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1 Center for Protein Engineering CIP and 2014 Activity Reports 2013 and 2014 Activity Reports Center for Protein Engineering Université de Liège Institut de Chimie B6a Sart Tilman B4000 Liège Belgium University of Liège

2 Model of peptidoglycan transpeptidation based on the crystal structure of Escherichia coli PBP3. Author: Eric Sauvage

3 CONTENTS INTRODUCTION 3 RESEARCH GROUPS 5 EXPERTISES 7 MAJOR EQUIPMENTS 9 HIGHLIGHTS OF THE YEAR 12 SCIENTIFIC SERVICES 32 SCIENTIFIC PRODUCTION 37 - Awards 37 - Invited speakers 38 - Oral presentations 39 - Patents 41 - PhD theses 42 - Publications 44 - Symposia 52 EDUCATION 53 - Academic courses 53 - Trainees and students 58 - General public activities 65 INTERNATIONAL EXCHANGES 68 - Postdocs In 68 - Collaborations 72 - Stays in other institutions 79 - Visitors 80 FUNDING 81 MISSIONS OF EXPERTISE 86 COMMITTEES AND SOCIETIES 87 COMPOSITION OF THE CENTER 88

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5 INTRODUCTION The Center for Protein Engineering (Centre d Ingénierie des Protéines CIP) is composed of 6 research teams that comprise 85 members. These teams work in close collaboration to apply their collective knowledge of advanced structural biology, biochemistry, genomics, molecular imaging and microbiology to bacteria and plant models. CIP members are currently exploring the molecular and cellular mechanisms involved in bacterial division and cell wall synthesis, bacterial resistance toward antibiotics, production of cryptic bioactive metabolites, metal homeostasis in plants, alternative splicing mechanisms in plants, folding and misfolding of proteins, and adaptation to extreme environments. Truly efficient research requires access to a broad range of technologies, specific state-of-the-art equipments and expertise. The CIP is also dedicated to the development of technological platforms which aim to: 1) meet the needs of the Center s scientists; and 2) maintain our strong commitment to support biomedical and biotechnological research in the industrial sector. With the help of the University, we developed the management system of these platforms and are currently developing their technical capabilities in order to provide a spectrum of services ranging from routine assays to specific and customized demands. Furthermore, motivated by our desire to offer a large set of skills to researchers, we constantly adapt and improve networks with other platforms of our institution or in other research institutes. Our ambition is to maintain access to state-of-the-art techniques and equipments to the CIP members and collaborators by maximizing our expertise and insuring a synergic development between platforms. Despite these exciting developments and opportunities, our structure requires a rigorous day-to-day management and ongoing practical respect of the rules we have collegially discussed and approved. The durability of the CIP depends not only on securing subventions, but more importantly on the willingness of all Center s members to dedicate time and energy to the benefit of all. Dr Dominique Dehareng left the CIP on July 2014 after more than 28 years of research in the Center. Dominique worked from the start in the unit dedicated to quantum chemistry. She developed expertise in the field of Molecular Mechanics and Molecular Dynamics, both theoretical approaches being used for the conformational studies of peptides, proteins and proteinligand complexes. Dominique has trained many students in this difficult field, always with enthusiasm and scientific rigor. For several years she was involved in the development of the Master degree in Bio-informatics and Modelling as secretary and, as such, she acted as an interface between the Faculties of Sciences and Applied Sciences of the University. She was one of the kingpins for the edition of our previous research reports and she also dedicated her time to create the first version of our web site. We were saddened by the death of Georges Dive on June 12 th Georges was one of the founding members of the CIP. He obtained his Master degree in Pharmaceutical Sciences in 1973 and his PhD in In 1980, he was appointed Chercheur Qualifié (Research Associate) by the FNRS, a permanent position that enabled him to develop his own research group in the CIP. This report describes the major activities of the Center during 2013 and We hope that it represents a good testimony to the dedication of its members to the initiation of new research lines and to the success of the older ones Moreno Galleni

6 INTRODUCTION - 4 -

7 RESEARCH GROUPS APPLIED QUANTUM CHEMISTRY AND MODELING Group leader Associate member Dr Georges Dive Dr Dominique Dehareng BACTERIAL DIVERSITY, PHYSIOLOGY AND GENETICS Group leader Permanent scientists Associate members Prof. Bernard Joris Dr Colette Duez Dr Colette Goffin Dr Mohammed Terrak Dr Annick Wilmotte Dr Ana Amoroso Dr Arabela Cuirolo Dr Michaël Delmarcelle Dr Adeline Derouaux Dr Dail Laughinghouse IV Dr Serge Leimanis Dr Samir Olatunji Dr Badrish Soni Mr Patrick Stefanic Mr Olivier Verlaine BIOLOGICAL MACROMOLECULES AND BIOCHEMISTRY Group leader Permanent scientist Associate members Prof. Moreno Galleni Dr Georges Feller Dr Sébastien Rigali Dr Paola Mercuri Dr Jean-Sébastien Sohier - 5 -

8 RESEARCH GROUPS BIOLOGICAL MACROMOLECULE CRYSTALLOGRAPHY Group leader Permanent scientist Associate members Prof. Paulette Charlier Dr Frédéric Kerff Dr Eric Sauvage Dr Meriem El Ghachi ENZYMOLOGY AND PROTEIN FOLDING Group leader Permanent scientist Associate members Prof. André Matagne Dr Mireille Dumoulin Dr Caroline Montagner Dr David Thorn Dr Julie Vandenameele FUNCTIONAL GENOMICS AND PLANT MOLECULAR IMAGING Group leader Permanent scientist Associate members Prof. Patrick Motte Dr Marc Hanikenne Dr Cécile Nouet Dr Sol Schvartzman - 6 -

9 EXPERTISES MOLECULAR BIOLOGY Activity screening Gene cloning in E. coli, Bacillus, Streptomyces and P. pastoris Site-directed mutagenesis Phage display Metagenomics Protein engineering (random mutagenesis, protein design) PROTEIN PRODUCTION In E. coli, Bacillus, Streptomyces, L. lactis, P. pastoris or in environmental strains From ml to 60 L In flasks or fermentors Optimisation of industrial processes 2 H, 13 C, 15 N enrichment for NMR studies Selenomethionyl enrichment for crystallography studies PROTEIN PURIFICATION Classical purification techniques (ion exchange, affinity, hydroxyapatite ) From mg to g HPLC, FPLC, Åkta prime, Åkta explorer, Profinia, Biopilot Membrane protein purification MACROMOLECULE CHARACTERISATION Biochemical characterisation Cellular localization of proteins: - Fluorescence microscopy 2D-DIGE DDGE ELISA EMSA Enzymology: - Steady and transient state kinetics - Stopped-flow & quenched-flow N-terminal sequencing Protein-protein interactions: - Bacterial two hybrids and immunoprecipitation Proteomics Western blot - 7 -

10 EXPERTISES Biophysical characterization Microcalorimetry (DSC and ITC) Dynamic/static light scattering Analysis of peptidoglycan by HPLC Protein stability, folding and aggregation: - Spectroscopy: UV-Vis, fluorescence and circular dichroïsm - Time-resolved spectroscopy X-Ray crystallography: - Crystallogenesis - de novo structure determination - Studies of ligand-protein complexes - 3D structure determination PLANT MOLECULAR IMAGING Plant physiology Plant genetic transformation Molecular imaging Plant genetics and genomics IN SILICO STUDIES Molecular modeling and applied quantum chemistry 16S rrna phylogenetic analysis Prokaryotic regulon predictions: web tool PREDetector (Prokaryotic Regulatory Elements Detector) - 8 -

11 MAJOR EQUIPMENTS GENETIC ENGINEERING AND MOLECULAR BIOLOGY 2 DNA sequencers: 1 ALF model (Pharmacia) 1 Gene Pulser electroporator (Biorad) Several PCR apparatus including: 1 MJ Mini Real Time Quantitative PCR PTC0148 (Biorad) 1 Nanovue (GE Healthcare) GREEN ALGAL CULTURES 1 Versatile environment test chamber (Sanyo) MICROBIAL CULTURES 2 Controlled environment incubator shakers (New Brunswick Scientific) 11 Incubator shakers: five G-25 (New Brunswick Scientific), one 25D (New Brunswick Scientific), one Excella E24 (New Brunswick Scientific), two Innova 44 (New Brunswick Scientific) and two Innova 4330 (New Brunswick Scientific) incubators 1 Gradient Table for crossed gradients of Temperature and Light (Labio chromatography) PLANT CULTURES 4 Climate-controlled chambers (Binder) for plant growth and cell cultures PROTEIN PRODUCTION Eigth fermentors including: one 5 L (Biostat, B. Braun Biotech International), three 10 L (1 Bioflow 3000, New Brunswick scientific and 2 Biostat B plus, Sartorius), two 20 L (Bioflow 4500, New Brunswick scientific) and one 80 L (Bioflow 5000, New Brunswick scientific). 1 Turbidimeter FSC402 (Mettler Toledo) 1 Steam generator Maxi 24 (Ghidini Benvenuto) 1 Microlab Starlet robot (Hamilton) with Hepa Filter PROTEIN PURIFICATION 1 Centrifugation system (SA model, Westfalia) 2 homogenizers: one Panda (GEA Process Technology) and one Emulsiflex-C3 (Avestin, Inc) 2 sonicators: one MSE and one Sonifer B-12 (Branson Sonic Power Company) and one Bioruptor Plus (Diagenode). A range of instruments to perform protein purification at low or high pressure The most remarkable include: 2 Åkta-explorer (10S 2D-LC and 100-Air), 1 Åkta- purifier, 2 Åkta prime and 2 Åkta prime plus (GE Healthcare) 2 LC210 purification systems (Isco) 1 Microlab Star robot (Hamilton) 1 P-6000 Bio-Pilot autosampler with a Unicorn controller (GE Healthcare) 2 Profinia purification systems (Bio-Rad) 1 Tangential filtration system (Sartoflow Alpha, Sartorius) 1 Table top ultracentrifuge model OPTIMA MAX XP equipped with TLA-100, MLA-55 and MLA-55 rotors - 9 -

12 MAJOR EQUIPMENTS ANALYTICAL STUDIES 1 Circular Dichroism spectrophotometer J-810 equipped with a peltier and a 6 cell holder (Jasco) 2 2D-electrophoresis GE Ettan IPGphor3 and Ettan DALTsix apparatus (GE Healthcare) 2 DGGE electrophoresis apparatus (Dcode, Biorad) 1 DynaPro NanoStar DLS/SLS recorder for Dynamic/Static Light Scattering (Wyatt Technology Corporation) 3 Fluorimeters: one SLM-Aminco 8100 (Spectrometric Instruments), one Carry Eclipse (Varian) and one LS50B (Perkin-Elmer) 1 HPLC Alliance with an auto-injection system equipped with 3 detector modes: UV/vis (diode array), fluorescence and refractometer and a fraction collector (Waters) 2 Microcalorimeters: ITC200 (isothermal titration calorimetry) and VP-DSC (differential scanning calorimetry) from GE-MicroCal 4 Microplate readers: one Labsystems Multiskan Multisoft (TechGen International), one PowerwaveX (Bio-Tek instruments, Inc), one Tecan Infinite 200 PRO UV/Vis + fluorescence, one Tecan Infinite 200 PRO UV/Vis + chemiluminescence with injectors. 1 Microplate Strip Washer EL X 50 (Bio-Tek Instruments, Inc) 1 Procise 492 N-terminus amino acid sequencer (Applied Biosystems, Perkin Elmer) 1 Quenched-flow QFM-5 (Bio-Logic) and 1 Quenched-Flow SFM 400 (Bio-Logic) 1 Rapid filtration system (Bio-Logic) Several spectrophotometers Uvikon (Bio-Tek Instruments, Inc,), one spectrophotometer Carry 100 Biomelt (Varian), two UV/Vis spectrophotometers: Specord 50 and 200 (Analytik Jena) 2 Stopped-flow apparatus: MOS 450 with UV/visible light, fluorescence and circular dichroism detection and MPS-51 with UV/visible light and fluorescence (Bio-Logic) 1 Fortebio Octet HTX (Pall) 1 Labchip GXII (Perkin Elmer) CRYSTALLOGRAPHY 1 Cryogenic AD41 cryosystem (Oxford) 4 Graphic-PC stations (Linux) 1 Imaging Plate Marresearch IPmar345 with an Incoatec IµS X-ray microfocus source (Rigaku) 1 Minstrel DT Imager: crystal imaging and protein crystal monitoring systems (Rigaku) 1 TTP Labtech Mosquito Crystallization robot (compact bench-top instrument for nanolitre liquid handling) (Cambridge UK) IMAGING 1 Axio Imager Z1 fluorescent microscope (Zeiss) 1 camera for digitalisation of images and analytical analyses (Olympus) 1 CKX 31 inversed microscope (Olympus) 1 DMLB2 microscope (Leica) 1 Molecular Imager FX system (Biorad) 1 Phase contrast microscope (Reichert) 1 binocular microscope (model SZ-6 PHOTO Bauch & Lomb) 1 binocular microscope with a digital camera (SMZ1500, Nikon) 1 microscope equipped for epifluorescence (Zeiss) 1 confocal inverted microscope (Leica TCS SP2 with Argon et 2 Helium/Neon lasers, AOTF, 3 PMTs + transmitted light and MicroLab software) for FRAP and FRET. 1 State-of-the-art Leica TCS SP5 II multiphoton confocal microscope: this microscope is equipped with an inverted electrophysiology microscope, full set of UV (diode laser with 405 nm excitation) and visible lasers (argon laser with nm excitation and Helium Neon lasers with

13 MAJOR EQUIPMENTS nm), coherent 2-photon infrared, tandem scanner with a resonant scanner (8000Hz). The system has 5 spectral internal detectors two of which for FLIM (Fluorescence Lifetime Imaging) measurements, 1 transmitted light detector, 2 NDD detectors, a Single Molecule Detection (SMD) platform for molecular dynamic analysis, FCS (Fluorescence Correlation Spectroscopy), FCCS (Fluorescence Cross-Correlation Spectroscopy) and FLCS (Fluorescence Lifetime Correlation Spectroscopy) measurements + high resolution and sensitivity digital cameras. 1 stereomicroscope Stemi 2000C, 10*/23 BR FOC ocular (Zeiss) 1 Typhoon Trio + scanner (GE Healthcare) MICROFLUIDIC PLATFORM 1 Microfluidic pump system (model MFCS-Flex Fluidgen) 1 Industrial camera (National instrument) 1 Labview station for real-time control and monitoring (National instrument) 2 Low pulse flow peristaltic pumps (Ismatec) 1 8-channel peristaltic pump (model 205C Watson Marlow) 1 Fastgene LED Transilluminator (Nippon Genetics) MISCELLANEOUS 1 Freeze-dryer (Christ)

14 HIGHLIGHTS OF THE YEAR Characterization of Amylolysin, a Novel Lantibiotic from Bacillus amyloliquefaciens GA1 Arguelles Arias A, Ongena M, Devreese B, Terrak M, Joris B and Fickers P PLoS One Dec 9; 8(12):e83037 BACKGROUND: Lantibiotics are heat-stable peptides characterized by the presence of thioether amino acid lanthionine and methyllanthionine. They are capable to inhibit the growth of Grampositive bacteria, including Listeria monocytogenes, Staphylococcus aureus or Bacillus cereus, the causative agents of food-borne diseases or nosocomial infections. Lantibiotic biosynthetic machinery is encoded by gene cluster composed by a structural gene that codes for a pre-lantibiotic peptide and other genes involved in pre-lantibiotic modifications, regulation, export and immunity. METHODOLOGY/FINDINGS: Bacillus amyloliquefaciens GA1 was found to produce an antimicrobial peptide, named amylolysin, active on an array of Gram-positive bacteria, including methicillin resistant S. aureus. Genome characterization led to the identification of a putative lantibiotic gene cluster that comprises a structural gene (amla) and genes involved in modification (amlm), transport (amlt), regulation (amlkr) and immunity (amlfe). Disruption of amla led to loss of biological activity, confirming thus that the identified gene cluster is related to amylolysin synthesis. MALDI-TOF and LC-MS analysis on purified amylolysin demonstrated that this latter corresponds to a novel lantibiotic not described to date. The ability of amylolysin to interact in vitro with the lipid II, the carrier of peptidoglycan monomers across the cytoplasmic membrane and the presence of a unique modification gene suggest that the identified peptide belongs to the group B lantibiotic. Amylolysin immunity seems to be driven by only two AmlF and AmlE proteins, which is uncommon within the Bacillus genus. CONCLUSION/ SIGNIFICANCE: Apart from mersacidin produced by Bacillus amyloliquefaciens strains Y2 and HIL Y-85, , reports on the synthesis of type B-lantibiotic in this species are scarce. This study reports on a genetic and structural characterization of another representative of the type B lantibiotic in B. amyloliquefaciens. 11 Figures. Left panel: Characterization of the RFB137 mutant. The RP-HPLC chromatogram of concentrated supernatant of GA1 is in redand that of the amla RFB137 mutant is in black. Insert: Antimicrobial activity of concentrated supernatant (10 µl) from GA1 and RFB137 strains on LB agar plates seeded with M. luteus ATCC 9341.Right panel: Interation with lipid. II. (A) Direct interaction of amylolysin with lipid II. Both compounds were incubated in DMSO/1-octanol mixture (60/40 v/v) for 1 hour at room temperature before being subjected to TCL using a butanol/acetic acid/pyridine/water mixture (15/3/12/10, v/v/v/v) as mobile phase. (B) Inhibition of the transglycosylation reaction. Amylolysin, lipid II and glycosyltransferase were incubated for one hour before being subjected to TLC using a mixture of ethanol/chloroform/ammoniac/water (88/48/10/1, v/v/v/v) as mobile phase. [14C] lipid II was detected with a Molecular Imager FX system. Direction of solvent migration during TLC is indicated by the arrow

15 HIGHLIGHTS OF THE YEAR Antimicrobial Activity of Bacillus amyloliquefaciens ANT1 Toward Pathogenic Bacteria and Mold: Effects on Biofilm Arguelles Arias A, Nastro R, Ongena M, Di Costanzo A, Trifuoggi M, Guida M and Fickers P. Probiotics & Antimicro. Prot. 5: , 2013 The intensive use and misuse of antibiotics over the last decades have generated a strong selective pressure for the emergence of multi-resistant strains and nosocomial infections. Biofilm has been demonstrated as a key parameter in spreading infections, especially in hospitals and healthcare units. Therefore, the development of novel anti-biofilm drugs is actually of the upmost importance. Here, the antimicrobial and antibiofilm activities toward pathogenic microorganisms of a set of non-ribosomal synthesized peptides and polyketides isolated from Bacillus amyloliquefaciens ANT1 culture supernatant are presented. A B Figures. Left panel: Metabolite production of B. amyloliquefaciens ANT1, detected by HPLC-ESI mass spectrometry. Right panel: A: Densities (CFU/mL) of planktonic cells in tryptic soy broth for selected pathogen test strains during monoculture (gray bars) or co-culture (white bars) with B. amyloliquefaciens ANT1. Data represent means of three independent experiments. B: Densities (CFU/cm2) of viable cells contained in biofilms of selected pathogen test strains during monoculture (gray bars) or co-culture (white bars) with B. amyloliquefaciens ANT1. Data represent means of three independent experiments

16 HIGHLIGHTS OF THE YEAR Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification Berlemont R., Spee O., Delsaute M., Lara Y., Schuldes J., Simon C., Power P., Daniel R. and Galleni M. Rev. Argent. Microbiol., 45, 3-12, 2013 In order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid prbest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/ß hydrolase subgroup (abh04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenylesters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at ph 6.5 and at temperatures ranging from 38 to 45 C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins. Figure. Neighbor Joining tree of the RBest1 sequence and related sequences. Bootstrap values were calculated for MP /ME / NJ method

17 HIGHLIGHTS OF THE YEAR A nanobody binding to non-amyloidogenic regions of the protein human lysozyme enhances partial unfolding but inhibits amyloidfibril formation De Genst E., Chan P-H., Pardon E., Hsu S-T.D., Kumita J.R, Christodoulou R., Menzer L., Chirgadze D.Y, Robinson C.V., Muyldermans S., Matagne A., Wyns L., Dobson C.M., Dumoulin M. J Phys Chem B., 117, , 2013 We report the effects of the interaction of two camelid antibody fragments, generally called nanobodies, namely cab-hul5 and a stabilized and more aggregation-resistant variant cab-hul5g obtained by protein engineering, on the properties of two amyloidogenic variants of human lysozyme, I56T and D67H, whose deposition in vital organs including the liver, kidney, and spleen is associated with a familial non-neuropathic systemic amyloidosis. Both NMR spectroscopy and X-ray crystallographic studies reveal that cab-hul5 binds to the α-domain, one of the two lobes of the native lysozyme structure. The binding of cab-hul5/cab-hul5g strongly inhibits fibril formation by the amyloidogenic variants; it does not, however, suppress the locally transient cooperative unfolding transitions, characteristic of these variants, in which the β-domain and the C-helix unfold and which represents key early intermediate species in the formation of amyloid fibrils. Therefore, unlike two other nanobodies previously described, cab-hul5/cab-hul5g does not inhibit fibril formation via the restoration of the global cooperativity of the native structure of the lysozyme variants to that characteristic of the wild-type protein. Instead, it inhibits a subsequent step in the assembly of the fibrils, involving the unfolding and structural reorganization of the α-domain. These results show that nanobodies can protect against the formation of pathogenic aggregates at different stages in the structural transition of a protein from the soluble native state into amyloid fibrils, illustrating their value as structural probes to study the molecular mechanisms of amyloid fibril formation. Combined with their amenability to protein engineering techniques to improve their stability and solubility, these findings support the suggestion that nanobodies can potentially be developed as therapeutics to combat protein misfolding diseases. Figure: Possible mechanism for lysozyme fibril formation and its inhibition by nanobodies. Partially unfolded intermediate species of the protein (I) in which the C helix and the β-domain are cooperatively unfolded self-associate through the unfolded regions to initiate oligomer formation. This provides a template for the further aggregation of the protein and for the development of the stable, mainly β- sheet, core structure of the protofilaments within an amyloid fibril. The binding of cab-hul5 to the loop connecting the A and B helices is likely to inhibit the formation of the first intermolecular interaction (1) or the unfolding and reorganization of the regions of the α-domain that are native-like in the amyloidogenic intermediate (2) but are disorganized in the fibrils. The stage at which aggregation is inhibited by the two nanobodies studied previously, cab- HuL6 and cab-hul22, is also shown for comparison

18 HIGHLIGHTS OF THE YEAR Three-dimensional structure of RBcel1, psychrotolerant family GH5 endoglucanase a metagenome-derived Delsaute M., Berlemont R., Dehareng D., Van Elder D., Galleni M., and C. Bauvois Acta Cryst. F69, [ doi: /s ], 2013 RBcel1 is an endoglucanase belonging to glycoside hydrolase family 5 subfamily 5 (GH5_5) that was recently identified from a soil metagenome library from the Antarctic. Unlike its closest structural homologue (Cel5A from Thermoascus aurantiacus), this enzyme was reported to be able to catalyze transglycosylation reactions and has putatively been implicated in the bacterial cellulose-synthesis process. Here, the structure of RBcel1 at 1.4 Å resolution, solved by molecular replacement, is reported. The structure and putative substrate-binding site are described and compared with those of other GH5_5 subfamily members. PDB reference: 4ee9 Figure. Overall structure and active site of RBcel1. (a) Aminoacid sequence of RBcel1. Secondary structures are shown above the sequence. (b) Ribbon stereo diagram showing the eightfold β/α barrel. (c) Stereoview of the molecular surface of RBcel1 showing the hypothetical 2, -1, +1 and +2 sugar-binding subsite positions. β-strands, α-helices and loops are shown in orange, blue and grey, respectively. The noncanonical elements of secondary structure are shown in green. The disulfide bond (dis) is shown in pink. Side chains of the active-site residues are represented as stick models and are coloured according to the amino-acid sequence in (a)

19 HIGHLIGHTS OF THE YEAR The Proline-Rich Motif of the proder p 3 Allergen Propeptide Is Crucial for Protease-Protease Interaction Dumez ME., Herman J., Campisi V., Bouaziz A., Rosu F., Luxen A., Vandenberghe I., de Pauw E., Frère JM., Matagne A., Chevigné A., and Galleni M. PLOSOne. DOI: /journal.pone , 2013 The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific inhibition of the enzyme. The propeptide of the house dust mite allergen Der p 3, NPILPASPNAT, contains a proline-rich motif (PRM), which is unusual for a trypsin-like protease. By truncating the propeptide or replacing one or all of the prolines in the non-glycosylated zymogen with alanine(s), we demonstrated that the full-length propeptide is not required for correct folding and thermal stability and that the PRM is important for the resistance of proder p 3 to undesired proteolysis when the protein is expressed in Pichia pastoris. Additionally, we followed the maturation time course of proder p 3 by coupling a quenched-flow assay to mass spectrometry analysis. This approach allowed to monitor the evolution of the different species and to determine the steady-state kinetic parameters for activation of the zymogen by the major allergen Der p 1. This experiment demonstrated that prolines 5 and 8 are crucial for proder p 3-Der p 1 interaction and for activation of the zymogen. Figure. Continuous mass spectrometry assay for proder p 3 maturation by Der p 1. Electrospray mass spectra (positive ion mode) for 16 µm proder p 3 activation by 0.16 µm Der p 1 in 25 mm ammonium acetate, ph 7.4, containing 1 mm DTT, obtained after 5 min (A) and 40 min (B). ProD, D and prop refer to proder p 3, Der p 3 and propeptide, respectively. C: The decrease in the proder p 3 and the increase in the Der p 3 relative concentrations were calculated as described in Material and Methods

20 HIGHLIGHTS OF THE YEAR Late Holocene changes in cyanobacterial community structure in maritime Antarctic lakes R. Fernandez-Carazo, E. Verleyen, D.A. Hodgson, S.J. Roberts, K. Waleron, W. Vyverman and A. Wilmotte J. Paleolimnol., 50, 15-31, 2013 Despite the dominance of cyanobacteria in polar freshwater aquatic ecosystems, little is known about their past biodiversity and response to climate and environmental changes. We explored the use of light microscopy of microfossils, high performance liquid chromatography of the fossil pigment composition and denaturing gradient gel electrophoresis of fossil 16S rrna genes to study past and present-day differences in cyanobacterial community structure in response to climate changes in two adjacent maritime Antarctic lakes (Beak-1 and Beak-2) with contrasting depths (4 and 26 m) and light climates. Light microscopy was of limited use because of degradation of cell structures. Fossil cyanobacterial pigment concentrations were below the detection limits of our method in several sediment samples in the deep lake, but abundant and diverse in the sediment core from the shallow pond, probably as a consequence of increased light availability and/or a more diverse and abundant benthic cyanobacterial flora. Total carotenoid and chlorophyll concentrations were highest in both lakes between ca. 2,950 and 1,800 cal yr BP, which coincides with the late Holocene climate optimum recognised elsewhere in maritime Antarctica. Cyanobacterial molecular diversity was higher in the top few centimeters of the sediments in both lakes. In deeper sediments, the taxonomic turnover of cyanobacteria appeared to be relatively small in response to past climate anomalies in both lakes, underscoring the broad tolerance of cyanobacteria to environmental variability. This, however, may in part be explained by the low taxonomic resolution obtained with the relatively conserved 16S rrna gene and/or the preferential preservation of particular taxa. Our results highlight the potential of fossil DNA in lake sediments to study colonization and succession dynamics of lacustrine cyanobacteria and warrant further investigation of the factors that affect preservation of cyanobacterial DNA

21 HIGHLIGHTS OF THE YEAR Hard Selective Sweep and Ectopic Gene Conversion in a Gene Cluster Affording Environmental Adaptation M. Hanikenne, J. Kroymann, A. Trampczynska, M. Bernal, P. Motte, S. Clemens, U. Krämer PLoS Genet 9:e doi: /journal.pgen , 2013 Among the rare colonizers of heavy-metal rich toxic soils, Arabidopsis halleri is a compelling model extremophile, physiologically distinct from its sister species A. lyrata, and A. thaliana. Both naturally selected metal hypertolerance and extraordinarily high leaf metal accumulation in A. halleri require Heavy Metal ATPase4 (HMA4) encoding a P IB -type ATPase that pumps Zn 2+ and Cd 2+ out of specific cell types. Strongly enhanced HMA4 expression results from a combination of gene copy number expansion and cis-regulatory modifications, when compared to A. thaliana. These findings were based on a single accession of A. halleri. Few studies have addressed nucleotide sequence polymorphism at loci known to govern adaptations. We thus sequenced 13 DNA segments across the HMA4 genomic region of multiple A. halleri individuals from diverse habitats. Compared to control loci flanking the three tandem HMA4 gene copies, a gradual depletion of nucleotide sequence diversity and an excess of low-frequency polymorphisms are hallmarks of positive selection in HMA4 promoter regions, culminating at HMA4-3. The accompanying hard selective sweep is segmentally eclipsed as a consequence of recurrent ectopic gene conversion among HMA4 protein-coding sequences, resulting in their concerted evolution. Thus, HMA4 coding sequences exhibit a network-like genealogy and locally enhanced nucleotide sequence diversity within each copy, accompanied by lowered sequence divergence between paralogs in any given individual. Quantitative PCR corroborated that, across A. halleri, three genomic HMA4 copies generate overall 20- to 130-fold higher transcript levels than in A. thaliana. Together, our observations constitute an unexpectedly complex profile of polymorphism resulting from natural selection for increased gene product dosage. We propose that these finding are paradigmatic of a category of multi-copy genes from a broad range of organisms. Our results emphasize that enhanced gene product dosage, in addition to neo- and sub-functionalization, can account for the genomic maintenance of gene duplicates underlying environmental adaptation. Figure. Nucleotide sequence diversity across the HMA4 genomic region in A. halleri. (A) Average pairwise nucleotide sequence diversity π across the HMA4 genomic region in A. halleri ssp. halleri. (B) Molecular population genetic statistical tests for the deviation from neutrality, Tajima's D, Fu and Li's D*, and Fu and Li's F* in A. halleri ssp. halleri. Differently colored symbols are defined by concordantly colored axis titles. Vertical arrows indicate positions of sequenced segments. The region of HMA4 triplication is shaded in grey. Marked in red color are stretches of sequence that are present in several, almost identical copies (horizontal bars; detected using NCBI MEGABLAST program with default settings and a word size of 256; see Table S3), and sequenced segments therein (arrows, fonts, red open symbols instead of closed black symbols in panel (A)). Direction of transcription (triangular arrow) is given for each gene (rectangle, Arabidopsis Genome Identifier code of A. thaliana ortholog), with genes of unknown position in A. halleri shown in grey

22 HIGHLIGHTS OF THE YEAR Activity of Ceftaroline against Enterococcus faecium PBP5 Henry X., Verlaine O., Amoroso A., Coyette J., Frère J-M., and Joris B. Antimicrob Agents Chemother. 57(12): , 2013 β-lactam antibiotics (penicillins, cephalosporins, carbapenems, and monobactams) represent the most important group of drugs prescribed to treat bacterial infections. They form stable acyl-enzymes with their targets, the membrane-bound DD-transpeptidases, which are essential enzymes in peptidoglycan biosynthesis. These proteins are usually referred to as penicillin-binding proteins (PBPs). The opportunistic human pathogen Enterococcus faecium overproduces the low-affinity PBP5 and mutations within this gene further reduce the acylation rate of PBP5 by altering its amino acid sequence. Ceftaroline is a cephalosporin with broad-spectrum activity against Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA), and many common Gramnegative bacteria which was recently approved by the U.S. Food and Drug Administration and by the European Medicines Agency (EMA) for similar indications. We have shown that ceftaroline inactivates the low-affinity E. faecium PBP5 more efficiently than benzylpenicillin. It exhibits bacterial killing against our laboratory-derived ampicillin-resistant E. faecium mutant that overproduces the wild-type PBP5. Figure. Left panel. PBP binding studies for ceftaroline with membrane fractions from D63 and D63r. The concentration of ceftaroline in the competition assay with fluorescent ampicillin is indicated in µg/ml. Right panel. Drug binding studies for ceftaroline with D63 and D63r membrane fractions. MICs of D63 (A) and D63r (B) for ceftaroline were 0.25 mg/liter and 2 mg/liter, respectively. The surviving bacteria were counted after 0, 4, and 24 h of incubation at 37 C by subculturing serial dilutions (at least 10-fold, to minimize drug carryover). For all data, the standard deviations were below 10% of the mean. Such a profile differs from those of most E. faecium clinical isolates, which were reported resistant to all β-lactams, including ceftaroline. Like other cephalosporins, ceftaroline is not indicated for treatment of enterococcal infections. It is likely that most E. faecium clinical isolates produce mutant PBP5 forms with reduced affinity. This suggests that simple overexpression of wild-type PBP5 is sufficient to moderately increase the MIC for ceftaroline but that amino acid substitutions in the protein are necessary to confer high-level resistance. Among the β-lactams tested to date, ceftaroline has the highest affinity for wild-type PBP5, which makes it a potentially useful tool for PBP5 studies

23 HIGHLIGHTS OF THE YEAR The crystal structure of the cell division amidase AmiC reveals the fold of the AMIN domain, a new peptidoglycan binding domain Rocaboy M, Herman R, Sauvage E, Remaut H, Moonens K, Terrak M, Charlier P, Kerff F. Mol Microbiol. 90(2):267-77, 2013 Binary fission is the ultimate step of the prokaryotic cell cycle. In Gram-negative bacteria like Escherichia coli, this step implies the invagination of three biological layers (cytoplasmic membrane, peptidoglycan and outer membrane), biosynthesis of the new poles and eventually, daughter cells separation. The latter requires the coordinated action of the N-acetylmuramyl-L-alanine amidases AmiA/B/C and their LytM activators EnvC and NlpD to cleave the septal peptidoglycan. We present here the 2.5 Å crystal structure of AmiC which includes the first report of an AMIN domain structure, a β-sandwich of two symmetrical four-stranded β-sheets exposing highly conserved motifs on the two outer faces. We show that this N-terminal domain, involved in the localization of AmiC at the division site, is a new peptidoglycan-binding domain. The C-terminal catalytic domain shows an autoinhibitory alpha helix obstructing the active site. AmiC lacking this helix exhibits by itself an activity comparable to that of the wild type AmiC activated by NlpD. We also demonstrate the interaction between AmiC and NlpD by microscale thermophoresis and confirm the importance of the active site blocking alpha helix in the regulation of the amidase activity. Figure. A. Sequence alignment of the two β-sheets composing the AMIN domain. β-strands are represented by red boxes. Surface residues conserved in the AMIN domains of amidases are highlighted: in yellow, for residues identical in β-sheet1 and β-sheet2 and in blue for residues of similar shape. Residues not conserved in AMIN domains but identical in the two β- sheets are represented in pink. B. Cartoon representation of the AMIN domain. The β-sheet1 (strands β ) and the β- sheet2 (strands β ) are depicted in orange and green respectively. Coils and helix α1 are shown in grey. C. Superposition of the two β-sheets of the AMIN domain. The colour code is the same as in panel B. Amino-acids highlighted in panel A are represented as sticks (nitrogens and oxygens are shown in blue and red respectively). D. Surface representation of the AMIN domain. Conservation of amino-acids of AmiC related proteins is mapped onto the surface from poorly conserved in blue to highly conserved in purple

24 HIGHLIGHTS OF THE YEAR Allosteric inhibition of VIM metallo-β-lactamases by a camelid nanobody Sohier JS., Laurent C., Chevigné A., Pardon E., Srinivasan V., Wernery U., Lassaux P., Steyaert J., and Galleni M. Biochem. J. 450, , doi: /bj , 2013 MβL (metallo-β-lactamase) enzymes are usually produced by multi-resistant Gram-negative bacterial strains and have spread worldwide. An approach on the basis of phage display was used to select single-domain antibody fragments (V H Hs, also called nanobodies) that would inhibit the clinically relevant VIM (Verona integron-encoded MβL)-4 MβL. Out of more than 50 selected nanobodies, only the NbVIM_38 nanobody inhibited VIM-4. The paratope, inhibition mechanism and epitope of the NbVIM_38 nanobody were then characterized. An alanine scan of the NbVIM_38 paratope showed that its binding was driven by hydrophobic amino acids. The inhibitory concentration was in the micromolar range for all β-lactams tested. In addition, the inhibition was found to follow a mixed hyperbolic profile with a predominantly uncompetitive component. Moreover, substrate inhibition was recorded only after nanobody binding. These kinetic data are indicative of a binding site that is distant from the active site. This finding was confirmed by epitope mapping analysis that was performed using peptides, and which identified two stretches of amino acids in the L6 loop and at the end of the α2 helix. Because this binding site is distant from the active site and alters both the substrate binding and catalytic properties of VIM-4, this nanobody can be considered as an allosteric inhibitor. Figure. Epitope of the NbVIM_38 nanobody (A) ELISA experiment with coated WT VIM-4 ( ) and coated VIM epitope ( ). (B) Alignment of the VIM-4 epitope sequence and the corresponding BlaB sequence. The amino acid stretches that are thought to be the main binding determinants are shaded in grey. (C) Crystallographic structure of VIM-4 (PDB code 2WHG). The epitope region Ile 99 Gly 133, is shown in orange. The amino acid stretches LPVTRA and VLRAAG are displayed in black. The Zn 2+ ions are represented by grey spheres. The Zn 2+ -co-ordinating amino acids of the L7 loop are shown as red sticks

25 HIGHLIGHTS OF THE YEAR Energetics of protein stability at extreme environmental temperatures in bacterial trigger factors Struvay C, Negro S., Matagne A. and Feller G. Biochemistry 52(17): , 2013 Trigger factor is the first molecular chaperone interacting co-translationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. The stability of this primary folding assistant was investigated using trigger factors from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli, and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by living organisms. We show that proteins adapt their stability in the whole range of biological temperatures via adjustments of the same fundamental mechanisms, involving increases in enthalpic stabilization and decreases in unfolding rates, in parallel with the environmental temperature. Enthalpic stabilization in trigger factors is characterized by large increases in the melting temperature Tm, ranging from 33 C to 96.6 C, associated with similarly large increases in unfolding enthalpy as revealed by differential scanning calorimetry. Stopped-flow spectroscopy shows that the folding rate constants for the three investigated proteins are similar, whereas the unfolding rate constants differ by several orders of magnitude, revealing that kinetic resistance toward unfolding drives adjustments of protein stability. While the unusual stability of hyperthermophilic proteins has attracted much attention, this study indicates that they are an extreme case of a more general continuum, the other extreme being represented by natively unstable proteins from psychrophiles. In the hyperthermophilic protein, resistance against unfolding, resulting in slow unfolding kinetics, is the main determinant of its high stability, while fast unfolding is responsible for the low stability of the psychrophilic protein

26 HIGHLIGHTS OF THE YEAR Use of Red Autofluorescence for Monitoring Prodiginine Biosynthesis Tenconi E., Guichard P., Motte P., Matagne A., Rigali S. J Microbiol Methods (2):138-43, 2013 Prodigiosin-like pigments or prodiginines (PdGs) are promising drugs owing to their reported antitumor, antibiotic, and immunosuppressive activities. These natural compounds are produced by several bacteria, including Streptomyces coelicolor and Serratia marcescens as most commonly studied models. The bright red color of these tripyrrole pigments made them excellent reporter molecules for studies aimed at understanding the molecular mechanisms that control secondary metabolite production in microorganisms. However, the natural red fluorescence of PdGs has only been rarely used as a biophysical parameter for detection and assessment of PdG biosynthesis. In this work, we used S. coelicolor in order to exemplify how intrinsic red fluorescence could be utilized for rapid, low-cost, sensitive, specific and accurate semi-quantitative analyses of PdG biosynthesis. Additionally, and contrary to the colorimetric-based approach, the fluorescence-based method allows in situ spatio-temporal visualization of PdG synthesis throughout a solid culture of S. coelicolor. As PdG production is related to cell differentiation, their red autofluorescence could be exploited, by means of confocal microscopy, as a natural marker of the entrance into a crucial developmental stage in the course of the S. coelicolor life cycle Figures. Left panel: in situ visualization of prodiginine biosynthesis in confluent culture of S. coelicolor. Red Auto Fluorescence production starts at the surface of a 30 h culture and progresses towards the deeper zone of the agar plate at later time points. The surface of the S. coelicolor M145 culture is indicated by a white arrow in both light and fluorescence pictures. Scale bars, 40µm. Right panel: Visualization of prodiginine production in filaments of S. coelicolor. Fluorescence confocal image (A) and light microscopy image (B) of isolated filaments. (C) 3D reconstruction of the original confocal image shown in (A), and of a specific filament (D)

27 HIGHLIGHTS OF THE YEAR A Pathway Closely Related to the D-Tagatose Pathway of Gram- Negative Enterobacteria Identified in the Gram-Positive Bacterium Bacillus licheniformis Van der Heiden E., Delmarcelle M., Lebrun S., Freichels R., Brans A., Vastenavond C., Galleni M., Joris B. Appl Environ Microbiol. 79 (11) : , 2013 We report the first identification of a gene cluster involved in D-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the D-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the D-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus. The functionality of the gene cluster has been demonstrated in E. coli and Bacillus subtilis by supplementation experiments. Pathways for catabolism of galactitol (Gat), D-tagatose (Tag), and lactose (Lac) in different bacteria. In Gram-negative bacteria, such as E. coli or K. oxytoca, galactitol is used as a carbohydrate source and Tag 6-P is an intermediate of the pathway. In K. oxytoca, tagatose is also used as a carbohydrate source by being transported and phosphorylated to yield Tag 1-P. Tag 1,6-BP formed by the tagatose 1-phosphate kinase TagK is further cleaved into DHAP and G3P with the class II tagatose aldolase GatYZ (6). An example of the D-tagatose 6-P pathway of the Gram-positive bacterium S. aureus is shown on the right. In this case, lactose is used as the carbohydrate source and Tag 6-P is an intermediate of the pathway. Glu, D- glucose; Gal, D-galactose. The B. licheniformis PTS-mediated D-tagatose catabolic pathway is related to that of K.oxytoca

28 HIGHLIGHTS OF THE YEAR Positive cooperativity between acceptor and donor sites of the peptidoglycan glycosyltransferase Bury D., Dahmane I., Derouaux A., Dumbre S., Herdewijn P., Matagne A., Breukink E. Mueller-Seitz E., Petz P. and Terrak M. Biochem. Pharmacol., 93, , 2014 The glycosyltransferases of family 51 (GT51) catalyze the polymerization of lipid II to form linear glycan chains, which, after cross linking by the transpeptidases, form the net-like peptidoglycan macromolecule. The essential function of the GT makes it an attractive antimicrobial target; therefore a better understanding of its function and its mechanism of interaction with substrates could help in the design and the development of new antibiotics. In this work, we have used a surface plasmon resonance Biacore( ) biosensor, based on an amine derivative of moenomycin A immobilized on a sensor chip surface, to investigate the mechanism of binding of substrate analogous inhibitors to the GT. Addition of increasing concentrations of moenomycin A to the Staphylococcus aureus MtgA led to reduced binding of the protein to the sensor chip as expected. Remarkably, in the presence of low concentrations of the most active disaccharide inhibitors, binding of MtgA to immobilized moenomycin A was found to increase; in contrast competition with moenomycin A occurred only at high concentrations. This finding suggests that at low concentrations, the lipid II analogs bind to the acceptor site and induce a cooperative binding of moenomycin A to the donor site. Our results constitute the first indication of the existence of a positive cooperativity between the acceptor and the donor sites of peptidoglycan GTs. In addition, our study indicates that a modification of two residues (L119N and F120S) within the hydrophobic region of MtgA can yield monodisperse forms of the protein with apparently no change in its secondary structure content, but this is at the expense of the enzyme function

29 HIGHLIGHTS OF THE YEAR Origin and evolution of metal P-type ATPases in Plantae (Archaeplastida) Hanikenne M. and Baurain D. Front Plant Sci, 4:544, 1-15, 2014 Metal ATPases are a subfamily of P-type ATPases involved in the transport of metal cations across biological membranes. They all share an architecture featuring eight transmembrane domains in pairs of two and are found in prokaryotes as well as in a variety of Eukaryotes. In Arabidopsis thaliana, eight metal P-type ATPases have been described, four being specific to copper transport and four displaying a broader metal specificity, including zinc, cadmium and possibly copper and calcium. So far, few efforts have been devoted to elucidating the origin and evolution of these proteins in Eukaryotes. In this work, we used large-scale phylogenetics to show that metal P-type ATPases form a homogenous group among P-type ATPases and that their specialisation into either monovalent (Cu) or divalent (Zn, Cd ) metal transport stems from a gene duplication that took place early in the evolution of Life. Then, we demonstrated that the four subgroups of plant metal ATPases all have a different evolutionary origin and a specific taxonomic distribution, only one tracing back to the cyanobacterial progenitor of the chloroplast. Finally, we examined the subsequent evolution of these proteins in green plants and concluded that the genes thoroughly characterised in model organisms are often the result of lineage-specific gene duplications, which calls for caution when attempting to infer function from sequence similarity alone in non-model organisms. Phylogeny of IB metal ATPases in prokaryotes and Eukaryotes. The tree was obtained with PhyloBayes (C20 model) from the analysis of a protein alignment of 1761 sequences x 340 AA. Tree branches were coloured based on homogeneous taxonomic composition and collapsed to highlight relationships between prokaryotic and eukaryotic proteins. Me + : Monovalent metals, Me 2+ : Divalent metals. The tree was rooted using a small group of architecture AII proteins as outgroup. Statistical support is provided as Posterior Probabilities

30 HIGHLIGHTS OF THE YEAR Der p 1 is the primary activator of Der p 3, Der p 6 and Der p 9 the proteolytic allergens produced by the house dust mite Dermatophagoides pteronyssinus Herman J., Thelen N., Smargiasso N., Mailleux AC, Luxen A., Cloes M., De Pauw E., Chevigné A., Galleni M., Dumez ME. Biochim Biophys Acta, 1840, , 2014 The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation cascade of their corresponding precursor forms and particularly to highlight the interconnection between proteases during this cascade. The cleavage of the four peptides corresponding to the mite zymogen activation sites was studied on the basis of the Förster Resonance Energy Transfer method. The proder p 6 zymogen was then produced in Pichia pastoris to elucidate its activation mechanism by mite proteases, especially Der p 1. The role of the propeptide in the inhibition of the enzymatic activity of Der p 6 was also examined. Finally, the Der p 1 and Der p 6 proteases were localised via immunolocalisation in D. pteronyssinus. All peptides were specifically cleaved by Der p 1, such as proder p 6. The propeptide of proder p 6 inhibited the proteolytic activity of Der p 6, but once cleaved, it was degraded by the protease. The Der p 1 and Der p 6 proteases were both localised to the midgut of the mite. Der p 1 in either its recombinant form or in the natural context of house dust mite extracts specifically cleaves all zymogens, thus establishing its role as a major activator of both mite cysteine and serine proteases. Immunolocalization of Der p 1 and Der p 6 in longitudinal sections of Dermatophagoides pteronyssinus body. (A-B) Localization of Der p 6 (red) in the posterior midgut and (C-D) colocalization of Der p 1 (yellow) in the anterior to posterior gut with Der p 6 only detected in posterior part. The scale bar is 50 µm. AMg, Anterior midgut; Hg, Hindgut; PMg, Posterior midgut. Proteases activation cascade in the digestive tract of the mite D. pteronyssinus. The box represents the activation cascade in the hindgut. AMg, Anterior midgut; FP, Fecal pellet; Hg, Hindgut; PMg, Posterior midgut

31 HIGHLIGHTS OF THE YEAR Altered desferrioxamine-mediated iron utilization is a common trait of bald mutants of Streptomyces coelicolor Lambert S., Traxler M., Craig M., Maciejewska M., Ongena M., van Wezel G., Kolter R. and Rigali S. Metallomics, 8, , 2014 Streptomyces coelicolor is an important model organism for developmental studies of filamentous GCrich actinobacteria. The genetic characterization of mutants of S. coelicolor blocked at the vegetative mycelium stage, the so-called bald (bld) mutants that are unable to erect spore-forming aerial hyphae, has opened the way to discovering the molecular basis of development in actinomycetes. Desferrioxamine (DFO) production and import of ferrioxamines (FO; iron-complexed DFO) are key to triggering morphogenesis of S. coelicolor and we show here that growth of S. coelicolor on the reference medium for Streptomyces developmental studies is fully dependent on DFO biosynthesis. UPLC-ESI-MS analysis revealed that all bld mutants tested are affected in DFO biosynthesis, with blda, bldj, and ptsh mutants severely impaired in DFO production, while bldf, bldk, crr and ptsi mutants overproduce DFO. Morphogenesis of bldk and bldj mutants was recovered by supplying exogenous iron. Transcript analysis showed that the bldj mutant is impaired in expression of genes involved in the uptake of FO, whereas transcription of genes involved in both DFO biosynthesis and FO uptake is increased in bldk mutants. Our study allows proposing altered DFO production and/or FO uptake as a novel phenotypic marker of many S. coelicolor bld mutants, and strengthens the role of siderophores and iron acquisition in morphological development of actinomycetes. Siderophore production is altered in bald mutants. Top: details of bald mutant colonies overlaid with a CAS agar solution to detect the extracellular production of iron-chelating molecules (orange halos). The diameter of the colonies varies between 1 and 0.5 cm. Bottom: histograms displaying coelichelin (CCLIN), desferrioxamine B (DFOB) and desferrioxamine E (DFOE) production quantified by UPLC-ESI-MS analysis. Relative amounts of siderophores produced by the mutants (Grey bars) are expressed as a percentage of levels produced by S. coelicolor M145 (wild-type strain, Black bars). A.U., arbitrary units

32 HIGHLIGHTS OF THE YEAR Control of chitin and N-acetylglucosamine utilization in Saccharopolyspora erythraea Liao C., Rigali S., Licona Cassani C., Marcellin E., Keld Nielsen L., and Ye BC. Microbiology, 160, , 2014 Chitin degradation and subsequent N-acetylglucosamine (GlcNAc) catabolism is thought to be a common trait of a large majority of actinomycetes. Utilization of aminosugars had been poorly investigated outside the model strain Streptomyces coelicolor A3(2), and we examined here the genetic setting of the erythromycin producer Saccharopolyspora erythraea for GlcNAc and chitin utilization, as well as the transcriptional control thereof. Sacch. erythraea efficiently utilize GlcNAc most likely via the phosphotransferase system (PTS GlcNAc ); however, this strain is not able to grow when chitin or N,N'-diacetylchitobiose [(GlcNAc) 2 ] is the sole nutrient source, despite a predicted extensive chitinolytic system (chi genes). The inability of Sacch. erythraea to utilize chitin and (GlcNAc) 2 is probably because of the loss of genes encoding the DasABC transporter for (GlcNAc) 2 import, and genes for intracellular degradation of (GlcNAc) 2 by β-n-acetylglucosaminidases. Transcription analyses revealed that in Sacch. erythraea all putative chi and GlcNAc utilization genes are repressed by DasR, whereas in Strep. coelicolor DasR displayed either activating or repressing functions whether it targets genes involved in the polymer degradation or genes for GlcNAc dimer and monomer utilization, respectively. A transcriptomic analysis further showed that GlcNAc not only activates the transcription of GlcNAc catabolism genes but also activates chi gene expression, as opposed to the previously reported GlcNAc-mediated catabolite repression in Strep. coelicolor. Finally, synteny exploration revealed an identical genetic background for chitin utilization in other rare actinomycetes, which suggests that screening procedures that used only the chitin-based protocol for selective isolation of antibiotic-producing actinomycetes could have missed the isolation of many industrially promising strains. The chitinolytic system and GlcNAc metabolism pathways in Strep. coelicolor and Sacch. erythraea. The functions and references associated with proteins and enzymes shown here are described in Table 1. Proteins/enzymes in black are present in both Strep. coelicolor and Sacch. erythraea. Proteins/enzymes in grey and light grey are only present in Strep. coelicolor and in Sacch. erythraea, respectively. Chitinases with diagonal stripes are present in both strains, but the architecture of orthologous genes is not fully conserved. (GlcNAc) 2-5, chitooligosaccharides; NH 3, ammonia; PEP, phosphoenolpyruvate

33 HIGHLIGHTS OF THE YEAR Crystal structure of penicillin-binding protein 3 (PBP3) from Escherichia coli Sauvage E., Derouaux A., Fraipont C., Joris M., Herman R., Rocaboy M., Schloesser M., Dumas J., Kerff F., Nguyen-Distèche M. and Charlier P. PlosOne, 9, e98042, 2014 In Escherichia coli, penicillin-binding protein 3 (PBP3), also known as FtsI, is a central component of the divisome, catalyzing cross-linking of the cell wall peptidoglycan during cell division. PBP3 is mainly periplasmic, with a 23 residues cytoplasmic tail and a single transmembrane helix. We have solved the crystal structure of a soluble form of PBP3 (PBP ) at 2.5 Å revealing the two modules of high molecular weight class B PBPs, a carboxy terminal module exhibiting transpeptidase activity and an amino terminal module of unknown function. To gain additional insight, the PBP3 Val88-Ser165 subdomain (PBP ), for which the electron density is poorly defined in the PBP3 crystal, was produced and its structure solved by SAD phasing at 2.1 Å. The structure shows a three dimensional domain swapping with a β-strand of one molecule inserted between two strands of the paired molecule, suggesting a possible role in PBP dimerization. Cartoon representation of the crystal structure of PBP3. A ribbon trace of modelled loops undefined in the crystal structure is shown in grey. The active site is indicated by a red sphere. Loops discussed in the text are indicated

34 SCIENTIFIC SERVICES CONTACTS BCCM/ULC: Culture collection for cyanobacteria : Manager: Dr Annick Wilmotte awilmotte@ulg.ac.be Tel: + 32 (0) / Technical assistance: Marine Renard Protein Factory : Manager: Dr Alain Brans abrans@ulg.ac.be Tel: + 32 (0) Collaborators: Fabrice Bouillenne, Anne-Marie Matton and Iris Thamm High throughput facility Dr Alain Brans : abrans@ulg.ac.be Tel: + 32 (0) Dr André Matagne : amatagne@ulg.ac.be Tel : + 32 (0) Dr Julie Vandenameele: Julie.Vandenameele@ulg.ac.be Tel: + 32 (0) Training: Techniques for protein production and purification Biotechnology Training Centre: Laurent Corbesier: forem.biotech@skynet.be Tel : + 32 (0) Dr Alain Brans : abrans@ulg.ac.be Tel: + 32 (0) Fabrice Bouillenne : F.Bouillenne@ulg.ac.be Tel : + 32 (0)

35 SCIENTIFIC SERVICES BCCM/ULC: A CULTURE COLLECTION OF (SUB)POLAR CYANOBACTERIA Since 2005, the BCCM (Belgian Co-ordinated Collections of Microorganisms) has supported the elaboration of a collection of (sub)polar cyanobacteria. The integration towards an official public collection, called BCCM/ULC, has realized in In 2010, the implementation of a Quality Management System was started and BCCM/ULC has obtained and maintained an ISO9001 certificate for the public deposit and distribution services since This is a part of the consolidation of the Belgian Biological Resource Centre (BRC ). The BCCM/ULC public collection is now holding over 200 cyanobacterial strains of various origins (freshwater planktonic, terrestrial habitats ) but with a focus on (sub)polar habitats. The catalogue is available on: It includes 120 (sub)polar unicyanobacterial strains coming from various regions of the Antarctic (South Victoria Land, East Antarctica, Transantarctic Mountains, James Ross Island) and the Arctic (North Canada, Arctic Ocean, Alaska, Svalbard), and different biotopes (microbial mats, lakes, ice shelves, dry valleys, cryptoendoliths, oceans). Twelve strains were isolated in the Sub-Arctic, in Siberian lakes. The most important cyanobacterial orders are represented: Chroococcales, Oscillatoriales, and Nostocales. Moreover, our laboratory is involved in projects for which new isolates are being purified, and will extend the geographic coverage of the collection. The majority of the strains is psychrotolerant and can be cultivated at C. They are available as living cultures, and 63 strains also have been cryopreserved (-70 C). A BRAIN-be project to improve the cryopreservation techniques for BCCM/ULC started in 2014 (PRESPHOTO) ( Genomic DNA is available on request. The molecular characterization is underway, on the basis of 16S rrna and ITS sequences. Exploration of the bioactivities A first screening by Biondi et al. (J. Applied. MIcrobiol., 2008) had already shown the bioactivity of several strains against the bacterium Staphylococcus aureus and the fungi Cryptococcus neoformans. During a bilateral cooperation project with Prof. Fiore (CENA, Piracicaba, Brazil), it was shown with bioassays that the methanol extracts of two strains could inhibit the growth of fungal strains (Candida kruzeii and Phoma sp.)

36 SCIENTIFIC SERVICES PROTEIN FACTORY An effective research requires access to a broad range of technologies, some of which require expertise and specific equipments. Protein Factory is a protein production platform for academic laboratories and private companies. One of the objectives of the platform is to provide laboratory and pilot-scaled equipment for on- and off-campus users. Services include protein production in bacterial, yeast and filamentous fungal strains, followed by their purification. The platform can provide many services including: The genetic engineering and cloning in bacterial strains such as E. coli, Bacillus subtilis, Bacillus amyloliquefaciens, Streptomyces lividans or in yeast such as Pichia pastoris The analytical scale or pilot scale recombinant protein production from these organisms in high cell density conditions The cell harvesting or supernatant cleaning using bucket or continuous centrifugation or hollow fiber filtration The cell disintegration to recover proteins produced in the intracellular compartment The protein purification at the analytical and pilot scales. For these purposes the platform is equipped with: Several shaking incubators for microtitre plates to 2 L flasks Wide range of computer controlled fermentors with working volumes from 1 to 80 L for batch and fed-batch cultures. Dissolved oxygen, ph, temperature, agitation and turbidity are controlled in all fermentors 1 bucket and 1 continuous centrifuges 1 crossflow filtration unit Several systems for semi- or totally automated purification of proteins including: ÅKTA prime, ÅKTA explorer, ÅKTA explorer (2D system), ÅKTA purifier, Biopilot, Profinia Protein Purification System. Protein Factory has provided proteins for: kinetic studies, protein structure determination by NMR or crystallography, protein-protein or protein-ligand interaction studies, secondary metabolite productions, enzyme-inhibitor studies, amyloid fibril formation and protein folding studies, immunoassay developments, vaccination studies and immuno test kit manufacturing

37 SCIENTIFIC SERVICES HIGH-THROUGHPUT FACILITY In September 2013, the CIP submitted an application to the DGO6 (Wallonia, Belgium), within the frame of the EQUIP 2013 call. The project (acronym: ROBOTEINE), coordinated by André Matagne, involves two academic partners, namely the CIP and the Structural Biology and Bioinformatics Centre (SBBC, represented by Erik Goormaghtigh) at the Université Libre de Bruxelles (ULB), and an academic associate partner, i.e. the Structural Biology Brussels (SBB, represented by Jan Steyaert) at the Vrije Universiteit Brussel (VUB)/VIB. Furthermore, the project is supported by three private companies, i.e. Eurogentec, Puratos and Progenosis. The aim of ROBOTEINE is to develop an automated platform for protein production and analysis, on the basis of original high-throughput (HT) methods developed in our laboratories. The project, which has been selected by the DGO6 for funding, started May 1 st, A post-doctoral researcher (Julie Vandenameele) was hired to develop part of the new facility at the CIP. With the help of Alain Brans, Julie is currently developing a platform that offers automated cloning and protein analysis, together with easy colony picking. Furthermore, it allows screening for expression of recombinant proteins in bacteria (both Gram negative and Gram positive) and yeast, and for optimal protein purification. The core of the facility consists of two HT liquid handling devices designed for flexible, fast and accurate sample preparation. Both robotic workstations, which are of the Microlab STAR line by Hamilton, use air displacement pipetting and the unique CO-RE system for forceless tip pickup and ejection. One of these liquid handlers is contained in a sterile housing with a laminar flow of air drawn through a HEPA filter, thus allowing dispensing of sterile liquids for culture media preparation. It is equipped with a high resolution CCD camera and a light table to image colonies, thus allowing fast and economical colony picking. The other liquid handling platform is linked to specialized automated analysis instruments to measure various relevant parameters in the wells (typically 96 or 384 but up to 1536) of microtiter plates. Thus, two microplate readers (Infinite 200 PRO by Tecan) allow UV/Vis absorbance and fluorescence measurements, together with chemiluminescence detection. A system (LabChip GXII) for automated electrophoretic separations of nucleic acids (RNA, DNA) and protein provide fast and easy way to analyze samples across a wide range of molecule and fragment sizes. Finally, an Octet platform allows HT analysis of biomolecular interactions in 96- and 384-well microplates. It enables real-time and label-free determination of affinity, kinetics and concentration and it is thus highly suitable for characterization of drug candidates and development of possible applications. Up to 96 samples can be analyzed in parallel and hence, protein quantitation of 96 samples can be completed in a relatively short time. Rapid kinetics, screening and epitope binding assays are enabled by the multiplexed biosensor formats. Furthermore, proteins can be assayed in crude mixtures such as cell lysates or hybridoma supernatants, glycerol, or in up to 10% DMSO, which drastically reduces sample preparation

38 SCIENTIFIC SERVICES Techniques for protein production and purification Since 2006, the CIP works in close collaboration with the Biotechnology Training Centre Forem- GIGA to develop and organize biotechnology training for jobseekers in the field of protein production and purification. At the CIP, the training includes the following technological modules: Bacterial transformation (Escherichia coli and Bacillus subtilis) Protein production in flasks and in 20 L fermentors (batch and fed-batch cultures) with E. coli, B. subtilis and P. pastoris Cell harvesting and cell disruption Protein purification by different chromatography technologies including: ion exchange, molecular sieve, hydrophobic and affinity chromatographies Protein identification by SDS-PAGE, enzymatic testing and Western blotting

39 SCIENTIFIC PRODUCTION AWARDS Vincenzo Campisi, Best Poster Award, Highlighting potential targets of House Dust Mite allergenic proteases EAACI-WAO Congress, Milan, Italy, June 22-26, 2013 Amandine Godin, Best Poster Award-Biotechnology, «Thermal adaptation of the ribosomal chaperone trigger factor» Bioforum 2013, Liège, Belgium, April 18, 2013 Céline Huynen, Protein Society Finn World Travel Award, 27 th Annual Symposium of the Protein Society, Boston, USA, July 20-23, 2013 Elodie Tenconi, best poster presentation award for Prodiginines, secondary metabolites associated with the programmed cell death of S. coelicolor, 17 th International Symposium on the Biology of Actinomycetes, Izmir, October 14, 2014 Elodie Tenconi, ASM special price: best poster presentation award for Prodiginines, secondary metabolites associated with the programmed cell death of S. coelicolor, Belgian Society for Microbiology, Brussels November 18,

40 SCIENTIFIC PRODUCTION INVITED SPEAKERS Dr Sébastien Dilly, GIGA-Neurosciences, ULg, Liege, Belgium, Etude in silico de l architecture des canaux SK et des modalités d interaction entre ces canaux et leurs ligands», May 24 Prof. Bertrand Garcia-Moreno, John Hopkins University, Baltimore, USA, Why, how, and whither biophysics, November 14 Prof. Stjepko Golubic, Boston University, USA, Cyanobacteria and carbonate deposits: construction and destruction, December 3 Prof. Tom Lenaerts, ULB, Brussels, Belgium, Entropy-driven communication in protein domains; computational prediction and NMR-based validation, September 20 Dr Philippe Minard, Université Paris Sud, France, Conception et caractérisation d une famille de protéines artificielles: les alpharep, December 4 Dr Kris Pauwels, VUB, Brussels, Belgium, Structural aspect of polypeptide aggregation in neurodegeneration: a case study of Abeta and the prion protein, February 15 Prof. Patricia Polverino de Laureto, CRIBI Biotechnology Centre, Padova, Italy, Erasmus courses Limited proteolysis of proteins: general concept and applications and Natively unfolded proteins, February 7-9 Dr Karen Stroobants, KUL, Leuven, Belgium, Surface charge fit, peptide bond snip: metalsubstituted polyoxometalates as region selective artificial proteases, November 22 Dr Matt Traxler, Harvard Medical School, Boston, USA, The interactive life of Streptomyces coelicolor, March 8 Prof. Wim Versees, VUB, Brussels, Belgium, Architecture and conformational plasticity of the MnmE-GidA complex, December 6 INVITED SPEAKERS Dr Jean-Denis Docquier, University of Siena, Italy, Antibiotiques et résistance: vers un retour à l ère pré-antibiotique, January 22 Dr Giovanni Maglia, University of Leuven, Belgium, Single-molecule enzymology with protein nanopores, September 19 Dr Emmanuel Oheix, University of Birmingham, UK, Development of polypyridine metaldependent switches as artificial regulation sites, September 10 Dr Nicolas Smargiasso, Laboratoire de Spectrométrie de Masse, ULg, Liege, Belgium, Overview of mass spectrometry applied to protein biochemistry, March

41 SCIENTIFIC PRODUCTION ORAL PRESENTATIONS M. Dumoulin, Investigation of the mechanism of aggregation of polyglutamine proteins which are associated with neurodegenerative amyloidoses, Department of Structural Biology and Chemistry, Institut Pasteur, Paris, France, January 24 M. Dumoulin, La formation des fibres amyloïdes pathogènes et fonctionnelles», Royal Academy of Sciences, Academy Palace, Brussels, Belgium, December 7 G. Feller, Cold adaptations in proteins from psychrophiles, Stephan Angeloff Institute of Microbiology, Sofia, Bulgaria, January 15 G. Feller, Analysis report on superoxide dismutases from the Antarctic fungus Aspergillus glaucus, Stephan Angeloff Institute of Microbiology, Sofia, Bulgaria, January 15 C. Huynen, Role of non-polyq regions on the aggregation of polyq proteins into amyloid fibrils triggered by polyq expansions, 27 th Annual symposium of the Protein Society, Boston, USA, July 21 B. Joris, Peptidoglycan recycling and induction of β-lactamase in Gram-positive bacteria Great Wall Symposium - 3rd Symposium: The Dynamics of Peptidoglycan Structure and Function: New Insights into the Great Wall, Paris, France, September A. Matagne, Protein Folding: theory, methods of study and importance in human diseases, Dipartimento di Biotecnologie Mediche, Scuola di Dottorato Biotecnologie Mediche, Scuola di Dottorato Scienze Chimiche e Farmaceutiche, Siena, Italy, September 27 A. Matagne, Le repliement des protéines expliqué aux Académiciens, Royal Academy of Sciences, Academy Palace, Brussels, Belgium, October 5 C. Montagner, Equilibrium unfolding of Bacillus cereus zinc β-lactamase gives insight into the role of loops in the enzyme activity, 24 th Regensburger Faltertage at the Thon-Dittmer-Palais, Regensburg, Germany, October C. Montagner, Equilibrium unfolding of Bacillus cereus zinc β-lactamase gives insight into the role of loops in the enzyme activity, Young Belgian Magnetic Resonance Scientists 2013, Blankenberg, Belgium, December 2-3 D. Thorn, Switching to the dark side: repositioning of polyglutamine repeat promotes amyloid fibril formation by the model protein, beta-lactamase BlaP, 4 th Scandinavian Meeting on Amyloid Proteins and Disease, Lund, Sweden, November A. Wilmotte, Human impacts on Antarctic ecosystems: do not forget the microorganisms!, XI th SCAR Biology Meeting, Barcelona, Spain, July ORAL PRESENTATIONS I. Dahmane, Positive cooperativity between acceptor and donor sites of the peptidoglycan glycosyltransferase, PhD Day of the SFMBBM doctoral school, Namur, Belgium, November 14 M. Dumoulin, Mechanism of aggregation of polyglutamine (polyq) proteins which are associated with neurodegenerative amyloidoses: creation and characterization of polyq hybrid proteins made from the beta-lactamase BlaP, Chemistry mini symposium: Protein Amyloid Degeneration Still an Open Challenge, Abu Dhabi, United Arab Emirates, October

42 SCIENTIFIC PRODUCTION JM Frère, CIP, ULg, Liege, Belgium, K i et K d Comment convaincre les membres d un jury?, February 7 JM Frère, The NDM-1 beta-lactamase: much ado about the wrong thing, 12 th beta-lactamasse meeting, San Bartolomé de Tirajana, Gran Canaria, Spain, June 28-July 1 JM Frère (with T. Keating and W. Nichols), Inhibition kinetics of beta-lactamase by avibactam and relation to human pharmacokinetics, 12 th beta-lactamase meeting, San Bartolomé de Tirajana, Gran Canaria, Spain, June 28-July 1 M. Hanikenne, Epigenetics and local adaptation, UniGR-Workshop Systems Biology, Epigenetics & Systems Analysis, Sarrebrück, Germany, January 22 M. Hanikenne, Natural selection at the HMA4 locus in Arabidopsis halleri: a glimpse of the evolution of a complex trait, INRA SupAgro, Montpellier, France, March 24 M. Hanikenne, HMA4 and metal hyperaccumulation in Arabidopsis halleri, GDRI LOCOMET meeting, University of Lille, France, July 4 S. Lambert, Fer, sidérophores, et induction du développement chez Streptomyces coelicolor, Journées Actinomycètes, Liège, Belgium, May 15 C. Huynen, Role of non-polyq regions on the aggregation process by polyq proteins into amyloid fibrils, Neurobiology of Brain Disorders, Gordon-Merck Research Seminar, Neurodegeneration and Aging- Related Disorders of the Nervous System, Girona, Spain, July C. Huynen, Role of the polyq length and non-polyq regions during the aggregation process into amyloid fibrils of polyq proteins using a model polyq protein, IAP 7/44 Meeting: Integrative Protein Science: from small molecules to complex biological systems, Liège, Belgium, November 28 M. Maciejewska, Subterranean life: Mining moonmilk-derived actinobacterial diversity, Journées Actinomycètes, Liège, Belgium, May 16 S. Rigali, Streptomyces in nature and medicine, Institut de Biologie Physico-Chimique, Paris, France, January 31 S. Rigali, Prédiction de régulons et recherche des mécanismes d induction de la production de métabolites secondaires, Journées Actinomycètes, Liège, Belgium, May 16 S. Rigali, Prodiginines: cell-death associated metabolites in Nature and Medicine, Symposium Life, death and survival in microorganisms, Bruxelles, Belgium, September 9 S. Rigali, Prediction of Regulatory Networks & Control of Secondary Metabolite Biosynthesis, 17 th International Symposium on the Biology of Actinomycetes (ISBA17), Izmir, Turkey, October 10 E. Tenconi, Prodiginines, métabolites associés à la mort cellulaire de Streptomyces coelicolor, Journées Actinomycètes, Liège, Belgium, May 16 D. Thorn, Switching to the dark side: repositioning of polyglutamine repeat promotes amyloid fibril formation by the model protein, beta-lactamase BlaP, Proteostasis and Disease Symposium, Wollongong, Australia, November 5 A. Wilmotte, The BCCM/ULC collection to safeguard and exploit cyanobacterial diversity, Symposium on Cyanobacteria, Göttingen, Germany, June

43 SCIENTIFIC PRODUCTION PATENTS Enzymatic method for rapid and continuous determination of beta-lactamic antibiotics A. Brans, B. Joris, M. Delmarcelle, J. Marchand, C. Hammaecher, P. Tulkens, E. Goormaghtigh, J. De Coninck Application: international patent WO Method for enhancing a sum frequency generation signal Yves Caudano, Francesca Cecchet, Dan Lis, Sophie Demoustier, Etienne Ferain, Marie Henry, Bernard Joris Application: EP Recombinant alpha-hemolysin polypeptide of Staphylococcus aureus, having a deletion in the stem domain and heterologous sequences inserted P. Filée, N. Rhazi, M. Galleni, B. Taminiau, O. Jolis, A. Collard, A. Jacquet WO (A1) 22/07/2010 Extension US 8,545,835,B2 01/10/2013 JP (A) 02/07/2013 ES (T3) 07/08/2014 Methods for thaxtomin production and modified Streptomyces with increased thaxtomin production R. Loria, S. Rigali, S. Jourdan, and I. Francis EFS ID: ; Application number: Attorney Docket Number: Receipt date: 19 Septembre

44 SCIENTIFIC PRODUCTION PHD THESIS 15/03/2013 Caroline Struvay (Biochemistry) Adaptation des protéines aux températures extrêmes : étude des contributions thermodynamiques et cinétiques à la stabilité de «trigger factors» bactériens 07/06/2013 Jessica Guillerm (Sciences) Etude des propriétés de repliement d une protéine hélice β parallèle droite : la pectine méthylesterase d Erwinia chrysanthemi /06/2013 Roya Barumandzadeh (Sciences) Etude des cinétiques de repliement de fragments d anticorps constitués d un seul domaine, V H H 25/09/2013 Jean-Sébastien Sohier (Biochemistry) Sélection et caractérisation de nanobodies inhibiteurs de métallo-β-lactamases 17/12/2013 Mathieu Rocaboy (Sciences) Structural and biochemical study of the proteins AmiC, NlpD and FtsW involved in the bacterial cell division 24/01/2014 Coralie Pain (Sciences) Nanobodies as tools to investigate the mechanism of aggregation of chimeric proteins made by the insertion of polyglutamine stretches into the beta-lactamase BlaP 10/04/2014 Julie Herman (Sciences) Etude du mécanisme d activation du zymogène proder p 6 de l acarien Dermatophagoides pteronyssinus et du rôle de son propeptide dans l inhibition, le repliement et l immunogénicité de l allergène 30/04/2014 Janice Dumont (Sciences) Etude des propriétés des variants amyloïdogéniques du lysozyme humain à l aide de fragments d anticorps à chaînes lourdes comme sondes structurales 08/05/2014 Anthony Arguelles Arias (Sciences) Identification, production et caractérisation de l amylolysine, un lantibiotique de Type-B produit par Bacillus amyloliquefaciens GA1 04/06/2014 Amandine Godin (Sciences) Adaptations fonctionnelles des protéines aux températures environnementales extrêmes : étude des activités chaperon et prolyl de «trigger factors» bactériens» 04/09/2014 Maud Delsaute (Sciences) Etude de l implication de cellulases dans la voie de la biosynthèse de cellulose chez les bactéries 12/09/2014 Yannick Lara (Sciences) Molecular tools applied to the study of microcystin-producing cyanobacteria in Belgian waterbodies

45 SCIENTIFIC PRODUCTION 09/12/2014 Jean-Benoît Charlier (Sciences) Régulation complexe de FRD3, un gène de l homéostasie des métaux, chez deux espèces d Arabidopsis 11/12/2014 Ahlem Bouaziz (Sciences) Mise au point d une forme stable de l allergène Der p 3 de Dermatophagoides pteronyssinus pour le diagnostic rapide et le développement de nouvelles approches d immunothérapie de l allergie aux acariens

46 SCIENTIFIC PRODUCTION PUBLICATIONS 2013 A. Arguelles Arias, M. Ongena, B. Devreese, M. Terrak, B. Joris and P. Fickers Characterization of amyloslysin, a novel lantibiotic from Bacillus amyloliquefaciens GA1 PLoS One, 8(12):e83037 R. Ben Naya, K. Matti, A. Guellier, A. Matagne, D. Boquet, D. Thomas, A. Friboulet, B. Avalle and S. Padiolleau-Lefèvre Efficient refolding of a recombinant abzyme. Structural and catalytic characterizations Appl. Microbiol. Biotechnol., 97, R. Berlemont, O. Spee, M. Delsaute, Y. Lara, J. Schuldes, P. Power, R. Daniel, M. Galleni and B. Joris Novel organic solvent-tolerant esterase isolated by metagenomics : insights into the lipase/esterase classification Rev. Argent. Microbiol., 45, 3-12 F. Bono, F. De Smet, C. Herbert, K. De Bock, M. Georgiadou, P. Fons, M. Tjwa, C. Alcouffe, A. Ny, M. Bianciotto, B. Jonckx, M. Murakami, A. A. Lanahan, C. Michielsen, D. Sibrac, F. Dol-Gleizes, M. Mazzone, S. Zacchigna, J.-P. Herault, C. Fischer, P. Rigon, C. Ruiz de Almodovar, F. Claes, I. Blanc, K. Poesen, J. Zhang, I. Segura, G. Gueguen, M.-F. Bordes, D. Lambrechts, R. Broussy, M. van de Wouwer, C. Michaux, T. Shimada, I. Jean, S. Blacher, A. Noel, P. Motte, E. Rom, J.-M. Rakic, S. Katsuma, P. Schaeffer, A. Yayon, A. Van Schepdael, H. Schwalbe, F.L. Gervasio, G. Carmeliet, J. Rozensky, M. Dewerchin, M. Simons, A. Christopoulos, J.-M. Herbert and P. Carmeliet Inhibition of tumor angiogenesis and growth by a small-molecule multi-fgf receptor blocker with allosteric properties Cancer Cell, 23, C. Brulé, J. Grugier, A. Brans, B. Joris, E. Sauvage, G. Dive and J. Marchand-Brynaert 2-nitrobenzyl esters of penam and cephem derivatives as inhibitors of penicillin-binding proteins Asian J. Org. Chem., 2, C. Commin, M. Aumont-Nicaise, G. Claisse, G. Feller and JL Da Lage Enzymatic characterization of recombinant alpha-amylase in the Drosophila melanogaster species subgroup: is there an effect of specialization on digestive enzyme? Genes Genet. Syst., 88, E. de Genst, P-H Chan, E. Pardon, S-T Hsu, J. Kumita, J. Christodoulou, L. Menzer, D.Y. Chirgadze, C.V. Robinson, S. Muyldermans, A. Matagne, L. Wyns, C.M. Dobson and M. Dumoulin A nanobody binding to non-amyloidogenic regions of the protein human lysozyme enhances partial unfolding but inhibits amyloid fibril formation J. Phys. Chem. B, 117, M. Delsaute, R. Berlemont, D. Dehareng, D. Van Elder, M. Galleni and C. Bauvois Three-dimensional structure of RBcel1, a metagenome-derived psychotolerant family GH5 endoglucanase Acta Cryst., 69, D. Delvaux, F. Kerff, M.R.S.V Murty, B. Lakaye, J. Czerniecki, G. Kohn, P. Wins, R. Herman, V. Gabelica, F. Heuze, X. Tordoir, R. Marée, A. Matagne, P. Charlier, E. De Pauw and L. Bettendorff Structural determinants of specificity and catalytic mechanism in mammalian 25-kDa thiamine triphosphatase Biochim. Biophys. Acta, 1830,

47 SCIENTIFIC PRODUCTION A. Derouaux, E. Sauvage and M. Terrak Peptidoglycan glycosyltransferase substrate mimics as templates for the design of new antibacterial drugs Frontiers in Immunology, 4, 1-6, doi: /fimmu G. Dive, C. Bouillon, A. Sliwa, B. Valet, O. Verlaine, E. Sauvage and J. Marchand-Brynaert Macrocycle-embedded beta-lactams as novel inhibitors of the Penicillin Binding Protein PBP2a from MRSA Eur. J. Med. Chem., 64, N. Dony, J.M. Crowet, B. Joris, R. Brasseur and L. Lins SAHBNET, an accessible surface-based elastic network: an application to membrane protein Int. J. Mol. Sci., 14, M.E. Dumez, J. Herman, V. Campisi, A. Bouaziz, F. Rosu, A. Luxen, I. Vandenberghe, E. De Pauw, J.M. Frère, A. Matagne, A. Chevigné and M. Galleni The proline-rich motif of the proder p 3 allergen propeptide is crucial for protease-protease interaction PlosOne, 8, e68014 L. Dzhekieva, S.A. Adediran, R. Herman, F. Kerff, C. Duez, P. Charlier, E. Sauvage et R.F. Pratt Inhibition of DD-peptidases by a specific trifluoroketone: crystal structure of a complex with the Actinomadura R39 DD-peptidase Biochemistry, 52, R. Fernandez-Carazo, E. Verleyen, D.A. Hodgson, S.J. Roberts, K. Waleron, W. Vyverman and A. Wilmotte Late Holocene changes in cyanobacterial community structure in maritime Antarctic lakes J. Paleolimnol., 50, M. Figueroa, N. Oliveira, A. Lejeune, KW Kaufmann, BM Dorr, A. Matagne, JA Martial, J. Meiler and C. Van de Weerdt Octarellin VI: using rosetta to design a putative artificial (β/α)8 protein PLoS One 8(8):e71858 M. Gahungu, A. Arguelles Arias, P. Fickers, A. Zervosen, B. Joris, C. Damblon and A. Luxen Synthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A Bioorg. Med. Chem., 21, A. Gustot, V. Raussens, M. Dehousse, M. Dumoulin, C.E. Bryant, J.M. Ruysschaert and C. Lonez Activation of innate immunity by lysozyme fibrils is critically dependent on cross-β sheet structure Cell. Mol. Life Sci, 70, M. Hanikenne, J. Kroymann, A. Trampczynska, M. Bernal, P. Motte, S. Clemens and U. Krämer Hard selective sweep and ectopic gene conversion in a gene cluster affording environmental adaptation PLOS Genetics, 9, e X. Henry, O. Verlaine, A. Amoroso, J. Coyette, JM Frère and B. Joris Activity of ceftaroline against Enterococcus faecium PBP5 Antimicrob. Agents Chemother., 57, V. Houbart, E. Rozet, A. Matagne, J. Crommen, A.C. Servais and M. Fillet Influence of sample and mobile phase composition on peptide retention behaviour and sensitivity in reversed-phase liquid chromatography/mass spectrometry Journal of Chromatography A, 1314,

48 SCIENTIFIC PRODUCTION MC Jonville, G. Dive, L. Angenot, J. Bero, M. Tits, E. Ollivier and M. Frédérich Dimeric bisindole alkaloids from the stem bark of Strychnos nux-vomica L Phytochemistry, 87, Y. Lara, A. Lambion, D. Menzel, G.A. Codd and A. Wilmotte A cultivation-independent approach for the genetic and cyanotoxin characterization of colonial cyanobacteria Aquat. Microb. Ecol., 69, JC Monbaliu, G. Dive, C.V. Stevens and A.R. Katritzky Governing parameters of long-range intramolecular S-to-N acyl transfers within (S)-acyl isopeptides J. Chem.Theory Comp., 9, M. Perilli, G. Celenza, P.S. Mercuri, M. Galleni, C. Pellegrini, B. Segatore and G. Amicosante R164H and V240H replacements by site-directed mutagenesis of TEM-149 extended-spectrum β- lactamase: kinetic analysis of TEM-149 H240 and TEM-149 H164-H240 laboratory mutants Antimicrobial. Agents and Chemotherapy, 57, M. Rocaboy, R. Herman, E. Sauvage, H. Remaut, K. Moonens, M. Terrak, P. Charlier and F. Kerff The crystal structure of the cell division amidase AmiC reveals the fold of the AMIN domain, a new peptidoglycan binding domain Mol. Microbiol., 90, G. Roussel, E.A. Perpète, A. Matagne, E. Tinti and C. Michaux Towards a universal method for protein refolding: the trimeric beta barrel membrane Omp2a as a test case Biotechnology and Bioengineering, 110, G. Schyns, C.R. Serra, A.O. Henriques, A. Arguelles-Arias, B. Joris and P. Fickers Isolation of the antimicrobial cyclic peptide subtilosin A from a gut-associated Bacillus subtilis strain Am. J. Biochem. Biotechnol., 9, L.J. Smith, A.M. Bowen, A. Di Paolo, A. Matagne and C. Redfield The dynamics of lysozyme from bacteriophage lambda in solution probed by NMR and MD simulations ChemBioChem, 14, J.S. Sohier, C. Laurent, A. Chevigné, E. Pardon, V. Srinivasan, U. Wernery, P. Lassaux, J. Steyaert and M. Galleni Allosteric inhibition of VIM metallo-beta-lactamase by a camelid nanobody Biochem. J., 450, C. Struvay, S. Negro, A. Matagne et G. Feller Energetics of protein stability at extreme environmental temperatures in bacterial trigger factors Biochemistry, 52, E. Tenconi, P. Guichard, P. Motte, A. Matagne and S. Rigali Use of red autofluorescence for monitoring prodiginine biosynthesis J. Microbiol. Methods, 93, E. Van der Heiden, M. Delmarcelle, S. Lebrun, R. Freichels, A. Brans, C.M. Vastenavond, M. Galleni and B. Joris A pathway closely related to the D-Tagatose pathway of Gram-negative enterobacteria identified in the Gram-positive bacterium Bacillus licheniformis

49 SCIENTIFIC PRODUCTION Appl. Environ. Microbiol., 79, N.V. Venkatesh, S.A. Adediran, D. Kinjal, C. Duez and R.F. Pratt Dual substrate specificity of Bacillus subtilis PBP4a Biochemistry, 52, N. Verbruggen, M. Hanikenne and S. Clemens A more complete picture of metal hyperaccumulation through next-generation sequencing technologies Front. Plant Sci., 4, 1-7 A. Zervosen, A. Zapun and JM Frère Inhibition of Streptococcus pneumoniae penicillin-binding protein 2x and Actinomadura R39 DDpeptidase activities by ceftaroline Antimicrobial. Agents and Chemotherapy, 57, BOOK CHAPTERS AND REVIEWS C. Bauvois, A. L. Huston and G. Feller The cold-active M1 aminopeptidase from the Arctic bacterium Colwellia psychrerythraea Handbook of Proteolytic Enzymes 3 rd edition, chapter 95, pp Eds N.D. Rawlings and G.S Salvesen Oxford Academic Press, Elsevier P. Charlier, J.M. Ghuysen and J.M. Frère Zinc D-Ala-D-Ala carboxypeptidase Handbook of Proteolytic Enzymes 3 rd edition, chapter 312, Eds N.D. Rawlings and G.S Salvesen Oxford Academic Press, Elsevier P. Charlier, J.M. Ghuysen and J.M. Frère Streptomyces K15 D-Ala-D-Ala transpeptidase Handbook of Proteolytic Enzymes 3 rd edition, chapter 765, pp Eds N.D. Rawlings and G.S Salvesen Oxford Academic Press, Elsevier J. Coyette and M. Mergeay Microbiologie Ed. De Boeck (2013 4ème édition) Traduction de Prescott s Microbiology ( th edition), J.M. Willey, L.M.Sherwood and C.J. Woolverton Mc Graw Hill G. Feller Psychrophilic enzymes: from folding to function and biotechnology Hindawi Publishing Corporation Scientifica Volume 2013, Article ID , pp J.M. Frère and J. Van Beeumen DmpA L-aminopeptidase D-Ala-D-Ala esterase/amidase of Ochrobactrum anthropi Handbook of Proteolytic Enzymes 3 rd edition, chapter 812, pp Eds N.D. Rawlings and G.S Salvesen

50 SCIENTIFIC PRODUCTION Oxford Academic Press, Elsevier C. Huynen, P. Filée, A. Matagne, M. Galleni and M. Dumoulin Class A beta-lactamase as versatile scaffolds to create hybrid enzymes: applications from basic research to medicine Hindawi Publishing Corporation BioMed Research International Volume 2013, Artilce ID , pp R.F. Pratt and J.M. Frère Streptomyces R61 D-Ala-D-Ala carboxypeptidase Handbook of Proteolytic Enzymes 3 rd edition, chapter 766, Eds N.D. Rawlings and G.S Salvesen Oxford Academic Press, Elsevier E. Sauvage and J.M. Frère Actinomadura R39 D-Ala-D-Ala carboxypeptidase Handbook of Proteolytic Enzymes 3 rd edition, chapter 771, Eds N.D. Rawlings and G.S Salvesen Oxford Academic Press, Elsevier PUBLICATIONS 2014 A. Arguelles-Arias, B. Joris and P. Fickers Dual mode of action of amylopsin, a type-b lantibiotic produced by Bacillus amyloliquefaciens GA1 Protein Pept. Lett., 4, N. Ben Achour, O. Belhadj, M. Galleni, M. Ben Moussa and PS Mercuri Study of a natural mutant SHV-type beta-lactamase, SHV-104 from Klebsiella pneumoniae Int. J. Microb., 2014, T. Denoël, A. Zervosen, T. Gerards, C. Lemaire, B. Joris, D. Blanot and A. Luxen Stereoselective synthesis of lanthionine derivatives in aqueous solution and their incorporation into the peptidoglycan of Escherichia coli Bioorg. Med. Chem., 22, T. Denoël, A. Zervosen, C. Lemaire, B. Joris, M. Hervé, D. Blanot, G. Zaragoza and A. Luxen Enantioselective synthesis of alpha-benzylated lanthionines and related tripeptides for biological incorporation into E. coli peptidoglycan Org. Biomol. Chem., 12, J. de Ruyck, M.W. Janczak, S.S. Neti, S.C. Rothman, H.L. Schubert, R.M. Cornish, A. Matagne, J. Wouters and C.D. Poulter Determination of kinetics and the crystal structure of a novel type 2 isopentenyl diphosphate: dimethylallyl diphosphate isomerase from Streptococcus pneumoniae ChemBioChem, 15, S. Golubic and A. Wilmotte The phycologist Pierre Compère: his contribution to cyanobacterial studies Plant Ecology and Evolution, 147,

51 SCIENTIFIC PRODUCTION M. Hanikenne and D. Baurain Origin and evolution of metal P-type in Plantae (Archaeplastida) Front. Plant Sci., 4, 1-15 J. Herman, N. Thelen, N. Smargiasso, AC Mailleux, A. Luxen, M. Cloes, E. De Pauw, A. Chevigné, M. Galleni and ME Dumez Der p 1 is the primary activator of Der p 3, Der p 6 and Der p 9 the proteolytic allergens produced by the house dust mite Dermatophagoides pteronyssinus Biochim. Biophys. Acta, 1840, J.F. Imhoff and A. Wilmotte International Committee on Systematics of Prokaryotes. Subcommittee on the taxonomy of phototrophic bacteria: Minutes of the meetings, 11 August 2009, Montreal, Canada Int. J. Syst. Evol. Microbiol, 64, S. Lambert, F.M. Traxler, M. Craig, M. Maciejewska, M. Ongena, G. P. van Wezel, R. Kolter and S. Rigali Altered desferrioxamine-mediated iron utilization is a common trait of bald mutants of Streptomyces coelicolor Metallomics, 8, K. Lepot, P. Compère, E. Gérard, Z. Namsaraev, E. Verleyen, I. Tavernier, D.A. Hodgson, W. Vyverman, B. Gilbert, A. Wilmotte and E.J. Javaux Organic and mineral imprints in fossil photosynthetic mats of an East Antarctic lake Geobiology, 12, (+ supplementary material) C. Liao, S. Rigali, C. Licona Cassani, E. Marcellin, L. Keld Nielsen and B-C Ye Control of chitin and N-acetylglucosamine utilization in Saccharopolyspora erythraea Microbiology, 160, M. Lismont, N. Vandewalle, B. Joris and L. Dreesen Fiber based optofluidic biosensors Appl. Phys. Lett., 105, R. Locht, D. Dehareng and B. Leyh The threshold photoelectron spectrum of the geminal chloro-fluoro-ethene (1, 1-C 2 H 2 FCl) isomer. Experiment and theory. J. Phys. B: At. Mol. Opt. Phys., 47, doi: / /47/8/ (+ supplementary material) A. Pérez, F.J. Pérez-Llarena, P. Garcia, F. Kerff, A. Beceiro, M. Galleni and G. Bou New mutations in ADC-type beta-lactamase from Acinetobacter spp affect cefotoxin and ceftazidime hydrolysis J.Antimicrob. Chemother, 69, F.J. Pérez-LLarena, L. Zamorano, F. Kerff, A. Beceiro, P. Garcia, E. Miró, N. Larrosa, F. Gómez- Bertomeu, J. Antonio Méndez, J.J. González-López, A. Oliver, M. Galleni, F. Navarro and G. Bou Genetic and kinetic characterization of the novel AmpC beta-lactamase DHA-6 and DHA-7 J.Antimicrob. Chemother, 58, M. Ruggiero, F. Kerff, R. Herman, F. Sapunaric, M. Galleni, G. Gutkind, P. Charlier, E. Sauvage and P. Power Crystal structure of the extended-spectrum β-lactamase PER-2 and insights into the role of specific residues in the interaction with β-lactams and β-lactamase inhibitors J. Antimicrob. Chemother., 58,

52 SCIENTIFIC PRODUCTION E. Sauvage, A. Derouaux, C. Fraipont, M. Joris, R. Herman, M. Rocaboy, M. Schloesser, J. Dumas, F. Kerff, M. Nguyen-Distèche and P. Charlier Crystal structure of penicillin-binding protein 3 (PBP3) from Escherichia coli PlosOne, 9, e98042 A. T. Tchinda, O. Jansen, J.N. Nyemb, M. Tits, G. Dive, L. Angenot and M. Frédérich Strychnobaillonine, an unsymmetrical bisindole alkaloid with an unprecedented skeleton from Strychnos icaja roots J. Nat. Prod., 77, (+ supporting information) E. Vassal-Stermann, M. Lacroix, E. Gout, E. Laffly, C.M. Pedersen, L. Martin, A. Amoroso, R.R. Schmidt, U. Zähringer, C. Gaboriaud, A.-M. Di Guilmi and N.M. Thielens Human L-ficolin recognizes phosphocholine moieties of pneumococcal teichoic acid J. Immunol., 193, L.L. Yao, C.H. Liao, G. Huang, Y. Zhou, S. Rigali, B. Zhang and B.C. Ye GlnR-mediated regulation of nitrogen metabolism in the actinomycete Saccharopolyspora erythraea Appl. Microbiol. Biotechnol., 98, BOOK CHAPTERS AND REVIEWS A. Derouaux, M. Terrak, T. den Blaauwen and W. Vollmer Bacterial Cell Wall Growth, Shape and Division Bacterial Membranes: Structural and Molecular Biology, chapter 1, 3-54 Eds H. Remaut and R. Fronzes Caister Academic Press ME Dumez, J. Herman, V. Campisi, M. Galleni, A. Jacquet and A. Chevigné Orchestration of an uncommon maturation cascade of the house dust mite protease allergen quartet Front. Immunol., 5, 138 JM Frère and M. Page Penicillin-binding proteins: evergreen drug targets Curr. Opin. Pharmacol., 18, M. Hanikenne, M. Bernal and EI Urzica Ion homeostasis in the chloroplast Plastid Biology, Advances in Plant Biology 5, chapter 17, Eds S.M. Theg, F.-A. Wollman Springer Science + Business Media, New-York

53 SCIENTIFIC PRODUCTION PROTEIN STRUCTURES DEPOSITED WITHIN THE PROTEIN DATA BANK PDB ID STRUCTURE TITLE AUTHOR 3ZNT 3ZNY 4BIN CRYSTAL STRUCTURE OF OXA-24 CLASS D BETA-LACTAMASE WITH TAZOBACTAM CRYSTAL STRUCTURE OF THE CLASS A EXTENDED-SPECTRUM BETA-LACTAMASE CTX- M-96, A NATURAL D240G MUTANT DERIVED FROM CTX-M-12 CRYSTAL STRUCTURE OF THE E. COLI N-ACETYLMURAMOYL-L- ALANINE AMIDASE AMIC Power, P., Sauvage, E., Herman, R., Kerff, F., Charlier, P. Power, P., Herman, R., Bouillenne, F., Ghiglione, B., Rodriguez, M.M., Galleni, M., Gutkind, G., Charlier, P., Sauvage, E. Kerff, F., Rocaboy, M., Herman, R., Sauvage, E., Charlier, P. 4BZE 4BZF CRYSTAL STRUCTURE OF GALACTOSE MUTAROTASE GALM FROM BACILLUS SUBTILIS CRYSTAL STRUCTURE OF GALACTOSE MUTAROTASE GALM FROM BACILLUS SUBTILIS WITH TREHALOSE Vanden Broeck, A., Sauvage, E., Herman, R., Kerff, F., Duez, C., Charlier, P. Vanden Broeck, A., Sauvage, E., Herman, R., Kerff, F., Duez, C., Charlier, P. 4BZG 4BZH CRYSTAL STRUCTURE OF GALACTOSE MUTAROTASE GALM FROM BACILLUS SUBTILIS IN COMPLEX WITH MALTOSE CRYSTAL STRUCTURE OF GALACTOSE MUTAROTASE GALM FROM BACILLUS SUBTILIS IN COMPLEX WITH MALTOSE AND TREHALOSE Vanden Broeck, A., Sauvage, E., Herman, R., Kerff, F., Duez, C., Charlier, P. Vanden Broeck, A., Sauvage, E., Herman, R., Kerff, F., Duez, C., Charlier, P. 4M3J STRUCTURE OF A SINGLE DOMAIN CAMELID ANTIBODY FRAGMENT CAB-H7S SPECIFIC OF THE BLAP BETA-LACTAMASE FROM BACILLUS LICHENIFORMIS Pain, C., Kerff, F., Herman, R., Sauvage, E., Preumont, S., Charlier, P., Dumoulin, M. 4M3K STRUCTURE OF A SINGLE DOMAIN CAMELID ANTIBODY FRAGMENT CAB-H7S IN A COMPLEX WITH THE BLAP BETA-LACTAMASE FROM BACILLUS LICHENIFORMIS Pain, C., Kerff, F., Herman, R., Sauvage, E., Preumont, S., Charlier, P., Dumoulin, M. 4BJP CRYSTAL STRUCTURE OF E. COLI PENICILLIN BINDING PROTEIN 3 Sauvage, E., Joris, M. Herman, R., Kerff, F., Rocaboy, M., Charlier, P. 4BJQ CRYSTAL STRUCTURE OF E. COLI PENICILLIN BINDING PROTEIN 3, DOMAIN V88-S165 Sauvage, E., Joris, M., Herman, R., Kerff, F., Rocaboy, M., Charlier, P. 4D2O CRYSTAL STRUCTURE OF THE CLASS A EXTENDED-SPECTRUM BETA-LACTAMASE PER-2 Power, P.,Herman, R., Ruggiero, M., Kerff, F., Galleni, M., Gutkind, G., Charlier, P., Sauvage, E. 4N1H STRUCTURE OF A SINGLE-DOMAIN CAMELID ANTIBODY FRAGMENT CAB-F11N IN COMPLEX WITH THE BLAP BETA-LACTAMASE FROM BACILLUS LICHENIFORMIS Pain, C., Kerff, F.,Herman, R., Sauvage, E., Preumont, S., Charlier, P., Dumoulin, M

54 SCIENTIFIC PRODUCTION SYMPOSIA 2013 The Bioforum of BioLiège, University of Liège, Belgium, April 18 Main organizers: Prof. J. Dommes, B. Joris, T. Dauvrin (BioLiège association) Eleventh Meeting of the Belgian Biophysical Society on Protein Folding and Stability, University of Liege, Belgium, August 30 Main organizer: Prof. A. Matagne SFMBBM doctoral school meeting University of Liège, Belgium, November 15 Main organizers: Prof A. Matagne, C. Laurent, C. Huynen, G. Lekeux and V. Campisi First meeting of the Belgian Interdisciplinary Biofilm Research (FRS-FNRS BIBR contact group) UCL, Louvain-La-Neuve, December 20 Main organizers: Dr C. Duez, T. Vanzieleghem and Prof. J. Mahillon SYMPOSIA 2014 The Bioforum of BioLiège, University of Liège, Belgium, May 15 Main organizers: Prof. J. Dommes, B. Joris, T. Dauvrin (BioLiège association) Journées Actinomycètes, University of Liège, Belgium, May Main organizer: Dr S. Rigali Session S22 at the XXXIII SCAR Biennal Meetings and Open Science Conference 2014, Scientific Advice for Policymakers and Evidence-based Conservation (AntEco), August 28 Main organizers: A. Wilmotte and K. Hughes Twelfth Meeting of the Belgian Biophysical Society on Protein Folding and Stability, University of Liege, Belgium, August 29 Main organizer: Prof. A. Matagne Joint meeting of the National Committee for Biophysics and the Belgian Biophysical Society on Modern Biophysical Techniques for the Life Sciences, Palais des Académies, Brussels, Belgium, October Organizing and scientific committee: Prof. A. Matagne Second meeting of the Belgian Interdisciplinary Biofilm Research (FRS-FNRS BIBR contact group) UCL, Louvain-La-Neuve, December 12 Main organizer: Dr C. Duez

55 ACADEMIC COURSES EDUCATION Bachelor and Preparation to Masters Biochimie, 30 h + 30 h Pr - BIOC P. Charlier Bac 2 Sciences de l'ingénieur, orientation ingénieur civil, option génie biomédical Biochimie, 30 h - CHIM A. Matagne Bac3 Sciences chimiques et année préparatoire aux Sciences Chimiques Biochimie et thermodynamique des systèmes biologiques, 40h + 20h Pr - BIOC M. Galleni Bac 2 Sciences biologiques. Biologie et introduction à la biochimie, 30h + 30h Pr - BIOL B. Joris Bac 2 Sciences Chimiques Chimie des macromolécules biologiques, 60h + 40h Pr + 4h de visite d usine - BIOC0209-3/4/6 - M. Galleni et A. Matagne Bac 3 Sciences biologiques et année préparatoire aux masters en Biochimie et Biologie Moléculaire et Cellulaire (BBMC) et Biologie des Organismes et Ecologie (BOE) Chimie des macromolécules biologiques etthermodynamique des systèmes biologiques, 70h + 40h Pr + 4h de visite d usine - BIOC M. Galleni et A. Matagne Année préparatoire aux masters en sciences biologiques Documentation, stages et séminaires (étudiants), 50h St. - STRA J. Dommes et P. Motte Bac 3 Sciences biologiques et année préparatoire aux masters BBMC et BOE Génétique, biologie moléculaire et chimie des macromolécules, 30h + 30h Pr - BIOC J. Dommes et M. Galleni. Bac 3 Sciences biologiques et année préparatoire au master BOE. Microbiologie - MICR Partim 2 : Bactériologie : 20h + 10h Pr B. Joris Bac 3 et années préparatoires aux masters BBMC et BOE Physiologie cellulaire et histologie végétales, 30h Th + 20h Pr - BIOL P. Motte et C. Périlleux - Bac 2 en Sciences biologiques Physiologie végétale, 40h Th + 25h Pr -BIOL P. Motte et C. Périlleux Bac 3 et année préparatoire aux masters en sciences biologiques Principes généraux de la biologie et de la biochimie, 15 h - CHIM P. Charlier 3 e année Ingénieur civil chimiste Masters Analyse des séquences des gènes et des protéines : partim a, 10h, 10h Pr - GBIO B. Joris Master 2 en Bioinformatique et modélisation, finalité approfondie et master 2 en Ingénieur civil biomédical, finalité approfondie Application des techniques spectroscopiques à l étude du repliement et de la stabilité des protéines, 20h+10h TD - BIOC0722 A. Matagne

56 EDUCATION Approches moléculaires de la diversité des microorganismes marins, 15h + 15h Pr BOT A0401- A. Wilmotte - Master 2 en Océanographie, finalité approfondie Aspects génétiques et biochimiques de l'évolution, 25h + 20h Pr - GENE M. Galleni et C. Remacle. Masters 1 BBMC et BOE Astrobiologie, 30h Th. GEOL Ph. Claeys, Véronique Dehant, M. Galleni, E. Javaux, Y. Nazé et A. Wilmotte. Master 2 en Biologie des Organismes et Ecologie, à finalité approfondie Biochimie, 30 h + 30 h Pr - BIOC P. Charlier Master 1 en Ingénieur civil biomédical, finalité approfondie Biochimie, 30 h + 40 h Pr - BIOC P. Charlier Master 1 en Bioinformatique et modélisation, finalité approfondie Biochimie et physiologie des microorganismes, 20h + 20h Pr - BIOC B. Joris Masters 1 BBMC et BOE Biochimie macromoléculaire, 30h + 30h Pr - BIOC M. Galleni - Master 1 Sciences chimiques Bioinformatique appliquée, 10h Th + 10h Pr - BIOC b - B. Joris - Master 2 BBMC Chimie des macromolécules biologiques, 60h + 40h Pr + 4h de visite d usine - BIOC0209-3/4 M. Galleni et A. Matagne - Master générique en Sciences biologiques Compléments de microbiologie : pathogénicité bactérienne, 15h Th - MICR a - B. Joris Master 1 BBMC Compléments de physiologie cellulaire végétale, 30h Th. - BIOL P. Motte - Master 2 BOE Compléments de physiologie moléculaire et cellulaire, 40h Th + 20h Pr - BIOL P. Motte, M. Muller et M. Thiry - Master 1 BBMC Développement des microorganismes, 15h Th. BIOL S. Rigali - Master 1 BBMC Enzymologie, 15h - BIOC A. Matagne - Master 1 Sciences chimiques Enzymologie, 15h + 25h Pr - BIOC A. Matagne - Master 1 Bioinformatique et modélisation Functional and Molecular Marine Microbiology, Molecular approaches to the diversity of marine microorganisms, 15h + 15h Pr. - OCEA A. Wilmotte - Master 2 en Océanographie à finalité approfondie (en 2014) Génomique, 20h + 20h Pr GENE M. Hanikenne - Master 1 BBMC Interactions dans les macromolécules biologiques, 20h + 20h Pr BIOC M. Galleni - Master 1 Bioinformatique et modélisation, finalité approfondie Introduction to systems and synthetic biology, 30h Th + 30h Pr - GBIO a - B. Joris - Master 2 en Bioinformatique et modélisation Introduction to synthetic biology. 10h Th + 20h Pr - GBIO a - B. Joris et F. Delvigne - Master 2 en Bioinformatique et modélisation

57 EDUCATION Méthodes de visualisation et de quantification en biologie cellulaire, 30h Th - BIOL P. Motte Master 2 BOE. Microorganismes extremophiles, 25h Th MICR G. Feller, M. Galleni et A. Wilmotte Master1 BBMC Molecular and cellular basis of disease: Protein misfolding and aggregation diseases, generalities, 1 h - SBIM M. Dumoulin - Master 2 Sciences Biomédicales Multidisciplinary English New therapeutic approaches to disease: Various uses of Nanobodies in diagnosis and treatment, 2 h - SBIM M. Dumoulin - Master 2 Sciences Biomédicales Multidisciplinary English Principes généraux de la biologie et de la biochimie, 15h - CHIM P. Charlier - Master 2 Ingénieur civil en chimie et sciences des matériaux, finalité approfondie Propriétés fonctionnelles des macromolécules biologiques, 20h+10h TD+ 20h Pr - BIOC A. Matagne - Master 1 BBMC et Sciences Biologiques Propriétés optiques des macromolécules biologiques, 15h + 20h Pr. BIOC0721-A C. Damblon et A. Matagne - Master 1 BBMC et BOE Relations structure-fonction dans les biomolécules, 15h + 25h Pr - BIOC M. Dumoulin Master 2 Ingénieur civil biomédical, finalité approfondie Structure des macromolécules biologiques, 20h + 10h Pr - CHIM P. Charlier - Master 2 Bioinformatique et modélisation, finalité approfondie Structure des macromolécules biologiques (RX, RMN), 15h + 10h Pr - CHIM P. Charlier. Master 2 Bioinformatique et modélisation, finalité approfondie Voies de signalisation chez les végétaux, 25h Th + 25h Pr. - BOTA J. Dommes, P. Motte et C. Périlleux - Master 2 BBMC Inter University Thematic Weeks Antibiotic resistance, 25h BIOC J.-M. Frère, M. Galleni et B. Joris - Master 2 BBMC Biologie cellulaire et méthodes de visualisation, h Pr - BIOL P. Motte et M. Thiry Master 2 BBMC. Microorganismes extrémophiles, h Pr - MICR M. Galleni, G. Feller et A. Wilmotte Master 2 BBMC. Structure et fonction des protéines, 25h + 25h Pr - BIOC P. Charlier et M. Dumoulin Master 2 BBMC, finalités approfondie, didactique et industrielle Visualisation et modélisation des protéines h Pr - BIOC P. Charlier, F. Kerff et E. Sauvage - Master 2 BBMC

58 EDUCATION Complementary Masters Biochimie, 30h + 30h Pr - BIOC P. Charlier - Master complémentaire en Nanotechnologie Chimie des macromolécules biologiques, 20h - BIOC0209-3/4/6 - M. Galleni et A. Matagne Master complémentaire en Nanotechnologie Génie génétique des bactéries, 15h GENE Biotechnologie et Biologie appliquée A. Brans - Master complémentaire en Microbiologie - MICR Partim 2 : Bactériologie : 20h + 10h Pr B. Joris - Master complémentaire en Biotechnologie et Biologie appliquée Propriétés fonctionnelles des macromolécules biologiques, 20h+10h TD+ 20h Pr - BIOC A. Matagne - Master complémentaire en Nanotechnologie Third Cycle Advanced course on «Protein purification» - 15h - SDOC E. Depauw, J.-M. Frère, M. Galleni, B. Joris et A. Matagne, May 6-8, 2013 Approches Moléculaires de la diversité des microorganismes marins, 15h + 15h Pr - BOTA0401 A. Wilmotte (2013) Production de protéines recombinantes en systèmes procaryotes, 15h - SDOC C. Duez Courses given abroad Bioinformatique, 35 h - A. Brans - Bac 3 DUT Génie biologique. IUT de Mont de Marsan, Université de Pau et des Pays de l Adour, France (novembre 2013 et novembre 2014) Enzyme kinetics, Protein folding and Protein Purification - A. Matagne, J.-M. Frère et M. Galleni. Masters en Biotechnologie et Microbiologie. Cycle de 3 ans, 15h/an. Università degli Studi di Siena, Siena, Italy Nanobodies or camelid antibody fragments: Properties and application, 7 h M. Dumoulin Department of Pharmaceutical Sciences, University of Padova, Italy, November 28-29, 2013 Production de protéines recombinantes, 8 h - M. Delmarcelle - Bac 3 DUT Génie biologique -IUT de Mont de Marsan. Université de Pau et des Pays de l Adour, France (novembre 2013 et novembre 2014) Nanobodies or camelid antibody fragments: Properties and application, 8 h M. Dumoulin Department of Pharmaceutical Sciences, University of Padova, Italy, December Courses given in another Belgian university Biologie végétale, 30h + 30 Pr P. Motte. Bac 2 Pharmacie et Bac 3 Biologie Université de Mons An introduction to microcalorimetric studies of proteins, University of Namur FUNDP, Belgium, G. Feller, 2h. December 10,

59 EDUCATION Mécanismes de résistance aux antibiotiques (Formation continue pour les pharmaciens hospitaliers en Belgique), University of Namur FUNDP, Belgium, JM Frère, 2h. February 21, 2013 Courses given in Technical High Schools Génie génétique et enzymatique, 30h Partim 1 - A. Brans. Master 1 en Sciences de l ingénieur industriel, finalité biochimie. Haute Ecole de la Province de Liège, Quai Gloesener 6, 4020 Liège (2013) Génie génétique et enzymatique, 30h Partim 2 - A. Brans. Master 2 en Sciences de l ingénieur industriel, finalité biochimie. Haute Ecole de la Province de Liège, Quai Gloesener, Liège (2013)

60 EDUCATION TRAINEES AND STUDENTS Bac 3 Trainees Bac 3 Trainees DEVLEMINCK Thomas WARIN Alicia GILLOT Lionel Master I Trainees Master I Trainees ABU JAHRUR Nora BARTHOLOME Odile COOLS Melody DESCHEEMAEKER Célia DEVAUX Floriane DJIAHA NOUSSIYA Sylviane DOMMES Stéphane FANARA Steven GENET Lidvine GILSOUL Maxime HOEBRECK Charline HORRION Bastien LEGRAND François LEPAIGE Ulric LUIS Géraldine MARIAVELLE Emeline NOIRFALISE Pauline POLLENUS Thomas SANJUAN PACHECO Wilmer SQUIFFLET Steeve VACCARELLA Marie-Ange VANDEN BROECK Arnaud WARNITZ Sarah WERA Odile ZUTTERMAN Pascal ADAM Delphine BEZZAOU Warda BIGURI Arrate BOES Adrien CASTELLANOS Oscar CORATO Amélie DEBLANDER Victor DOMMES Stéphane DURAY Elodie LEGRAND François MANI-VANGELIS Mahel MEUNIER Loïc MICHAUX Stéphane POUDEVIGNE Loïc ROBLAIN Quentin ROMPEN Stephan VANDAMME François VANDERVELDEN Geoffrey VAN VLIERBERGHE Mick WARNITZ Sarah Master II Students ALLEGRO Lucas LAMBERT Damien Master Ingénieur Industriel, option chimie. HEPL, ISIL, Quai Gloesener, 6, 4020 Liège Conception d un système asservi pour le suivi de la croissance microbienne lors d une fermentation continue et automatisation d une unité de séparation de protéines Master Ingénieur Industriel, option chimie. HEPL, ISIL, Quai Gloesener, 6, 4020 Liège Développement et mise en œuvre d'un système de monitoring à l'aide du logiciel LabView dans le cadre d'une électrophorèse en chip microfluidique

61 EDUCATION LECLERCQ Sophie NEMERY Quentin PIERRE Barbara RICH Bryan TRUS Caroline UNGARO Benjamin Master BBMC à finalité approfondie, ULg Purification des protéines FtsW et PBP3 d E. coli et étude de leur effet sur la synthèse du peptidoglycane Master BBMC à finalité approfondie, ULg Etude de la régulation de la protéine PBP5 résistante chez Enterococcus hirae Bachelier-technologue de laboratoire, option cytologie, biologie médicale. HEPL Quai du Barbou, 2, 4020 Liège Caractérisation de souches de cyanobactéries Master en sciences Biomédicales à finalité approfondie en cancérologie et immunologie, ULg Construction génétique, production et caractérisation des deux domaines isolés de la β-lactamase de Bacillus cereus 569/H/9 Master en sciences Biomédicales à finalité approfondie en analyses biologiques, ULg Mise en évidence de la présence d'antigènes spécifiques et conservés à la surface des différents sérotypes de Streptococcus agalactiae Master en sciences de l ingénieur industriel à finalité biochimie, ISIL, Quai Gloesener, 6, 4020 Liège Conception et automatisation d une unité pilote de fermentation en vue d une production continue d enzyme intracellulaire Master II Students BEZOUICH Imen BREBAN Kevin FANARA Steven HOEBRECK Charline Master 2 Ingénieur génie protéique, biologie moléculaire et biochimie. Université de Lorraine, Nancy, France Analyse structurale et fonctionnelle de Cav p 6 et Cav p 1, deux lipocalines décrites comme allergènes majeurs du cobaye domestique Master en Sciences de l'ingénieur Industriel finalité Biochimie. HEPL ISIL. Quai Gloesener, Liège Contribution au développement d'un système de fermentation en continu pour la production de la L-arabinose isomérase Master BBMC à finalité approfondie, ULg Étude fonctionnelle des facteurs d épissage SR de la sousfamille RS2Z d Arabidopsis thaliana Master BBMC à finalité approfondie, ULg Etude du rôle des prodiginines sur le développement de Streptomyces coelicolor

62 EDUCATION KABANYANA Françoise KAY Jennifer LAMBERT Damien LE PAIGE Ulric NERA Quentin NOIRFALISE Pauline SCHEEPERS Maxime VANDEN BROECK Arnaud VREVEN Aurélie Master BBMC à finalité spécialisée en bioindustrie Etude de l induction de la β-lactamase BlaP chez Bacillus licheniformis Master BBMC à finalité approfondie, ULg Investigation of the effects of the insertion of a polyq tract increasingly long on the structure and dynamics of BlaP using NMR spectroscopy Master Ingénieur Industriel, option chimie. ISIL, quai Gloesner 6, 4020 Liège. Développement et mise en œuvre d'un système de monitoring à l'aide du logiciel LabView dans le cadre d'une électrophorèse en chip microfluidique Master BBMC, à finalité approfondie, ULg Etudes structurales par RMN de la métallo beta-lactamase CAU-1 Master en science de l'ingénieur industriel, finalité chimie. ISIL, Quai Gloesner 6, 4020 Liège Mise au point, informatisation et automatisation d une installation de filtration tangentielle. Master BBMC à finalité approfondie, ULg Genome Mining for Polysaccharide-degrading Enzymes from Karstic Actinomycetes Master BBMC, ULg Etude de l expression du gène FRD3 chez deux espèces d Arabidopsis Master BBMC à finalité approfondie, ULg Etude des racémases RacX et YlmE et de la protéine PBP4* de Bacillus subtilis en relation avec la désintégration de biofilms. Master Bioingénieur en Chimie et Bio-industries, Gemboux Agro Bio Tech, ULg Expression, purification et caractérisation d un inhibiteur de trypsine de type Kunitz, issu de la papaye (PaPI) Erasmus Students - Master II COLARUSSO Andrea Graduate in Biomolecular and Industrial Biotechnology, Faculty of Biotechnological sciences, University Federico II of Napoli, Italy Investigation into the activity and stereoselectivity of D- tagatose 1,6-bisphosphate aldolases

63 EDUCATION LAVECCHIA Francesco Graduate in Biomolecular and Industrial Biotechnology, Faculty of Biotechnological sciences, University Federico II of Napoli, Italy Functional and structural characterisation of Arabidopsis halleri HMA4 N-terminal domain MARITAN Martina Master in Pharmaceutical Biotechnologies, Faculty of Biotechnological sciences, University of Padova, Italy Investigation of the mechanism of fibril formation by characterizing of chimeric proteins made of the betalactamase BlaP and polyq sequences PARENTE Raffaella Graduate in Biomolecular and Industrial Biotechnology, University Federico II of Napoli, Italy Functional characterization of chitin binding domain from human macrophage chitotriosidase based on ELISA and ITC experiments TIEPPO Paola Master in Pharmaceutical Biotechnologies, Faculty of Biotechnological sciences, University of Padova, Italy Production and characterization of human lysozyme produced in E. coli: a study to investigate the effects of a methionine as first amino acid Erasmus Students - Master II 2014 BERTOLDI Loris CALLEGARO Sara CASTALDO Anna LITIME Djihad PALAZON Estefania Master II, Faculty of Pharmacy. University of Padova, Italy Design and characterization of nanotools for the investigation of amyloid fibril formation Master II, Faculty of Pharmacy. University of Padova, Italy Effects of a series of ligands on the aggregation of model polyq proteins made of the beta-lactamase BlaP and polyq tracts of different lengths Bachelor 3 in: Biotecnologie Biomolecolari ed Industriali. Università degli studi di Napoli Frederico II, Napoli, Italy Recombinant production and purification of human alternative splicing factor ZRanB2 Master en BioSciences et Ingénierie de la Santé, Université de Lorraine, Nancy, France Clonage, Production et purification du canal potassique SK2. Graduat en Biotechnologie. University of Leon, Spain Etude de l'implication des protéases dans l'allergie aux acariens

64 EDUCATION Technical high schools 2013 BONDY Pauline DOL Mélanie HOLLANGE Elodie MAYNARD Benjamin NGUYEN THANH GIANG V. NONNON Delphine ROLAND Lilian VAN DEN BOSSCHE François YENAULT Graziella Licence Professionnelle de Biotechnologie, option biologie moléculaire appliquée à la sécurité alimentaire. IUT de Mont de Marsan, Université de Pau et des pays de l'adour, France Construction de plasmides pour la production continue de protéines recombinantes Bachelier-Technologue de laboratoire médical. Haute Ecole HELMO, 4000 Liège Caractérisation de souches de Cyanobactéries Bachelier-Technologue de laboratoire médical. Haute Ecole Charlemagne. CHU-B36/Tour 4 Etude de la régulation de l opéron ftsw-psr-pbp5 chez Enterococcus hirae Licence Professionnelle de Biotechnologie, option biologie moléculaire appliquée à la sécurité alimentaire. IUT de Mont de Marsan, Université de Pau et des pays de l'adour, France Mise en place d un système de maintien de plasmide basé sur l auxotrophie à la proline Bachelier-Technologue de laboratoire médical, HEPL, Quai du Barbou, 2, 4020 Liège Développement d une technologie permettant la production de lait délactosé Ecole de Biologie Industrielle (E.B.I) Cergy-Pontoise, France Essai de production du PBP4a de Bacillus amyloliquefaciens Etude du domaine III du PBP4a de Bacillus subtilis Recherche des métabolites secondaires produits par Streptomyces coelicolor Licence professionnelle en biologie moléculaire appliquée à la sécurité alimentaire, IUT Mont de Marsan, Université de Pau et des pays de l'adour, France Etudes de la résistance des plantes aux métaux Bachelier en Biochimie. HEPL Rennequin Sualem, Quai Gloesener, Liège Identification des gènes impliqués dans l autolyse bactérienne Licence Professionnelle de Biotechnologie, option biologie moléculaire appliquée à la sécurité alimentaire, IUT de Mont de Marsan, Université de Pau et des pays de l'adour, France Construction d un plasmide pour l amélioration de la production de la Green fluorescent Protein (GFP)

65 Technical high schools 2014 EDUCATION CHAPONNAY Lucille DANGEL Thomas DESCHAMPS Maxime FOTSO Albert GRISARD Johanna JOIRIS Olivier Bachelor 2 - DUT en Génie Biologique. IUT Louis Pasteur, Schiltigheim, France Etude de la bactérie Bacillus subtilis et caractérisation de sa galactose mutarotase GalM Bachelor 2 - DUT en Génie Biologique, IUT Louis Pasteur, Université de Strasbourg, France Contribution à l étude de deux transporteurs potentiellement impliqués dans l induction de la production de thaxtomine chez Streptomyces scabies Bachelier en biochimie. HEPL Rennequin Sualem, Liège Production, purification et caractérisation préliminaire d une estérase de Bacillus licheniformis 749/I. Contribution au séquençage du génome de souches de Bacillus licheniformis Bachelier-Technologue de laboratoire médical. Haute Ecole Charlemagne CHU-B36/Tour Etude de l activité promotrice au sein de l opéron ftsw-psrpbp5 chez Enterococcus hirae. Licence professionnelle en Biotechnologie, option biologie moléculaire, IUT Pau et Pays de l Adour, France Surexpression de la tagatose-1p kinase de Bacillus licheniformis en vue de sa caractérisation biochimique et mutagenèse dirigée d un opéron bicistronique codant pour un fragment d anticorps (Fab) dans le but de développer un test de qualité Bachelier en biochimie. HEPL Rennequin Sualem, Liège Caractérisation de souches de cyanobactéries LAFARGUE Paul DUT Génie Biologique option Industrie alimentaires et biologiques, IUT Pau et Pays de l Adour, France Clonage de fragments d anticorps et d un répresseur en vue de leurs caractérisations LAMBERT Marine LICOPS Maxime PAULUS Thomas Bachelier-Technologue de laboratoire médical. Haute Ecole Charlemagne CHU-B36/Tour 4 Induction de la β-lactamase BlaP chez Bacillus licheniformis. Bachelier-Technologue de laboratoire médical. HELMo Saint- Laurent Recherche et caractérisation d actinomycètes karstiques producteurs de nouveaux métabolites secondaires Bachelier - Technologue de Laboratoire Biomédical option cytologie, Haute Ecole André Vésale, Liège Détection de la microcystine, cylindrospermopsine et saxitoxine chez les cyanobactéries provenant de régions polaires

66 EDUCATION PALUSSIERE Simon Licence professionnelle Biotechnologies option biologie moléculaire, IUT Pau et Pays de l Adour, France Clonage, production et purification d une phénylalanine déshydrogénase et d une leucine déshydrogénase. TOUILLAUX Kathleen ZWAENEPOEL Manon Bachelier-Technologue de laboratoire médical option cytologie. HEPL André Vésale. Quai du Barbou 2, 4020 Liège Production, purification et caractérisation biochimique de la racémase YlmE de Bacillus subtilis ATCC Bachelier-Technologue de laboratoire médical, HELMo Saint- Laurent Contribution à l étude de l induction de la virulence de Streptomyces scabies

67 EDUCATION GENERAL PUBLIC ACTIVITIES Printemps des Sciences 2013 (March 18-22). Dr A. Wilmotte : atelier 'hands on' d'extraction des pigments de cyanobactéries "LL42 : Les capteurs solaires des végétaux " Printemps des Sciences 2014 (March 24-28). Dr A. Wilmotte, E. Javaux, M. Renard. Atelier 'hands on' LL11 : Les débuts de la vie et l'importance des cyanobactéries. Activities for students of secondary schools Atelier d'extraction de pigments pour la visite de l école St Roch de Theux, January 11, 2013 et April 4, Dr A. Wilmotte et M. Renard Atelier d'extraction des pigments de cyanobactéries et chromatographie sur couche mince de silice dans le cadre des laboratoires de biologie à la Haute Ecole Rennequin Sualem (Prof. Mme Frédérique Hubin) February 27, 2013 Atelier «Journée Enseignement Secondaire 4 ème édition» February 5, 2013 Accueil des Rhétoriciens au CIP February 4, 2014 par J.-M. Frère, F. Kerff, S. Leclercq et A. Wilmotte Journée «Portes Ouvertes : Destination avenir» - ULg - October 18, M. Vandevenne Articles and interviews Prof. B. Joris. A Broadcast on ULg.TV: Les bactéries vaincront-elles les antibiotiques? Prof. B. Joris and M. Galleni. A Broadcast on ULg.TV: Comprendre la protéine pour mieux saisir le vivant. Interview du Dr A. Wilmotte on WebTV sur le Traité Antarctique:

68 EDUCATION Interview du Dr A. Wilmotte dans le magazine 'Le 15e Jour' sur le Traité sur l Antarctique : Interview du Dr A. Wilmotte during the TV Broadcast 'Le Jardin Extraordinaire': Interview of Dr A. Wilmotte during the Radio broadcast 'Nuwa' sur l'antarctique à la RTBF, May 1, 2013 Article du Dr A. Wilmotte sur le site Réflexion de l'ulg: Outreach article 'Out of sight, out of mind, Antarctic microbial diversity as an additional criterion for conservation purposes', Verleyen E, Obbels D, De Wever A, Souffreau C, Vanormelingen P, Sabbe K, Vyverman W, Peeters K, Tytgat B, Willems A, Ertz D, Van de Vijver B, Mano M-J, De Carvalho Maalouf P, Fernández-Carazo R, Namsaraev Z & Wilmotte A. Science Connection (2013) 41, p Lecture given by Dr A. Wilmotte during the Science Fair of the Association for Polar Early Career Scientists (APECS) at the Academy in Brussels: "The forgotten heroes of Antarctica (microbial life)", May 26, 2013 Communiqué de presse. L ULg partenaire d un programme européen pour le développement de nouveaux antibiotiques. 13 février 2014 Interview du Prof. M. Galleni Jouranl télévisé de RTC Liège du 17/02/ Interview des Prof. P. Charlier et M. Galleni le 28 février 2014 pendant l émission O Positif (RTB1) de 11 à 12h: «Les nouveaux antibiotiques contre les pathogènes à Gram négatif», dans le cadre du projet ENABLE Article dans Le 15 e jour du mois, mars 2014 /232. L urgence antibiotique, le CIP au cœur d un consortium européen. Interview des Prof. P. Charlier et M. Galleni Article dans Le Vif, 31/03/2014. Présentation du projet Européen Mon4Strat. Interview du Prof. B. Joris. Interview du Prof. B. Joris par RTL-TVI le 1/04/2014. Présentation du projet Européen Mon4Strat aux journaux télévisés de 13h et 19h. Interview du Prof. B. Joris en radio : RTBF Vivacité, 01/04/2014. Présentation du projet Européen Mon4Strat au journal parlé de 7h30. Articles dans la presse écrite: Présentation du projet Européen Mon4Strat coordonné par le Prof. B. Joris

69 EDUCATION La Meuse 1/04/2014, page 10 La Libre Belgique 1/04/2014, page 24 Le Soir 4/04/2014, page 26 Article dans La Libre Belgique, 23/04/2014, page 25. Commentaire du Prof. B. Joris sur une étude ayant identifié la présence de 80 gènes de bactéries résistantes aux antibiotiques dans le fumier de vaches laitières. Interview des Prof. P. Charlier et M. Galleni le 18/05/2014 dans l émission «Matière grise» de la RTBF : «Les nouveaux antibiotiques contre les bactéries résistantes» Article dans Le 15 e jour du mois, mai 2014 /234. La bonne dose, un projet européen MON4STRAT pour mieux dispenser les antibiotiques. Interview du Prof. B. Joris. Article dans Le 15 e jour du mois, septembre 2014 /236. Robotéine : au CIP, une plateforme d innovation unique en Wallonie. Interviews du Dr A. Brans et du Prof. A. Matagne Interview du Dr A. Wilmotte during the TV Broadcast Devoir d enquête, November 26, 2014 WIDE AUDIENCE CONFERENCES JM Frère, «La résistance aux antibiotiques : la fin de l état de grâce?», Association des Professeurs émérites et honoraires de l ULg, Colonster, Belgium, January 8, 2013 JM Frère, «Mécanismes de résistance aux antibiotiques», formations continues pour les pharmaciens hospitaliers en Belgique, UNamur, February 21, 2013 Cours-conférence :" Le traité sur l'antarctique : une gouvernance originale pour un continent exceptionnel" au Collège Belgique (March 28 and May 28, 2013), Palais des Académies, Brussels. Coordinator : Dr A. Wilmotte JM Frère, «La résistance aux antibiotiques : une nouvelle maladie?», Connaissance et vie aujourd hui, Mol, Belgium, March 24,

70 INTERNATIONAL EXCHANGES POSTDOCS IN 2013 Dr Sol Schvartzman Research group: Functional Genomics and Plant Molecular Imaging Project leader: M. Hanikenne My name is Sol Schvartzman and I am working in Bioinformatics in the Functional Genomics and Plant Molecular Imaging laboratory (Plant Biology Institute) with a postdoctoral fellowship since July I am originally from Argentina but I lived in Spain most of my life where I studied Biology. I then moved to Ireland to carry out doctoral studies at the School of Biosystems Engineering at the University College Dublin. My PhD explored the field of mathematical modeling to predict bacterial behavior. I worked with Listeria monocytogenes and was interested in finding the combination of primary and secondary models that would best predict whether Listeria s populations would grow, survive or inactivate depending on environmental conditions such as ph, water activity, lactic acid concentration or temperature. I also did some work on detecting growth/no growth boundaries. When I finished my PhD I pursued a theoretical MSc in Bioinformatics at the University College Cork in the south of Ireland. I am currently working with Arabidopsis halleri, a plant species known to hyperaccumulate Cd and Zn. This ecotype can be found in sites with contaminated soils but it also lives in healthy grounds. It is thought that adaptation to contaminated soils may have occurred fairly recently. The objective of my postdoc is to find genetic determinants of adaptations to high Zn concentrations, and for this I work with RNA-Seq and epigenetic sequencing data

71 INTERNATIONAL EXCHANGES Dr Samir Olatunji Research group: Bacterial diversity, physiology and genetics Project leader: Dr M. Terrak I did my PhD in Protein Engeneering and Therapeutical Target (October March 2013) with Dr A. Bouhss as Supervisor, in the team of Dr D. Mengin-Lecreux, at the University Paris-Sud 11 in France. The field of this thesis work was structural and functional studies of MraY, a membrane protein, member of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, involved in the biosynthesis of bacterial peptidoglycan. On one hand, we have undertaken the structural characterization of MraY by different biochemical and biophysical approaches like X-ray crystallography, electron microscopy and small angle X-ray scattering (SAXS). On the other hand, we have elucidated the catalytic mechanism of this enzyme. In July 2013, I joined the group of Dr Mohammed Terrak to work on structural characterization of the membrane lipid II transferase FtsW (production, purification and structure determination) and study of its interactions with other proteins of the bacterial divisome. Working in the CIP is an experience both rewarding and enjoyable. It is a pleasant environment with many helpful and available colleagues. And if occasionally a rain falls on us, because yes, we are in Belgium, there is certainly a Trappist club or other joy in the Liege city on the horizon to make us feel on top of the world

72 INTERNATIONAL EXCHANGES Dr Julia Kleinteich Research group: Bacterial diversity, physiology and genetics Group leader: Dr A. Wilmotte Cyanobacteria produce a range of secondary metabolites. Very little is known about the bioactive potential and toxicity of cyanobacteria of the Polar Regions where they are the dominant phototrophs and are exposed to extreme environmental pressure. In my current research at the CIP, I identify and characterize toxic strains and their secondary metabolites in one of the largest culture collection of Antarctic cyanobacteria available in the group of Dr A. Wilmotte or in the BCCM/ULC collection. Various gene clusters involved in secondary metabolite and toxin production will be investigated. In parallel, extracts are analysed for secondary metabolites using HPLC and LC-MS. Strains identified as bioactive will be genetically described using Whole Genome Sequencing and compared with cyanobacteria from other regions. The identification of geographically separated and genetically different toxin producing cyanobacteria would give valuable information about the distribution and evolution of cyanobacterial toxicity. We plan also to study the physiology of the selected organisms and the production of the toxins under variable parameters such as light, temperature, or nutrient concentrations. After my graduation in Environmental Toxicology and the completion of my PhD, both at the University of Konstanz in Germany, I worked at the Natural History Museum in London. In my previous research I focused on the physiology and toxicology of Arctic and Antarctic cyanobacteria, but most studies were performed on mixed environmental samples so that an identification of the toxic/bioactive species was not possible to date. The unialgal cultivation of the respective strains at the BCCM collection will facilitate detailed genetic, phylogenetic and physiological descriptions. Newly detected bioactive or cytotoxic compounds may have an application in industrial or medical services. Furthermore the outcomes of this study would allow the development of functional screening methods to identify toxin producers in many water bodies worldwide

73 POSTDOCS IN 2014 INTERNATIONAL EXCHANGES Dr Borjana Arsova Research group: Functional Genomics and Plant Molecular Imaging Group leader: Marc Hanikenne In an interesting turn of events, I am starting in Belgium 2 years later than planned. My position- an FNRS Chargé de recherche, was awarded in 2012 roughly at the same time that I received an independent postdoc position at the University of Düsseldorf. Since both positions allowed me to follow up personally suggested projects I was happy that the FNRS position could be postponed for a while. I spent focusing on the nitrogen use efficiency of plants with C3 and C4 type of photosynthesis using the genus Flaveria that includes species with C3, intermediate and C4 photosynthesis. I analyzed the adaptations to varying nitrogen conditions using proteomics and next generation sequencing approaches. I am originally from Macedonia, but most of my scientific experience has been obtained in Germany. After my PhD at the Max-Planck Institute of Molecular Plant Physiology in Potsdam (Germany), nutrient sensing and signaling became the main model for studying protein post-translational modifications as one of the first responses of plants to changing environmental conditions. I find it intriguing, how a sedentary organism like the plant can adapt and survive in a constantly changing environment. How information from the plasma membrane is transferred to the nucleus in a cell, or signals from the root are transferred to the shoot (and vice versa) and as a result the whole organism changes its behavior: produces more or less sugars, takes up more or less of a specific nutrient or changes its growth pattern. I want to help determine as many of the molecular mechanisms behind these sensing/signaling pathways as possible. My hope is that in the long run this work will help in breeding/producing plants that will need lower amount of fertilizers or grow on poorer soils. This direction of work also continues at ULg. Here, my main lab is the Functional Genomics and Plant Molecular Imaging where with Dr Marc Hanikenne we will try to detangle some of the zinc sensing and signaling pathways in Arabidopsis thaliana. I am co-hosted by the GIGA proteomics lab (Prof. E. de Pauw) where we will perform the proteomic analyses and look for changes in protein quantities and phosphorylation. Based on my initial few months, I am looking forward to further work in this interdisciplinary and international environment

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