Identification of the Serratia marcescens hemolysin determinant by cloning into Escherichia coli

Abstract

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 (18; W. Konig, Y. Faltin, J. Scheffer, B. Konig, H. Schoffler, and V. Braun, unpublished data).Hemolysis required actively metabolizing cells and could be inhibited by various energy poisons (4).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...

Characterization of a Novel Two-Partner Secretion System in Escherichia coli O157:H7

“…OtpA does not display adhesive or hemolytic activity. A variety of TPS exoproteins have been shown to lyse red blood cells when they are secreted either from their native hosts or from E. coli K-12 strains (3,4,16,28,40). In each case the synthesis of the TPS exoprotein and its cognate transporter is sufficient to produce hemolytic activity in E. coli K-12; no other factors from the native organism are required.…”

“…One group of TPS exoproteins, which includes Serratia marcescens ShlA, Proteus mirabilis HpmA, Photorhabdus luminescens PhlA, Haemophilus ducreyi HhdA, and Edwardsiella tarda EthA, have hemolytic/cytolytic activities and share extensive sequence homology (3,4,16,28,40,45). A hemolysin-like protein from Pseudomonas putida (HlpA) has recently been shown to affect root colonization and iron uptake and may also be a member of this family (25).…”

The cell-bound hemolysin of Serratia marcescens contributes to uropathogenicity

“…8 The transformant showed a narrow zone of faint hemolysis on sheep blood agar similar to that of Seratia marcescens. With respect to other possible virulence factors such as serum resistance, fimbriae, cell wall antigens and growth rates strain 536/21 and transformant 536/21 pES2 were identical.…”

Cloning and expression in Escherichia coli of the Serratia marcescens metalloprotease gene: secretion of the protease from E. coli in the presence of the Erwinia chrysanthemi protease secretion functions

“…The S. marcescens 365 chromosomal DNA library used in this work has been described elsewhere (3). The recombinant cosmids were present in E. coli K5 and were plated either directly on skimmed milk agar, with ampicillin resistance selection, or after transformation with pRUW4, with ampicillin and chloramphenicol resistance selection.…”