Sherbet GV and Lakshmi MS. (1974). Characterisation of cell surfaces by isoelectric equilibrium analysis. In: Isoelectric focusing, eds. Arbuthnott JP and Bealy JA, Butterworth, London, pp.338-346 (original) (raw)
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Biochimica et Biophysica Acta
A characterisation of the lipopolysaccharide (outermost) layer of Escherichia coli cells has been made by isoelectric equilibrium analysis. Unmodified E. coli cells show a surface isoelectric point (pl) of 5.6. Cells treated with ethyleneimine in order to esterify the carboxy[ groups are isoelectric at pH 8.55. When amino groups are blocked the bacterial surface has a p[ of 3.85. An analysis of these results suggests that the ionisable groups occurring in the isoelectric zone i.e. the zone amenable to investigation by the isoelectric equilibrium method are: carboxyl groups and amino groups of polysaccharide and protein components. The carboxyl groups have a pK between 3.2 and 4.5 and the amino groups have a pK of 7.5. e-Amino groups, phenolic hydroxyl groups and guanidyl groups do not occur, and phosphate and amino groups of the phospholipid complex are not detected. The number of thiol groups in the isoelectric zone has been determined using 6,6'-dithiodinicotinic acid. The number of anionogenic and cationogenic groups has been determined. From the density of the negative charges on the surface it is estimated that the isoelectric zone might extend up to 60/~ below the cell surface. The data discussed in this paper relate to the outermost layer of the bacterial cell wall composed of lipopolysaccharidephospholipid-protein complex. Since reactive groups of the phospholipid component of the complex have not been detected in the isoelectric zone, it is suggested that the arrangement of lipopolysaccharide-phospholipid protein complex is such that the phospholipids are located at a depth of more than 60 ~ from the bacterial surface.
Characterisation of Escherichia coli cell surface by isoelectric equilibrium analysis
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1973
A characterisation of the lipopolysaccharide (outermost) layer of Escherichia coli cells has been made by isoelectric equilibrium analysis. Unmodified E. coli cells show a surface isoelectric point (pl) of 5.6. Cells treated with ethyleneimine in order to esterify the carboxy[ groups are isoelectric at pH 8.55. When amino groups are blocked the bacterial surface has a p[ of 3.85. An analysis of these results suggests that the ionisable groups occurring in the isoelectric zone i.e. the zone amenable to investigation by the isoelectric equilibrium method are: carboxyl groups and amino groups of polysaccharide and protein components. The carboxyl groups have a pK between 3.2 and 4.5 and the amino groups have a pK of 7.5. e-Amino groups, phenolic hydroxyl groups and guanidyl groups do not occur, and phosphate and amino groups of the phospholipid complex are not detected. The number of thiol groups in the isoelectric zone has been determined using 6,6'-dithiodinicotinic acid. The number of anionogenic and cationogenic groups has been determined. From the density of the negative charges on the surface it is estimated that the isoelectric zone might extend up to 60/~ below the cell surface. The data discussed in this paper relate to the outermost layer of the bacterial cell wall composed of lipopolysaccharidephospholipid-protein complex. Since reactive groups of the phospholipid component of the complex have not been detected in the isoelectric zone, it is suggested that the arrangement of lipopolysaccharide-phospholipid protein complex is such that the phospholipids are located at a depth of more than 60 ~ from the bacterial surface.
Langmuir : the ACS journal of surfaces and colloids, 2016
Understanding the electrostatic interactions between bacterial membranes and exogenous proteins is crucial to designing effective antimicrobial agents against Gram-negative bacteria. Here we study, using neutron reflecometry (NR) under multiple isotopic contrast conditions, the role of the uncharged sugar groups in the outer core region of lipopolysaccharide (LPS) in protecting the phosphate rich inner core region from electrostatic interactions with antimicrobial proteins. Models of the asymmetric Gram negative outer membrane on silicon were prepared with phopshatidylcholine (PC) in the inner leaflet (closest to the silicon), whereas rough LPS was used to form the outer leaflet (facing the bulk solution). We show how salt concentration can be used to reversibly alter the binding affinity of a protein antibiotic colicin N (ColN) to the anionic LPS confirming that the interaction is electrostatic in nature. By examining the interaction of ColN with two rough LPS types with different ...
Colloids and Surfaces B: Biointerfaces, 1995
The use of the isoelectric point (IEP) of a bacterium as a measure of the ability of bacterial surface polymers to inhibit adhesion was tested. This inhibition is attributed to repulsive steric interactions and not to electrostatic repulsion as accounted for by the DLVO theory of colloid stability. IEP values were compared with literature data on cell wall composition and with adhesion results, obtained at pH 7 and an ionic strength of 0.1 M. The literature data demonstrate that an IEP~<2.8 indicates the presence of significant amounts of cell surface polysaccharides containing negatively charged phosphate and/or carboxyl groups. The experimental results showed that these polymers inhibit adhesion onto both hydrophilic (glass) and hydrophobic (Teflon) surfaces. The coryneform Rhodococcus strain C125 with an |EP of 3.0 possesses amphiphilic cell surface components which inhibit adhesion onto glass and promote deposition onto Teflon. Bacteria with an IEP/>3.2 appear to be free from polymer coatings that inhibit adhesion. They adhere in large amounts onto Teflon and in slightly lower amounts onto glass. Our findings therefore indicate that the IEP is a suitable parameter complementary to hydrophobicity in predicting the affinity of bacterial surface polymers for substrata with different hydrophobicities.
Effect of external electric fields on membrane proteins: The bacteriorhodopsin
Bioelectrochemistry and Bioenergetics, 1984
One of the possible mechanisms of the effect of external electric fields on biological systems is through their membrane proteins. At present, contradicting reports exist about the effect of external electric fields on the behaviour of the bacteriorhodopsin, the only protein molecule in the purple membrane fragments. Complex electro-optic studies (electric light scattering; electric dichroism and electric birefringence) and detailed investigations of the electric field strength dependence of the electro-optic effects on different fractions of purple membranes provide no evidence of alterations in the behaviour of bacteriorhodopsin. The effects observed are explained well by the orientations of the whole purple membrane fragments. The faster process detected in the electric light scattering decay curve could be attributed to changes in the form and/or the volume of purple membrane aggregates. The importance of these studies in understanding the dependence of the membrane properties on the variation of the transmembrane potential, which are essential for membrane pumping and channel behaviour, is discussed. Further possibilities of the application of the methods described in the study of other membrane proteins and other membrane problems are also discussed.
Electrokinetic Potential of Bacterial Cells
Langmuir, 1997
Microelectrophoresis studies are of relevance in the characterization of the electrical double layer of bacterial cell surfaces. In order to interpret the electrophoretic mobility in terms of the-potential, the classical Helmholtz-Smoluchowski equation is regularly used. However, this equation has been derived under several more or less restrictive conditions, which are easily violated by complex colloidal systems, such as bacterial cell suspensions. In recent theories as derived by Dukhin, O'Brien, and Fixman, the effect of double layer polarization on the electrophoretic mobility of colloidal particles is accounted for. These theories predict that, at high surface charge densities, the electrophoretic mobility may be strongly retarded compared to the Helmholtz-Smoluchowski equation. In this paper the effect of the mobile charge in the bacterial wall on the electrophoretic mobility is considered. For this purpose a comprehensive equation for the electrophoretic mobility has been derived, which also includes surface conduction within the hydrodynamically stagnant layer. To that end, Fixman's theory, valid for large κa, has been modified. It is shown that cell wall conduction can have a considerable effect on the electrophoretic mobility of bacterial cells, especially at low salt concentrations. In 1 and 10 mM electrolyte solution, the classical Helmholtz-Smoluchowski equation underestimates the-potential by approximately a factor of 2 and 1.3, respectively. Obviously a full description of the composition of the electrical double layer of bacterial cell surfaces cannot be based on electrophoretic mobility measurements only but should be obtained from a combination of experimental techniques, including titration and conductivity measurements.
Pulsed electric field (PEF) treatments, a nonthermal process, have been reported to injure and inactivate bacteria in liquid foods. However, the effect of this treatment on bacterial cell surface charge and hydrophobicity has not been investigated. Apple juice (pH 3.8) purchased from a wholesale distributor was inoculated with cocktail of Escherichia coli O157:H7 at 7.4 log CFU/mL, processed with a PEF at a field strength of 18.4 kV/cm and 32.2 kV/ cm at 25°C, 35°C, and 45°C with a treatment time of 160 ls and a flow rate of 120 mL/min. Bacterial cell surface charge and hydrophobicity of untreated and PEF-treated E. coli O157:H7 were determined immediately and after storage at 5°C and 23°C using hydrophobic and electrostatic interaction chromatography. Similarly, the populations surviving the PEF treatments including injured cells were determined by plating 0.1 mL of the sample on sorbitol MacConkey agar and tryptic soy agar (TSA) plates. The surviving populations of E. coli cells after PEF treatment varied depending on field strength and treatment temperature used. Percent injury in the surviving populations was high immediately after PEF treatment and varied among treatment temperatures. Cell surface charge of E. coli bacteria before PEF treatment averaged 32.10 -8.12. PEF treatments at 25°C, 35°C, and 45°C reduced the above surface charge to 26.34 -1.24, 14.24 -3.30, and 6.72 -2.82, respectively. Similarly, the surface hydrophobicity of untreated E. coli cells at 0.194 -0.034 was increased to an average of 0.268 -0.022, 0.320 -0.124, and 0.586 -0.123 after PEF treatments at 25°C, 35°C, and 45°C, respectively. The results of this study indicate that PEF treatment affects the outer cell envelope of E. coli bacteria as evidenced by the changes in surface hydrophobicity and cell surface charge leading to injury and subsequent inactivation of the cells.