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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method of identifying plasmids containing genes responsible for synthesis of nucleotide sugar:lipopolysaccharide glycosyltransferases is described. Hybrid ColE1 plasmids containing random fragments of the chromosome of Escherichia coli K12 were introduced into an indicator strain of Salmonella typhimurium which lacks UDP-glucose:lipopolysaccharide glucosyltransferase I due to an rfaG mutation. Plasmids capable of correcting the transferase defect were identified by their ability to convert the bacteriophage sensitivity pattern of the recipient strain from Ffm-sensitive to Ffm-resistant. Analysis of the lipopolysaccharide of the S. typhimurium/ColE1 hybrid strains and assay of cell extracts defined the new enzyme activities. Two plasmids were identified which carried the rfaG+ gene; one of these plasmids also contained genetic information for a second glucosyltransferase, the E. coli glucosyltransferase II, which normally is not present in S. typhimurium.
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PMID:Cloning of genes for bacterial glycosyltransferases. I. Selection of hybrid plasmids carrying genes for two glucosyltransferases. 36 61

R-prime plasmids carrying the pyrE-rfa-cysE region of the chromosome of Salmonella typhimurium were isolated by using the vector pULB113 (RP4::mini-Mu). One of the R-prime plasmids was used as a source of DNA to clone the rfa genes for lipopolysaccharide synthesis to pBR322. The following three hybrid plasmids were constructed: pKZ15, with a 4.0-kilobase EcoRI fragment of S. typhimurium DNA, containing the rfaG gene; pKZ27, a 9-kilobase BglII fragment with the rfaG, rfaB, and rfaI genes; and pKZ26, a 7.7-kilobase HindIII fragment with the rfaG, rfaB, rfaI, and rfaJ genes. We propose that these cloned genes code for four glycosyltransferases used for synthesis of the lipopolysaccharide core region (rfaG for glucosyltransferase I; rfaI for galactosyltransferase I; rfaB for galactosyltransferase II; and rfaJ for glucosyltransferase II). For all four genes, mutants which lacked the appropriate enzyme activity were complemented by the plasmids to give completed core lipopolysaccharide with O (somatic) side chains; for rfaG, rfaB, and rfaI, mutants gave restored or even amplified levels of the appropriate glycosyltransferase in in vitro assays. We show that the order of genes in the region is pyrE-rfaG-(rfaB-rfaI)-rfaJ-rfaL-rfaF -cysE.
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PMID:Cloning of rfaG, B, I, and J genes for glycosyltransferase enzymes for synthesis of the lipopolysaccharide core of Salmonella typhimurium. 315 16

The role of sfrB and rfaH genes in the regulation of expression of membrane glycosyltransferases was studied in Escherichia coli and Salmonella typhimurium. The transferase enzymes form part of a multienzyme system involved in biosynthesis of the polysaccharide core of Gram-negative bacterial lipopolysaccharides. Several sfrB mutants of E. coli showed reductions of 90-98% in the activities of two of the glycosyltransferases (UDP-galactose:(glucosyl)lipopolysaccharide 1,6-galactosyltransferase and UDP-glucose: (glucosyl)lipopolysaccharide 1,3-glucosyltransferase). Introduction of a recombinant ColE1 plasmid restored the transferase levels to normal and simultaneously corrected the F-factor defects that also characterize sfrB mutants; recombinant plasmids containing other regions of the E. coli chromosome were ineffective. An amber mutation of the S. typhimurium rfaH gene (thought to be the homologue of the E. coli sfrB gene) resulted in 97% loss of activity of the Salmonella UDP-galactose:(glucosyl)lipopolysaccharide galactosyltransferase. Antibody precipitation studies showed that the loss of enzyme activity in the amber mutant was associated with a corresponding decrease in amount, but not in size, of the transferase protein, indicating that the gene is not the structural gene for the S. typhimurium galactosyltransferase. Taken together, the results indicate that the sfrB(rfaH) gene acts as a positive regulatory element in expression of multiple glycosyltransferases in E. coli and S. typhimurium.
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PMID:Regulation of membrane glycosyltransferases by the sfrB and rfaH genes of Escherichia coli and Salmonella typhimurium. 623 Mar 55

A Drosophila UDP-glucose:glycoprotein glucosyltransferase was isolated, cloned and characterized. Its 1548 amino acid sequence begins with a signal peptide, lacks any putative transmembrane domains and terminates in a potential endoplasmic reticulum retrieval signal, HGEL. The soluble, 170 kDa glycoprotein occurs throughout Drosophila embryos, in microsomes of highly secretory Drosophila Kc cells and in small amounts in cell culture media. The isolated enzyme transfers [14C]glucose from UDP-[14C]Glc to several purified extracellular matrix glycoproteins (laminin, peroxidasin and glutactin) made by these cells, and to bovine thyroglobulin. These proteins must be denatured to accept glucose, which is bound at endoglycosidase H-sensitive sites. The unusual ability to discriminate between malfolded and native glycoproteins is shared by the rat liver homologue, previously described by A.J. Parodi and coworkers. The amino acid sequence presented differs from most glycosyltransferases. There is weak, though significant, similarity with a few bacterial lipopolysaccharide glycotransferases and a yeast protein Kre5p. In contrast, the 56-68% amino acid identities with partial sequences from genome projects of Caenorhabditis elegans, rice and Arabidopsis suggest widespread homologues of the enzyme. This glucosyltransferase fits previously proposed hypotheses for an endoplasmic reticular sensor of the state of folding of newly made glycoproteins.
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PMID:Drosophila UDP-glucose:glycoprotein glucosyltransferase: sequence and characterization of an enzyme that distinguishes between denatured and native proteins. 772 8

Lactococcus lactis strain NIZO B40 produces an extracellular phosphopolysaccharide containing galactose, glucose, and rhamnose. A 40 kb plasmid encoding exopolysaccharide production was isolated through conjugal transfer of total plasmid DNA from strain NIZO B40 to the plasmid-free L. lactis model strain MG1614 and subsequent plasmid curing. A 12 kb region containing 14 genes with the order epsRXABCDEFGHIJKL was identified downstream of an iso-IS982 element. The predicted gene products of epsABCDEFGHIJK show sequence homologies with gene products involved in exopolysaccharide, capsular polysaccharide, lipopolysaccharide, or teichoic acid biosynthesis of other bacteria. Transcriptional analysis of the eps gene cluster revealed that the gene cluster is transcribed as a single 12 kb mRNA. The transcription start site of the promoter was mapped upstream of the first gene epsR. The involvement of epsD in exopolysaccharide (EPS) biosynthesis was demonstrated through a single gene disruption rendering an exopolysaccharide-deficient phenotype. Heterologous expression of epsD in Escherichia coli showed that its gene product is a glucosyltransferase linking the first sugar of the repeating unit to the lipid carrier.
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PMID:Molecular characterization of the plasmid-encoded eps gene cluster essential for exopolysaccharide biosynthesis in Lactococcus lactis. 915 24

Acyl carrier protein participates in a number of biosynthetic pathways in Escherichia coli: fatty acid biosynthesis, phospholipid biosynthesis, lipopolysaccharide biosynthesis, activation of prohemolysin, and membrane-derived oligosaccharide biosynthesis. The first four pathways require the protein's prosthetic group, phosphopantetheine, to assemble an acyl chain or to transfer an acyl group from the thioester linkage to a specific substrate. By contrast, the phosphopantetheine prosthetic group is not required for membrane-derived oligosaccharide biosynthesis, and the function of acyl carrier protein in this biosynthetic scheme is currently unknown. We have combined biochemical and molecular biological approaches to investigate domains of acyl carrier protein that are important for membrane-derived oligosaccharide biosynthesis. Proteolytic removal of the first 6 amino acids from acyl carrier protein or chemical synthesis of a partial peptide encompassing residues 26 to 50 resulted in losses of secondary and tertiary structure and consequent loss of activity in the membrane glucosyltransferase reaction of membrane-derived oligosaccharide biosynthesis. These peptide fragments, however, inhibited the action of intact acyl carrier protein in the enzymatic reaction. This suggests a role for the loop regions of the E. coli acyl carrier protein and the need for at least two regions of the protein for participation in the glucosyltransferase reaction. We have purified acyl carrier protein from eight species of Proteobacteria (including representatives from all four subgroups) and characterized the proteins as active or inhibitory in the membrane glucosyltransferase reaction. The complete or partial amino acid sequences of these acyl carrier proteins were determined. The results of site-directed mutagenesis to change amino acids conserved in active, and altered in inactive, acyl carrier proteins suggest the importance of residues Glu-4, Gln-14, Glu-21, and Asp-51. The first 3 of these residues define a face of acyl carrier protein that includes the beginning of the loop region, residues 16 to 36. Additionally, screening for membrane glucosyltransferase activity in membranes from bacterial species that had acyl carrier proteins that were active with E. coli membranes revealed the presence of glucosyltransferase activity only in the species most closely related to E. coli. Thus, it seems likely that only bacteria from the Proteobacteria subgroup gamma-3 have periplasmic glucans synthesized by the mechanism found in E. coli.
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PMID:Domains of Escherichia coli acyl carrier protein important for membrane-derived-oligosaccharide biosynthesis. 917 19

Escherichia coli K-12 WaaO (formerly known as RfaI) is a nonprocessive alpha-1,3 glucosyltransferase, involved in the synthesis of the R core of lipopolysaccharide. By comparing the amino acid sequence of WaaO with those of 11 homologous alpha-glycosyltransferases, four strictly conserved regions, I, II, III, and IV, were identified. Since functionally related transferases are predicted to have a similar architecture in the catalytic sites, it is assumed that these four regions are directly involved in the formation of alpha-glycosidic linkage from alpha-linked nucleotide diphospho-sugar donor. Hydrophobic cluster analysis revealed a conserved domain at the N termini of these alpha-glycosyltransferases. This domain was similar to that previously reported for beta-glycosyltransferases. Thus, this domain is likely to be involved in the formation of beta-glycosidic linkage between the donor sugar and the enzyme at the first step of the reaction. Site-directed mutagenesis analysis of E. coli K-12 WaaO revealed four critical amino acid residues.
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PMID:Conserved structural regions involved in the catalytic mechanism of Escherichia coli K-12 WaaO (RfaI). 976 61

The major core oligosaccharide biosynthesis operons from prototype Escherichia coli strains displaying R1 and R4 lipopolysaccharide core types were polymerase chain reaction-amplified and analyzed. Comparison of deduced products of the open reading frames between the two regions indicate that all but two share total similarities of 94% or greater. Core oligosaccharide structures resulting from nonpolar insertion mutations in each gene of the core OS biosynthesis operon in the R1 strain allowed assignment of all of the glycosyltransferase enzymes required for outer core assembly. The difference between the R1 and R4 core oligosaccharides results from the specificity of the WaaV protein (a beta1, 3-glucosyltransferase) in R1 and WaaX (a beta1, 4-galactosyltransferase) in R4. Complementation of the waaV mutant of the R1 prototype strain with the waaX gene of the R4 strain converted the core oligosaccharide from an R1- to an R4-type lipopolysaccharide core molecule. Aside from generating core oligosaccharide specificity, the unique beta-linked glucopyranosyl residue of the R1 core plays a crucial role in organization of the lipopolysaccharide. This residue provides a novel attachment site for lipid A-core-linked polysaccharides and distinguishes the R1-type LPS from existing models for enterobacterial lipopolysaccharides.
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PMID:The assembly system for the outer core portion of R1- and R4-type lipopolysaccharides of Escherichia coli. The R1 core-specific beta-glucosyltransferase provides a novel attachment site for O-polysaccharides. 979 56

Escherichia coli K-12 WaaR is a non-processive alpha-1,2 glucosyltransferase, involved in the synthesis of the R-core of lipopolysaccharide. WaaR possesses the four conserved structural regions I, II, III and IV, each presumably involved in the mechanistic function in catalysis. Regions I and III contain the pair of strictly conserved Asp residues. Asp-129, 131 (region I) and 215, 217 (region III) of WaaR were individually converted to Asn by the site-directed mutagenesis of the waaR gene. All mutated enzymes were inactive, supporting the model for an alpha-glycosyl transfer reaction where the pair of strictly conserved aspartic acid residues in regions I and III play a critical role in the catalytic function.
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PMID:Four critical aspartic acid residues potentially involved in the catalytic mechanism of Escherichia coli K-12 WaaR. 1023 27

In Escherichia coli, phosphoryl substituents in the lipopolysaccharide core region are essential for outer membrane stability. Mutation of the core glucosyltransferase encoded by waaG (formerly rfaG) resulted in lipopolysaccharide truncated immediately after the inner core heptose residues, which serve as the sites for phosphorylation. Surprisingly, mutation of waaG also destabilized the outer membrane. Structural analyses of waaG mutant lipopolysaccharide showed that the cause for this phenotype was a decrease in core phosphorylation, an unexpected side effect of the waaG mutation.
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PMID:Mutation of the lipopolysaccharide core glycosyltransferase encoded by waaG destabilizes the outer membrane of Escherichia coli by interfering with core phosphorylation. 1098 72


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