1 Lehrstuhl für Mikrobiologie, Biozentrum der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany
2 Zentralinstitut für Ernährungs- und Lebensmittelforschung, Abteilung Mikrobiologie, D-85354 Freising, Germany
3 Zentrum für Molekulare Biologie, Universität Heidelberg, D-69120 Heidelberg, Germany
Correspondence
Roy Gross
roy.gross{at}mail.uni-wuerzburg.de
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ABSTRACT |
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Present address: Ingenium Pharmaceuticals AG, Fraunhofer Str. 13, D-82152 Martinsried, Germany.
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INTRODUCTION |
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Most members of the genus Bordetella are assumed to be obligatorily associated with host organisms (Gerlach et al., 2001; Weiss, 1992
). Bordetella bronchiseptica causes chronic and often asymptomatic respiratory infections in a variety of animals, but only rarely in humans (Goodnow, 1980
; Weiss, 1992
). It is closely related to Bordetella pertussis and Bordetella parapertussis, the aetiological agents of whooping cough. Recently, comparative genomics revealed reductive genome evolution to occur in B. pertussis and the closely related pathogen B. parapertussis, which may have developed from a B. bronchiseptica-like ancestor (Parkhill et al., 2003
). So far, only a single Bordetella species, Bordetella petrii, has been isolated from an environmental location (von Wintzingerode et al., 2001
). Interestingly, B. bronchiseptica also has the capacity to survive harsh environmental conditions, and may therefore be able to exist outside a host organism (Porter et al., 1991
). There appears to be an evolutionary trend in the pathogenic members of the genus Bordetella to lose factors possibly involved in survival outside of the host upon specialization to specific host organisms (Gerlach et al., 2001
).
Despite the reductive evolution during adaptation to an apparently obligate association with warm-blooded animals or man, the genes encoding cold-shock proteins are conserved among all Bordetella species. In fact, the family of CSPs consists of five members (CspA to CspE) and all five genes are found in the genomes of all Bordetella species sequenced so far (B. pertussis, B. parapertussis, B. bronchiseptica, Bordetella avium and B. petrii). In this study, the phenotype of csp mutants and the pattern of differential expression of the cold-shock genes in response to different stress conditions including cold shock, salt stress, heat shock and treatment with antibiotics were investigated. By proteome analysis, we show that B. bronchiseptica has a cold shock response similar to that of E. coli and other bacteria. In addition, we analysed the expression of cspB and obtained evidence for transcriptional control of its expression in B. bronchiseptica and for a posttranscriptional regulatory function of the long non-translated upstream region preceding the coding region of the cspB gene.
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METHODS |
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Computer analysis.
For the analysis of nucleotide and amino acid sequences, we used the HUSAR program package (http://genome.dkfz-heidelberg.de/), which is based on the GCG program package described by Devereux et al. (1984), the databases of the Sanger Institute (http://www.sanger.ac.uk/), and the pedant database of the Munich 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 and cspB-KO-3-PstI/cspB-KO-3-HindIII, two DNA fragments of 404 and 429 bp in size flanking the cspB gene were amplified from chromosomal B. bronchiseptica BB7865 DNA (Table 2). The amplified 404 and 429 bp DNA fragments were digested with BamHI/PstI and PstI/HindIII, respectively, and cloned into pBluescriptKS. Following linearization of the resulting construct with PstI, a kanamycin-resistance cassette derived from pUC4K was cloned between the regions flanking the cspB gene on the chromosome. Both flanking sequences and the kanamycin cassette were then amplified by PCR using the primers cspB-KO-5-BamHI and cspB-KO-3-BamHI to introduce a second BamHI restriction site. The resulting BamHI fragment was then cloned into the allelic exchange vector pSS1129. The resulting construct, pSS1129-
cspB, was transferred by conjugation into B. bronchiseptica, and selection for double-crossover events was carried out as described elsewhere (Carbonetti et al., 1994
; Stibitz & Yang, 1991
). The resulting cspB deletion mutant was termed BB7865
cspB.
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To investigate the role of the 5'-UTR of the cspB gene, three additional 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/DelD containing the desired mutated sequence and the plasmid pMMB206-IV-gfp as template. The oligonucleotides were extended by Pfu DNA polymerase (Promega) during temperature cycling (PCR programme: 95 °C/30 s; 18 cycles of 95 °C/30 s, 55 °C/1 min, 68 °C/10 min; 72 °C/10 min). Following PCR amplification the DNA fragment was treated with DpnI (New England Biolabs), and E. coli strain DH5 was then transformed with the nicked vector DNA. The mutated cspB gene fragment was digested with BamHI and HindIII, confirmed by DNA sequencing, and cloned into the pMMB206 vector for conjugation in B. bronchiseptica strain BB7865. FACS analysis was done as described previously (Schneider et al., 2002
).
Construction of pLAFR2 derivatives containing different csp genes.
The coding regions of five cold-shock genes were amplified together with their 5'-UTR and their entire promoter regions from chromosomal B. bronchiseptica DNA by PCR using the following primer 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 template to amplify cspB with the oligonucleotides cspB-I/cspB-II. The cspA gene of Bacillus cereus was amplified with the oligonucleotides Bc1 and Bc2. The PCR fragments were digested with BamHI and cloned into the BamHI-digested low-copy vector pLAFR2. The resulting constructs were conjugated into BB7865 as described above.
RNA isolation.
Whole-cell RNA of Bordetella strains was isolated as described previously (Gross & Rappuoli, 1989). Briefly, bacteria grown 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 containing 100 mM Tris/HCl (pH 7·5), 1 % SDS and 2 mM EDTA. To test the response to several stress conditions, cells were grown to OD590 0·6 and then subjected to the following treatment: for heat shock the cultures were incubated at 48 °C for 20 min, for salt shock 2 M NaCl was added and the cultures were further incubated for 30 min. Finally, to test the response to translation inhibitors, tetracycline (50 µg ml1) or chloramphenicol (120 µg ml1) was added to the cultures, and cultivation was continued for 30 min. After boiling, the samples were kept on ice in the presence of 80 mM KCl for 5 min. This step was followed by the removal of cellular debris by centrifugation. To 3·5 ml of each sample, 4·56 g CsCl was added, and the RNA was pelleted by centrifugation in a Beckman SW50Ti rotor at 35 000 r.p.m. at room temperature for 20 h. Finally, the pellet was resuspended in 500 µl TE buffer [10 mM Tris/HCl (pH 8·0), 1 mM EDTA], extracted with an equal volume of a solution of 1 : 1 phenol/chloroform (v/v), precipitated once with ethanol, resuspended in 50 µl TE buffer and finally stored at 20 °C.
Primer extension analysis.
For the various csp genes, and the cyaA and bvgA genes, this was done with the appropriate PE primers listed in Table 2. The oligonucleotides were labelled with [
-32P]ATP using T4 polynucleotide kinase, then hybridized with their respective templates. Primer extension reactions were performed with 25 µg total RNA for 45 min at 46 °C using avian myeloblastosis virus reverse transcriptase (Amersham). Primer extension products were resolved on polyacrylamide gels (6 %) containing 6 M urea. Sequencing reactions were done in parallel using the various pLAFR2 derivatives containing the respective csp genes as a template. Radiolabelled bands were quantified using a PhosphorImager system (Amersham Biosciences) and the corresponding software (Molecular Dynamics).
RT-qPCR.
For each reaction several dilutions of cDNA reverse transcribed from 5 µg total RNA with random primers and the SuperScriptII RNaseH Reverse Transcriptase (Invitrogen) were used, with 1 pg to 2 ng of genomic B. bronchiseptica BB7865 DNA as a standard. RT-PCR reactions were performed using a DNA Engine Opticon System (MJ Research) and the qPCR Core Kit for Sybr Green I (Eurogentec) according to the manufacturer's recommendations. From each of two independent RNA preparations two separate cDNA preparations were made. These cDNA preparations were characterized three times each by RT-qPCR, resulting in an overall replicate number of 12 experiments. The threshold cycle number was set to a fluorescence of 0·01. For this analysis the for/rev primers listed in Table 2 and named according to the respective genes were used.
Rifampicin treatment.
To investigate the half-life of transcripts, the B. bronchiseptica wild-type strain BB7865 was grown at 37 °C in SS medium to OD590 0·6. Rifampicin was then added to a final concentration of 0·25 mg ml1, and the culture was divided into two equal samples, one of which was further incubated at 37 °C and the other was cold shocked in an ice bath and further incubated at 15 °C. At different time points, total RNA was isolated from 50 ml aliquots of the cultures as described above.
Preparation of protein extracts and 2D protein gel electrophoresis.
Cultures (50 ml) of B. bronchiseptica were grown to late mid-exponential phase (OD578 1·0) and cold shocked as described above. At different time points after cold shock the cells were harvested by centrifugation. Equal amounts of up to 150 µg protein were separated by 2D-PAGE according to the method of O'Farrell (1975). Briefly, the proteins were separated in the first dimension by isoelectric focusing in a pH gradient ranging from 3 to 10; in the second dimension, the proteins were separated according to their molecular masses by SDS-PAGE, and visualized by Coomassie blue or silver staining. The cellular proteins of each tested strain were isolated in at least three separate experiments, and 2D-PAGE of each protein preparation was performed at least in triplicate. Only those protein spots that showed a reproducible and significant increase in their amount after cold shock were further analysed by mass spectrometry as described previously (Kimmel et al., 2000
). Evaluation and quantification of the 2D gels were performed using the DELTA2D program from Decodon.
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RESULTS |
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Transcript analysis of cold-shock genes
To characterize the promoters of the five B. bronchiseptica csp genes and to analyse the effect of cold stress on their transcriptional response, we conducted primer extension experiments with RNA preparations from wild-type strain BB7865 cultivated at 37 °C or cold shocked for 30 min at 15 °C. All five B. bronchiseptica cold-shock genes comprise extremely long 5'-UTRs ranging from 109 bp (T1cspD) to 167 bp (T2cspB), a feature that appears to be characteristic for many of the cold-shock genes of eubacteria (Fig. 2) (Bae et al., 1997
; Brandi et al., 1996
; Fang et al., 1997
; Jones & Inouye, 1994
). Within the 5'-UTRs of four of the csp genes (cspA, cspB, cspC and cspE), a highly conserved 9 bp sequence motif, the 9 bp box, with the consensus sequence 5'-TCCTTGATT-3' was identified (Fig. 2
). In all cases, this motif was found to be located approximately 5157 bp upstream of the respective start codon. This sequence motif does not show any similarity with the cold box present in the 5'-UTR of transcripts of the csp genes of E. coli, for which a regulatory function was suggested previously (Jiang et al., 1996
).
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Involvement of the upstream region of the cspB gene in transcriptional regulation of cspB expression
To investigate the function of the upstream region of cspB in the transcriptional control of its expression, the wild-type sequence up to position 105 as counted from T1, and a deletion derivative lacking parts of the upstream region between position 105 and 74, were cloned in front of a promoterless gfp gene, resulting in the constructs pMMB206-I-gfp and pMMB206-II-gfp, respectively (Fig. 5a). Primer extension analysis using a gfp specific oligonucleotide demonstrated that both cspB specific transcripts, T1 and T2, were significantly reduced in the deletion variant. The further deletion of the upstream region up to sequence position 57 in the construct pMMB206-III-gfp resulted in the complete disappearance of the T2 transcript, while the amount of the T1 transcript was not further diminished (Fig. 5b
). Interestingly, the deletion of parts of the upstream region did not affect the cold-inducibility of the T1 transcript, which still accumulated at the low temperature even in cells harbouring the construct pMMB206-III-gfp, although the general expression level was much lower as compared to that observed with the pMMB206-I-gfp construct (Fig. 5b
). This suggests that synthesis of both transcripts of the cspB gene is under transcriptional control by an unknown factor interacting with the upstream region of the cspB gene; however, the upstream region is not essential for the cold induction of the T1 transcript. Moreover, the presence of the T1 transcript also in the strain harbouring pMMB206-III-gfp in which the T2 transcript disappeared indicates that cspB is transcribed from two independent promoters.
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Interference of the cold-shock stimulon and the virulence regulon of B. bronchiseptica
Transcriptional gfp fusions have been generated previously in B. bronchiseptica, by means of transposon mutagenesis, resulting in the identification of several gene loci that were negatively regulated by the virulence regulatory BvgAS two-component system, the so-called virulence-repressed genes (VRGs) (Knapp & Mekalanos, 1988; Schneider et al., 2002
). Here we show that two of the CSPs also belong to the vrg regulon of B. bronchiseptica. In fact, primer extension experiments performed with mRNA isolated from the bvg-negative (phase variant) strain BB7866 revealed an accumulation of the T3cspC and T1cspD transcripts (Figs 3 and 9
). The amount of these cspC- and cspD-derived transcripts was found to be significantly increased in the bvg mutant grown at 37 °C as compared to the wild-type strain, indicating that BvgAS is involved in the repression of CspC and CspD at high temperature. To confirm the impact of the two-component system in the regulation of these two loci, we investigated the influence of the modulating compounds nicotinic acid and MgSO4, known to be recognized by BvgS and leading to the inactivation of this histidine kinase. The amount of the transcripts increased significantly under these conditions in the wild-type strain BB7865, thus confirming the classification of cspC and cspD as members of the vrg regulon (data not shown). In the case of the BvgAS-repressed cspC-specific transcript T3, this effect is particularly prominent in bacteria harvested in stationary phase at an OD578 of 1·2 (data not shown). In contrast, as mentioned above, none of the other csp transcripts were found to be significantly affected by the transition to the stationary growth phase of the bacteria.
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DISCUSSION |
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Although CSPs may be endowed with activities required for growth under optimal environmental conditions, their specific function during stress adaptation is not well understood. Even the production of knock-out mutants did not contribute much to the understanding of their specific function, possibly because of compensatory effects by related cold-shock proteins. For example, in Bacillus subtilis all three csp genes must be deleted to obtain a lethal phenotype (Graumann et al., 1997), while four out of 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 cspB deletion in B. bronchiseptica led to a growth-impaired phenotype at low (15 °C) and elevated temperature (37 °C). Thus, CspB appears to play an important role in the normal growth of the bacteria independent of temperature.
It has been observed that the cold-shock response can be induced by antibiotics like chloramphenicol and tetracycline, suggesting that ribosomes can act as temperature sensors in E. coli (VanBogelen & Neidhardt, 1990). The investigation of the effect of translation inhibitors in B. bronchiseptica revealed a complex pattern of csp gene expression. The cspA specific transcripts were found to be most responsive to the presence of such inhibitors (Fig. 4
). Other stress conditions including heat shock and salt stress also resulted in the prevalence of certain CSP transcripts. For example, the amount of the T3cspA transcript is strongly increased after heat shock, while one CSP encoding gene, cspC, was found to be responsive to salt stress (Fig. 4
). Similar to certain CSPs of E. coli and Lactobacillus plantarum (Derzelle et al., 2000
; Yamanaka & Inouye, 1997
), the growth phase can affect CSP expression in B. bronchiseptica, since the T3cspC transcript was found to accumulate strongly in stationary phase (data not shown).
The regulation of expression of cold-shock proteins is complex, and transcriptional and posttranscriptional mechanisms appear to be involved (Fang et al., 1997; Gualerzi et al., 2003
). Here we provide evidence for the contribution of both levels of regulation in the control of CSP expression in B. bronchiseptica. For instance, there is evidence that an unknown transcriptional activator is involved in the transcriptional control of the cspB gene. Moreover, the cspC and cspD genes are apparently under the negative transcriptional control of the BvgAS two-component system. The repression of transcription of these genes at 37 °C may occur either directly via the BvgA response regulator or indirectly by a repressor such as the BvgR protein, which is itself under the positive control of the BvgAS system (Bock & Gross, 2001
; Cotter & DiRita, 2000
). However, despite these indications for transcriptional control in the case of the cspB, cspC and cspD genes, it is not known so far whether the strong accumulation of several specific transcripts, in particular that observed for the cspA, cspB and cspC genes under various stress conditions, is due mainly to transcriptional induction or to posttranscriptional mechanisms.
In fact, in the case of the cspB gene we provide evidence that additional control mechanisms contribute to its expression. The data indicate that the 5'-UTR of the cspB gene is important for efficient translation of the mRNA at low temperature, since the deletion of part of the 5'-UTR interfered negatively with translation of the respective transcript. Within this 5'-UTR, the 9 bp box present in the transcripts of four of the five cold-shock genes may contribute to translational efficiency, since its mutation to an unrelated sequence interfered significantly with the translation of the transcript. Finally, the stability of cspB mRNA differs strongly under different growth conditions, since the half-life of the T1cspB and T2cspB transcripts was found to be increased more than ten-fold during cold shock.
We analysed the changes in the pattern of proteins synthesized by B. bronchiseptica up to 3 h after cold shock by 2D-PAGE, and we determined the identity of 18 of these CIPs (Fig. 8, Table 3
). Among them, several proteins were found that were also identified to be cold inducible in Listeria monocytogenes (Phan-Thanh & Gormon, 1997
), in E. coli (Gualerzi et al., 2003
; Jones et al., 1987
) and in Bacillus subtilis (Graumann et al., 1996
). Examples other than the CSPs are the general stress protein GroES, the translation initiation factor IF-2, and IlvC. The latter enzyme is cold inducible also in Bacillus subtilis and catalyses the second step in valine and isoleucine synthesis. An interesting difference between the cold-shock response of E. coli and B. bronchiseptica is the induction of the universal stress protein UspA, which is induced under various stress conditions, but not after cold shock, in E. coli (Ferianc et al., 1998
). Finally, it is interesting to note that despite the reductive genome evolution in B. pertussis, all factors identified to be cold inducible in B. bronchiseptica have been retained in the streamlined genome of the highly specialized human pathogen, a fact that once more may indicate a role of the cold-shock factors during infection.
Most of the known virulence factors expressed by B. pertussis and B. bronchiseptica are positively regulated by the BvgAS two-component system (Bock & Gross, 2001; Cotter & DiRita, 2000
). In addition to the virulence factors that are activated by the bvg locus, a second class of factors encoded by the VRGs are directly or indirectly repressed by the bvg locus (Knapp & Mekalanos, 1988
; Schneider et al., 2002
; Stenson & Peppler, 1995
). Environmental stimuli such as a temperature below 30 °C or the presence of compounds like nicotinic acid and sulfate ions cause a reversible inactivation of the system, and result in the lack of expression of virulence factors, but in the induction of VRGs, a phenomenon termed phenotypic modulation (Cotter & DiRita, 2000
). B. bronchiseptica cells expressing VRGs are more resistant to toxic reagents and harmful growth conditions, 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 Bvg phase may have been relevant for an ancestor of the mammalian pathogens occupying environmental niches (Gerlach et al., 2001
). On the other hand, the fine-tuned down-regulation of virulence gene expression, e.g. by a mild reduction of temperature, leads to the so-called intermediate phase, which is characterized by a lower level of VAG (virulence-activated gene) expression and by the appearance of novel antigens. This intermediate phase may contribute to aerosol-mediated transmission of the bordetellae in late infection phases (Cotter & DiRita, 2000
). Interestingly, the cold-shock stimulon of pathogenic bordetellae and the virulence regulon are somehow interconnected. In fact, cspC and cspD expression was found to be controlled by the BvgAS system in a negative manner (Fig. 9
), indicating that CspC and CspD belong to the Bordetella VRGs. A second interference with the cold-shock-responsive system and the virulence regulon was found after a slight overexpression of CspB. In both B. pertussis and B. bronchiseptica, mild overexpression of CspB interferes negatively with the transcription of the adenylate cyclase toxin, leading to a non-haemolytic phenotype of the bacteria. Since some increase of CspB expression is already observed at a temperature leading to the expression of intermediate antigens of B. pertussis, the data may indicate an in vivo role for the CspB-mediated control of CyaA expression. This is reminiscent of the previously described negative effect on toxin expression by the Tex protein of B. pertussis (Fuchs et al., 1996
; König et al., 2002
). Interestingly, the RNA-binding protein Tex is also responsive to cold shock in B. bronchiseptica and therefore also belongs to the family of the CIP proteins.
In conclusion, we show that the cold-shock response of B. bronchiseptica resembles that of E. coli but differs in several aspects, e.g. the composition of the CIP family of proteins and the requirement of an individual cold-shock protein, CspB, for normal growth of the bacteria. It appears that the cold-shock stimulon is conserved between B. bronchiseptica and its closely related human pathogen, B. pertussis, indicating that CSP-mediated responses are not an evolutionary relict, but part of a genetic programme that may still be relevant also for the human pathogen despite its reductive genome evolution. Since several of the CSPs were found to be induced by stress signals other than cold shock, and cold stress is not considered to be relevant for the obligate host-associated species, the CSP regulon may be engaged in defence against other stress conditions encountered during infection, and cold-shock factors may be involved in a differential fine-tuning of virulence gene expression.
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ACKNOWLEDGEMENTS |
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Received 24 November 2004;
revised 22 February 2005;
accepted 7 March 2005.
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