Figure 5.

ArsR2 interacts with the vjbR promoter region and inhibits vjbR transcription. (A) ChIP-seq reveals potential target genes of ArsR2. B. abortus A19 CΔarsR2 in stationary phase was cross-linked, washed, and sonicated to produce sheared chromosomal DNA. DNA was purified were utilized for library construction to conduct ChIP-seq. (B) Analysis of RNA-Seq and ChIP-Seq data identifies the ArsR2 regulon. RNA-seq and ChIP-seq data were analyzed further to evaluate genes that potentially are regulated directly by ArsR2. The set of peak-associated genes was filtered using the list of differentially-expressed genes as determined by RNA-seq. The 58 differentially-expressed genes were identified as potential target genes for ArsR2 regulation. (C) Expression levels of were determined by qRT-PCR and normalized to gene expression in B. abortus S2308, A19 and derivatives. (D) Activity of the pvjbR-lacZ transcriptional reporter in S2308 and A19 with or without ATc. The pControl and pvjbR-lacZ plasmids or pArsR2 and pvjbR-lacZ were electroporated into B. abortus S2308 and A19, respectively, and β-galactosidase activity was detected in the presence or absence of 200 ng/mL ATc. pControl indicates pBB-PZT1 empty vector and PvjbR indicates pBB-PZT1-VjbR plasmid. Strains were grown in TSB to OD₆₀₀ 0.6 in the absence or presence of 100 nM ATc. (E) Activity of the pvjbR-lacZ transcriptional reporter in B. abortus S2308, A19 and derivatives. β-galactosidase activity was assayed in strains grown in TSB to OD₆₀₀ 0.6. (F) B1H assays for the interaction between ArsR2 and vjbR promoter. E. coli XR reporter strains containing the pTRG, pBX plasmids and derivatives were cultured and spotted on LB agar in the presence or absence of streptomycin and 3-AT. The pBRG and pBX empty vectors were used as a negative control, pBRG-ArsR6 and pBX-ArsR6 were used as a positive control, and pBRG and pBX-ArsR2 was used as a self-activation control. (G) EMSA of His-ArsR2 binding to the vjbR promoter. His-tag was incubated with the promoter as a negative control. Unlabelled arsR2 promoter sequence was tested for competition with the biotin-labelled vjbR promoter. Results are representative of at least three independent experiments. (H) The effect of metal ions on DNA binding activity of ArsR2. The arsR6 promoter region probe was incubated with ArsR6 (2 µg) in the presence or absence of metal ions. Results are representative of at least three independent experiments. (I) Activity of a plasmid-borne pvjbR-lacZ transcriptional reporter in B. abortus S2308 and A19 in the present or absence of copper ions. The pvjbR-LacZ plasmid was electroporated into the strains, and β-galactosidase activity was assayed in strains grown in TSB to OD₆₀₀ 0.6. (J) Schematic depiction of the outcome of ArsR2 regulation of T4SS genes by VjbR in the wild-type strain, ΔarsR2, and in the presence of copper ions.