Scanning iron response regulator binding sites using RNA-seq and Dap-seq in the Brucella genome

Brucella spp. are gram-negative, facultative intracellular pathogens that cause brucellosis in humans and animals. Iron is an essential element required for all organisms. Iron response regulator (Irr) is a crucial transcriptional regulator and can affect the growth and iron uptake of Brucella. The growth rate of Brucella melitensis M5-90 irr mutant was significantly lower than that of B. melitensis M5-90 under normal or iron-sufficient conditions, however, the growth rate of the B. melitensis M5-90 irr mutant was significantly higher than that of B. melitensis M5-90 under iron-limited conditions. In addition, irr mutation significantly reduced iron uptake under iron-limited conditions. Currently, bhuA (a heme transporter), bhuQ (heme utilization oxygenase Q), and RirA (iron-responsive regulator) have been identified as iron metabolism genes and are regulated by the Irr protein. These results suggest that the Irr protein has multiple target genes in the Brucella genome that are involved in iron metabolism. Therefore, a combination of RNA-seq and Dap-seq was used to investigate the other iron metabolism genes that are also regulated by the Irr protein in Brucella. A total of seven genes were identified as target genes for Irr in this study and the expression levels of these seven genes were similar in both the RNA-seq and qRT-PCR results. Furthermore, the DNA bases C and G and amino acids asparagine, arginine, histidine, and serine were predicted as predominant docking sites for DNA and Irr using the HDOCK server. The electrophoretic mobility shift assay confirmed that six out of the seven genes, namely rirA (BME_RS13665), membrane protein (BME_RS01725), hypothetical protein (BME_RS09560), Iron transporter (BME_RS14525), cation-transporting P-type ATPase (BME_RS10660), and 2Fe-2S binding protein (BME_RS13655), interact with the Irr protein. In summary, our results identified six genes regulated by the Irr protein that may participate in iron metabolism. In addition, from the RNA-seq results, the mutation of irr resulted in the down/upregulation of many genes that are associated with the bacterial secretion system, lipopolysaccharides, and flagellar assembly, and these three components are the main virulence factors for Brucella. Collectively, these results provide valuable insights for the exploration of Brucella iron metabolism and its effects on virulence. Overall design: In this study, transcriptome and DNA affinity purification sequencing were used to scan the potential targets of Irr protein in Brucella genome. Next, HDOCK was used to predict the interface of Irr protein and the identifid genes targets. EMSA was used to further confirm the direct interaction of Irr with the potential target genes.