Daniel Gigot

Purification and Characterization of Sa-Lrp, a DNA-Binding Protein from the Extreme Thermoacidophilic Archaeon Sulfolobus acidocaldarius Homologous to the Bacterial Global Transcriptional Regulator Lrp

Archaea, constituting the third primary domain of life, harbor a basal transcription apparatus of the eukaryotic type, whereas curiously, a large fraction of the potential transcription regulation factors appear to be of the bacterial type. To date, little information is available on these predicted regulators and on the intriguing interplay that necessarily has to occur with the transcription machinery. Here, we focus on Sa-lrp of the extremely thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, encoding an archaeal homologue of the Escherichia coli leucine-responsive regulatory protein Lrp, a global transcriptional regulator and genome organizer. Sa-lrp was shown to produce a monocistronic mRNA that was more abundant in the stationary-growth phase and produced in smaller amounts in complex medium, this down regulation being leucine independent. We report on Sa-Lrp protein purification from S. acidocaldarius and from recombinant E. coli, both identified by N-terminal amino acid sequence determination. Recombinant Sa-Lrp was shown to be homotetrameric and to bind to its own control region; this binding proved to be leucine independent and was stimulated at high temperatures. Interference binding experiments suggested an important role for minor groove recognition in the Sa-Lrp-DNA complex formation, and mutant analysis indicated the importance for DNA binding of the potential helix-turn-helix motif present at the N terminus of Sa-Lrp. The DNA-binding capacity of purified Sa-Lrp was found to be more resistant to irreversible heat inactivation in the presence of L-leucine, suggesting a potential physiological role of the amino acid as a cofactor.

Ss‐LrpB, a novel Lrp‐like regulator of Sulfolobus solfataricus P2, binds cooperatively to three conserved targets in its own control region

Ss‐LrpB, a novel Lrp‐like DNA‐binding protein from the hyperthermophilic crenarchaeon Sulfolobus solfataricus, was shown to bind cooperatively to three regularly spaced targets in its own control region, with as consensus the 15 bp palindrome 5′‐TTGYAW WWWWTRCAA‐3′. Binding to the border sites occurred with high affinity; the target in the middle proved to be a low affinity site which is stably bound only when both flanking sites are occupied. Ss‐LrpB contacts two major groove segments and the intervening minor groove of each site, all aligned on one face of the helix. The operator shows intrinsic bending and is increasingly deformed upon binding of Ss‐LrpB to one, two and three targets. Complex formation relies therefore on DNA conformability, protein–DNA and protein–protein contacts. Mobility‐shift assays and in gel footprinting indicate that Ss‐LrpB and the transcription factors TATA‐box binding protein (TBP) and transcription factor B (TFB) can bind simultaneously to the control region. Based on these findings we present a model for the construction of the higher order nucleoprotein complexes and a hypothesis for the autoregulatory process. The latter is based on the concentration‐dependent formation of distinct complexes exhibiting different stoichiometries and conformations, which could positively and negatively affect promoter activity.

Pathways of glutathione degradation in the yeast Saccharomyces cerevisiae

Abstract The degradation of glutathione (GSH) in the yeast Saccharomyces cerevisiae appears to be mediated only by γ-glutamyltranspeptidase and cysteinylglycine dipeptidase. Other enzymes of the γ-glutamyl cycle, γ-glutamyl cyclotransferase and 5-oxo- l -prolinase, are not present in the yeast. In vivo transpeptidation was shown in the presence of a high intracellular level of γ-glutamyltranspeptidase, but only when the de-repressing nitrogen source was a suitable acceptor of the transferase reaction. In contrast, when the de-repressing source was not an acceptor of the transferase reaction (e.g. urea), only glutamate was detected. Intracellular GSH is virtually inert when the level of γ-glutamyltranspeptidase is low. Possible roles for in vivo transpeptidation are discussed.

The highly thermostable arginine repressor of Bacillus stearothermophilus: gene cloning and repressor–operator interactions

We report here the cloning of the arginine repressor gene argR of Bacillus stearothermophilus and the characterization and purification to homogeneity of its product. The deduced amino acid sequence of the 16.8‐kDa ArgR subunit shares 72% identity with its mesophilic homologue AhrC of Bacilus subtilis. Sequence analysis of B. stearothermophilus ArgR and comparisons with mesophilic arginine repressors suggest that the thermostable repressor comprises an N‐terminal DNA‐binding and a C‐terminal oligomerization and arginine‐binding region. B. stearothermophilus ArgR has been overexpressed in E. coli and purified as a 48.0‐kDa trimeric protein. The repressor inhibits the expression of a B. stearothermophilus argC–lacZ fusion in E. coli cells. In the presence of arginine, the purified protein binds tightly and specifically to the argC operator, which largely overlaps the argC promoter. The purified B. stearothermophilus repressor proved to be very thermostable with a half‐life of approximately 30 min at 90°C, whereas B. subtilis AhrC was largely inactivated at 65°C. Moreover, ArgR operator complexes were found to be remarkably thermostable and could be formed efficiently at up to 85°C, well above the optimal growth temperature of the moderate thermophile B. stearothermophilus. This pronounced resistance of the repressor–operator complexes to heat treatment suggests that the same type of regulatory mechanism could operate in extreme thermophiles.

Physiology and genetics of carbamoylphosphate synthesis in Escherichia coli K12

Summary76 mutants have been isolated in which the function of the single carbamoylphosphate synthetase of Escherichia coli K 12 is affected. A wide variety of phenotypes have been observed among these mutants, the most typical ones being: requirement for arginine and uracil, arginineless behaviour, sensitivity towards arginine and sensitivity towards uracil. The mutations have been localized by reciprocal transduction and deletion mapping; all are clustered in the same locus, car. The study of carbamoylphosphate synthesizing activities of these mutants and the combination of car mutations in various in vivo as well as in vitro complementation tests lead to the conclusion that car contains two genes: carA, covering the left part of the locus and coding for the “glutamine subunit” of the enzyme; carB, to the right, governing the synthesis of the heavy subunit of the enzyme.

Role of γ-glutamyltranspeptidase in detoxification of xenobiotics in the yeasts Hansenula polymorpha and Saccharomyces cerevisiae

GGT1 gene of the methylotrophic yeast Hansenula polymorpha appears to be a structural and functional homologue of Saccharomyces cerevisiae CIS2/ECM38 gene encoding γ‐glutamyltranspeptidase (γGT). This is confirmed by the absence of the corresponding activity of γGT in the mutant with disrupted GGT1 gene. It was shown that γGT of both H. polymorpha and S. cerevisiae are involved in detoxification of electrophilic xenobiotics, as the corresponding mutants appeared to be defective in the disappearance of the fluorescent vacuolar complex of GSH with xenobiotic bimane and the further diffuse distribution of this complex in the cytosol. We hypothesize that metabolism of electrophilic xenobiotics in the yeasts H. polymorpha and S. cerevisiae occurs through a γGT‐dependent mercapturic acid pathway of GSH‐xenobiotic detoxification, similar to that known for mammalian cells, with cysteine‐xenobiotics and/or N‐acetylcysteine‐xenobiotics as the end products.

Crystal structure of Bacillus subtilis TrmB, the tRNA (m7G46) methyltransferase

The structure of Bacillus subtilis TrmB (BsTrmB), the tRNA (m7G46) methyltransferase, was determined at a resolution of 2.1 Å. This is the first structure of a member of the TrmB family to be determined by X-ray crystallography. It reveals a unique variant of the Rossmann-fold methyltransferase (RFM) structure, with the N-terminal helix folded on the opposite site of the catalytic domain. The architecture of the active site and a computational docking model of BsTrmB in complex with the methyl group donor S-adenosyl-l-methionine and the tRNA substrate provide an explanation for results from mutagenesis studies of an orthologous enzyme from Escherichia coli (EcTrmB). However, unlike EcTrmB, BsTrmB is shown here to be dimeric both in the crystal and in solution. The dimer interface has a hydrophobic core and buries a potassium ion and five water molecules. The evolutionary analysis of the putative interface residues in the TrmB family suggests that homodimerization may be a specific feature of TrmBs from Bacilli, which may represent an early stage of evolution to an obligatory dimer.

High resolution contact probing of the Lrp-like DNA-binding protein Ss-Lrp from the hyperthermoacidophilic crenarchaeote Sulfolobus solfataricus P2.

Ss‐Lrp, from Sulfolobus solfataricus, is an archaeal homologue of the global bacterial regulator Lrp (Leucine‐responsive regulatory protein), which out of all genome‐encoded proteins is most similar to Escherichia coli Lrp (E‐value of 5.6 e−14). The recombinant protein has been purified as a 68 kDa homotetramer. The specific binding of Ss‐Lrp to its own control region is suggestive of negative autoregulation. A high resolution contact map of Ss‐Lrp binding was established by DNase I and hydroxyl radical footprinting, small non‐intercalating groove‐specific ligand‐binding interference, and various base‐specific premodification and base removal binding interference techniques. We show that Ss‐Lrp binds one face of the DNA helix and establishes the most salient contacts with two major groove segments and the intervening minor groove, in a region that overlaps the TATA‐box and BRE promoter elements. Therefore, Ss‐Lrp most likely exerts autoregulation by preventing promoter recognition by TBP and TFB. Moreover, the results demonstrate profound Ss‐Lrp induced structural alterations of sequence stretches flanking the core contact site, and reveal that the deformability of these regions significantly contributes to binding selectivity.

Integration Host Factor (IHF) modulates the expression of the pyrimidine-specific promoter of the carAB operons of Escherichia coli K12 and Salmonella typhimurium LT2

SummaryWe report the identification of Integration Host Factor (IHF) as a new element involved in modulation of P1, the upstream pyrimidine-specific promoter of the Escherichia coli K12 and Salmonella typhimurium carAB operons. Band-shift assays, performed with S-30 extracts of the wild type and a himA, hip double mutant or with purified IHF demonstrate that, in vitro, this factor binds to a region 300 by upstream of the transcription initiation site of P1 in both organisms. This was confirmed by deletion analysis of the target site. DNase I, hydroxyl radical and dimethylsulphate footprinting experiments allowed us to allocate the IHF binding site to a 38 bp, highly A + T-rich stretch, centred around nucleotide −305 upstream of the transcription initiation site. Protein-DNA contacts are apparently spread over a large number of bases and are mainly located in the minor groove of the helix. Measurements of carbamoyl-phosphate synthetase (CPSase) and β-galactosidase specific activities from car-lacZ fusion constructs of wild type or IHF target site mutants introduced into several genetic backgrounds affected in the himA gene or in the pyrimidine-mediated control of P1 (carP6 or pyrH±), or in both, indicate that, in vivo, IHF influences P1 activity as well as its control by pyrimidines. IHF stimulates P1 promoter activity in minimal medium, but increases the repressibility of this promoter by pyrimidines. These antagonistic effects result in a two- to threefold reduction in the repressibility of promoter P 1 by pyrimidines in the absence of IHF binding. IHF thus appears to be required for maximal expression as well as for establishment of full repression. IHF could exert this function by modulating the binding of a pyrimidine-specific regulatory molecule.

CarP, a novel gene regulating the transcription of the carbamoylphosphate synthetase operon of Escherichia coli☆

The carAB operon, encoding carbamoylphosphate synthetase (CPSase; EC 6.3.5.5) is transcribed from two tandem promoters. The upstream promoter (P1) is controlled by pyrimidines and the downstream promoter (P2) is controlled by arginine. We have isolated a new type of constitutive mutation (carP) that specifically affects the control of the pyrimidine-sensitive promoter but does not appear to influence other genes of the pyrimidine pathway. The carP mutation acts in trans and is dominant, which suggests that the carP product is an activator of car transcription. The downstream promoter P2, which is repressed by arginine, overlaps two operator modules characteristic of the arginine regulon. We have isolated two operator-constitutive mutations that specifically affect P2; both map in the upstream ARG box at a strongly conserved position.