Peptidoglycan-associated outer membrane proteins in gramnegative bacteria (original) (raw)
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Soluble nascent peptidoglycan in growing Escherichia coli cells
Journal of Biological Chemistry
Two homologous strains of Escherichia coli, one of which completely lacked the cell envelope Braun's lipoprotein, were compared with respect to their peptidoglycan synthesis and assembly. Both strains were auxotrophic for diaminopimelic acid and their uptake of radiolabeled diaminopimelic acid was comparable. Analysis of subcellular fractions obhined after mechanical disruption of the cells in a French pressure cell and sedimentation of the cell envelopes showed the existence of a soluble, chromatographically immobile macromolecular peptidoglycan. This labeled peptidoglycan contained a reduced degree of peptide side chain cross-linkages (19 mol % of labeled residues as compared to that present in the insoluble cell sacculus, 27 mol 8). In addition, approximately 20% of its peptide side chains terminated in pentapeptide structures uersus 1 to 4% in the sacculus. Furthermore, the soluble peptidoglycan of the parent strain also contained covalently bound lipoprotein (4.6%). Extraction of the cell envelope fraction with detergents afforded an additional amount of soluble peptidoglycan. This material was quite similar, in its degree of cross-linkage and amount of covalently bound lipoprotein, to the peptidoglycan present in the detergent-insoluble sacculus. These results indicate that peptidoglycan strands which are, in part, covalently linked to lipoprotein are late stage synthesis intermediates which subsequently become covalently attached to the preexisting sacculus.
Variability of peptidoglycan structural parameters in Gram-negative bacteria
FEMS Microbiology Letters, 1995
Mnropeptide composition of peptidoglycan from the Gram-negative bacteria Aeromonas sp., Acinetobacter acetouceticus, Agrobacterium tumejaciens, Enterobacter cloacae, Proteus morganii, Pseudomonas aeruginosa, Pseudomonas putida, Vibrio parahaemolyticus Yersinia enterocolitica and Escherichia coli, was analyzed by HPLC In all instances peptidoglycan was built up from the same subunits. A wide disparity in the relative abundance of muropeptides and all structural parameters was observed. The contribution of LD+Upm-A2pm cross-linked muropeptides was extremely variable; from 1 to 45% of cross-linked muropeptides. Muropeptides with the dipeptides Lys-Lys or Arg-Lys, indicative of murein-bound (lipojproteins, were detected in all instances although abundance was very variable.
Amount of peptidoglycan in cell walls of gram-negative bacteria
Journal of bacteriology, 1991
The amount of diaminopimelic acid (Dap) in the cell wall of Escherichia coli was measured in two ways. A radiochemical method first described by us in 1985 (F. B. Wientjes, E. Pas, P. E. M. Taschner, and C. L. Woldringh, J. Bacteriol. 164:331-337, 1985) is based on the steady-state incorporation of [3H]Dap during several generations. Knowing the cell concentration and the specific activity of the [3H]Dap, one can calculate the number of Dap molecules per sacculus. The second method measures the Dap content chemically in sacculi isolated from a known number of cells. With both methods, a value of 3.5 x 10(6) Dap molecules per sacculus was obtained. Combined with electron microscopic measurements of the surface area of the cells, the data indicate an average surface area per disaccharide unit of ca. 2.5 nm2. This finding suggests that the peptidoglycan is basically a monolayered structure.
Genetics and biochemistry of the peptidoglycan-associated proteins b and c of Escherichia coli K12
MGG Molecular & General Genetics, 1979
To collect information on synthesis and regulation of the peptidoglycan-associated pore-forming outer membrane proteins b and c, mutants resistant to phages Mel and TuIa were analyzed. Genetic analysis showed three linkage groups, corresponding with the genes tolF (phenotype b-c+), meoA (phenotype b+c -) and ompB (phenotypes b c , b-c +, b++c -and b++c~). It has recently been described that also a b+c -phenotype can occur in the latter linkage group [Chai, T., Foulds, J., J. Bacteriol. 130, 781 786 (1977)]. Among ompB (b-c+)/meoA (b+c -) double mutants strains were found with the b+c phenotype, showing that ompB is not the structural gene for protein b. Studies on purified proteins b and c showed profound differences between the two proteins with respect to the electrophoretic mobility of fragments obtained by treatment with cyanogen bromide, trypsin and chymotrypsin. The amino acid in position three of the amino-termini of proteins b and c, isolated from isogenic strains, were identified as isoleucine and valine respectively. Both the genetic and biochemical results are consistent with a model recently published [Ichihara, S., Mizushima, S., J. Biochem. (Japan) 83, 1095-1100 (1978)] which predicts that tolF and meoA are the structural genes for the proteins b and c respectively and that ompB is a regulatory gene whose product regulates the levels of both proteins.
Genetics and biochemistry of the peptidoglycan-associated proteins b and c of Escherichia coli</i
Mol Gen Genet, 1979
To collect information on synthesis and regulation of the peptidoglycan-associated pore-forming outer membrane proteins b and c, mutants resistant to phages Mel and TuIa were analyzed. Genetic analysis showed three linkage groups, corresponding with the genes tolF (phenotype b-c+), meoA (phenotype b+c-) and ompB (phenotypes b c , b-c +, b++c-and b++c~). It has recently been described that also a b+c-phenotype can occur in the latter linkage group [Chai, T., Foulds, J., J. Bacteriol. 130, 781 786 (1977)]. Among ompB (b-c+)/meoA (b+c-) double mutants strains were found with the b+c phenotype, showing that ompB is not the structural gene for protein b. Studies on purified proteins b and c showed profound differences between the two proteins with respect to the electrophoretic mobility of fragments obtained by treatment with cyanogen bromide, trypsin and chymotrypsin. The amino acid in position three of the amino-termini of proteins b and c, isolated from isogenic strains, were identified as isoleucine and valine respectively. Both the genetic and biochemical results are consistent with a model recently published [Ichihara, S., Mizushima, S., J. Biochem. (Japan) 83, 1095-1100 (1978)] which predicts that tolF and meoA are the structural genes for the proteins b and c respectively and that ompB is a regulatory gene whose product regulates the levels of both proteins.
Peptidoglycan (PG) is a trademark of bacteria and constitutes its most important part that shields it from every possible external environmental hazard. It is known to play pivotal roles in maintaining cell shape, the right surface to volume ratio, toxicity control, pathogenesis, motility, protein transport, osmotic lysis to name a few and is vital for its very survival and this is also why its chemical composition is evolutionarily conserved in all bacteria. It is worth thinking if this particular part is so important for the survival of the cell, why its regulation remains even as of today, a rather poorly understood mechanism and the reason why the only time this "bacterial life line" comes into the lime light is when we think of the beta-lactam antibiotics such as the revolutionary penicillin which acts on the bacterial PG and breaks it open like an overinflated balloon. But, this very PG that was degraded by Penicillin way back in 1928 is still very much an unconquered territory. The reason for this could be the lack of the much needed tools that are required to study this rather dynamic structure. Fortunately, this has become possible today with the advent of tools such as super resolution microscopy. Introduction:
Expression of membrane-associated proteins by strains of enteroaggregative Escherichia coli
FEMS Microbiology Letters, 1998
Certain strains of enteroaggregative Escherichia coli express an outer membrane-associated protein, involved with the adhesion of these bacteria to HEp-2 cells. Strains of enteroaggregative E. coli hybridising with DNA probes for aggregative adhesion, diffuse adhesion and aggregative adhesion fimbriae II expressed an outer membrane-associated protein of 18 kDa regulated by magnesium ions. Strains hybridising with the aggregative adhesion probe only expressed a 20-kDa outer membrane-associated protein regulated by calcium and magnesium. The present study describes two populations of enteroaggregative E. coli which appear to adhere to HEp-2 cells by expressing antigenically distinct, negatively charged membrane-associated proteins. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
Analytical Biochemistry, 2004
Currently, reversed-phase high-performance liquid chromatography (HPLC) is the method of choice for determining the types and amounts of muropeptide subunits comprising bacterial peptidoglycan. Although effective and sensitive, the technique does not lend itself to high throughput screening, and its complexity and equipment requirements may dissuade some investigators from pursuing certain types of cell wall experiments. Previously, we showed that muropeptides can be labeled with a fluorescent dye and separated by fluorophore-assisted carbohydrate electrophoresis (FACE), a simple and rapid gel procedure that might serve as a prelude to more intense analysis by HPLC. To validate the utility of FACE, we used both techniques to perform a side-by-side analysis of the peptidoglycan of eight mutants and their Escherichia coli parent strain. FACE and HPLC both detected the seven major muropeptides, which represent more than 95% of the total muropeptides present in this organism. In addition, FACE returned the same relative and quantitative results in 92% of 72 measurements, indicating that the procedure gives an accurate overview of peptidoglycan composition. The results also suggest a possible biochemical activity for the AmpC and AmpH proteins of E. coli, and the use of FACE as an in vitro enzyme assay detected possible substrate preferences for the endopeptidase penicillin binding protein 4.