Figure 2

Introduction of a clyA ⁺ locus in UPEC strains. (A) Schematic representation of the clyA loci in E. coli K-12 and in derivatives of UPEC strain 536. In strain JON53 the clyA locus was restored in UPEC isolate 536 as described in Materials and Methods and a kanamycin resistance gene was inserted downstream of clyA. The strain COE2 is a derivative of JON53 with a chimeric clyA::luxAB operon included adjacent to the restored clyA ⁺ locus. (B) Hemolysis activity tests with E. coli K-12 and UPEC derivatives on blood plates under Ca²⁺ depleted conditions due to addition of Na-oxalate (final concentration 2mM). Strains: 536 (UPEC, ΔclyA), JON53 (UPEC, clyA ⁺), J96 (UPEC, ΔclyA), JON47 (UPEC, clyA ⁺) MC1061 (K-12; clyA ⁺), MG1655 ( K-12; clyA ⁺), MWK7 (K-12; ΔclyA). Tests were performed in absence (i) and in presence (ii) of Mitomycin C applied onto the center of horizontally streaked rows of the E. coli strains. The images show the center part of the blood agar plates. (C) Detection of ClyA proteins by Western immunoblotting using a polyclonal ClyA antiserum. The immunoreactive bands corresponding to ClyA are indicated with an arrow. Whole cell lysates (lanes 15) and periplasmic fractions (lanes 610; prepared using osmolysis as described in Materials and Methods) were obtained from samples collected at OD₆₀₀=2.0. Lanes 1 & 6: UPEC strain 536. Lanes 2 & 7: strain JON53. Lanes 3 & 8: strain COE2. Lanes 4 & 9: strain MG1655. Lanes 5 & 10: strain MG1655 hns.