Identiﬁcation and regulation of cold-inducible factors of … · 2015-07-28 · Identiﬁcation...

Identification and regulation of cold-induciblefactors of Bordetella bronchiseptica

Dorothee Stubs,1 Thilo M. Fuchs,2 Boris Schneider,13 Armin Bosserhoff3

and Roy Gross1

Correspondence

Roy Gross

[email protected]

1Lehrstuhl fur Mikrobiologie, Biozentrum der Universitat Wurzburg, Am Hubland, D-97074Wurzburg, Germany

2Zentralinstitut fur Ernahrungs- und Lebensmittelforschung, Abteilung Mikrobiologie, D-85354Freising, Germany

3Zentrum fur Molekulare Biologie, Universitat Heidelberg, D-69120 Heidelberg, Germany

Received 24 November 2004

Revised 22 February 2005

Accepted 7 March 2005

The expression of bacterial cold-shock proteins (CSPs) is highly induced in response to cold

shock, and some CSPs are essential for cells to resume growth at low temperature. Bordetella

bronchiseptica encodes five CSPs (named CspA to CspE) with significant amino acid homology

to CspA of Escherichia coli. In contrast to E. coli, the insertional knock-out of a single csp gene

(cspB) strongly affected growth of B. bronchiseptica independent of temperature. In the

case of three of the csp genes (cspA, cspB, cspC) more than one specific transcript could be

detected. The net amount of cspA, cspB and cspC transcripts increased strongly after cold

shock, while no such effect could be observed for cspD and cspE. The exposure to other stress

conditions, including translation inhibitors, heat shock, osmotic stress and nutrient deprivation

in the stationary phase, indicated that the csp genes are also responsive to these conditions. The

coding regions of all of the cold-shock genes are preceded by a long non-translated upstream

region (59-UTR). In the case of the cspB gene, a deletion of parts of this region led to a significant

reduction of translation of the resulting truncated transcript, indicating a role of the 59-UTR in

translational control. The cold-shock stimulon was investigated by 2D-PAGE and mass

spectrometric characterization, leading to the identification of additional cold-inducible proteins

(CIPs). Interestingly, two cold-shock genes (cspC and cspD) were found to be under the negative

control of the BvgAS system, the main transcriptional regulator of Bordetella virulence genes.

Moreover, a negative effect of slight overexpression of CspB, but not of the other CSPs, on the

transcription of the adenylate cyclase toxin CyaA of Bordetella pertussiswas observed, suggesting

cross-talk between the CSP-mediated stress response stimulon and the Bordetella virulence

regulon.

INTRODUCTION

Almost all single-celled organisms respond to a suddendecrease in temperature with a specific adaptive programmethat results in a decrease of membrane fluidity, an increasein the negative supercoiling of DNA and an inhibition oftranslational initiation of bulk mRNA (Jones & Inouye,1994). During this cold-adaptation phase, the transientinduction of a subset of proteins termed cold-inducedproteins (CIPs) is observed (Graumann & Marahiel, 1998;Gualerzi et al., 2003). Among these CIPs there is a family of

small and mostly quite acidic proteins, the cold-shockproteins (CSPs). These CSPs mainly consist of the cold-shock domain, which is found in many nucleic acidbinding proteins of prokaryotic and eukaryotic organisms(Graumann & Marahiel, 1998). Escherichia coli K-12encodes nine csp genes, but only four of them are stronglyinduced after cold shock (Wang et al., 1999). Accordingly, itis assumed that the other members of the CSP family areinvolved in the protection against stresses other than lowtemperature; e.g. transcription of the cspD gene is responsiveto nutrient deprivation (Yamanaka et al., 1998). Theresponse to cold shock has been extensively studied inE. coli, where a specific set of 26–27 CIPs is induced underthese conditions (Gualerzi et al., 2003). The majorityof these proteins bind nucleic acids and contribute tofundamental functions such as DNA packaging, transcrip-tion, RNA degradation, translation or ribosome assembly.

3Present address: Ingenium Pharmaceuticals AG, Fraunhofer Str. 13,D-82152 Martinsried, Germany.

Abbreviations: CIP, cold-inducible protein; CSP, cold-shock protein;RT-qPCR, real-time quantitative PCR; 59-UTR, upstream untranslatedregion; VRG, virulence-repressed gene.

0002-7785 G 2005 SGM Printed in Great Britain 1895

Microbiology (2005), 151, 1895–1909 DOI 10.1099/mic.0.27785-0

The production of the major cold-shock protein CspA istransient and drops to a basal level after a few hours(Jones et al., 1987). CspA expression appears to be regu-lated mainly on the posttranscriptional level, e.g. bydifferential mRNA stability (Brandi et al., 1996). CspAacts as transcriptional antiterminator of certain genes thatare upregulated in the cold (Bae et al., 2000) as well as anRNA chaperone, which prevents the formation of secondarystructures in RNA, and thus ensures efficient translationof mRNA at low temperature (Jiang et al., 1997). Fourhours after a cold shock, the cold-adaptation phase iscompleted, and regular protein synthesis and growth areresumed.

Most members of the genus Bordetella are assumed to beobligatorily associated with host organisms (Gerlach et al.,2001; Weiss, 1992). Bordetella bronchiseptica causes chronicand often asymptomatic respiratory infections in a varietyof animals, but only rarely in humans (Goodnow, 1980;Weiss, 1992). It is closely related to Bordetella pertussisand Bordetella parapertussis, the aetiological agents ofwhooping cough. Recently, comparative genomics revealedreductive genome evolution to occur in B. pertussis andthe closely related pathogen B. parapertussis, which mayhave developed from a B. bronchiseptica-like ancestor(Parkhill et al., 2003). So far, only a single Bordetellaspecies, Bordetella petrii, has been isolated from anenvironmental location (von Wintzingerode et al., 2001).Interestingly, B. bronchiseptica also has the capacity tosurvive harsh environmental conditions, and may there-fore be able to exist outside a host organism (Porter et al.,1991). There appears to be an evolutionary trend in thepathogenic members of the genus Bordetella to lose factorspossibly involved in survival outside of the host uponspecialization to specific host organisms (Gerlach et al.,2001).

Despite the reductive evolution during adaptation to anapparently obligate association with warm-blooded animalsor man, the genes encoding cold-shock proteins are con-served among all Bordetella species. In fact, the family ofCSPs consists of five members (CspA to CspE) and all fivegenes are found in the genomes of all Bordetella speciessequenced so far (B. pertussis, B. parapertussis, B. bronchi-septica, Bordetella avium and B. petrii). In this study,the phenotype of csp mutants and the pattern of differ-ential expression of the cold-shock genes in response todifferent stress conditions including cold shock, salt stress,heat shock and treatment with antibiotics were inves-tigated. By proteome analysis, we show that B. bronchisepticahas a cold shock response similar to that of E. coli and otherbacteria. In addition, we analysed the expression of cspBand obtained evidence for transcriptional control of itsexpression in B. bronchiseptica and for a posttranscrip-tional regulatory function of the long non-translatedupstream region preceding the coding region of the cspBgene.

METHODS

Bacterial strains and growth conditions. The strains used inthis study are listed in Table 1. All Bordetella strains were grownat 37 uC either on Stainer–Scholte (SS) agar plates containingdefibrinated horse blood [5% (v/v)] or on charcoal (CC) agar plates(Difco). Liquid cultures of bordetellae were grown in SS broth(Stainer & Scholte, 1970), and E. coli strains were cultivated inLuria–Bertani (LB) broth. Antibiotics were used at the follow-ing concentrations: streptomycin sulfate, 100 mg ml21; ampicillin,100 mg ml21; gentamicin, 15 mg ml21; kanamycin, 25 mg ml21;tetracycline, 30 mg ml21; chloramphenicol, 30 mg ml21.

General techniques. DNA manipulations, isolation of chromoso-mal DNA and b-galactosidase assays were performed as described instandard protocols (Sambrook & Russell, 2001). DNA sequencingwas done using the T7Sequencing Kit (Amersham Pharmacia Biotech)based on a variant of the dideoxynucleotide method (Sanger et al.,1977) as recommended by the manufacturer. For Western hybridiza-tions, an ECL direct labelling and detection kit (Amersham) was used.Conjugational DNA transfer into B. pertussis and B. bronchisepticawas carried out as described previously (Gross & Rappuoli, 1988)with E. coli strain SM10 as a donor (Simon et al., 1983).

Computer analysis. For the analysis of nucleotide and amino acidsequences, we used the HUSAR program package (http://genome.dkfz-heidelberg.de/), which is based on the GCG program packagedescribed by Devereux et al. (1984), the databases of the SangerInstitute (http://www.sanger.ac.uk/), and the pedant database of theMunich information center for protein sequences (mips; http://pedant.gsf.de/).

Construction of a B. bronchiseptica cspB deletion mutant.Using the primer pairs cspB-KO-5-BamHI/cspB-KO-5-PstI andcspB-KO-3-PstI/cspB-KO-3-HindIII, two DNA fragments of 404and 429 bp in size flanking the cspB gene were amplified from chro-mosomal B. bronchiseptica BB7865 DNA (Table 2). The amplified404 and 429 bp DNA fragments were digested with BamHI/PstI andPstI/HindIII, respectively, and cloned into pBluescriptKS. Follow-ing linearization of the resulting construct with PstI, a kanamycin-resistance cassette derived from pUC4K was cloned between theregions flanking the cspB gene on the chromosome. Both flankingsequences and the kanamycin cassette were then amplified by PCRusing the primers cspB-KO-5-BamHI and cspB-KO-3-BamHI tointroduce a second BamHI restriction site. The resulting BamHIfragment was then cloned into the allelic exchange vector pSS1129.The resulting construct, pSS1129-DcspB, was transferred by conjuga-tion into B. bronchiseptica, and selection for double-crossover eventswas carried out as described elsewhere (Carbonetti et al., 1994;Stibitz & Yang, 1991). The resulting cspB deletion mutant wastermed BB7865DcspB.

Construction of cspB : :gfp fusions and determination ofGFP fluorescence. A DNA fragment containing a promoterless gfpgene was excised with XbaI and HindIII from plasmid pKEN2 andligated into plasmid pBluescriptSK, giving rise to plasmid pSK-gfp.A 96 bp DNA fragment containing part of the upstream region ofthe cspB gene was amplified by PCR from chromosomal B. bronchi-septica DNA using the primer pair cspB-57-BamHI/cspB+39-XbaI.The resulting fragment was cloned into pSK-gfp to obtain plasmidpSK-I-gfp. The DNA fragment comprising the cspB upstream regionfused to gfp was then excised with BamHI and HindIII, and sub-sequently ligated into plasmid pMMB206. E. coli SM10 wastransformed with the resulting construct pMMB206-I-gfp and theconstruct was transferred into B. bronchiseptica wild-type strainBB7865 by conjugation. The plasmids pMMB206-II-gfp and

1896 Microbiology 151

D. Stubs and others

pMMB206-III-gfp were constructed in an analogous manner using

the primer pairs cspB-74-BamHI/cspB+39-XbaI and cspB-105-

BamHI/cspB+39-XbaI, respectively.

To investigate the role of the 59-UTR of the cspB gene, three addi-

tional plasmids, pMMB206-IV-gfp, pMMB206-V-gfp and pMMB206-

VI-gfp, were constructed. The construction of these plasmids was

performed essentially as described above, using the primer pairs cspB-

105-BamHI/cspB+136-XbaI to generate construct IV and the primer

pair cspB-105-BamHI/cspB+87-XbaI to obtain construct V. In the

case of pMMB206-VI-gfp, the sequence of the conserved ‘9 bp box’

was changed to GGGGAATTC by site-directed mutagenesis. For this

purpose, a PCR was performed using the primer pair DelC/DelDcontaining the desired mutated sequence and the plasmid pMMB206-IV-gfp as template. The oligonucleotides were extended by Pfu DNApolymerase (Promega) during temperature cycling (PCR programme:95 uC/30 s; 18 cycles of 95 uC/30 s, 55 uC/1 min, 68 uC/10 min; 72 uC/10 min). Following PCR amplification the DNA fragment was treatedwith DpnI (New England Biolabs), and E. coli strain DH5a was thentransformed with the nicked vector DNA. The mutated cspB genefragment was digested with BamHI and HindIII, confirmed by DNAsequencing, and cloned into the pMMB206 vector for conjugation inB. bronchiseptica strain BB7865. FACS analysis was done as describedpreviously (Schneider et al., 2002).

Table 1. Bacterial strains and plasmids

Strain or plasmid Relevant feature Source or reference

E. coli K-12

DH5a High-efficiency transformation Gibco-BRL

SM10 Mobilizing strain Simon et al. (1983)

B. pertussis

Tohama I Dtox Derivative of B. pertussis Tohama I, but rpsL and Dptx Weiss & Falkow (1984)

B. bronchiseptica

BB7865 Wild-type, but rpsL Arico et al. (1997)

BB7866 Phase variant of BB7865 with mutation in bvgS Monack et al. (1989)

BB7865DcspB As for BB7865, but DcspB This study

BB7865cspC : : Tn5 As for BB7865, but cspC : : Tn5 Schneider (2001)

18323A4 Derivative of B. pertussis 18323 carrying a fusion

of lacZ with the PcyaA promoter

Goyard & Ullmann (1991)

Plasmids

pBluescript High-copy-number cloning vector Stratagene

pSL1180 High-copy-number cloning vector Pharmacia

pUC4K Vector encoding a kanamycin cassette Amersham

pKEN High-copy-number cloning vector containing a promoterless gfp gene Cormack et al. (1996)

pMMB206 Broad-host-range vector Morales et al. (1991)

pSS1129 Vector for allelic exchange Stibitz & Yang (1991)

pLAFR2 Broad-host-range vector Friedman et al. (1982)

pLAFR2-cspA pLAFR2 containing a 0?7 kb DNA fragment of B. bronchiseptica encoding cspA This study

pLAFR2-cspABC pLAFR2 containing a 0?4 kb DNA fragment of Bacillus cereus encoding cspA This study; Mayr et al. (1996)

pLAFR2-cspB pLAFR2 containing a 0?5 kb DNA fragment of B. bronchiseptica encoding the

cspB locus

This study

pLAFR2-cspC pLAFR2 containing a 0?9 kb DNA fragment of B. bronchiseptica encoding cspC This study

pLAFR2-cspD pLAFR2 containing a 0?7 kb DNA fragment of B. bronchiseptica encoding cspD This study

pLAFR2-cspE pLAFR2 containing a 0?5 kb DNA fragment of B. bronchiseptica encoding cspE This study

pSS1129-DcspB pSS1129 containing the kanamycin gene flanked by the upstream and

downstream sequences of cspB of B. bronchiseptica

This study

pMMB206-I-gfp pMMB206 containing the cspB 59-UTR of B. bronchiseptica (257 to +39)

fused to a promoterless gfp reporter gene

This study

pMMB206-II-gfp pMMB206 containing the cspB 59-UTR of B. bronchiseptica (274 to +39)


This study

pMMB206-III-gfp pMMB206 containing the cspB 59-UTR of B. bronchiseptica (2105 to +39)


This study

pMMB206-IV-gfp pMMB206 containing the cspB 59-UTR of B. bronchiseptica (2105 to +136)


This study

pMMB206-V-gfp pMMB206 containing the cspB 59-UTR of B. bronchiseptica (2105 to +87)


This study

pMMB206-VI-gfp pMMB206 containing the cspB 59-UTR of B. bronchiseptica (2105 to +136)

fused to a promoterless gfp reporter gene, but containing a mutated 9 bp

box in the 59-UTR

This study

http://mic.sgmjournals.org 1897

Cold-inducible factors of B. bronchiseptica

Table 2. Oligonucleotides

Name Sequence

cspA-PE 59-TTC ACC AAG TTA CGG CTT TTC CC-39

cspB-PE 59-GCA GCA ATT CAC CAA GTT GC-39

cspC-PE 59-GTT CCA GTT CAC CAA GTT GC-39

cspD-PE 59-GTC CCC TTG GAC AGC GGT TGC GG-39

cspE-PE 59-CCC TTC TCG TCG TTG AAC CAC TTG-39

cyaA-PE 59-CGG CGG CGT TTG CGT AAC CAG CCT GAT GCG-39

bvgA-PE 59-ATA TGC TTT CAG TTT CG-39

cspB-KO-5-BamHI 59-GGC GGA TCC CAG CCA CCT GCT TGC CGA AC-39

cspB-KO-5-PstI 59-GGC CTG CAG CTG CCG TGC CAC GAT GC-39

cspB-KO-3-PstI 59-GGC CTG CAG GCA CTT CTC GGA AAT-39

cspB-KO-3-HindIII 59-GGC AAG CTT GAA TTG ATC CAG CAG-39

cspB-KO-3-BamHI 59-GGC GGA TCC GAA TTG ATC CAG CAG-39

cspB-5-KO-PCR 59-GGT CAT GCG CAC ACC TTA TAA GC-39

cspB-3-KO-PCR 59-CGT CGC CTG AGG ACC CTT CGG-39

cspA-I 59-GGC GGA TCC GCT GCA CGC CGA AAC CTT GC-39

cspA-II 59-GGC GGA TCC TCA AGC GAT GAC TTT TAT GT-39

cspB-I 59-GGC GGA TCC CCG ACA TTC ACG CGG CGG-39

cspB-II 59-GGC GGA TCC GCT TCG AAA CGC ATG G-39

cspC-I 59-GGC GGA TCC ACC TGT CGG ATC CAG GTG CCG-39

cspC-II 59-GGC GGA TCC GCA TGT AGG GAT CCT GGT TGT GCC-39

cspD-I 59-GGC GGA TCC GGT TCC ATA ATG CAC TGC AG-39

cspD-II 59-GGC GGA TCC TCA GGC AGA CGT AAT ATT GA-39

cspE-I 59-GGC GGA TCC CGA TCT GTT CGG GTG CAT GC-39

cspE-II 59-GGC GGA TCC TCA CAA CGG GGT GAT CTT GG-39

tex for 59-TGC AGG ATC CTT TGG CCG AAC T-39

tex rev 59-GTT CAC GCA GTC CTC GAC CA-39

cspB for 59-TTA TGG CTT CAT CAC CCC-39

cspB rev 59-TGG ACG CGC TCA GAT TCT TA-39

gltA for 59-TGG AAT ACA CCA TCG TGG T-39

gltA rev 59-AGG CCT GCT TGG TCA GGT CGT-39

atpA for 59-ACA AGT ATT CGC AAG GCC A-39

atpA rev 59-TGC AGG ATG AAG ATG CGA T-39

sbp for 59-TCA TCA TGG CGA CCT TGA-39

sbp rev 59-TCG ATG GCC TCG AAA AGC T-39

gmk for 59-ATG CGC AAC AGC TGC TCG AAT-39

gmk rev 59-AGG ACG AAT ATG CCG AT-39

groES for 59-AAG CCG GAT CAA GGT GAA GT-39

groES rev 59-ATG ACG AGC AGC TCT T-39

uspA for 59-ACA TCG ACA AGT CCA TGC T-39

uspA rev 59-TGC CGA TGA AGA TCA GGT-39

pti for 59-ACC TGG TGT CCG GAG AAT TTC GA-39

pti rev 59-AAC TTG TTC AGG TCC GTC AGG A-39

infB for 59-TGA ATC TCC CAA GAG CGC CAA-39

infB rev 59-TTG CGC GGA CGG CTG AGC ATC T-39

cspB-105-BamHI 59-GGG GGA TCC GCG CAA AAA ATA AGC ATC GTG-39

cspB-74-BamHI 59-GGG CCC GGA TCC GTG TGT TTT AAT ATT GGC ACG-39

cspB-57-BamHI 59-GGG GGA TCC TTA TGT TGA CTT GTG AGG G-39

cspB+39-XbaI 59-GGG TCT AGA GGC ACT GAA CAA ACA TTG CAA CG-39

cspB+136-XbaI 59-GGG TCT AGA CTG TAT TTC CTA GAA ATT AAT GGG-39

cspB+87-XbaI 59-GGG GAA TTC CCC GCC TCG CGC CAG AAC GC-39

DelC 59-CCG GGA CCA CGA ATC GAA TTC CCC CGG GGC A-39

DelD 59-TTC TAC TGC CCC GGG GGA ATT CGA TTC G-39

Bc1 59-GGA TTG TGG ATC CTA TTT TAA G-39

Bc2 59-AAA AAA GGA TCC CAT AAG CCA TC-39


D. Stubs and others

Construction of pLAFR2 derivatives containing different cspgenes. The coding regions of five cold-shock genes were amplifiedtogether with their 59-UTR and their entire promoter regions fromchromosomal B. bronchiseptica DNA by PCR using the followingprimer pairs: cspA-I/cspA-II, cspC-I/cspC-II, cspD-I/cspD-II, cspE-I/cspE-II (Table 2). B. pertussis chromosomal DNA was used as a tem-plate to amplify cspB with the oligonucleotides cspB-I/cspB-II. ThecspA gene of Bacillus cereus was amplified with the oligonucleotidesBc1 and Bc2. The PCR fragments were digested with BamHI andcloned into the BamHI-digested low-copy vector pLAFR2. Theresulting constructs were conjugated into BB7865 as describedabove.

RNA isolation. Whole-cell RNA of Bordetella strains was isolatedas described previously (Gross & Rappuoli, 1989). Briefly, bacteriagrown under appropriate conditions were harvested in the mid-exponential phase (OD590 0?6) or in the stationary phase (OD590

1?2), and then lysed by the addition of a solution containing100 mM Tris/HCl (pH 7?5), 1% SDS and 2 mM EDTA. To test theresponse to several stress conditions, cells were grown to OD590

0?6 and then subjected to the following treatment: for heat shockthe cultures were incubated at 48 uC for 20 min, for salt shock2 M NaCl was added and the cultures were further incubated for30 min. Finally, to test the response to translation inhibitors,tetracycline (50 mg ml21) or chloramphenicol (120 mg ml21) wasadded to the cultures, and cultivation was continued for 30 min.After boiling, the samples were kept on ice in the presence of80 mM KCl for 5 min. This step was followed by the removal ofcellular debris by centrifugation. To 3?5 ml of each sample, 4?56 gCsCl was added, and the RNA was pelleted by centrifugation in aBeckman SW50Ti rotor at 35 000 r.p.m. at room temperature for20 h. Finally, the pellet was resuspended in 500 ml TE buffer [10 mMTris/HCl (pH 8?0), 1 mM EDTA], extracted with an equal volumeof a solution of 1 : 1 phenol/chloroform (v/v), precipitated oncewith ethanol, resuspended in 50 ml TE buffer and finally stored at220 uC.

Primer extension analysis. For the various csp genes, and thecyaA and bvgA genes, this was done with the appropriate PE pri-mers listed in Table 2. The oligonucleotides were labelled with[c-32P]ATP using T4 polynucleotide kinase, then hybridized withtheir respective templates. Primer extension reactions were per-formed with 25 mg total RNA for 45 min at 46 uC using avian myelo-blastosis virus reverse transcriptase (Amersham). Primer extensionproducts were resolved on polyacrylamide gels (6%) containing 6 Murea. Sequencing reactions were done in parallel using the variouspLAFR2 derivatives containing the respective csp genes as a template.Radiolabelled bands were quantified using a PhosphorImager system(Amersham Biosciences) and the corresponding software (MolecularDynamics).

RT-qPCR. For each reaction several dilutions of cDNA reversetranscribed from 5 mg total RNA with random primers and theSuperScriptII RNaseH Reverse Transcriptase (Invitrogen) were used,with 1 pg to 2 ng of genomic B. bronchiseptica BB7865 DNA as astandard. RT-PCR reactions were performed using a DNA EngineOpticon System (MJ Research) and the qPCR Core Kit for SybrGreen I (Eurogentec) according to the manufacturer’s recommenda-tions. From each of two independent RNA preparations twoseparate cDNA preparations were made. These cDNA preparationswere characterized three times each by RT-qPCR, resulting in anoverall replicate number of 12 experiments. The threshold cyclenumber was set to a fluorescence of 0?01. For this analysis the ‘for/rev’ primers listed in Table 2 and named according to the respectivegenes were used.

Rifampicin treatment. To investigate the half-life of transcripts,the B. bronchiseptica wild-type strain BB7865 was grown at 37 uC in

SS medium to OD590 0?6. Rifampicin was then added to a final con-centration of 0?25 mg ml21, and the culture was divided into twoequal samples, one of which was further incubated at 37 uC and theother was cold shocked in an ice bath and further incubated at15 uC. At different time points, total RNA was isolated from 50 mlaliquots of the cultures as described above.

Preparation of protein extracts and 2D protein gel electro-phoresis. Cultures (50 ml) of B. bronchiseptica were grown to latemid-exponential phase (OD578 1?0) and cold shocked as describedabove. At different time points after cold shock the cells were har-vested by centrifugation. Equal amounts of up to 150 mg proteinwere separated by 2D-PAGE according to the method of O’Farrell(1975). Briefly, the proteins were separated in the first dimensionby isoelectric focusing in a pH gradient ranging from 3 to 10; in thesecond dimension, the proteins were separated according to theirmolecular masses by SDS-PAGE, and visualized by Coomassie blueor silver staining. The cellular proteins of each tested strain were iso-lated in at least three separate experiments, and 2D-PAGE of eachprotein preparation was performed at least in triplicate. Only thoseprotein spots that showed a reproducible and significant increase intheir amount after cold shock were further analysed by mass spec-trometry as described previously (Kimmel et al., 2000). Evaluationand quantification of the 2D gels were performed using the DELTA2Dprogram from Decodon.

RESULTS

The family of B. bronchiseptica CSPscomprises five members

Within the genome sequence of B. bronchiseptica RB50(Parkhill et al., 2003), five ORFs were identified exhibiting ahighly significant amino acid similarity to the major cold-shock protein CspA of E. coli (Fig. 1). The five putativeCSPs show a sequence identity of 65–80% at the aminoacid level among each other and of 63–73% with CspA ofE. coli. According to their decreasing similarity with E. coli

Fig. 1. Amino acid sequences of CspA, CspB, CspC, CspDand CspE. The CLUSTALW program was used to align thesequences. Black boxes, amino acids that are identical in atleast four proteins; grey boxes, similar or identical amino acidsin at least three of the Csp proteins. The groups of aminoacids considered similar in this analysis were I, L, M and V; A,S, G, P and T; H, K and R; D, E, N and Q; F, W and Y; andC. Dashes mark the gaps placed in the sequences by theCLUSTALW program to optimize alignment. E.c., E. coli; B.b.,B. bronchiseptica.



CspA, the Bordetella CSPs were termed CspA, CspB, CspC,CspD and CspE. The calculated molecular masses of theB. bronchiseptica CSPs range from 7?3 (CspC) to 8?6 kDa(CspD). In comparison to the E. coli CspA protein (iso-electric point, pI=4?3), the pI values of the CSPs of B.bronchiseptia are significantly higher, with an unusually highpI value of 9?15 for CspD. The genomic organization of theCSP-encoding genes in B. bronchiseptica and the predictionof promoter and termination sites indicate that all of themare transcribed monocistronically (data not shown). ThecspC, cspD and cspE genes were found to be scattered overthe chromosome, while the cspA and cspB genes are clusteredand separated only by a single ORF encoding a putativeprotein of unknown function (data not shown).

Phenotypic characterization of mutants in thecspB and cspC genes

In E. coli, the deletion of single csp genes did not result in anobvious phenotype, possibly due to back-up mechanismsbased on the presence of closely related CSPs (Xia et al.,2001). In B. bronchiseptica, we investigated the pheno-typic effects of mutations in the cspB and cspC genes. Forthis purpose we constructed a cspB deletion mutant byreplacement of the gene with a kanamycin-resistancecassette, resulting in strain BB7865DcspB. In addition, weinvestigated a transposon-insertion mutant in the cspCgene described previously. The mutant, BB7865cspC : : Tn5,carries a transposon inserted at the beginning of the cspCcoding region (Schneider, 2001). The wild-type strain and

the two isogenic mutants were cultivated at 37 and 15 uC.The generation time of each strain at the different growthtemperatures was then calculated. The wild-type strain andthe cspC mutant could not be distinguished from eachother, and showed a generation time of 0?9 and 3?2 h at 37and 15 uC, respectively. Since cspC is expressed maximallyunder modulating conditions inactivating the BvgAS two-component system (see below), the experiments were alsoperformed in the presence of modulating agents such asMgSO4 and nicotinic acid, but again no difference betweenthe wild-type and the cpsC mutant could be observed. Incontrast, the cspB mutant grew much more slowly under allgrowth conditions tested and a generation time of 3?7 and12?6 h could be calculated at 37 and 15 uC, respectively (datanot shown). Thus, the generation time of the cspB mutantwas prolonged about fourfold independently of the tem-perature conditions applied.

Transcript analysis of cold-shock genes

To characterize the promoters of the five B. bronchisepticacsp genes and to analyse the effect of cold stress on theirtranscriptional response, we conducted primer extensionexperiments with RNA preparations from wild-type strainBB7865 cultivated at 37 uC or cold shocked for 30 min at15 uC. All five B. bronchiseptica cold-shock genes compriseextremely long 59-UTRs ranging from 109 bp (T1cspD) to167 bp (T2cspB), a feature that appears to be characteristicfor many of the cold-shock genes of eubacteria (Fig. 2) (Baeet al., 1997; Brandi et al., 1996; Fang et al., 1997; Jones &

Fig. 2. Nucleotide sequences of the promo-ter and upstream regions of the five csp

genes of B. bronchiseptica. The start sitesof the transcripts were determined by primerextension experiments and are marked byarrows, and in case of more than one tran-script, the corresponding number. Putativeribosome-binding sites (Shine–Dalgarnosequences) are underlined and the con-served sequence motif present in the 59-UTR of four of the five csp genes, the 9 bpbox, is shaded.


D. Stubs and others

Inouye, 1994). Within the 59-UTRs of four of the cspgenes (cspA, cspB, cspC and cspE), a highly conserved 9 bpsequence motif, the 9 bp box, with the consensus sequence59-TCCTTGATT-39 was identified (Fig. 2). In all cases, thismotif was found to be located approximately 51–57 bpupstream of the respective start codon. This sequence motifdoes not show any similarity with the ‘cold box’ present inthe 59-UTR of transcripts of the csp genes of E. coli, for whicha regulatory function was suggested previously (Jiang et al.,1996).

In the case of the cspA, cspB and cspC genes more than onetranscript could be detected. In the case of the cspB gene, twotranscripts were identified, which according to the deletionanalysis of the promoter region of this gene are likely tobe transcribed from two independent promoters (seebelow) (Fig. 3). The main transcript of the cspB gene at37 uC starts 167 nucleotides upstream of the start codon(T2cspB), while a strong increase in the amount of theslightly smaller T1cspB transcript and, concomitantly, aweak decrease of the T2cspB transcript was observed aftercold shock (Fig. 3). The amount of mRNA reached amaximum between 60 and 90 min after the cold-shocktreatment and decreased after 150 min (data not shown). Atemperature-dependent change in the amount of tran-scripts was also observed for the cspA gene, which revealedtwo strongly enhanced transcripts at 15 uC as compared to37 uC (T3cspA and T1cspA), and a third transcript (T2cspA) thatwas not significantly affected by the temperature decrease(Fig. 3). In contrast to the cspA and cspB transcripts, onlylow amounts of cspC-specific transcripts (T1-3cspC) weredetected in bacteria cultivated at 37 uC. A temperaturedownshift resulted in the accumulation of two of thesetranscripts, T1cspC and T2cspC. It is not clear so far whetherthese transcripts are transcribed from different promotersor whether some of them are specific degradation products

of a precursor mRNA generated under the various stressconditions. In contrast to the cspA, cspB and cspC genes, inthe case of the cspD and cspE genes a single transcript couldbe detected, and the amount of these transcripts was notaltered significantly after cold shock (Fig. 3 and data notshown). A fourfold to ten-fold increase of total transcriptfollowing cold shock was determined for each of the cold-induced csp genes by phosphorimager quantification of theprimer extension products and by RT-qPCR (data notshown). To narrow down the temperature spectrum leadingto induction of the cold-shock stimulon, we investigated theenhancement of the amount of the cspB transcript after atemperature downshift from 37 uC to 30, 26, 20 and 15 uC. Asignificant twofold cold-induced accumulation of the cspBT1 transcript could already be observed at a temperature of26 uC but maximal transcript accumulation occurred only ata temperature of 20 uC or below (data not shown).

To examine whether stress conditions other than cold shockalso affect csp transcription in B. bronchiseptica, whole-cellRNA was prepared from bacteria grown under salt stresscaused by 2 M NaCl, after antibiotic treatment with thetranslation inhibitors chloramphenicol and tetracycline,and after heat shock to 48 uC as described in Methods. Thepreviously identified cspA transcripts showed a complexresponse pattern to the different stress conditions. TheT2cspA transcript was only slightly affected by all of the stressconditions applied, while the transcripts T1cspA and T3cspAstrongly accumulated in the presence of the two translationinhibitors (Fig. 4). In addition, the amount of the T3cspAtranscript strongly increased after heat shock. In the case ofthe cspB gene the transcript T2cspB was found to be inde-pendent of all tested stress conditions, while the transcriptT1cspB accumulated in response not only to cold shock, butalso to the translation inhibitors (Fig. 4). Two of the threecspC-specific transcripts, T1cpsC and T2cspC, were found to

Fig. 3. Determination of the transcript 59-ends of cspA, cspB, cspC, cspD and cspE

in B. bronchiseptica by primer extensionexperiments. RNA was prepared from wild-type strain BB7865 (+) and the bvg mutantBB7866 (”) grown at 37 6C, and from thesame culture after cold shock (CS) to15 6C for 30 min. The corresponding DNAsequence reactions are shown on the leftfor each gene. The various transcripts (T)identified by primer extension were num-bered according to their increasing distanceto the ATG start codon of the respectivegene and are indicated by arrows.

A C G T + _ + _CScspA

A C G T +_+_CScspB

T2T1T2

T1

T3

A C G T + _ + _CScspC

T2T1

T3

A C G T + _ + _CScspD

A C G T + _ + _CScspE

T1

T1



accumulate under cold shock, while only the T2cspC tran-script was also affected by the translation inhibitors (Fig. 4).With the exception of the T3cspC transcript, which showed amild response to osmotic stress, none of the other tran-scripts of the various csp genes were affected by salt stress(Fig. 4). Moreover, the growth phase had little effect ontranscription of the csp genes. In fact, a significant effect ofthe growth phase could be detected only in the case of thecspC transcript T3 and the respective signal was maximal inthe stationary phase at an OD578 of 1?2 (data not shown).

We also investigated cold induction of cspB gene expres-sion in B. pertussis by primer extension analysis. Since theresults were virtually identical in the two species (data notshown), they suggest that an analogous cold-shock responseis also occurring in B. pertussis despite its reductive genomeevolution.

Involvement of the upstream region of the cspBgene in transcriptional regulation of cspBexpression

To investigate the function of the upstream region of cspBin the transcriptional control of its expression, the wild-type sequence up to position 2105 as counted from T1,and a deletion derivative lacking parts of the upstreamregion between position 2105 and 274, were cloned infront of a promoterless gfp gene, resulting in the con-structs pMMB206-I-gfp and pMMB206-II-gfp, respectively(Fig. 5a). Primer extension analysis using a gfp specificoligonucleotide demonstrated that both cspB specifictranscripts, T1 and T2, were significantly reduced in thedeletion variant. The further deletion of the upstream regionup to sequence position 257 in the construct pMMB206-III-gfp resulted in the complete disappearance of the T2transcript, while the amount of the T1 transcript was notfurther diminished (Fig. 5b). Interestingly, the deletionof parts of the upstream region did not affect the cold-inducibility of the T1 transcript, which still accumulated atthe low temperature even in cells harbouring the constructpMMB206-III-gfp, although the general expression levelwas much lower as compared to that observed with thepMMB206-I-gfp construct (Fig. 5b). This suggests thatsynthesis of both transcripts of the cspB gene is undertranscriptional control by an unknown factor interactingwith the upstream region of the cspB gene; however, theupstream region is not essential for the cold induction ofthe T1 transcript. Moreover, the presence of the T1 tran-script also in the strain harbouring pMMB206-III-gfp inwhich the T2 transcript disappeared indicates that cspB istranscribed from two independent promoters.

Posttranscriptional control mechanisms affectthe expression of the cspB gene

The primer extension experiments showed that all of the cspgenes are preceded by a long 59-UTR. Within this non-translated region of the three cold-responsive genes cspA,cspB and cspC of B. bronchiseptica, a nucleotide sequencewith the consensus 59-TCCTTGATT-39 is present, while the59-UTRs of the other two cold-shock genes, which are notinduced at 15 uC, carry a sequence with onemismatch withinthis 9 bp box (cspE) or lack the entire box (cspD) (Fig. 2).The conservation of this sequence and its similar location inthe 59-UTR of the csp genes suggested that it might play arole in the differential regulation of expression of theBordetella cold-shock genes. In order to determine regionsof the transcripts possibly involved in the regulation of cspBexpression at low temperature, various DNA fragmentscomprising different parts of the upstream region of theORF encoding CspB were fused to a promoterless gfpreporter gene (Fig. 6a). The control fragment contains theentire 59-UTR up to position +136 with respect to thetranscriptional start site T1cspB. In the second construct,the 59-UTR was shortened at its 39 side by 49 nt, resulting ina truncated 59-UTR that ends at position+87 and lacks the9 bp box. The third construct comprised the entire 59-UTR,

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Fig. 4. Relative amounts of csp specific transcripts (T1–T3) ofthe cspA, cspB and cspC genes of strain BB7865 grown atdifferent growth and stress conditions. The transcript numbers(T1–T3) are the same as in Fig. 3. The different growth condi-tions (see Methods) are indicated below the respective bars:normal growth at 37 6C; cold shock at 15 6C; heat shock at48 6C, presence of translation inhibitors tetracycline (Tet) andchloramphenicol (Cm); osmostress caused by 2 M NaCl.The means of the amounts of transcripts detected in fourindependent primer extension experiments are shown; errorbars indicate SD.


D. Stubs and others

but the sequence of the 9 bp box was changed to theunrelated sequence 59-GGGGAATTC-39 by site-directedmutagenesis. Transcripts deriving from the three differentconstructs were characterized by primer extension analy-sis of whole-cell RNA isolated from BB7865 derivativesharbouring these plasmids. No significant differences couldbe observed in the amount of the transcripts from thevarious constructs at low and high temperature (data notshown). However, the amount of GFP synthesized in thestrains harbouring the different constructs showed inter-esting differences as determined by FACS and by Westernblot analysis with a GFP-specific antiserum. A very signi-ficant reduction in GFP expression was observed in thestrain carrying the deletion construct (pMMB206-V-gfp)as compared to the strain harbouring the wild-type con-struct (pMMB206-IV-gfp). Interestingly, a weaker but stillsignificant reduction in GFP expression (P¡0?03) canalso be observed in the strain carrying the mutated 9 bpbox (pMMB206-VI-gfp) (Fig. 6b). This indicates thatthe 59-UTR is involved in the control of translation effi-ciency of GFP expression and that the 9 bp box may be arelevant sequence motif required for maximal translationefficiency.

To address the question of whether additional posttran-scriptional mechanisms contribute to the regulation of cspBexpression, the half-life of the cspB transcripts was deter-mined in B. bronchiseptica cultures grown at 37 uC and incold-shocked cultures by addition of rifampicin and sub-sequent detection of the transcripts by RT-qPCR and primerextension analysis. RT-qPCR revealed an increase of about10-fold in the half-life of the cspB transcripts in culturesexposed to low temperature as compared to the culturegrown at elevated temperature. In agreement with thisfinding, the primer extension analysis also showed that thehalf-life of the T1 and T2 transcripts is increased about 10-to 15-fold at 15 uC as compared to their half-life at 37 uC

(Fig. 7). The increase in the half-life of these transcriptsappears to be specific for the cspB gene, since the stability ofother transcripts, for instance that of the guanylate kinase

pMMB206-I-gfp

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pMMB206-III-gfp

T2 T1

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p

Fig. 5. (a) Deletion analysis of the region upstream of the transcriptional start sites of the cspB gene. On the top, the full-length fragment cloned in the vector pMMB206 resulting in the construct pMMB206-I-gfp is shown. The upstream anddownstream regions of the cspB gene are counted from the start site of transcript T1. The promoterless gfp gene and itsShine–Dalgarno sequence is fused to the 59-UTR of the cspB gene at position +39. In the construct pMMB206-II-gfp theupstream region is shorter and ends at position ”74. In the third construct pMMB206-III-gfp the upstream region was furthershortened and ends at position ”57. T1 and T2, transcriptional start sites. (b) Primer extension experiments carried out withwhole-cell RNA prepared from strain BB7865 grown either at 37 6C or cold shocked at 15 6C carrying the constructs shownin (a). I-gfp, BB7865/pMMB206-I-gfp; II-gfp, BB7865/pMMB206-II-gfp; III-gfp, BB7865/pMMB206-III-gfp. T1 and T2indicate the cspB-specific transcripts shown in Figs 3 and 5(a).

pMMB206-IV-gfp T2 T1

-105 -30 +1 +136gfp

(a)

pMMB206-V-gfp T2 T1

-105 -30 +1 +87gfp

pMMB206-VI-gfp T2 T1

-105 -30 +1 +136gfp

9-bp box

9-bp box

10080

6040

20

2 4 6 8 10

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cent

age

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essi

on

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Fig. 6. Deletion analysis of the 59-UTR of the cspB gene toinvestigate a possible role of the 59-UTR in translation effi-ciency. (a) Plasmid pMMB206-IV-gfp contains the entire pro-moter region and the entire 59-UTR region of the cspB genecloned in front of the promoterless gfp gene. ConstructpMMB206-V-gfp has a deletion of 49 bp from the 39 end ofthe 59-UTR destroying part of the 9 bp box. ConstructpMMB206-VI-gfp is a derivative of pMMB206-IV-gfp made bysite-directed mutagenesis with a changed 9 bp box sequence.(b) GFP expression at low temperature from the three con-structs after conjugation in strain BB7865. GFP expressionwas determined by FACS analysis as described in Methods,and the means±SD of at least three independent experimentsare shown. X, BB7865; &, BB7865(pMMB206-IV-gfp); m,BB7865(pMMB206-V-gfp); %, BB7865(pMMB206-VI-gfp).



encoding gmk gene of B. bronchiseptica, was not alteredsignificantly under the same experimental conditions (datanot shown).

Proteome analysis of the cold-shock response

Protein expression during cold adaptation was investigatedby the 2D-PAGE of proteins from B. bronchiseptica culturesharvested at various time points after cold shock. Asexpected, an elevated synthesis of distinct cytosolic proteinscould be observed upon cold shock (Fig. 8). Eighteendifferent protein spots (CIPs) were excised from preparativegels loaded with cell extracts of bacteria that were cultivatedat 15 uC for up to 3 h. The proteins were then identified bymass spectrometry (Table 3). Interesting examples of thesecold-inducible proteins include a chaperone (Cpn10) withsimilarities to GroES and the putative translation inhibitorBB2940 (Table 3). Among these proteins we also found thecold-shock proteins CspA, CspB and CspC, as well as a lowmolecular mass protein with significant similarities to theuniversal stress protein UspA of E. coli, which is known tomodify at least six other E. coli proteins in response tostress and starvation (Nystrom & Neidhardt, 1996). OtherB. bronchiseptica CIPs apparently are involved in aminoacid metabolism, such as the LysA diaminopimelate decar-boxylase, the IlvC and TrpE proteins involved in isoleu-cine and tryptophan biosynthesis, and the 5-oxoprolinaseOplaH. Two cold-inducible proteins, the citrate synthaseGltA and the ATP synthase a-chain AtpA, are involved inrespiration. Interestingly, the superoxide dismutase SodB

100

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Fig. 7. Half-life of the T1 (&) and T2(X) transcripts at 37 (top)and 15 6C (bottom) as determined by primer extension experimentsof rifampicin-treated cultures (see Methods for details). Themeans±SD of three independent experiments are shown.

Fig. 8. Representative silver stained 2D-PAGE gels are shown. The gels were loaded with 75 mg protein of whole-cell lysatesprepared from cells grown at 37 6C (0 h) and further incubated under cold-shock conditions for 1, 2 or 3 h. Thirteen proteinspots with increased amount after cold shock are marked by arrows. The identities of the corresponding proteins are listed inTable 3. The gels were evaluated using the DELTA2D software (Decodon).


D. Stubs and others

was found to be cold inducible in B. bronchiseptica, as wellas the c-glutamyltranspeptidase precursor encoded by theggt gene, which is involved in the control of glutathionemetabolism (Table 3).

In addition to the proteins identified by 2D-PAGE, weinvestigated the cold-inducibility of IF-2, which in E. coli isstrongly induced after cold shock. The recently identifiedRNA-binding protein Tex was also included in this analysisbecause it contains an S1 domain (Fuchs et al., 1996; Koniget al., 2002). S1 domains are evolutionarily related to cold-shock domains since both types of domains show a similarthree-dimensional structure (Bycroft et al., 1997). Asdemonstrated by quantitative RT-PCR, the expression ofthe genes encoding IF-2 and Tex was found to be stronglyinduced upon cold shock (data not shown), classifying themas CIPs in B. bronchiseptica (Table 3).

Interference of the cold-shock stimulon and thevirulence regulon of B. bronchiseptica

Transcriptional gfp fusions have been generated previouslyin B. bronchiseptica, by means of transposon mutagenesis,resulting in the identification of several gene loci thatwere negatively regulated by the virulence regulatory BvgAStwo-component system, the so-called virulence-repressedgenes (VRGs) (Knapp & Mekalanos, 1988; Schneider et al.,2002). Here we show that two of the CSPs also belong tothe vrg regulon of B. bronchiseptica. In fact, primer extension

experiments performed with mRNA isolated from thebvg-negative (phase variant) strain BB7866 revealed anaccumulation of the T3cspC and T1cspD transcripts (Figs 3and 9). The amount of these cspC- and cspD-derivedtranscripts was found to be significantly increased in the bvgmutant grown at 37 uC as compared to the wild-type strain,indicating that BvgAS is involved in the repression of CspCand CspD at high temperature. To confirm the impact ofthe two-component system in the regulation of these two

Table 3. Factors of B. bronchiseptica induced by cold shock

Gene product Synonym Gene Spot* Detected byD

Universal stress protein A BB4260 uspA 2 RT-qPCR, 2D-PAGE

Cpn10 BB0963 cpn10 3 RT-qPCR, 2D-PAGE

CspA BB2251 cspA PE, 2D-PAGE

CspB BB2249 cspB PE, RT-qPCR, 2D-PAGE

CspC BB5013 cspC PE, 2D-PAGE

RNA binding protein Tex BB3601 tex RT-qPCR

Superoxide dismutase BB2012 sodB 1 PE, 2D-PAGE

IF-2 BB3246 infB RT-qPCR

Putative translation inhibitor BB2940 RT-qPCR, 2D-PAGE

Ketol-acid reductoisomerase BB0791 ilvC 5 2D-PAGE

Diaminopimelate decarboxylase BB0068 lysA 8 2D-PAGE

Citrate synthase BB3677 gltA 7 RT-qPCR, 2D-PAGE

c-Glutamyl transpeptidase precursor BB0978 ggt 9 2D-PAGE

5-Oxoprolinase BB2978 oplaH 13 2D-PAGE

Anthranilate synthase component BB4625 trpE 11 2D-PAGE

Probable carboxymuconolactone decarboxylase BB0458 2D-PAGE

Phosphoglycerate mutase 1 BB0398 gpmA 4 2D-PAGE

Solute-binding protein BB0185 6 RT-qPCR, 2D-PAGE

Putative peptidase BB3096 12 2D-PAGE

ATP synthase a-subunit BB4607 atpA 10 RT-qPCR, 2D-PAGE

*The spot number refers to the protein spots in Fig. 8.

DPE, primer extension.

300

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cspA cspB cspC cspD cspE

+ _ + _ + _ + _ + _ + _ + _ + _ + _ + _T1 T2 T3 T1 T2 T1 T2 T1 T1T3

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Fig. 9. BvgAS-dependent transcription of B. bronchiseptica

csp genes investigated by primer extension analysis. The col-umns represent the relative amount of different transcripts ofthe five csp genes in the wild-type strain BB7865 (+) and inthe bvg mutant strain BB7866 (”). At least three independentexperiments were carried out for each csp gene, and the errorbars indicate SD.



loci, we investigated the influence of the modulating com-pounds nicotinic acid and MgSO4, known to be recognizedby BvgS and leading to the inactivation of this histidinekinase. The amount of the transcripts increased significantlyunder these conditions in the wild-type strain BB7865, thusconfirming the classification of cspC and cspD as membersof the vrg regulon (data not shown). In the case of theBvgAS-repressed cspC-specific transcript T3, this effect isparticularly prominent in bacteria harvested in stationaryphase at an OD578 of 1?2 (data not shown). In contrast, asmentioned above, none of the other csp transcripts werefound to be significantly affected by the transition to thestationary growth phase of the bacteria.

These data suggested the possibility that the CSPs may alsoplay a role in the regulation of Bordetella virulence genes. Toinvestigate this question, we cloned four B. bronchiseptica-derived cold-shock genes, cspA, cspC, cspD and cspE, the cspBgene of B. pertussis, and the cspA orthologue of Bacilluscereus into the low-copy vector pLAFR2. A non-haemolyticphenotype of transconjugants of both B. pertussis andB. bronchiseptica was observed with plasmid pLAFR2-cspBBP, but not after introduction of the other csp genes.This demonstrates a negative regulatory effect of CspBoverexpression on the expression of the adenylate cyclasetoxin CyaA. In fact, a B. pertussis strain harbouring a fusionof the cyaA promoter to the lacZ reporter gene (Goyard &Ullmann, 1991) showed an approximately fourfold decreasein b-galactosidase activity after introduction of the addi-tional cspB copy as compared to the wild-type (data notshown). In agreement with the lacZ reporter gene data, cyaAexpression investigated by primer extension analysis wasfound to be significantly lower in the strain carrying thepLAFR2-cspB plasmid than in the control strain harbour-ing pLAFR2 only. This effect appears to be specific forcyaA expression since no alteration of the expression ofthe bvgAS two-component system could be observed byprimer extension analysis in bacteria cultivated at 37 uC inthe presence of the additional cspB gene copies (data notshown).

DISCUSSION

Cold-inducible genes have been identified in a wide varietyof psychrophilic and mesophilic bacteria (Graumann et al.,1997; Wouters et al., 1998), and are known to play an essen-tial role in the adaptation to low temperature. Genomicsequencing revealed that the three mammalian pathogensB. bronchiseptica, B. parapertussis and B. pertussis encodefive proteins homologous to CspA of E. coli (Parkhill et al.,2003). Orthologues of the same five proteins are alsoencoded by the bird pathogen B. avium (http://www.sanger.ac.uk/Projects/B_avium/) and by B. petrii, the onlyknown environmental species of the genus Bordetella (R.Gross, C. Guzman, U. Gobel & H. Blocker, unpublishedresults; von Wintzingerode et al., 2001). Therefore, despitethe reductive genome evolution that has streamlined thegenetic equipment of the specialized pathogens B. pertussis,

B. parapertussis and B. avium, all bordetellae conserved thesame set of CSPs, indicating a relevant role for these factorsalso during infection.

Although CSPs may be endowed with activities requiredfor growth under optimal environmental conditions, theirspecific function during stress adaptation is not wellunderstood. Even the production of knock-out mutantsdid not contribute much to the understanding of theirspecific function, possibly because of compensatory effectsby related cold-shock proteins. For example, in Bacillussubtilis all three csp genes must be deleted to obtain alethal phenotype (Graumann et al., 1997), while four outof nine csp genes had to be inactivated before a cold-sensitive phenotype was achieved in E. coli (Bae et al., 1997;Xia et al., 2001). In contrast to these data, the single cspBdeletion in B. bronchiseptica led to a growth-impairedphenotype at low (15 uC) and elevated temperature (37 uC).Thus, CspB appears to play an important role in thenormal growth of the bacteria independent of temperature.

It has been observed that the cold-shock response can beinduced by antibiotics like chloramphenicol and tetra-cycline, suggesting that ribosomes can act as temperaturesensors in E. coli (VanBogelen & Neidhardt, 1990). Theinvestigation of the effect of translation inhibitors inB. bronchiseptica revealed a complex pattern of csp geneexpression. The cspA specific transcripts were found to bemost responsive to the presence of such inhibitors (Fig. 4).Other stress conditions including heat shock and saltstress also resulted in the prevalence of certain CSP tran-scripts. For example, the amount of the T3cspA transcript isstrongly increased after heat shock, while one CSP encod-ing gene, cspC, was found to be responsive to salt stress(Fig. 4). Similar to certain CSPs of E. coli and Lactobacillusplantarum (Derzelle et al., 2000; Yamanaka & Inouye,1997), the growth phase can affect CSP expression in B.bronchiseptica, since the T3cspC transcript was found toaccumulate strongly in stationary phase (data not shown).

The regulation of expression of cold-shock proteins is com-plex, and transcriptional and posttranscriptional mechan-isms appear to be involved (Fang et al., 1997; Gualerziet al., 2003). Here we provide evidence for the contributionof both levels of regulation in the control of CSP expressionin B. bronchiseptica. For instance, there is evidence thatan unknown transcriptional activator is involved in thetranscriptional control of the cspB gene. Moreover, the cspCand cspD genes are apparently under the negative tran-scriptional control of the BvgAS two-component system.The repression of transcription of these genes at 37 uC mayoccur either directly via the BvgA response regulator orindirectly by a repressor such as the BvgR protein, which isitself under the positive control of the BvgAS system (Bock& Gross, 2001; Cotter & DiRita, 2000). However, despitethese indications for transcriptional control in the caseof the cspB, cspC and cspD genes, it is not known so farwhether the strong accumulation of several specific tran-scripts, in particular that observed for the cspA, cspB and


D. Stubs and others

cspC genes under various stress conditions, is due mainlyto transcriptional induction or to posttranscriptionalmechanisms.

In fact, in the case of the cspB gene we provide evidencethat additional control mechanisms contribute to its expres-sion. The data indicate that the 59-UTR of the cspB gene isimportant for efficient translation of the mRNA at lowtemperature, since the deletion of part of the 59-UTRinterfered negatively with translation of the respectivetranscript. Within this 59-UTR, the 9 bp box present in thetranscripts of four of the five cold-shock genes may con-tribute to translational efficiency, since its mutation to anunrelated sequence interfered significantly with the transla-tion of the transcript. Finally, the stability of cspB mRNAdiffers strongly under different growth conditions, sincethe half-life of the T1cspB and T2cspB transcripts was found tobe increased more than ten-fold during cold shock.

We analysed the changes in the pattern of proteinssynthesized by B. bronchiseptica up to 3 h after cold shockby 2D-PAGE, and we determined the identity of 18 ofthese CIPs (Fig. 8, Table 3). Among them, several proteinswere found that were also identified to be cold induciblein Listeria monocytogenes (Phan-Thanh & Gormon, 1997),in E. coli (Gualerzi et al., 2003; Jones et al., 1987) and inBacillus subtilis (Graumann et al., 1996). Examples otherthan the CSPs are the general stress protein GroES, thetranslation initiation factor IF-2, and IlvC. The latterenzyme is cold inducible also in Bacillus subtilis and catalysesthe second step in valine and isoleucine synthesis. Aninteresting difference between the cold-shock responseof E. coli and B. bronchiseptica is the induction of theuniversal stress protein UspA, which is induced undervarious stress conditions, but not after cold shock, in E. coli(Ferianc et al., 1998). Finally, it is interesting to notethat despite the reductive genome evolution in B. pertussis,all factors identified to be cold inducible in B. bronchisep-tica have been retained in the streamlined genome of thehighly specialized human pathogen, a fact that once moremay indicate a role of the cold-shock factors duringinfection.

Most of the known virulence factors expressed by B. pertussisand B. bronchiseptica are positively regulated by the BvgAStwo-component system (Bock & Gross, 2001; Cotter &DiRita, 2000). In addition to the virulence factors that areactivated by the bvg locus, a second class of factors encodedby the VRGs are directly or indirectly repressed by the bvglocus (Knapp & Mekalanos, 1988; Schneider et al., 2002;Stenson & Peppler, 1995). Environmental stimuli such as atemperature below 30 uC or the presence of compounds likenicotinic acid and sulfate ions cause a reversible inactiva-tion of the system, and result in the lack of expressionof virulence factors, but in the induction of VRGs, aphenomenon termed phenotypic modulation (Cotter &DiRita, 2000). B. bronchiseptica cells expressing VRGs aremore resistant to toxic reagents and harmful growthconditions, e.g. the action of antimicrobial compounds,

intracellular survival in phagocytes or survival in lake water(Banemann et al., 1997; Porter et al., 1991; Schneider et al.,2000). It has been suggested that the Bvg2 phase may havebeen relevant for an ancestor of the mammalian pathogensoccupying environmental niches (Gerlach et al., 2001). Onthe other hand, the fine-tuned down-regulation of viru-lence gene expression, e.g. by a mild reduction of tem-perature, leads to the so-called intermediate phase, which ischaracterized by a lower level of VAG (virulence-activatedgene) expression and by the appearance of novel antigens.This intermediate phase may contribute to aerosol-mediated transmission of the bordetellae in late infectionphases (Cotter &DiRita, 2000). Interestingly, the cold-shockstimulon of pathogenic bordetellae and the virulenceregulon are somehow interconnected. In fact, cspC andcspD expression was found to be controlled by the BvgASsystem in a negative manner (Fig. 9), indicating that CspCand CspD belong to the Bordetella VRGs. A secondinterference with the cold-shock-responsive system andthe virulence regulon was found after a slight overexpressionof CspB. In both B. pertussis and B. bronchiseptica, mildoverexpression of CspB interferes negatively with thetranscription of the adenylate cyclase toxin, leading to anon-haemolytic phenotype of the bacteria. Since someincrease of CspB expression is already observed at atemperature leading to the expression of intermediateantigens of B. pertussis, the data may indicate an in vivo rolefor the CspB-mediated control of CyaA expression. This isreminiscent of the previously described negative effect ontoxin expression by the Tex protein of B. pertussis (Fuchset al., 1996; Konig et al., 2002). Interestingly, the RNA-binding protein Tex is also responsive to cold shock inB. bronchiseptica and therefore also belongs to the family ofthe CIP proteins.

In conclusion, we show that the cold-shock response ofB. bronchiseptica resembles that of E. coli but differs inseveral aspects, e.g. the composition of the CIP family ofproteins and the requirement of an individual cold-shockprotein, CspB, for normal growth of the bacteria. It appearsthat the cold-shock stimulon is conserved between B.bronchiseptica and its closely related human pathogen, B.pertussis, indicating that CSP-mediated responses are not anevolutionary relict, but part of a genetic programme thatmay still be relevant also for the human pathogen despite itsreductive genome evolution. Since several of the CSPs werefound to be induced by stress signals other than cold shock,and cold stress is not considered to be relevant for theobligate host-associated species, the CSP regulon may beengaged in defence against other stress conditions encoun-tered during infection, and cold-shock factors may beinvolved in a differential fine-tuning of virulence geneexpression.

ACKNOWLEDGEMENTS

We thank Dagmar Beier and Geraldine Bresolin for critical reading ofthe manuscript. We are grateful to Klaus Neuhaus and Siegfried



Scherer for discussions, and for providing us the cspA gene ofBacillus cereus. This work was supported by grant SFB479/A2and by a postdoctoral fellowship to T.M. F. from the DeutscheForschungsgemeinschaft.

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