|
| |
Journal of Bacteriology, July 2004, p . 4813-4817, Vol . 186,
No . 14
Transcriptional Analysis of the groES-groEL1, groEL2, and
dnaK genes in Corynebacterium glutamicum: Characterization of Heat
Shock-Induced Promoters
Carlos Barreiro,1 Eva González-Lavado,1
Miroslav Pátek,2 and Juan-Francisco Martín1,3*
Institute of Biotechnology of León, León, 24006,1 Area of
Microbiology, Faculty of Biologic and Environmental Sciences, University of
León, León, 24071, Spain,3 Institute of Microbiology, Academy of
Sciences of the Czech Republic, 14220 Prague 4, Czech Republic2
Received 11 February 2004/ Accepted 13 April 2004
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 (2x TY plus 2%
glucose) as described by Barreiro and coworkers (2) .
|
FIG . 1 . 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 .
|
FIG . 2 . 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 (2x
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 .
|
FIG . 3 . 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) .
|
FIG . 4 . 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
 R-dependent
S . coelicolor promoters and of
H-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 .
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 . Ne vera
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 .
* Corresponding author . Mailing address: Institute of
Biotechnology of León (INBIOTEC) . Science Park of León . Av . Real, 1, León,
24006, Spain . Phone: 34 987 291 505 . Fax: 34 987 291 506 . E-mail: degjmm@unileon.es .
- Ahmad, S., A . Selvapandiyan, and R . K . Bhatnagar. 2000 .
Phylogenetic analysis of Gram-positive bacteria based on grpE, encoded
by the dnaK operon . Int . J . Syst . Evol . Microbiol . 50:1761-1766 .
- Barreiro, C., E . González-Lavado, and J . F . Martín. 2001 .
Organization and transcriptional analysis of a six-gene cluster around the
rplK-rplA operon of Corynebacterium glutamicum encoding the
ribosomal proteins L11 and L1 . Appl . Environ . Microbiol . 67:2183-2190 .
- Cadenas, R . F., J . F . Martín, and J . A . Gil. 1991 .
Construction and characterization of promoter-probe vectors for Corynebacteria
using the kanamycin-resistance reporter gene . Gene 98:117-121.
- Chastanet, A., J . Fert, and T . Msadek. 2003 . Comparative
genomics reveal novel heat shock regulatory mechanisms in Staphylococcus
aureus and other Gram-positive bacteria . Mol . Microbiol . 47:1061-1073.
- Grandvalet, C., V . de Crecy-Lagard, and P . Mazodier.
1999 . The ClpB ATPase of Streptomyces albus G belongs to the HspR heat
shock regulon . Mol . Microbiol . 31:521-532.
- Grandvalet, C., P . Servant, and P . Mazodier. 1997 .
Disruption of hspR, the repressor gene of the dnaK operon in
Streptomyces albus G . Mol . Microbiol . 23:77-84.
- Hermann, T. 2003 . Industrial production of amino acids by
coryneform bacteria . J . Biotechnol . 104:155-172.
- Ikeda, M., and S . Nakagawa. 2003 . The Corynebacterium
glutamicum genome: features and impacts on biotechnological processes .
Appl . Microbiol . Biotechnol . 62:99-109.
- Karlin, S., and L . Brocchieri. 2000 . Heat shock protein
60 sequence comparisons: duplications, lateral transfer, and mitochondrial
evolution . Proc . Natl . Acad . Sci . USA 97:11348-11353 .
- Kirchner, O., and A . Tauch. 2003 . Tools for genetic
engineering in the amino acid-producing bacterium Corynebacterium
glutamicum. J . Biotechnol . 104:287-299.
- Manganelli, R., M . I . Voskuil, G . K . Schoolnik, E . Dubnau,
M . Gomez, and I . Smith. 2002 . Role of the extracytoplasmic-function
factor
H
in Mycobacterium tuberculosis global gene expression . Mol . Microbiol.
45:365-374.
- Martín, J . F. 1989 . Molecular genetics of amino
acid-producing corynebacteria, 25-59 . In S . Baumberg, I . Hunter, and M .
Rhodes (ed.), Microbial products: new approaches . Cambridge University Press,
Cambridge, United Kingdom.
- Martín, J . F., and J . A . Gil. 1999 . Corynebacteria, p .
379-391 . In A . L.Demain, J . E . Davies, R . M . Atlas, G . Cohen, C . L .
Hershberger, W.-S . Hu, D . H . Sherman, R . C . Willson, and J . H . D . Wu (ed.),
Manual of industrial microbiology and biotechnology, 2nd ed . ASM Press,
Washington, D.C.
- Muffler, A., S . Bettermann, M . Haushalter, A . Horlein, U .
Neveling, M . Schramm, and O . Sorgenfrei. 2002 . Genome-wide transcription
profiling of Corynebacterium glutamicum after heat shock and during
growth on acetate and glucose . J . Biotechnol . 98:255-268.
- Nishio, Y., Y . Nakamura, Y . Kawarabayasi, Y . Usuda, E .
Kimura, S . Sugimoto, K . Matsui, A . Yamagishi, H . Kikuchi, K . Ikeo, and T .
Gojobori. 2003 . Comparative complete genome sequence analysis of the amino
acid replacements responsible for the thermostability of Corynebacterium
efficiens. Genome Res . 13:1572-1579 .
- Paget, M . S., V . Molle, G . Cohen, and Y . Aharonowitz.
2001 . Defining the disulphide stress response in Streptomyces coelicolor
A3(2): identification of the
R
regulon . Mol . Microbiol . 42:1007-1020.
- Pátek, M., B . J . Eikmanns, J . Pátek, and H . Sahm. 1996 .
Promoters from Corynebacterium glutamicum: cloning, molecular analysis
and search for a consensus motif . Microbiology 142:1297-1309.
- Pátek, M., G . Muth, and W . Wohlleben. 2003 . Function of
Corynebacterium glutamicum promoters in Escherichia coli,
Streptomyces lividans, and Bacillus subtilis. J . Biotechnol .
104:325-334.
- Pátek, M., J . Nevera,
A . Guyonvarch, O . Reyes, and G . Leblon. 2003 . Promoters of
Corynebacterium glutamicum. J . Biotechnol . 104:311-323.
- Raman, S., T . Song, X . Puyang, S . Bardarov, W . R . Jacobs,
Jr., and R . Husson. 2001 . The alternative sigma factor SigH regulates
major components of oxidative and heat stress responses in Mycobacterium
tuberculosis. J . Bacteriol . 183:6119-6125 .
- Roberts, R . C., C . Toochinda, M . Avedissian, R . L . Baldini,
S . L . Gomes, and L . Shapiro. 1996 . Identification of a Caulobacter
crescentus operon encoding hrcA, involved in negatively regulating
heat-inducible transcription, and the chaperone gene grpE. J .
Bacteriol . 178:1829-1841.
- Rosen, R., and E . Y . Ron. 2002 . Proteome analysis in the
study of the bacterial heat-shock response . Mass Spectrom . Rev . 21:244-265.
- Servant, P., and O . Mazodier. 2001 . Negative regulation
of the heat shock response in Streptomyces. Arch . Microbiol . 176:237-242.
- Simic, P., H . Sahm, and L . Eggeling. 2001.
L-Threonine export: use of peptides to identify a new
translocator from Corynebacterium glutamicum. J . Bacteriol . 183:5317-5324 .
- Van Helden, J. 2003 . Regulatory sequence analysis tools .
Nucleic Acids Res . 31:3593-3596 .
- Vaicová,
P., Z . Abrhámová, J . Nevera,
M . Pátek, H . Sahm, and B . Eikmanns. 1998 . Integrative and autonomously
replicating vectors for analysis of promoters in Corynebacterium
glutamicum. Biotechnol . Tech . 12:743-746.
- Viala, J., G . Rapoport, and P . Mazodier. 2000 . The
clpP multigenic family in Streptomyces lividans: conditional
expression of the clpP3 and clpP4 operon is controlled by PopR,
a novel transcriptional activator . Mol . Microbiol . 38:602-612.
- Zuber, U., and W . Schumann. 1994 . CIRCE, a novel heat
shock element involved in regulation of heat shock operon dnaK of
Bacillus subtilis. J . Bacteriol . 176:1359-1363.
Free Online Full-text Article
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
|
