Title Participants Abstract "Evaluation of different strategies to produce Vibrio cholerae ParE2 toxin" "Yana Girardin, Margot Galle, Yaël Vanden Abeele, Henri De Greve, Remy Loris" "Toxin-antitoxin (TA) systems are small operons that are omnipresent in bacteria and archaea with suggested roles in stabilization of mobile genetic elements, bacteriophage protection, stress response and possibly persister formation. A major bottleneck in the study of TA toxins is the production of sufficient amounts of well-folded, functional protein. Here we examinate alternative approaches for obtaining the VcParE2 toxin from Vibrio cholerae. VcParE2 can be successfully produced via bacterial expression in presence of its cognate antitoxin VcParD2, followed by on-column unfolding and refolding. Alternatively, the toxin can be expressed in Spodoptera frugiperda (Sf9) insect cells. The latter requires disruption of the VcparE2 gene via introduction of an insect cell intron. Both methods provide protein with similar structural and functional characteristics." "Structure-Based Design and Synthesis of Stapled 10Panx1 Analogues for Use in Cardiovascular Inflammatory Diseases" "Arthur Lamouroux, Malaury Tournier, Debora Iaculli, Anne Caufriez, Olga M Rusiecka, Charlotte Martin, Viviane Bes, Laureano E Carpio, Yana Girardin, Remy Loris, Andres Tabernilla Garcia, Filippo Molica, Rafael Gozalbes, María Dolores Mayán Santos, Mathieu Vinken, Brenda R. Kwak, Steven Ballet" "Following a rational design, a series of macrocyclic (""stapled"") peptidomimetics of 10Panx1, the most established peptide inhibitor of Pannexin1 (Panx1) channels, were developed and synthesized. Two macrocyclic analogues SBL-PX1-42 and SBL-PX1-44 outperformed the linear native peptide. During in vitro adenosine triphosphate (ATP) release and Yo-Pro-1 uptake assays in a Panx1-expressing tumor cell line, both compounds were revealed to be promising bidirectional inhibitors of Panx1 channel function, able to induce a two-fold inhibition, as compared to the native 10Panx1 sequence. The introduction of triazole-based cross-links within the peptide backbones increased helical content and enhanced in vitro proteolytic stability in human plasma (>30-fold longer half-lives, compared to 10Panx1). In adhesion assays, a ""double-stapled"" peptide, SBL-PX1-206 inhibited ATP release from endothelial cells, thereby efficiently reducing THP-1 monocyte adhesion to a TNF-α-activated endothelial monolayer and making it a promising candidate for future in vivo investigations in animal models of cardiovascular inflammatory disease." "PrsQ2, a small periplasmic protein involved in increased uranium resistance in the bacterium Cupriavidus metallidurans" "Kristel Mijnendonckx, Tom Rogiers, Francisco Javier Giménez del Rey, Mohamed L. Merroun, Adam Williamson, Md Muntasir Ali, Daniel Charlier, Natalie Leys, Nico Boon, Rob Van Houdt" "Uranium contamination is a widespread problem caused by natural and anthropogenic activities. Although microorganisms thrive in uranium-contaminated environments, little is known about the actual molecular mechanisms mediating uranium resistance. Here, we investigated the resistance mechanisms driving the adaptation of Cupriavidus metallidurans NA4 to toxic uranium concentrations. We selected a spontaneous mutant able to grow in the presence of 1 mM uranyl nitrate compared to 250 µM for the parental strain. The increased uranium resistance was acquired via the formation of periplasmic uranium-phosphate precipitates facilitated by the increased expression of a genus-specific small periplasmic protein, PrsQ2, regulated as non-cognate target of the CzcS2-CzcR2 two-component system. This study shows that bacteria can adapt to toxic uranium concentrations and explicates the complete genetic circuit behind the adaptation." "Determination of RNA Structure with In Vitro SHAPE Experiments" "Rani Baes, Daniel Charlier, Eveline Peeters" "The structure of an RNA molecule is often critical for its correct functioning, post-transcriptional regulation, and/or translation. Predicting RNA secondary structures with in silico tools is relatively straightforward with the large array of software and webservers available. However, for long RNAs and RNA at high temperatures, in silico predictions are less accurate and require experimental validation. To this end, a variety of structural probing reagents are commonly used, both for in vitro and in vivo mapping of RNA structure. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments make use of a nonbase-specific modifying reagent, acylating all conformationally flexible (mainly single-stranded or unpaired) nucleotides and have been employed both for in vitro and in vivo modification of RNA. Here, we describe a protocol for an in vitro SHAPE experiment, starting from in vitro transcribed RNA using a T7 polymerase system. This RNA is folded and subsequently modified in vitro with the SHAPE-reagent N-methyl isatoic anhydride (NMIA). Primer extension employing a radioactive 32P-labeled primer that binds the RNA downstream of the structure of interest will generate cDNA until the reverse transcriptase enzyme is halted by the introduced SHAPE modifications. Denaturing acrylamide gel electrophoresis of the pool of 32P-labeled cDNAs and the corresponding sequencing ladders, followed by autoradiography, will expose these stops in reverse transcription (RT) and will therefore enable to identify single-stranded nucleotides in the RNA of interest. These RT stops and NMIA-modification efficiencies can be quantified with ImageJ software and can be used to validate or increase the accuracy of RNA secondary structure predictions." "In Vitro Transcription Assay for Archaea Belonging to Sulfolobales" "David Sybers, Daniel Charlier, Eveline Peeters" "Archaeal transcription and its regulation are characterized by a mosaic of eukaryotic and bacterial features. Molecular analysis of the functioning of the archaeal RNA polymerase, basal transcription factors, and specific promoter-containing DNA templates allows to unravel the mechanisms of transcription regulation in archaea. In vitro transcription is a technique that allows the study of this process in a simplified and controlled environment less complex than the archaeal cell. In this chapter, we present an in vitro transcription methodology for the study of transcription in Sulfolobales. It is described how to purify the RNA polymerase and the basal transcription factors TATA-binding protein and transcription factor B of Saccharolobus solfataricus and how to perform in vitro transcription reactions and transcript detection. Application of this protocol for other archaeal species could require minor modifications to protein overexpression and purification conditions." "Chemical Protection and Premodification-Binding Interference for the Identification of Phosphate and Base-Specific Contacts in Protein-DNA Complexes" "Daniel Charlier" "The specificity and strength of protein-DNA complexes rely on tight interactions between side- and main chain atoms of amino acid residues and phosphates, sugars, and base-specific groups. Various (in-gel) footprinting methods (for more information, see Chapter 11 ) allow the identification of the global-binding region but do not provide details on the contribution to complex formation of individual sequence-specific constituents of the DNA-binding site. Here, we describe how various chemicals can be used to randomly and sparingly modify specific bases or phosphates and allow the identification of those residues that are specifically protected against modification upon protein binding (protection studies) or interfere with complex formation when modified or removed prior to protein binding (premodification-binding interference). Each one of these complementary approaches has its advantages and shortcomings and results have to be interpreted with caution, having in mind the precise chemistry of the modification. However, used in combination, these methods provide an accurate and high-resolution image of the protein-DNA contacts." "DNA-Binding and Transcription Activation by Unphosphorylated Response Regulator AgrR From Cupriavidus metallidurans Involved in Silver Resistance" "Md Muntasir Ali, Ann Provoost, Kristel Mijnendonckx, Rob Van Houdt, Daniel Charlier" "Even though silver and silver nanoparticles at low concentrations are considered safe for human health, their steadily increasing use and associated release in nature is not without risk since it may result in the selection of silver-resistant microorganisms, thus impeding the utilization of silver as antimicrobial agent. Furthermore, increased resistance to metals may be accompanied by increased antibiotic resistance. Inactivation of the histidine kinase and concomitant upregulation of the cognate response regulator (RR) of the AgrRS two-component system was previously shown to play an important role in the increased silver resistance of laboratory adapted mutants of Cupriavidus metallidurans. However, binding of AgrR, a member of the OmpR/PhoP family of RRs with a conserved phosphoreceiver aspartate residue, to potential target promoters has never been demonstrated. Here we identify differentially expressed genes in the silver-resistant mutant NA4S in non-selective conditions by RNA-seq and demonstrate sequence-specific binding of AgrR to six selected promoter regions of upregulated genes and divergent operons. We delimit binding sites by DNase I and in gel copper-phenanthroline footprinting of AgrR-DNA complexes, and establish a high resolution base-specific contact map of AgrR-DNA interactions using premodification binding interference techniques. We identified a 16-bp core AgrR binding site (AgrR box) arranged as an imperfect inverted repeat of 6 bp (ATTACA) separated by 4 bp variable in sequence (6-4-6). AgrR interacts with two major groove segments and the intervening minor groove, all aligned on one face of the helix. Furthermore, an additional in phase imperfect direct repeat of the half-site may be observed slightly up and/or downstream of the inverted repeat at some operators. Mutant studies indicated that both inverted and direct repeats contribute to AgrR binding in vitro and AgrR-mediated activation in vivo. From the position of the AgrR box it appears that AgrR may act as a Type II activator for most investigated promoters, including positive autoregulation. Furthermore, we show in vitro binding and in vivo activation with dephosphomimetic AgrR mutant D51A, indicating that unphosphorylated AgrR is the active form of the RR in mutant NA4S." "Regulatory mechanisms behind the activities of bacterial HEPN ribonuclease RnlA and ParE2 gyrase poison" "Gabriela Garcia Rodriguez" "The staggering biodiversity of microbes stands as a testament to the long evolutionary history of the oldest lifeforms on Earth. We are only now beginning to unravel, as technological breakthroughs open new horizons for science, the magnificent molecular machineries that constitute the basis of microbial structures and hence function. Horizontal gene transfer events in prokaryotic cells, as well as the arms race between bacteriophages and their hosts, assisted in the wide spread of molecular weapons that target many vital cell processes and their counteracting entities that provide a natural defense. These prokaryotic toxins are ubiquitous and abundant in the genomes of almost all species, and yet they exist under tight regulation to protect the cell against self-harm under homeostasis. The molecular mechanisms by which prokaryotes accomplish this task take place at many levels of regulation, i.e., direct inhibition of protein activity by protein-protein interactions, transcriptional regulation by repressor binding to DNA promoter to abrogate the activity of RNA polymerase, etc. By looking at the molecular level into the activity and regulation of the Escherichia coli RnlA-RnlB toxin-antitoxin (TA) system I have unveiled a novel regulatory mechanism exerted on the toxin RnlA by its cognate chromosomic antitoxin RnlB. This toxin functions as an endoribonuclease first reported as the host-encoded RNase responsible for the T4 phage late gene silencing when a dmd- mutant of the phage infects the cells, halting its propagation. Here I show that RnlA is a HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain) ribonuclease with broad sequence specificity in vitro. Comparative bioinformatics led to the identification of the catalytic residues in RnlA, which upon mutation into alanine residues permitted the mapping of the active site of this enzyme. The drastic conformational changes on the structure of RnlA exerted by its antitoxin RnlB and detected by X-ray crystallography and SAXS, constitute the basis for its inhibition and the first example of a mechanism involving quaternary structural changes halting the activity of a ribonuclease known to the scientific community." "Regulation of arginine biosynthesis, catabolism and transport in Escherichia coli" "Daniel Charlier, Indra Bervoets" "Already very early, the study of microbial arginine biosynthesis and its regulation contributed significantly to the development of new ideas and concepts. Hence, the term ""repression"" was proposed by Vogel (The chemical basis of heredity, The John Hopkins Press, Baltimore, 1957) (in opposition to induction) to describe the relative decrease in acetylornithinase production in Escherichia coli cells upon arginine supplementation, whereas the term ""regulon"" was coined by Maas and Clark (J Mol Biol 8:365-370, 1964) for the ensemble of arginine biosynthetic genes dispersed over the E. coli chromosome but all subjected to regulation by the trans-acting argR gene product. Since then, unraveling of the molecular mechanisms controlling arginine biosynthesis, catabolism, and transport in and out the cell, have revealed moonlighting activities of enzymes and transcriptional regulators that generate unexpected interconnections between at first sight totally unrelated cellular processes, and have continued to replenish scientific knowledge and stimulated creative thinking. Furthermore, arginine is much more than just a common amino acid for protein synthesis. It may also be used as sole source of nitrogen by E. coli and a source of nitrogen, carbon and energy by many other bacteria. It is a substrate for the synthesis of polyamines, and important for the extreme acid resistance of E. coli. Furthermore, the guanidino group of arginine is well suited to engage in multiple interactions involving hydrogen bonds and ionic interactions with proteins and nucleic acids. Here, we combine major historical discoveries with current state of the art knowledge on arginine biosynthesis, catabolism and transport, and especially the regulation of these processes in E. coli, with reference to other microorganisms." "Competitive Repression of the artPIQM Operon for Arginine and Ornithine Transport by Arginine Repressor and Leucine-Responsive Regulatory Protein in Escherichia coli" "Oscar Torres Montaguth, Indra Bervoets, Eveline Peeters, Daniel Charlier" "Two out of the three major uptake systems for arginine in Escherichia coli are encoded by the artJ-artPIQM gene cluster. ArtJ is the high-affinity periplasmic arginine-specific binding protein (ArgBP-I), whereas artI encodes the arginine and ornithine periplasmic binding protein (AO). Both ArtJ and ArtI are supposed to combine with the inner membrane-associated ArtQMP2 transport complex of the ATP-binding cassette-type (ABC). Transcription of artJ is repressed by arginine repressor (ArgR) and the artPIQM operon is regulated by the transcriptional regulators ArgR and Leucine-responsive regulatory protein (Lrp). Whereas repression by ArgR requires arginine as corepressor, repression of P artP by Lrp is partially counteracted by leucine, its major effector molecule. We demonstrate that binding of dimeric Lrp to the artP control region generates four complexes with a distinct migration velocity, and that leucine has an effect on both global binding affinity and cooperativity in the binding. We identify the binding sites for Lrp in the artP control region, reveal interferences in the binding of ArgR and Lrp in vitro and demonstrate that the two transcription factors act as competitive repressors in vivo, each one being a more potent regulator in the absence of the other. This competitive behavior may be explained by the partial steric overlap of their respective binding sites. Furthermore, we demonstrate ArgR binding to an unusual position in the control region of the lrp gene, downstream of the transcription initiation site. From this unusual position for an ArgR-specific operator, ArgR has little direct effect on lrp expression, but interferes with the negative leucine-sensitive autoregulation exerted by Lrp. Direct arginine and ArgR-dependent repression of lrp could be observed with a 25-bp deletion mutant, in which the ArgR binding site was artificially moved to a position immediately downstream of the lrp transcription initiation site. This finding is reminiscent of a previous observation made for the carAB operon encoding carbamoylphosphate synthase, where ArgR bound in overlap with the downstream promoter P2 does not block transcription initiated 67 bp upstream at the P1 promoter, and further supports the hypothesis that ArgR does not act as an efficient roadblock."