Certain strains of biocontrol bacterium 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 and it is still unclear how the pathway-specific regulator within this gene cluster regulates the metabolism of 2,4-DAPG. Here, we found that PhlH in strain 2P24 represses the expression of the gene encoding the 2,4-DAPG hydrolase by binding to a sequence motif overlapping with the -35 site recognized by σ 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 Comparison of 2,4-DAPG production between the Δ, Δ, and Δ mutants confirmed that and 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 levels of 2,4-DAPG, the genes could dynamically modulate the metabolic loads attributed to 2,4-DAPG production and potentially contribute to rhizosphere adaptation. 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 () have been characterized, it is still not clear how the pathway-specific regulator 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 by PhlH offers a way to dynamically modulate the metabolic loads attributed to 2,4-DAPG production.

Summary 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)2‐4AGGTT‐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)3AGGGTA‐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.

Blockade of the PD-1/PD-L1 immunologic checkpoint using monoclonal antibodies has provided breakthrough therapies against cancer in the recent years. Nevertheless, intrinsic disadvantages of therapeutic antibodies may limit their applications. Thus, blocking of the PD-1/PD-L1 interaction by small molecules may be a promising alternative for cancer immunotherapy. We used a docking-based virtual screening strategy to rapidly identify new small molecular inhibitors targeting PD-L1. We demonstrated that a small molecule compound (N-[2-(aminocarbonyl)phenyl][1,1′-biphenyl]-4-carboxamide [APBC]) could effectively interrupt the PD-1/PD-L1 interaction by directly binding to PD-L1, presenting the K D and IC 50 values at low-micromolar level. Molecular docking study revealed that APBC may have function through a PD-L1 dimer-locking mechanism, occluding the PD-1 interaction surface of PD-L1. We further confirmed the ligand blocking activity and T cell-reinvigoration potency of APBC using cell-based assays. APBC could dose-dependently elevate cytokine secretions of the primary T-lymphocytes that are cocultured with cancer cells. Importantly, APBC displayed superior antitumor efficacy in hPD-L1 knock-in B16F10-bearing mouse model without the induction of observable liver toxicity. Analyses on the APBC-treated mice further revealed drastically elevated levels of infiltrating CD4 + and CD8 + T cells, and inflammatory cytokines production in tumor microenvironment. The APBC compound could serve as a privileged scaffold in the design of improved PD pathway modulators, thus providing us promising drug candidates for tumor immunotherapy.

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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.