inge

neex

pres

sion












001

01

1

10

100

11168

CJNSSR1

CJNSSRC

Cha

nge

inge

neex

pres

sion

Cj0761Cj0830Cj0465c

001

01

1

10

100

11168

CJNSSR1

CJNSSRC

Cha

nge

inge

neex

pres

sion

Cj0761Cj0830Cj0465c








Discussion















0

20

40

60

80

100

120

140

160

180

pMW10 pKE117 pKE120

Reporter construct

b-ga

lact

osid

ase

activ

ity (

Mil

ler

unit


100 mM 1 h

100 mM 2 h

200 mM 1h

200 mM 2 h

1 2 3 4 5 6 7 8

Wild type

200 mM 2 h CJNSSR1




































StrainsC jejuni











Microarray analysis






























Acknowledgements


References


















































































C jejuni 737



57

735ndash750


tert


Campylobacter

(Purdy

et al

1999) The

perR


et al

1999) Notably the

fur


The effect of the

nssR



ndash


nssR


cgb


Microarray analysis


cgb




Wild type

AV17 (Fur-)

AV63 (PerR-)

CJNSSR1 (Cj0466-)


0 25 45


CJNSSRC

Wild type

AV17 (Fur-)

AV63 (PerR-)

CJNSSR1 (Cj0466-)


0 25 45


CJNSSRC


Stress inducer



NCTC 11168(n = 6)





118 plusmn 08(P = 051)

147 plusmn 04(P = 89 yen 10-5)


348 plusmn 08(P = 00003)

422 plusmn 48(P = 059)


323 plusmn 08(P = 002)

397 plusmn 06(P = 0001)


250 plusmn 00(P = 48 yen 10-6)

385 plusmn 35(P = 041)








0 05 1 15 2

GSNO (mM)

cfu

ml-1

108

107

106

105

104

103



Gene
















001

01

1

10

100


Cha

nge

inge

neex

pres

sion



001

01

1

10

100


Cha

nge

inge

neex

pres

sion












001

01

1

10

100

11168

CJNSSR1

CJNSSRC

Cha

nge

inge

neex

pres

sion

Cj0761Cj0830Cj0465c

001

01

1

10

100

11168

CJNSSR1

CJNSSRC

Cha

nge

inge

neex

pres

sion

Cj0761Cj0830Cj0465c








Discussion















0

20

40

60

80

100

120

140

160

180

pMW10 pKE117 pKE120

Reporter construct

b-ga

lact

osid

ase

activ

ity (

Mil

ler

unit


100 mM 1 h

100 mM 2 h

200 mM 1h

200 mM 2 h

1 2 3 4 5 6 7 8

Wild type

200 mM 2 h CJNSSR1




































StrainsC jejuni











Microarray analysis






























Acknowledgements


References























































































0 05 1 15 2

GSNO (mM)

cfu

ml-1

108

107

106

105

104

103



Gene
















001

01

1

10

100


Cha

nge

inge

neex

pres

sion



001

01

1

10

100


Cha

nge

inge

neex

pres

sion












001

01

1

10

100

11168

CJNSSR1

CJNSSRC

Cha

nge

inge

neex

pres

sion

Cj0761Cj0830Cj0465c

001

01

1

10

100

11168

CJNSSR1

CJNSSRC

Cha

nge

inge

neex

pres

sion

Cj0761Cj0830Cj0465c








Discussion















0

20

40

60

80

100

120

140

160

180

pMW10 pKE117 pKE120

Reporter construct

b-ga

lact

osid

ase

activ

ity (

Mil

ler

unit


100 mM 1 h

100 mM 2 h

200 mM 1h

200 mM 2 h

1 2 3 4 5 6 7 8

Wild type

200 mM 2 h CJNSSR1




































StrainsC jejuni











Microarray analysis






























Acknowledgements


References


























































































001

01

1

10

100


Cha

nge

inge

neex

pres

sion



001

01

1

10

100


Cha

nge

inge

neex

pres

sion












001

01

1

10

100

11168

CJNSSR1

CJNSSRC

Cha

nge

inge

neex

pres

sion

Cj0761Cj0830Cj0465c

001

01

1

10

100

11168

CJNSSR1

CJNSSRC

Cha

nge

inge

neex

pres

sion

Cj0761Cj0830Cj0465c








Discussion















0

20

40

60

80

100

120

140

160

180

pMW10 pKE117 pKE120

Reporter construct

b-ga

lact

osid

ase

activ

ity (

Mil

ler

unit


100 mM 1 h

100 mM 2 h

200 mM 1h

200 mM 2 h

1 2 3 4 5 6 7 8

Wild type

200 mM 2 h CJNSSR1




































StrainsC jejuni











Microarray analysis






























Acknowledgements


References

























































































001

01

1

10

100

11168

CJNSSR1

CJNSSRC

Cha

nge

inge

neex

pres

sion

Cj0761Cj0830Cj0465c

001

01

1

10

100

11168

CJNSSR1

CJNSSRC

Cha

nge

inge

neex

pres

sion

Cj0761Cj0830Cj0465c








Discussion















0

20

40

60

80

100

120

140

160

180

pMW10 pKE117 pKE120

Reporter construct

b-ga

lact

osid

ase

activ

ity (

Mil

ler

unit


100 mM 1 h

100 mM 2 h

200 mM 1h

200 mM 2 h

1 2 3 4 5 6 7 8

Wild type

200 mM 2 h CJNSSR1




































StrainsC jejuni











Microarray analysis






























Acknowledgements


References























































































Discussion















0

20

40

60

80

100

120

140

160

180

pMW10 pKE117 pKE120

Reporter construct

b-ga

lact

osid

ase

activ

ity (

Mil

ler

unit


100 mM 1 h

100 mM 2 h

200 mM 1h

200 mM 2 h

1 2 3 4 5 6 7 8

Wild type

200 mM 2 h CJNSSR1




































StrainsC jejuni











Microarray analysis






























Acknowledgements


References























































































0

20

40

60

80

100

120

140

160

180

pMW10 pKE117 pKE120

Reporter construct

b-ga

lact

osid

ase

activ

ity (

Mil

ler

unit


100 mM 1 h

100 mM 2 h

200 mM 1h

200 mM 2 h

1 2 3 4 5 6 7 8

Wild type

200 mM 2 h CJNSSR1




































StrainsC jejuni











Microarray analysis






























Acknowledgements


References



















































































































StrainsC jejuni











Microarray analysis






























Acknowledgements


References






















































































Microarray analysis






























Acknowledgements


References





































































































Acknowledgements


References

















































































NssR, a member of the Crp-Fnr superfamily from Campylobacter jejuni, regulates a nitrosative...

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Transcript of NssR, a member of the Crp-Fnr superfamily from Campylobacter jejuni, regulates a nitrosative...