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At least 19 recordsLinked to original sources
PubMed1992

The rfaC gene of Salmonella typhimurium. Cloning, sequencing, and enzymatic function in heptose transfer to lipopolysaccharide.

We have cloned a gene from a Salmonella typhimurium with the ability to complement the rfaC mutation (heptose-deficient lipopolysaccharide, sensitivity to rough-specific bacteriophages, and susceptibility to hydrophobic antibiotics). A 1018-base pair EcoRV-Tth111I fragment, subcloned into the pBluescriptKS+ vector to yield pKZ103, retains complementing activity. Nucleotide sequencing revealed an open reading frame corresponding to a protein of 317 amino acids (M(r) approximately 35,100). The plasmid pKZ103, which has a properly aligned T7 promoter, can overexpress a protein of M(r) = 31,000 when T7 RNA polymerase is supplied. An in vitro system was established for analysis of heptose addition to the precursor [4'-32P](KDO)2-IVA (Brozek, K. A., Hosaka, K., Robertson, A. D., and Raetz, C. R. H. (1989) J. Biol. Chem, 264, 6956-6966). Soluble fractions from wild-type or heptose-deficient rfa mutants were tested for their ability to convert [4'-32P](KDO)2-IVA to more polar substances. In wild-type extracts, these conversions required addition of ATP or ADP-heptose. In extracts of rfaC-, rfaD-, or rfaE-deficient strains, no polar products were observed with ATP. ADP-heptose restored synthesis in rfaD and rfaE but not rfaC extracts, indicating that rfaD and rfaE are involved in ADP-heptose formation. When the cloned rfaC gene was introduced into an rfaC-deficient mutant, extracts from such cells regained the ability to metabolize [4'-32P](KDO)2-IVA, showing that rfaC encodes the enzyme that attaches the proximal heptose to lipopolysaccharide.

Amino Acid Sequence↗
PubMed1976

Heptose-deficient mutants of Escherichia coli K12 deficient in up to three major outer membrane proteins.

Mutants of Escherichia coli K12, deficient in up to three major outer membrane proteins b, c and d have been constructed. Mutants that lack the lipopolysaccharide sugar heptose are deficient in protein b. All heptose-deficient strains are supersensitive to lysozyme, various antibiotics and detergents. They excrete the periplasmic enzyme ribonuclease I. Mutants deficient in proteins c and/or d have the same sensitivity towards these compounds as the parent strain. Cells of single, double and triple mutants are all rod-shaped. Electrophoretic analysis of cell envelope proteins indicates that in some mutants the protein deficiency is partially compensated for by increased amounts of one or two of the other major outer membrane proteins. Heptose-deficient strains have an increased amount of 2-keto-3-deoxyoctonate.

Anti-Bacterial Agents↗
PubMed1975

Alterations in envelope structure of heptose-deficient mutants of Escherichia coli as revealed by freeze-etching.

The surface of freeze-etched E. coli strain GR467, a heptose-deficient ("deep rough") mutant derived from CR34, was studied by electron microscopy. The outer membrane of GR467 has an increased ratio of phospholipid to protein, mainly due to a decreased protein content. Freeze-etched CR34 showed structural features indistinguishable for wild-type E. coli, i.e., the primary cleavage occurring in the inner membrane with only minor appearance of cleavage within the outer membrane. In contrast to this, in mutant GR467 most of the freeze-cleavages had taken place along a new plane, presumably in a hydrophobic region of the outer membrane. In this cleavage plane numerous particles were seen. Often the cleavage extended over the entire exposed cell surface; occasionally only a few large plateaus were visible, around which the next deeper cleavage plane, that of the protoplasmic or inner membrane, was discernible. Two spontaneous revertants (R11 and R16) with protein and lipid A levels similar to wild-type cells showed mostly freeze fractures with wild-type characteristics, and only a few cells had retained fracturing properties of GR467. A partial revertant revealed intermediate characteristics. Thus, there appears to be a morphological correlation with the chemical data relating the amount of outer membrane protein with the heptose content of the lipopolysaccharide.

Cell Membrane↗
PubMed1992

Biological activity of chemically synthesized core sugar linked lipid A analog, heptose-(alpha 1----5)-2-keto-3-deoxyoctonic acid-(alpha 2----6)-2,3-diacyloxyacylglucosamine-4-phosphate.

The mitogenicity, lethal toxicity and antitumor activity against Meth A fibrosarcoma and the induction of tumor necrosis factor (TNF) of chemically synthesized compounds designated as A-103, 2,3-diacyloxyacylglucosamine-4-phosphate (GlcN-4-P), and A-503), heptose-(alpha 1----5)-2-keto-3-deoxyoctonic acid (KDO)-linked GlcN-4-P (A-103), were determined. Compound A-103 induced significant incorporation of [3H]thymidine of C57BL/6 mice at 25-100 micrograms/ml, and A-503 showed the highest incorporation of [3H]thymidine at 100 micrograms/ml. The mitogenicity of A-503 exhibited a lower activity than of A-103. Compound A-503 showed no lethality at high doses of 25 and 50 micrograms/mouse in C57BL/6 mice loaded with D-galactosamine, whereas A-103 caused the death of one of three mice at a dose of 50 micrograms/mouse. Although, the two compounds with or without muramyl dipeptide showed weak antitumor activity against Meth A fibrosarcoma in BALB/c mice, but there were no remarkable differences between the compounds on antitumor activity. Peritoneal macrophages, stimulated with A-103 or A-503 caused no production of TNF which induces L929 cell lysis in vitro. These findings indicate that the addition of heptose and KDO to GlcN-4-P seems not to affect mitogenic activity, lethal toxicity, antitumor activity and TNF-production of the GlcN-4-P compound (A-103).

Animals↗
PubMed1976

Conjugation deficient E. coli K12 F- mutants with heptose-less lipopolysaccharide.

Two F- mutants deficient in conjugation with F-type donors are isolated and characterized. Phenotypically, these mutants are similar; they have heptose-less lipopolysaccharide and lack some outer membrane protein. Genotypically, they are different. One mutant harbors a point mutation in the 70 to 74 min region, while the other is deleted for the chromosomal region 6.5 to 8.5 min. Comparison of the properties of the conjugation-deficient mutants described in this paper with other such mutants suggests that an outer membrane protein is the receptor for the f-pilus.

Binding Sites↗
PubMed1976

7-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-heptose. A constituent of the endotoxin of Bordetella pertussis.

Hydrolysis of the Bordetella pertussis endotoxin, extracted from both "phase I" and "phase IV" bacteria, with 4 M HCl for 1 h at 100 degrees C, released the disaccharide named in the title; it was isolated by paper electrophoresis or by ion-exchange chromatography in about 1% yield (w/w). The structure of the heptose could be rigorously established by chemical degradation; the facts that the glucosaminidic linkage was hydrolysed by an enzyme preparation containing both, alpha and beta-N-acetylglucosaminidase activities, whereas it was resistant to cleavage by pure beta-N-acetylglucosaminidase strongly support the assumption that the disaccharide contains an alpha-D-glucosaminide linkage.

Bordetella pertussis↗
PubMed1975

Comparison of the cell envelope structure of a lipopolysaccharide-defective (heptose-deficient) strain and a smooth strain of Salmonella typhimurium.

The cell envelope structure of Salmonella typhimurium LT2, which has a heptose-deficient lipopolysaccharide (LPS), is significantly different from that of an isogenic strain with a normal LPS. The rough strain, when examined by freeze-etching, lacks most surface structures that are routinely present in the smooth strain (surface particles and flagella) and has few transmemberane studs in the cytoplasmic membrane (those present are generally found in aggregates), and the outer membrane cleavage is substantially stronger than that of the smooth strain. These envelope differences were independent of both growth temperature and culture age. Examination of ultrathin sections indicated that the rough strain has an outer membrane which forms a much more defined double-track artifact than the smooth strain. The addition of MgCl2 to the growth medium of the rough strain decreased the extent of outer membrane cleavage, and flagella became evident in freeze-etched preparations. The presence of supplemental MgCl2 in the growth medium, which resulted in these morphological changes in the rough strain, also produced growth at a previously restrictive temperature and a decrease in the leakage of periplasmic enzymes. The smooth strain was unaltered morphologically or physiologically by MgCl2 under identical conditions. It is suggested that the outer membrane of the rough strain is more planar.

Cell Wall↗
PubMed1979

Structure of the lipopolysaccharide from an Escherichia coli heptose-less mutant. I. Chemical degradations and identification of products.

The structure of lipopolysaccharide from a heptose-less mutant of Escherichia coli K-12 has been investigated. Lipopolysaccharide isolated from 32P-labeled cells was treated with mild alkali to yield two separable components: [OH-LPS]-I (approximately 70%) and [OH-LPS]-II (approximately 30%). Mild acidic treatment of [OH-LPS]-I gave mainly a product which was identified as (4-O-phosphoryl-N-beta-hydroxymyristyl-D-glucosaminyl)-beta(1 leads to 6)-N-beta-hydroxymyristyl-D-glucosamine 1-phosphate (Compound I). Further acidic hydrolysis of both [OH-LPS]-I and [OH-LPS]-II yielded as the main product (4-O-phosphoryl-N-beta-hydroxymyristyl-D-glucosaminyl)-beta(1 leads to 6)-N-beta-hydroxymyristyl-D-glucosamine (Compound II). The structures of the above products were deduced by a combination of compositional analyses, sensitivity to phosphomonoesterase, rates of hydrolysis of the phosphate groups and alkali-catalyzed beta elimination of the phosphate residues following appropriate oxidation of hydroxyl groups. These studies together with work reported in the accompanying papers have led to the identification of two species of lipopolysaccharide in the E. coli strain both of which contain a single glucosamine dissacharide unit but differ in having monosubstituted phosphate or pyrophosphate groups at the glycosidic position. Each species of lipopolysaccharide also appeared to be heterogeneous with respect to the number of esterified fatty acyl groups.

Alkaline Phosphatase↗
PubMed1979

Structural and biochemical examination of ghosts derived from a deep rough (heptose-deficient lipopolysaccharide) strain and a smooth strain of Escherichia coli.

Outer membrane derived 'ghosts' can be readily generated from both smooth and deep rough (heptose-deficient LPS) strains of Escherichia coli 08. MORPHOlogical and biochemical studies confirmed that 'ghosts' of both strains are composed of protein (four major proteins), LPS, and phospholipid (cardiolipin and phosphatidylethanolamine) in the form of a single membrane of roughly the same shape as intact normal cells. The ghost membrane cleaves only slightly in freeze-etch preparations of ghosts derived from the smooth strain as compared to the extensive cleavage plane of ghosts derived from the rough strain. The asymmetrical distribution of ghost proteins was visualized, by critical point drying and shadowing with platinum, as a relatively smooth outer surface with some discernible particles (10-15 nm) and an extremely particulate inner surface (10-15-mm particles. Ghosts derived from the smooth strain retained their structure following chloroform-methanol extraction, while ghosts derived from the rough strain fragmented with chloroform-methanol extraction. Evidence is presented that LPS-protein interactions as well as protein-protein interactions are significant in maintaining the ghost structure.

Bacterial Proteins↗
PubMed1988

[Antigenic polysaccharides of bacteria. 25. Structure of the O-specific polysaccharide chain of Pseudomonas cepacia 673/2 lipopolysaccharide containing L-glycero-D-manno-heptose].

O-Specific polysaccharide, consisting of D-rhamnose and L-glycero-D-manno-heptose (LD-Hep) in a 2 : 1 ratio, was obtained on the mild acid degradation of the Pseudomonas cepacia IMV 673/2 lipopolysaccharide; monosaccharide LD-Hep has not previously been found in O-specific chains of lipopolysaccharides. On the basis of methylation and 13C-NMR data, it was concluded that the polysaccharide is composed of trisaccharide repeating units having the following structure: ----3)-alpha-D-Rha-(1----3)-alpha-D-Rha-(1----2)-alpha-LD-Hep-(1----

Antigens, Bacterial↗
PubMed1979

The structure of lipopolysaccharide from an Escherichia coli heptose-less mutant. III. Two fatty acyl amidases from Dictyostelium discoideum and their action on lipopolysaccharide derivatives.

Two fatty acyl amidases have been partially purified from the slime mold, Dictyostelium discoideum. Their action on lipopolysaccharide derivatives, especially Compound I, has been studied. Amidase I removes specifically the beta-hydroxymyristyl group, which is present on the amino group adjacent to the C-1 phosphate. The product, Compound V, is then a substrate for Amidase II, which removes the remaining beta-hydroxymyristyl group from the amino group in the distal glucosamine ring to give Compound VI. Compound I itself is resistant to Amidase II. Thus, the two enzymes show a high degree of structural specificity. The structure of lipopolysaccharide from the E. coli K-12 mutant is concluded in the light of studies reported in this and the accompanying papers, and this structure is discussed in relation to other bacterial lipopolysaccharides.

Amidohydrolases↗
PubMed1992

Synthesis of 2-(4-trifluoroacetamidophenyl)ethyl O-(L-glycero-alpha-D-manno-heptopyranosyl)-(1----7)-O-(L-glycero-alpha- D-manno- heptopyranosyl)-(1----3)-L-glycero-alpha-D-manno-heptopyranoside, corresponding to the heptose region of the Salmonella Ra core structure.

The title trisaccharide was synthesized from methyl 2,3,4-tri-O-benzyl-L-glycero-alpha-D-manno-heptopyranoside by acetolysis, followed by conversion into ethyl thioglycosides and also glycosyl bromides, which were both used in glycosylation reactions. In glycosylations using thioglycosides as glycosyl donors, N-iodosuccinimide-silver triflate and dimethyl(methylthio)sulfonium triflate were used as promoters, and in glycosylations with glycosyl bromides silver triflate was used. The protecting groups introduced into intermediates during the synthesis of the title trisaccharide were designed to allow later glycosylation at O-3' to give larger oligosaccharide fragments of the Salmonella LPS core region, and also to allow the introduction of phosphate groups at O-4 and O-4', a structural element that is suggested to be present in the Ra core.

Carbohydrate Sequence↗
PubMed1976

Leakage of periplasmic enzymes from lipopolysaccharide-defective mutants of Salmonella typhimurium.

Mutants of Salmonella typhimurium with defects in the heptose region of the lipopolysaccharide (LPS) molecule (heptose-deficient, chemotype Re) leak periplasmic enzymes (acid phosphatase (EC 3.1.3.2), cyclic phosphodiesterase, ribonuclease I (EC 3.1.4.22), and phosphoglucose isomerase (EC 5.3.1.9) (PGI is at least partially periplasmic in E. coli and S. typhimurium; see below)) and do not leak an internal enzyme (glucose-6-phosphate dehydrogenase) into the growth medium. The extent of this leakage is markedly increased at higher temperature (42 degrees C). Leakage of periplasmic enzymes from the strains lacking units distal to heptose I in the LPS molecule (chemotype Rd2) occurs only at 42 degrees C, and not at 30 or 37 degrees C. The extent of leakage of these enzymes from smooth strain and mutants of other LPS chemotypes (Rc, Rd1) is not significant, and is not influenced by growth temperatures. The kinetics of leakage of periplasmic enzymes after shift to 42 degrees C in nutrient broth reveal an accelerated release into the medium from heptose-deficient strains of cyclic phosphodiesterase and ribonuclease I after 30 min at 42 degrees C, and phosphoglucose isomerase after 60 min at 42 degrees C; at 30 degrees C the rate of release of cyclic phosphodiesterase and ribonuclease I is relatively slower. After 60 min at 42 degrees C in nutrient broth, growth of these strains has either slowed down or stopped. In L-broth, which permits the growth of the heptose-deficient strain (SA1377) at 42 degrees C, leakage of cyclic phosphodiesterase and phosphoglucose isomerase occurs, whereas there is no detectable leakage of these enzymes from the isogenic smooth strain (SA1355). Thus, leakage of the periplasmic enzymes from the heptose-deficient strain occurs with or without growth. Mg2+ (0.75 mM), sodium chloride (50 mM), and sucrose (100 mM) in nutrient broth at 42 degrees C prevent the leakage of these enzymes. The shedding of LPS from the heptose-deficient as well as the smooth strains is enhanced by high temperature (42 degrees C), whereas considerable leakage of protein occurs only in the heptose-deficient strain at 42 degrees C and not in the smooth strain. The smooth and heptose-deficient strains are equally sensitive to osmotic shock although a significant proportion of acid phosphatase and cyclic phosphodiesterase activities from the heptose-deficient cells grown at 42 degrees C comes off in the Tris-NaCl wash step suggesting a rather loose attachment of these enzymes onto the cell surface.

Acid Phosphatase↗
PubMed1992

Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12.

Deletions which removed rfa genes involved in lipopolysaccharide (LPS) core synthesis were constructed in vitro and inserted into the chromosome by linear transformation. The deletion delta rfa1, which removed rfaGPBI, resulted in a truncated LPS core containing two heptose residues but no hexose and a deep rought phenotype including decreased expression of major outer membrane proteins, hypersensitivity to novobiocin, and resistance to phage U3. In addition, delta rfa1 resulted in the loss of flagella and pili and a mucoid colony morphology. Measurement of the synthesis of beta-galactosidase from a cps-lacZ fusion showed that the mucoid phenotype was due to rcsC-dependent induction of colanic acid capsular polysaccharide synthesis. Complementation of delta rfa1 with rfaG+ DNA fragments resulted in a larger core and restored the synthesis of flagella and pili but did not reverse the deep rough phenotype or the induction of cps-lacZ, while complementation with a fragment carrying only rfaP+ reversed the deep rough phenotype but not the loss of flagella and pili. A longer deletion which removed rfaQGPBIJ was also constructed, and complementation studies with this deletion showed that the product of rfaQ was not required for the functions of rfaG and rfaP. Thus, the function of rfaQ remains unknown. Tandem mass spectrometric analysis of LPS core oligosaccharides from complemented delta rfa1 strains indicated that rfaP+ was necessary for the addition of either phosphoryl (P) or pyrophosphorylethanolamine (PPEA) substituents to the heptose I residue, as well as for the partial branch substitution of heptose II by heptose III. The substitution of heptose II is independent of the type of P substituent present on heptose I, and this results in four different core structures. A model is presented which relates the deep rough phenotype to the loss of heptose-linked P and PPEA.

Carbohydrate Sequence↗