Fig. 5.

Intracellular phtA mutants fail to differentiate. (A) WT, phtA mutants, and phtA mutants supplemented with 3 mM threonine obtained from macrophages after 1 h (black bars) are sensitive to 100 mM NaCl. By 2 h (white bars) or 10 h (gray bars) after infection, WT and threonine-supplemented phtA mutants are NaCl-resistant, but without supplementation, phtA mutants remain sodium-sensitive. Experiments were performed in triplicate, and means and standard deviations are presented. (B) WT, phtA mutants, and phtA mutants supplemented with 3 mM threonine were lysed from host cells 1 h or 10 h postinfection; then their expression of the replicative phase rpoB gene and the transmissive phase flaA gene was analyzed by RT-PCR. As a reference for the replicative (WT EE) and transmissive (WT PE) differentiation state, RNA purified from broth cultures was analyzed; to control for genomic DNA contamination, non-reverse-transcribed RNA samples also were analyzed (no RT). 16s rRNA was analyzed as a loading control.