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Anurag Agrawal, James C Weisshaar
The outermost layer of Gram negative bacteria is composed of a lipopolysaccharide (LPS) network that forms a dense protective hydrophilic barrier against entry of hydrophobic drugs. At low μM concentrations, a large family of cationic polypeptides known as antimicrobial peptides (AMPs) is able to penetrate the LPS layer and permeabilize the outer membrane (OM) and the cytoplasmic membrane (CM), causing cell death. Cecropin A is a well-studied cationic AMP from moth. Here a battery of time-resolved, single-cell microscopy experiments explores how deletion of sugar layers and/or phosphoryl negative charges from the core oligosaccharide layer (core OS) of K12 E...
April 20, 2018: Biochimica et Biophysica Acta
Dustin J Little, Roland Pfoh, François Le Mauff, Natalie C Bamford, Christina Notte, Perrin Baker, Manita Guragain, Howard Robinson, Gerald B Pier, Mark Nitz, Rajendar Deora, Donald C Sheppard, P Lynne Howell
Poly-β(1,6)-N-acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in Escherichia coli and Bordetella species require the protein products encoded by the pgaABCD operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show that the C-terminal domains of Bordetella bronchiseptica PgaB (PgaBBb) and E...
April 23, 2018: PLoS Pathogens
Francesca Mensitieri, Federica De Lise, Andrea Strazzulli, Marco Moracci, Eugenio Notomista, Valeria Cafaro, Emiliano Bedini, Matthew Howard Sazinsky, Marco Trifuoggi, Alberto Di Donato, Viviana Izzo
α-L-Rhamnosidases (α-RHAs, EC are glycosyl hydrolases (GHs) hydrolyzing terminal α-L-rhamnose residues from different substrates such as heteropolysaccharides, glycosylated proteins and natural flavonoids. Although the possibility to hydrolyze rhamnose from natural flavonoids has boosted the use of these enzymes in several biotechnological applications over the past decades, to date only few bacterial rhamnosidases have been fully characterized and only one crystal structure of a rhamnosidase of the GH106 family has been described...
April 17, 2018: Archives of Biochemistry and Biophysics
Consuelo Badilla, Thomas H Osborne, Ambrose Cole, Cameron Watson, Snezana Djordjevic, Joanne M Santini
Arsenic contamination of drinking water affects more than 140 million people worldwide. While toxic to humans, inorganic forms of arsenic (arsenite and arsenate), can be used as energy sources for microbial respiration. AioX and its orthologues (ArxX and ArrX) represent the first members of a new sub-family of periplasmic-binding proteins that serve as the first component of a signal transduction system, that's role is to positively regulate expression of arsenic metabolism enzymes. As determined by X-ray crystallography for AioX, arsenite binding only requires subtle conformational changes in protein structure, providing insights into protein-ligand interactions...
April 19, 2018: Scientific Reports
Kathryn M Schultz, Matthew A Fischer, Elizabeth L Noey, Candice S Klug
Lipopolysaccharide (LPS) is an essential element of nearly all Gram-negative bacterial outer membranes and serves to protect the cell from adverse environmental stresses. Seven members of the lipopolysaccharide transport (Lpt) protein family function together to transport LPS from the inner membrane (IM) to the outer leaflet of the outer membrane of bacteria such as Escherichia coli. Each of these proteins has a solved crystal structure, including LptC, which is a largely periplasmic protein that is associated with the IM LptB2 FG complex and anchored to the membrane by an N-terminal helix...
April 19, 2018: Protein Science: a Publication of the Protein Society
Chao Peng, Anneli Sundman, Casey Bryce, Charlotte Catrouillet, Thomas Borch, Andreas Kappler
Fe(II)-organic-matter (Fe(II)-OM) complexes are abundant in the environment and may play a key role for the behavior of Fe and pollutants. Mixotrophic nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOx) reduce nitrate coupled to the oxidation of organic compounds and Fe(II). Fe(II) oxidation may occur enzymatically or abiotically by reaction with nitrite that forms during heterotrophic denitrification. However, it is unknown whether Fe(II)-OM complexes can be oxidized by NRFeOx. We used cell-suspension experiments with the mixotrophic nitrate-reducing Fe(II)-oxidizing bacterium Acidovorax sp...
April 19, 2018: Environmental Science & Technology
Yi-Wei Chang, Carrie L Shaffer, Lee A Rettberg, Debnath Ghosal, Grant J Jensen
The type IV secretion system (T4SS) is a versatile nanomachine that translocates diverse effector molecules between microbes and into eukaryotic cells. Here, using electron cryotomography, we reveal the molecular architecture of the Helicobacter pylori cag T4SS. Although most components are unique to H. pylori, the cag T4SS exhibits remarkable architectural similarity to other T4SSs. Our images revealed that, when H. pylori encounters host cells, the bacterium elaborates membranous tubes perforated by lateral ports...
April 17, 2018: Cell Reports
Kristin Støen Gunnarsen, Lene Støkken Høydahl, Ralf Stefan Neumann, Kaare Bjerregaard-Andersen, Nicolay Rustad Nilssen, Ludvig Magne Sollid, Inger Sandlie, Geir Åge Løset
There is a quest for production of soluble protein of high quality for the study of T-cell receptors (TCRs), but expression often results in low yields of functional molecules. In this study, we used an E. coli chaperone-assisted periplasmic production system and compared expression of 4 different soluble TCR formats: single-chain TCR (scTCR), two different disulfide-linked TCR (dsTCR) formats, and chimeric Fab (cFab). A stabilized version of scTCR was also included. Additionally, we evaluated the influence of host (XL1-Blue or RosettaBlueTM) and the effect of IPTG induction on expression profiles...
2018: PloS One
A A Tyurin, K V Kabardaeva, O N Mustafaev, O S Pavlenko, N S Sadovskaya, V S Fadeev, E A Zvonova, I V Goldenkova-Pavlova
A recombinant DNA in which the interferon αA (IFN-αA) gene sequence is integrated into a loop region of the gene coding thermostable lichenase was constructed. This approach of insertion fusion with thermostable lichenase is advantageous in terms of increasing the solubility, stability, and production of the fusion partner in soluble form in general and in the periplasm of bacterial cells in particular. Thus, the insertion of IFN-αA into the loop (53 a.a.) of thermostable lichenase from Clostridium thermocellum resulted in effective expression of the soluble form of the recombinant protein in the periplasm of Escherichia coli without any compromise in biological activity of IFN-αA, while the thermostable lichenase retained its ability for functional folding without dramatic loss of its basic activity and thermostability...
March 2018: Biochemistry. Biokhimii︠a︡
Leticia S Guidolin, Vilma Arce-Gorvel, Andres A Ciocchini, Diego J Comerci, Jean-Pierre Gorvel
Cyclic β-1,2-D-glucans (CβG) are natural bionanopolymers present in the periplasmic space of many Proteobacteria. These molecules are sugar rings made of 17 to 25 D-glucose units linked exclusively by β-1,2 glycosidic bonds. CβG are important for environmental sensing and osmoadaptation in bacteria, but most importantly, they play key roles in complex host-cell interactions such as symbiosis, pathogenesis and immunomodulation. In the last years, the identification and characterization of the enzymes involved in the synthesis of CβG allowed to know in detail the steps necessary for the formation of these sugar rings...
April 6, 2018: Cellular Microbiology
Yue Zhang, Weikang Gong, Yan Wang, Yang Liu, Chunhua Li
Human P-glycoprotein (P-gp), a kind of ATP-Binding Cassette transporter, can export a diverse variety of anti-cancer drugs out of the tumor cell. Its overexpression is one of the main reasons for the multidrug resistance (MDR) of tumor cells. It has been confirmed that during the substrate transport process, P-gp experiences a large-scale structural rearrangement from the inward- to outward-facing states. However, the mechanism of how the nucleotide-binding domains (NBDs) control the transmembrane domains (TMDs) to open towards the periplasm in the outward-facing state has not yet been fully characterized...
April 5, 2018: Journal of Biomolecular Structure & Dynamics
Petru-Iulian Trasnea, Andreea Andrei, Dorian Marckmann, Marcel Utz, Bahia Khalfaoui-Hassani, Nur Selamoglu, Fevzi Daldal, Hans-Georg Koch
PccA and SenC are periplasmic copper chaperones required for the biogenesis of cbb3-type cytochrome c oxidase (cbb3-Cox) in Rhodobacter capsulatus at physiological Cu concentrations. However, both proteins are dispensable for cbb3-Cox assembly when external Cu concentration is high. PccA and SenC bind Cu using Met and His, and Cys and His residues as ligands, respectively, and both proteins form a complex during cbb3-Cox biogenesis. SenC also interacts directly with cbb3-Cox as shown by chemical crosslinking...
April 3, 2018: ACS Chemical Biology
Vicente T Sedano-Núñez, Sjef Boeren, Alfons J M Stams, Caroline M Plugge
Syntrophobacter fumaroxidans is a sulfate-reducing bacterium able to grow on propionate axenically or in syntrophic interaction with methanogens or other sulfate-reducing bacteria. We performed a proteome analysis of S. fumaroxidans growing with propionate axenically with sulfate or fumarate, and in syntrophy with Methanospirillum hungatei, Methanobacterium formicicum or Desulfovibrio desulfuricans. Special attention was put on the role of hydrogen and formate in interspecies electron transfer (IET) and energy conservation...
April 3, 2018: Environmental Microbiology
Ricardo Monteiro, Ingrid Chafsey, Sabine Leroy, Christophe Chambon, Michel Hébraud, Valérie Livrelli, Mariagrazia Pizza, Alfredo Pezzicoli, Mickaël Desvaux
Surface proteins are the major factor for the interaction between bacteria and its environment, playing an important role in infection, colonisation, virulence and adaptation. However, the study of surface proteins has proven difficult mainly due to their hydrophobicity and/or relatively low abundance compared with cytoplasmic proteins. To overcome these issues new proteomic strategies have been developed, such as cell-surface protein labelling using biotinylation reagents. Sulfo-NHS-SS-biotin is the most commonly used reagent to investigate the proteins expressed at the cell surface of various organisms but its use in lipopolysaccharidic diderm bacteria (archetypical Gram-negative bacteria) remains limited to a handful of species...
March 30, 2018: Journal of Proteomics
Thibault Meyer, Armelle Vigouroux, Magali Aumont-Niçaise, Gilles Comte, Ludovic Vial, Celine Lavire, Solange Morera
The bacterial plant pathogen Agrobacterium fabrum uses periplasmic binding proteins (PBPs) along with ABC transporters to import a wide variety of plant molecules as nutrients. Nonetheless, how A. fabrum acquires plant metabolites is incompletely understood. Using genetic approaches and affinity measurements, we identified here the PBP MelB and its transporter as being responsible for the uptake of the raffinose family of oligosaccharides (RFOs), which are the most widespread Dgalactose containing oligosaccharides in higher plants...
March 30, 2018: Journal of Biological Chemistry
Bradley R Clarke, Olga G Ovchinnikova, Steven D Kelly, Monica L Williamson, Jennifer E Butler, Bin Liu, Lu Wang, Xi Gou, Rainer Follador, Todd L Lowary, Chris Whitfield
Klebsiella pneumoniae is a major health threat. Vaccination and passive immunization are considered as alternative therapeutic strategies for managing Klebsiella infections. Lipopolysaccharide O antigens are attractive candidates because of the relatively small range of known O-antigen polysaccharide structures, but immunotherapeutic applications require a complete understanding of the structures found in clinical settings. Currently, the precise number of Klebsiella O antigens is unknown because available serological tests have limited resolution, and their association with defined chemical structures is sometimes uncertain...
March 30, 2018: Journal of Biological Chemistry
Irina Smirnova, Vladimir Kasho, Xiaoxu Jiang, Hong-Ming Chen, Stephen G Withers, H Ronald Kaback
Binding kinetics of α-galactopyranoside homologs with fluorescent aglycones of different sizes and shapes were determined with the lactose permease (LacY) of Escherichia coli by FRET from Trp151 in the binding site of LacY to the fluorophores. Fast binding was observed with LacY stabilized in an outward-open conformation ( k on = 4-20 μM-1 ·s-1 ), indicating unobstructed access to the binding site even for ligands that are much larger than lactose. Dissociation rate constants ( k off ) increase with the size of the aglycone so that K d values also increase but remain in the micromolar range for each homolog...
March 30, 2018: Proceedings of the National Academy of Sciences of the United States of America
Lisa M Stiller, Erwin A Galinski, Elisabeth M H J Witt
It has been firmly established that organic osmolytes (compatible solutes) of halophilic Bacteria and Archaea have positive effects on conformation and activity of proteins, and may therefore improve their functional production. In particular, the amino acid derivative ectoine is known for its conformational stabilization, aggregation suppression, and radical protection properties. The natural producer and industrial production strain Halomonas elongata accumulates ectoine in the cytoplasm, and as a result offers a unique stabilizing environment for recombinant proteins...
March 28, 2018: Genes
Jinli Chen, Lulu Wang, Fei Shang, Yuesheng Dong, Nam-Chul Ha, Ki Hyun Nam, Chunshan Quan, Yongbin Xu
The ZnuABC ATP-binding cassette transporter found in gram-negative bacteria has been implicated in ensuring adequate zinc import into Zn(II)-poor environments. ZinT is an essential component of ZnuABC and contributes to metal transport by transferring metals to ZnuA, which delivers them to ZnuB in periplasmic zinc recruitment. Although several structures of E. coli ZinT have been reported, its zinc-binding sites and oligomeric state have not been clearly identified. Here, we report the crystal structure of E...
March 26, 2018: Biochemical and Biophysical Research Communications
Weiqing Zhang, Jing Lu, Shuwen Zhang, Lu Liu, Xiaoyang Pang, Jiaping Lv
BACKGROUND: Thermostable lipases from microbial sources have been substantially overexpressed in E. coli, however, these enzymes are often produced with low-level enzymatic activity and mainly in the form of inclusion bodies. Several studies have reported that the secretory production of recombinant proteins fused their N-terminus to a signal peptide has been employed to resolve the problem. In general, the feasibility of this approach largely depends on the secretory pathway of signal peptide and the type of target protein to be secreted...
March 28, 2018: Microbial Cell Factories
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