Frédéric Colland

The bacteriophage T4 AsiA protein: a molecular switch for sigma 70-dependent promoters

The AsiA protein, encoded by bacteriophage T4, inhibits Eσ70‐dependent transcription at bacterial and early‐phage promoters. We demonstrate that the inhibitory action of AsiA involves interference with the recognition of the −35 consensus promoter sequence by host RNA polymerase. In vitro experiments were performed with a C‐terminally labelled sigma factor that is competent for functional holoenzyme reconstitution. By protease and hydroxyl radical protein footprinting, we show that AsiA binds region 4.2 of σ70, which recognizes the −35 sequence. Direct interference with the recognition of the promoter at this locus is supported by two parallel experiments. The stationary‐phase sigma factor containing holoenzyme, which can initiate transcription at promoters devoid of a −35 region, is insensitive to AsiA inhibition. The recognition of a galP1 promoter by Eσ70 is not affected by the presence of AsiA. Therefore, we conclude that AsiA inhibits transcription from Escherichia coli and T4 early promoters by counteracting the recognition of region 4.2 of σ70 with the −35 hexamer.

Identification of the Helicobacter pylori anti‐σ28 factor

Flagellar motility is essential for colonization of the human gastric mucosa by Helicobacter pylori. The flagellar filament is composed of two subunits, FlaA and FlaB. Transcription of the genes encoding these proteins is controlled by the σ28 and σ54 factors of RNA polymerase respectively. The expression of flagellar genes is regulated, but no σ28‐specific effector was identified. It was also unclear whether H. pylori possessed a checkpoint for flagellar synthesis, and no gene encoding an anti‐σ28 factor, FlgM, could be identified by sequence similarity searches. To investigate the σ28‐dependent regulation, a new approach based on genomic data was used. Two‐hybrid screening with the H. pylori proteins identified a protein of unknown function (HP1122) interacting with the σ28 factor and defined the C‐terminal part of HP1122 (residues 48–76) as the interaction domain. HP1122 interacts with region 4 of σ28 and prevents its association with the β‐region of H. pylori RNA polymerase. Thus, HP1122 presented the characteristics of an anti‐σ28 factor. This was confirmed in H. pylori by RNA dot‐blot hybridization and electron microscopy. The level of σ28‐dependent flaA transcription was higher in a HP1122‐deficient strain and was decreased by the overproduction of HP1122. The overproduction of HP1122 also resulted in H. pylori cells with highly truncated flagella. These results demonstrate that HP1122 is the H. pylori anti‐σ28 factor, FlgM, a major regulator of flagellum assembly. Potential anti‐σ28 factors were identified in Campylobacter jejuni, Pseudomonas aeruginosa and Thermotoga maritima by sequence homology with the C‐terminal region of HP1122.

σ factor selectivity of Escherichia coli RNA polymerase: role for CRP, IHF and Lrp transcription factors

osmY is a stationary phase‐induced and osmotically regulated gene in Escherichia coli that requires the stationary phase RNA polymerase (EσS) for in vivo expression. We show here that the major RNA polymerase, Eσ70, also transcribes osmY in vitro and, depending on genetic background, even in vivo. The cAMP receptor protein (CRP) bound to cAMP, the leucine‐responsive regulatory protein (Lrp) and the integration host factor (IHF) inhibit transcription initiation at the osmY promoter. The binding site for CRP is centred at −12.5 from the transcription start site, whereas Lrp covers the whole promoter region. The site for IHF maps in the −90 region. By mobility shift assay, permanganate reactivity and in vitro transcription experiments, we show that repression is much stronger with Eσ70 than with EσS holoenzyme. We conclude that CRP, Lrp and IHF inhibit open complex formation more efficiently with Eσ70 than with EσS. This different ability of the two holoenzymes to interact productively with promoters once assembled in complex nucleoprotein structures may be a crucial factor in generating σS selectivity in vivo.

Development of Inducible Systems To Engineer Conditional Mutants of Essential Genes of Helicobacter pylori

ABSTRACT The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacIq-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacIq repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring β-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-β-d-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds.

Synthesis and Biological Evaluation of 9-Oxo-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile Analogues as Potential Inhibitors of Deubiquitinating Enzymes

High‐throughput screening highlighted 9‐oxo‐9H‐indeno[1,2‐b]pyrazine‐2,3‐dicarbonitrile (1) as an active inhibitor of ubiquitin‐specific proteases (USPs), a family of hydrolytic enzymes involved in the removal of ubiquitin from protein substrates. The chemical behavior of compound 1 was examined. Moreover, the synthesis and in vitro evaluation of new compounds, analogues of 1, led to the identification of potent and selective inhibitors of the deubiquitinating enzyme USP8.

Positioning of σS, the stationary phase σ factor, in Escherichia coli RNA polymerase–promoter open complexes

The σS subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and is required for promoter recognition of many stationary phase genes. We have analysed open complexes of EσS RNA polymerase, using σS derivatives carrying single cysteine residues at nine different positions to which the reagent FeBABE has been tethered. All holoenzymes but one formed transcriptionally active open complexes at three different promoters (osmY, galP1 and lacUV5). The chemical nuclease FeBABE can cleave DNA in proximity to the chelate. The overall cutting pattern of EσS open complexes does not depend on the nature of the promoter and is similar to that obtained with Eσ70, but extends towards the downstream part of the promoter. The strongest cleavages are observed with FeBABE positioned on cysteines in regions 2.2 to 3.1. In contrast to σ70, region 2.1 of σS appears to be far from DNA. Region 4.2 of σS appears less accessible than its counterpart in σ70 and FeBABE positioned in the turn of the helix–turn–helix (HTH) motif in region 4.2 reacts only weakly with the −35 promoter element. This provides a structural basis for the minor role of the −35 sequence in σS‐dependent promoter recognition.

The interaction between sigmaS, the stationary phase sigma factor, and the core enzyme of Escherichia coli RNA polymerase.

Background: The RNA polymerase holoenzyme of Escherichia coli is composed of a core enzyme (subunit structure α2ββ′) associated with one of the σ subunits, required for promoter recognition. Different σ factors compete for core binding. Among the seven σ factors present in E. coli, σ70 controls gene transcription during the exponential phase, whereas σS regulates the transcription of genes in the stationary phase or in response to different stresses. Using labelled σS and σ70, we compared the affinities of both σ factors for core binding and investigated the structural changes in the different subunits involved in the formation of the holoenzymes.

Identification of PCTA, a TGIF antagonist that promotes PML function in TGF‐β signalling

The TGIF homoeodomain protein functions as an important negative regulator in the TGF‐β signalling pathway. The inhibitory function of TGIF is executed in part through its ability to sequester the tumour suppressor cytoplasmic promyelocytic leukaemia (cPML) in the nucleus, thereby preventing the phosphorylation of Smad2 by the activated TGF‐β type I receptor. Here, we report on the identification of PCTA (PML competitor for TGIF association), a TGIF antagonist that promotes TGF‐β‐induced transcriptional and cytostatic responses. We provide evidence that PCTA functions in TGF‐β signalling by relieving the suppression of Smad2 phosphorylation by TGIF. Furthermore, we demonstrate that PCTA selectively competes with cPML for TGIF association, resulting in the accumulation of cPML in the cytoplasm, where it associates with SARA and coordinates the access of Smad2 for phosphorylation by the activated TGF‐β type I receptor. Thus, our findings on the mode of action of PCTA provide new and important insights into the molecular mechanism underlying the antagonistic interplay between TGIF and cPML in the TGF‐β signalling network.

Characterization of the elongasome core PBP2 : MreC complex of Helicobacter pylori.

The definition of bacterial cell shape is a complex process requiring the participation of multiple components of an intricate macromolecular machinery. We aimed at characterizing the determinants involved in cell shape of the helical bacterium Helicobacter pylori. Using a yeast two‐hybrid screen with the key cell elongation protein PBP2 as bait, we identified an interaction between PBP2 and MreC. The minimal region of MreC required for this interaction ranges from amino acids 116 to 226. Using recombinant proteins, we showed by affinity and size exclusion chromatographies and surface plasmon resonance that PBP2 and MreC form a stable complex. In vivo, the two proteins display a similar spatial localization and their complex has an apparent 1:1 stoichiometry; these results were confirmed in vitro by analytical ultracentrifugation and chemical cross‐linking. Small angle X‐ray scattering analyses of the PBP2 : MreC complex suggest that MreC interacts directly with the C‐terminal region of PBP2. Depletion of either PBP2 or MreC leads to transition into spherical cells that lose viability. Finally, the specific expression in trans of the minimal interacting domain of MreC with PBP2 in the periplasmic space leads to cell rounding, suggesting that the PBP2/MreC complex formation in vivo is essential for cell morphology.

Functional interaction between the ubiquitin-specific protease 25 and the SYK tyrosine kinase.

The SYK non-receptor tyrosine kinase is a key effector of immune receptors signaling in hematopoietic cells. Here, we identified and characterized a novel interaction between SYK and the ubiquitin-specific protease 25 (USP25). We report that the second SH2 domain of SYK physically interacts with a tyrosine-rich, C-terminal region of USP25 independently of tyrosine phosphorylation. Moreover, we showed that SYK specifically phosphorylates USP25 and alters its cellular levels. This study thus uncovers a new SYK substrate and reveals a novel SYK function, namely the regulation of USP25 cellular levels.