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Structural and Biochemical Analysis of Escherichia coli ObgE, a Central Regulator of Bacterial Persistence Vrije Universiteit Brussel KU Leuven

Sotirios Gkekas, Ranjan Kumar Singh, Alexander V Shkumatov, Joris Messens, Maarten Fauvart, Natalie Verstraeten, Jan Michiels, Wim Versées

The Obg protein family belongs to the TRAFAC (translation factor) class of P-loop GTPases and is conserved from bacteria to eukaryotes. Essential roles in many different cellular processes have been suggested for the Obg protein from Escherichia coli (ObgE), and we recently showed that it is a central regulator of bacterial persistence. Here, we report the first crystal structure of ObgE at 1.85 Å resolution in the GDP-bound state, showing ...

Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli Ghent University

Bradley Walters Biggs, Chin Giaw Lin, Kristen Saglinani, Smriti Shankar, Gregory Stephanopoulos, Marjan De Mey, Parayil Kumaran Ajikumar
Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set ...

An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation Hasselt University

Duy Tien TA, Erik STEEN REDEKER, Brecht BILLEN, Gunter REEKMANS, Josephine Sikulu, Jean-Paul NOBEN, Wanda GUEDENS, Peter ADRIAENSENS
In this study, several expression strategies were investigated in order to develop a generic, highly productive and efficient protocol to produce nanobodies modified with a clickable alkyne function at their C-terminus via the intein-mediated protein ligation (IPL) technique. Hereto, the nanobody targeting the vascular cell adhesion molecule 1 (NbVCAM1) was used as a workhorse. The highlights of the protocol can be ascribed to a cytoplasmic ...

Structural Basis of the Subcellular Topology Landscape of Escherichia coli Vrije Universiteit Brussel KU Leuven

Maria S Loos, Reshmi Ramakrishnan, Wim Vranken, Alexandra Tsirigotaki, Evrydiki-Pandora Tsare, Valentina Zorzini, Jozefien De Geyter, Biao Yuan, Ioannis Tsamardinos, Maria Klappa, et al.

Cellular proteomes are distributed in multiple compartments: on DNA, ribosomes, on and inside membranes, or they become secreted. Structural properties that allow polypeptides to occupy subcellular niches, particularly to after crossing membranes, remain unclear. We compared intrinsic and extrinsic features in cytoplasmic and secreted polypeptides of the Escherichia coli K-12 proteome. Structural features between the cytoplasmome and ...

Heptyl α-D-mannosides grafted on a β-cyclodextrin core to interfere with Escherichia coli adhesion Vrije Universiteit Brussel KU Leuven

Julie Bouckaert, Zhaoli Li, Catarina Xavier, Mehdi Almant, Vicky Caveliers, Tony Lahoutte, Stephen D Weeks, José Kovensky, Sébastien G Gouin

n-Heptyl α-D-mannoside (HM) has previously been identified as a nanomolar FimH antagonist able to prevent Escherichia coli adhesion. We have designed mono- and heptavalent glycoconjugates in which HM is tethered to β-cyclodextrin (β-CD) through short and long spacers. One-pot click or co-clicking procedures were developed to directly obtain the glycoconjugates from unprotected HM and β-CD precursors. These FimH antagonists were examined ...

Mutant Variants of the Substrate-Binding Protein DppA from Escherichia coli Enhance Growth on Nonstandard gamma-Glutamyl Amide-Containing Peptides KU Leuven

Puneet Srivastava, Piet Herdewyn
The import of nonnatural molecules is a recurring problem in fundamental and applied aspects of microbiology. The dipeptide permease (Dpp) of Escherichia coli is an ABC-type multicomponent transporter system located in the cytoplasmic membrane, which is capable of transporting a wide range of di- and tripeptides with structurally and chemically diverse amino acid side chains into the cell. Given this low degree of specificity, Dpp was previously ...

Exploring the conformational changes of the ATP binding site of gyrase B from Escherichia coli complexed with different established inhibitors by using molecular dynamics simulation: protein-ligand interactions in the light of the alanine scanning and fre KU Leuven

Liane Saiz, Mathy Froeyen
Currently, bacterial diseases cause a death toll around 2 million people a year encouraging the search for new antimicrobial agents. DNA gyrase is a well-established antibacterial target consisting of two subunits, GyrA and GyrB, in a heterodimer A(2)B(2). GyrA is involved in DNA breakage and reunion and GyrB catalyzes the hydrolysis of ATP. The GyrB subunit from Escherichia coli has been investigated, namely the ATP binding pocket both ...

α-amino diphenyl phosphonates as novel inhibitors of Escherichia coli ClpP protease University of Antwerp

Carlos Moreno Cinos, Elisa Sassetti, Irene Garcìa Salado, Gesa Witt, Siham Benramdane, Laura Reinhardt, Cristina D. Cruz, Jurgen Joossens, Pieter Van Der Veken, Heike Broetz-Oesterhe, et al.
Increased Gram-negative bacteria resistance to antibiotics is becoming a global problem, and new classes of antibiotics with novel mechanisms of action are required. The caseinolytic protease subunit P (ClpP) is a serine protease conserved among bacteria that is considered as an interesting drug target. ClpP function is involved in protein turnover and homeostasis, stress response, and virulence among other processes. The focus of this study was ...

Prevalence of enterotoxigenic Escherichia coli virulence genes from scouring piglets in Zimbabwe Vrije Universiteit Brussel

Evelyn Madoroba, Edilbert Van Driessche, Henri De Greve, Jan Mast, Ignatious Ncube, John S. Read, Sonia Beeckmans
World-wide, enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC)-induced diarrhea are economically important for porcine producers. Our aim was to investigate the prevalence of toxin and fimbrial genes among E. coli isolated from diarrheic piglets from randomly selected piggeries in Zimbabwe.We used multiplex PCR for screening STa, STb, LT, and Stx-2e toxins. Subsequently F4, F5, F6, F18 and F41 fimbriae genes were screened ...

Identification and characterization of approved drugs and drug-like compounds as covalent Escherichia coli ClpP inhibitors University of Antwerp

Elisa Sassetti, Cristina Durante Cruz, Paeivi Tammela, Mathias Winterhalter, Koen Augustyns, Philip Gribbon, Bjoern Windshuegel
The serine protease Caseinolytic protease subunit P (ClpP) plays an important role for protein homeostasis in bacteria and contributes to various developmental processes, as well as virulence. Therefore, ClpP is considered as a potential drug target in Gram-positive and Gram-negative bacteria. In this study, we utilized a biochemical assay to screen several small molecule libraries of approved and investigational drugs for Escherichia coli ClpP ...