Determination of the total charge in the cell walls of Gram-positive bacteria

The charge in the bacterial wall originates from the dissociation of acidic groups such as carboxyl, phosphate and amino groups. The degree of dissociation of these chargeable groups is a function of the pH and the activity of the surrounding electrolyte solution. In this study the cell wall charge density of Gram-positive bacterial strains, including four coryneforms and a Bacillus brevis, is assessed by proton titrations of whole bacterial cells and isolated cell walls at different electrolyte concentrations. At neutral pH rather high values, between 0.5 and 1.0 C m −2, for the cell wall surface charge density are found. The titration curves for the isolated cell walls are free of hysteresis allowing a rigorous (thermodynamic) analysis. For the coryneform bacteria these curves have a common intersection point between pH 3 and 4, which is identified as the point of zero charge. The carboxyl and phosphate groups are titrated in distinct pH regions, allowing accurate estimation of their numbers. These numbers compare very well with those based on a chemical analysis of the isolated cell walls. The uncertainty in the estimated number of amino groups is somewhat higher, because these groups are only partly titrated within the pH range accessible by proton titrations. At electrolyte concentrations below 0.01 M maximum expulsion of co-ions from the cell walls already occurs at relatively low charge densities. At these low electrolyte concentrations the compensating countercharge predominantly consists of counterions that penetrate into the porous cell wall matrix and to a much lower and constant extent by the exclusion of co-ions.