Non-lamellar Structure and Negative Charges of Lipopolysaccharides Required for Efficient Folding of Outer Membrane Protein PhoE of Escherichia coli

Cock, Hans de; Brandenburg, Klaus; Wiese, Andre; Holst, Otto; Seydel, Ulrich

doi:10.1074/jbc.274.8.5114

Cited by 79 publications

(68 citation statements)

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“…Performing in vitro folding studies, it has been shown that LPS is required for correct and efficient folding of PhoE into a folded monomeric intermediate (13) and that LPS influences folding and trimerization of OmpF protein (25). In the …”

Section: Discussionmentioning

confidence: 99%

“…For PhoE a decrease of 95% was found for a Re mutant in comparison to a Ra mutant (12). Furthermore, it has been shown in an in vitro model that the chemical structure and the biophysical properties of LPS are important for a correct and efficient folding of PhoE into a native-like, trimerization-competent, folded monomeric form (13). The folding of in vitro synthesized non-native PhoE protein into a native-like monomeric form was more efficient with LPS of chemotype Ra (Ra LPS) as compared to Re LPS, lacking a large part of the core.…”

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confidence: 99%

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Pore Formation and Function of Phosphoporin PhoE of Escherichia coli Are Determined by the Core Sugar Moiety of Lipopolysaccharide

Hagge

Cock

Gutsmann

et al. 2002

Journal of Biological Chemistry

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The lipid matrix of the outer membrane of Gram-negative bacteria is an asymmetric bilayer composed of a phospholipid inner leaflet and a lipopolysaccharide outer leaflet. Incorporated into this lipid matrix are, among other macromolecules, the porins, which have a sieve-like function for the transport or exclusion of hydrophilic substances. It is known that a reduced amount of porins is found in the outer membrane of rough mutants as compared with wild-type bacteria. This observation was discussed to be caused by a reduced number of insertion sites in the former. We performed electrical measurements on reconstituted planar bilayers composed of lipopolysaccharide on one side and a phospholipid mixture on the other side using lipopolysaccharide from various rough mutant strains of Salmonella enterica serovar Minnesota. We found that pore formation by PhoE trimers that were added to the phospholipid side of the bilayers increased with the increasing length of the lipopolysaccharide core sugar moiety. These results allow us to conclude that the length of the sugar moiety of lipopolysaccharide is the parameter governing pore formation and that no particular insertion sites are required. Furthermore, we found that the voltage gating of the porin channels is strongly dependent on the composition of the lipid matrix.The cell envelope of Gram-negative bacteria consists of the cytoplasmic membrane, the peptidoglycan layer, and an additional barrier, the outer membrane (OM), 1 (1) which is strictly asymmetric with respect to its lipid composition. Whereas the inner membrane (IM) is composed on both sides of phospholipids, the OM consists of a phospholipid inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The LPS consists of an oligo-or polysaccharide portion covalently linked to a lipid component termed lipid A, which anchors the molecule in the membrane (2). In wild-type strains, the polysaccharide portion consists of an O-specific chain and the core oligosaccharide. Rough mutant strains do not express the O-specific chain, but retain core oligosaccharides of varying length. The LPS of various rough mutants are characterized by chemotypes in a sequence of decreasing length of the core sugar as Ra (complete core), Rb, Rc, Rd, and Re. Deep-rough LPS (Re LPS) represents the minimal structure of LPS consisting of only lipid A and two 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) monosaccharides (3) (Fig. 1).The OM protects the cell from harmful agents like antibiotics and toxins and against changes in osmotic pressure. Transmembrane proteins, the porins, allowing the uptake and disposal of small hydrophilic compounds such as nutrients and waste products (4), are assembled in the OM. In Escherichia coli OmpF and OmpC represent the general diffusion pores. The phosphoporin PhoE is synthesized when cells are grown under phosphate limitation (5). PhoE has a molecular weight of M r 36,822 and an exclusion size of M r ϳ600 and is weakly anion selective (6). The crystal structure of PhoE has been solved (7). The channel-formin...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Pore Formation and Function of Phosphoporin PhoE of Escherichia coli Are Determined by the Core Sugar Moiety of Lipopolysaccharide

Hagge

Cock

Gutsmann

et al. 2002

Journal of Biological Chemistry

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“…They further showed that LPS of deep rough mutant strains of E. coli is much less efficient than wild-type LPS, indicating that the core region of LPS plays an important role in the formation of folded PhoE monomers. In a subsequent study with a range of different types of lipopolysaccharides, de Cock et al [58] demonstrated that the negative charges in the inner core region of LPS contribute to high folding efficiencies of PhoE into LPS/Triton-X-100 mixtures. LPS with a lipid A part with more flexible hydrophobic chains is more efficient in inducing the formation of folded monomers than LPS with more rigid chains.…”

Section: Role Of Lipopolysaccharidementioning

confidence: 99%

Membrane protein folding on the example of outer membrane protein A of Escherichia coli

Kleinschmidt¹

2003

Cellular and Molecular Life Sciences (CMLS)

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Abstract. The biophysical principles and mechanisms by which membrane proteins insert and fold into a biomembrane have mostly been studied with bacteriorhodopsin and outer membrane protein A (OmpA). This review describes the assembly process of the monomeric outer membrane proteins of Gram-negative bacteria, for which OmpA has served as an example. OmpA is a two-domain outer membrane protein composed of a 171-residue eight-stranded b-barrel transmembrane domain and a 154-residue periplasmic domain. OmpA is translocated in an unstructured form across the cytoplasmic membrane into the periplasm. In the periplasm, unfolded OmpA is kept in solution in complex with the molecular chaperone Skp. After binding of periplasmic lipopolysaccharide, OmpA insertion and folding occur spontaneously upon interaction of the complex with the phospholipid bilayer. Insertion and folding of the b-barrel transmembrane domain into the lipid bilayer are highly synchronized, i.e. the formation of large amounts of b-sheet secondary structure and b-barrel tertiary structure take place in parallel with the same rate constants, while OmpA inserts into the hydrophobic core of the membrane. In vitro, OmpA can successfully fold into a range of model membranes of very different phospholipid compositions, i.e. into bilayers of lipids of different headgroup structures and hydrophobic chain lengths. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers. Their formation was monitored by time-resolved distance determinations by fluorescence quenching, and they were structurally distinguished by the relative positions of the five tryptophan residues of OmpA in projection to the membrane normal. Recent studies indicate a chaperone-assisted, highly synchronized mechanism of secondary and tertiary structure formation upon membrane insertion of b-barrel membrane proteins such as OmpA that involves at least three structurally distinct folding intermediates.

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“…Modifications in LPS structure have been associated with changes in assembly and composition of OM proteins (Ames et al, 1974;Ried et al, 1990;de Cock et al, 1999), in A. brasilense Sp7 also (Lerner et al, 2009). Therefore, we examined whether the mutant strains differed in their OM protein profiles relative to the wild-type.…”

Section: Cp and Om Protein Analysismentioning

confidence: 99%

The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis

et al. 2009

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The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis Azospirillum brasilense is a plant root-colonizing bacterium that exerts beneficial effects on the growth of many agricultural crops. Extracellular polysaccharides of the bacterium play an important role in its interactions with plant roots. The pRhico plasmid of A. brasilense Sp7, also named p90, carries several genes involved in synthesis and export of cell surface polysaccharides. We generated two Sp7 mutants impaired in two pRhico-located genes, noeJ and noeL, encoding mannose-6-phosphate isomerase and GDP-mannose 4,6-dehydratase, respectively. Our results demonstrate that in A. brasilense Sp7, noeJ and noeL are involved in lipopolysaccharide and exopolysaccharide synthesis. noeJ and noeL mutant strains were significantly altered in their outer membrane and cytoplasmic/periplasmic protein profiles relative to the wild-type strain. Moreover, both noeJ and noeL mutations significantly affected the bacterial responses to several stresses and antimicrobial compounds. Disruption of noeL, but not noeJ, affected the ability of the A. brasilense Sp7 to form biofilms. The pleiotropic alterations observed in the mutants could be due, at least partially, to their altered lipopolysaccharides and exopolysaccharides relative to the wild-type.

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Non-lamellar Structure and Negative Charges of Lipopolysaccharides Required for Efficient Folding of Outer Membrane Protein PhoE of Escherichia coli

Cited by 79 publications

References 55 publications

Pore Formation and Function of Phosphoporin PhoE of Escherichia coli Are Determined by the Core Sugar Moiety of Lipopolysaccharide

Pore Formation and Function of Phosphoporin PhoE of Escherichia coli Are Determined by the Core Sugar Moiety of Lipopolysaccharide

Membrane protein folding on the example of outer membrane protein A of Escherichia coli

The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis

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