Influence of growth temperature and lipopolysaccharide on hemolytic activity of Serratia marcescens

Abstract

Log-phase cells of Serratia marcescens cultured at 30 degrees C were approximately 10-fold more hemolytic than those grown at 37 degrees C. By using a cloned gene fusion of the promoter-proximal part of the hemolysin gene (shlA) to the Escherichia coli alkaline phosphatase gene (phoA), hemolysin gene expression as a function of alkaline phosphatase activity was measured at 30 and 37 degrees C. No difference in alkaline phosphatase activity was observed as a function of growth temperature, although more hemolysin was detectable immunologically in whole-cell extracts of cells grown at 30 degrees C. The influence of temperature was, however, growth phase dependent, because the hemolytic activities of cells cultured to early log phase at 30 and 37 degrees C were comparable. Given the outer membrane location of the hemolysin, lipopolysaccharide (LPS) was examined as a candidate for mediating the temperature effect on hemolytic activity. Silver staining of LPS in polyacrylamide gels revealed a shift towards shorter O-antigen molecules at 37 degrees C relative to 30 degrees C. Moreover, there was less binding of O-antigen-specific bacteriophage to S. marcescens with increasing growth temperature, a finding consistent with temperature-mediated changes in LPS structure. Smooth strains of S. marcescens were 20- to 30-fold more hemolytic than rough derivatives, a result confirming that changes in LPS structure can influence hemolytic activity. The alkaline phosphatase activity of rough strains harboring the shlA-phoA fusion was threefold lower than that of smooth strains harboring the fusion plasmids, a result consistent with a decrease in hemolysin gene expression in rough strains. The absence of a similar effect of temperature on gene expression may be related to less-marked changes in LPS structure as a function of temperature compared with a smooth-to-rough mutational change.