2-oxogluatarate (2-OG), a metabolite of the highly conserved Krebs cycle, not only plays a critical role in metabolism, but also constitutes a signaling molecule in a variety of organisms ranging from bacteria to plants and animals. In cyanobacteria, the accumulation of 2-OG constitutes the signal of nitrogen starvation and NtcA, a global transcription factor, has been proposed as a putative receptor for 2-OG. Here we present three crystal structures of NtcA from the cyanobacterium Anabaena : the apoform, and two ligand-bound forms in complex with either 2-OG or its analogue 2,2-difluoropentanedioic acid. All structures assemble as homodimers, with each subunit composed of an N-terminal effector-binding domain and a C-terminal DNA-binding domain connected by a long helix (C-helix). The 2-OG binds to the effector-binding domain at a pocket similar to that used by cAMP in catabolite activator protein, but with a different pattern. Comparative structural analysis reveals a putative signal transmission route upon 2-OG binding. A tighter coiled-coil conformation of the two C-helices induced by 2-OG is crucial to maintain the proper distance between the two F-helices for DNA recognition. Whereas catabolite activator protein adopts a transition from off-to-on state upon cAMP binding, our structural analysis explains well why NtcA can bind to DNA even in its apoform, and how 2-OG just enhances the DNA-binding activity of NtcA. These findings provided the structural insights into the function of a global transcription factor regulated by 2-OG, a metabolite standing at a crossroad between carbon and nitrogen metabolisms.

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The compatible solute ABC (ATP-binding cassette) transporters are indispensable for acquiring a variety of compatible solutes under osmotic stress in Bacillus subtilis. The substrate-binding protein OpuCC (Opu is osmoprotectant uptake) of the ABC transporter OpuC can recognize a broad spectrum of compatible solutes, compared with its 70% sequence-identical paralogue OpuBC that can solely bind choline. To explore the structural basis of this difference of substrate specificity, we determined crystal structures of OpuCC in the apo-form and in complex with carnitine, glycine betaine, choline and ectoine respectively. OpuCC is composed of two α/β/α globular sandwich domains linked by two hinge regions, with a substrate-binding pocket located at the interdomain cleft. Upon substrate binding, the two domains shift towards each other to trap the substrate. Comparative structural analysis revealed a plastic pocket that fits various compatible solutes, which attributes themultiple-substrate binding property to OpuCC. This plasticity is a gain-of-function via a single-residue mutation of Thr⁹⁴ in OpuCC compared with Asp⁹⁶ in OpuBC.

ORCID IDs: 0000-0002-6213-9791 (D.Q.); 0000-0002-5178-0700 (Y.X.)Functional divergence in paralogs is an important genetic source of evolutionary innovation. Actin-depolymerizing factors (ADFs) are among the most important actin binding proteins and are involved in generating and remodeling actin cytoskeletal architecture via their conserved F-actin severing or depolymerizing activity. In plants, ADFs coevolved with actin, but their biochemical properties are diverse. Unfortunately, the biochemical function of most plant ADFs and the potential mechanisms of their functional divergence remain unclear. Here, in vitro biochemical analyses demonstrated that all 11 ADF genes in Arabidopsis thaliana exhibit opposing biochemical properties. Subclass III ADFs evolved F-actin bundling (B-type) function from conserved F-actin depolymerizing (D-type) function, and subclass I ADFs have enhanced D-type function. By tracking historical mutation sites on ancestral proteins, several fundamental amino acid residues affecting the biochemical functions of these proteins were identified in Arabidopsis and various plants, suggesting that the biochemical divergence of ADFs has been conserved during the evolution of angiosperm plants. Importantly, N-terminal extensions on subclass III ADFs that arose from intron-sliding events are indispensable for the alteration of D-type to B-type function. We conclude that the evolution of these N-terminal extensions and several conserved mutations produced the diverse biochemical functions of plant ADFs from a putative ancestor.

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.