Expression of outer membrane protein e of <i>escherichia coli</i> K12 by phosphate limitation

Overbeeke, Nico; Lugtenberg, Ben

doi:10.1016/0014-5793(80)80186-4

Cited by 119 publications

(58 citation statements)

References 30 publications

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“…PhoE was induced only in phosphate-limited minimal medium cultures and appeared as a band migrating just above OmpF (Fig. 3 A) as previously reported [21]. The antiserum directed against OmpF also recognizes PhoE, the two porins sharing 70% homology [22,231.…”

Section: Preparation Of Antiserasupporting

confidence: 83%

Production and purification of human growth-hormone-releasing factor from continuous cultures of recombinant-plasmid-containing Escherichia coli

Pagès¹,

Belaı̈ch²,

Anba³

et al. 1987

Eur J Biochem

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A recombinant gene comprising phoS (the gene for the phosphate‐binding protein PhoS) fused to a synthetic gene for a modified human growth‐hormone‐releasing factor (mhGRF) has been constructed. This gene was highly expressed in cells growing under conditions of phosphate starvation. Various conditions of continuous culture, varying in phosphate concentrations and dilution rates, have been tested to optimize the expression of the hybrid gene product (PhoS‐mhGRF). Conditions were obtained such that a large amount of the hybrid protein was no longer exported as a result of saturation of export sites, which also induce the inhibition of processing of pre‐PhoE and pre‐OmpA. The pre‐PhoS‐mhGRF, accumulated in the cell, was recovered mainly in the particulate fraction after cell fractionation. This protein was purified. Besides the methionine residues located within the signal sequence, the only other one is located in the fusion joint of the hybrid protein. Thus cyanogen bromide treatment allowed the isolation of pure mhGRF. The yield obtained is about of 1 mg/1 culture.

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Section: Preparation Of Antiserasupporting

confidence: 83%

Production and purification of human growth-hormone-releasing factor from continuous cultures of recombinant-plasmid-containing Escherichia coli

Pagès¹,

Belaı̈ch²,

Anba³

et al. 1987

Eur J Biochem

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“…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).…”

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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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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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“…A new major outer membrane protein called Ic [30], e [31] or E [21] can be present in pseudo revertants of mutants lacking the ompF and ompC proteins [21,26,30,31]. We have recently observed that this protein is induced in wildtype E. coli K12 cells by phosphate limitation [17] and is coregulated with alkaline phosphatase [32]. Also protein e is peptidoglycan-associated and has porin properties [31].…”

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confidence: 99%

Antigenic Relationships between Pore Proteins of Escherichia coli K12

Overbeeke

Scharrenburg

Lugtenberg

1980

European Journal of Biochemistry

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Antisera were raised against the purified Esckerichia coli K 12 outer membrane proteins ompA-, ompC-and ompF proteins and protein e. Several immunological methods were used to investigate the specificity of the antisera and the immunological relationship between the major outer membrane proteins. Although the antisera had been raised against highly purified proteins, several of them contained activity against lipopolysaccharide and lipoprotein due to minor impurities in the immunogens. The three general porins ompF protein, ompC protein and protein e were shown to be cross-reactive. Anti-(ompA protein) serum only reacted with the homologous protein. None of these antisera reacted with the phage lambda receptor protein or with protein 111. Pore protein preparations isolated from SaImonella typhimurium, Klebsiella aerogenes, Enterobacter cloaceae and Proteus mirabilis were found to be structurally related to the E. coli K12 porins as they reacted with the antisera raised against E. coli K12 porins.The outer membrane of Enterobacteriaceae contains phospholipids, which are mainly or exclusively present in the inner monolayer [1,2], lipopolysaccharide, which is exclusively located in the outer monolayer [3] and proteins [4-71, many of which transverse the lipid bilayer [8]. The most abundant protein in the outer membrane of Escherichia coli is the lipoprotein (7 x lo5 copies per cell) [9, lo]. Wildtype cells of E. coli K12 contain several other major proteins. For the proteins designated as b, c and d in our laboratory [5], other nomenclatures were also used [4,7,11], but it is now agreed that the proteins will be named according to the designation of their structural genes [12], which are ompF, ompC, and ompA respectively. Although these proteins are often called 'major outer membrane proteins', this term can be misleading, as the relative amounts of these proteins are dependent on the growth conditions [ 13 -181.The ompA protein has a function in the F-pilusmediated conjugation [19,20] and might play a role in the uptake of colicin L [21] and ferrichrome iron [22]. The ompF and ompC proteins form tight complexes with the peptidoglycan layer, which resists incubation in 2 % sodium dodecyl sulphate at 60°C [15,23,24]. These proteins form aqueous pores through which small molecules up to a molecular weight of about 700 can pass the outer membrane [25-291. can be present in pseudo revertants of mutants lacking the ompF and ompC proteins [21,26,30,31]. We have recently observed that this protein is induced in wildtype E. coli K12 cells by phosphate limitation [17] and is coregulated with alkaline phosphatase [32]. Also protein e is peptidoglycan-associated and has porin properties [31]. The ompF and ompC proteins and protein e are very similar with respect to their total amino acid composition [31,33,34] and their amino termini [30], but considerable differences were found in the fragment patterns obtained by incubation of the purified proteins with cyanogen bromide and proteolytic enzymes [31,33,34]. Protein I11 usuall...

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Expression of outer membrane protein e of escherichia coli K12 by phosphate limitation

Cited by 119 publications

References 30 publications

Production and purification of human growth-hormone-releasing factor from continuous cultures of recombinant-plasmid-containing Escherichia coli

Production and purification of human growth-hormone-releasing factor from continuous cultures of recombinant-plasmid-containing Escherichia coli

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

Antigenic Relationships between Pore Proteins of Escherichia coli K12

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