Figure 4.

ArsR2 negatively regulates expression of T4SS products. (A) Volcano plot of differentially-expressed genes (Log₂ FC > 1 and P value < 0.05) in B. abortus A19 and A19 ΔarsR2. The horizontal axis is the log₂ FC between these strains. The negative log10 of the P value of Fisher’s exact test is plotted on the vertical axis. Each gene is represented by one point on the graph. (B) Schematic representation of Brucella T4SS encoded by the virB operon which comprises 12 open reading frames (virB1 to virB12). (C) RNA-seq analysis of expression of virB1-virB12 (Log₂ FC > 1 and P value < 0.05) in B. abortus A19. (D) Expression of virB1-virB12 was determined by qRT-PCR and normalized to gene expression in B. abortus S2308, A19 or deletion derivatives. (E) Activity of a plasmid-borne pvirBp-lacZ transcriptional reporter in S2308 and A19 with or without ATc induction of ArsR2. Plasmids pControl and pvirBp-lacZ or pArsR2 and pvirBp-lacZ were electroporated into B. abortus S2308 and A19, respectively. The β-galactosidase activity was detected in the presence or absence of 200 ng/mL ATc. pControl indicates pBB-PZT1 empty vector and PvirBp indicates pBB-PZT1-VirBp plasmid. Strains were grown in TSB to OD₆₀₀ 0.6 in the absence or presence of 100 nM ATc. (F) Activity of a plasmid-borne pvirBp-lacZ transcriptional reporter in B. abortus S2308, A19 strain or deletion derivatives. Relevant plasmids were electroporated into the required host strains and β-galactosidase activity was assessed in strains grown in TSB to OD₆₀₀ 0.6. (G) B1H assays for the interaction between ArsR2 and virB 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 pTRG and pBX empty vectors were used as negative controls. The pBRG-ArsR6 and pBX-ArsR6 plasmids were used as positive controls and pTRG and pBX-ArsR2 were used as self-activation controls. (H) EMSA of His-ArsR2 binding to the virB promoter region. His-tag was used as a negative control and ArsR6 was incubated with arsR6p as a positive control. (I) Model for indirect regulation of virB1-virB12 by ArsR2. Data shown in panels F-H are mean and standard deviation. *<0.05, **<0.01 by unpaired two-tailed t tests with Welch’s correction.