Structure of the core regions in lipopolysaccharides from Escherichia coli K12 W2252-11U−, the Ter-15 mutant,and Ter-15 (F′-lac) and Ter-15 (F+) cells

From Escherichia coli K12 W2252-11U^? cells, the Ter-15 mutant, the Ter-15 (F′-lac) and the Ter-15 (F⁺) cells, lipopolysaccharides were isolated and the primary structure of its core oligosaccharides was elucidated. When the F′-lac episome is transferred to the Ter-15 mutant by conjugation, the structure of the glucose III(1 → 3)glucose II(1 → 3)glucose I residue and the galactose I(1 → 2)-linked to the glucose I residue in the core oligosaccharide from the Ter-15 mutant changes into the structure of the glucose IV(1 → 6)glucose III(1 → 2)glucose II(1 → 3)glucose I residue and the galactose I (1 → 6)-linked to the glucose I residue in the core oligosaccharide from the Ter-15 (F′-lac) cells, but the core oligosaccharide in the Ter-15 (F⁺) cells is the same structure with that of the core oligosaccharide from the Ter-15 mutant when F⁺ episome is transferred to the Ter-15 mutant. Also, the core oligosaccharide from the Ter-15 (F′-lac) cells shows the same structure with that of the core oligosaccharide from E. coli K12 W2252-11U^? cells (the parent cells). As the result, the ability to produce the structure of the core oligosaccharide in E. coli K12 W2252-11U^? cells is recovered in the Ter-15 (F′-lac) cells by the dominant expression of lac gene or its containing DNA segment in F′-lac episome.     

Blocking of bacteriophages phi W and phi 5 with lipopolysaccharides from Escherichia coli K-12 mutants.

In the preceding paper we presented a formula for the composition of lipopolysaccharides (LPS) from Escherichia coli K-12. This formula contains four regions defined from analyses of LPS from four key strains, the parent and mutants which had lost one, two, or three regions of their carbohydrates. Support for the formula was derived from the susceptibility of the key mutants to several bacteriophages. One of these, phage phi W, was found specific for strains which had lost region 4. In this paper we described inactivation in vitro of phage phi W and its host-range mutant phi 5, using LPS devoid of regions 2 to 4. The blocking of phi W was found to require about 0.15 M concentrations of monovalent cations and to be inhibited by low concentrations of calcium and magnesium ions. One particle of phage phi W required 2 times 10-16 g of LPS devoid of region 4 for stoichiometric inactivation. Phage phi 5 was blocked by both heptose-less LPS (devoid of regions 2 to 4) and glucose-less LPS (devoid of regions 3 to 4) but was unaffected by LPS devoid of region 4. LPS from a heptose-less mutant of Salmonella minnesota showed the same inactivation ability as did LPS from heptose-less strains of E. coli K-12. Lipid A was prepared from LPS containing all four regions. Such lipid A was found to inactivate phi 5, whereas both the polysaccharide moiety as well as the intact LPS were without effect. It is suggested that lipid A is part of the receptor site for phage phi 5.     

电离辐射对3T3细胞膜钙依赖性钾通道的影响及康复新的作用

肉芽组织增生是创伤愈合的中心环节 ,成纤维细胞不仅是这一环节的重要组成部分 ,而且还分泌一些细胞因子促进肉芽组织增生、调节伤口愈合。放射损伤使伤口愈合延迟与其抑制成纤维细胞的分化、增殖与分泌功能密切相关 ,但对其机理的深入研究的文献报道较少。近年来 ,细胞膜离子流的作用日益受到人们的重视 ,因为它与细胞的分化、增殖及细胞内大分子物质的合成与分泌等功能均有密切关系。本文研究 6Ｇｙ照射对 3Ｔ3成纤维细胞株钙依赖性钾离子通道活动的影响及促进伤口愈合药物康复新的作用 ,以期从离子通道角度探讨放射损伤后创伤愈合延迟及…     

The structures of the carbohydrate backbones of the lipopolysaccharides from Escherichia coli rough mutants F470 (R1 core type) and F576 (R2 core type).

The lipopolysaccharides (LPS) from Escherichia coli rough mutant strains F470 (R1 core type) and F576 (R2 core type) were deacylated yielding in each case a mixture of oligosaccharides with one predominant product which was isolated using high-performance anion-exchange chromatography. In addition, one oligosaccharide present in minor quantities was isolated from LPS of E. coli strain F576 (R2 core type). The structures of the oligosaccharides were determined by chemical analyses and NMR spectroscopic experiments. Furthermore, de-O-acylated and dephosphorylated LPS preparations were investigated by fast-atom bombardment and collision induced dissociation tandem mass spectrometry. The combined data allow us to deduce the following carbohydrate backbones of the E. coli R1 and R2 core types which share the following structure (Scheme 1): but differ in the substituents R1 and R2 which for the R1 core type are predominantly: and to a minor extent: and for the R2 core type predominantly: and to a minor extent: in which all sugars are d-pyranoses (l,d-Hep, lglycerodmanno-heptopyranose; P, phosphate).     

The effect of composition of the core region of Escherichia coli K-12 lipopolysaccharides on the surface properties of cells

The pH dependences of electrokinetic potentials (EKP) of the cells of two Escherichia coli K-12 strains (D21 and JM 103) with known lipopolysaccharide (LPS) core composition have been determined by the method of microelectrophoresis. At pH 4.6-5.2, the negative surface charge of the cells with Re core LPS was reliably higher. It was shown that the interaction of bacteria with lysozyme results in a decrease of optical density of suspensions due to higher sensitivity of the cells with complete LPS core to hypotonic shock. LPS release from bacterial cell wall depended also on bacterial LPS core composition and increased with LPS core extension. Electrokinetic measurements and the study of the interaction of cells with lysozyme suggest that higher negative surface charge of E. coli JM 103 cells (Re type LPS) is associated with higher quantity and density of LPS packing in the cell wall as compared with the cells of E. coli D21 (Ra type LPS).     

Molecular basis for structural diversity in the core regions of the lipopolysaccharides of Escherichia coli and Salmonella enterica

Bacterial lipopolysaccharides (LPS) are unique and complex glycolipids that provide characteristic components of the outer membranes of Gram-negative bacteria. In LPS of the Enterobacteriaceae, the core oligosaccharide links a highly conserved lipid A to the antigenic O-polysaccharide. Structural diversity in the core oligosaccharide is limited by the constraints imposed by its essential role in outer membrane stability and provides a contrast to the hypervariable O-antigen. The genetics of core oligosaccharide biosynthesis in Salmonella and Escherichia coli K-12 have served as prototypes for studies on the LPS and lipo-oligosaccharides from a growing range of bacteria. However, despite the wealth of knowledge, there remains a number of unanswered questions, and direct experimental data are not yet available to define the precise mechanism of action of many gene products. Here we present a comparative analysis of the recently completed sequences of the major core oligosaccharide biosynthesis gene clusters from the five known core types in E. coli and the Ra core type of Salmonella enterica serovar Typhimurium and discuss advances in the understanding of the related biosynthetic pathways. Differences in these clusters reflect important structural variations in the outer core oligosaccharides and provide a basis for ascribing functions to the genes in these model clusters, whereas highly conserved regions within these clusters suggest a critical and unalterable function for the inner region of the core.     

Structure and function of an OmpC deletion mutant porin from Escherichia coli K-12.

Escherichia coli K-12 strain RAM122 contains a mutation in the ompC gene that results in an eight amino acid deletion, delta 103-110, in the porin protein. Since this strain is capable of growing on maltodextrins in the absence of a functional lamB gene, the mutant protein is thought to have a larger channel size. The stability and structure/function properties of the mutant OmpC porin were investigated and compared to wild-type porin. Isolated unheated RAM122 porin was characterized as a trimer on sodium dodecyl sulfate-polyacrylamide gels. The RAM122 trimer was less stable to temperature when compared to the wild-type porin. In addition, the overall enthalpy for thermal denaturation was lower for the mutant than the wild-type porin as determined by using differential scanning microcalorimetry. Both the proteins' secondary structures, monitored by circular dichroism, were high in beta-sheet content, but the spectra were slightly different in their crossover points as well as their minima. When the proteins were reconstituted and channel activity was assayed by using a liposome swelling technique, the size-exclusion limit of the mutant porin was twice that of the wild-type porin. Conductance measurements across bilayer lipid membranes showed that the mutant porin was voltage gated at much lower membrane potentials, 50 and 75 mV, than the wild-type sample. The closing events of the mutant porin were predominantly of monomer size. The channels detected by using the mutant protein were larger in size than those measured for the wild-type porin monomer. These data suggest that the OmpC mutant porin has a channel size capable of allowing maltodextrins to enter and that this channel is highly voltage regulated.     

Nucleoside transport in cells and membrane vesicles from Escherichia coli K12.

Osmotic shock treatment of cells of Escherichia coli K12 caused a reduction in the transport of nucleosides into the cells. The strains used carried mutations in the nucleoside catabolizing enzymes. This indicated that the decrease in transport capacity was not due to loss of these enzymes during the shock treatment. Membrane vesicles, prepared from the same strains, showed a limited transport of cytidine, deoxycytidine, and uridine. Transport of purine nucleosides and of thymidine was very low in vesicles lacking the appropriate nucleoside phosphorylases and no significant stimulation was observed if the nucleoside phosphorylases were present in the membrane vesicles. These results all indicate that components outside the cytoplasmic membrane are important for nucleoside transport. Selection for resistance to fluorodeoxycytidine yielded mutants which were unable to transport any nucleoside, even when the nucleoside phosphorylases were present in high amounts. This finding is consistent with a requirement for a specific transport process prior to the initial enzymatic attack on the incoming nucleoside.     

Sequence of the mglB gene from Escherichia coli K12: comparison of wild-type and mutant galactose chemoreceptors

Summary The mglB gene of Escherichia coli codes for a galactose-binding protein (GBP) that serves both as the galactose chemoreceptor and as the recognition component of the -methylgalactoside transport system. The mglB551 mutation eliminates the chemotactic function of GBP without altering its transport or substrate-binding properties. To investigate the interaction between GBP and Trg, the chemotactic signal transducer for galactose, we sequenced the mglB genes from wild-type and mglB551 mutant strains. The mutation causes the replacement of Gly₇₄ of GBP by Asp. This residue is located in alpha-Helix III at the tip of the P domain in the GBP tertiary structure farthest removed from the substrate-binding cleft between the P and Q domains. We conclude that Helix III must be part of, or at least adjacent to, the recognition site for Trg. Our sequence also included part of the mglA gene, which is immediately distal to mglB. The amino acid sequence deduced for the beginning of the MglA protein showed homology with a family of polypeptides that contain an ATP-binding site and are components of binding-protein-dependent transport systems.     

Structural analysis of oligosaccharides from lipopolysaccharide (LPS) of Escherichia coli K12 strain W3100 reveals a link between inner and outer core LPS biosynthesis

Lipopolysaccharide (LPS) from Escherichia coli K12 W3100 is known to contain several glycoforms, and the basic structure has been investigated previously by methylation analyses (Holst, O. (1999) in Endotoxin in Health and Disease (Brade, H., Opal, S. M., Vogel, S. N., and Morrison, D., eds) pp. 115-154; Marcel Dekker, Inc., New York). In order to reveal dependences of gene activity and LPS structure, we have now determined the composition of de-O-acylated LPS by electrospray ionization-Fourier transform ion cyclotron-mass spectrometry (ESI-FT-MS) and identified 11 different LPS molecules. We have isolated the major glycoforms after de-O- and de-N-acylation and obtained four oligosaccharides that differed in their carbohydrate structure and phosphate substitution. The main oligosaccharide accounted for approximately 70% of the total and had a molecular mass of 2516 Da according to ESI-FT-MS. The dodecasaccharide structure (glycoform I) as determined by NMR was consistent with MS and compositional analysis. One minor oligosaccharide (5%) of the same carbohydrate structure did not contain the 4'-phosphate of the lipid A. Two oligosaccharides contained the same phosphate substitution but differed in their carbohydrate structure, one (5%) which contained an additional beta-D-GlcN in 1-->7 linkage on a terminal heptose residue (glycoform II) which was N-acetylated in LPS. A minor amount of a molecule lacking the terminal L-alpha-D-Hep in the outer core but otherwise identical to the major oligosaccharide (glycoform III) could only be identified by ESI-FT-MS of the de-O-acylated LPS. The other oligosaccharide (20%) contained an alpha-Kdo-(2-->4)-[alpha-l-Rha-(1-->5)]-alpha-Kdo-(2-->4)-alpha-Kdo branched tetrasaccharide connected to the lipid A (glycoform IV). This novel inner core structure was accompanied by a truncation of the outer core in which the terminal disaccharide L-alpha-D-Hep-(1-->6)-alpha-D-Glc was missing. The latter structure was identified for the first time in LPS and revealed that changes in the inner core structure may be accompanied by structural changes in the outer core.     

Analysis of Escherichia coli K12 F factor transfer genes: traQ, trbA, and trbB

The genes that encode the transfer properties of plasmid F, the fertility factor of Escherichia coli K12, are known to be clustered over a large, 33.3-kb segment of F DNA. As the central segment of the transfer region has not previously been well characterized, we constructed a detailed restriction map of the large F EcoRI DNA fragment, fl, and isolated a series of plasmid derivatives that carry various overlapping segments of this F tra operon DNA. We also analyzed the protein products of those clones that carried DNA segments extending over the region between traF and traH. This region was known to include traQ, a gene required for efficient conversion of the direct product of traA to the 7000-Da pilin polypeptide. We identified the traQ product as a polypeptide that migrates as a 12,500-Da protein on sodium dodecyl sulfate-polyacrylamide gels. We also detected the products of two other new genes that we have named trbA and trbB. These polypeptides migrate with apparent molecular weights of 14,200 and 18,400, respectively. Analysis of plasmid deletion derivatives that we constructed in vitro shows that these genes map in the order traF trbA traQ trbB traH. The presence of a plasmid carrying a small 0.43-kb fragment that expressed only the 12,500 traQ product caused the traA product of a co-resident compatible plasmid to be converted to the 7000-Da pilin polypeptide, demonstrating that TraQ is the only tra operon product required for this step of F-pilin biosynthesis.     

Mutations in the promoter regions of the malEFG and malK-lamB operons of Escherichia coli K12

The malB region of Escherichia coli is composed of two operons, malEFG and malK-lamB, transcribed divergently from a control region located between the malE and malK genes. Expression of the malB operons is under the positive control of the malT gene product (MalT) and maltose and of the crp gene product (CRP) and cyclic AMP. Strains in which the lac genes have been fused to malE or malK are unable to use lactose as carbon source if they have been deleted for malT or crp. Mutations in the malB region allowing such fusion strains to grow on lactose have been isolated. These and previously isolated mutations were genetically characterized. As regards the malEp promoter mutations, malEp9, malEp1 and malEp6 create new promoters that are MalT and CRP independent. malEp9 and malEp1 change residues -1 and -2, respectively, of malEp without altering its activity. malEp6 duplicates six base-pairs between residues -22 and -23. malEp3 improves the -10 region hexamer. malEp5 deletes residues -29 to -62. It creates a new promoter that is MalT independent, CRP dependent, likely by fusing together functional regions of malEp that are normally apart. malEp5 also reduces the expression of malK-lamB, suggesting the existence of a link between the malEp and malKp promoters. As regards the malKp mutations, malKp6 changes residue -81 of malKp without altering its activity. It creates a new promoter, which is MalT independent, CRP dependent, likely by using a pre-existing cyclic AMP/CRP binding site. malKp102 changes residue -36, two bases upstream of the -35 region hexamer. It decreases the activity of malKp by at least four orders of magnitude and likely alters the MalT binding site. These results are discussed in terms of regulatory interactions within the malB control region.     

Study of the lipopolysaccharide from a thermosensitive mutant CR-34 T 83 of Escherichia coli K 12]

The structure of the core of the lipopolysaccharide from T 83 mutant of Escherichia coli K 12 CR 34 was partially determined. Using dephosphorylation, enzymic hydrolysis, Smith degradation, methylations and analysis by gas chromatography/mass spectrometry an oligosaccharide sequence was determined with D-glucose, D-galactose and L-glycero-D-mannoheptose as sugar components. The structure which was demonstrated could be that of the characteristic core fragment of the K 12 type lipopolysaccharides from Escherichia coli.     

Structure of the core part of the lipopolysaccharides from Proteus penneri strains 7, 8, 14, 15, and 21

The core-lipid A region of the lipopolysaccharides from Proteus penneri strains 7, 8, 14, 15, and 21 was studied using NMR spectroscopy, ESI MS, and chemical analysis after alkaline deacylation, deamination, and mild-acid hydrolysis of the lipopolysaccharides. The following general structure of the major core oligosaccharides is proposed: [abstract: see text] where all sugars are in the pyranose form and have the D configuration unless otherwise stated, Hep and DDHep=L-glycero- and D-glycero-D-manno-heptose, respectively, K=H, and Q=H in strain 8 or alpha-Glc in strains 7, 14, 15, and 21. In addition, several minor structural variants are present, including those lacking Ara4N in strains 7 and 15 and having the alpha-GlcN residue N-acylated to a various degree with glycine in strains 7, 8, 14, and 21. In strain 14, there are also core oligosaccharides with K=amide of beta-D-GalpA with putrescine, spermidine, or 4-azaheptane-1,7-diamine; remarkably, these structural variants lack either the PEtN group or the alpha-Hep-(1-->2)-alpha-DDHep disaccharide fragment at alpha-D-GalpA. While structural features of the inner core part are shared by Proteus strains studied earlier, the outermost Q-(1-->4)-alpha-GalNAc-(1-->2)-alpha-DDHep-(1-->6)-alpha-GlcN oligosaccharide unit has not been hitherto reported.     

Structure of the 2-keto-3-deoxy-D-manno-octonic-acid-containing capsular polysaccharide (K12 antigen) of the urinary-tract-infective Escherichia coli O4:K12:H-

The primary structure of the K12 antigenic capsular polysaccharide (K12 antigen) of Escherichia coli O4:K12:H- was elucidated by composition, nuclear magnetic resonance spectroscopy, methylation, periodate oxidation and oligosaccharide analysis. The polysaccharide consists of repeating trisaccharide alpha-rhamnosyl-1,2-alpha-rhamnosyl-1,5-dOclA units (dOclA = 2-keto-3-deoxy-D-manno-octonic acid) which are joined through beta-2,3-linkages. About 50% of the dOclA units are O-acetylated at C7 or C8. The sequence of acetylated and non-acetylated dOclA residues is not known. As had been reported before, the polysaccharide is linked to a phosphatidic acid at the reducing end (dOclA) via a phosphodiester bridge. The serologically specific part of the K12 antigen is its polysaccharide moiety.