Institut für Mikrobiologie, Ernst-Moritz-Arndt-Universität Greifswald,F.-L.-Jahn-Str. 15, D-17487 Greifswald, Germany1
Author for correspondence: Thomas Schweder. Tel: +49 3834 864212. Fax: +49 3834 864238. e-mail: schweder{at}uni-greifswald.de
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ABSTRACT |
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Keywords: temperature downshift, transcriptome, proteome, mRNA, DNA array
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INTRODUCTION |
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Although synthesis of the majority of proteins decreases after a temperature downshift, the level of a specific group of proteins increases under these conditions in B. subtilis (Lottering & Streips, 1995 ; Graumann et al., 1996
). Using two-dimensional (2D) protein gel electrophoresis, it has been demonstrated that the synthesis of at least 37 proteins of B. subtilis increases after a temperature shift from 37 to 15 °C (Graumann et al., 1996
). It is supposed that these proteins play important roles in the adaptation of the cellular machinery to low-temperature conditions (Graumann & Marahiel, 1999
). One of the highest accumulating protein groups of B. subtilis after a temperature downshift is formed by CspB, CspC and CspD (Graumann et al., 1996
). It is assumed that these cold-shock proteins are able to bind mRNA and thus act as RNA chaperones, keeping critical mRNAs accessible for the ribosomes under low-temperature conditions (Graumann et al., 1997
; Schindler et al., 1999
). Furthermore, it has been suggested that cold-shock-induced proteins associated with translation, e.g. the ribosomal proteins S6 and L7/L12, are involved in the adaptation of ribosome function to low-temperature conditions (Graumann & Marahiel, 1999
). There are also indications that prolyl isomerization represents a problem that cells have to cope with at low temperatures (Kandror & Goldberg, 1997
; Graumann & Marahiel, 1999
). Increasing levels of the peptidyl-prolyl cis-trans isomerases PpiB and Tig of B. subtilis in response to cold shock have been described by Graumann et al. (1996)
. Finally, it has been observed that the level of several enzymes required for general cellular metabolism, e.g. amino acid synthesis or glycolysis, increases after a temperature downshift (Graumann & Marahiel, 1999
). It was suggested that the higher protein level compensates for the lower activity of these essential enzymes under low-temperature conditions.
It is supposed that the expression of most cold-shock-induced proteins is regulated mainly at the post-transcriptional and translational level. However, examples of regulation of cold-inducible genes at the transcriptional level have also been described. One example is the des gene of B. subtilis (Aguilar et al., 1998 ). This gene encodes a
5-lipid desaturase and exhibits one of the highest induction ratios during cold shock (Aguilar et al., 1999
). The des mRNA level strongly increases 30 min after a temperature downshift. Recently, it has been described that expression of des is regulated by a two-component signal transduction system composed of the sensor kinase DesK and the response regulator DesR (Aguilar et al., 2001
).
Besides direct transcriptional induction of cold-shock gene expression, mRNA stabilization after a temperature downshift has also been addressed as a mechanism for increased synthesis of cold-shock-induced proteins. It has been demonstrated that the overall mRNA half- life increases sixfold when B. subtilis cells are shifted from 37 to 20 °C (Aguilar et al., 1999 ). For the genes of the major cold-shock proteins of B. subtilis, CspB and CspC, an approximately 40-fold increase in mRNA stability after a temperature downshift was shown (Kaan et al., 1999
), which is significantly greater than the increase in bulk mRNA stability. A similar dramatic stabilization has been described for the mRNA of the major cold-shock protein of Escherichia coli, CspA (Brandi et al., 1996
; Goldenberg et al., 1996
; Fang et al., 1997
). Thus, besides the translational regulation of the synthesis of cold-shock proteins, transcriptional and post-transcriptional events are of importance for the adaptational response of bacterial cells under cold-shock conditions.
The aim of this study was to obtain a more comprehensive view of the cold-shock response of B. subtilis. 2D protein gel electrophoresis allows analysis of distinct groups of proteins from various cellular compartments with some limitations. However, global gene expression profiling by means of DNA arrays allows the transcriptional analysis of all genes of a specific genome at one defined time point. We analysed the transcriptome of B. subtilis after a temperature downshift from 37 to 18 °C. This approach allowed the identification of 46 genes exhibiting increased mRNA levels and 52 genes exhibiting decreased mRNA levels under cold-shock conditions. The contribution of altered mRNA levels to the varying amounts of proteins after cold shock was analysed by comparing transcriptome and proteome data.
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METHODS |
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Growth experiments were performed with B. subtilis strains BFS4469, BFS4564, YHEAd, BFS4794 and BFS4226 carrying pMUTIN integrations (Vagner et al., 1998 ) in ydeB, ydjO, yheA, yqgA and yacK, respectively. Strains were cultivated in the minimal medium described above as well as in Spizizens minimal medium (Spizizen, 1958
) supplemented with 0·2% glucose and 0·08 mM tryptophan. Erythromycin and lincomycin were added to final concentrations of 1 and 10 µg ml-1, respectively.
RNA extraction.
For RNA extraction, B. subtilis cells were disrupted in liquid-nitrogen-chilled vessels using a Micro Dismembrator S (Braun Biotech International) for 2 min at 2600 r.p.m. Total RNA was isolated by using the single step method as described by Chomczynski & Sacchi (1987) . The RNA pellet was resuspended in sterile water and the RNA concentration was determined spectrophotometrically.
Preparation of labelled cDNA and hybridization to macroarrays.
Reverse transcription and hybridization of the purified labelled target cDNA to B. subtilis nylon arrays were performed as described by Wiegert et al. (2001) with the following modifications: 10 µg total RNA was used, and macroarrays and specific cDNA primers were purchased from Eurogentec. Hybridization signals were detected by exposure of the arrays to a storage phosphor screen (Molecular Dynamics). Each analysis was carried out twice using two independently isolated RNA preparations and two different array batches.
Data analysis.
After scanning of the phosphor screens using a Storm 840 Phosphor Imager (Molecular Dynamics), the resulting images were analysed using ArrayVision 5.1 software (Imaging Research). Calculation of normalized intensity values of the individual spots was performed using the overall-spot-normalization function of ArrayVision. To avoid extreme intensity ratios for genes close to or below the detection limit, signal intensity values corresponding to a signal to noise (S/N) ratio <1·0 were scaled up to a value corresponding to an S/N ratio=1·0. Further analyses were carried out using GeneSpring 3.2.12 software (Silicon Genetics). Genes exhibiting S/N ratios 3 under at least one growth condition were considered to be significantly expressed. All genes specifying signals below this significance threshold were excluded from further data analysis. Then, the mean of the normalized intensity values of the duplicate spots of each gene was used to calculate the expression level ratios. Induction or repression ratios
2 in both experiments were considered as significant.
Final evaluation of the macroarray data included the consideration of putative operons derived from the genome sequence using the SubtiList database (http://genolist.pasteur.fr/SubtiList/) as well as previously known transcriptional units. Genes exhibiting significant expression ratios were checked for their transcriptional organization. If these genes were members of polycistronic transcriptional units, further members of these operons were also included in the resulting tables even if their expression parameters did not fulfil the significance criteria.
RNA slot-blot analysis.
Digoxigenin-labelled probes were obtained by in vitro transcription with T7 RNA polymerase using PCR-generated DNA fragments as templates. Primers used for PCR are listed in Table 1. Reverse primers contained the T7 RNA polymerase recognition sequence. The probes were generated with the Dig Chem Link Labelling and Detection Set (Roche Diagnostics).
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Hybridization signals were detected according to the manufacturers instructions (Roche Diagnostics). Chemiluminographs were analysed with a Lumi-Imager and the Lumi-Analyse-software package (Boehringer Mannheim). Signals corresponding to mRNA samples taken from B. subtilis cells at 37 °C served as standards and were set to 1.
2D PAGE analyses.
Pulse labelling of the bacterial culture for 5 min with 10 µCi L-[35S]methionine ml-1 during exponential growth was carried out as described by Schmid et al. (1997) . After sonication of the harvested cells, the amount of protein was determined by using Roti-Nanoquant (Roth). Crude protein extracts (50 µg protein) were loaded onto IPG strips for the first dimension of 2D gel electrophoresis (Völker et al., 1994
). The second dimension was carried out as described by Bernhardt et al. (1997)
. After fixation and silver staining, the wet gels were scanned with a Hewlett Packard ScanJet 6000 in transmission mode at a resolution of 300 d.p.i. and an 8 bit/256 greys colour depth. For autoradiography of the radiolabelled protein pattern (after separation of 2 million c.p.m.), the dried gels were exposed to storage phosphor screens (Molecular Dynamics Storage Phosphor Screen; 20x25 cm) for 17 h and scanned with a STORM 840 Phosphor Imager (Molecular Dynamics) at a resolution of 200 µm and a colour depth of 16 bit/65536 greys.
For computer-aided analysis of the gels, Delta2D 1.1 software (Decodon) was used as described by Bernhardt et al. (1999) . The protein spots were relocated from the Sub2D database (Bernhardt & Werner, 1999
).
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RESULTS AND DISCUSSION |
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Besides genes encoding proteins involved in amino acid biosynthesis, nucleotide and coenzyme metabolism, other down-regulated genes can be functionally classified in different groups. The mRNA levels of the opuCACBCCCD operon strongly decreased 60 min after the temperature downshift. This operon encodes a glycine betaine/carnitine/choline ABC transporter involved in osmoprotection (Kappes et al., 1999 ). The monocistronic yocS and yodF genes most probably also encode transport/binding proteins, in particular a sodium-dependent transporter and a proline permease, respectively. Both genes revealed maximally decreased mRNA levels 60 min after the temperature downshift.
Two down-regulated transcriptional units specify proteins fulfilling cell-wall-associated functions: the bicistronic wapA-yxxG operon and the monocistronic ywtD gene. Whereas wapA encodes a cell-wall-associated protein precursor (Foster, 1993 ), the function of the co-transcribed yxxG gene is unknown. Due to its sequence similarity, the gene product of ywtD most probably represents a murein hydrolase.
The bicistronic ahpC-ahpF operon, encoding the small and large subunits of the alkyl hydroperoxide reductase, was also strongly down-regulated by 60 min after cold shock. This enzyme fulfils an essential function in detoxification under conditions of oxidative stress (Antelmann et al., 1996 ). The transcript level of the yjbC gene encoding a general stress protein of unknown function (Petersohn et al., 2001
) exhibited the maximal decrease by 30 min after cold shock.
The physiological function of the down-regulated genes is partially evident. In the case of ywtD, which most probably encodes a murein hydrolase, the reduction of the mRNA level could ensure the adjustment of the enzyme amount to the reduced growth rate under cold-shock conditions. Murein hydrolases are essentially involved in cell growth and cell division. Similarly, reduced expression of wapA, encoding a cell-wall-associated protein precursor (Foster, 1993 ), could function to adapt the amount of this large membrane protein to the general decline in membrane- and cell-wall-located functions during growth in the cold. The down-regulation of the ahpC-ahpF operon could reflect the lower activity of the respiratory chain enzymic components at low temperatures, resulting in a reduced production of potentially toxic, highly reactive side products of respiratory chain reactions.
The reduced expression of the following transcriptional units specifying biosynthetic enzymes can also be interpreted as consequence of the lower growth rate after cold shock: bioWAFDBI, thiA, ymaA-nrdEF-ymaB, purEKBC-yexA-purLQFMNHD, guaB, metC, ilvD and ilvBHCleuABCD. All encoded enzymes from these genes are involved in major biosynthetic processes, such as the biosynthesis of amino acids, nucleotides and coenzymes. Therefore, it is obvious to speculate that the observed decreased mRNA levels reflect the cellular reaction to the reduced growth rate at lower temperature.
The physiological relevance of the down-regulation of the glycine betaine ABC transporter opuCACBCCCD as well as of yocS and yodF, encoding transporter/permease proteins, remains unclear. The opuC operon functions in coping with osmotic stress (Kappes et al., 1999 ). The operon encodes an ABC transporter responsible for the uptake of the compatible solutes glycine betaine, carnitine and choline. It is transcriptionally induced under conditions of increased osmolarity, but also expressed at a low level under conditions of moderate osmolarity. The adaptive effect of a lowering of the basal expression of the opuC operon under cold-shock conditions is not obvious.
Transcriptome analysis: genes exhibiting significantly increased mRNA levels
Approximately 46 genes exhibited significantly increased mRNA levels after the temperature downshift (Table 3). According to the time profile of the mRNA levels, these genes could be assigned to groups of early-and late-induced genes. Most of the induced genes exhibited significantly elevated mRNA levels 60 or 90 min after the temperature downshift. However, a small group of genes revealed significantly increased mRNA levels by 30 min.
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The majority of the cold-shock-induced genes was characterized by significantly increased mRNA levels after 60 or 90 min. Two of these genes encode proteins with metabolic functions: the fbaA gene, encoding fructose-1,6-bisphosphate aldolase (Tobisch et al., 1999 ), and the yrpG gene, encoding a protein similar to sugar-phosphate dehydrogenase. Among the late-induced cold-shock genes, several exhibited only around twofold increased mRNA levels after 60 as well as 90 min: rpoE, encoding the
-subunit of RNA polymerase, cspC and cspD, encoding cold-shock proteins, and yopU, encoding a protein of unknown function. The operon yydFGHIJ, specifying a protein with similarity to an ATP-binding protein of an ABC transporter and four genes encoding products of unknown function, followed the same expression pattern. The late-induced monocistronic genes listed below exhibited a continuous increase in their mRNA levels up to 90 min after cold shock: yugI, encoding a protein similar to polyribonucleotide nucleotidyltransferase, and yheA and ydeB, encoding proteins of unknown function. The monocistronic genes yomT, yorD and ypaA exhibited a transient increased mRNA pattern with a maximum after 60 min. The late-induced ylbG and yneF genes most probably represent members of the bicistronic operons ylbF-ylbG and yneE-yneF, respectively. However, the promoter-proximal genes of these putative operons, ylbF and yneE, specified signals below the significance threshold. Therefore, it remains unclear if ylbG and yneF are monocistronically transcribed from promoters immediately upstream of these genes.
Besides the known cold-shock genes cspB, cspC and cspD, there are further candidates, which possibly play roles in the adaptational response of B. subtilis cells to low-temperature conditions. In addition to the genes mentioned so far, one group of cold-inducible genes could function in the adaptation of the protein synthesis machinery. After the temperature downshift, we found increased mRNA levels of the following genes encoding ribosomal proteins: rpmA (encoding L27), rpmE (L31), rpsO (S15) and rpmF (L32). The rpmF gene forms a bicistronic operon together with the promoter-proximal ylbN gene, which encodes a protein with unknown function. Both genes exhibited elevated mRNA levels 60 min after the cold shock and a further increase in their mRNA levels after 90 min. The genes rpmE and rpsO are also co-transcribed as a bicistronic operon. There was a doubling in their mRNA levels 60 min after the temperature downshift and this was still the case after 90 min. Finally, according to the genome sequence, rpmA represents the promoter-distal gene of a tricistronic operon rplU-ysxB-rpmA. There was a doubling in the rpmA-specific mRNA level after 60 and 90 min, whereas the amount of the rplU-ysxB transcript was not significantly altered. Possibly, cold-dependent regulation of rpmA is mediated by an internal promoter upstream of the gene.
The cold-shock-induced genes identified in this study for which putative functions have been addressed can be affiliated to two main groups. The genes belonging to the first group encode proteins with functions in the adaptation of the cells to low-temperature conditions. The second group encompasses genes encoding proteins required for normal vegetative cellular activities.
The genes cspB, cspC and cspD belong to the first group of cold-shock-induced genes. For cspB, the increase in the mRNA level was verified by slot-blot experiments (see below). Furthermore, the up-regulation of cspB and cspD mRNA levels was also reflected by a strong increase in the amount of the corresponding proteins as revealed by proteome analysis (see below). CspB and its homologous proteins carry conserved motifs that are essential for binding to single-stranded DNA and RNA (Schröder et al., 1995 ; Lopez et al., 1999
, 2001
). For CspB and related proteins, an RNA chaperone function has been proposed, facilitating translation at low temperature by blocking the formation of secondary structures in the mRNA (Graumann & Marahiel, 1999
). Recently, Weber et al. (2001b
) demonstrated that CspB of B. subtilis is localized to zones of newly synthesized RNA, most probably coupling transcription with initiation of translation.
The strongest cold-shock-induced gene according to the array analysis was des, encoding a desaturase, which forms unsaturated fatty acids from saturated phospholipid precursors, thereby increasing membrane fluidity (Aguilar et al., 1999 ; Weber et al., 2001a
). Membrane fluidity is one of the most important demands for cells during growth at low temperatures to maintain substrate transport via the cell envelope. The strong cold-inducibility of des was also verified by slot-blot experiments (see below).
The genes encoding the ribosomal proteins L27, L31, L32 and S15 exhibited increased mRNA levels under cold-shock conditions. It is tempting to speculate that these proteins play a role in the low-temperature adaptation of the B. subtilis translational machinery, as suggested for the ribosomal proteins S6 and L7/L12 by Graumann et al. (1996) .
The second group of cold-shock-induced genes is very diverse. This group includes genes fulfilling functions in the metabolism of carbohydrates (fbaA, yrpG), nucleotides (purA), transport processes (mntABC, yydFGHIJ), RNA synthesis (rpoE) and RNA modification (yugI). The mntABC operon encodes an ABC transporter for manganese uptake (Que & Helmann, 2000 ). Transcription of this operon is controlled by the positive transcriptional regulator MntR which is activated in the absence of manganese ions. Possibly, manganese complexes with other ions like phosphates at low temperature and is thus limited. This could in turn activate MntR and consequently induce mntABC expression. In this respect it is noteworthy that no increased mntABC mRNA levels could be observed after a temperature downshift in Luria Broth (LB) complex medium (data not shown).
In the case of fbaA, encoding fructose-1,6-bisphosphate aldolase, there was a significant increase in the mRNA amount after 90 min cold shock. Interestingly, the proteome approach revealed a significant decrease in the FbaA protein level 30, 60 and 90 min after the temperature downshift. A possible explanation could be that the fbaA-specific mRNA is translationally blocked under cold-shock conditions, causing a reduction in the amount of the encoded protein. The late increase in the RNA level would then indicate the cellular reaction to equalize the lack in fructose-1,6-bisphosphate aldolase. A similar mechanism might be responsible for the increase in purA mRNA. This gene encodes the adenylosuccinate synthetase involved in purine biosynthesis and belongs to the genes with the highest increased mRNA levels under the conditions tested in this study. On the other hand, as in the case of fbaA, the proteome approach revealed a decreased level of the corresponding PurA protein after 30, 60 and 90 min cold shock.
The cold-shock-dependent regulation of the heptacistronic operon ptb-bcd-buk-lpd-bkdA1-bkdA2-bkdB is of special interest. The enzymes encoded by this operon catalyse the degradation of the branched-chain amino acids isoleucine and valine to branched-chain carboxylic acids (Debarbouille et al., 1999 ). Transcription of this operon has been shown to be strongly dependent on the alternate sigma factor SigL and positively regulated by the regulator BkdR. Furthermore, there is also a negative regulation via the transcriptional repressor CodY. The degradation intermediates of isoleucine and valine,
-methylbutyryl-CoA and isobutyryl-CoA, are utilized for synthesis of anteiso-branched fatty acids. These fatty acids, as components of membrane lipids, contribute essentially to sufficient fluidity of the membrane under low-temperature conditions. At low temperature, the content of anteiso-branched fatty acids in B. subtilis increases and the level of iso-branched fatty acid species decreases (Klein et al., 1999
). Besides the activity of the fatty acid desaturase, this system also contributes to the reorganization of the cell membrane at low temperature. However, the mechanism mediating the increase of the mRNA level of the ptb-bcd-buk-lpd-bkdA1-bkdA2-bkdB operon under cold-shock conditions remains unclear.
The increased mRNA levels identified by the transcriptome analyses in this study could be due to either an indirect post-transcriptional effect, such as the strong stabilization of the cspB or cspC mRNAs (Kaan et al., 1999 ) or a direct positive regulation at the transcriptional level. Recently, a two-component signal transduction system has been determined, which positively regulates the expression of the des gene, encoding the desaturase of B. subtilis under low-temperature conditions (Aguilar et al., 2001
). These authors suggested that the DesK/DesR two-component system functions like a cold-shock thermometer. It could be demonstrated by footprinting experiments that the response regulator DesR binds to a specific region upstream of the des promoter. To verify whether additional cold-shock-induced genes could be regulated by this two-component system, we performed a sequence pattern search with the suggested binding region of DesR. However, none of the cold-shock-induced genes identified in this study was preceded by a similar sequence motif.
Verification of DNA macroarray results by RNA slot-blot analysis
To check the significance of the DNA array data, follow-up experiments were carried out. First, the RNA levels of selected cold-inducible genes derived from the transcriptome study were analysed by slot-blot experiments (Fig. 1). Altogether seven genes, all exhibiting significantly increased mRNA levels under cold-shock conditions, were chosen: mntA, purA, yydG, yvcE, des, cspB and cspC. The mRNA level of the first gene of the mntABC operon, mntA, was maximally increased 2·5-fold in the array experiment after 60 min. The slot-blot analysis revealed a corresponding maximal induction factor of 3·5 after 60 min. The purA gene, showing a maximally fivefold increased mRNA level after 60 min, according to the DNA array, was increased about 20-fold at the same time point in the slot-blot experiment and reached a maximal mRNA level after 90 min (about 40-fold). This represented the highest mRNA level ratio of all cold-shock-sensitive mRNAs checked by slot-blot analysis and the largest discrepancy between global (DNA macroarray) and detailed (slot-blot) approaches. The yydG gene represents the second gene of the pentacistronic yydFGHIJ operon, whose mRNA level was increased about twofold after 60 and 90 min in the array experiment. The slot-blot analysis gave comparable mRNA ratios of 1·9 and 3·2. A similarly good agreement was found in the case of the monocistronic yvcE gene: the array analysis revealed mRNA ratios of 2·7, 4·9 and 4·6 after 30, 60 and 90 min cold stress; the respective slot-blot values were 2·1, 2·2 and 4·1. The des gene encoding desaturase exhibited the strongest induction in the array experiments: a 2·5-fold increase in des-specific mRNA after 30 min was followed by the peak of induction (around 10-fold) after 60 min and a subsequent decrease in the mRNA level to a ratio of 3·3 after 90 min. This transient induction pattern was also reflected in the slot-blot experiments, although the absolute values for the induction ratios were higher: 12·1, 18·2 and 15·8, respectively. A transient increase in the mRNA level was also verified in the case of the cspB gene encoding the major cold-shock protein CspB. The array analysis resulted in mRNA ratios of 2·6 (30 min), 4·6 (60 min) and 1·9 (90 min), corresponding to ratios of 5·3, 3·8 and 1·5 in the slot-blot experiment. In the case of the cspC mRNA, the macroarray analysis indicated a transient increase after 60 min. The slot-blot experiments revealed an increase by 30 min, followed by similar high mRNA levels after 60 and 90 min.
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Comparison of DNA macroarray results with proteome data
The DNA array results were checked further by a corresponding proteome analysis using a 2D PAGE technique. Comparison of [35S]methionine-labelled cytoplasmic protein fractions prepared from cells cultivated at 37 °C and at different times after the temperature downshift demonstrated that the protein synthesis capacity of the cold-shocked cells is drastically decreased. The incorporation rate of [35S]methionine was reduced by a factor of five after the temperature downshift from 37 to 18 °C. Interestingly, the transcriptome approach resulted in the identification of only 52 genes exhibiting decreased mRNA levels, whereas the proteome approach revealed the down-regulation of the synthesis of significantly more proteins: about 30% of the protein spots visible in the control gel were not detectable in the 2D protein gels of the cold-shocked cells. This discrepancy verifies the earlier idea that the strong global down-regulation of gene expression under cold-shock conditions results primarily from inhibition of translation due to stabilized secondary structures of the mRNA. Among the proteins whose synthesis was strongly reduced after cold shock were IlvB, LeuA, LeuB, LeuC and IlvD, all identified as down-regulated by the transcriptomics approach, as shown in Table 2. The decreased mRNA levels of guaB, purF, thiA, ykrT, ykrS and yjbC were also reflected at the protein level (Table 2
). In all cases, apart from IlvC, the down-regulated genes also showed a decreased expression at the protein level, if detectable on the 2D gels. In the macroarray analysis, all genes of the ilvBHCleuABCD operon revealed decreased mRNA levels, but the gene product of ilvC increased at the protein level.
About 43 proteins demonstrating increased levels after the temperature downshift were identified by means of the 2D PAGE approach. The most dominant protein spots after the temperature downshift were represented by the cold-shock proteins CspB and CspD. The increased amount of these cold-shock proteins was observed 30 min after the temperature downshift. In the case of CspB, this expression pattern corresponds to the transcriptome analysis (Table 3), whereas in the case of CspD, an increased mRNA level was only detectable after 90 min. Furthermore, the increased mRNA level of the yugI gene, encoding a putative polyribonucleotide nucleotidyltransferase, was also verified by the proteomics approach. In contrast, the proteins PurA and FbaA were present in lower amounts under cold-shock conditions, although their mRNA levels were increased.
Besides the cold-shock proteins CspB and CspD, the ribosomal proteins S6 and L7/L12 belonged to the most highly accumulating proteins under cold-shock conditions in B. subtilis. The number of cold-shock-increased proteins is pretty consistent with results from Graumann et al. (1996) who identified 37 cold-shock proteins by the 2D PAGE approach. However, there are slight differences between these two studies. We did not find significantly increased protein levels of the prolyl-isomerases PpiB and Tig, the cysteine synthase CysK or the glyceraldehyde phosphate dehydrogenase Gap under the conditions examined in this study. Although there are slight differences in the experimental design of Graumann et al. (1996)
in comparison to our study, the reasons for these differences are not clear.
A considerable part of the proteins encoded by genes showing significantly increased mRNA levels in the DNA array experiments was not visible in the 2D PAGE analyses. In most cases, this was due to either an unfavourable alkaline pH or a low protein level.
Whereas the mRNA levels of most of the cold-shock-induced genes reached their maximum 60 min after the temperature downshift, the level of the majority of the cold-shock proteins was highest after 90 min. This is consistent with the data of Graumann et al. (1996) who demonstrated that the cold-shock response in B. subtilis indeed represents a transient regulated phenomenon.
Growth experiments of strains carrying null mutations in cold-shock-induced genes
The mutant strains BFS4469, BFS4564, YHEAd and BFS4794 carry pMUTIN integrations in the genes ydeB, ydjO, yheA and yqgA, which were identified as strongly cold-shock-induced in the transcriptome analysis. These mutants were analysed for a cold-shock-sensitive growth phenotype.
Strains were inoculated with an exponentially growing preculture to an initial OD500 of 0·04 and cultivated at 37 °C until reaching an OD500 of 0·3. Cultures were then shifted to 15 °C by transferring them to a precooled shaker incubator and incubated for 30 h. Two different minimal media were used (see Methods). The experiments were carried out several times and generated highly reproducible results. All mutants revealed a weak but significant growth disadvantage in comparison to the wild-type strain B. subtilis 168. These decreased growth rates were most probably due to the necessity to cultivate the mutant strains in the presence of erythromycin and lincomycin to stabilize the single-crossover integration of the pMUTIN. Therefore, BFS4226, carrying a pMUTIN integration in yacK was used as an additional control. This strain was chosen because its expression was not changed by the cold shock. The yacK mutant showed the same growth behaviour as the other mutant strains (data not shown). Consequently, it has to be concluded that, at least under the conditions tested in this study, inactivation of the cold-inducible genes ydeB, ydjO, yheA and yqgA does not significantly impair the fitness of B. subtilis at low temperature.
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ACKNOWLEDGEMENTS |
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Received 15 February 2002;
revised 1 July 2002;
accepted 23 July 2002.