Identification of the genes regulated by the two-component system response regulator NarP of Actinobacillus pleuropneumoniae by DNA-affinity-purified (DAP) sequencing

ABSTRACT: 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.

ORGANISM(S): Actinobacillus pleuropneumoniae

PROVIDER: GSE107526 | GEO | 2019/11/30

REPOSITORIES: GEO