Downloaded from www.microbiologyresearch.org by IP: 54.162.190.106 On: Wed, 03 Feb 2016 11:31:37 Microbiohgy (1994), 140,797-805 Printed in Great Britain Isolation and characterization of the outer- membrane proteins of Burkholderia (Pseudomonas) pseudomallei Naomasa Gotoh,' Nicholas J. White,2 Wipada Chaowagr~l~ and Donald E. Woods' Author for correspondence: Donald E. Woods. Tel: + 1 403 220 6885. Fax: + 1 403 270 2772. Department of Microbiology and Infectious Diseases, The University of Calgary Health Sciences Centre, Calgary, Alberta, Canada T2N 4N1 Wellcome-Ma hidol-Oxford Tropical Medicine Research Programme, Hospital for Tropical Diseases, Bangkok, Thailand Department of Medicine, Sappasitprasong Hospital, Ubon Ratchatani, Thailand Membranes obtained from whole-cell lysates of Burkholderia (Pseudomonas) pseudomallei (strain 319a) were separated into four fractions by sucrose density gradient centrifugation. Membranes were characterized by enzymic and chemical analyses, and by SDS-PAGE. Cytoplasmic membranes and two forms of outer membranes (OM-l,OM-2) were detected. The major outer- membrane proteins had M, values of 70000,38000,31000,24000 and 17000. To determine which outer-membrane proteins were common to B. pseudomallei strains, OM-I fractions from 12 different strains were 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, 110000 protein, which is oligomeric in that it migrates as an M, 38000 protein upon heating at 95 "C and which is peptidoglycan-associated, serves as a porin in B. pseudomallei. Using proteoliposomes reconstituted from this protein 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, 38000 protein will be important in delineating the role of this molecule in the permeability of the B. pseudomallei outer membrane. Keywords : Burkholderia (Pseudomonas) pseudomallei, outer-membrane proteins, porin INTRODUCTION Btlrkholderia psetldomallei (formerly Psetldomonas psetldo- mallei: see Yabuuchi et al., 1992) is the causative agent of melioidosis, cases of which have been documented in Korea, the Philippines, Central and Southern America, the West Indies, Turkey, Malaysia, Madagascar, Guam and Australia (Beamer et al., 1954; Dance, 1991). Most cases occur in the 20" N to 20" S latitudes. The disease is rare in the Western Hemisphere, but has been identified in Panama, Ecuador, Haiti, Brazil, Peru and Guyana. Some investigators feel that the organism will cause disease only in endemic areas ; whereas others (Bremmelgaard, 1975 ; Thomas et al., 1979), feel that because of its nutritional versatility, the organism and the disease due to it should be found in many other areas of the world. Melioidosis can be manifested in several ways. It can be Abbreviations: DOC, sodium deoxycholate; KDO, 2-keto-3-deoxyoctonic acid (3-deoxy-D-mano-2-octulosonic acid); 2-ME, 2-mercaptoethanol; p- OG, n-octyl p-D-glucopyranoside; SDH, succinate dehydrogenase. seen as an inapparent infection, asymptomatic pulmonary infiltration, acute localized suppurative infection, acute pulmonary infection (most common), acute septicaemic infection or chronic suppurative infection (Sanford, 1985, 1987). With better diagnosis and more prolonged appro- priate therapy, mortality in forms except the septicaemic should be low (Chaowagul et al., 1989; Eickhoff et al., 1970). B. pseudomallei is a motile Gram-negative rod and grows on most standard media (Wetmore & Gochenour, 1956). It can grow at temperatures of 18-42 "C. Routine laboratory identification of the organism is difficult due principally to problems in differentiating this organism from Burkholderia (Psetldomonas) cepacia (Howe et al., 1971). A low but significant level of DNA homology between B. cepacia and B. pseudomallei has been reported (Ballard et al., 1970 ; Rogul et al., 'I 970). Various biochemical testing procedures have been employed for identification of B. psetldomallei; however, these have not proved to be totally satisfactory (Ashdown, 1979 ; Thomas, 1983). The pathogenic determinants of B. psetldomallei have not 0001-8621 0 1994SGM 797