scholarly journals Lipopolysaccharide-Linked Enterobacterial Common Antigen (ECALPS) Occurs in Rough Strains of Escherichia coli R1, R2, and R4

2020 ◽  
Vol 21 (17) ◽  
pp. 6038
Author(s):  
Anna Maciejewska ◽  
Marta Kaszowska ◽  
Wojciech Jachymek ◽  
Czeslaw Lugowski ◽  
Jolanta Lukasiewicz
Keyword(s):  
Escherichia Coli ◽  
Surface Antigen ◽  
Structural Data ◽  
Outer Core ◽  
Permeability Barrier ◽  
Common Antigen ◽  
Enterobacterial Common Antigen ◽  
Specific Polysaccharide ◽  
K 12 ◽  
Outer Membrane Permeability

Enterobacterial common antigen (ECA) is a conserved surface antigen characteristic for Enterobacteriaceae. It is consisting of trisaccharide repeating unit, →3)-α-d-Fucp4NAc-(1→4)-β-d-ManpNAcA-(1→4)-α-d-GlcpNAc-(1→, where prevailing forms include ECA linked to phosphatidylglycerol (ECAPG) and cyclic ECA (ECACYC). Lipopolysaccharide (LPS)-associated form (ECALPS) has been proved to date only for rough Shigella sonnei phase II. Depending on the structure organization, ECA constitutes surface antigen (ECAPG and ECALPS) or maintains the outer membrane permeability barrier (ECACYC). The existence of LPS was hypothesized in the 1960–80s on the basis of serological observations. Only a few Escherichia coli strains (i.e., R1, R2, R3, R4, and K-12) have led to the generation of anti-ECA antibodies upon immunization, excluding ECAPG as an immunogen and conjecturing ECALPS as the only immunogenic form. Here, we presented a structural survey of ECALPS in E. coli R1, R2, R3, and R4 to correlate previous serological observations with the presence of ECALPS. The low yields of ECALPS were identified in the R1, R2, and R4 strains, where ECA occupied outer core residues of LPS that used to be substituted by O-specific polysaccharide in the case of smooth LPS. Previously published observations and hypotheses regarding the immunogenicity and biosynthesis of ECALPS were discussed and correlated with presented herein structural data.

scholarly journals Cyclic Enterobacterial Common Antigen Maintains the Outer Membrane Permeability Barrier ofEscherichia coliin a Manner Controlled by YhdP

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Angela M. Mitchell ◽  
Tharan Srikumar ◽  
Thomas J. Silhavy
Keyword(s):  
Membrane Permeability ◽  
Outer Membrane ◽  
Unknown Function ◽  
Permeability Barrier ◽  
Dependent Manner ◽  
Common Antigen ◽  
Gram Negative ◽  
Enterobacterial Common Antigen ◽  
K 12 ◽  
Outer Membrane Permeability

ABSTRACTGram-negative bacteria have an outer membrane (OM) impermeable to many toxic compounds that can be further strengthened during stress. InEnterobacteriaceae, the envelope contains enterobacterial common antigen (ECA), a carbohydrate-derived moiety conserved throughoutEnterobacteriaceae, the function of which is poorly understood. Previously, we identified several genes inEscherichia coliK-12 responsible for an RpoS-dependent decrease in envelope permeability during carbon-limited stationary phase. For one of these,yhdP, a gene of unknown function, deletion causes high levels of both vancomycin and detergent sensitivity, independent of growth phase. We isolated spontaneous suppressor mutants ofyhdPwith loss-of-function mutations in the ECA biosynthesis operon. ECA biosynthesis gene deletions suppressed envelope permeability fromyhdPdeletion independently of envelope stress responses and interactions with other biosynthesis pathways, demonstrating suppression is caused directly by removing ECA. Furthermore,yhdPdeletion changed cellular ECA levels andyhdPwas found to co-occur phylogenetically with the ECA biosynthesis operon. Cells make three forms of ECA: ECA lipopolysaccharide (LPS), an ECA chain linked to LPS core; ECA phosphatidylglycerol, a surface-exposed ECA chain linked to phosphatidylglycerol; and cyclic ECA, a cyclized soluble ECA molecule found in the periplasm. We determined that the suppression of envelope permeability withyhdPdeletion is caused specifically by the loss of cyclic ECA, despite lowered levels of this molecule found withyhdPdeletion. Furthermore, removing cyclic ECA from wild-type cells also caused changes to OM permeability. Our data demonstrate cyclic ECA acts to maintain the OM permeability barrier in a manner controlled by YhdP.IMPORTANCEEnterobacterial common antigen (ECA) is a surface antigen made by all members ofEnterobacteriaceae, including many clinically relevant genera (e.g.,Escherichia,Klebsiella,Yersinia). Although this surface-exposed molecule is conserved throughoutEnterobacteriaceae, very few functions have been ascribed to it. Here, we have determined that the periplasmic form of ECA, cyclic ECA, plays a role in maintaining the outer membrane permeability barrier. This activity is controlled by a protein of unknown function, YhdP, and deletion ofyhdPdamages the OM permeability barrier in a cyclic ECA-dependent manner, allowing harmful molecules such as antibiotics into the cell. This role in maintenance of the envelope permeability barrier is the first time a phenotype has been described for cyclic ECA. As the Gram-negative envelope is generally impermeable to antibiotics, understanding the mechanisms through which the barrier is maintained and antibiotics are excluded may lead to improved antibiotic delivery.

scholarly journals Crystal Structure of TDP-Fucosamine Acetyltransferase (WecD) from Escherichia coli, an Enzyme Required for Enterobacterial Common Antigen Synthesis

2006 ◽  
Vol 188 (15) ◽  
pp. 5606-5617 ◽  
Author(s):  
Ming-Ni Hung ◽  
Erumbi Rangarajan ◽  
Christine Munger ◽  
Guy Nadeau ◽  
Traian Sulea ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Coenzyme A ◽  
Structural Data ◽  
Enteric Bacteria ◽  
Common Antigen ◽  
Acetyl Coa ◽  
Enterobacterial Common Antigen ◽  
Thiolate Anion ◽  
Acetyl Transfer

ABSTRACT Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose, organized into trisaccharide repeating units having the sequence →3)-α-d-Fuc4NAc-(1→4)-β-d-ManNAcA-(1→4)-α-d-GlcNAc-(1→. While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coli (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-d-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD). We have determined the crystal structure of WecD in apo form at a 1.95-Å resolution and bound to acetyl-CoA at a 1.66-Å resolution. WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars. The crystal structure of WecD, however, represents the first structure of a GNAT family member that acts on nucleotide sugars. Based on this cocrystal structure, we have used flexible docking to generate a WecD-bound model of the acetyl-CoA-TDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. Our structural data show that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of coenzyme A.

scholarly journals O Acetylation of the Enterobacterial Common Antigen Polysaccharide Is Catalyzed by the Product of the yiaH Gene of Escherichia coli K-12

2006 ◽  
Vol 188 (21) ◽  
pp. 7542-7550 ◽  
Author(s):  
Junko Kajimura ◽  
Arifur Rahman ◽  
James Hsu ◽  
Matthew R. Evans ◽  
Kevin H. Gardner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Water Soluble ◽  
Hydroxyl Group ◽  
Insertion Mutagenesis ◽  
Amino Sugars ◽  
Common Antigen ◽  
Putative Gene ◽  
E Coli ◽  
Enterobacterial Common Antigen ◽  
K 12

ABSTRACT The carbohydrate component of the enterobacterial common antigen (ECA) of Escherichia coli K-12 occurs primarily as a water-soluble cyclic polysaccharide located in the periplasm (ECACYC) and as a phosphoglyceride-linked linear polysaccharide located on the cell surface (ECAPG). The polysaccharides of both forms are comprised of the amino sugars N-acetyl-d-glucosamine (GlcNAc), N-acetyl-d-mannosaminuronic acid (ManNAcA), and 4-acetamido-4,6-dideoxy-d-galactose (Fuc4NAc). These amino sugars are linked to one another to form trisaccharide repeat units with the structure →3-α-d-Fuc4NAc-(1→4)-β-d-ManNAcA-(1→4)-α-d-GlcNAc-(1→. The hydroxyl group in the 6 position of the GlcNAc residues of both ECACYC and ECAPG are nonstoichiometrically esterified with acetyl groups. Random transposon insertion mutagenesis of E. coli K-12 resulted in the generation of a mutant defective in the incorporation of O-acetyl groups into both ECACYC and ECAPG. This defect was found to be due to an insertion of the transposon into the yiaH locus, a putative gene of unknown function located at 80.26 min on the E. coli chromosomal map. Bioinformatic analyses of the predicted yiaH gene product indicate that it is an integral inner membrane protein that is a member of an acyltransferase family of enzymes found in a wide variety of organisms. The results of biochemical and genetic experiments presented here strongly support the conclusion that yiaH encodes the O-acetyltransferase responsible for the incorporation of O-acetyl groups into both ECACYC and ECAPG. Accordingly, we propose that this gene be designated wecH.

scholarly journals An “Uncommon” Role for Cyclic Enterobacterial Common Antigen in Maintaining Outer Membrane Integrity

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Corey S. Westfall
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Membrane Integrity ◽  
Physiological Role ◽  
Permeability Barrier ◽  
Common Antigen ◽  
Enterobacterial Common Antigen ◽  
Inner Membrane Protein ◽  
Outer Membrane Permeability

ABSTRACTAlthough discovered over 50 years ago, the physiological role of enterobacterial common antigen, a surface antigen produced by all members of theEnterobacteriaceae, has been poorly understood. In the work of Mitchell et al. (mBio 9:e01321-18, 2018,https://doi.org/10.1128/mBio.01321-18), the cyclized version of enterobacterial common antigen has been shown to play a role in maintaining the outer membrane permeability barrier, possibly through the inner membrane protein YhdP. This work also provides the tests needed to separate true effects from the numerous possible artifacts possible with mutations in enterobacterial common antigen synthesis.

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.

scholarly journals Genome-Wide Screening of Genes Required for Swarming Motility in Escherichia coli K-12

2006 ◽  
Vol 189 (3) ◽  
pp. 950-957 ◽  
Author(s):  
Tetsuyoshi Inoue ◽  
Ryuji Shingaki ◽  
Shotaro Hirose ◽  
Kaori Waki ◽  
Hirotada Mori ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Iron Acquisition ◽  
Tricarboxylic Acid Cycle ◽  
Common Antigen ◽  
Surface Acting ◽  
Swarming Motility ◽  
E Coli ◽  
Comprehensive Collection ◽  
Enterobacterial Common Antigen ◽  
K 12

ABSTRACT Escherichia coli K-12 has the ability to migrate on semisolid media by means of swarming motility. A systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection) was used to identify the genes involved in the swarming motility of this bacterium. Of the 3,985 nonessential gene mutants, 294 were found to exhibit a strongly repressed-swarming phenotype. Further, 216 of the 294 mutants displayed no significant defects in swimming motility; therefore, the 216 genes were considered to be specifically associated with the swarming phenotype. The swarming-associated genes were classified into various functional categories, indicating that swarming is a specialized form of motility that requires a wide variety of cellular activities. These genes include genes for tricarboxylic acid cycle and glucose metabolism, iron acquisition, chaperones and protein-folding catalysts, signal transduction, and biosynthesis of cell surface components, such as lipopolysaccharide, the enterobacterial common antigen, and type 1 fimbriae. Lipopolysaccharide and the enterobacterial common antigen may be important surface-acting components that contribute to the reduction of surface tension, thereby facilitating the swarm migration in the E. coli K-12 strain.

scholarly journals Assembly of Cyclic Enterobacterial Common Antigen in Escherichia coli K-12

2005 ◽  
Vol 187 (20) ◽  
pp. 6917-6927 ◽  
Author(s):  
Junko Kajimura ◽  
Arifur Rahman ◽  
Paul D. Rick
Keyword(s):  
Escherichia Coli ◽  
Subcellular Location ◽  
Dry Weight ◽  
Water Soluble ◽  
Cell Fractionation ◽  
Common Antigen ◽  
Enterobacterial Common Antigen ◽  
Repeat Units ◽  
Cell Extracts ◽  
K 12

ABSTRACT We describe here the purification and quantification of a water-soluble cyclic form of enterobacterial common antigen (ECACYC) from Escherichia coli K-12 as well as information regarding its subcellular location and the genetic loci involved in its assembly. Structural characterization of purified ECACYC molecules obtained from E. coli K-12 revealed that they uniformly contained four trisaccharide repeat units, and they were substituted with from zero to four O-acetyl groups. Cells from overnight cultures contained approximately 2 μg ECACYC per milligram (dry weight), and cell fractionation studies revealed that these molecules were localized exclusively in the periplasm. The synthesis and assembly of ECACYC were found to require the wzxE and wzyE genes of the wec gene cluster. These genes encode proteins involved in the transmembrane translocation of undecaprenylpyrophosphate-linked ECA trisaccharide repeat units and the polymerization of trisaccharide repeat units, respectively. Surprisingly, synthesis of ECACYC was dependent on the wzzE gene, which is required for the modulation of the polysaccharide chain lengths of phosphoglyceride-linked ECA (ECAPG). The presence of ECACYC in extracts of several other gram-negative enteric organisms was also demonstrated; however, it was not detected in cell extracts of Pseudomonas aeruginosa. These data suggest that in addition to ECAPG, ECACYC may be synthesized in many, if not all, members of the Enterobacteriaceae.

scholarly journals Outer Membrane Permeability Barrier in Escherichia coli Mutants That Are Defective in the Late Acyltransferases of Lipid A Biosynthesis

1999 ◽  
Vol 43 (6) ◽  
pp. 1459-1462 ◽  
Author(s):  
Martti Vaara ◽  
Marjatta Nurminen
Keyword(s):  
Escherichia Coli ◽  
Membrane Permeability ◽  
Outer Membrane ◽  
Lipid A ◽  
Enteric Bacteria ◽  
Normal Function ◽  
Permeability Barrier ◽  
Core Oligosaccharide ◽  
Hydrophobic Solutes ◽  
Outer Membrane Permeability

ABSTRACT The tight packing of six fatty acids in the lipid A constituent of lipopolysaccharide (LPS) has been proposed to contribute to the unusually low permeability of the outer membrane of gram-negative enteric bacteria to hydrophobic antibiotics. Here it is shown that theEscherichia coli msbB mutant, which elaborates defective, penta-acylated lipid A, is practically as resistant to a representative set of hydrophobic solutes (rifampin, fusidic acid, erythromycin, clindamycin, and azithromycin) as the parent-type control strain. The susceptibility index, i.e., the approximate ratio between the MIC for the msbB mutant and that for the parent-type control, was maximally 2.7-fold. In comparison, the rfa mutant defective in the deep core oligosaccharide part of LPS displayed indices ranging from 20 to 64. The lpxA and lpxD lipid A mutants had indices higher than 512. Furthermore, the msbBmutant was resistant to glycopeptides (vancomycin, teicoplanin), whereas the rfa, lpxA, and lpxDmutants were susceptible. The msbB htrB double mutant, which elaborates even-more-defective, partially tetra-acylated lipid A, was still less susceptible than the rfa mutant. These findings indicate that hexa-acylated lipid A is not a prerequisite for the normal function of the outer membrane permeability barrier.

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