The cyclic AMP receptor protein (CRP) homolog, GlxR, controls the expression of several genes involved in the regulation of diverse physiological processes in
Corynebacterium glutamicum. In silico
analysis has revealed the presence of
glxR
binding sites upstream of genes
ptsG, adhA,
and
ald
, encoding glucose-specific phosphotransferase system protein, alcohol dehydrogenase (ADH), and acetaldehyde dehydrogenase (ALDH), respectively. However, the involvement of the GlxR-cAMP complex on the expression of these genes has been explored only
in vitro.
In this study, the expressions of
ptsG, adhA
, and
ald
were analyzed in detail using an adenylate cyclase gene (
cyaB
) deletion mutant and
glxR
deletion mutant. The specific activities of ADH and ALDH were increased in both the mutants in glucose and glucose plus ethanol media, in contrast to the wild type. In accordance, the promoter activities of
adhA
and
ald
were derepressed in the
cyaB
mutant, indicating that
glxR
acts as a repressor of
adhA
. Similarly, both the mutants exhibited derepression of
ptsG
regardless of the carbon source. These results confirm the involvement of GlxR on the expression of important carbon metabolic genes;
adhA, ald,
and
ptsG
.
Introduction
Corynebacterium glutamicum
is a gram-positive soil bacterium, widely used in the industrial production of amino acids such as lysine and glutamic acid. This organism is able to grow on a variety of carbon and energy sources such as sugars, sugar alcohols, and organic acids
[9
,
21
,
23]
. In addition, ethanol can be used as a sole or an additional carbon source, and the major enzymes involved in the breaking down of ethanol in
C. glutamicum
are alcohol dehydrogenase (ADH, encoded by
adhA
) and acetaldehyde dehydrogenase (ALDH, encoded by
ald
)
[2
,
19]
.
C. glutamicum
co-metabolizes glucose with other sugars or with organic acids and exhibits monophasic growth behavior
[6
-
8
,
12
,
20
,
30
,
33]
. In contrast, the growth of
C. glutamicum
on a mixture of glucose and ethanol shows biphasic growth behavior, which is due to the sequential utilization of glucose before ethanol
[1
,
19]
. It has been reported that this biphasic growth behavior is accompanied by relatively low ADH and ALDH activities in the first and higher activities in the second growth phases, indicating that ethanol catabolism in
C. glutamicum
is subject to carbon catabolite control
[1]
. In addition, it was revealed that the expressions of
adhA
and
ald
are under the control of transcriptional regulators, RamA and RamB
[2]
. Whereas RamA is essential for the expression of
adhA
and
ald
, RamB exerts a negative control on
adhA
and
ald
expression in the presence of glucose or acetate in the growth medium. In a RamB deletion mutant, the glucose- and acetate-dependent down-regulation of
adhA
was partially released, indicating the involvement of an additional regulator in the catabolite repression of
adhA
[2]
. In addition, they observed a binding motif for the cAMP-dependent regulator, GlxR in the promoter region of
adhA
, 21 bp downstream of the transcriptional start site. In 2008, Kohl
et al
.
[17]
verified the binding of GlxR upstream of the
adhA
gene by electrophoretic mobility shift assay (EMSA). In the case of
ald
, putative GlxR binding motifs were detected upstream of the gene
[3]
, and the binding of GlxR was verified by EMSA
[18]
. However, there are no
in vivo
data showing the direct involvement of GlxR on the expression of ethanol-catabolic genes
adhA
and
ald
in
C. glutamicum.
The
in vivo
evaluation of the regulatory effect of GlxR is limited, as the
glxR
deletion mutant shows severe growth defect
[26]
.
In
C. glutamicum
, the preferred carbon source, glucose, is taken up
via
the phosphoenolpyruvate-dependent phosphotransferase system (PTS)
[22
,
24]
. It has been reported that the expression of
ptsG
, which encodes the glucose-specific PTS enzyme Ⅱ is repressed by the DeoR-type regulator SugR
[11
,
31]
. In contrast, the transcriptional regulators GntR1 and GntR2 activate the expression of
ptsG
[12]
. In addition, a putative binding site for the transcriptional regulator RamB is seen in the promoter region of
ptsG
[13]
. The putative binding site of RamB is located downstream of the transcriptional start site of
ptsG
, indicating that RamB might function as a negative regulator of
ptsG
[11]
. In 2008, Kohl
et al
.
[17]
reported the presence of a putative binding site of GlxR in the promoter region of
ptsG
, which was verified by EMSA. Since the binding motif is located downstream of the transcriptional initiation site of
ptsG
, GlxR might act as a repressor of
ptsG
expression. Even though there are
in vitro
data showing the involvement of GlxR and RamB on the expression of
ptsG
, there are no
in vivo
results showing the regulation of
ptsG
by these regulators. Therefore, we have carried out
in vivo
experiments in order to confirm the role of the global regulator GlxR and the master regulator RamB on the expression of
ptsG
in
C. glutamicum.
As reported earlier, the ethanol metabolic pathway and glucose-uptake system in
C. glutamicum
are under the control of a number of regulators
[2
,
3
,
11
-
13
,
17
,
18
,
31]
. However, the studies revealed that there might be the involvement of additional regulators controlling the expression of ethanol metabolic genes and
ptsG
. In the present study, we investigated the role of GlxR in the regulation of
adhA, ald
, and
ptsG
, and of RamB on the expression of
ptsG
in
C. glutamicum
. We present
in vivo
experimental evidence indicating a negative regulation of GlxR on
ptsG
,
adhA,
and
ald
. In addition, the
in vivo
data confirm the repressive effect of RamB on the expression of
ptsG
.
Materials and Methods
- Bacterial Strains, Plasmids, Oligonucleotides, and Growth Conditions
The bacterial strains, plasmids, and oligonucleotides used in this study are listed in
Table 1
. The
E. coli
strain was grown in LB (Luria-Bertani) medium (10 g/l tryptone, 5g/l yeast extract, 10 g/l NaCl) at 37℃, whereas the
C. glutamicum
ATCC 13032 was grown at 30℃ in LB or minimal medium. The minimal medium contained 5g/l (NH
4
)
2
SO
4
, 5g/l urea, 0.5g/l KH
2
PO
4
, 0.5g/l K
2
HPO
4
, 21 g/l MOPS, 0.25g/l MgSO
4
·7H
2
O, 10 mg/l CaCl
2
·H
2
O, 10 mg/l MnSO
4
·H
2
O, 10 mg/l FeSO
4
·7H
2
O, 1 mg/l ZnSO
4
·7H
2
O, 0.2 mg/l CuSO
4
, and 0.2 mg/l biotin
[5]
. As the carbon source, glucose and/or acetate, ethanol and/or glucose were added to the media at a level of 1% (w/v). BHIS (BHI Sorbitol) medium comprised 3.7% (w/v) brain heart infusion (BHI) medium, and 9.1% sorbitol was used for the preparation of competent cells of
C. glutamicum
[10]
.
C. glutamicum
strains used in this work were precultured in BHI medium overnight in a rotary shaker at 200 rpm. The cells were harvested and washed with LB or a buffer containing 5g/l (NH
4
)
2
SO
4
, 5g/l urea, 0.5g/l KH
2
PO
4
, 0.5g/l K
2
HPO
4
, and 21 g/l MOPS. These cells were used to inoculate main cultures of LB or minimal medium containing apposite carbon sources. When appropriate, kanamycin and chloramphenicol were added at concentrations of 50 and 10 μg/ml, respectively. The oligonucleotides used in this study were purchased from Cosmo Genetech Co., Ltd, Korea.
- DNA Manipulation and Transformation
Standard molecular cloning procedures were followed in this study
[27]
. The chromosomal DNA from the
C. glutamicum
cells was isolated using a genomic DNA purification kit (SolGent Co.), and the DNA fragments from the agarose gel were eluted using a gel extraction kit (SolGent Co.). The plasmids were introduced into
C. glutamicum
by electroporation as described before
[32]
using MicroPulser (Bio-Rad Laboratories Inc.).
- Construction of Transcriptional Fusion Vectors
The promoter probe transcriptional fusion plasmids were constructed using the vector pSK1CAT
[25]
, which harbors a promoterless
cat
(chloramphenicol acetyltransferase) gene encoding chloramphenicol acetyltransferase (CAT) enzyme. The promoter fragments of the
adhA, ald, ptsG
, and
glxR
genes were generated by PCR using primers tagged with
Bam
HI and cloned in front of the
cat
gene of pSK1CAT. The recombinant plasmid pAD contained the promoter fragment of
adhA,
383 bp from the translational start site (255 bp from the transcriptional start site) and 18 bp in the
adhA
gene. The promoter fragment, 318 bp upstream of the translational start site (227 bp from the transcriptional start site) and 23 bp in the
ald
gene were taken for constructing the fusion plasmid pAL. DNA fragments containing 657 bp from the translational start site (399 bp from the transcriptional start site) and 11 bp in the
ptsG
gene were used to construct the fusion plasmid pGP. The promoter fragment that includes 520 bp upstream of the translational start site (400 bp from the transcriptional start site) and 14 bp in the
glxR
gene was amplified to construct the recombinant plasmid p
glxR
. The primers used to amplify the promoter sequences are listed in
Table 1
.
Bacterial strains, plasmids, and oligonucleotides used in this study.
Underlined sequences indicate the recognition sites for restriction enzyme.
- Enzyme Assays
For all the assays, except for CAT assay with
ptsG
promoter, the strains were cultivated in minimal medium containing acetate or ethanol or/and glucose. To determine the CAT activity with
ptsG
promoter, the strains were cultivated in LB medium containing glucose or/and acetate. The cells were harvested in the exponential phase, washed twice in 50 mM potassium phosphate, pH 7.0 (for ADH assay) or 100 mM Tris-HCl, pH 7.5 (for ALDH assay) or 50 mM Tris-HCl, pH 7.0 (for CAT assay), and resuspended in the same buffer containing 10 mM MgCl
2
, 1 mM EDTA, and 30% (v/v) glycerol. The cell suspension was mixed with glass beads (Sigma-Aldrich) and subjected to mechanical disruption using a Mini-Beadbeater (Biospec Products, USA) with intermittent cooling on ice for 1 min. After the disruption, the glass beads and cellular debris were removed by centrifugation (13,000 ×
g
, 4℃, 15min for CAT, ADH, and ALDH assays, and 45,000 ×
g
, 4℃, 60 min for ADH and ALDH assays), and the supernatant was used for the assay. The protein concentration was measured using the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin as the standard. The ADH activity was assayed following the increase of NADH at 340 nm in 1 ml of 86 mM sodium pyrophosphate buffer (pH 9.0), 19 mM glycin, 6.2 mM semicarbacide-hydrochloride (pH 6.5), 1.8 mM NAD
+
, 1 mM glutathione, and 600 mM ethanol, as described previously
[4]
. One unit of ADH activity corresponds to the production of 1 μmol of NADH per minute at 30℃. ALDH activity was measured according to a modified protocol described previously
[4]
following the formation of NADH at 340 nm. One unit of ALDH activity is defined as 1 μmol of NADH formed per minute at 30℃. CAT activity was assayed as described by Shaw
[29]
with modifications outlined by Schreiner
et al
.
[28]
. Briefly, the assay mixture (1 ml) contained 100 mM Tris-HCl (pH7.8), 0.1 mM acetyl-CoA, 1 mM 5,5-dithiobis-2-nitrobenzoic acid, 0.25 mM chloramphenicol, and crude extract. The formation of free 5-thio-2-nitrobenzoate was measured photometrically at 412 nm. One unit of CAT activity corresponds to 1 μmol of chloramphenicol acetylated per minute at 37℃.
Results
- Involvement of GlxR in the Expression of Alcohol Dehydrogenase and Acetaldehyde Dehydrogenase
In vitro
studies have revealed the binding of GlxR to upstream of the
adhA
and
ald
genes
[1
,
2]
and this regulator protein together with RamA and RamB is responsible for carbon catabolite repression in
C. glutamicum
.
In silico
analysis has revealed the presence of two putative GlxR binding sites, -75 and -104 upstream of the translational start site of the
adhA
gene. Two possible GlxR binding motifs, 72 bp and 44 bp upstream of the start codon of the
ald
gene, have also been reported by
in silico
analysis. These observations suggested a direct interaction of GlxR with the
adhA
and
ald
promoters and prompted us to determine specific ADH and ALDH activities in the
glxR
and
cyaB
deletion mutants. It has been reported that GlxR is a cAMPdependent transcriptional regulator
[16]
, and thus the experiments with
cyaB
deletion mutant could provide additional indirect evidence for the involvement of GlxR on the expression of genes. The
C. glutamicum
wild type, and
glxR
and
cyaB
deletion mutants were grown in minimal medium containing 1% ethanol and/or 1% glucose, and ADH and ALDH activities were measured. As reported earlier
[1
,
2]
, the expressions of ADH and ALDH were repressed when the wild-type strain was grown in medium containing glucose, or glucose plus ethanol, when compared with the expression in ethanol-grown wild type (
Tables 2
and
3
). In contrast, the deletion mutants derepressed the expression of ADH in glucose, or glucose plus ethanol (
Table 2
). In the case of ALDH, the activity was derepressed in deletion mutants irrespective of the carbon source in the medium. The specific activity of ADH and ALDH in the
glxR
mutant was increased 3-4- and 9-12-folds, respectively, in glucose and glucose plus ethanol medium compared with those in wild-type. In addition, the ALDH activity of
glxR
deletion mutant in ethanol medium was 2-times higher than that of wild type. Similarly, in the
cyaB
mutant, the ADH and ALDH activities were increased 2-fold in glucose and glucose plus ethanol medium compared with those in the wild type. Thus, when taken together, these results confirm that utilization of ethanol is regulated by CCR, and GlxR represses the expression of the genes
adhA
and
ald
.
Specific ADH activity of wild type (WT), mutant CgYA (△cyaB),glxRdeletion mutant (△glxR), and complemented mutants.
aThe strains were grown in minimum medium containing 1% ethanol and/or 1% glucose, and enzyme activity was assayed in cell extracts from early exponential phase. All the values are from at least three independent cultivations and two determinations per experiment with standard deviations.
Specific ALDH activity of wild type (WT), mutant CgYA (∆cyaB),glxRdeletion mutant (∆glxR), and complemented utants.
aThe strains were grown in minimum medium containing 1% ethanol and/or 1% glucose, and enzyme activity was assayed in cell extracts from early exponential phase. All the values are from at least three independent cultivations and two determinations per experiment with standard deviations.
To investigate the carbon-source-dependent expressions of
adhA
and
ald
in more detail, the wild type and
cyaB
deletion mutant harboring the recombinant plasmids pAD and pAL were cultivated in minimal medium containing 1% ethanol and/or 1% glucose and the CAT activities determined. In accordance with the ADH enzyme assay results,
cyaB
mutant showed increased specific CAT (promoter) activities, with glucose and with glucose plus ethanol, when compared with wild type (
Fig. 1
). The specific CAT activity was 4 times higher in the deletion mutant in glucose and glucose plus ethanol media, compared with those in wild type. In the case of
ald
promoter, the
cyaB
mutant showed increased specific CAT (promoter) activities, when compared with wild type (
Fig. 2
), irrespective of the carbon source. The specific CAT activity was 2-4 times higher in the deletion mutant in all the media tested, compared with those in wild type. These results conclude that the regulation of
adhA
and
ald
genes occurs by the binding of the cAMP-GlxR complex. Furthermore, we can conclude that GlxR exerts a negative effect on the expression of the
adhA
and
ald
genes.
Specific CAT activities of C. glutamicum wild type (WT) and cyaB deletion mutant (ΔcyaB) carrying the adhA promoter fragment in the transcriptional fusion vector pSK1CAT. The strains were grown in minimal medium containing 1% ethanol and/or 1% glucose. The cells were harvested in the early exponential phase to measure the enzyme activity. All the values are from at least three independent cultivations and two determinations per experiment with standard deviations.
Specific CAT activities of C. glutamicum wild type (WT) and cyaB deletion mutant (ΔcyaB) carrying the ald promoter fragment in the transcriptional fusion vector pSK1CAT. The cells were harvested in the early exponential phase, after growing in minimal medium containing 1% ethanol and/or 1% glucose. All the values are from at least three independent cultivations and two determinations per experiment with standard deviations.
- Expression of ptsG Is Controlled by GlxR and RamB
It has previously been reported that a DeoR-type transcriptional regulator, SugR, controls the expression of
ptsG
and other PTS genes during growth on gluconeogenic substrates in
C. glutamicum
[11]
. In addition, it has been pointed out that the regulation of
ptsG
in
C. glutamicum
does not depend solely on SugR, but seems to be complex. Another study suggested a putative GlxR binding site upstream of
ptsG
, and this finding was supported by electrophoretic mobility shift assay
[17]
. To test the transcriptional regulation of
ptsG
by GlxR
in vivo
, the transcriptional fusion plasmid (pGP) carrying the promoter region of
ptsG
was transformed into
C. glutamicum
wild type,
cyaB
deletion mutant, and
glxR
deletion mutant. The strains were cultivated in minimal medium containing glucose (1%), a glucose-acetate substrate mixture (1%), and acetate (1%), and CAT activities were determined. The specific CAT activity was relatively low during growth on acetate compared with glucose-acetate or glucose. With all tested substrates, the specific CAT activities in both the deletion mutants were higher when compared with that of wild type (
Fig. 3
). The
glxR
deletion mutant showed 67 times higher specific activity in acetate medium compared with that of wild type. In medium containing glucose or glucose plus acetate, the
glxR
deletion mutant showed 5 and 10 times higher levels of expression compared with that in wild type, respectively. In acetate medium, the specific activity in the
cyaB
deletion mutant was 13 times higher than that of wild type
C. glutamicum
. The
cyaB
deletion mutant expressed 5 times higher
ptsG
expression in medium containing glucose or glucose plus acetate, compared with that of wild type. These results conclude that GlxR acts as an essential repressor of the
ptsG
gene in
C. glutamicum.
Specific CAT activities of C. glutamicum wild type (WT), mutant CgYA (ΔcyaB), glxR deletion mutant (ΔglxR) and ramB deletion mutant (ΔramB) carrying the ptsG promoter fragment in the transcriptional fusion vector pSK1CAT. The cells were grown in LB medium containing 1% glucose and/or 1% acetate, and enzyme activity was assayed in cell extracts from early exponential phase. All the values are from at least three independent cultivations and two determinations per experiment with standard deviations.
In addition to the GlxR binding site, two putative binding sites for the master regulator RamB was reported in the promoter region of
ptsG
[13]
. Since the putative binding sites of RamB (AAATTTTTGCCAA, CAAATTGTGCAAT) are located downstream of the transcriptional start site (+77,+87), it was assumed that RamB might function as a negative regulator of
ptsG
[13]
. To test for the effect of RamB on
ptsG
transcription, promoter fusion experiments were performed with the
ramB
deletion mutant and wild-type strain of
C. glutamicum.
For this purpose, plasmid pGP carrying the promoter region of
ptsG
was transformed into the wild type and mutant strain, and after growth in LB containing 1% glucose and/or 1% acetate, the specific CAT activities (
i.e
., the
ptsG
promoter activities) were determined. As described in
Fig. 3
, the specific CAT activity was relatively low in wild-type
C. glutamicum
during growth on medium containing acetate. In contrast, the
ramB
deletion mutant showed higher specific CAT activities compared with wild type irrespective of the carbon source (
Fig. 3
). The promoter activity of
ramB
mutant was found to be 28fold higher in acetate medium when compared with that of wild type. Four-to 10-fold higher CAT activities were observed in the
ramB
deletion mutant when cultivated with glucose and glucose plus acetate, respectively. These results indicate that RamB is a repressor of the
ptsG
under all the tested carbon sources.
Discussion
GlxR, being the global regulator, controls the expression of over 400 genes in
C. glutamicum
[18]
. It was first characterized as a protein that represses the gene
aceB
of the glyoxylate pathway. In
C. glutamicum
, the genes involved in the catabolism of ethanol include
ald
, encoding acetaldehyde dehydrogenase, and
adhA
, encoding alcohol dehydrogenase. Bioinformatic studies have revealed the presence of putative GlxR binding sites upstream of the genes
adhA
and
ald
[18]
. Enzyme assay of alcohol dehydrogenase and acetaldehyde dehydrogenase along with the transcriptional fusion experiments of the
adhA
and
ald
promoter regions revealed that GlxR acts as a repressor of these genes. The proteome analysis of wild-type
C. glutamicum
and
glxR
deletion mutant revealed that the alcohol dehydrogenase and acetaldehyde dehydrogenase enzymes are derepressed in the
glxR
deletion mutant compared with that of wild-type (data not shown). Taken together, these results confirm that the ethanol metabolic genes
ald
and
adhA
in
C. glutamicum
are regulated by GlxR, depending on the availability of different carbon sources.
The unpublished data from our laboratory reveals that the cAMP level and
glxR
promoter activity are very high in ethanol medium compared with that in glucose and acetate media
[5]
, thereby suppressing the genes in the glycolytic and gluconeogenic pathways. It has been reported earlier that GlxR represses the master regulator RamB
[15]
. It could be concluded that the expression of RamB is repressed by the high level of GlxR in ethanol medium, and this might remove the repressive effect of RamB over the genes;
adhA
and
ald
encoding the ethanol catabolic enzymes alcohol dehydrogenase and acetaldehyde dehydrogenase, respectively. In the case of acetate medium, as the
glxR
promoter activity is low, the expressions of glyoxylate and glycolytic pathway enzymes are functional. This might be the reason why
C. glutamicum
is able to utilize glucose along with acetate, in contrast to the case with ethanol, where glucose or acetate cannot be catabolized along with ethanol. In glucose medium, the acetate and ethanol catabolic genes might be repressed because of the high GlxR activity.
In vitro
studies have pointed out the involvement of transcriptional regulators, GlxR and RamB, on the expression of
ptsG
in
C. glutamicum.
Gerstmeir
et al
.
[13]
reported the presence of putative binding sites of RamB, downstream of the transcriptional start site of
ptsG
. Another study revealed the presence of putative GlxR binding sites in the promoter region of
ptsG
, suggesting the involvement of GlxR controlling the expression of
ptsG
[17]
. We have carried out
in vivo
experiments to elucidate the role of GlxR and RamB on the expression of
ptsG
in more detail. It has been noticed that in
glxR,
cyaB
, and
ramB
deletion mutants, the
ptsG
expression was derepressed irrespective of the carbon sources, indicating that GlxR and RamB act as a repressor of
ptsG
in
C. glutamicum
. Earlier studies have revealed the involvement of other regulators, SugR (repressor) and GntR1 and GntR2 (activators), on the expression of
ptsG
[11
,
12]
. It was noticed that SugR regulates
ptsG
expression in a carbon-source-dependent manner and represses
ptsG
under gluconeogenic carbon sources. GntR1 and GntR2 are functionally redundant transcriptional regulators that activate the expression of
ptsG
in the absence of gluconate. There is a compiled effect of all the five regulators (SugR, GntR1, GntR2, GlxR, and RamB) for the fine-tuning of the expression of
ptsG
. These regulators are the most important players in a complex regulatory network that controls the uptake and metabolism of different carbon sources, allowing the most favorable combination of the available substrates.
As outlined in the introduction, the genes
adhA, ald
, and
ptsG
have important roles in the carbon metabolism of
C. glutamicum
. However, there was no complete information on the functional characterization of these genes. In the present study, we have provided
in vivo
data confirming the binding of different regulators to the promoter region controlling the expression of these genes.
Acknowledgements
This work was supported by the Next-Generation BioGreen 21 Program (No. PJ 009486) under the Rural Development Administration, Korea. We thank Prof. Heung-Shick Lee of Korea University for providing us the pSK1CAT vector.
Arndt A
,
Auchter M
,
Ishige T
,
Wendisch VF
,
Eikmanns BJ
2008
Ethanol catabolism in Corynebacterium glutamicum.
J. Mol. Microbiol. Biotechnol.
15
222 -
233
DOI : 10.1159/000107370
Arndt A
,
Eikmanns BJ
2007
The alcohol dehydrogenase gene adhA in Corynebacterium glutamicum is subject to carbon catabolite repression.
J. Bacteriol.
189
7408 -
7416
DOI : 10.1128/JB.00791-07
Auchter M
,
Arndt A
,
Eikmanns BJ
2009
Dual transcriptional control of the acetaldehyde dehydrogenase gene ald of Corynebacterium glutamicum by RamA and RamB.
J. Biotechnol.
140
84 -
91
DOI : 10.1016/j.jbiotec.2008.10.012
Bernt E
,
Gutmann I
1974
Aethanol, In Bergmeyer HU (ed.), Methoden der Enzymatischen Analyse.
Verlag Chemie
Weinheim
1545 -
1548
Cha PH
,
Park SY
,
Moon MW
,
Subhadra B
,
Oh TK
,
Kim E
,
Kim JF
,
Lee JK
2010
Characterization of an adenylate cyclase gene (cyaB) deletion mutant of Corynebacterium glutamicum ATCC 13032.
Appl. Microbiol. Biotechnol.
85
1061 -
1068
DOI : 10.1007/s00253-009-2066-9
Claes WA
,
Pühler A
,
Kalinowski J
2002
Identification of two prpDBC gene clusters in Corynebacterium glutamicum and their involvement in propionate degradation via the 2-methylcitrate cycle.
J. Bacteriol.
184
2728 -
2739
DOI : 10.1128/JB.184.10.2728-2739.2002
Cocaign M
,
Monnet C
,
Lindley ND
1993
Batch kinetics of Corynebacterium glutamicum during growth on various carbon substrates: use of substrate mixtures to localize metabolic bottlenecks.
Appl. Microbiol. Biotechnol.
40
526 -
530
DOI : 10.1007/BF00175743
Dominguez H
,
Cocaign-Bousquet M
,
Lindley ND
1993
Simultaneous consumption of glucose and fructose from sugar mixtures during batch growth of Corynebacterium glutamicum.
Appl. Microbiol. Biotechnol.
47
600 -
603
DOI : 10.1007/s002530050980
Eggeling L
,
Bott M
2005
Handbook of Corynebacterium glutamicum.
CRC Press
Boca Raton, USA
Eggeling L
,
Reyes O
2004
Experiments, In Eggeling L, Bott M (ed.), Handbook of Corynebacterium glutamicum
Taylor & Francis
Boca Raton, FL
535 -
566
Engels V
,
Wendisch VF
2007
The DeoR-type regulator SugR represses expression of ptsG in Corynebacterium glutamicum.
J. Bacteriol.
189
2955 -
2966
DOI : 10.1128/JB.01596-06
Frunzke J
,
Engels V
,
Hasenbein S
,
Gätgens C
,
Bott M
2008
Co-ordinated regulation of gluconate catabolism and glucose uptake in Corynebacterium glutamicum by two functionally equivalent transcriptional regulators, GntR1 and GntR2.
Mol. Microbiol.
67
305 -
322
DOI : 10.1111/j.1365-2958.2007.06020.x
Gerstmeir R
,
Cramer A
,
Dangel P
,
Schaffer S
,
Eikmanns BJ
2004
RamB, a novel transcriptional regulator of genes involved in acetate metabolism of Corynebacterium glutamicum.
J. Bacteriol.
186
2798 -
2809
DOI : 10.1128/JB.186.9.2798-2809.2004
Jungwirth B
,
Emer D
,
Brune I
,
Hansmeier N
,
Pühler A
,
Eikmanns BJ
,
Tauch A
2008
Triple transcriptional control of the resuscitation promoting factor 2 (rpf2) gene of Corynebacterium glutamicum by the regulators of acetate metabolism RamA and RamB and the cAMP-dependent regulator GlxR.
FEMS Microbiol. Lett.
281
190 -
197
DOI : 10.1111/j.1574-6968.2008.01098.x
Kim HJ
,
Kim TH
,
Kim Y
,
Lee HS
2004
Identification and characterization of glxR, a gene involved in regulation of glyoxylate bypass in Corynebacterium glutamicum.
J. Bacteriol.
186
3453 -
3460
DOI : 10.1128/JB.186.11.3453-3460.2004
Kohl TA
,
Baumbach J
,
Jungwirth B
,
Pühler A
,
Tauch A
2008
The GlxR regulon of the amino acid producer Corynebacterium glutamicum: in silico and in vitro detection of DNA binding sites of a global transcription regulator.
J. Biotechnol.
135
340 -
350
DOI : 10.1016/j.jbiotec.2008.05.011
Kohl TA
,
Tauch A
2009
The GlxR regulon of the amino acid producer Corynebacterium glutamicum: detection of the corynebacterial core regulon and integration into the transcriptional regulatory network model.
J. Biotechnol.
143
239 -
246
DOI : 10.1016/j.jbiotec.2009.08.005
Kotrbova-Kozak A
,
Kotrba P
,
Inui M
,
Sajdok J
,
Yukawa H
2007
Transcriptionally regulated adhA gene encodes alcohol dehydrogenase required for ethanol and n-propanol utilization in Corynebacterium glutamicum R.
Appl. Microbiol. Biotechnol.
76
1347 -
1356
DOI : 10.1007/s00253-007-1094-6
Lee HW
,
Pan JG
,
Lebeault JM
1998
Enhanced l-lysine production in threonine-limited continuous culture of Corynebacterium glutamicum by using gluconate as a secondary carbon source with glucose.
Appl. Microbiol. Biotechnol.
49
9 -
15
DOI : 10.1007/s002530051130
Merkens H
,
Beckers G
,
Wirtz A
,
Burkovski A
2005
Vanillate metabolism in Corynebacterium glutamicum.
Curr. Microbiol.
51
59 -
65
DOI : 10.1007/s00284-005-4531-8
Mori A
,
Shiio I
1987
Phosphoenol pyruvate: sugar phosphotransferase systems and sugar metabolism in Brevibacterium flavum.
Agric. Biol. Chem.
51
2671 -
2678
DOI : 10.1271/bbb1961.51.2671
Netzer R
,
Krause M
,
Rittmann D
,
Peters-Wendisch PG
,
Eggeling L
,
Wendisch VF
,
Sahm H
2004
Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis.
Arch. Microbiol.
182
354 -
363
DOI : 10.1007/s00203-004-0710-4
Parche S
,
Burkovski A
,
Sprenger GA
,
Weil B
,
Krämer R
,
Titgemeyer F
2001
Corynebacterium glutamicum: a dissection of the PTS.
J. Mol. Microbiol. Biotechnol.
3
423 -
428
Park SD
,
Lee SN
,
Park IH
,
Choi JS
,
Jeong WK
,
Kim YH
,
Lee HS
2004
Isolation and characterization of transcriptional elements from Corynebacterium glutamicum.
J. Microbiol. Biotechnol.
14
789 -
795
Park SY
,
Moon MW
,
Subhadra B
,
Lee JK
2010
Functional characterization of the glxR deletion mutant of Corynebacterium glutamicum ATCC 13032: involvement of GlxR in acetate metabolism and carbon catabolite repression.
FEMS Microbiol. Lett.
304
107 -
115
DOI : 10.1111/j.1574-6968.2009.01884.x
Sambrook J
,
Fritsch EF
,
Maniatis T
1989
Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor Laboratory Press
Cold Spring Harbor, NY
Schreiner ND
,
Fiur D
,
Holátko J
2005
E1 enzyme of the pyruvate dehydrogenase complex in Corynebacterium glutamicum: molecular analysis of the gene and phylogenetic aspects.
J. Bacteriol.
187
6005 -
6018
DOI : 10.1128/JB.187.17.6005-6018.2005
Shaw WV
1975
Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria.
Methods Enzymol.
43
737 -
755
Stansen C
,
Uy D
,
Delaunay S
,
Eggeling L
,
Goergen JL
,
Wendisch VF
2005
Characterization of a Corynebacterium glutamicum lactate utilization operon induced during temperature-triggered glutamate production.
Appl. Environ. Microbiol.
71
5920 -
5928
DOI : 10.1128/AEM.71.10.5920-5928.2005
Tanaka Y
,
Teramoto H
,
Inui M
,
Yukawa H
2008
Regulation of expression of general components of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) by the global regulator SugR in Corynebacterium glutamicum.
Appl. Microbiol. Biotechnol.
78
309 -
318
DOI : 10.1007/s00253-007-1313-1
Tauch A
,
Kirchner O
,
Löffler B
,
Götker S
,
Pühler A
,
Kalinowski J
2002
Efficient electrotransformation of Corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1.
Curr. Microbiol.
45
362 -
367
DOI : 10.1007/s00284-002-3728-3
Wendisch VF
,
de Graaf AA
,
Sahm H
,
Eikmanns BJ
2000
Quantitative determination of metabolic fluxes during coutilization of two carbon sources: comparative analyses with Corynebacterium glutamicum during growth on acetate and/or glucose.
J. Bacteriol.
182
3088 -
3096
DOI : 10.1128/JB.182.11.3088-3096.2000