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 IS50L::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 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 carboxy-terminal end were partially hemolytic.
Hemolysis was progressively inhibited by saccharides with increasing molecular weights from
maltotriose (Mr 504) to
maltoheptaose (Mr 1,152) and was totally abolished by
dextran 4 (Mr 4,000). This result and the observed influx of [14C]
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.