scholarly journals Model for the Controlled Synthesis of O-Antigen Repeat Units Involving the WaaL Ligase

mSphere ◽  
2015 ◽  
Vol 1 (1) ◽  
Author(s):  
Yaoqin Hong ◽  
Peter R. Reeves
Keyword(s):  
Repeat Unit ◽  
Feedback Mechanism ◽  
Growth Defect ◽  
Controlled Synthesis ◽  
Inner Membrane ◽  
O Antigen ◽  
Repeat Units

ABSTRACT The Wzx/Wzy O-antigen pathway involves synthesis of a repeat unit (O unit) consisting of 3 to 8 sugars on an inner-membrane-embedded lipid carrier. These O units are translocated across the membrane to its periplasmic face by Wzx, while retaining linkage to the carrier, and then polymerized by Wzy to O-antigen polymer, which WaaL ligase transfers to a lipopolysaccharide precursor to complete lipopolysaccharide synthesis, concomitantly releasing the lipid carrier. This lipid carrier is also used for peptidoglycan assembly, and sequestration is known to be toxic. Thus, O-unit synthesis must involve precise regulation to meet demand but avoid overproduction. Here we show that loss of WaaL reverses a known growth defect in a Salmonella mutant that otherwise accumulates O-unit intermediates and propose that WaaL is also involved in a novel feedback mechanism to regulate O-unit synthesis, based on the availability of O units on the periplasmic face of the membrane.

scholarly journals Bacteriophage-encoded glucosyltransferase GtrII of Shigella flexneri: membrane topology and identification of critical residues

2005 ◽  
Vol 389 (1) ◽  
pp. 137-143 ◽  
Author(s):  
Adele M. LEHANE ◽  
Haralambos KORRES ◽  
Naresh K. VERMA
Keyword(s):  
Sequence Similarity ◽  
Repeat Unit ◽  
Shigella Flexneri ◽  
Membrane Topology ◽  
Transmembrane Helices ◽  
O Antigen ◽  
Repeat Units ◽  
Related Group ◽  
Critical Residues ◽  
O Antigens

The Shigella flexneri serotypes differ in the nature of their O-antigens. The addition of glucosyl or O-acetyl groups to the common backbone repeat units gives rise to the different serotypes. GtrII glucosylates rhamnose III of the O-antigen repeat unit, thus converting serotype Y (which has no modifications to the basic O-antigen repeat unit) into serotype 2a, the most prevalent serotype. In the present study, the topology of GtrII has been determined. GtrII has nine transmembrane helices, a re-entrant loop and three large periplasmic regions. Four critical residues (Glu40, Phe414, Cys435 and Lys478) were identified in two of the periplasmic regions. Despite the lack of sequence similarity between GtrII and the Gtrs from other serotypes, three of the critical residues identified are conserved in the remaining Gtrs. This is consistent with some degree of mechanistic conservation in this functionally related group of proteins.

scholarly journals A Mutation of the Yeast Gene Encoding PCNA Destabilizes Both Microsatellite and Minisatellite DNA Sequences

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 511-519 ◽  
Author(s):  
Robert J Kokoska ◽  
Lela Stefanovic ◽  
Andrew B Buermeyer ◽  
R Michael Liskay ◽  
Thomas D Petes
Keyword(s):  
Dna Polymerase ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Repeat Unit ◽  
Nuclear Antigen ◽  
Temperature Sensitive ◽  
Dinucleotide Repeats ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Polymerase Δ ◽  
Repeat Units

AbstractThe POL30 gene of the yeast Saccharomyces cerevisiae encodes the proliferating cell nuclear antigen (PCNA), a protein required for processive DNA synthesis by DNA polymerase δ and ϵ. We examined the effects of the pol30-52 mutation on the stability of microsatellite (1- to 8-bp repeat units) and minisatellite (20-bp repeat units) DNA sequences. It had previously been shown that this mutation destabilizes dinucleotide repeats 150-fold and that this effect is primarily due to defects in DNA mismatch repair. From our analysis of the effects of pol30-52 on classes of repetitive DNA with longer repeat unit lengths, we conclude that this mutation may also elevate the rate of DNA polymerase slippage. The effect of pol30-52 on tracts of repetitive DNA with large repeat unit lengths was similar, but not identical, to that observed previously for pol3-t, a temperature-sensitive mutation affecting DNA polymerase δ. Strains with both pol30-52 and pol3-t mutations grew extremely slowly and had minisatellite mutation rates considerably greater than those observed in either single mutant strain.

scholarly journals Tim18p Is a New Component of the Tim54p-Tim22p Translocon in the Mitochondrial Inner Membrane

2000 ◽  
Vol 11 (1) ◽  
pp. 103-116 ◽  
Author(s):  
Oliver Kerscher ◽  
Naresh B. Sepuri ◽  
Robert E. Jensen
Keyword(s):  
Growth Defect ◽  
Inner Membrane ◽  
New Members ◽  
Mitochondrial Inner Membrane ◽  
Native Electrophoresis ◽  
Multiple Copies ◽  
New Gene ◽  
Synthetically Lethal ◽  
Blue Native Electrophoresis ◽  
Blue Native

The mitochondrial inner membrane contains two separate translocons: one required for the translocation of matrix-targeted proteins (the Tim23p-Tim17p complex) and one for the insertion of polytopic proteins into the mitochondrial inner membrane (the Tim54p-Tim22p complex). To identify new members of the Tim54p-Tim22p complex, we screened for high-copy suppressors of the temperature-sensitivetim54-1 mutant. We identified a new gene,TIM18, that encodes an integral protein of the inner membrane. The following genetic and biochemical observations suggest that the Tim18 protein is part of the Tim54p-Tim22p complex in the inner membrane: multiple copies of TIM18 suppress thetim54-1 growth defect; thetim18::HIS3 disruption is synthetically lethal with tim54-1; Tim54p and Tim22p can be coimmune precipitated with the Tim18 protein; and Tim18p, along with Tim54p and Tim22p, is detected in an ∼300-kDa complex after blue native electrophoresis. We propose that Tim18p is a new component of the Tim54p-Tim22p machinery that facilitates insertion of polytopic proteins into the mitochondrial inner membrane.

scholarly journals A structure hierarchy for silicate minerals: chain, ribbon, and tube silicates

2020 ◽  
Vol 84 (2) ◽  
pp. 165-244 ◽  
Author(s):  
Maxwell C. Day ◽  
Frank C. Hawthorne
Keyword(s):  
Repeat Unit ◽  
Structural Connectivity ◽  
The Other ◽  
Silicate Minerals ◽  
One Dimensional ◽  
Compositional Range ◽  
Repeat Units ◽  
Sheet Silicates ◽  
Sheet Silicate ◽  
Connectivity Degree

AbstractA structure hierarchy is developed for chain-, ribbon- and tube-silicate based on the connectedness of one-dimensional polymerisations of (TO4)n− tetrahedra, where T = Si4+ plus P5+, V5+, As5+, Al3+, Fe3+, B3+, Be2+, Zn2+ and Mg2+. Such polymerisations are described by a geometrical repeat unit (with ng tetrahedra) and a topological repeat unit (or graph) (with nt vertices). The connectivity of the tetrahedra (vertices) in the geometrical (topological) repeat units is denoted by the expression cTr (cVr) where c is the connectivity (degree) of the tetrahedron (vertex) and r is the number of tetrahedra (vertices) of connectivity (degree) c in the repeat unit. Thus cTr = 1Tr12Tr23Tr34Tr4 (cVr = 1Vr12Vr23Vr34Vr4) represents all possible connectivities (degrees) of tetrahedra (vertices) in the geometrical (topological) repeat units of such one-dimensional polymerisations. We may generate all possible cTr (cVr) expressions for chains (graphs) with tetrahedron (vertex) connectivities (degrees) c = 1 to 4 where r = 1 to n by sequentially increasing the values of c and r, and by ranking them accordingly. The silicate (sensu lato) units of chain-, ribbon- and tube-silicate minerals are identified and associated with the relevant cTr (cVr) symbols. Following description and association with the relevant cTr (cVr) symbols of the silicate units in all chain-, ribbon- and tube-silicate minerals, the minerals are arranged into decreasing O:T ratio from 3.0 to 2.5, an arrangement that reflects their increasing structural connectivity. Considering only the silicate component, the compositional range of the chain-, ribbon- and tube-silicate minerals strongly overlaps that of the sheet-silicate minerals. Of the chain-, ribbon- and tube-silicates and sheet silicates with the same O:T ratio, some have the same cVr symbols (vertex connectivities) but the tetrahedra link to each other in different ways and are topologically different. The abundance of chain-, ribbon- and tube-silicate minerals decreases as O:T decreases from 3.0 to 2.5 whereas the abundance of sheet-silicate minerals increases from O:T = 3.0 to 2.5 and decreases again to O:T = 2.0. Some of the chain-, ribbon- and tube-silicate minerals have more than one distinct silicate unit: (1) vinogradovite, revdite, lintisite (punkaruaivite) and charoite have mixed chains, ribbons and/or tubes; (2) veblenite, yuksporite, miserite and okenite have clusters or sheets in addition to chains, ribbons and tubes. It is apparent that some chain-ribbon-tube topologies are favoured over others as of the ~450 inosilicate minerals, ~375 correspond to only four topologically unique graphs, the other ~75 minerals correspond to ~46 topologically unique graphs.

scholarly journals Proton-Dependent Gating and Proton Uptake by Wzx Support O-Antigen-Subunit Antiport Across the Bacterial Inner Membrane

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Salim T. Islam ◽  
Paul D. W. Eckford ◽  
Michelle L. Jones ◽  
Timothy Nugent ◽  
Christine E. Bear ◽  
...  
Keyword(s):  
Cell Surface ◽  
Bacterial Cell ◽  
Electrical Potential ◽  
Three Dimensional ◽  
Structural Data ◽  
Inner Membrane ◽  
Content Type ◽  
Bacterial Cell Surface ◽  
Repeat Units ◽  
Dependent Transport

ABSTRACTWzx flippases are crucial for bacterial cell surface polysaccharide assembly as they transport undecaprenyl pyrophosphate-linked sugar repeat units from the cytoplasmic to the periplasmic leaflets of the inner membrane (IM) for final assembly. Our recently reported three-dimensional (3D) model structure of Wzx fromPseudomonas aeruginosaPAO1 (WzxPa) displayed a cationic internal vestibule and functionally essential acidic amino acids within transmembrane segment bundles. Herein, we examined the intrinsic transport function of WzxPafollowing its purification and reconstitution in phospholipid liposomes. WzxPawas capable of mediating anion flux, consistent with its cationic interior. This flux was electrogenic and modified by extraliposomal pH. Mutation of the above-mentioned acidic residues (E61, D269, and D359) reduced proton (H+)-modified anion flux, showing the role of these amino acid side chains in H+-dependent transport. Wzx also mediated acidification of the proteoliposome interior in the presence of an outward anion gradient. These results indicate H+-dependent gating and H+uptake by WzxPaand allow for the first H+-dependent antiport mechanism to be proposed for lipid-linked oligosaccharide translocation across the bacterial IM.IMPORTANCEMany bacterial cell surface polysaccharides that are important for survival and virulence are synthesized at the periplasmic leaflet of the inner membrane (IM) using precursors produced in the cytoplasm. Wzx flippases are responsible for translocation of lipid-linked sugar repeat units across the IM and had been previously suggested to simply facilitate passive substrate diffusion. Through our characterization of purified Wzx in a reconstitution system described herein, we have observed protein-dependent intrinsic transport producing a change in the electrical potential of the system, with H+identified as the coupling ion. These results provide the first evidence for coupled (i.e., secondary active) transport by these proteins and, in conjunction with structural data, allow for an antiport mechanism to be proposed for the directed transport of lipid-linked sugar substrates across the IM. These findings bring our understanding of lipid-linked polysaccharide transporter proteins more in line with the efflux pumps to which they are evolutionarily related.

Structural analysis of the lipopolysaccharide O-antigen from Fusobacterium nucleatum strain CC 7/3 JVN3 C1 and development of a mouse monoclonal antibody specific to the O-antigen

2020 ◽  
Vol 66 (9) ◽  
pp. 529-534
Author(s):  
Chantelle M. Cairns ◽  
Frank St. Michael ◽  
Perry Fleming ◽  
Evgeny V. Vinogradov ◽  
Andrew D. Cox
Keyword(s):  
Colorectal Cancer ◽  
Repeat Unit ◽  
Fusobacterium Nucleatum ◽  
Cross Reactivity ◽  
Emerging Pathogen ◽  
O Antigen ◽  
Still Births ◽  
Structural Identity ◽  
Potential Factor ◽  
Surface Carbohydrates

Fusobacterium nucleatum is becoming increasingly recognised as an emerging pathogen, gaining attention as a potential factor for exacerbating colorectal cancer and is strongly linked with pregnancy complications including pre-term and still births. Little is known about the virulence factors of this organism; thus, we have initiated studies to examine the bacterium’s surface glycochemistry. In an effort to characterise the surface carbohydrates of F. nucleatum, the aims of this study were to investigate the structure of the lipopolysaccharide (LPS) O-antigen of the cancer-associated isolate F. nucleatum strain CC 7/3 JVN3 C1 (hereafter C1) and to develop monoclonal antibodies (mAbs) to the LPS O-antigen that may be beneficial to the growing field of F. nucleatum research. In this study, we combined several technologies, including nuclear magnetic resonance (NMR) spectroscopy, to elucidate the structure of the LPS O-antigen repeat unit as -[-4-β-Gal-3-α-FucNAc4N-4-α-NeuNAc-]-. We have previously identified this structure as the LPS O-antigen repeat unit from strain 10953. In this present study, we developed a mAb to the C1 LPS O-antigen and confirmed the mAbs cross-reactivity to the 10953 strain, thus confirming the structural identity.

scholarly journals Isolation of a Pericentromeric Satellite DNA Family in Chnootriba argus (Henosepilachna argus) with an Unusual Short Repeat Unit (TTAAAA) for Beetles

Insects ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 306 ◽  
Author(s):  
Pablo Mora ◽  
Jesús Vela ◽  
Areli Ruiz-Mena ◽  
Teresa Palomeque ◽  
Pedro Lorite
Keyword(s):  
Repetitive Dna ◽  
Satellite Dna ◽  
Repeat Unit ◽  
Pericentromeric Region ◽  
Fish Analysis ◽  
Agricultural Pests ◽  
Base Pairs ◽  
Satellite Dnas ◽  
Repeat Units

Ladybird beetles (Coccinellidae) are one of the largest groups of beetles. Among them, some species are of economic interest since they can act as a biological control for some agricultural pests whereas other species are phytophagous and can damage crops. Chnootriba argus (Coccinellidae, Epilachnini) has large heterochromatic pericentromeric blocks on all chromosomes, including both sexual chromosomes. Classical digestion of total genomic DNA using restriction endonucleases failed to find the satellite DNA located on these heterochromatic regions. Cloning of C0t-1 DNA resulted in the isolation of a repetitive DNA with a repeat unit of six base pairs, TTAAAA. The amount of TTAAAA repeat in the C. argus genome was about 20%. Fluorescence in situ hybridization (FISH) analysis and digestion of chromosomes with the endonuclease Tru9I revealed that this repetitive DNA could be considered as the putative pericentromeric satellite DNA (satDNA) in this species. The presence of this satellite DNA was tested in other species of the tribe Epilachnini and it is also present in Epilachna paenulata. In both species, the TTAAAA repeat seems to be the main satellite DNA and it is located on the pericentromeric region on all chromosomes. The size of this satDNA, which has only six base pairs is unusual in Coleoptera satellite DNAs, where satDNAs usually have repeat units of a much larger size. Southern hybridization and FISH proved that this satDNA is conserved in some Epilachnini species but not in others. This result is in concordance with the controversial phylogenetic relationships among the genera of the tribe Epilachnini, where the limits between genera are unclear.

scholarly journals Synthesis and Preliminary Characterization of a PPE-Type Polymer Containing Substituted Fullerenes and Transition Metal Ligation Sites

2015 ◽  
Vol 2015 ◽  
pp. 1-11
Author(s):  
Corinne A. Basinger ◽  
Kaitlin Sullivan ◽  
Sarah Siemer ◽  
Stuart Oehrle ◽  
Keith A. Walters
Keyword(s):  
Transition Metal ◽  
Excited States ◽  
Conjugated Polymer ◽  
Repeat Unit ◽  
Sonogashira Coupling ◽  
Polymeric Systems ◽  
Preliminary Characterization ◽  
Repeat Units ◽  
Future Work

A substituted fullerene was incorporated into a PPE-conjugated polymer repeat unit. This subunit was then polymerized via Sonogashira coupling with other repeat units to create polymeric systems approaching 50 repeat units (based on GPC characterization). Bipyridine ligands were incorporated into some of these repeat units to provide sites for transition metal coordination. Photophysical characterization of the absorption and emission properties of these systems shows excited states located on both the fullerene and aromatic backbone of the polymers that exist in a thermally controlled equilibrium. Future work will explore other substituted polyaromatic systems using similar methodologies.

Synthesis of the tetrasaccharide repeat unit of the O-antigen polysaccharide from Escherichia coli O77 employing the 2′-carboxybenzyl glycoside

2006 ◽  
Vol 84 (4) ◽  
pp. 506-515 ◽  
Author(s):  
Bo Ram Lee ◽  
Joo Mi Jeon ◽  
Jae Hyuk Jung ◽  
Heung Bae Jeon ◽  
Kwan Soo Kim
Keyword(s):  
Escherichia Coli ◽  
Repeat Unit ◽  
O Antigen ◽  
Glycosyl Donor ◽  
Glycosyl Donors

The synthesis of the suitably protected form (1) of the tetrasaccharide repeat unit, →2)-α-D-Manp-(1→2)-β-D-Manp-(1→3)-α-D-GlcpNAc-(1→6)-α-D-Manp-(1→ (A), of the O-antigen polysaccharide of the lipopolysaccharide from Escherichia coli O77 has been accomplished by latent–active glycosylation employing the 2′-carboxybenzyl (CB) gly coside method. In addition to previously used latent glycosyl donors, 2′-(benzyloxycarbonyl)benzyl (BCB) glycosides, new latent glycosyl donors, 2'-(allyloxycarbonyl)benzyl (ACB) glycosides, have been introduced as a direct precursor for the active CB glycosides. We also demonstrate that 4,6-O-benzylidene-2-azido-2-deoxy-α-D-mannopyranoside (7) has been readily prepared from D-glucosamine in good yield.Key words: Escherichia coli O77, glycosylation, 2′-carboxybenzyl (CB) glycosides, 2′-(allyloxycarbonyl)benzyl (ACB) glycosides, glycosyl donor.

scholarly journals Genetic Analysis of the Mode of Interplay between an ATPase Subunit and Membrane Subunits of the Lipoprotein-Releasing ATP-Binding Cassette Transporter LolCDE

2006 ◽  
Vol 188 (8) ◽  
pp. 2856-2864 ◽  
Author(s):  
Yasuko Ito ◽  
Hitomi Matsuzawa ◽  
Shin-ichi Matsuyama ◽  
Shin-ichiro Narita ◽  
Hajime Tokuda
Keyword(s):  
Abc Transporters ◽  
Atp Hydrolysis ◽  
Dominant Negative ◽  
Growth Defect ◽  
Atp Binding ◽  
Inner Membrane ◽  
Atpase Subunit ◽  
E Coli ◽  
Atp Binding Cassette Transporter ◽  
Atp Binding Cassette

ABSTRACT The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the inner membrane. Thus, functional interaction between LolD and LolC/E is critically important for coupling of ATP hydrolysis to the lipoprotein release reaction. LolD contains a characteristic sequence called the LolD motif, which is highly conserved among LolD homologs but not other ABC transporters of E. coli. The LolD motif is suggested to be a region in contact with LolC/E, judging from the crystal structures of other ABC transporters. To determine the functions of the LolD motif, we mutagenized each of the 32 residues of the LolD motif and isolated 26 dominant-negative mutants, whose overexpression arrested growth despite the chromosomal lolD + background. We then selected suppressor mutations of the lolC and lolE genes that correct the growth defect caused by the LolD mutations. Mutations of the lolC suppressors were mainly located in the periplasmic loop, whereas ones of lolE suppressors were mainly located in the cytoplasmic loop, suggesting that the mode of interaction with LolD differs between LolC and LolE. Moreover, the LolD motif was found to be critical for functional interplay with LolC/E, since some LolD mutations lowered the ATPase activity of LolCDE without affecting that of LolD.

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