scholarly journals Functional Analysis of the Galactosyltransferases Required for Biosynthesis of d-Galactan I, a Component of the Lipopolysaccharide O1 Antigen of Klebsiella pneumoniae

2001 ◽  
Vol 183 (11) ◽  
pp. 3318-3327 ◽  
Author(s):  
Shukui Guan ◽  
Anthony J. Clarke ◽  
Chris Whitfield
Keyword(s):  
Klebsiella Pneumoniae ◽  
Abc Transporter ◽  
Genetic Locus ◽  
Repeat Unit ◽  
Chain Extension ◽  
Transferase Activity ◽  
Unit Structure ◽  
Enterobacterial Common Antigen ◽  
K 12

ABSTRACT d-Galactan I is an O-antigenic polymer with the repeat unit structure [→3)-β-d-Galf-(1→3)-α-d-Galp-(1→], that is found in the lipopolysaccharide of Klebsiella pneumoniae O1 and other gram-negative bacteria. A genetic locus containing six genes is responsible for the synthesis and assembly ofd-galactan I via an ATP-binding cassette (ABC) transporter-dependent pathway. The galactosyltransferase activities that are required for the processive polymerization ofd-galactan I were identified by using in vitro reactions. The activities were determined with endogenous lipid acceptors in membrane preparations from Escherichia coli K-12 expressing individual enzymes (or combinations of enzymes) or in membranes reconstituted with specific lipid acceptors. Thed-galactan I polymer is built on a lipid acceptor, undecaprenyl pyrophosphoryl-GlcpNAc, a product of the WecA enzyme that participates in the biosynthesis of enterobacterial common antigen and O-antigenic polysaccharide (O-PS) biosynthesis pathways. This intermediate is directed intod-galactan I biosynthesis by the bifunctionalwbbO gene product, which sequentially adds one Galp and one Galf residue from the corresponding UDP-sugars to form a lipid-linked trisaccharide. The two galactosyltransferase activities of WbbO are separable by limiting the UDP-Galf precursor. Galactosyltransferase activity in membranes reconstituted with exogenous lipid-linked trisaccharide acceptor and the known structure of d-galactan I indicate that WbbM catalyzes the subsequent transfer of a single Galp residue to form a lipid-linked tetrasaccharide. Chain extension of the d-galactan I polymer requires WbbM for Galp transferase, together with Galftransferase activity provided by WbbO. Comparison of the biosynthetic pathways for d-galactan I and the polymannose E. coli O9a antigen reveals some interesting features that may reflect a common theme in ABC transporter-dependent O-PS assembly systems.

scholarly journals Klebsiella pneumoniae O1 and O2ac antigens provide prototypes for an unusual strategy for polysaccharide antigen diversification

2019 ◽  
Vol 294 (28) ◽  
pp. 10863-10876 ◽  
Author(s):  
Steven D. Kelly ◽  
Bradley R. Clarke ◽  
Olga G. Ovchinnikova ◽  
Ryan P. Sweeney ◽  
Monica L. Williamson ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Repeat Unit ◽  
Limited Range ◽  
Polysaccharide Antigen ◽  
Core Element ◽  
Unit Structure ◽  
Repeat Structure ◽  
Catalytic Sites ◽  
Glcnac Transferase ◽  
Enzymatic Pathways

A limited range of different structures is observed in O-antigenic polysaccharides (OPSs) from Klebsiella pneumoniae lipopolysaccharides. Among these, several are based on modifications of a conserved core element of serotype O2a OPS, which has a disaccharide repeat structure [→3)-α-d-Galp-(1→3)-β-d-Galf-(1→]. Here, we describe the enzymatic pathways for a highly unusual modification strategy involving the attachment of a second glycan repeat-unit structure to the nonreducing terminus of O2a. This occurs by the addition of the O1 [→3)-α-d-Galp-(1→3)-β-d-Galp-(1→] or O2c [→3)-β-d-GlcpNAc-(1→5)-β-d-Galf-(1→] antigens. The organization of the enzyme activities performing these modifications differs, with the enzyme WbbY possessing two glycosyltransferase catalytic sites solely responsible for O1 antigen polymerization and forming a complex with the O2a glycosyltransferase WbbM. In contrast, O2c polymerization requires glycosyltransferases WbmV and WbmW, which interact with one another but apparently not with WbbM. Using defined synthetic acceptors and site-directed mutants to assign the activities of the WbbY catalytic sites, we found that the C-terminal WbbY domain is a UDP-Galp–dependent GT-A galactosyltransferase adding β-(1→3)–linked d-Galp, whereas the WbbY N terminus includes a GT-B enzyme adding α-(1→3)–linked d-Galp. These activities build the O1 antigen on a terminal Galp in the O2a domain. Using similar approaches, we identified WbmV as the UDP-GlcNAc transferase and noted that WbmW represents a UDP-Galf–dependent enzyme and that both are GT-A members. WbmVW polymerizes the O2c antigen on a terminal Galf. Our results provide mechanistic and conceptual insights into an important strategy for polysaccharide antigen diversification in bacteria.

scholarly journals Identification and Biosynthesis of Cyclic Enterobacterial Common Antigen in Escherichia coli

2003 ◽  
Vol 185 (6) ◽  
pp. 1995-2004 ◽  
Author(s):  
Paul J. A. Erbel ◽  
Kathleen Barr ◽  
Ninguo Gao ◽  
Gerrit J. Gerwig ◽  
Paul D. Rick ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Repeat Unit ◽  
Enteric Bacteria ◽  
Water Soluble ◽  
Ionization Mass ◽  
Common Antigen ◽  
Esi Ms ◽  
Enterobacterial Common Antigen ◽  
Repeat Units ◽  
K 12

ABSTRACT Phosphoglyceride-linked enterobacterial common antigen (ECAPG) is a cell surface glycolipid that is synthesized by all gram-negative enteric bacteria. The carbohydrate portion of ECAPG consists of linear heteropolysaccharide chains comprised of the trisaccharide repeat unit Fuc4NAc-ManNAcA-GlcNAc, where Fuc4NAc is 4-acetamido-4,6-dideoxy-d-galactose, ManNAcA is N-acetyl-d-mannosaminuronic acid, and GlcNAc is N-acetyl-d-glucosamine. The potential reducing terminal GlcNAc residue of each polysaccharide chain is linked via phosphodiester linkage to a phosphoglyceride aglycone. We demonstrate here the occurrence of a water-soluble cyclic form of enterobacterial common antigen, ECACYC, purified from Escherichia coli strains B and K-12 with solution nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and additional biochemical methods. The ECACYC molecules lacked an aglycone and contained four trisaccharide repeat units that were nonstoichiometrically substituted with up to four O-acetyl groups. ECACYC was not detected in mutant strains that possessed null mutations in the wecA, wecF, and wecG genes of the wec gene cluster. These observations corroborate the structural data obtained by NMR and ESI-MS analyses and show for the first time that the trisaccharide repeat units of ECACYC and ECAPG are assembled by a common biosynthetic pathway.

Activité antibactérienne de l’huile essentielle de Pelargonium x asperum et son potentiel synergique avec la nisine

2019 ◽  
Vol 17 (3) ◽  
pp. 140-148 ◽  
Author(s):  
A. Ouelhadj ◽  
L. Ait Salem ◽  
D. Djenane
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Klebsiella Pneumoniae ◽  
Nous Avons ◽  
Activité Antibactérienne

Ce travail vise l’étude de l’activité antibactérienne de l’huile essentielle (HE) de Pelargoniumx asperum et de la bactériocine, la nisine seul et en combinaison vis-à-vis de six bactéries dont quatre sont multirésistantes d’origine clinique. L’activité antibactérienne in vitro a été évaluée par la méthode de diffusion sur gélose. La concentration minimale inhibitrice (CMI) est aussi déterminée pour HE. Les résultats ont révélé une activité antibactérienne significative exercée par HE visà-vis de Staphylococcus aureus (ATCC 43300), Staphylococcus aureus et Escherichia coli avec des diamètres d’inhibition de 36,00 ; 22,50 et 40,00 mm, respectivement. Cependant, l’HE de Pelargonium asperum a montré une activité antibactérienne supérieure par rapport à la nisine. Les valeurs des CMI rapportées dans cette étude sont comprises entre 1,98–3,96 μl/ml. Les combinaisons réalisées entre HE et la nisine ont montré un effet additif vis-à-vis de Escherichia coli (ATCC 25922) avec (50 % HE Pelargonium asperum + 50 % nisine). Par contre, nous avons enregistré une synergie vis-à-vis de Klebsiella pneumoniae avec (75 % HE Pelargonium asperum + 25 % nisine) et contre Pseudomonas aeruginosa avec les trois combinaisons testées. Les résultats obtenus permettent de dire que l’HE de Pelargonium asperum possède une activité antibactérienne ainsi que sa combinaison avec la nisine pourrait représenter une bonne alternative pour la lutte contre l’antibiorésistance.

Hyperbaric oxygen suppresses growth of Klebsiella pneumoniae and enhances effect of tigecycline in vitro

2017 ◽  
Vol 44 (6) ◽  
pp. 543-549
Author(s):  
Lina Zhang ◽  
◽  
Qing Sun ◽  
Di Wu ◽  
Ying Yang ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Hyperbaric Oxygen

scholarly journals Heterodimer Formation of the Homodimeric ABC Transporter OpuA

2021 ◽  
Vol 22 (11) ◽  
pp. 5912
Author(s):  
Patricia Alvarez-Sieiro ◽  
Hendrik R. Sikkema ◽  
Bert Poolman
Keyword(s):  
Abc Transporter ◽  
Conformational Dynamics ◽  
Host Organism ◽  
Affinity Tag ◽  
Practical Applications ◽  
Fundamental Research ◽  
Protein Multimerization ◽  
Biochemical Analyses

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.

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