GSE107526
Identification of the genes regulated by the two-component system response regulator NarP of Actinobacillus pleuropneumoniae by DNA-affinity-purified (DAP) sequencing
Identification of the direct target genes of a response regulators (RRs) of a bacterial two-component system (TCS) is critical to understand the roles of the TCS in bacterial environmental adaption and pathogenesis. A. pleuropneumoniae is an important respiratory bacterial pathogen causing great economic losses to swine industry worldwide. The triggering signal(s) and targets of the RR NarP belonging to the TCS NarQ/NarP of A. pleuropneumoniae are still unknown. In the present study, a DNA-affinity-purified sequencing (DAP-Seq) approach was established. The upstream regions of a total of 131 candidate genes from the genome of A. pleuropneumoniae were shown to be co-purified with the activated NarP protein. Electrophoretic mobility shift assay results confirmed the interactions of NarP with the promoter regions of five selected target genes, including dmsA, pgaA, ftpA, cstA and ushA. Then, the EMSA-confirmed target genes were shown to be significantly up-regulated in the narP deleted mutant in the presence of additional nitrate, while the transcriptional changes were restored in the complemented strain. The NarP binding motif in the upstream regions of the target gene dmsA and ftpA were further identified and confirmed by EMSA using the truncated binding motif. The NarP binding sites were present in a total of 25.2% of the DNA fragments captured by DAP-Seq. These results demonstrated that the DAP-Seq method established in this study was effective to explore the direct targets of RRs of bacterial TCSs, and indicated that the A. pleuropneumoniae NarP could be a repressor in response to nitrate. Overall design: The potential NarP binding sites were acquired by DNA-affinity-purified sequencing (DAP-Seq). The genomic DNA were extracted and fragmented, the paired-end adapters were added on both ends. The adapter-containing DNA fragments were then subject to DNA affinity purification by incubation with the purified activated protein NarP with His tag and applied to Ni-NTA resin. The resin was washed and the protein was eluted. The DNA bound to NarP was purified and sequenced. A incubation using DNA fragments without any protein was used as a control. Afterwards, the peaks generated by the sequenced data analysis were used to predict binding sites of NarP and be further validated.