Hiroyasu Onaka

Abstract Microorganisms in nature form communities through diverse interactions, such as mutualism and competition, to adapt to their ecological environments. These interactions seem to be mediated by extracellular metabolites (exometabolites), yet the chemical and biological diversity underlying these processes remains largely unexplored. In this study, we examined the chemical basis of cell-cell communication in the fission yeast Schizosaccharomyces pombe by a genome-wide screen employing 3,420 viable gene deletion mutants. We identified 37 strains that exhibited growth defects in monoculture on a minimal medium but exhibited growth recovery in the vicinity of wild-type colonies (co-culture), suggesting that exometabolites secreted by wild-type cells compensated for the gene deletion. Both lipophilic and water-soluble fractions obtained by solvent partitioning of the wild-type culture supernatant promoted growth recovery. Among the 11 mutants rescued by the water-soluble fraction, six were cysteine auxotrophs, prompting analyses of thiol-containing metabolites by liquid chromatography-mass spectrometry (LC-MS), revealing the presence of glutathione (GSH) in the culture supernatant. GSH restored growth in most strains as a nutrient source. In contrast, GSH rescued cell morphology defects in the hob3 Δ mutant, lacking the Bin/Amphiphysin/Rvs (BAR) adaptor protein Hob3, through a mechanism independent of nutrition. This research advances understanding of exometabolite-mediated interactions in S. pombe by highlighting the role of GSH as one of the communication molecules that influence cellular processes and shape microbial communities. Author summary Microorganisms secrete a wide range of metabolites that control microbial community behavior. These extracellular metabolites (exometabolites) include not only well-studied signaling molecules but also diverse primary and secondary metabolites, suggesting complex interactions among microbes. However, the molecular basis of these interactions remains poorly understood, partly due to challenges in detecting them experimentally. In this study, we surveyed exometabolites involved in cell-cell interactions in the model eukaryotic microorganism Schizosaccharomyces pombe . S. pombe secretes a wide variety of metabolites, including previously reported nitrogen signaling factors (NSFs) and glutathione identified in this work. By analyzing gene deletion mutants that depend on GSH for growth, we provide new insights into how microbes regulate collective behavior by sharing exometabolites.

Goadsporin is one of linear azole-containing peptides (LAPs) that form a subgroup within ribosomally synthesized and post-translationally modified peptides (RiPPs). It contains two dehydroalanine residues formed through the action of two enzymes, GodF and GodG, in a two-step process involving serine O-glutamylation followed by elimination. Here, we report the X-ray crystal structure of GodF, which catalyzes the tRNA-dependent glutamylation of target serine residues, resolved at a 2.34-Å resolution. Although GodF exhibits low homology at the primary sequence level, its overall structure closely resembles that of TbtB, a tRNAGlu-dependent enzyme involved in thiopeptide biosynthesis, as well as the O-glutamylation domains of NisB and MibB, which serve as dehydroalanine synthases in lanthipeptide biosynthesis. The residues and structural elements forming the active site are well-aligned among these enzymes, while regions outside the active site are poorly conserved. Like TbtB, GodF features a coiled-coil subdomain at its N-terminus, and AlphaFold3 predicts this region plays a key role in recognizing the substrate tRNAGlu. GodF also contains a typical RiPP recognition element (RRE) motif; however, the spatial arrangement of the secondary structural elements comprising this motif differs notably from those in other O-glutamylating enzymes. These structural characteristics of GodF highlight the diversity of substrate-binding pockets among RiPP-modifying enzymes, reflecting the variability in their substrate peptides and the necessity to accommodate distinct conformational and physicochemical properties.

Contractile injection systems (CISs) are derivatives of phage tails and widely distributed in prokaryotes. CISs load cognate effectors and eject them through contractile actions resembling those of phage tails. Ejected effectors play central roles in CIS functionality by acting on target cells and mediating various biological processes. Here, we report a novel group of CIS effectors related to phage tapemeasure protein, the transmembrane component of the phage infection machinery. This group is broadly distributed within the class actinobacteria, one of the bacterial classes in which CIS gene clusters are highly conserved, and is represented by Sle1, a cognate effector of the intracellularly localised Streptomyces lividans phage tail-like nanoparticle (SLP). This effector is associated with Sle2, which contains a CIS effector core domain and interacts with the SLP core component. Sle1 is packaged inside SLP and is translocated to lipid membranes along with SLPs. The functional domain of Sle1, probably through interactions with ribosome-containing subcellular fractions, upregulates the membrane-associated proteome in S. lividans and E. coli . This effect modifies the physiological properties of S. lividans , ultimately enhancing its adaptation to microbial competition. In addition, we revealed that Sle1-type effectors conserved among actinobacterial species are structurally and functionally diverse in their functional domains. One of them from Micromonospora eburnea constitutes a novel toxin-antitoxin system and introducing its functional domain into Sle1 reprogrammes the phenotypic responsiveness of S. lividans to neighbouring bacteria. Our findings illustrate that phage elements can be incorporated into CISs as reconfigurable platforms for bacterial adaptation to various environmental conditions.

Backbone thiazole (Thz) moieties prevail in bioactive peptidic natural products and play important roles in their biological functions. However, the de novo discovery of artificial Thz-containing peptide ligands remains challenging. Here, we report an mRNA display-based selection platform for Thz-containing macrocyclic peptides (ThzteMP), established through a dedicated posttranslational chemoenzymatic transformation. This method exploits the unique reactivity of ribosomally incorporated thioamides, enabling enzyme-free spontaneous heterocyclization to form thiazoline (Thn), which is further oxidized using the substrate-tolerant azoline dehydrogenase (GodE) to yield a Thz moiety. By integrating this chemoenzymatic process with chloroacetyl-mediated thioether macrocyclization and mRNA display, we have successfully discovered Thz-containing macrocyclic peptide ligands with high binding affinities against p21-activated kinase 4 (PAK4). This study establishes a robust system to expedite ligand discovery of pseudo-natural peptides and to investigate the functional benefit of their backbone Thzs.