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

Journal Article · Fri May 01 04:00:00 EDT 1987 · J. Bacteriol.; (United States)

OSTI ID:6220832

Braun, V; Neuss, B; Ruan, Y; Schiebel, E; Schoeffler, H; Jander, G

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 Tn1000 and Tn5 IS50/sub L/::phoA (TnphoA), the coding region was assigned to a DNA fragment, designated hyl, 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 extracellular medium concomitant with hemolytic activity. The genes encoding the 61K and the 160K proteins were transcribed in the same direction. Mutants expressing a 160K protein truncated at the carboxyl-terminal end were partially hemolytic. Hemolysis was progressively inhibited by saccharides with increasing molecular weights from maltotriose (M/sub r/ 504) to maltoheptaose (M/sub r/ 1152) and as totally abolished by dextran 4 (M/sub r/ 4000). This result and the observed influx of (/sup 14/C)sucrose into erythrocytes in the presence of hemolytic E. coli transformants under osmotically protective conditions suggest the formation of defined transmembrane channels by the hemolysin.

• Research Organization: Universitaet Tuerbingen, West Germany
• OSTI ID: 6220832
• Journal Information: J. Bacteriol.; (United States), Journal Name: J. Bacteriol.; (United States) Vol. 169:5; ISSN JOBAA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANTIBODIES
BACTERIA
BIOLOGICAL MATERIALS
BIOSYNTHESIS
BLOOD
BLOOD CELLS
BODY FLUIDS
CARBOHYDRATES
CARBON 14 COMPOUNDS
CELL CONSTITUENTS
CLONING
DNA
DNA-CLONING
ENZYMES
ERYTHROCYTES
ESCHERICHIA COLI
GENE REPRESSORS
GENES
HEMOLYSINS
HEMOLYSIS
INHIBITION
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LYSIS
MATERIALS
MICROORGANISMS
MOLECULAR WEIGHT
NUCLEIC ACIDS
NUCLEOPROTEINS
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
PATHOLOGICAL CHANGES
PHOSPHORUS-GROUP TRANSFERASES
PLASMIDS
POLYMERASES
PROTEINS
RNA POLYMERASES
SACCHARIDES
SERRATIA
SYNTHESIS
TRACER TECHNIQUES
TRANSCRIPTION
TRANSFERASES