B. melitensis 16M genome analysis revealed the presence of six putative sigma factor-encoding genes: rpoD, rpoH1, rpoH2, rpoE1, rpoE2, and rpoN. We mutated all these genes except rpoD. Phenotypic analysis of the mutants reveals that a strain carrying an rpoH2 null mutation (ΔrpoH2) is impaired for growth at 21 and 42°C and shows increased sensitivity to hydrogen peroxide. Compared to the wild-type strain, the ΔrpoH2 mutant is attenuated in all virulence models tested. Three other null mutants (ΔrpoH1, ΔrpoE1, and ΔrpoE2 mutants) are also defective for survival in mice at 4 weeks postinfection. We also demonstrated that rpoH2 deletion strongly reduces the expression of two major virulence factors in B. melitensis, the type IV secretion system and the flagellum.