Transcriptome analysis from the BvrR/BvrS two-component regulatory system. also depended around the TCS BvrRS. In addition, we demonstrate a direct interaction between the promoter region of the VirB operon and the response regulator BvrR. Altogether these data demonstrate that this TCS BvrRS controls the expression of the T4SS VirB through direct and indirect mechanisms. organisms are intracellular bacteria infecting animals and humans (21, 23). The pathogenesis exerted by members of the genus is usually critically dependent on the establishment of chronic intracellular infections (3, 21). Among the various systems and molecules known to participate in virulence, the two-component regulatory system BvrR/BvrS (TCS BvrRS) and the type IV secretion system VirB (T4SS VirB) are critical. The TCS BvrRS, composed of a histidine kinase sensor located in the cell membrane (BvrS) and a cytoplasmic regulator (BvrR), participates in the homeostasis of the outer membrane (OM) controlling the structure of the lipopolysaccharide (LPS) and the expression of Oxytocin Acetate periplasmic and OM proteins (Omp) (12, 15, 20). Mutants with mutations in this regulatory system are nonvirulent in mice, displaying increased sensitivity to bactericidal peptides and complement, deficient cell invasion, and altered intracellular trafficking (30). The T4SS VirB is usually devoted to the control of intracellular trafficking; as a consequence, bacterial mutants defective in this system are impaired in their ability to multiply within cultured cells or YKL-06-061 to persist in mice (6, 22, 29). It has been proposed that this T4SS YKL-06-061 VirB extends from the inner membrane to the OM and delivers effectors into the host cell in order to control the biogenesis of the intracellular compartment where the bacteria will eventually reside (9). Although there has been some controversy, the VirB mutants do not show altered cell invasion (7, 11). The expression of the T4SS VirB is usually tightly regulated both YKL-06-061 and T4SS VirB and the TCS BvrRS have homologs present in alphaproteobacterial endosymbionts and pathogens of plants and animals such as (4, 19, 30). Indeed, ChvG/ChvI and ExoS/ChvI are also two-component systems devoted to the control of critical functions during parasitism and endosymbiosis, respectively. The ChvG/ChvI system regulates the acid-induced expression of the gene, coding for an Omp, and the expression of the and genes, coding for T4SS proteins responsible for the transfer of transfer DNA (T-DNA) to host cells (13, 16, 33). ChvG/ChvI mutants are nonvirulent and display increased sensitivity to detergents, antibiotics, and low pH (8). Similarly, the ExoS/ChvI system controls the expression of the flagellum and the production of succinoglycan, components required for the invasion of legume plants, and the TCS BatR/BatS of regulates in a pH-dependent manner several virulence genes of this intracellular pathogen (25). Due to the unfavorable impact that mutations in the TCS BvrRS exert in intracellular trafficking, we have hypothesized that this system controls the expression of the T4SS VirB. Indeed we have found that the TCS BvrRS exerts a direct transcriptional control around the expression of VirB. MATERIALS AND METHODS Bacterial strains and growth conditions. 2308 NaIr is usually a virulent smooth-LPS strain described elsewhere (26). Nonvirulent and mutants are smooth-LPS strains derived from 2308 NaIr with a mini-Tninsertion YKL-06-061 in the and genes, respectively. The mutant transformed with the p(30). strains were produced in tryptic soy broth (TSB), and BL21 was grown on Luria-Bertani (LB) or 2 yeast extract-tryptone (YT) medium supplemented with 50 g/ml ampicillin or 30 g/ml chloramphenicol when required. Expression of the VirB promoter in -galactosidase assay. To analyze the direct effect of BvrR around the transcription of the VirB promoter (PVirB), a -galactosidase transcriptional fusion approach was used (9). Briefly, strain BL21 (Cmr) and with plasmid.