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Characterization of a novel two-component system TmrA/TmrB involved in multi-drug resistance of Pseudomonas fluorescens

ABSTRACT: It has been widely reported that bacteria can sense environment changes and coordinates its behaviors via two-component systems （TCSs） to survive in the complicated niche. A TCS was mainly composed of a sensor kinase and a transcriptional factor, working as a signaling-sensor and a gene transcription regulator respectively. Thriving in diverse ecological niches, Pseudomonas fluorescens inherently resists a wide spectrum of antibiotics and the operon emhABC encoding an efflux pump plays a major role in the resistance to antibiotic and toxic compounds. Here, we identified that TmrA, a responsive regulator of the TmrA/TmrB two-component system, directly activated the expression of emhABC in P. fluorescens and a consensus motif recognized by TmrA was identified upstream of the emhABC promoter. Consistently, the tmrA deletion mutant (ΔtmrA) conferred significant decrease of resistance to multiple antibiotics. Moreover, deletion of intra-cellular kinase domain of TmrB resulted in decreased drug resistance similar to ΔTmrA mutant, inferring that phosphorylation of TmrA is essential for activation of emhABC expression. It was further demonstrated that in vitro phosphorylation of TmrA led to enhanced (>10 fold) binding affinity to the promoter region of emhABC. The knock-in TmrAD52A mutant, which bore the mutation of the conserved phosphorylation site across the TmrA homologues, showed partially decreased antibiotic resistance and in vitro phosphorylation of TmrAD52A protein also improved the binding to the emhABC promoter, suggesting there should be other unidentified phosphorylation sites yet essential to the regulation role of TmrA. Lastly, we performed the label-free quantification analysis of the whole proteome of the wild-type and ΔtmrA mutant strains and found that TmrA is likely to be a global regulator participating in multiple physiological processes including the multidrug resistance, secondary metabolite synthesis and nitrate assimilation. The characterization of other potential phosphorylation sites of TmrA is currently being undertaken.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Pseudomonas Fluorescens F113

TISSUE(S): Cell Culture

SUBMITTER: Diyin Li

LAB HEAD: Yongxing He

PROVIDER: PXD018793 | Pride | 2021-10-08

REPOSITORIES: pride

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Project description:Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part, by inducing production of damaging reactive species. This notion has been supported by many groups, but recently challenged. Here we robustly test the hypothesis using biochemical, enzymatic and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular hydrogen peroxide (H2O2) sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species, to demonstrate that antibiotics broadly induce redox stress. Subsequent gene expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supra-physiological levels of H2O2. We next developed a method to dynamically quantify cellular respiration and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that catalase or DNA mismatch repair enzyme overexpression, as well as antioxidant pre-treatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions, but could be enhanced by exposure to molecular oxygen or addition of alternative electron acceptors, suggesting that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that bactericidal antibiotics, downstream of their target-specific interactions, induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality. Here, we used microarrays to analyze oxidative stress responses to bactericidal antibiotic treatment in wildtype and mutant E coli WT or mutant E coli cells were grown to OD ~0.3. Untreated cells were harvested at time 0 as controls. Treated cells given the appropriate chemical perturbation and harvested 1 hour post-treatment. All experiments were performed in technical triplicate.

			Action	DRS
	2p24.fasta	Fasta
	d52a-1.raw	Raw
	d52a-2.raw	Raw
	d52a-3.raw	Raw
	d52a-wt_Proteins.txt	Txt

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Characterization of a novel two-component system TmrA/TmrB involved in multi-drug resistance of Pseudomonas fluorescens

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