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Research Institute of Innovative Technology for the Earth (RITE), 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan
Correspondence
Hideaki Yukawa
mmg-lab{at}rite.or.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A supplementary figure showing real-time RT-PCR analysis of pts gene expression is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Ikeda, 2003; Kelle et al., 2005). We are investigating the C. glutamicum R strain, which can provide high yields of lactate and succinate from sugar (Inui et al., 2004a; Okino et al., 2005). To improve the production of organic acids, understanding the regulatory systems of sugar transport and metabolism is important.
In many bacteria, the phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS) catalyses the transport of carbohydrates by coupling carbohydrate translocation and phosphorylation (Postma et al., 1993; Kotrba et al., 2001a). The PTS consists of two common cytoplasmic proteins, enzyme I and HPr, and an array of sugar-specific enzyme II complexes (EIIs). The phosphoryl group from phosphoenolpyruvate (PEP) is sequentially transferred to enzyme I, HPr, EIIs and finally to the substrate as it is translocated across the membrane. In addition to carbohydrate uptake, the PTS regulates the expression of many catabolic genes, depending on its phosphorylation state (Postma et al., 1993; Kotrba et al., 2001a).
Expression of pts genes has been studied in many bacteria (Postma et al., 1993; Vadeboncoeur et al., 2000; Kotrba et al., 2001b; Deutscher et al., 2006). Generally, EII expression is induced in the presence of its substrate sugar. Expression of enzyme I and HPr, encoded by ptsI and ptsH, increases in the presence of different PTS sugars. In many bacteria, glucose is reported to be the most effective inducing sugar of ptsI and ptsH expression (De Reuse & Danchin, 1988; Stülke et al., 1997; Viana et al., 2000; Nothaft et al., 2003). Regulation of pts gene expression is mainly controlled at the stage of transcription initiation or at transcription elongation. The mechanism for control of pts gene expression differs for the respective pts genes. For example, Mlc protein represses the expression of the ptsHIcrr operon, which encodes general components of PTS, HPr and enzyme I, and the EIIA component of the glucose-PTS in Escherichia coli (Kim et al., 1999; Plumbridge, 1999; Tanaka et al., 1999). This repression is relieved by dephosphorylated glucose-PTS, which is generated by the transport of PTS sugar. In Bacillus subtilis, ptsGHI operon expression is controlled by the GlcT antiterminator. GlcT is active in its dephosphorylated form, which is generated by the transport of glucose. Activated GlcT inhibits the rho-independent terminator that precedes the ptsG ORF, and transcription proceeds to the 3' end of ptsGHI (Stülke et al., 1997).
Expression of the fructose-pts operon is negatively regulated by Cra, which belongs to the GalR–LacI-type regulator family. Repression is relieved by fructose 1-phosphate in E. coli. In B. subtilis, expression of the lev operon, which is a fructose-PTS permease forming fructose 6-phosphate, is regulated by the antitermination mechanism mediated by LevR. This operon is also controlled by the CcpA–HPr complex. Ser-phosphorylated HPr binds to CcpA, which belongs to the GalR–LacI-type regulator family, and represses operon transcription. HPr phosphorylation at Ser is catalysed by HPr (Ser) kinase, which is activated in the presence of ATP and fructose 1,6-bisphosphate (Vadeboncoeur et al., 2000). In other low-GC Gram-positive bacteria, induction of the fructose-pts operon is mediated by FruR, which is a deoxyribonucleoside repressor (DeoR)-type regulator encoded by the first gene of the fructose-pts operon. In Spiroplasma citri, FruR is an activator, whereas in Streptococcus gordonii and Lactococcus lactis, FruR is a repressor (Gaurivaud et al., 2001; Loo et al., 2003; Barrière et al., 2005). It is proposed that fructose 1-phosphate modulates FruR activity in L. lactis.
In C. glutamicum, the presence of general components (enzyme I and HPr), as well as glucose-, fructose- and sucrose-specific EIIs of the PTS, has been demonstrated in several biochemical and genetic studies (Mori & Shiio, 1987; Dominguez & Lindley, 1996; Parche et al., 2001a; Moon et al., 2005), and there are additional pts genes for unknown substrates (NCgl2933 and NCgl2934 of C. glutamicum ATCC 13032). C. glutamicum R has a β-glucoside-specific PTS in addition to these pts genes (Kotrba et al., 2001b). The genes that encode general components of the PTS are located near the fructose-pts gene, which is peculiar to Corynebacterium species (Fig. 3a).
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; Parche et al., 2001a). Expression of the pts genes of C. glutamicum has been examined in only a few studies (Gerstmeir et al., 2003; Kotrba et al., 2003). In this study, we investigated the expression of the general components of the PTS and the glucose-, fructose- and sucrose-specific EIIs of the PTS. We found that PTS sugars induced the expression of pts genes. In particular, fructose induced the expression of all the pts genes examined. We also investigated the role of FruR in the control of pts gene expression.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) supplemented with 1.0 % (w/v) acetate, and various PTS sugars were added at 1.0 % (w/v) as carbon sources. Antibiotics were added at the following final concentrations: kanamycin, 50 µg ml–1; chloramphenicol, 5 µg ml–1. Bacterial growth was monitored by determining OD610.
Bacterial strains and plasmids.
Construction of recombinant plasmid pCRC800 containing the fruR gene was carried out as follows. The fruR gene was amplified from C. glutamicum R genomic DNA by PCR using primers EcoRI-fruR-20F (5'-GCGGAATTCAACATCAGCGAGGTTAAGCATG-3') and HindIII-fruR-840R (5'-GCGAAGCTTGCGACAGCGTGGAATCAATAC-3'). The amplified DNA fragment was digested with EcoRI and HindIII, and cloned into the corresponding sites on pCRB1 (Nakata et al., 2003) to construct pCRC800.
C. glutamicum R (Yukawa et al., 2007) was used as a wild-type strain. The ptsI-FLAG strain was constructed as follows. First, the 3' region of the ptsI gene was amplified from C. glutamicum R genomic DNA by PCR using primers SacI-ptsI-707F (5'-CGCGAGCTCGCGGACGAAGCTGAAGCAACCAAG-3') and SalI-1762-25F (5'-CGCGTCGACGTATCTGTTGAGCACCAATGAGCTTGAC-3'). The amplified DNA fragment was digested by SacI and SalI, and cloned into the corresponding site on plasmid pCRA925 (Inui et al., 2004b). SpeI and NotI restriction endonuclease sites were introduced at the 3' terminus of the cloned ptsI by PCR using primers SpeI-ptsI-1704R (5'-CGGACTAGTGACTGCTGCGTCGATCACTGC-3') and NotI-ptsI-1707F (5'-GAGGCGGCCGcACCACTGTTGAGCTAAAAAAGCCTC-3') to construct pCRC801. DNA fragments coding for FLAG-epitope tag and chloramphenicol-resistance genes were amplified by PCR using primers SpeI-FLAG-CmF (5'-CGGACTAGTGCAGACTACAAGGATGACGATGACAAGTAAGGCCCTTCCGGTTTTGGGGTAC-3') and Cm-NotIR1 (5'-CGCGCGGCCGCGGCTCTTCCTGTTTTAGAGTGCATTGATC-3'). The amplified DNA fragment was digested by SpeI and NotI, and cloned into the corresponding site on pCRC801 to construct pCRC802. C. glutamicum R was transformed with pCRC802, and KmS and CmR colonies were selected. Integration of the FLAG-Cm fragment into the C. glutamicum R genome by double cross-over was confirmed by colony PCR using primers mini-CmL (5'-GCGAAGTGATCTTCCGTTCG-3') and ptsI-1 F (5'-GTGGCTACTGTGGCTGATGT-3'), or mini-CmR (5'-CACGACAGGTTTCCCGACTGG-3') and EcoRI-1761-23F (5'-GCGGAATTCGGCTTTTTGCTTTAAGGAGTGACATG-3').
Disruption of the fruR gene was achieved by transposon-mediated mutagenesis, as described previously (Suzuki et al., 2006). Disruption of the fruR gene was confirmed by DNA sequencing. The transposon was inserted at 463 bases downstream from the 5' end of the fruR ORF.
The fruR promoter–lacZ strain was constructed as follows. The promoter region of fruR was amplified by PCR using primers SmaI-fruR (5'-GCGCCCGGGAAGCAATTGCATGCTGTCTTTCCGTTTG-3') and SmaI-ptsI (5'-GCGCCCGGGCACATCAGCCACAGTAGCCAC-3'). The amplified fragment was digested with SmaI and cloned into the DraI site of the pCRA741 reporter plasmid, which has been described previously (Inui et al., 2007). These promoter–lacZ fusion plasmids were then digested with KpnI and SmaI, and the DNA fragment containing the fruR promoter–lacZ gene was cloned into the corresponding site on pHSG398 (Takara) to construct pCRC804. pCRC804 was used to transform wild-type C. glutamicum R or fruR-disruptant cells, and recombinant cells were selected for chloramphenicol resistance. Insertion of the promoter–lacZ fusion gene between CgR0734 and CgR0735 was confirmed by PCR using primers LlacZLR-4354F (5'-ATAACCGGGCAGGGGTCTAG-3') and Ind7insert-checkR (5'-GCGTCACGAACAACAGACAGC-3'), or LlacZLR-6425R (5'-CGACGGCCAGTGCCAAGC-3') and Ind7insert-checkF (5'-CGAGACTGGAATTGAGGCTC-3').
Northern blotting analysis.
Total RNA was isolated using the RNeasy Kit (Qiagen) from exponentially growing cells (OD610 1.2) in BT medium supplemented with 1.0 % (w/v) carbon source. Ten micrograms of total RNA were resolved by 1.25 % (w/v) agarose gel electrophoresis in the presence of formaldehyde and blotted onto Hybond-N+ membranes (GE Healthcare). The mRNAs were visualized using a non-radioactive nucleic acid labelling and detection system (GE Healthcare), according to the procedure specified by the manufacturer. The fluorescein-11-dUTP-labelled DNA probe was synthesized by a random-labelling kit (GE Healthcare). DNA fragments covering each pts gene were used as probes. The signal was scanned by a luminescent image analyser (Fuji model LAS-1000 CH).
Immunoblot analysis.
A 10 ml aliquot of cell culture grown to OD610 1.0 was collected by centrifugation, and pellets were mixed with glass beads and 1.0 ml buffer (4 % SDS, 5 % 2-mercaptoethanol, 40 mM Tris/HCl, pH 6.8, 8 M urea, 0.1 mM EDTA). Cells were disrupted by vigorous vortexing and samples were centrifuged. Crude extract (10 µl) was loaded onto 0.1 % (w/v) SDS–10 % (w/v) polyacrylamide gels and electrophoresed. Western blotting was performed using anti-FLAG M2 mAb (Sigma) and horseradish peroxidase-conjugated anti-mouse antibody (GE Healthcare). Chemiluminescence reactions were done using the ECL Plus Western blotting detection system (GE Healthcare). The signal was scanned by a luminescent image analyser (Fuji model LAS-1000 CH).
Primer-extension analysis.
Primer extension was carried out using appropriate gene-specific primers (Table 1) and total RNA (10–50 µg). Briefly, 1.5 pmol IRD700-labelled fluorescein primer was hybridized to RNA at 52 °C for 20 min, and cDNA was synthesized at 42 °C for 30 min using the AMV Reverse Transcriptase Primer Extension System (Promega) by the method described by the manufacturer. A 1.65 µl aliquot of sample was heat-denatured and loaded onto a denaturing 5.5 % (w/v) polyacrylamide sequencing gel in parallel with the sequencing ladder and run in the LI-COR DNA sequencer (Aloka model 4000).
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β-Galactosidase assay.
C. glutamicum R was grown to OD610 1.2, and 1 ml culture sample was harvested and dissolved in 1 ml Z buffer (Na2HPO4/NaH2PO4, pH 7.0, 10 mM KCl, 1 mM MgSO4, 50 mM β-mercaptoethanol) with 2 % toluene to permeabilize the cells. β-Galactosidase activity was determined with permeabilized cells by the method described by Miller (1972).
Affinity purification of promoter binding protein.
Biotin-labelled ptsI–fruR promoter DNA fragment was generated by PCR using primers promoter-ptsI (5'-GCGCCCGGGCACATCAGCCACAGTAGCCAC-3') and bio-promoter-fruR (5'-AAGCAATTGCATGCTGTCTTTCCGTTTG-3'). The 5' terminus of the bio-promoter-fruR primer was labelled with biotin. PCR product (100 pmol) was coupled to 3 mg Dynabeads streptavidin (Dynal). C. glutamicum R was grown in BT medium supplemented with 2.0 % (w/v) fructose to OD610 3.0, harvested, washed with 50 ml cell wash buffer (50 mM Tris/HCl, pH 7.5, 50 mM NaCl) and suspended in 6 ml binding buffer (50 mM Tris/HCl, pH 7.5, 1 mM EDTA, 10 %, w/v, glycerol, 1 mM DTT, 100 mM NaCl, 0.05 % (w/v) Triton X-100) with 1.5 g glass beads. Cells were disrupted by vigorous vortexing, and cell debris was removed by centrifugation at 12 000 g for 10 min and then at 50 000 g for 60 min. Supernatants were concentrated by using Amicon Ultra-15 centrifugal filter devices (Millipore) to 1.5 ml, and incubated with Dynabeads or ptsI–fruR promoter-coupled Dynabeads for 2 h at room temperature. Beads were washed six times with 300 µl binding buffer and twice with binding buffer containing 200 mM NaCl. Bound proteins were eluted with 50 µl binding buffer containing 1 M NaCl. Eluted fractions of 20 µl were loaded onto 0.1 % (w/v) SDS–12 % (w/v) polyacrylamide gels and electrophoresed. Proteins were transferred to Immobilon-P PVDF membranes (Millipore) and Coomassie Brilliant Blue-stained. The protein bands that appeared only for affinity-purified samples were excised, and the N-terminal protein sequence was determined by APRO Life Science Institute.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In the absence of PTS sugars, a very faint band was detected with the ptsF probe (Fig. 1a, lane 1), and addition of glucose did not increase the level of ptsF mRNA (Fig. 1a, lane 2). In contrast, the addition of fructose increased the expression of ptsF (Fig. 1a, lane 3), and sucrose also increased the ptsF mRNA signal (Fig. 1a, lane 4). The expression pattern of ptsS was similar to that of ptsF, that is, the expression of ptsS was induced by both fructose and sucrose (Fig. 1b). Glucose-specific pts showed a different expression pattern. Significant ptsG mRNA was detected in the absence of a PTS sugar (Fig. 1c, lane 1), and ptsG expression increased in the presence of glucose (Fig. 1c, lane 2). Surprisingly, in the presence of fructose and sucrose, ptsG expression increased to almost the same level as that seen with glucose induction (Fig. 1c, lanes 3 and 4). For general components of the PTS, expression of ptsH was observed in the absence of PTS sugar (Fig. 1d, lane 1), but very little expression was observed for ptsI mRNA (Fig. 1e, lane 1). Both ptsH and ptsI expression increased in the presence of PTS sugar. Fructose was the most effective inducing sugar for ptsH and ptsI expression (Fig. 1d, e, lane 3). The results in Fig. 1(a, d) show that several transcriptional products are present for ptsF and ptsH genes. In summary, fructose and sucrose induced the expression of all pts genes tested. We observed the induction of pts genes by PTS sugars when cells were cultured in a rich medium with yeast extract and no added acetate (data not shown), indicating that a decrease in acetate usage is not the signal for the induction of pts gene expression by PTS sugars.
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, lanes 4 and 5). A further increase in enzyme I protein level was observed in the presence of fructose (Fig. 2, lane 6). No band corresponding to enzyme I was observed in wild-type cells (Fig. 2, lanes 1–3). This result corresponds to the results of the mRNA study of the ptsI gene (Fig. 1e
), and suggests that the regulation of ptsI expression by PTS sugars occurs mainly at the transcriptional level.
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), the ptsF and ptsH genes are adjacent to each other (Fig. 3a). The gene order and intergenic spacing suggest that ptsF and ptsH are transcribed as a polycistronic mRNA. ptsI is also located near ptsF. Between ptsF and ptsI there is the fruR gene, encoding a protein of 264 aa that shows homology to a member of the DeoR family of transcriptional regulatory proteins, and the pfkB1 gene, encoding a protein of 330 aa that shows homology to fructose-1-phosphate kinase. The fruR, pfkB1, ptsF and ptsH genes are oriented in the same direction, while ptsI and the ORF that encodes a hypothetical protein located downstream of ptsH are oriented in the opposite direction (Fig. 3a). A rho-independent terminator structure is present downstream of the ptsH gene. This gene cluster is also conserved among C. glutamicum ATCC 13032, Corynebacterium diphtheriae and Corynebacterium efficiens (Parche et al., 2001b; Yokota & Lindley, 2005).
We analysed the operon structure of this region by Northern blotting using probes specific for fruR, pfkB1, ptsF and ptsH (Fig. 3b). A product of about 4.7 kb (product a) was detected by all four probes, indicating that fruR, pfkB1, ptsF and ptsH are transcribed as a polycistronic message. Using a ptsH probe, an intense 0.5 kb transcript (product d) was detected. The ptsH gene therefore seems to have its own promoter, as the expression pattern of ptsH mRNA was different from that of the other products. It is significant that product d was seen in the absence of fructose, and the amount of product d was more abundant than that of other mRNA products. For products b and c, the expression pattern was the same as that for product a, and we observed many bands by primer-extension analysis using a primer specific for ptsF. This suggests therefore that these products are the result of the nuclease digestion.
Determination of the transcriptional start sites of the pts genes
For further detailed analysis of the regulation of pts gene expression, we examined the transcriptional initiation site of each pts gene. Total RNA prepared from wild-type cells grown in a minimal medium in the presence or absence of PTS sugars (glucose or fructose) was analysed by primer-extension analysis using fluorescently labelled primers specific for each pts gene (Table 1). The position of the transcriptional start point was confirmed by experiments using two different primers for each pts gene (Table 1). A major transcriptional start site of ptsH (ptsH-P1 promoter) is the G residue located 102 nt upstream of the start codon of ptsH (Fig. 4a), and we observed a minor signal corresponding to the T residue located 82 nt upstream of the start codon of ptsH (ptsH-P2 promoter). Using a ptsI-specific primer, we detected three bands (Fig. 4b, c). The signals corresponded to T residues located at 103 and 105 nt upstream of the GTG start codon (ptsI-P1 promoter), and 37 nt upstream of the start codon (ptsI-P2 promoter). Using a ptsG-specific primer, we detected a single band corresponding to a T residue, located 256 nt upstream of the start codon (Fig. 4d). The transcriptional start site of ptsG has recently been reported in C. glutamicum ATCC 13032 (Engels & Wendisch, 2007). Those authors reported the start site at a G residue located 258 nt upstream of the start codon. The difference from our result may come from the slight difference in DNA sequence at the pstG promoter. The A residue located 262 nt upstream of the start codon in C. glutamicum R is a G residue in the ATCC 13032 strain.
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). Nucleotide sequences of the pts promoter regions are compared in Fig. 5. We found putative –10 and –35 hexamers. Comparison of each promoter showed that a TGTTT(TT)G sequence was conserved. The ptsI-P1 promoter region and the fruR-P1 promoter region were found to overlap.
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). We also determined the effect of glucose on ptsG mRNA expression, and an increase in ptsG mRNA level was detected (Fig. 4d). These data correspond to the results of Northern blotting assays (Fig. 1).
Effect of fruR gene disruption on the expression of the pts genes
We were interested in how fructose regulates the expression of pts genes, as fructose has the strongest inducing effect on pts gene expression, and the general component of the PTS is located near the fructose-pts operon, which differs from that of other bacteria. The fruR gene encodes a DeoR-type regulator that is located upstream of ptsF (Fig. 3a). This arrangement is conserved in many bacteria, and the regulator is responsible for the induction of the fructose-pts operon (Gaurivaud et al., 2001; Loo et al., 2003; Barriere et al., 2005). Thus, we examined the role of FruR on the expression of the pts genes.
We compared the expression of pts genes in wild-type and fruR cells. In cells grown in the presence of fructose, the mRNA levels of ptsI and ptsH were higher in the fruR mutant than in the wild-type (Fig. 6a, b). In the absence of PTS sugars, or in the presence of glucose, the bands for the mRNAs were faint in both the fruR mutant and the wild-type. The exposure time for detection of bands in the Northern blotting analysis was shorter in Fig. 6 than in Fig. 1 to avoid saturation of the band intensity. These results showed that fructose increased the pts mRNA level in the fruR strain as in the wild-type, and that the induced level in response to fructose was increased by disruption of fruR. Expression of ptsG was hardly affected by the fruR mutation with all PTS sugars (data not shown).
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We then complemented the fruR mutant with pCRC800, which has the fruR gene expressed under the control of the lac promoter (Fig. 6d). We observed that induction of ptsI by fructose returned to the wild-type level in the strain containing pCRC800 (Fig. 6d, lane 6; Supplementary Fig. S1). These results suggested that FruR functions as a repressor of the pts gene.
To examine whether FruR controls the expression of the fruR-pfkB1-ptsF operon, we constructed a fruR promoter–lacZ fusion and then introduced this construct into the wild-type or the fruR mutant in a region of the genome known to be non-essential in C. glutamicum R (Yukawa et al., 2007). We determined β-galactosidase activity in the presence or absence of fructose (Fig. 6f). In wild-type cells, a twofold increase in fruR promoter activity was observed in the presence of fructose (Fig. 6f, columns 1 and 2). The induction by fructose seemed weaker than that observed by Northern analysis. This might have been a consequence of the fact that the fruR–lacZ promoter construct included only 135 bp of DNA upstream from the fruR-P1 promoter, which may be insufficient for full promoter activity. Alternatively, the difference might have been caused by post-transcriptional regulation. In fruR-deficient cells, fruR promoter activity increased to a level threefold higher than that in the wild-type (Fig. 6f, columns 3 and 4). In the absence of fructose, fruR promoter activity in fruR-deficient cells was the same as that in the wild-type (Fig. 6f, columns 1 and 3). These results suggested that FruR reduces the induction effect of fructose on the fructose-pts operon. By affinity-purification experiments using ptsI-fruR promoter DNA as a bait (Fig. 6g), we isolated two proteins of about 28 kDa that bound specifically to the promoter region. The determined N-terminal amino acid sequence of one protein was VSQTE, which was identical to the N-terminal amino acid sequence of FruR lacking the first amino acid. This result demonstrated that FruR interacts directly with the promoter and suggested that FruR directly represses the expression of the pts gene.
In addition, the second protein we isolated that bound specifically to the promoter region was identified as SugR, which has recently been reported to bind the fruR promoter region (Engels & Wendisch, 2007).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Dominguez & Lindley, 1996; Parche et al., 2001a; Moon et al., 2005). The genes that encode the general PTS and the enzyme II specific for glucose, fructose and sucrose are known. By transport assay, it was demonstrated that glucose-PTS activity is relatively constitutive and that fructose-PTS activity is induced by fructose. However, the regulation of pts gene expression is largely unknown. In this paper we determined the expression patterns of pts genes in the presence of PTS sugars and determined the location of promoter regions, and finally we investigated the role of FruR on pts expression.
Expression of ptsF and ptsS showed similar patterns; low-level expression of these genes was detected in acetate or glucose cultures, and the expression was induced by both fructose and sucrose (Fig. 1a, b). On the other hand, significant expression of ptsG was observed in the absence of PTS sugars, and ptsG expression was increased by glucose, fructose and sucrose (Fig. 1c). Glucose-PTS has the ability to transport fructose, although the contribution of glucose-PTS to fructose uptake is only minor (Dominguez et al., 1998; Kiefer et al., 2004). Thus, there might be some advantage for C. glutamicum R to increase ptsG expression in the presence of fructose. The expression of the general component of the PTS increased in the presence of PTS sugar (Fig. 1d, e). Interestingly, fructose is the most effective inducing sugar, which is different from that which has been observed for other bacteria (De Reuse & Danchin, 1988; Stülke et al., 1997; Viana et al., 2000; Nothaft et al., 2003). In other bacteria, expression of the general PTS is induced most significantly by glucose. It is also noteworthy that ptsH and ptsI are located near the fructose-pts operon in C. glutamicum, which also differs from the situation in E. coli and B. subtilis, in which ptsH and ptsI are co-transcribed with the glucose-pts genes. In C. glutamicum, the uptake rate of fructose is faster than that of glucose (Kiefer et al., 2004), which may be explained by the higher expression of the general component of PTS.
In C. glutamicum, sucrose metabolism produces fructose and glucose 6-phosphate, and fructose is exported to the outside of the cell. Fructose re-enters the cell via the fructose-PTS (Dominguez & Lindley, 1996; Pátek et al., 2003; Moon et al., 2005). This means that the effect of sucrose on the expression of pts genes may be caused by fructose generated inside the cell. By comparing the sucrose induction of pts genes for the wild-type and the ptsF mutant, it might be possible to determine whether sucrose induces pts gene expression directly or indirectly.
To investigate how pts gene expression is controlled, we determined the transcriptional start site of each pts gene. The nucleotide sequence that resembles the –10 hexamer is located upstream of each transcriptional start site. Alignment of pts promoters through their –10 regions is shown in Fig. 5. The sequence tgngnTA(c/t)aaTgg is proposed as an extended –10 consensus sequence for C. glutamicum (Pátek et al., 2003). We compared this sequence with each pts promoter. Core hexamers are well conserved in all pts promoters, especially the first T and last T, while extended regions are poorly conserved. Only the G residue that is 2 nt upstream of the core hexamer and the G residue 1 nt downstream from the hexamer are conserved. We also found –35-like sequences that are separated by 15–18 bp from the –10 regions. The hexamer ttGcca is proposed as a –35 consensus sequence (Patek et al., 2003). The first three nucleotides ttG are well conserved among pts promoters, but the last three nucleotides are less well conserved. Among the pts promoters, the TGTTT(TT)G sequence is conserved, and may be an operator site for the putative common transcriptional regulator for pts. This sequence is unlikely to be the binding site for FruR because FruR does not regulate the expression of ptsG, which has the sequence TGTTT(TT)G.
In many bacteria, the gene that encodes the transcriptional regulator located in front of the fructose-pts genes is responsible for the fructose-dependent induction of the fructose-pts operon (Gaurivaud et al., 2001; Loo et al., 2003; Barrière et al., 2005). The regulatory mechanism is either derepression or activation of transcription in the presence of fructose. C. glutamicum also has a regulator FruR for the fructose-pts operon; hence, we determined the effect of fruR disruption on the expression of the pts genes. We found that the expression levels of ptsI, ptsH and the fructose-pts operon increased more in a fruR mutant than in wild-type cells in the presence of fructose, indicating that FruR decreases the induction effect of fructose. This is different from the role of FruR in other bacteria. These results also indicate that transcriptional regulator(s) other than FruR exist, as the increase in pts expression was still observed in the presence of PTS sugar in the fruR mutant. In general, the expression of a catabolic gene is controlled so as not to exceed the metabolic capacity. The role of FruR seems to be to adjust the expression level of pts genes to prevent overflow of PTS sugars.
During the preparation of this manuscript, Engels & Wendisch (2007) reported that EII components of pts genes (ptsG, fruR-pfkB1-ptsF and ptsS) are regulated by a DeoR-type regulator SugR. SugR represses the expression of EII components of the PTS in the absence of PTS sugar, and addition of PTS sugar relieves this repression. Our results showed that the expression of cytoplasmic components of the PTS (ptsH and ptsI) is increased in the presence of PTS sugar. We also found that a conserved motif is found in pts promoters. One possibility is that SugR also controls the expression of ptsH and ptsI. We are now investigating whether SugR controls the expression of cytoplasmic components of the PTS.
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ACKNOWLEDGEMENTS |
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Edited by: T. Nihira
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REFERENCES |
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Received 5 April 2007;
revised 11 September 2007;
accepted 12 September 2007.
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