Isolation of the outer membrane of Branhamella catarrhalis

The emergence of Branhamella catarrhalis as an important human pathogen has stimulated interest in investigations of the outer membrane (OM) of the bacterium. In this study, the OM of B. catarrhalis was isolated and partially characterized. Radiolabelled cells were lysed and fractionated by isopycnic centrifugation in a continuous sucrose gradient. Five fractions were identified. Fraction A consisted of OM fragments of varying density. Fractions B and C were OM of a discrete density containing some cytoplasmic membrane. Fraction D was cytoplasmic membrane and Fraction E contained smaller less dense fragments of cytoplasmic membrane. The protein composition of the Branhamella OM is typical for that of Gram-negative bacteria in that approximately 10 to 20 proteins were present with six to eight of these proteins predominating. Having isolated and partially characterized the OM by sucrose density centrifugation, five simpler techniques for isolating OM were employed and the preparations compared to OM isolated on the gradient. Techniques that are based on differential detergent solubility of OM and cytoplasmic membrane were ineffective in isolating OM of B. catarrhalis. By contrast, techniques that involved collection of OM vesicles were successful in isolating OM of B. catarrhalis. Collection of vesicles from broth culture supernatants and EDTA-heat-induced vesicles were identified as convenient and reliable methods for isolating OM. Isolating and partially characterizing the OM of B. catarrhalis represents an initial step in a systematic study of outer membrane antigens of the bacterium.