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1 Molecular Microbiology and Biotechnology Group, Research Institute of Innovative Technology for the Earth (RITE), Kyoto 619-0292, Japan
2 College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of Korea
Correspondence
Hideaki Yukawa
mmg-lab{at}rite.or.jp
Sung Ok Han
samhan{at}korea.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Liebl, 2005; Nishimura et al., 2007). In contrast to closely related, medically important pathogenic species, such as Corynebacterium diphtheriae and Mycobacterium tuberculosis, C. glutamicum is generally recognized as a non-hazardous organism (Dover et al., 2004; Funke et al., 1997). Furthermore, this organism has gained increasing interest as a suitable model organism for high-G+C-content Gram-positive bacteria in general and for Corynebacterineae, a suborder of the Actinomycetes, in particular.
One of the central metabolic pathways in C. glutamicum and in other aerobic bacteria is the tricarboxylic acid (TCA) cycle, which is responsible for the complete oxidation of acetyl-CoA derived from various substrates and for the provision of precursors for amino acid biosynthesis. TCA cycle intermediates are commonly used by other metabolic reactions in a wide variety of cell types. Due to the commercial importance of the amino acids and organic acids produced by C. glutamicum, the control of TCA cycle enzyme activities has been the subject of intensive studies, and the phenotypes of mutants affecting TCA cycle genes have been analysed (Eikmanns et al., 1994, 1995; Molenaar et al., 1998, 2000; Usuda et al., 1996; Wittmann & De Graaf, 2005). However, limited work has been devoted to the regulation of the expression of C. glutamicum TCA cycle genes in response to different growth phases and carbon sources. Recently we have reported the transcription of C. glutamicum genes involved in the TCA cycle and glyoxylate bypass (Han et al., 2008). In fact, aspects of the regulation of only three TCA cycle enzymes in C. glutamicum, citrate synthase (encoded by gltA), aconitase (encoded by acn), and isocitrate dehydrogenase (encoded by icd), have been described (Eikmanns et al., 1994, 1995; Krug et al., 2005).
C. glutamicum can use a variety of carbohydrates and organic acids as carbon and energy sources (Kinoshita & Tanaka, 1972; Liebl, 1991). As carbon sources, glucose and acetate have been shown to serve as substrates for amino acid production by C. glutamicum. The genes required for the utilization of a specific carbon source are, in most cases, expressed only if that carbon source is present in the medium and if preferred carbon sources, such as acetate and glucose, are absent from the growth medium. These two processes in C. glutamicum are referred to as substrate induction and carbon catabolite repression (CCR), respectively (Letek et al., 2006). CCR is an environment-sensing mechanism used by bacteria to establish priorities in carbon metabolism. Transcriptional regulation is modulated by the level of cAMP and cAMP receptor protein (Crp), a global transcriptional regulator. It has been reported that the activities of Bacillus subtilis TCA cycle enzymes, particularly citrate synthase and aconitase, are severely depressed when glucose is supplied (Cox & Hanson, 1968; Hanson & Cox, 1967; Ohne, 1974) and that the dyad symmetry sequence located in the aconitase gene promoter region is the target for CCR (Fouet et al., 1990; Fouet & Sonenshein, 1990; Jin & Sonenshein, 1994; Rosenkrantz et al., 1985). Expression of Escherichia coli aconitase genes is also subject to CCR mediated by Crp (Cunningham et al., 1997; Gruer & Guest, 1994; Gruer et al., 1997). The transcriptional regulator GlxR contains a domain with similarity to the cAMP-binding motifs of Crp from E. coli and, in fact, cAMP is essential for binding of GlxR to the aceA/aceB intergenic region in C. glutamicum (Kim et al., 2004; Letek et al., 2006). The regulation of C. glutamicum metabolism in the presence of various carbon sources clearly differs from that of E. coli or B. subtilis (Gerstmeir et al., 2003; Hayashi et al., 2002; Letek et al., 2006; Muffler et al., 2002). No direct evidence has been found of a CCR system in C. glutamicum (Bruckner & Titgemeyer, 2002; Gerstmeir et al., 2003; Han et al., 2007), although a CCR-like phenomenon has been reported from cells grown on medium containing glucose with either glutamate (Gerstmeir et al., 2003; Kramer et al., 1990; Kronemeyer et al., 1995) or ethanol (Arndt & Eikmanns, 2007; Kotrbova-Kozak et al., 2007). However, mechanisms of true induction and repression in the TCA cycle have not been studied in depth.
To add to our previous findings on transcription of TCA cycle genes (Han et al., 2008) and to define the regulatory networks responsive to the TCA cycle, we performed a detailed transcriptional analysis of TCA cycle genes in various growth phases and with various carbon sources to assess their effects on mRNA levels of different sets of genes. We uncovered a unique pattern of gene expression that alternates between elevated and diminished levels of contiguous pairs of TCA cycle genes in response to culture conditions. The data indicate that the TCA cycle is regulated by the availability of carbon sources such as acetate and glucose, and that CCR protein might be involved in this global regulation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Liebl, 2005; Yukawa et al., 2007) was used as the source of genomic DNA and total RNA. The organism was cultured at 33 °C with constant agitation (1000 r.p.m.) and aeration (1 volume per volume min–1) in 500 ml mineral salts medium described previously (Han et al., 2007) in a 1-litre jar fermenter (BMJ01PI, Biott). The sole carbon and energy source was provided by glucose, acetate, pyruvate, lactate, citrate, succinate or malate (200 mM each). Cells were subcultured in medium containing the same carbon source before they were added to fresh medium as inoculants. The pH was monitored using a pH controller (DT-1023, Biott) and maintained at 7.6 by supplementing the medium with 5 M NH3 and 1 M HCl. Cell growth was monitored continuously by measuring OD610. Cultured samples were centrifuged (10 000 g, 4 °C, 3 min), and the supernatants were analysed for sugars and organic acids. Organic acids were quantified by high-performance liquid chromatography (8020 chromatograph, Tosoh Corporation) equipped with a UV detector and conductivity meter, and a TSKgel OApak-A column (Tosoh Corporation) operating at 40 °C with a 0.75 mM H2SO4 mobile phase at a flow rate of 1.0 ml min–1. The concentrations of glucose were measured by an enzyme electrode glucose sensor (BF-4, Oji Scientific Instruments).
Nucleic acid isolation.
Chromosomal DNA was isolated using a Genomic DNA Purification kit (Promega) according to the manufacturer's instructions. Total RNA was extracted from broth cultures using a Qiagen RNeasy kit (Qiagen) as previously described (Han et al., 2007).
Quantitative real-time reverse transcription PCR (qRT-PCR).
mRNA was quantified with an ABI Prism 7000 RT-PCR system (Applied Biosystems). A 50 ng total RNA sample was used per RT-PCR with each primer pair using a QuantiTect SYBR Green RT-PCR kit (Qiagen) according to the manufacturer's instructions. Negative controls with no reverse transcriptase were included with each RNA sample to rule out DNA contamination. DNA templates without reverse transcriptase were included as positive controls. Amplicons were run on a 2 % (w/v) agarose gel to verify that only a single band was produced. The target gene transcripts were normalized to the reference gene transcript (16S rRNA) from the same RNA sample. Each gene was analysed using RNA isolated from three independent samples. The comparative threshold cycle (Ct) method (Applied Biosystems) was used to quantify relative expression, with a Ct being defined as the cycle at which the reporter fluorescence is distinguishable from the background in the extension phase of the PCR. The Ct values were computed as described previously (Bustin, 2002; Winer et al., 1999). The relative change in transcription rate was determined as 
, with 
Ct equal to the difference of the measured crossing points for the test and the control condition.
Purification of GlxR.
Primers glxR-TF (5'-caccGTGGAAGGTGTACAGGAGAT) and glxR-TR (5'-TCGAGCGCGACGTGCCAAAT) were used to amplify the glxR gene from C. glutamicum. The amplified glxR gene was cloned into pET100/D-TOPO, which allows translational fusion between a C-terminal 6-His tag and the target gene, and then transformed into E. coli BL21 Star for overexpression (Invitrogen). The enzyme was purified using a nickel-nitrilotriacetic acid column (Invitrogen) and dialysed against 20 mM Tris (pH 7.0) and 1 mM dithiothreitol. Where indicated, the 6-His tag was removed by treatment with EnterokinaseMax (Invitrogen) after purification. Samples taken at different stages of the purification process and the purified protein were analysed by SDS–10 % PAGE as described by Laemmli (1970). Samples mixed with Tris/glycine SDS sample buffer (Invitrogen) (1 : 1, v/v) were denatured at 100 °C for 5 min and applied to a 10 % polyacrylamide gel using a mini-Protean II system (Bio-Rad). Gels were stained with colloidal Coomassie Blue stain (Genomic Solutions) to visualize proteins.
Electrophoretic mobility shift assay (EMSA).
To test the binding of GlxR to putative target promoters, purified GlxR protein (50–500 ng) was mixed with DNA fragments (mostly 200 bp; final concentration, 10–100 ng) in a total volume of 10 µl. The binding buffer (pH 7.4) contained 150 mM KCl, 0.1 mM dithiothreitol, 0.1 mM EDTA, 10 mM Tris and 0.2 mM cAMP. The reaction mixture was incubated at room temperature for 30 min and then loaded onto a 5 % native polyacrylamide gel. Electrophoresis was performed at room temperature and 200 V using 0.5xTBE buffer (44.5 mM Tris base, 44.5 mM boric acid and 1 mM EDTA) supplemented with 0.2 mM cAMP. The gels were subsequently stained using the EMSA kit (Invitrogen) as described by the manufacturer and photographed. All PCR products used in EMSA were purified with the PCR purification kit (Qiagen) and eluted in distilled water.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) or glucose (Fig. 2) as the sole carbon and energy source. RNA was prepared from the cultures at different growth stages (Figs 1N and 2N). The RNA was subjected to qRT-PCR analyses using specific primer sets for the TCA cycle genes (Table 1). To ensure that the resulting PCR products were amplified from cDNA, instead of from contaminating chromosomal DNA, reverse transcriptase or total RNA was omitted in control experiments. In these controls, no signal was detected (data not shown).
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). Although the errors in expression level were wide, the levels of initial (0 h) transcription of gltA and sucCD were considerably higher compared with those of the other TCA genes. In the early stages of growth (1 h), most TCA cycle gene transcripts were induced in the presence of acetate; the levels of gltA and aceA mRNA were highest in TCA cycle and glyoxylate bypass, respectively. As the culture entered the mid-exponential growth phase at 2–3 h, the transcription of most TCA cycle genes increased simultaneously and reached maximum levels. After this, the transcription level decreased as the cells adapted to the late exponential phase. Generally, the degree of expression at each growth phase, especially stationary phase, appeared to correlate with the acetate consumption rate (Fig. 1N and O). Unlike that of other genes, the expression levels of sucCD and mdhB mRNA were maximal when the cells had completely consumed the acetate in the medium at stationary phase and then gradually decreased as the cells reached the late stationary phase (Fig. 1F and J). There was a 12-fold increase in sucCD transcripts associated with simultaneous depletion of acetate in the stationary phase (9 h) relative to the values observed in the mid-exponential phase (3 h) (Fig. 1F). In addition, there was a sevenfold increase in mdhB mRNA in the stationary phase (9 h) compared with the value measured in the exponential phase (2 h) (Fig. 1J). These observations indicate that most TCA cycle genes of C. glutamicum are constitutively expressed but are induced to express at higher levels in the presence of acetate after a rather short lag period. Reduced expression was observed at later growth stages. The close correlation between the transcription time-courses of TCA cycle genes suggests that these genes are expressed simultaneously to enable acetate utilization.
To determine whether glucose activates TCA cycle gene transcription, RNA was isolated from C. glutamicum at different growth phases in the presence of glucose. The levels of most TCA cycle gene transcripts increased simultaneously from the mid-exponential phase to the early stationary phase, and decreased during the mid-stationary phase (Fig. 2). The pattern of glucose induction was similar among most TCA cycle genes. However, the mRNA levels of sucCD, mdh, mdhB, aceA and aceB increased simultaneously when the cells had completely consumed the glucose in the medium during the stationary phase (Fig. 2). In particular, the highest expression of the sucCD operon, which encodes succinyl-CoA synthetase, was observed when the cells were starved, regardless of whether the cells were grown on acetate or glucose (Figs 1F and 2F). In addition, glucose depletion most strongly induced aceA and aceB (Fig. 2L and M). The levels of aceA and aceB transcripts in acetate-grown cells were up to 30-fold higher than those in glucose-grown cells (Figs 1L, and M, 2L and 2M). These data also indicated that either acetate (approx. 30 mM) or lactate (approx. 400 mM) accumulated in the medium at 16 h induced these genes, when the glucose has been completely consumed (Fig. 2O). However, these accumulated carbon sources may not affect the transcripts of genes strongly when glucose remains abundant (Gerstmeir et al., 2003). Interestingly, glucose induced high levels of expression of TCA cycle genes during the early stationary phase, whereas the transcript levels were highest at the mid-exponential phase in acetate medium (Figs 1 and 2).
Effect of carbon depletion on expression of TCA cycle genes
To determine whether carbon source withdrawal induces or represses TCA cycle gene expression, transcript levels were determined in C. glutamicum before and after starvation of cells grown on acetate (Fig. 3I) or glucose (Fig. 3II) as the sole carbon and energy source. First of all, a carbon-starvation condition was created when exponential-phase cells from medium containing either acetate or glucose were washed with the same medium lacking any carbon source. Expression of most TCA cycle genes, except sucCD, mdh and mdhB, which were clearly observed in acetate-fed cells, dramatically decreased when the cells were starved for 2 h (Fig. 3I). In agreement with the previous section, the genes that were most positively regulated by acetate depletion were sucCD and mdhB (Figs 3I F and J). Acetate starvation for about 1.5 h provoked an approximately threefold increase in sucCD mRNA (Fig. 3I F). Expression of mdhB was also stimulated by acetate starvation, increasing 1.5-fold after 2 h (Fig. 3I J). After about 1 h acetate starvation, transcripts of mdh were depressed, but later markedly increased until about 2 h of starvation. The levels of sdhCAB transcripts increased during the initial 0.5 h of starvation, but decreased during the next 1 h (Fig. 3I G). We next explored whether this induction was specific to acetate withdrawal or was a general consequence of carbon depletion. Accordingly, cells were grown on fermentable glucose and then washed in medium not containing the carbon source. Upon glucose depletion, the expression pattern of TCA cycle genes differed from that upon acetate depletion (Fig. 3). In glucose-starved cells, transcripts of all TCA cycle genes were depressed during the initial 0.5 h of starvation, but increased during the next 1 h (Fig. 3II), with the only variation being in the level of expression. Upon further starvation (2 h), most transcripts decreased (Fig. 3II). However, up to threefold increases of mdh, mdhB and mqo transcripts were observed after 2 h starvation (Fig. 3II K).
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). However, most transcripts, except sucCD, were repressed after the culture had been incubated for 1 h. These transcripts were slightly induced again during the next 1.5 h incubation, but were almost completely repressed upon further incubation (2 h) (Fig. 4). The expression of the sucCD operon was most strongly regulated by glucose supplementation (Fig. 4F). Glucose addition provoked about a 10-fold increase in sucCD transcripts during the 1–1.5 h incubation, and the expression was then clearly reduced to basal level after 2 h incubation. In contrast to other genes, the levels of mdh and mdhB transcripts increased during the 2 h incubation (Figs 4I and J). Glucose addition was associated with decreased icd, sucB and fum expression, after about 0.5–1 h (Figs 4C, E and H). After 1.5 h incubation, sucB transcripts regained pre-incubation levels (Fig. 4E), while icd and fum transcripts were slightly induced (Fig. 4C and H). However, levels of all of these transcripts were depressed after further incubation (2 h) (Fig. 4). In this study, transcription of most TCA cycle genes was induced when glucose was present with acetate for 0.5–1.5 h, but was completely repressed after further incubation with co-utilization of glucose and acetate. This analysis demonstrates that glucose represses most genes of TCA cycle metabolism in C. glutamicum.
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). Out of the 13 sites, 6 exhibited high similarity (up to two mismatches) with the CBS consensus motif in the upstream regions of gltA, acn, sucCD, sdhCAB, aceA and aceB (Fig. 5A), with sucC having the highest homology with the consensus motif (no mismatch). The nucleotides of the GlxR-binding motifs in the aceB promoter region have been described previously (Kim et al., 2004; Letek et al., 2006), with two mismatches (Fig. 5A).
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); its molecular mass coincides with the expected size (25 kDa) deduced from the gene sequence. Treating His-tagged GlxR with EnterokinaseMax (Invitrogen) to remove the His tag had no effect on binding (data not shown). In agreement with previous reports (Kim et al., 2004; Letek et al., 2006), the presence of retarded bands was directly dependent on the presence of cAMP in the reaction mixture. No retarded bands were observed in the absence of cAMP or His-tagged GlxR (data not shown). Different amounts of purified GlxR protein (50–500 ng) and DNA fragments (10–100 ng) were used to optimize the binding reaction (data not shown).
As indicated in the previous sections, expression of sucCD was subject to tight carbon regulation. Hence, we first tested whether GlxR recognizes and binds to the promoter region of the sucCD operon by performing mobility shift assays with a 200 bp DNA fragment containing the sucC promoter region (Fig. 5D). When cAMP was added to the binding mixture, a clear band shift was observed for sucC as it was with the positive control aceB (Fig. 5D). In addition, DNA fragments containing the sequences upstream of gltA, acn, sdhCAB, aceA and aceB (Fig. 5C) appear to bind with GlxR (Fig. 5D). When the DNA fragment of the sequence upstream of fum was used, clear retardation was observed (Fig. 5D), although all possible binding sites upstream of fum exhibited relatively low similarity (three mismatches) with the consensus motif.
Induction of TCA cycle genes in response to various carbon sources
To further our understanding of gene expression regulation by various carbon sources, we measured the amount of TCA cycle gene transcripts in C. glutamicum cells grown in medium containing different carbon substrates as the sole carbon and energy source. Cells were subcultured in the medium containing the same carbon source before they were added to fresh medium as inocula (OD610 1.8–2.0). After 1 h incubation, cell growth in the medium was similar with the various carbon sources tested (Fig. 6xiv), although glucose culture showed the highest cell growth. The levels of the TCA cycle gene transcripts varied considerably depending on the nature of the carbon source (Fig. 6i–xiii). Some carbon sources clearly induced stronger expression of certain sets of genes. Acetate caused the expression of all the TCA cycle genes, including gltA, acn, fum, mdh, mqo, aceA and aceB, most strongly. In the presence of other non-fermentable carbon sources such as pyruvate or lactate, most TCA cycle genes (acn, icd, odhA, sucB, fum, mdh, mdhB, mqo, aceA and aceB) were induced to their highest levels (Fig. 6). With non-fermentable carbon along with acetate, the expression of aceA and aceB was particularly high (Fig. 6xii and xiii). Significant expression of some TCA cycle genes was observed with glucose, but acn, sucCD, sdhCAB, aceA and aceB had the lowest expression levels (Fig. 6vi, vii, xii and xiii). TCA intermediates, such as citrate and succinate, stimulated the expression of most of the genes, including gltA, acn, icd, odhA, sucB, sucCD, sdhCAB, fum, mdh, mdhB and mqo, although less effectively (Fig. 6). The sucCD operon of C. glutamicum showed a markedly different pattern of expression from that of other TCA cycle genes. On succinate-containing medium, mRNA levels of sucCD remained at least twofold higher than on acetate, glucose, pyruvate and lactate medium (Fig. 6vi–vii). Similar to sucCD, succinate induction of sdhCAB was observed, and citrate allowed the most significant expression of the sdhCAB gene (Fig. 6vii). Low expression levels of most TCA cycle genes were detected on medium containing malate (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Wendisch et al., 2000). Similar upregulation of TCA cycle genes in acetate has been reported in B. subtilis (Nakano et al., 1998). In contrast, TCA cycle genes are repressed at high glucose concentrations in the presence of acetate. The transcriptional analysis provided an overall view of the expression of almost all TCA cycle genes and was in good agreement with previous reports of enzyme activities, metabolic arrays and DNA microarrays (Bott, 2007; Kromer et al., 2004; Zhao & Lin, 2002). The results presented in this work suggest that a common and specific mechanism may exist at the transcriptional level for regulation of TCA cycle genes by different carbon sources, and that this pathway is coordinately regulated to balance the need for energy production and the generation of precursors for anabolism.
TCA cycle genes are regulated by several of the following regulators: Crp, the catabolite repressor activated by cAMP (Saier & Ramseier, 1996; Zhao & Lin, 2002); Fnr, whose modulon encodes proteins that are involved in cellular adaptation to growth in anoxic environments (Lynch & Lin, 1996; Unden & Schirawski, 1997; Unden et al., 2002); and ArcA/ArcB, a signal transduction system that regulates gene expression in response to redox conditions (Liu & De Wulf, 2004; Lynch & Lin, 1996; Perrenoud & Sauer, 2005). However, expression of genes encoding enzymes of the TCA cycle and glyoxylate bypass in C. glutamicum has been shown previously to be subject to at least the following five transcriptional regulators: AcnR, a TetR-type repressor of the acn gene (Krug et al., 2005); RipA, a repressor of the acn gene under iron limitation (Wennerhold et al., 2005); RamB, a negative regulator of glyoxylate bypass genes (aceA and aceB) involved in acetate metabolism (Cramer et al., 2006; Gerstmeir et al., 2004); DtxR, a transcriptional regulator of the sdhCAB gene in iron metabolism (Brune et al., 2006); and GlxR, the repressor of the glyoxylate bypass genes (Kim et al., 2004). It is possible that multiple regulators control TCA cycle genes to manifest a complex response to the environment. The glyoxylate bypass genes are transcriptionally regulated by a dual mechanism with GlxR and RamB (Gerstmeir et al., 2004; Kim et al., 2004). Krug et al. (2005) reported that the increased aconitase activity of cells grown on acetate is partially because of decreased repression by AcnR and that an additional transcriptional regulator (besides AcnR) or another regulatory mechanism for aconitase might exist. The only regulatory system that has ubiquitous effects on many different genes is catabolite derepression by cAMP receptor protein (Eraso & Gancedo, 1984; Kolb et al., 1993; Saier et al., 1996). This regulator activates many genes in the presence of acetate.
One of the features of transcriptional analysis is its utility in predicting gene functions. Although the first three enzymes (GltA, Acn and Icd) at the entry of the TCA cycle are needed for oxoglutarate generation, the regulation of the first two enzymes (GltA and Acn) is somewhat different from that of Icd (Krug et al., 2005; Nakano et al., 1998). In contrast to the mRNA levels of the icd gene, the level of gltA transcripts on acetate-containing medium remained about 1.2-fold higher than on glucose medium, whereas the mRNA level of acn was at least twofold higher on acetate medium than on glucose medium (Fig. 6). These results suggest that gltA and acn, but not always icd, are closely regulated, which may be due in part to the fact that citrate is an inducer of acn. Furthermore, EMSA showed that proteins such as AcnR (Krug et al., 2005) and GlxR (Fig. 5) bind to the dyad symmetry sequences in the acn promoter region. In addition, both gltA and acn have GlxR operator sites, the target sequence for probable catabolite repression, in their regulatory regions that bind to GlxR. Taken together, these results suggest that gltA and acn regulation might be controlled by the same mechanism. The malate : quinine oxidoreductase gene, mqo, is downregulated by acetate depletion at exponential phase, in contrast to malate dehydrogenase, mdh, which is upregulated. In addition, the two malate dehydrogenase genes, mdh and mdhB, are non-regulated and upregulated, respectively, in acetate depletion at stationary phase. This result suggests that mdh plays a different role from mdhB. The expression pattern also suggests that the mqo gene may serve other functions. The inconsistencies between regulation of gene expression and presumed function call for further investigations.
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ACKNOWLEDGEMENTS |
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Edited by: M. Hecker
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 21 April 2008;
revised 23 June 2008;
accepted 27 June 2008.
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), acetate* (
), pyruvate* (
), lactate* (
, 10x) and malate* (x) (* indicates use of y-axis scale on right of graph).
