We have isolated outer and inner membranes of Serpulina hyodysenteriae by using discontinuous sucrose density gradients. The outer and inner membrane fractions contained less than 1 and 2%, respectively, of the total NADH oxidase activity (soluble marker) in the cell lysate. Various membrane markers including lipooligosaccharide (LOS), the 16-kDa outer membrane lipoprotein (SmpA), and the C subunit of the F 1 F 0 ATPase indicated that the lowest-density membrane fraction contained outer membranes while the high-density membrane fraction contained inner membranes and that both are essentially free of contamination by the periplasmic flagella, a major contaminant of membranes isolated by other techniques. The outer membrane fractions ( ؍ 1.10 g/cm 3 ) contained 0. Swine dysentery, caused by Serpulina (formerly Treponema) hyodysenteriae (9, 10), is a highly infectious, acute to chronic disease that can result in a debilitating mucohemorrhagic diarrhea and death. The first stage of the disease is spirochetal colonization and proliferation in the enteric epithelium, resulting in a significant decrease in the absorption of nutrients, ions, and water (12). However, the spirochetes do not invade beyond the lamina propria, and putative virulence factors, such as hemolysins and lipooligosaccharide (LOS), probably cause the cellular damage (16,(21)(22)(23)(24)35). Because a close association of the bacteria with the intestinal lining is critical in the development of disease, surface components of the spirochetes must play a pivotal role in the process.Previously, different groups have attempted to isolate outer membranes (OMs) from S. hyodysenteriae with less than favorable results. This was mainly due to the contamination of OM fractions by periplasmic flagella. For example, Joens et al. (11) found seven major proteins, ranging from 42 to 32 kDa in size, in OMs isolated from S. hyodysenteriae B204 by a Sarkosyl extraction technique. However, six of the seven bands appeared to be derived from the periplasmic flagella. Similarly, Chatfield et al. (5) and Wannemuehler et al. (39) found that the majority of the putative OM proteins isolated with detergent ranged in size from 45 to 29 kDa and strongly resembled the protein profile observed for the flagellar proteins. These data suggested that membranes isolated by these techniques were heavily contaminated by flagella. Therefore, a more effective technique is needed to better define the OM of S. hyodysenteriae.We have developed a membrane separation technique that permits the isolation of S. hyodysenteriae OMs and inner membranes (IMs) with minimal contamination by flagella. Analysis of the OMs indicated that they are quite interesting. First, the OM of S. hyodysenteriae has a lower density than the IM, which is very unusual for bacteria. In addition, the OM contains significant levels of cholesterol. Previously, Mycoplasma species (6), Borrelia hermsii (15), and S. hyodysenteriae (30, 33) had been described as having sterols in their membranes; however, the distributions ...
