Disruption of Bordetella pertussis in the Ribi cell fractionator. 1. Effect of different pressures.

Abstract

Exposure of B. pertussis in pH 7.0 phosphate buffer to disruption in the Ribi Cell Fractionator (RCF) at a series of increasing pressures caused progressive lysis as judged by the decrease of optical density and by the release of protein and DNA into the 20 000 g supernatant. A plateau of maximum lysis was reached around 20 000 psi, although DNA continued to be released from the cell residues up to 30 000 psi. The 20 000 g residues (cell envelopes) from 30 000 psi lysates comprised, after pH 7.0 washing, about 30% of the dry weight of the original cells and contained about 30% of the original protein and 14–27% of the original DNA. They contained the greater part (80–90%) of the original lipopolysaccharide (LPS) and between one-half and two-thirds of the histamine-sensitizing factor(HSF)and protective antigen (PA). However, the 20 000 g supernatant fractions also contained HSF and PA. Thus although HSF and PA were not destroyed by RCF treatment, it was not possible to get efficient cell disruption in pH 7.0 phosphate buffer without causing release of one-third or more of the HSF and PA into the cytoplasmic fraction.