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.

The antitoxin MqsA homologue in Pseudomonas fluorescens 2P24 has a rewired regulatory circuit through evolution.

The mqsRA operon encodes a toxin-antitoxin pair that was characterized to participate in biofilm and persister cell formation in Escherichia coli. Notably, the antitoxin MqsA possesses a C-terminal DNA-binding domain that recognizes the [5'-AACCT(N)2-4 AGGTT-3'] motif and acts as a transcriptional regulator controlling multiple genes including the general stress response regulator RpoS. However, it is unknown how the transcriptional circuits of MqsA homologues have changed in bacteria over evolutionary time. Here, we found mqsA in Pseudomonas fluorescens (PfmqsA) is acquired through horizontal gene transfer and binds to a slightly different motif [5'-TACCCT(N)3 AGGGTA-3'], which exists upstream of the PfmqsRA operon. Interestingly, an adjacent GntR-type transcriptional regulator, which was termed AgtR, is under negative control of PfMqsA. It was further demonstrated that PfMqsA reduces production of biofilm components through AgtR, which directly regulates the pga and fap operons involved in the synthesis of extracellular polymeric substances. Moreover, through quantitative proteomics analysis, we showed AgtR is a highly pleiotropic regulator that influences up to 252 genes related to diverse processes including chemotaxis, oxidative phosphorylation and carbon and nitrogen metabolism. Taken together, our findings suggest the rewired regulatory circuit of PfMqsA influences diverse physiological aspects of P. fluorescens 2P24 via the newly characterized AgtR.

Scanning for New BRI1 Mutations via TILLING Analysis.

The identification and characterization of a mutational spectrum for a specific protein can help to elucidate its detailed cellular functions. BRASSINOSTEROID INSENSITIVE1 (BRI1), a multidomain transmembrane receptor-like kinase, is a major receptor of brassinosteroids in Arabidopsis (Arabidopsis thaliana). Within the last two decades, over 20 different bri1 mutant alleles have been identified, which helped to determine the significance of each domain within BRI1. To further understand the molecular mechanisms of BRI1, we tried to identify additional alleles via targeted induced local lesions in genomes. Here, we report our identification of 83 new point mutations in BRI1, including nine mutations that exhibit an allelic series of typical bri1 phenotypes, from subtle to severe morphological alterations. We carried out biochemical analyses to investigate possible mechanisms of these mutations in affecting brassinosteroid signaling. A number of interesting mutations have been isolated via this study. For example, bri1-702, the only weak allele identified so far with a mutation in the activation loop, showed reduced autophosphorylation activity. bri1-705, a subtle allele with a mutation in the extracellular portion, disrupts the interaction of BRI1 with its ligand brassinolide and coreceptor BRI1-ASSOCIATED RECEPTOR KINASE1. bri1-706, with a mutation in the extracellular portion, is a subtle defective mutant. Surprisingly, root inhibition analysis indicated that it is largely insensitive to exogenous brassinolide treatment. In this study, we found that bri1-301 possesses kinase activity in vivo, clarifying a previous report arguing that kinase activity may not be necessary for the function of BRI1. These data provide additional insights into our understanding of the early events in the brassinosteroid signaling pathway.

Transcriptional Regulator PhlH Modulates 2,4-Diacetylphloroglucinol Biosynthesis in Response to the Biosynthetic Intermediate and End Product.

Certain strains of biocontrol bacterium Pseudomonas fluorescens produce the secondary metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) to antagonize soilborne phytopathogens in the rhizosphere. The gene cluster responsible for the biosynthesis of 2,4-DAPG is named phlACBDEFGH and it is still unclear how the pathway-specific regulator phlH within this gene cluster regulates the metabolism of 2,4-DAPG. Here, we found that PhlH in Pseudomonas fluorescens strain 2P24 represses the expression of the phlG gene encoding the 2,4-DAPG hydrolase by binding to a sequence motif overlapping with the -35 site recognized by σ70 factors. Through biochemical screening of PhlH ligands we identified the end product 2,4-DAPG and its biosynthetic intermediate monoacetylphloroglucinol (MAPG), which can act as signaling molecules to modulate the binding of PhlH to the target sequence and activate the expression of phlG Comparison of 2,4-DAPG production between the ΔphlH, ΔphlG, and ΔphlHG mutants confirmed that phlH and phlG impose negative feedback regulation over 2,4-DAPG biosynthesis. It was further demonstrated that the 2,4-DAPG degradation catalyzed by PhlG plays an insignificant role in 2,4-DAPG tolerance but contributes to bacterial growth advantages under carbon/nitrogen starvation conditions. Taken together, our data suggest that by monitoring and down-tuning in situ levels of 2,4-DAPG, the phlHG genes could dynamically modulate the metabolic loads attributed to 2,4-DAPG production and potentially contribute to rhizosphere adaptation.IMPORTANCE 2,4-DAPG, which is synthesized by biocontrol pseudomonad bacteria, is a broad-spectrum antibiotic against bacteria, fungi, oomycetes, and nematodes and plays an important role in suppressing soilborne plant pathogens. Although most of the genes in the 2,4-DAPG biosynthetic gene cluster (phl) have been characterized, it is still not clear how the pathway-specific regulator phlH is involved in 2,4-DAPG metabolism. This work revealed the role of PhlH in modulating 2,4-DAPG levels by controlling the expression of 2,4-DAPG hydrolase PhlG in response to 2,4-DAPG and MAPG. Since 2,4-DAPG biosynthesis imposes a metabolic burden on biocontrol pseudomonads, it is expected that the fine regulation of phlG by PhlH offers a way to dynamically modulate the metabolic loads attributed to 2,4-DAPG production.

Discovery of pentapeptide-inhibitor hits targeting FKBP51 by combining computational modeling and X-ray crystallography.

FKBP51 is well-known as a cochaperone of Hsp90 machinery and implicated in many human diseases including stress-related diseases, tau-mediated neurodegeneration and cancers, which makes FKBP51 an attractive drug target for the therapy of FKBP51-associated diseases. However, it has been reported that only nature product rapamycin, cyclosporine A, FK506 and its derivatives exhibit good binding affinities when bound to FKBP51 by now. Given the advantages of peptide-inhibitors, we designed and obtained 20 peptide-inhibitor hits through structure-based drug design. We further characterized the interaction modes of the peptide-inhibitor hits on the FK1 domain of FKBP51 by biochemical and structural biology methods. Structural analysis revealed that peptide-inhibitor hits form U-shaped conformations and occupy the FK506 binding pocket and share similar interaction modes with FK506. Using molecular dynamics simulations, we delved into the interaction dynamics and found that hits are anchored to the FK506 binding pocket in a quite stable conformation. Meanwhile, it was shown that interactions between FK1 and peptide-inhibitor hits are mainly attributed to the hydrogen bond networks comprising I87 and Y113 and FPF cores of peptide-inhibitors involved extensive hydrophobic interactions. We presumed that the peptide design strategy based on the small molecule structure probably shed new lights on the peptide-inhibitor discovery of other targets. The findings presented here could also serve as a structural basis and starting point facilitating the optimization and generation of FKBP51 peptide-inhibitors with better bio-activities.

The Two-Component System RstA/RstB Regulates Expression of Multiple Efflux Pumps and Influences Anaerobic Nitrate Respiration in Pseudomonas fluorescens.

Multidrug resistance (MDR) efflux pumps are involved in bacterial intrinsic resistance to multiple antimicrobials. Expression of MDR efflux pumps can be either constitutive or transiently induced by various environmental signals, which are typically perceived by bacterial two-component systems (TCSs) and relayed to the bacterial nucleoid, where gene expression is modulated for niche adaptation. Here, we demonstrate that RstA/RstB, a TCS previously shown to control acid-induced and biofilm-related genes in Escherichia coli, confers resistance to multiple antibiotics in Pseudomonas fluorescens by directly regulating the MDR efflux pumps EmhABC and MexCD-OprJ. Moreover, we show that phosphorylation of the conserved Asp52 residue in RstA greatly enhances RstA-DNA interaction, and regulation of the multidrug resistance by RstA/RstB is dependent on the phosphorylation of the RstA Asp52 residue by RstB. Proteome analysis reveals RstA/RstB also positively regulates the efflux pump MexEF-OprN and enzymes involved in anaerobic nitrate respiration and pyoverdine biosynthesis. Our results suggest that, by coupling the expression of multiple efflux pumps and anaerobic nitrate respiration, RstA/RstB could play a role in defense against nitrosative stress caused by anaerobic nitrate respiration. IMPORTANCE Microenvironmental hypoxia typically increases bacterial multidrug resistance by elevating expression of multidrug efflux pumps, but the precise mechanism is currently not well understood. Here, we showed that the two-component system RstA/RstB not only positively regulated expression of several efflux pumps involved in multidrug resistance, but also promoted expression of enzymes involved in anaerobic nitrate respiration and pyoverdine biosynthesis. These results suggested that, by upregulating expression of efflux pumps and pyoverdine biosynthesis-related enzymes, RstA/RstB could play a role in promoting bacterial tolerance to hypoxia by providing protection against nitrosative stress.

Discovery and structural analysis of a phloretin hydrolase from the opportunistic human pathogen Mycobacterium abscessus.

The family of PhlG proteins catalyses the hydrolysis of carbon-carbon bonds and is widely distributed across diverse bacterial species. Two members of the PhlG family have been separately identified as 2,4-diacetylphloroglucinol (2,4-DAPG) hydrolase and phloretin hydrolase; however, the extent of functional divergence and catalytic substrates for most members of this family is still unknown. Here, using sequence similarity network and gene co-occurrence analysis, we categorized PhlG proteins into several subgroups and inferred that PhlG proteins from Mycobacterium abscessus (MaPhlG) are likely to be functionally equivalent to phloretin hydrolase. Indeed, we confirmed the hydrolytic activity of MaPhlG towards phloretin and its analog monoacetylphloroglucinol (MAPG), and the crystal structure of MaPhlG in complex with MAPG revealed the key residues involved in catalysis and substrate binding. Through mutagenesis and enzymatic assays, we demonstrated that H160, I162, A213 and Q266, which are substituted in 2,4-DAPG hydrolase, are essential for the activity towards phloretin. Based on the conservation of these residues, potential phloretin hydrolases were identified from Frankia, Colletotrichum tofieldiae and Magnaporthe grisea, which are rhizosphere inhabitants. These enzymes may be important for rhizosphere adaptation of the producing microbes by providing a carbon source through anaerobic degradation of flavonoids. Taken together, our results provided a framework for understanding the mechanism of functional divergence of PhlG proteins.

The environmental endocrine disruptor p-nitrophenol interacts with FKBP51, a positive regulator of androgen receptor and inhibits androgen receptor signaling in human cells.

The compound p-nitrophenol, which shows the anti-androgenic activity, can easily become anthropogenic pollutants and pose a threat to the environment and human health. Previous work indicates that the anti-androgenic mechanism of p-nitrophenol is complex and may involve several components in the AR signaling pathway, but the molecular details of how p-nitrophenol inhibits AR signaling are still not quite clear. Here, we characterized p-nitrophenol binds to the FK1 domain of an AR positive regulator FKBP51 with micromolar affinity and structural analysis of FK1 domain in complex with p-nitrophenol revealed that p-nitrophenol occupies a hydrophobic FK1 pocket that is vital for AR activity enhancement. Molecular dynamics simulation indicated that p-nitrophenol is stably bound to the FK1 pocket and the hotspot residues that involved p-nitrophenol binding are mainly hydrophobic and overlap with the AR interaction site. Furthermore, we showed that p-nitrophenol inhibits the androgen-dependent growth of human prostate cancer cells, possibly through down-regulating the expression levels of AR activated downstream genes. Taken together, our data suggests that p-nitrophenol suppresses the AR signaling pathway at least in part by blocking the interaction between AR and its positive regulator FKBP51. We believe that our findings could provide new guidelines for assessing the potential health effects of p-nitrophenol.