ABSTRACT
The appropriate conditions to switch on the heat shock promoters in Corynebacterium glutamicum were defined by Northern blot analysis. Transcriptional patterns were characterized for the groEL2 gene and the groES-groEL1 and dnaK operons. Transcriptional start points of these genes were determined by primer extension analysis, allowing the identification of CIRCE and HAIR boxes close to the −10 and −35 regions of the promoters. The presence of both CIRCE and HAIR sequences within a single promoter (P-groEL2) in bacteria is described for the first time. In addition, the dnaK promoter showed −10 and −35 sequences similar to those recognized by SigH of Mycobacterium and SigR of Streptomyces close to a second transcription start region with −10 and −35 boxes typical of promoters for housekeeping genes.
Corynebacterium glutamicum is a gram-positive soil bacterium particularly efficient for secreting large amounts of some amino acids and vitamins and in the bioconversions of organic compounds (7). Significant advances have been made over the last two decades in determining the molecular genetics of corynebacteria (10, 13). The complete genome sequence of C. glutamicum has been fully elucidated (8), and increasing attention has been paid to promoters of corynebacteria in order to understand the control of gene expression in these microorganisms (17, 18, 19).
Overexpression of biosynthetic genes from regulated promoters, which can be induced or repressed at will without addition of chemical inducers to the culture, is a useful tool for increasing the production of amino acids. The heat shock-induced promoters of the genes groEL, groES, and dnaK are attractive candidates for easily regulated transcription.
We report in this article a study of the groES, groEL1, groEL2, and dnaK promoters from C. glutamicum, a transcriptional analysis of the heat shock response, and identification of regulatory signals present in these promoters.
Transcriptional analysis of groEL1 and groEL2 under repeated heat shock conditions.
As a result of initial studies, a temperature of 40°C was chosen for long-term heat inductions (data not shown) since higher temperatures were suitable only for short-term inductions (14). Indeed, Nishio and coworkers (15) reported 40°C as the upper limit for growth of C. glutamicum.
In order to study the duration of the stress response, samples were taken at 0, 20, 40, 60, and 120 min following the heat shock (40°C) from cultures grown previously at 30°C until an optical density at 600 nm of 3.5 was reached. In addition, to elucidate whether mRNA levels respond to repeated heat shock inductions, cultures were returned to 30°C for 30 min and heat shocked again for 45 min (Fig. 1A). Total RNA was extracted from cultures in TYG broth (2× TY plus 2% glucose) as described by Barreiro and coworkers (2).
Heat shock induction of groES and groEL genes. (A) Pattern of heat shock induction. Heat shocks were initiated at 0 and 150 min (vertical arrows). The culture was returned to normal temperature at 120 min and kept at 30°C for 30 min. (B) Transcriptional maps and Northern hybridizations of samples taken at the indicated times. The heat shock pattern was as indicated for panel A. The probes are shown by solid lines. The sizes of the hybridizing bands (in kilobases) are indicated by arrowheads on the left and right of the panels. Note that there is a reinduction of expression after the second heat shock. 16S rRNA was hybridized as a control.
Two copies of the groEL gene (named groEL1 and groEL2) were found in the genome of C. glutamicum (accession number NC_003450 ). The groEL1 gene is located immediately downstream of the cochaperone-encoding groES gene. To test if both groES and groEL1 are transcribed into a single mRNA, total RNA was hybridized with an internal probe (320 bp) to groES (probes internal to groEL1 gave positive bands with both groEL1 and groEL2 transcripts). A transcript of 2.3 kb was found (Fig. 1B) that correlates well with the expected size of a bicistronic groES-groEL1 mRNA (open reading frames of 315 and 1,617 bp). Formation of the 2.3-kb transcript responded clearly to heat shock induction (40°C), and the transcript level was maximal 40 min after induction. A high level of the 2.3-kb transcript was maintained while the culture was incubated at 40°C (Fig. 1B). When the culture was reverted to the standard growth temperature (30°C) for 30 min, the level of the 2.3-kb transcript returned to the noninduced control level. However, following a second heat shock at 40°C for 45 min, there was a new induction, indicating that this promoter may be repeatedly switched on and off by changing the temperature of the culture.
For the analysis of the groEL2 transcript, we took advantage of a specific 15-nucleotide (nt) stretch within groEL2, which is not present in groEL1. When an antisense 15-mer oligonucleotide (5′GTTCAGCTTGCCACC 3′) was used as a probe, a clear transcript of 1.8 kb that corresponds to groEL2 (open reading frame of 1647 nt) was observed. This transcript follows the same induction pattern as that of the groES-groEL1 operon (Fig. 1B). The heat shock did not change the level of the 16S rRNA transcript used as a control.
Transcriptional analysis of dnaK.
C. glutamicum has one copy of the dnaK gene, which is clustered with the cochaperone genes grpE and dnaJ and the regulatory protein gene hspR in the order dnaK-grpE-dnaJ-hspR (Fig. 2). Northern analysis, using a 671-bp fragment internal to the dnaK gene as probe, showed two transcripts: a major 2.8-kb mRNA and a less abundant one of 4.7 kb. To characterize both transcripts, other hybridization was performed with a 940-bp probe corresponding to the 3′ end of dnaJ and the 5′ end of hspR. Northern analysis with this probe showed the same 4.7-kb band already observed in the hybridizations with the dnaK probe but did not highlight the 2.8-kb transcript.
Heat shock induction of dnaK. Transcriptional map and Northern hybridization of RNAs from samples taken at the indicated times. The heat shock pattern was as indicated for Fig. 1A. The probes are indicated by solid lines. The sizes of the hybridizing bands (in kilobases) are indicated by arrows on the left and right of the panels. Putative transcriptional terminators are shown by stem-loop structures. 16S rRNA was hybridized as a control. Observe the heterogeneity present in the smear because of the interference with the rRNAs. The “omega” symbols (palindromes) represent putative terminators found in the 3′ ends of the grpE and hspR genes. The medium used for RNA extraction was TYG (2× TY plus 2% glucose [see the text]).
These results suggest that the 4.7-kb transcript corresponds to the entire dnaK-grpE-dnaJ-hspR operon. The dnaK-hybridizing 2.8-kb band corresponds to a dnaK-grpE transcript terminating at a stem-and-loop structure (putative transcriptional terminator) found between grpE and dnaJ (Fig. 2).
In addition, a 1.6-kb transcript was observed when hybridization was done with the dnaJ-hspR probe, suggesting that this small 1.6-kb band corresponds to a separate transcript of dnaJ-hspR (Fig. 2). The transcripts of 2.8 and 1.6 kb responded clearly to heat shock induction. In addition, the 2.8-kb transcript was reinduced after another heat shock, but the second induction of the 1.6-kb transcript from the dnaJ promoter could not be clearly observed (Fig. 2).
The long 4.7-kb transcript was barely detectable in nonstressed cells, but it was clearly induced by heat shock and showed an apparent decay after 60 min of heat treatment. The same RNA samples were used in Fig. 1B and 2, but an intense smear was observed in the dnaK operon transcript. This smear indicates the instability or partial processing of that operon transcript, which has been described for Streptomyces albus G and Bacillus subtilis (6).
Transcription initiation sites of the groES, groEL2, and dnaK genes.
The transcription initiation abilities of the groES, groEL2, and dnaK promoters were studied in both C. glutamicum and Escherichia coli by coupling these promoters to the promoterless chloramphenicol resistance gene (cat) as a reporter (a well known reporter in corynebacteria) in the plasmid pET2 (26). All the promoters showed promoter activity in both C. glutamicum and E. coli, as happens with some C. glutamicum promoters (3). Transcription start points (TSPs) corresponding to the analyzed promoters in C. glutamicum were determined by primer extension analysis as described by Pátek et al. (17). The areas of the primer extension peaks provided a semiquantitative measurement of the induction factor (IF), calculated as the ratio of the area of the primer extension peaks using RNA from the heat-induced culture (40 min at 40°C) versus that from the noninduced culture (30°C).
A single TSP at a thymine 81 bp upstream of the translation start codon of the groES gene was detected (Fig. 3A). The IF was 8.7 (Fig. 3A). The −10 box (TAGAGT) and the −35 box (TTGAGT) of the promoter were located. An identical −10 hexamer was found in promoters of the corynebacterial genes ask and thrE (17, 24). The TSP of the groEL2 promoter was located at an adenine 146 bp upstream of its ATG translation start codon (Fig. 3B). The putative −10 box (TAGAAA) and the −35 box (TTGCAC) were then identified. Also, for the groEL2 promoter, the peak signal obtained using RNA from the heat-induced culture was 12-fold higher than that obtained using the noninduced culture.
Primer extension analysis. The reaction sequences of the promoter region, T, G, C, and A, were compared with that of the primer extension (PE) reaction product. The peak area (right) is shown for the induced (I) and noninduced (NI) conditions. The transcription start point is indicated by +1 in the nucleotide sequence. The CIRCE sequences are boxed, and the HAIR sequences are in reverse-type letters. Ribosome binding sites are shaded. The −10 and −35 sequences are underlined and in italic letters. (A) Analysis of promoter region of the groES-groEL1 operon. (B) Analysis of the groEL2 promoter region. (C) Analysis of the dnaK promoter. Two products of the primer extension +1(P1) and +1(P2) are indicated. The Fragment Manager Program (Pharmacia Biotech) was used to analyze the reactions.
Two transcription start signals were clearly identified in the dnaK promoter region. The major TSP, at a thymine, suggests that −10 TAAGCT and −35 TGGCAT regions form the core of dnaK P1 promoter. According to the peaks resulting from primer extension analysis (Fig. 3C), transcription from TSP1 was seven to ninefold more intensive than transcription from TSP2, which was located at an adenine 117 bp upstream of the translation initiation codon. A significant similarity of the −10 and −35 sequences upstream of TSP2 of dnaK with the promoter sequences of the dnaK gene and other heat shock genes from Mycobacterium tuberculosis (11, 20), was observed (Fig. 4). Since it is known that these promoters are recognized by the stress sigma factor SigH in M. tuberculosis, we suggest that a homologous sigma factor from C. glutamicum recognizes the upstream dnaK promoter P2. On the basis of this information, the dnaK gene of C. glutamicum might be expressed from the housekeeping promoter P1, negatively controlled by the HspR/HAIR (HspR-associated inverted repeat) system, and from promoter P2, positively regulated by the alternative sigma factor homologous to SigH of M. tuberculosis. This double transcriptional control has been suggested for the dnaK gene of M. tuberculosis (20).
Promoter regions of the dnaK gene from C. glutamicum, C. diphtheriae, C. efficiens, M. tuberculosis, and S. coelicolor. The promoter sequences are aligned with the consensus sequence of σR-dependent S. coelicolor promoters and of σH-dependent M. tuberculosis promoters (27). The conserved putative −10 and −35 sequences and the HAIR sequences are in boldface. Positions with identical nucleotides in all sequences are shaded. Gaps were introduced to achieve alignment of homologous regions.
CIRCE and HAIR elements in the C. glutamicum genome.
In Streptomyces, the groES-groEL1 and hrcA-dnaJ2 operons and the groEL2 gene comprise tandem copies of CIRCE (controlling inverted repeat of chaperone expression) elements (28). These elements are part of the CIRCE/HrcA (heat shock regulator at the CIRCE element [21]) regulatory system. Another regulation system of heat shock proteins in Streptomyces species is the HspR/HAIR regulon. The HAIR elements appear in the promoter region of the dnaK, clpB, and lon genes (23). Since both these regulatory genes are present in the C. glutamicum genome, we screened it for the CIRCE and HAIR elements within the 500-bp upstream region of the translation initiation codon of each gene using the “regulatory sequence analysis tools” (25). We found two partially overlapping CIRCE sequences in the groES-groEL1 promoter (Fig. 3A) that agree with the Streptomyces consensus CIRCE box (23). Also in the groEL2 promoter region two inverted repeats coinciding with the CIRCE motif were observed (Fig. 3B). We found in the groEL2 promoter one HAIR sequence (23) partially overlapping with the CIRCE box. This HAIR motif, separated by an abnormal distance of 18 bp, is a unique case in the C. glutamicum genome. This is the first report of the simultaneous presence of CIRCE and HAIR sequences in a single promoter, which suggests a dual regulation of groEL2 expression by HrcA and HspR. Recently, a similar dual heat shock regulation by the repressors CtsR and HrcA was found in streptococci and staphylococci (4).
A single HAIR sequence was found in the promoter of the dnaK gene, two HAIR elements were located in the promoter region of the clpB gene (5), and one element was found in the promoter of the popR-like gene, coding for a transcriptional regulator. In contrast to what occurs in Streptomyces, no CIRCE or HAIR sequences were found in the promoter regions of lon and hrcA (22).
All the regulatory motifs described are located in the region that contains the −10 and −35 boxes of the promoters. The alignment of all the CIRCE and HAIR sequences present in the C. glutamicum genome allowed us to establish the specific CIRCE [(C/T)TaGCACtCN9GAGTGC(C/T)a(A/G)] and HAIR [(C/A)TTGAgTN7ACTCAA(t/c)] consensus sequences for this microorganism.
In this work, the transcriptional patterns of the dnaK and the groES-groEL1 operons and the unlinked (monocistronic) groEL2 gene have been established. The presence of two groEL genes in several microorganisms has been described, but a clear explanation for this duplication phenomenon has not been provided (9). The arrangement of the dnaK operon has been found to be the same as in other high-G+C gram-positive bacteria (dnaK-grpE-dnaJ-hspR). This arrangement and the lack of the hrcA gene in this cluster in C. glutamicum, typically present in the dnaK operon in gram-positive bacteria with low G+C content (1), support the evolutionary relationship of corynebacteria with high-G+C actinomycetes (e.g., Mycobacterium and Streptomyces species).
We determined the transcription start points of the studied promoters by primer extension analysis. The related −10 and −35 sequences agreed with the consensus sequences reported for corynebacterial promoters (12, 18), indicating that the analyzed genes are expressed from housekeeping promoters. Moreover, in the upstream region of the dnaK gene a second promoter was found showing the −10 and −35 motifs described for the promoters of heat shock genes of M. tuberculosis recognized by SigH (20) or for the SigR-dependent promoters of S. coelicolor (16). We have found the same motifs within the dnaK promoters of Corynebacterium efficiens, Corynebacterium diphtheriae, M. tuberculosis and S. coelicolor (Fig. 4).
The arrangement of the CIRCE boxes in the C. glutamicum genome (groES-groEL1, groEL2) suggests a different HrcA-mediated regulation without the autogenous feedback control described for the hrcA-dnaJ2 operon in Streptomyces species (22). On the other hand, the HAIR boxes found in the promoter regions of clpB (5), dnaK, groEL2, and the popR-like transcriptional activator, which controls the expression of the clpP3-clpP4 operon in Streptomyces lividans (27), suggest a cascade expression of heat shock-regulated genes clustered under the control of HspR.
A significant IF was observed when the primer extension peak areas of the heat-shocked and noninduced genes were compared. The studied promoters respond clearly to a second induction, which suggests that these genes may be switched on several times, following a specific temperature program.
ACKNOWLEDGMENTS
This work was supported by a grant of the European Union (QLRT-2000-00497) and by grant 204/01/0998 from the Grant Agency of the Czech Republic. C. Barreiro received a fellowship of the Ministry of Science and Technology, Madrid, Spain, and E. González-Lavado was supported by a fellowship of the Basque Government (Vitoria, Spain).
We thank J. Nešvera for carefully reading the manuscript, A. Sánchez and M. Barriuso for support of the Corynebacterium group, and B. Martín, J. Merino, A. Casenave, and M. Álvarez for excellent technical assistance.
FOOTNOTES
- Received 11 February 2004.
- Accepted 13 April 2004.
- Copyright © 2004 American Society for Microbiology
