Novel dual regulators of Pseudomonas aeruginosa essential for productive biofilms and virulence

Abstract

Gene regulation network in Pseudomonas aeruginosa is complex. With a relatively large genome (6.2 Mb), there is a significant portion of genes that are proven or predicted to be transcriptional regulators. Many of these regulators have been shown to play important roles in biofilm formation and maintenance. In this study, we present a novel transcriptional regulator, PA1226, which modulates biofilm formation and virulence in P. aeruginosa. Mutation in the gene encoding this regulator abolished the ability of P. aeruginosa to produce biofilms in vitro, without any effect on the planktonic growth. This regulator is also essential for the in vivo fitness and pathogenesis in both Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA1226 regulates many essential virulence genes/pathways, including those involved in alginate, pili, and LPS biosynthesis. Genes/operons directly regulated by PA1226 and potential binding sequences were identified via ChIP-seq. Attempts to confirm the binding sequences by electrophoretic mobility shift assay led to the discovery of a co-regulator, PA1413, via co-immunoprecipitation assay. PA1226 and PA1413 were shown to bind collaboratively to the promoter regions of their regulons. A model is proposed, summarizing our finding on this novel dual-regulation system.

Two Regulators, PA3898 and PA2100, Modulate the Pseudomonas aeruginosa Multidrug Resistance MexAB-OprM and EmrAB Efflux Pumps and Biofilm Formation

“…E. coli EPMax10B-lacI q -pir was routinely used as a cloning strain. The P. aeruginosa wild-type strain PAO1 and its derivatives were cultured in Luria-Bertani (LB) medium with appropriate antibiotics, as previously described (38,49).…”

“…Gene fusion and mutant strain construction. All standard genetic manipulations were performed as previously described (38,49,52,54,55,57,58). All single mutant strains used throughout this study, including mdrR1::Tn, mdrR2::Tn, mexR::Tn, emrA::Tn, emrB::Tn, and all the mutant strains shown in Fig.…”

“…Phenotypic studies. A crystal violet biofilm assay and confocal microscopy for static biofilms were carried out in triplicate as previously described (38,49). Briefly, wild-type PAO1 and PA3898::Tn mutant strains were first grown overnight in LB medium with shaking.…”

“…Taking these outcomes together, we suspected that other proteins or factors in P. aeruginosa are necessary for the interaction between PA3898 and the DNA binding motif. To further evaluate this possibility, we employed a protein coimmunoprecipitation screen, as previously described (49), to search for a potential protein partner(s) necessary for the interaction of PA3898 and the binding motif (Fig. 3D).…”

“…Taken together, the ChIP-seq and EMSA results indicate that MdrR1 and MdrR2 bind to DNA sequences upstream of genes and operons that they directly regulate, and LSPR validates the specific interaction between MdrR1, MdrR2, and DNA. Furthermore, to verify the exact binding motif predicted by ChIP-seq, DNA footprinting was performed with an automated DNA analyzer as previously described (49,50). A DNA fragment upstream of P mexA (from bases Ϫ189 to ϩ224 relative to the start codon of mexA) was coincubated with PsrA (PA3006) (a regulator, as a negative control), MdrR1 alone, MdrR2 alone, and MdrR1 and MdrR2 together (Fig.…”