Abstract
An essential element required for B. abortus intracellular replication is the two-component system BvrR/BvrS. Intracellularly, the regulatory protein BvrR is activated by phosphorylation in response to cues found in early and late compartments. We generated two-point mutations in the BvrR putative phosphorylated aspartate 58 to understand the BvrR-regulated transcriptional response. Aspartate 58 was substituted witheither an alanine (BvrRD58A-dominant negative) or a glutamic acid (BvrRD58E-dominant positive), and the sequences were inserted in plasmids. We introduced these plasmids expressing bvrR mutants and the wildtype version into a B. abortus bvrR- and analyzed the strains’ phenotypes. The wild type and the BvrRD58E mutant restored the phenotypes affected in B. abortus bvrR- according to polymyxin assays, ex vivo replication, and expression of proteins such as Omp25, VirB8, and VjbR. In contrast to B. abortus bvrR-, we observed that the BvrRD58A mutant is not as attenuated in several phenotypes tested. For example, VjbR expression was recovered in the BvrRD58A mutant compared to B. abortus bvrR- confirming that unphosphorylated BvrR exerts transcriptional control. Interestingly, although BvrRD58A binds to the virb promoter, VirB8 expression was not restored. Based on these results, we propose three regulatory patterns defined by BvrR phosphorylation, (i) unphosphorylated BvrR binds and regulates the expression of genes directly, (ii) phosphorylated BvrR binds and regulates the expression of genes directly, and (iii) unphosphorylated BvrR binds to gene promoters, but additional regulators are needed to promote gene expression. These findings serve as a working model for understanding how the response regulators of two-component systems control gene expression.
