A cosmid bank of Serratia marcescens was established from which DNA fragments were cloned into the plasmid pBR322, which conferred the chromosomally encoded hemolytic activity to Escherichia coli K-12. By transposon mutagenesis with TnlO00 and Tn5 ISSOL::phoA (TnphoA), the coding region was assigned to a DNA fragment, designated hly, comprising approximately 7 kilobases. Two proteins with molecular weights of 61,000 (61K protein) and 160,000 (160K protein) were expressed by the pBR322 derivatives and by a plasmid which contained the hly genes under the control of a phage T7 promoter and the T7 RNA polymerase. When strongly overexpressed the 160K protein was released by E. coli cells into the extraceliular medium concomitant with hemolytic activity. The genes encoding the 61K and the 160K proteins were transcribed in the same direction. Mutants We characterized an exoprotease of Serratia marcescens and studied its secretion (24). The strains we used were all strongly hemolytic in a liquid assay but caused only very narrow lysis zones on blood agar. Hemolysis was unrelated to the formation of the exoprotease (4). The hemolytic activity resided in the membrane fraction (4). Hemolytic bacteria have been shown to induce the release of the leukotrienes LTC4 and LTB4 from polymorphnuclear leukocytes, the release of histamine from rat mast cells, and chemiluminescence of neutrophils (2, 3). It was concluded that via these inflammatory mediators hemolysin may increase vascular permeability, edema formation, and granulocyte accumulation and thus contribute to the pathogenicity of Serratia spp. (18; W. Konig, Y. Faltin, J. Scheffer, B. Konig, H. Schoffler, and V. Braun, unpublished data).Hemolysis required actively metabolizing cells and could be inhibited by various energy poisons (4). Hemolytic activity was not found in the culture supernatant of various Serratia strains grown under different conditions, nor could it be released from cells (4). Therefore, we used a genetic approach to characterize the hemolytic determinant of S. marcescens. MATERIALS AND METHODSBacterial strains and culture conditions. The strains used are listed in Table 1. The rough mutant SN8 was obtained as a partially bacitracin-resistant derivative by using a method developed by H. Rotering (personal communication, this institute). Bacitracin affects 0-antigen synthesis by inhibiting recycling of the 0-antigen polyisoprenoid carrier (25).Colonies of S. marcescens W1436 that grew on 1/8 TY agar plates near a filter paper disk which contained 10 mg of bacitracin were examined by gel electrophoresis to determine whether they expressed smooth or rough lipopolysac-* Corresponding author.charide (27). Of 20 colonies tested, 4 lacked the ladder of bands characteristic of 0-antigen heterogeneity.Cells were routinely grown in TY medium (0.8% tryptone [Difco Laboratories], 0.5% yeast extract, 0.5% sodium chloride, pH 7). The antibiotic ampicillin (25 or 50 jig/ml) or kanamycin (50 jig/ml) was added to maintain the plasmids.Cosmid cloning. Chromosomal DNA o...

A cell-bound hemolytic activity was found in several strains of Serratia marcescens. One Serratia cell per ten erythrocytes was sufficient to cause complete lysis of human erythrocytes within 2 h in the liquid assay. The hemolytic activity resided in the membrane fraction and could be inactivated by incubating cells with proteases. The hemolytic activity was greatly enhanced in actively metabolizing Serratia cells and was partially controlled by the iron supply. Hemolysis was accompanied by degradation of erythrocyte membrane proteins (band 3 and 6, glycophorin) and was independent of the blood group. The exoprotease secreted by S. marcescens in large amounts was not involved in hemolysis. Comparison with various hemolytic strains of Escherichia coli showed that hemolysis of erythrocytes was more pronounced with S. marcescens than with E. coli. In contrast to hemolysis by E. coli, lysis of erythrocytes by S. marcescens was not enhanced by Ca2+ ions.