Genome-Wide Mapping Reveals Complex Regulatory Activities of BfmR in Pseudomonas aeruginosa

Abstract

BfmR is a response regulator that modulates diverse pathogenic phenotypes and induces an acute-to-chronic virulence switch in Pseudomonas aeruginosa, an important human pathogen causing serious nosocomial infections. However, the mechanisms of action of BfmR remain largely unknown. Here, using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), we showed that 174 chromosomal regions of P. aeruginosa MPAO1 genome were highly enriched by coimmunoprecipitation with a C-terminal Flag-tagged BfmR. Integration of these data with global transcriptome analyses revealed that 172 genes in 106 predicted transcription units are potential targets for BfmR. We determined that BfmR binds to and modulates the promoter activity of genes encoding transcriptional regulators CzcR, ExsA, and PhoB. Intriguingly, BfmR bound to the promoters of a number of genes belong to either CzcR or PhoB regulon, or both, indicating that CzcRS and PhoBR two-component systems (TCSs) deeply feed into the BfmR-mediated regulatory network. In addition, we demonstrated that phoB is required for BfmR to promote the biofilm formation by P. aeruginosa. These results delineate the direct BfmR regulon and exemplify the complexity of BfmR-mediated regulation of cellular functions in P. aeruginosa.

Determination of the Two-Component Systems regulatory network reveals core and accessory regulations across Pseudomonas aeruginosa lineages

“…The comparison to this set extracted from 22 previous studies and composed of 59 known binding sites concerning 21 RRs showed that the vast majority were effectively retrieved, often resulting in very highly enriched peaks (Figure S2), further validating our approach. We then compared our results to the previously reported ChIP-seq studies on RRs for which we detected binding sites (PhoB, GacA, GltR, PhoP, CzcR, DsbR and BfmR) (Bielecki et al, 2015;Fan et al, 2021;Huang et al, 2019;Xu et al, 2021;Yang et al, 2021;Yu et al, 2021). Here again, there was a strong overlap in detected binding sites between the DAP-seq data and the six different ChIP-seq studies, as well as highly similar identified DNA motifs (Figure S3).…”

“…Peak lists from ChIP-seq experiments with PhoB, GacA, CzcR, GltR, PhoP, DsbR and BfmR were used to retrieve all peak summit locations (Bielecki et al, 2015;Fan et al, 2021;Huang et al, 2019;Xu et al, 2021;Yang et al, 2021;Yu et al, 2021). Peak summit positions were averaged between replicates and used to infer gene targets using the genome-wide promoter regions defined above with the Intersect tool from BEDTools (Quinlan and Hall, 2010) on the corresponding genomes: PAO1 (Fan et al, 2021;Huang et al, 2019;Xu et al, 2021;Yu et al, 2021) or PA14 (Bielecki et al, 2015;Yang et al, 2021). For DNA motif determination, peak regions were extended by 50bp on each side of the summits and retrieved using the GetFastaBed tool from BEDTools (Quinlan and Hall, 2010).…”

“…However, while bringing new information on selected phenotypes, this approach does not globally address direct RR-target regulatory interactions. Among all RRs encoded in the PAO1 genome, 51 are predicted TFs and the binding sites of only 8 of them (GacA, AlgR, PhoB, PhoP, CzcR, GltR, DsbR and BfmR) have been determined at a genome scale so far (Bielecki et al, 2015;Fan et al, 2021;Huang et al, 2019;Xu et al, 2021;Yang et al, 2021;Yu et al, 2021).…”

“…This is notably due to the fact that RRs are only active in vivo in response to specific signals that are unknown in most cases, making it challenging to assess their role, let alone determining their entire regulons (Rajeev et al, 2020). Additionally, some RRs seem to be involved in crossor co-regulations (Bielecki et al, 2015;Fan et al, 2021;Huang et al, 2019), which represent pivotal features of the network topology and thus call for a large-scale investigation of this phenomenon. P. aeruginosa is a fast-evolving bacterium with a large intraspecies genetic diversity, gifting this pathogen with numerous different ways of establishing infection and resisting treatments.…”

Determination of the two-component systems regulatory network reveals core and accessory regulations across Pseudomonas aeruginosa lineages

“…Peak lists from ChIP-seq experiments with PhoB, GacA, CzcR, GltR, PhoP, DsbR and BfmR were used to retrieve all peak summit locations ( 23–28 ). Peak summit positions were averaged between replicates and used to infer gene targets using the genome-wide promoter regions defined above with the Intersect tool from BEDTools ( 49 ) on the corresponding genomes: PAO1 ( 24–26 , 28 ) or PA14 ( 23 , 27 ). For DNA motif determination, peak regions were extended by 50 bp on each side of the summits and retrieved using the GetFastaBed tool from BEDTools ( 49 ).…”