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1 Biotechnology Research Center, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2 RIKEN Spring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan
Correspondence
Makoto Nishiyama
umanis{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-galactosidase reporter assay for this promoter indicated that arginine repressed the promoter in an argR-dependent manner. These results indicate that lysine biosynthesis is regulated by arginine in T. thermophilus.
-aminoadipate; EMSA, electrophoretic mobility shift assay; TCA, tricarboxylic acid |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-aminoadipate (AAA) pathway found in fungi and yeasts. Our recent studies revealed that the extremely thermophilic bacterium Thermus thermophilus synthesizes lysine through AAA. The pathway differs from the known AAA pathway found in lower eukaryotes. The Thermus pathway starts from the condensation of 2-oxoglutarate with acetyl-CoA to synthesize homocitrate, and proceeds in a manner similar to that in lower eukaryotes until the step of AAA synthesis (Kobashi et al., 1999
; Kosuge & Hoshino, 1999). However, the subsequent part of the pathway is totally different from that in lower eukaryotes, but similar to ornithine synthesis from glutamate, a step in arginine biosynthesis (Fig. 1) (Miyazaki et al., 2001, 2002).
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, 2002, 2003, 2004; Wulandari et al., 2002), and 2-oxoglutarate, which is provided through the TCA cycle, is a precursor of both arginine and lysine. This suggests that lysine biosynthesis of T. thermophilus is regulated by arginine as well as lysine. Homocitrate synthase (Hcs), which catalyses the first reaction in the pathway, is strictly inhibited by lysine (Wulandari et al., 2002). In addition to the inhibition by lysine, we also found moderate inhibition of Hcs by arginine (Wulandari et al., 2002). As for transcriptional control, we recently demonstrated that the promoter of the homocitrate synthase gene (hcs promoter), which is the promoter of the major lysine biosynthetic gene cluster, is regulated by lysine through leader-peptide-mediated transcriptional attenuation (Tsubouchi et al., 2005). A remaining question is whether the hcs promoter is also regulated by arginine. In this paper, we report the involvement of ArgR, a global arginine regulator, in the transcriptional regulation of the hcs promoter, depending on the availability of arginine. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-galactosidase derived from T. thermophilus HB27) was constructed by methods described previously (Fridjonsson & Mattes, 2001; Fridjonsson et al., 2002), and used as the host for the reporter assay. T. thermophilus cells were cultivated in TM medium (Koyama et al., 1986) or MM medium (Tsubouchi et al., 2005) supplemented with or without various additions. Escherichia coli JM105 [endA1 thi rpsL sbcB15 hsdR4 
(lac–proAB)/F' traD36 proAB lacIqZM15] was used for DNA manipulation, and E. coli BL21(DE3) [F– ompT hsdSB (
) dcm gal 
(DE3)] was used as the host for gene expression. For the cultivation of E. coli, 2x YT medium, containing Bactotryptone 1.6 %, yeast extract 1.0 % and NaCl 0.5 %, was generally used. All chemicals were purchased from Wako Pure Chemical, Kanto Chemicals or Sigma. Enzymes for DNA manipulation were purchased from Takara Shuzo.
Knockout of the arginase and argR genes in T. thermophilus.
The nucleotide sequences of oligonucleotides used in this study are shown in Table 1. The plasmids for the knockout of arginase were constructed as follows. Two independent PCRs using ANSM1/ANSM2 for amplifying 500 bp of a 5'-portion of the arginase gene TTC1132 of T. thermophilus HB27 and ANSU1/ANSU2 for amplifying 500 bp of a 3'-portion of the arginase gene as primers were performed with T. thermophilus TSU130 chromosomal DNA as the template. After the amplified 5'- and 3'-fragments had been cloned into pT7Blue (Novagen), the resulting plasmids were digested with XbaI/EcoRI and HindIII/HincII, respectively. Two fragments were cloned into pBluescript II SK(+) (Stratagene) to yield pRNSMU. An additional PCR for amplifying the heat-stable hygromycin B phosphotransferase gene was performed using HygR-F/HygR-R as the primers and p8S-T31-hph5 (Nakamura et al., 2005) as the template. An amplified fragment was cloned into pT7Blue to produce pHygRT. pRNSMU was digested with EcoRI/HindIII, and the larger DNA fragment was blunt-ended and ligated with the KpnI–HindIII smaller DNA fragment of pHygRT, which was also blunt-ended. The resulting plasmid, pRNMHygU, was used to knock out the arginase gene of T. thermophilus TSU130 by the method described previously (Hidaka et al., 1994). An arginase gene knockout mutant of T. thermophilus TSU130 verified by PCR with a set of primers ANSCN-F/ANSCN-R was named T. thermophilus ADK1.
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) containing the heat-stable kanamycin nucleotidyltransferase gene (Liao et al., 1986). The resulting plasmid, named pWRAB-KmRF, was used to knock out the argR gene of T. thermophilus TSU130 in the same way. Disruption of the argR gene in the resulting mutant was confirmed by PCR with primers RRKOCN-F and RRKOCN-R. pWRAB was also used to remove the heat-stable kanamycin nucleotidyltransferase gene from the chromosomal DNA of the argR-knockout mutant. Three kanamycin-sensitive colonies were isolated from about 1000 colonies. Deletion of the heat-stable kanamycin nucleotidyltransferase gene was confirmed by PCR with the same primers. The resulting argR-knockout mutant was named T. thermophilus WRK07. T. thermophilus ADK2 (argR, arginase gene) was generated by the knockout of the arginase gene of T. thermophilus WRK07 by using pRNMHygU.
Phenotypic analysis of the arginase gene knockout mutant.
The arginase gene knockout mutant, ADK1, was cultured for 10 h in 5 ml TM medium containing 1.6 mM CaCl2 and 1.6 mM MgCl2. A small portion (1 ml) was taken from the culture, centrifuged at 15 000 g for 30 s, and the precipitate was washed with MM medium three times. Cell suspension equivalent to 10 µl culture was added to 5 ml MP medium (MM medium supplemented with 100 µM proline) and incubated at 70 °C. Proline was added to MM medium to accelerate the growth. Growth was analysed by monitoring the OD600 of the culture at appropriate intervals.
Overexpression and preparation of His-tagged ArgR protein.
To express the argR gene in a form with a His8-tag at the COOH terminus, PCR was performed with the primer pairs TtArgRN and TtArgRHisC, using pBM4K710 as the template. The PCR product with the correct nucleotide sequence was chosen and cloned as an NdeI–HindIII fragment into pET26b(+) (Novagen) to yield pET-ArgRHis. E. coli BL21(DE3) cells harbouring pET-ArgRHis were precultured in 5 ml 2x YT medium with kanamycin at 50 µg ml–1 overnight. The culture was transferred to 1 l LB medium containing kanamycin at 50 µg ml–1. When the culture had grown to an OD600 of 0.6, IPTG was added at a final concentration of 0.4 mM. After cultivation for 14 h at 25 °C, cells were harvested, suspended in 30 ml STU buffer (500 mM NaCl, 20 mM Tris/HCl pH 8.0, 500 mM urea), disrupted by sonication, and heated at 70 °C for 20 min. Debris was removed by centrifugation at 40 000 g for 30 min and phenylmethylsulfonyl fluoride (final 2 mM) was added to the supernatant. The enzyme solution was applied onto a Ni-NTA resin (Novagen), and washed with 10 vols STU buffer followed by washing with 2 vols STU buffer containing 20 mM imidazole. Elution was done with STR buffer (150 mM NaCl, 20 mM Tris/HCl pH 8.0, 5 mM arginine) supplemented with 0.4 M imidazole. The eluted fractions were dialysed against STR buffer, and used as purified ArgR. The purity of ArgR was verified by SDS-PAGE. Protein concentration was determined by the method of Bradford (1976). Gel filtration was performed to estimate the quaternary structure, with a Superose 12HR 10/30 gel exclusion column (GE Healthcare Bio-Sciences) equilibrated in ST buffer with or without 5 mM arginine.
Electrophoretic mobility shift assays (EMSAs).
A 205 bp fragment containing the hcs promoter region and a 290 bp fragment containing the argF promoter region were amplified from T. thermophilus TSU130 chromosomal DNA by PCR with primers HCSUP/HCSDWN and RFUP/RFDWN, and cloned into pT7Blue to yield pHcsUT and pRFUT, respectively. To construct the hcs promoter mutant, two independent PCRs were performed using RPHCSR/CLMUT and RPHCSF/CLMUT-C as primers, and cloned into pT7Blue. Portions of the two reaction products were mixed together and subjected to additional PCR using RPHCSF and RPHCSR as primers. An amplified fragment was cloned into pT7Blue to produce pHCSUTmut. The hcs promoter region, the mutated hcs promoter region and the argF promoter region were amplified by PCRs from pHcsUT, pRFUT and pHcsUTmut with primers HCSUP/HCSDWN, HCSUP/HCSDWN and RFUP/RFDWN, respectively. After purification these fragments were endlabelled with [
-32P]ATP. The reaction mixture (total 20 µl) containing Tris/HCl pH 7.8, 500 µM arginine, 2 mM MgCl2, 1 mM EDTA, 50 mM NaCl, 1 mM dithiothreitol, 250 µg bovine serum albumin ml–1, 10 % (v/v) glycerol, 1 % sucrose, Nonidet P-40 and 0.05 µg poly(dI-dC) ml–1 was mixed with the labelled probe (1 nM) and 0–400 nM ArgR. Binding of ArgR to the labelled DNA fragment was started by adding the labelled fragment to the reaction mixture preincubated for 10 min at 70 °C and the mixture was further incubated at 70 °C at least 30 min. The reaction mixture was used for 5 % PAGE in TAE (40 mM Tris/acetate pH 8.0, 2 mM EDTA) buffer. Arginine was added at 5 mM to the electrophoresis buffer only in the experiment for Fig. 3(a). Band shift on the gel was analysed by an FLA-3000G fluoro/image analyser (Fuji Film). In the competition assay, 2.5 µM unlabelled probe was added. Each competition probe was prepared by annealing each complement pair of 35 bp nucleotides, CLWT/CLWT-C and CLMUT/CLMUT-C, by incubation at 95 °C for 5 min, 65 °C for 5 min and 37 °C for 5 min.
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-galactosidase assays.) previously digested with NdeI/NotI. An EcoRI–BglII fragment from the resulting plasmid was cloned into the appropriate portion of pOF5714 (Fridjonsson & Mattes, 2001) to yield pTtRF. T. thermophilus cells harbouring reporter plasmids, pTthcs-plp (Tsubouchi et al., 2005) or pTtRF were grown at 60 °C in TM medium supplemented with 1.6 mM CaCl2, 1.6 mM MgCl2 and 50 µg kanamycin ml–1 until the mid-exponential phase. Cells were harvested by centrifugation at 15 000 g and washed with MM medium three times. Cells were suspended in MM medium or MP medium containing 50 µg kanamycin ml–1 and various additives, and the culture was continued for an additional 24 h. Cells were harvested by centrifugation at 15 000 g for 30 s, washed and suspended in 0.1 M HEPES buffer (pH 7.5). Crude extract was prepared by sonication, and debris was removed by centrifugation at 15 000 g for 10 min. Proteins were determined by the method described above. -Galactosidase activity was determined by the methods previously described (Tsubouchi et al., 2005), except that 5 mM 4-nitrophenyl -D-galactopyranoside was used as the substrate. One unit of activity was defined as the amount of enzyme required to release 1 µmol p-nitrophenol min–1, and the specific activity of crude extract was expressed as units (mg protein)–1.
DNase I footprinting.
DNase I footprinting was performed using non-radioactive probes containing the IRD800 label at the 5'-end. DNA probes were prepared as follows. pHcsUT was used as a template for two PCRs with primers IRD800-labelled T7 promoter primer (MWG-Biotech)/M4p and IRD800-labelled M13-29 primer (Li-Cor)/T7p. These PCRs produced DNA fragments that contain an 188 bp hcs promoter region from –73 to +115 with respect to the transcriptional start point. The DNA fragment also contains an approximately 80 bp or 60 bp extension originating from pT7Blue at the upstream and the downstream ends, respectively, at which the IRD800 label was attached. After purification by Microspin S-400 HR columns (GE Healthcare Bioscience), 50 ng of the resulting DNA was used for DNase I footprinting. Binding of ArgR (0.06–7 µM) to the DNA fragment was performed as in the EMSA experiments (see above) with minor modification: the total volume was changed to 50 µl, poly(dI-dC) was omitted, and the arginine concentration was set to 10 mM. DNase I cleavage was done by adding 3 µl of a solution containing 160 mM MgCl2 and 0.25 units DNase I. After 1 min incubation at room temperature, the DNase I reaction was stopped by addition of 50 µl 5 M ammonium acetate and 30 mM EDTA. The DNA was extracted with 100 µl phenol and 100 µl chloroform, precipitated with ethanol, and washed with 70 % ethanol. The pellet, dissolved in 1 µl of formamide loading buffer, was heated at 95 °C for 10 min, rapidly chilled on ice, and applied to a Li-Cor automated DNA sequencer (model 4000L) using a 6 % KBPlus 41 cm denaturing sequence gel (Li-Cor) at 50 °C.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Czaplewski et al., 1992; Dimova et al., 2000; Dion et al., 1997; Larsen et al., 2004, 2005; Lim et al., 1987; Lu et al., 1992; Morin et al., 2003; Tian et al., 1994; Xu et al., 2003). For T. thermophilus, Sanchez et al. (2000) previously suggested that the argF promoter is regulated by ArgR. For the initial investigation, we aligned the sequences between the argF promoter region containing the ARG box and the hcs promoter region (Fig. 2a). The result of the alignment suggested the existence of an ARG box in the hcs promoter region, suggesting that the hcs promoter is regulated by ArgR. The whole genome sequence determined for T. thermophilus HB27 (Henne et al., 2004) indicates that T. thermophilus possesses only a single homologue of argR as TTC1194. We have already cloned a DNA fragment containing the argR homologue of T. thermophilus HB27 as a different research project, and therefore used the gene for the following expression experiment.
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). We carried out gel filtration to estimate the quaternary structure of the ArgR homologue using a column equilibrated with ST buffer with or without 5 mM arginine. The argR homologue encodes an 18 kDa protein with 164 amino acid residues. According to the elution volume, the ArgR homologue has a molecular mass of 60 kDa without arginine, suggesting the configuration of a trimer; however, in the presence of 5 mM arginine the ArgR homologue was eluted in the fraction with a molecular mass of 100 kDa. This result shows that arginine affects the quaternary structure of the ArgR homologue. ArgR proteins, such as AhrC from Bacillus subtilis (Holtham et al., 1999) and ArgRs from Bacillus stearothermophilus (Dion et al., 1997) and Thermotoga neapolitana (Dimova et al., 2000) are isolated as trimers and are in rapid equilibrium with hexamers. The oligomeric state of some ArgR proteins might be concentration dependent and affected by the presence of arginine and/or target DNA. Binding of six arginine molecules per hexamer has been shown for the E. coli and B. stearothermophilus ArgR proteins by X-ray crystallographic analysis (Ni et al., 1999; Van Duyne et al., 1996). Therefore, we assume that the ArgR homologue from T. thermophilus is as the hexamer in the arginine-bound form.
To examine binding of the ArgR homologue to the argF promoter in vitro, EMSA was performed with DNA fragments covering the argF promoter as the probe. The ArgR homologue was able to shift the target probe DNA in a dose-dependent manner with a Kd of approximately 30–50 nM (in monomer equivalent) (Fig. 2c). Then, to confirm that the argR homologue is involved in arginine regulation in vivo, we constructed an argR homologue knockout mutant (named WRK07) of TSU130, and used it as the host for the reporter assay. In TSU130 harbouring pTtRF, which carries the -galactosidase gene under the control of the argF promoter, -galactosidase activity was decreased by the addition of 0.5 mM arginine. In the argR knockout mutant WRK07, however, 50-fold increased -galactosidase activity that was not affected by the addition of 0.5 mM arginine was detected (Table 2). Similar deregulated overexpression was observed for arginine regulons in argR mutants of E. coli and Lactococcus lactis (Larsen et al., 2004; Tian et al., 1994). Thus, the argR homologue is shown to be an argR orthologue. We hereafter call the gene argR. The ArgR protein from T. thermophilus shows identity in amino acid sequence to the orthologues of E. coli, B. stearothermophilus, B. subtilis and Thermotoga neapolitana of 30.2, 43.3, 41.5, and 38.8 %, respectively.
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). The addition of higher concentrations of ArgR caused the formation of a more slowly migrating complex that might correspond to the hexamer–operator complex, whereas the faster-migrating band might correspond to the trimer–operator complex, as suggested previously by others (Dimova et al., 2000; Morin et al., 2003). When electrophoresis for EMSA was done in electrophoresis buffer containing 5 mM arginine, the slower-migrating band appeared at lower concentrations of ArgR (Fig. 3a). Apparent equilibrium dissociation constants (Kd) were around 30 nM and 100 nM in the presence and absence of 5 mM arginine in the buffer, respectively. Thus, arginine enhances the affinity of ArgR to the target DNA to some extent. In addition, in the presence of arginine, the complex forms a sharper band compared to the broad and diffuse band observed in its absence, suggesting that the complex is more stable in the presence of arginine. To elucidate the specificity of the binding, we prepared DNA fragments with 4 bp substitutions in the putative ARG box around the hcs promoter –10 region (Fig. 3b), and used them for EMSA. In contrast with the fragments with a wild-type sequence, the 4 bp-substituted DNA fragment showed lower affinity to ArgR (Fig. 3c). The observation that no fast-migrating band was formed for the mutant operator suggests that ArgR protein may bind to the mutant ARG box stably only in a hexameric form. In any case, it should be noted that ArgR from T. thermophilus binds to both hcs and argF promoters with very similar affinity.
To determine the region of ArgR binding more precisely, a 35 bp DNA fragment around the hcs promoter –10 region was used as a competitive probe. The band shifts observed without the competitive probe were diminished by the addition of the 35 bp DNA fragment with the wild-type sequence, whereas the 35 bp DNA fragment with 4 bp mutations had little effect on the shift band of ArgR (Fig. 3d). Furthermore, DNase I footprinting indicated that ArgR protected regions from –23 to –5 for the sense strand and from –19 to –1 for the antisense strand with respect to the transcriptional start point (Fig. 4a, b). These results show that ArgR specifically binds to the hcs promoter –10 region. ArgR also protected the upstream region (site B). This will be discussed below.
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-galactosidase specific activity of 40.9±7.9 mU (mg protein)–1. When a similar assay was performed with the argR mutant WRK07, the level of -galactosidase activity was increased to 95.2±15.2 mU (mg protein)–1, and no enhanced expression [activity 113.4±13.5 mU (mg protein)–1] was observed by addition of arginine. From these results, it can be concluded that ArgR is involved in the regulation (repression) of hcs promoter activity.
When 0.1 mM arginine was added to the culture of T. thermophilus TSU130 carrying pTthcs-plp, the response to arginine varied, with large deviation (data not shown). This observation suggests that a subtle change in culture conditions affects the hcs promoter in T. thermophilus TSU130. Considering that arginine degradation in cells might be the possible main reason for the large deviation of the response, we constructed an arginase gene knockout mutant, T. thermophilus ADK1, of T. thermophilus TSU130 and used it as the host for the same reporter assay. As expected, in the arginase-minus genetic background, a reproducible response to arginine was observed. In ADK1, with the knockout of the arginase gene, the -galactosidase activity [36.6±1.2 mU (mg protein)–1] obtained in the absence of arginine was decreased threefold [to 11.9±3.4 mU (mg protein)–1] by addition of arginine. On the other hand, when a different mutant, ADK2, defective in both argR and arginase genes was constructed and used as the host for the reporter assay, -galactosidase activity of 113.2±16.5 mU (mg protein)–1 was obtained in the absence of arginine. Addition of arginine did not decrease -galactosidase activity but increased the activity twofold, to 234.2±10.4 mU (mg protein)–1.
Growth inhibition of the arginase knockout mutant by arginine
The above results indicate the involvement of ArgR in hcs promoter activity. These results also suggest that T. thermophilus may show a lysine-auxotrophic phenotype when arginine is added to the culture, due to repression of hcs promoter activity. To examine this possibility, growth of T. thermophilus TSU130, ADK1 and ADK2 was monitored. As expected, the growth of the arginase mutant T. thermophilus ADK1 was inhibited by the addition of arginine to the minimal medium (Fig. 5), which contrasted with the cases of the wild-type strain T. thermophilus TSU130 and the mutant ADK2, defective in both arginase and argR genes, where no inhibition by arginine was observed (Table 3). Ornithine is an intermediate of arginine biosynthesis, and its synthesis is therefore controlled by arginine. Furthermore, ornithine is a peptidoglycan component in T. thermophilus (Quintela et al., 1995). We anticipated that an increase in the intracellular arginine pool could activate the ArgR function to repress the arginine biosynthetic gene expression and, as a result, lead to the short supply of ornithine as a cell wall component. We therefore added 0.1 mM ornithine to MP medium, and examined its effect on the growth of the arginase mutant ADK1; however, it had only a slight effect on growth (Fig. 5). We next examined the growth of ADK1 in MP medium supplemented with 0.1 mM lysine or both 0.1 mM lysine and 0.1 mM ornithine. The slow growth of ADK1 was partially restored by the addition of 0.1 mM lysine, and fully restored by the addition of both 0.1 mM lysine and 0.1 mM ornithine. When generation times were compared, addition of arginine prolonged the generation time of ADK1 significantly (Table 3). The prolonged generation time was partially shortened by addition of lysine and completely normalized by simultaneous addition of lysine and ornithine. On the other hand, the generation time of ADK2, with the knockout of both argR and arginase genes, was not affected by arginine. These results also support the idea that ArgR regulates lysine biosynthesis in vivo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, the bands in the EMSA were somewhat broad and smeary in the absence of arginine, and addition of arginine in the electrophoresis buffer gave clear band profiles. Thus, arginine may assure the formation of a stable ArgR–DNA complex. However, apparent Kd was not so significantly affected by arginine (30 nM and 100 nM in the presence or in the absence of arginine, respectively). Recently Charlier and colleagues classified arginine regulators into three major types (Charlier, 2004; Morin et al., 2003). Among them, ArgR from Thermotoga, which is the only ArgR classified into class III, is defined as serving as a general regulator exhibiting broad target specificity and low arginine-dependent activation of DNA-binding capacity. In T. thermophilus ArgR, a Gln residue, which is highly conserved in class I ArgR and involved in the highly arginine-dependent activation of DNA-binding ability, is replaced by Ser (Fig. 6). Substitution of Gln for the Ser of ArgR from T. neapolitana resulted in a mutant protein exhibiting a marked need for arginine as co-factor and an enhanced target specificity (Morin et al., 2003). It is also shown that the T. thermophilus ArgR is different from other class ArgR proteins in that it can interact with a single ARG box sequence. Arg from T. thermophilus also has a serine residue at the corresponding position and shows low arginine-dependent activation of DNA-binding capacity. The observation that the band shift in EMSA was diminished by addition of a short competitor of 35 bp having only a single ArgR-binding site around the hcs promoter –10 region may also suggest that ArgR interacts with a single ARG box sequence. DNase I footprinting indicates that ArgR binds to the hcs promoter –10 region (site A) as expected. The analysis revealed that another region (site B) was also protected by ArgR (Fig. 4a, b). However, the protection was found at a region connecting the hcs promoter upstream sequence with the cloning site of the vector pT7Blue. Actually, only a small portion, from –73 to –68 in the sense strand and from –73 to –70 in the antisense strand, was protected for the hcs promoter upstream region, and the other protection was found in the vector region. This observation indicates that the putative ArgR-binding site in the hcs promoter upstream region is artificially generated in our DNase I footprinting experiment. Although the DNase I footprinting could not rule out the possibility that ArgR may bind to other regions which were not tested in this study, our results are consistent with there being a single ARG box covering the hcs promoter –10 region, which regulates hcs promoter activity. From these observations, we may conclude that ArgR of T. thermophilus is a class III regulator.
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). Addition of lysine partially overcomes the growth inhibition by arginine, and the simultaneous addition of both lysine and ornithine restores the growth of ADK1 completely, even in the presence of arginine (Fig. 5). It could be that addition of arginine to ADK1 causes diversion of the excess arginine to a pathway that produces a toxic compound and that this pathway is repressed by lysine. However, in both the wild-type and the double mutant, ADK2, defective in arginase and argR, arginine inhibition was not observed (Table 3). The most likely explanation of these phenomena is that the growth inhibition by arginine in the arginase mutant is attributable to the function of ArgR, therefore suggesting that lysine biosynthesis as well as ornithine synthesis is regulated by ArgR in T. thermophilus. Lysine biosynthesis through AAA is also found in fungi, and AAA synthesis from 2-oxoglutarate proceeds in the same way in fungi and T. thermophilus; however, subsequent synthesis, from AAA to lysine, is totally different between these organisms. The T. thermophilus pathway is similar to a step in arginine biosynthesis, while the pathway in fungi starts from adenylation of the -amino group of AAA and contains saccharopine as an intermediate. It should be noted that similar growth inhibition by arginine has been reported in an arginase mutant of the fungus Neurospora crassa, but inhibition is restored by only ornithine (Davis et al., 1970). This suggests the presence of a different regulatory system in Neurospora, although it should be taken into account that the fungus has compartmentalization and a specific transport system, and therefore the situation may be totally different.
By EMSA and DNase I footprinting, we demonstrated that ArgR specifically bound to the DNA fragment containing the hcs promoter –10 region (Fig. 3). For further elucidation of the role of ArgR in hcs promoter regulation in vivo, reporter gene assays were carried out. In ADK1, with the knockout of the arginase gene, the addition of arginine repressed the hcs promoter. In ADK2, with the additional mutation of argR, no repression was observed, whereas the addition of arginine activated the hcs promoter. In WRK07, with an argR mutation, no response to arginine was observed. These results may indicate not only the involvement of ArgR in the regulation of lysine biosynthesis in T. thermophilus, but also the presence of some other regulatory systems that sense arginine or its metabolites to activate lysine biosynthesis. As described in Results, the involvement of ArgR in the regulation of the hcs promoter was not verified in the parent strain of T. thermophilus; in some cultures, arginine seemed to enhance -galactosidase expression, while it decreased the expression in others. Since such a variable effect was not observed with the addition of other amino acids in the reporter assay, we assume that there are multiple systems to sense arginine and/or its metabolites, and therefore arginine might play a central role in the metabolic flux of related amino acids in T. thermophilus. We have very recently found a transcriptional factor, which also regulates lysine biosynthesis, and are analysing its regulatory mechanism (unpublished results). Through detailed research of at least two transcriptional factors, regulatory mechanisms for lysine biosynthesis should be elucidated in the near future.
The lysine biosynthesis of T. thermophilus has an evolutionary relationship with the biosynthesis of arginine and leucine, and the TCA cycle (Nishida et al., 1999). Furthermore, the fact that a similar lysine biosynthesis pathway is also present in archaea (Brinkman et al., 2002; Lombo et al., 2004; Nishida et al., 1999), which are phylogenetically positioned close to the root of the universal tree of evolution (Woese et al., 1990), suggests that lysine biosynthesis was present in the earliest organisms. We may assume that the ancestral enzymes involved in the processes related to current Thermus lysine biosynthesis functioned in lysine and arginine biosynthesis as well as the TCA cycle. In previous studies, we have shown that T. thermophilus lysine biosynthetic enzymes have the ability to recognize intermediates of the TCA cycle and arginine biosynthesis as substrates, suggesting that the enzymes have additional roles in those metabolisms even now. It is of interest that an enzyme involved in lysine biosynthesis may play a role in biosynthesis of another basic amino acid, arginine; however, there are other reported cases of such functional overlap. Ledwidge & Blanchard (1999) showed that the argD gene encoding N-acetylornithine aminotransferase in the arginine biosynthesis pathway is the most probable candidate for dapC gene encoding N-succinyl-L,L-diaminopimelate : 2-oxoglutarate aminotransferase in a major root (DAP pathway) of the lysine biosynthesis pathway in E. coli. The argD gene is regulated by ArgR (Charlier et al., 1992). However, it is unclear whether this gene is regulated by lysine. In this study, we showed that ArgR, a repressor of arginine biosynthesis, has the ability to regulate lysine biosynthesis in T. thermophilus, although the regulation is apparent in an arginase gene knockout background. Cumulative data on the expression profile of lysine biosynthetic and related genes of T. thermophilus will elucidate the complicated regulatory mechanisms managed by multiple transcriptional machinery.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 15 June 2006;
revised 23 August 2006;
accepted 30 August 2006.
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) and the arginase-deficient mutant ADK1 (
) in MP medium. Growth of ADK1 in MP medium supplemented with 0.1 mM arginine (
), both 0.1 mM lysine and 0.1 mM arginine (
), both 0.1 mM ornithine and 0.1 mM arginine (
), and all three amino acids (lysine, arginine and ornithine, 0.1 mM each) (
) is also shown.