The heat shock protein DnaK is essential for intramacrophagic replication of Brucella suis. The replacement of the stress-inducible, native dnaK promoter of B. suis by the promoter of the constitutively expressed bla gene resulted in temperature-independent synthesis of DnaK. In contrast to a dnaK null mutant, this strain grew at 37 degrees C, with a thermal cutoff at 39 degrees C. However, the constitutive dnaK mutant, which showed high sensitivity to H(2)O(2)-mediated stress, failed to multiply in murine macrophage-like cells and was rapidly eliminated in a mouse model of infection, adding strong arguments to our hypothesis that stress-mediated and heat shock promoter-dependent induction of dnaK is a crucial event in the intracellular replication of B. suis.