ISOLATION AND CHARACTERIZATION OF THE OUTER-MEMBRANE PROTEINS OF BURKHOLDERIA (PSEUDOMONAS) PSEUDOMALLEI

Membranes obtained from whole-cell lysates of Burkholderia (Pseudomona s) pseudomallei (strain 319a) were separated into four fractions by su crose density gradient centrifugation. Membranes were characterized by enzymic and chemical analyses, and by SDS-PAGE. Cytoplasmic membranes and two forms of outer membranes (OM-1, OM-2) were detected. The majo r outer-membrane proteins had M(r) values of 70000, 38000, 31000, 2400 0 and 17000. To determine which outer-membrane proteins were common to B. pseudomallei strains, OM-1 fractions from 12 different strains wer e prepared. SDS-PAGE analysis of these fractions demonstrated that the five major outer-membrane proteins were common to the strains tested. Further studies have shown that an M(r) 110000 protein, which is olig omeric in that it migrates as an M(r) 38000 protein upon heating at 95 degrees C and which is peptidoglycan-associated, serves as a porin in B. pseudomallei. Using proteoliposomes reconstituted from this protei n and phospholipid, it was demonstrated by the liposome-swelling assay that this protein acts as a porin through which small saccharides may diffuse. Further characterization of this M(r) 38000 protein will be important in delineating the role of this molecule in the permeability of the B, pseudomallei outer membrane.