FIG. 5.

LcrG is located intracellularly. (A) Cell fractionation of yersiniae. Y. enterocolitica W22703 (wild type) was grown in the presence of 5 mM calcium (+Ca²⁺) or 5 mM EGTA (−Ca²⁺) for 3 h at 37°C. Bacteria were lysed in a French pressure cell. Unbroken cells were removed by low-speed centrifugation, and crude extracts were subjected to ultracentrifugation at 100,000 × g. The supernatant (S), containing soluble cytoplasmic contents, was separated from the insoluble (membrane) sediment (I). Samples were analyzed by SDS-PAGE and immunoblotting with antibodies raised against LcrG, LcrV, LcrH, YopE, and YopD. (B) Protease protection assay. Y. enterocolitica W22703 was grown in the presence or absence of calcium. Four 6-ml culture aliquots (10⁸ CFU/ml) were incubated at 37°C for 30 min with or without 20 μg of proteinase K per ml, 1% SDS, or 1 mM PMSF as indicated. Samples were precipitated with chloroform-methanol, dried, solubilized in sample buffer, and analyzed by SDS-PAGE and immunoblotting.