Antitoxin MqsA decreases antibiotic susceptibility through the global regulator AgtR in Pseudomonas fluorescens.

Type II toxin-antitoxin systems are highly prevalent in bacterial genomes and play crucial roles in the general stress response. Previously, we demonstrated that the type II antitoxin PfMqsA regulates biofilm formation through the global regulator AgtR in Pseudomonas fluorescens. Here, we found that both the C-terminal DNA-binding domain of PfMqsA and AgtR are involved in bacterial antibiotic susceptibility. Electrophoretic mobility shift assay (EMSA) analyses revealed that AgtR, rather than PfMqsA, binds to the intergenic region of emhABC-emhR, in which emhABC encodes an resistance-nodulation-cell division efflux pump and emhR encodes a repressor. Through quantitative real-time reverse-transcription PCR and EMSA analysis, we showed that AgtR directly activates the expression of the emhR by binding to the DNA motif [5´-CTAAGAAATATACTTAC-3´], leading to repression of the emhABC. Furthermore, we demonstrated that PfMqsA modulates the expression of EmhABC and EmhR. These findings enhance our understanding of the mechanism by which antitoxin PfMqsA contributes to antibiotic susceptibility.

EmhR is an indole-sensing transcriptional regulator responsible for the indole-induced antibiotic tolerance in Pseudomonas fluorescens.

Indole is well known as an interspecies signalling molecule to modulate bacterial physiology; however, it is not clear how the indole signal is perceived and responded to by plant growth promoting rhizobacteria (PGPR) in the rhizosphere. Here, we demonstrated that indole enhanced the antibiotic tolerance of Pseudomonas fluorescens 2P24, a PGPR well known for its biocontrol capacity. Proteomic analysis revealed that indole influenced the expression of multiple genes including the emhABC operon encoding a major multidrug efflux pump. The expression of emhABC was regulated by a TetR-family transcription factor EmhR, which was demonstrated to be an indole-responsive regulator. Molecular dynamics simulation showed that indole allosterically affected the distance between the two DNA-recognizing helices within the EmhR dimer, leading to diminished EmhR-DNA interaction. It was further revealed the EmhR ortholog in Pseudomonas syringae was also responsible for indole-induced antibiotic tolerance, suggesting this EmhR-dependent, indole-induced antibiotic tolerance is likely to be conserved among Pseudomonas species. Taken together, our results elucidated the molecular mechanism of indole-induced antibiotic tolerance in Pseudomonas species and had important implications on how rhizobacteria sense and respond to indole in the rhizosphere.

Flavonoids repress the production of antifungal 2,4-DAPG but potentially facilitate root colonization of the rhizobacterium Pseudomonas fluorescens.

In the well-known legume-rhizobia symbiosis, flavonoids released by legume roots induce expression of the Nod factors and trigger early plant responses involved in root nodulation. However, it remains largely unknown how the plant-derived flavonoids influence the physiology of non-symbiotic beneficial rhizobacteria. In this work, we demonstrated that the flavonoids apigenin and/or phloretin enhanced the swarming motility and production of cellulose and curli in Pseudomonas fluorescens 2P24, both traits of which are essential for root colonization. Using a label-free quantitative proteomics approach, we showed that apigenin and phloretin significantly reduced the biosynthesis of the antifungal metabolite 2,4-DAPG and further identified a novel flavonoid-sensing TetR regulator PhlH, which was shown to modulate 2,4-DAPG production by regulating the expression of 2,4-DAPG hydrolase PhlG. Although having similar structures, apigenin and phloretin could also influence different physiological characteristics of P. fluorescens 2P24, with apigenin decreasing the biofilm formation and phloretin inducing expression of proteins involved in the denitrification and arginine fermentation processes. Taken together, our results suggest that plant-derived flavonoids could be sensed by the TetR regulator PhlH in P. fluorescens 2P24 and acts as important signalling molecules that strengthen mutually beneficial interactions between plants and non-symbiotic beneficial rhizobacteria.

Bacterial quorum sensing quenching activity of Lysobacter leucyl aminopeptidase acts by interacting with autoinducer synthase.

Acyl-homoserine lactone (AHL) is the most studied autoinducer in gram-negative bacteria controlling infections of various pathogens. Quenching of AHL signaling by inhibiting AHL synthesis or AHL-receptor binding via small molecular chemicals or enzymatically degrading AHL is commonly used to block bacterial infections. Here, we describe a new quorum-quenching strategy that directly "acquires" bacterial genes/proteins through a defined platform. We artificially expressed a typical AHL synthase gene pcoI from the biocontrol Pseudomonas fluorescens 2P24 in the antifungal bacterium Lysobacter enzymogenes OH11 lacking AHL production. This step led to the discovery of multiple PcoI interacting protein candidates from L. enzymogenes. The individual expression of these candidate genes in 2P24 led to the identification of Le0959, which encodes leucyl aminopeptidase, an effective protein that inhibits AHL synthesis in 2P24. Therefore, we define Le0959 as LqqP (Lysobacterquorum-quenching protein). The expression of pcoI in E. coli could produce AHL, and the introduction of lqqP into E. coli expressing pcoI could prevent the production of AHL. LqqP directly binds to PcoI, and this protein-protein binding reduced the abundance of free PcoI (capable of AHL synthesis) in vivo, thereby blocking PcoI-dependent AHL production. Overall, this study highlights the discovery of LqqP in quenching AHL quorum sensing by binding to AHL synthase via developing a previously-uncharacterized screening technique for bacterial quorum quenching.

Proteomic analysis reveals the mechanism of different environmental stress-induced tolerance of Pseudomonas aeruginosa to monochloramine disinfection.

Although drinking water disinfection proved to be an effective strategy to eliminate many pathogens, bacteria can still show disinfection tolerance in drinking water distribution systems. To date, the molecular mechanisms on how environmental stress affects the tolerance of Pseudomonas aeruginosa to monochloramine are not well understood. Here, we investigated how three stress conditions, namely starvation, low temperature, and starvation combined with low temperature, affected the monochloramine tolerance of Pseudomonas aeruginosa, an opportunistic pathogen commonly found in drinking water distribution systems. All stress conditions significantly promoted monochloramine tolerance, among which starvation had the most drastic effects. Proteomic analyses suggested that the three conditions not only triggered a positive antioxidant defense against oxidative damages but also prepared the bacteria to employ a passive defense mechanism against disinfectants via dormancy. Moreover, the expression of antioxidant enzymes reached the maximum under the starvation condition and further low temperature treatment had little effect on bacterial response to oxidative stress. Instead, we found further treatment of the starved cells with low temperature decreased the osmotic stress response and the stringent response, which generally play pivotal roles in disinfection tolerance. Taken together, these findings shed light on how abiotic factors influence the bacterial disinfection tolerance and will aid design of efficient strategies to eliminate Pseudomonas aeruginosa from drinking water.