Characterisation of Escherichia coli cell surface by isoelectric equilibrium analysis

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 (pI) of 5.6. Cells treated with ethyleneimine in order to esterify the carboxyl groups are isoelectric at pH 8.55. When amino groups are blocked the bacterial surface has a pI 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. ε-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 Å below the cell surface. The data discussed in this paper relate to the outermost layer of the bacterial cell wall composed of lipopolysaccharide-phospholipid-protein complex. Since reactive groups of the phosphilipid 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 Å from the bacterial surface.