Extracellular secretion and hemolytic activity of <i>E. coli</i> HlyA and of HlyA mutants.

Abstract

<p>(A) SDS-PAGE of extracellular proteins from <i>E. coli</i> 5K containing different plasmids. Lane 1, molecular mass markers given in kDa; lane 2, <i>E. coli</i> 5K/pACYC184 (vector control); lane 3, <i>E. coli</i> 5K/pANN202–312* overproducing HlyA; lane 4, isogenic strain overproducing HlyA_Δ71–110; lane 5, isogenic strain overproducing HlyA_Δ158–167; lane 6, isogenic strain overproducing HlyA_Δ180–203; lane 7, isogenic strain overproducing HlyA_Δ264–286. The proteins in cell-free culture supernatants (harvested in the late log phase) were precipitated by addition of ice-cold trichloroacetic acid (final concentration, 10%), pelleted by centrifugation at 12,000×g, washed with acetone, dried under vacuum, and dissolved in sample buffer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112248#pone.0112248-Ludwig2" target="_blank">[11]</a>. Proteins from 100 µl culture supernatant were separated on the gel and visualized by silver staining. (B) Hemolytic phenotype of <i>E. coli</i> 5K/pANN202–312* overproducing HlyA and of isogenic strains overproducing the HlyA mutants with the indicated deletions. Bacteria from individual colonies were picked onto a sheep blood/Cm agar plate that was subsequently incubated for 24 hours at 37°C.</p