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Laboratorium voor Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium
Correspondence
Raymond Cunin
rcunin{at}vub.ac.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Complete microarray data have been deposited at the NCBI Gene Expression Omnibus (www.ncbi.nlm.nih.gov/projects/geo/index.cgi) with the accession number GSE4724.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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to describe the genetic and regulatory organization of the genes of arginine biosynthesis in Escherichia coli. The arg genes are not organized into one single operon, in contrast to what was observed at that time for several other pathways, but they are scattered over different chromosomal loci. Their expression is repressed by arginine to different extents, but in a coordinated fashion through the action of the arginine repressor ArgR. The regulon type of organization allows differential repression of genes or groups of genes within one metabolic way, as well as multiple regulation of individual genes – cumulative or cross-pathways regulation. Since then, regulons have been found to be widespread in E. coli and in other micro-organisms. The molecular basis for differential repression of arginine genes is the interaction of a unique complex of aporepressor (ArgR)/co-repressor (arginine) with operator loci differing in base composition, number and localization with respect to the promoter of conserved 18 bp elements called ARG boxes (Cunin et al., 1983; Charlier et al., 1992; Charlier & Glansdorff, 2004). The cis- and trans-acting elements of this regulatory mechanism are remarkably well conserved through the bacterial domain (Charlier, 2004).
Arginine is synthesized in eight enzymic steps, starting from glutamate. The synthesis of carbamyl phosphate, a metabolite common to arginine and pyrimidine biosynthesis, is regulated jointly by arginine and pyrimidines through transcriptional repression of a tandem pair of promoters and allosteric control of carbamoyl-phosphate synthase (CPSase) activity (Piérard et al., 1965; Piette et al., 1984; Devroede et al., 2004). Although information on genes, enzymes and regulation of the arginine regulon is substantial (for reviews, see Cunin et al., 1986; Charlier & Glansdorff, 2004), it is incomplete and, as it was obtained by different teams using different approaches often dealing with specific limited objectives, it does not always lend itself to integration into a coherent description of the system.
As a prerequisite to the development of a mathematical model of arginine biosynthesis, including its dynamics, it is necessary to identify all the components of the arginine regulon and to investigate their regulation, using a global approach to obtain a set of coherent values for key parameters of the system. The response to arginine of mRNA expression profiles was analysed using hybridization to chromosomal DNA microarrays and real-time quantitative (RTq) PCR, to obtain derepression/repression ratios for the transcription of individual genes and/or operons, including those for which such information was lacking, and to discover potential new members of the regulon on the basis of their expression profile. For such newly identified genes, the presence and functionality of ARG boxes was investigated.
In addition to biosynthetic genes, this analysis has revealed which other genes, e.g. arginine uptake genes, are subject to specific regulation by arginine, thus identifying them as components of the regulon.
Another important point, pertaining to the dynamics of the regulon, is the time response of its constituents to changes in the environment. The time course of repression by arginine was monitored by RTq-PCR for a representative set of genes, including newly discovered members of the regulon, and by parallel assays of selected enzymic activities.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Strains and growth conditions.
P4X (Hfr, metB) and its genetically derepressed derivative strain P4XB2 (Hfr, metB argR) were used in this study. P4X is a derivative of E. coli K-12, through 58-161 and W6 (Bachmann, 1987), and it is the strain in which previous studies of arginine synthesis were performed by this laboratory. It has been designated wild-type for arginine synthesis. Cells used for expression analysis were grown in minimal medium (Glansdorff, 1965) supplemented with 0.5 % (w/v) glucose, L-methionine (100 µg ml–1) and, when specified, with L-arginine (100 µg ml–1). Repression studies of hisJQMP with lacZ fusions used strains CSH100, FW102 (Whipple, 1998) and FW102 
argR (Xu et al., 2003) grown on rich medium (Glansdorff, 1965) or on minimal medium in the presence of appropriate antibiotics (kanamycin, 100 µg ml–1; streptomycin, 25 µg ml–1; chloramphenicol, 25 µg ml–1).
For transcription profile analysis, cells were grown in a rotary shaker at 37 °C and harvested by centrifugation at mid-exponential phase (OD660=0.5) before quenching their metabolism in liquid nitrogen. No RNA-protecting agent was used in this standard procedure. However, to verify that cold shock and other stresses related to the sampling procedure did not affect expression profiles through transcriptional induction or RNA degradation, RNAprotect (Qiagen) was added to ‘freeze’ transcriptional profiles in a series of samples prior to centrifugation, and extraction was performed in parallel with that of unprotected samples. The expression profiles of a representative set of genes (argA, argC, argD, artJ, hisJ and argR) were compared between protected and unprotected samples using RTq-PCR. The difference, when observed, was below or about the SD, showing that profiles are not modified during sampling and extraction. For these genes, derepression/repression ratios obtained by both procedures are shown in Table 4.
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RNA isolation.
Total RNA was isolated using the RNeasy RNA isolation kit, according to the manufacturer's specifications (Qiagen), and stored in RNase-free water at –80 °C. RNA concentration was determined by UV spectrometry and its quality was controlled by agarose gel electrophoresis.
Microarray analysis and data processing.
The GeneChip E. coli Genome 2.0 Array (Affymetrix) was used.
After UV spectrometry and agarose gel electrophoresis checks, total RNA was further controlled for integrity and purity using a Bioanalyser 2100 (Agilent) and a Nanodrop spectrophotometer. Total RNA (10 µg) showing no signs of degradation or impurities, was supplemented with poly-A RNA controls and reverse transcribed to cDNA using random primers. After RNA degradation with NaOH, cDNA was purified (MinElute PCR Purification Kit; Qiagen), analysed for yield (30–120 µg) and purity (260/280 nm and 260/230 nm absorption ratios >1.8), then fragmented using DNase I (Amersham Pharmacia Biosciences) and labelled terminally with biotinylated GeneChip DNA Labelling Reagent (Affymetrix). A minimum of 1 µg fragmented probe was resuspended in 80 µl hybridization buffer containing 3 nM control oligo B2 (Affymetrix). Hybridization was performed in a rotisserie oven at 45 °C for 16 h. The chips were washed and stained in a GeneChip Fluidics Station 400 (Affymetrix) and subsequently scanned with the GeneChip Scanner 3000 (Affymetrix). Initial data analysis was performed using GeneChip Operating Software. Microarray quality control parameters were as follows: noise (RawQ) less than 5, background signal less than 40 (100 target intensity for array scaling), consistent numbers of genes detected as present across arrays.
Data were processed using the robust multiarray average algorithm (RMA) for normalization, background correction and expression value calculation (Irizarry et al., 2003). Expression levels obtained by three independent replicates of every condition were compared using the Limma package of the Bioconductor software (Gentleman et al., 2004). The robustness of the data were further enhanced by Bayes adjustment and the P values were corrected for the false discovery rate (Benjamini & Hochberg, 1995).
RTq-PCR.
First-strand cDNA was generated from 2 µg RNA in a reaction (20 µl final vol.) using random hexamer primers and Superscript II reverse transcriptase (Invitrogen), followed by RNase H treatment (Fermentas). PCR reactions were prepared in a 25 µl volume containing SYBR Green PCR Master Mix (Applied Biosystems), 0.5 µl cDNA and 150 nM each forward and reverse primer. Amplicon size was always between 100 and 250 bp. Amplification cycles were carried out in an iCycler IQ (Bio-Rad) using the following protocol: 95 °C for 10 min, 40 cycles of 15 s at 95 °C, 30 s at 58 °C and 30 s at 72 °C, followed by melting curve analysis.
All runs included a primer pair for the detection of the rpoS gene. This gene was chosen as internal reference because it showed exactly the same significant expression levels across the strains and the conditions used in the microarrays. It was previously reported that rpoS is poorly expressed during exponential growth (Jishage et al., 1996). However, in the conditions used, this was not observed: the expression level of rpoS was comparable to that of the other genes studied and, as expected from the microarray analysis, stable, in contrast to the expression of rpoN or gyrB which are frequently used as references. The relative expression ratios were calculated using the ‘Delta-Delta Method’ (Perkin Elmer). For each gene tested, the expression measured in the wild-type on unsupplemented minimal medium was chosen as the reference value (100 %). Forward (Fw) and reverse (Rv) primers are listed in Table 1.
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N-Acetylglutamate synthase (NAGSase) was assayed according to Abadjieva et al. (2001), with the following modifications: prior to the assay, the extract was dialysed instead of being run on a Sephadex G-25 column and the reaction was incubated for 5 min at 37 °C.
Ornithine transcarbamylase (OTCase) activity was determined as described by Glansdorff & Sand (1965). The samples were incubated for 15 min at 37 °C in the presence of 2 mM ornithine and 10 mM CP in 50 mM Tris/HCl, pH 8.
CPSase was assayed according to Simmons et al. (1999) with modifications. The reaction mixture contained 10 mM bicarbonate, including 2 µCi (74 kBq) [14C]bicarbonate, and 10 mM ATP/MgCl2. The reaction was started by the addition of 100 µl cellular extract, allowed to proceed for 5 min at 37 °C and terminated by adding 100 µl 1 M ammonium sulfate. Each experiment contained a control without glutamine. Protein concentrations were determined by the Lowry method.
Mobility shift electrophoresis.
Mobility shift experiments were performed by the method of Fried & Crothers (1983). 32P-labelled DNA fragments were prepared by PCR amplification with a pair of oligonucleotides, of which one was 5'-end-labelled with 32P, and purified by PAGE. Binding experiments were performed at 37 °C, by incubating different amounts of purified ArgR repressor protein and a constant amount of labelled DNA (15 000 c.p.m.) in the presence of an excess of non-specific competitor DNA (sonicated calf thymus DNA). ArgR binding took place in 20 mM Tris/HCl, pH 7.4, 500 mM KCl, 10 mM MgCl2, 1 mM DTT, 20 mM L-arginine and 5 mM CaCl2. Separation of free and bound DNA was performed on 5 % polyacrylamide gels. Migration was for 3 h at 8 V cm–1 in arginine running buffer (89 mM Tris, 89 mM boric acid, 10 mM MgCl2 and 5 mM L-arginine).
Assay of hisJQMP repression, using fusions with lacZ.
To construct the wild-type hisJQMP promoter-lacZ reporter fusion (WT phisJQMP), the complete promoter region was amplified by PCR as an EcoRI–BamHI fragment using the primers Mc1Fw (5'-GGGAATTCTACGTCGCTGGGAAGCTG-3') and Mc2Rv (5'-GGGGATCCAATGTCCTCAACTGACAGTC-3'), and cloned into the promoterless plasmid pFW11-null (Kmr Cmr) which contains the 3' end of lacI and the 5' end of lacZ. To knock out the identified ARG box, a Kmr cassette was generated by PCR using primers with 50 nt homology extensions and introduced in a P4X strain expressing the 
Red recombinase. After selection, the resistance gene could be eliminated by using a helper plasmid expressing the FLP recombinase which acts on the FRT sites flanking the resistance gene as described by Datsenko & Wanner (2000). The ARG box knock-out promoter region obtained was then cloned into the pFW11-null plasmid following the same procedure as for the wild-type promoter. Two truncated promoters were also generated by PCR. The first one, 1 phisJQMP, was amplified starting from the ARG box using primer Mc3Fw (5'-GGGAATTCTTAATGAAAATAATACGTCTAAC-3') in combination with Mc2Rv. The second construct, 2 phisJQMP, starts downstream from the ARG box and was generated using primers Mc4Fw (5'-GGGAATTCTAACGGGGCGGGATATTTG-3') and Mc2Rv.
The different plasmids were introduced into strain CSH100 (Sms) which bears an F' episome containing a truncated lac operon. The transfer of the cassette from pFW11 occurred via a double homologous recombination. Strain construction was completed upon conjugation with the FW102 recipient strain (Smr) or its FW102 argR derivative, followed by screening for Smr Kmr Cms transconjugants as described by Whipple (1998).
-Galactosidase specific activity was assayed in cell-free extracts of cultures grown in minimal medium with or without arginine. Assays were performed as described by Miller (1972). Enzyme activities are expressed as µmol h–1 (mg protein)–1.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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A list of regulon genes and their cognate proteins – enzymes or binding proteins – is given in Table 2.
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). For argA, argD and argG, these are the first transcriptional repression data ever obtained. E. coli K-12 possesses two genes encoding OTCase: argI and argF: for these too, the first transcriptional repression values were obtained.
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The P4X(min) to P4X(arg) ratio for the carAB operon was below the cut-off value as expected from the only partial (40 %) repression of carAB by arginine (Piette et al., 1984). Indeed, liganded ArgR efficiently represses initiation at the downstream promoter carP2, but has no effect on transcription initiated at the upstream promoter carP1, which is mainly repressed by excess pyrimidines. Similarly, the weak autorepression of argR, the repressor gene, was below the detection level.
Only a few genes outside those of arginine biosynthesis were identified by the microarray analysis: the art genes encoding the arginine-specific uptake system and the hisJQMP genes of the histidine transporter complex (see below). Each one of these genes or gene clusters is preceded by one ARG box, the ArgR-binding component of arginine operators. It should be pointed out that a screening of the genomic sequence for ARG boxes (Makarova et al., 2001) identified only 11 operators or putative operator sites: those of the biosynthetic genes, of argR and of artPIQM and artJ, but failed to detect the box upstream of the hisJQMP cluster as well as the 6 or 7 putative boxes in the promoter region of the arginine catabolism ast operon (Lu, 2006). Since ARG boxes are partially conserved, the results of such a screening depend very much on the stringency of the search. Clearly, transcriptome analysis with DNA microarrays is a more sensitive approach, even with the high cut-off value applied in this study. Besides, the presence of an ARG box cannot by itself establish that a gene is part of the regulon without supporting evidence of ArgR-dependent regulation.
Analysis of the transcriptional repression of biosynthetic genes by the more sensitive RTq-PCR assay gave higher derepression/repression ratios than the microarrays (Table 4
), closer to values obtained from enzyme specific activities (last column of Table 4 and below). The derepression/repression ratios range from 3 and 1.6 for carAB to 150 for argF, illustrating the flexibility conferred to the system by the regulon type of organization. When these ratios are put in context with the position of the genes and the cognate enzymic reactions in the pathway, a rough regulatory logic emerges. The pathway can be divided into three modules (Fig. 1): (i) the synthesis of ornithine from glutamate (argA–argE), (ii) the synthesis of CP (carAB), and (iii) the synthesis of arginine from ornithine and CP (argF/I–argH). (i) A substantial repression of argA (43-fold) and feedback inhibition of NAGSase by arginine (Marvil & Leisinger, 1977; Caldara et al., 2005) combine to control the metabolic flow through the first part of the pathway and thus the production of ornithine. (ii) Repression of carAB by arginine is only partial (about threefold) but the activity of CPSase is subject to a complex set of allosteric regulations, among which are a strong feedback inhibition by UMP and a counteracting activation by ornithine (Piérard et al., 1965; Robin et al., 1989; Braxton et al., 1992). Limitation of ornithine production by arginine therefore enhances the feedback inhibition of CPSase by UMP. (iii) The strong repression of argF (150-fold) and argI (27-fold) makes OTCase synthesis the most repressible step of the pathway (475-fold repression of total OTCase activity; Table 4) and the major control point of the metabolic flux from ornithine to arginine. Arginine itself does not directly regulate the activity of OTCase, but it limits the availability of CP, the second substrate of the enzyme, which amounts to an inhibition of this enzymic step, and enhances its control of the metabolic flux to arginine.
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Amplitudes of transcriptional repression parallel those calculated from enzyme specific activities, but they are smaller, with the exception of carAB, as observed previously (Cunin et al., 1986), argA and argC (Table 4). The discrepancy between transcriptional and enzymic repression amplitudes led us to consider for a while the existence of a post-transcriptional control, e.g. attenuation. However, systematic investigations of the transcription of the argECBH operon and the sequences of the biosynthetic genes control regions did not support the existence of such a control (Beny et al., 1982; Charlier et al., 1992). The better correspondence obtained when transcription is measured by RTq-PCR indicates that, next to fine adjustments in response to global or cross-pathway signals, one cause of the discrepancy between ratios is probably methodological rather than related to post-transcriptional regulation.
The autorepression of argR was detected by RTq-PCR, with a ratio of 5 between the wild-type on minimal medium and on arginine-supplemented medium (Table 4).
No arginine catabolic gene was found to respond to arginine in the microarray experiments, which is not surprising since these genes respond to nitrogen limitation (Reitzer, 1996), a condition not examined here since all media contained ammonia. Only when arginine is the sole nitrogen source – again not the case here – is there an ArgR-dependent component to the induction of the arginine succinyltransferase (AST) pathway (a two- to threefold effect) (Kiupakis & Reitzer, 2002).
Arginine uptake genes.
Three arginine uptake systems have been identified in E. coli, which use distinctive periplasmic binding proteins: (i) the arginine-specific system encoded by the artPIQM and artJ genes (Wissenbach et al., 1995); (ii) the basic amino acid uptake system, designated LAO (lysine, arginine, ornithine) (Rosen, 1971; Celis et al., 1973); and (iii) the less well characterized AO system transporting arginine and ornithine (Celis, 1977a).
(i) The arginine-specific system.
This system is encoded by the artPIQM and artJ genes (Wissenbach et al., 1993) (Fig. 2). ArtJ is an arginine-binding protein, and ArtP, ArtQ and ArtM are similar to the membraneous and membrane-associated components of binding-protein-dependent carrier systems (Wissenbach et al., 1995). The artPIQM and artJ transcription units of the arginine-specific system are differentially repressed by arginine, with derepression/repression coefficients of 6 for artP, the first gene of the artPIQM operon, and 53 for artJ (Table 4). Both artPIQM and artJ are preceded by a single ARG box (Makarova et al., 2001) and repression is indeed mediated by ArgR, as shown by the increased mRNA levels in P4XB2 (Tables 3 and 4). This establishes unambiguously that the genes of this uptake system are organized in at least two distinct transcription units which are integral components of the arginine regulon. The repression control of artJ is strong (53-fold), an apparent regulatory logic behind this being that intracellular arginine controls the uptake of extracellular arginine by strongly repressing the first component of the system, the periplasmic binding protein. This is consistent with this system being the major arginine uptake system of E. coli under aerobic growth conditions and in the presence of sufficient nitrogen.
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; Liu & Ames, 1998). HisJ is a periplasmic histidine-binding protein. HisP is involved in histidine as well as arginine transport (Kustu & Ames, 1973; Kustu et al., 1979). In E. coli, the genes are organized in two adjacent transcription units of identical polarity, argT and hisJQMP (Fig. 2), whose expression is positively controlled by the nitrogen regulatory protein NtrC in response to nitrogen starvation (Zimmer et al., 2000). The NtrC regulatory site is located upstream of argT. The present analysis reveals that the hisJQMP genes are subject to ArgR-mediated repression (Table 3) with a derepression/repression coefficient of 3–4 for hisJ (Tables 3 and 4). RTq-PCR analysis shows that argT is not repressed by arginine and that its transcription remains unchanged in the argR mutant (Table 3). Therefore, the repression of hisJQMP by arginine is specific to this operon, which makes it a member of the regulon. A putative ARG box was identified between argT and hisJQMP, upstream of the promoter, and its involvement in repression was investigated (see next section). The histidine transporter appears to be the only histidine uptake system in E. coli. This could explain its comparatively weak response to arginine, leaving sufficient residual expression to ensure the uptake of histidine. Arginine transport by the LAO system appears to be essentially confined to the scavenging of a nitrogen-rich amino acid under conditions of nitrogen starvation.
(iii) The AO system.
This consists of the abpS and argK genes, AbpS (AO) being a binding protein and ArgK an ATP hydrolase whose activity is required for transport (Celis et al., 1998). The regulation of abpS and argK is ArgR-independent (Celis, 1977b), yet uptake by this system is repressed by arginine (Celis et al., 1973). Since the exact location of abpS is still not known and the level of argK expression is very low, making it difficult to quantify it correctly, we focussed on argP, the transcription activator of the system. The contention that ArgP represses its own synthesis in the presence of arginine (Celis, 1999) is not supported by our data (Table 4), concurring with more recent evidence obtained with argP-lacZ fusions (Nandineni & Gowrishankar, 2004). It was shown previously that single mutants affected arginine uptake by both the AO and the LAO systems, suggesting that these systems share one or more elements downstream of the periplasmic binding step (Celis et al., 1973; Rosen, 1973). In S. enterica serovar Typhimurium, HisP participates in both histidine and arginine transport (Kustu & Ames, 1973; Kustu et al., 1979). The similarity of the Salmonella and E. coli histidine transporters (Liu & Ames, 1998) suggests a similar dual involvement of HisP in E. coli. In that case, arginine could control the uptake of histidine by the AO system through the repression of hisJQMP.
Arginine repression of the histidine transporter genes
The functionality of the putative ARG box AATGAAAAATAATACGTC located 120–103 bp upstream of the hisJ start codon was tested by measuring arginine repression in a set of reporter fusions with lacZ, and by electrophoretic mobility shift experiments in the presence of pure ArgR repressor.
Repression assays.
In vivo repression of the hisJ promoter was assayed with single-copy F'-borne hisJ-lacZ reporter fusion constructs. The derepression/repression ratio for -galactosidase (3.8-fold, in good agreement with the transcriptome data, 3.8 and 3.0) was observed only when the whole 220 bp region between argT and hisJQMP, containing the hisJQMP promoter, was inserted (WT phisJQMP; Fig. 3). This repression was abolished in a strain containing a deletion of the argR gene (FW102argR). Disrupting the ARG box by the insertion of an FRT sequence (ARG box-KO), deleting the region upstream of the ARG box (1 phisJQMP), or the same region plus the ARG box (2 phisJQMP) abolished repression (Fig. 3). This indicates that in addition to the ARG box, part of the 100 bp upstream region is also required for repression, although, based on in silico analysis, it does not contain any sequence remotely similar to an ARG box.
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). In contrast, binding to the equivalent fragment bearing the ARG box-KO region required higher protein concentrations and resulted in the formation of unstable and non-specific complexes as indicated by the intense smearing and the ArgR concentration-dependent position of the retarded DNA (Fig. 4b). These results show that the ARG box is a major recognition element for ArgR in the hisJQMP operator and suggest that part of the 100 bp region upstream of the box may also interact with the repressor. Taken together with the repression assays, these results strongly suggest that full arginine-dependent repression of hisJQMP requires binding of a repressor not only to the ARG box, but also to as-yet-unidentified upstream sequences to strengthen and stabilize the interaction. Further analysis of ArgR interaction with DNA (e.g. by using footprinting) is needed to elucidate the role of the upstream region.
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. Initial experiments with measurements every minute over a 15 min range (data not shown) showed that transcriptional repression was already complete 2 min after arginine addition. Therefore, the time course was followed with measurements every 30 s over a 2 min range (Fig. 5). In contrast, it took 45–60 min for the NAGSase and OTCase activities to reach 50 % of their initial level, and 90 min for CPSase activity to reach its full repression level (Fig. 6). A 60 min period of time corresponds to one generation time of the chemostat culture. Taken together, these results show that while the ArgR-mediated response at the gene level is very fast, the enzymes are rather stable, their activities being either diluted pro rata of the cells doubling time in the case of NAGSase and OTCase, or possibly following a slightly different course in the case of CPSase. Whatever the molecular mechanism(s) involved in these different cases, the fact remains that a considerable residual enzymic activity is still present after the addition of arginine. Thus feedback inhibition by arginine of the first biosynthetic enzyme, NAGSase, must play a major role in controlling the metabolic flux in the first module of the biosynthesis immediately after the onset of repression with an impact, through reduced ornithine production, on CPSase activity (second module) (less antagonizing of UMP inhibition by ornithine) and, in turn, through limitation of CP production, on the conversion of ornithine to arginine (third module).
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; Kelker & Eckhardt, 1977; Zidwick et al., 1984) and the effect of cAMP/CAP on argG (Krin et al., 2003) fall into this category of controls.
Hybridization to microarrays identified the histidine-transporter-encoding hisJQMP operon as a new member of the arginine regulon, subject to weak ArgR-mediated repression by arginine. The corresponding gene products are part of the LAO basic amino acids uptake system, part of a scavenging defence against nitrogen limitation (Zimmer et al., 2000). In contrast, the specific arginine uptake system (Wissenbach et al., 1993) is strongly repressed by arginine. The comparatively low response of the hisJQMP genes to arginine is probably related to the scavenging function of their products and also to the fact that this could be the only histidine uptake system operating in E. coli, and therefore a stronger arginine-dependent repression would be incompatible with histidine uptake. No evidence was found for ArgR-mediated regulation of the genes of the less well characterized AO uptake system (Celis et al., 1973). ArgR-independent regulation of the AO system by arginine has been proposed before (Celis, 1977a, b, 1999). Since the AO and LAO systems share at least one element downstream of the binding step (Celis et al., 1973), possibly HisP (Kustu et al., 1979), the present results suggest that the AO system could be regulated by arginine through the repression of the hisJQMP component of the LAO system. Altogether, this study clarifies the respective roles of the arginine uptake systems in E. coli.
The time course of repression experiments showed that maximal response of transcription to arginine is reached in 2 min, whereas the cognate enzymic activities decrease more slowly (pro rata of cell division in the case of NAGSase and OTCase). This emphasizes the importance of feedback inhibition of the first biosynthetic enzyme, NAGSase, in controlling the metabolic flux in biosynthesis immediately after the onset of repression.
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ACKNOWLEDGEMENTS |
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Received 24 April 2006;
revised 20 July 2006;
accepted 28 July 2006.
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