A mutualistic bacterium rescues a green alga from an antagonist.

Photosynthetic protists, known as microalgae, are key contributors to primary production on Earth. Since early in evolution, they coexist with bacteria in nature, and their mode of interaction shapes ecosystems. We have recently shown that the bacterium Pseudomonas protegens acts algicidal on the microalga Chlamydomonas reinhardtii. It secretes a cyclic lipopeptide and a polyyne that deflagellate, blind, and lyse the algae [P. Aiyar et al., Nat. Commun. 8, 1756 (2017) and V. Hotter et al., Proc. Natl. Acad. Sci. U.S.A. 118, e2107695118 (2021)]. Here, we report about the bacterium Mycetocola lacteus, which establishes a mutualistic relationship with C. reinhardtii and acts as a helper. While M. lacteus enhances algal growth, it receives methionine as needed organic sulfur and the vitamins B1, B3, and B5 from the algae. In tripartite cultures with the alga and the antagonistic bacterium P. protegens, M. lacteus aids the algae in surviving the bacterial attack. By combining synthetic natural product chemistry with high-resolution mass spectrometry and an algal Ca2+ reporter line, we found that M. lacteus rescues the alga from the antagonistic bacterium by cleaving the ester bond of the cyclic lipopeptide involved. The resulting linearized seco acid does not trigger a cytosolic Ca2+ homeostasis imbalance that leads to algal deflagellation. Thus, the algae remain motile, can swim away from the antagonistic bacteria and survive the attack. All three involved genera cooccur in nature. Remarkably, related species of Pseudomonas and Mycetocola also act antagonistically against C. reinhardtii or as helper bacteria in tripartite cultures.

Streptomyces polyketides mediate bacteria-fungi interactions across soil environments.

Although the interaction between prokaryotic and eukaryotic microorganisms is crucial for the functioning of ecosystems, information about the processes driving microbial interactions within communities remains scarce. Here we show that arginine-derived polyketides (arginoketides) produced by Streptomyces species mediate cross-kingdom microbial interactions with fungi of the genera Aspergillus and Penicillium, and trigger the production of natural products. Arginoketides can be cyclic or linear, and a prominent example is azalomycin F produced by Streptomyces iranensis, which induces the cryptic orsellinic acid gene cluster in Aspergillus nidulans. Bacteria that synthesize arginoketides and fungi that decode and respond to this signal were co-isolated from the same soil sample. Genome analyses and a literature search indicate that arginoketide producers are found worldwide. Because, in addition to their direct impact, arginoketides induce a secondary wave of fungal natural products, they probably contribute to the wider structure and functioning of entire soil microbial communities.

Small-molecule activation of OGG1 increases oxidative DNA damage repair by gaining a new function.

Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed ß,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.

Bacterial marginolactones trigger formation of algal gloeocapsoids, protective aggregates on the verge of multicellularity.

Photosynthetic microorganisms including the green alga Chlamydomonas reinhardtii are essential to terrestrial habitats as they start the carbon cycle by conversion of CO2 to energy-rich organic carbohydrates. Terrestrial habitats are densely populated, and hence, microbial interactions mediated by natural products are inevitable. We previously discovered such an interaction between Streptomyces iranensis releasing the marginolactone azalomycin F in the presence of C. reinhardtii Whether the alga senses and reacts to azalomycin F remained unknown. Here, we report that sublethal concentrations of azalomycin F trigger the formation of a protective multicellular structure by C. reinhardtii, which we named gloeocapsoid. Gloeocapsoids contain several cells which share multiple cell membranes and cell walls and are surrounded by a spacious matrix consisting of acidic polysaccharides. After azalomycin F removal, gloeocapsoid aggregates readily disassemble, and single cells are released. The presence of marginolactone biosynthesis gene clusters in numerous streptomycetes, their ubiquity in soil, and our observation that other marginolactones such as desertomycin A and monazomycin also trigger the formation of gloeocapsoids suggests a cross-kingdom competition with ecological relevance. Furthermore, gloeocapsoids allow for the survival of C. reinhardtii at alkaline pH and otherwise lethal concentrations of azalomycin F. Their structure and polysaccharide matrix may be ancestral to the complex mucilage formed by multicellular members of the Chlamydomonadales such as Eudorina and Volvox Our finding suggests that multicellularity may have evolved to endure the presence of harmful competing bacteria. Additionally, it underlines the importance of natural products as microbial cues, which initiate interesting ecological scenarios of attack and counter defense.

The bacterium Pseudomonas protegens antagonizes the microalga Chlamydomonas reinhardtii using a blend of toxins.

The unicellular alga Chlamydomonas reinhardtii and the bacterium Pseudomonas protegens serve as a model to study the interactions between photosynthetic and heterotrophic microorganisms. P. protegens secretes the cyclic lipopeptide orfamide A that interferes with cytosolic Ca2+ homeostasis in C. reinhardtii resulting in deflagellation of the algal cells. Here, we studied the roles of additional secondary metabolites secreted by P. protegens using individual compounds and co-cultivation of algae with bacterial mutants. Rhizoxin S2, pyrrolnitrin, pyoluteorin, 2,4-diacetylphloroglucinol (DAPG) and orfamide A all induce changes in cell morphology and inhibit the growth of C. reinhardtii. Rhizoxin S2 exerts the strongest growth inhibition, and its action depends on the spatial structure of the environment (agar versus liquid culture). Algal motility is unaffected by rhizoxin S2 and is most potently inhibited by orfamide A (IC50  = 4.1 µM). Pyrrolnitrin and pyoluteorin both interfere with algal cytosolic Ca2+ homeostasis and motility whereas high concentrations of DAPG immobilize C. reinhardtii without deflagellation or disturbance of Ca2+ homeostasis. Co-cultivation with a regulatory mutant of bacterial secondary metabolism (ΔgacA) promotes algal growth under spatially structured conditions. Our results reveal how a single soil bacterium uses an arsenal of secreted antialgal compounds with complementary and partially overlapping activities.

Helper bacteria halt and disarm mushroom pathogens by linearizing structurally diverse cyclolipopeptides.

The bacterial pathogen Pseudomonas tolaasii severely damages white button mushrooms by secretion of the pore-forming toxin tolaasin, the main virulence factor of brown blotch disease. Yet, fungus-associated helper bacteria of the genus Mycetocola (Mycetocola tolaasinivorans and Mycetocola lacteus) may protect their host by an unknown detoxification mechanism. By a combination of metabolic profiling, imaging mass spectrometry, structure elucidation, and bioassays, we found that the helper bacteria inactivate tolaasin by linearizing the lipocyclopeptide. Furthermore, we found that Mycetocola spp. impair the dissemination of the pathogen by cleavage of the lactone ring of pseudodesmin. The role of pseudodesmin as a major swarming factor was corroborated by identification and inactivation of the corresponding biosynthetic gene cluster. Activity-guided fractionation of the Mycetocola proteome, matrix-assisted laser desorption/ionization (MALDI) analyses, and heterologous enzyme production identified the lactonase responsible for toxin cleavage. We revealed an antivirulence strategy in the context of a tripartite interaction that has high ecological and agricultural relevance.

Diiron monooxygenases in natural product biosynthesis.

Covering: up to 2017 The participation of non-heme dinuclear iron cluster-containing monooxygenases in natural product biosynthetic pathways has been recognized only recently. At present, two families have been discovered. The archetypal member of the first family, CmlA, catalyzes ß-hydroxylation of l-p-aminophenylalanine (l-PAPA) covalently linked to the nonribosomal peptide synthetase (NRPS) CmlP, thereby effecting the first step in the biosynthesis of chloramphenicol by Streptomyces venezuelae. CmlA houses the diiron cluster in a metallo-ß-lactamase protein fold instead of the 4-helix bundle fold of nearly every other diiron monooxygenase. CmlA couples O2 activation and substrate hydroxylation via a structural change caused by formation of the l-PAPA-loaded CmlP:CmlA complex. The other new diiron family is typified by two enzymes, AurF and CmlI, which catalyze conversion of aryl-amine substrates to aryl-nitro products with incorporation of oxygen from O2. AurF from Streptomyces thioluteus catalyzes the formation of p-nitrobenzoate from p-aminobenzoate as a precursor to the biostatic compound aureothin, whereas CmlI from S. venezuelae catalyzes the ultimate aryl-amine to aryl-nitro step in chloramphenicol biosynthesis. Both enzymes stabilize a novel type of peroxo-intermediate as the reactive species. The rare 6-electron N-oxygenation reactions of CmlI and AurF involve two progressively oxidized pathway intermediates. The enzymes optimize efficiency by utilizing one of the reaction pathway intermediates as an in situ reductant for the diiron cluster, while simultaneously generating the next pathway intermediate. For CmlI, this reduction allows mid-pathway regeneration of the peroxo intermediate required to complete the biosynthesis. CmlI ensures specificity by carrying out the multistep aryl-amine oxygenation without dissociating intermediate products.

Use of Isotopes and Isotope Effects for Investigations of Diiron Oxygenase Mechanisms.

Isotope effects of four broad and overlapping categories have been applied to the study of the mechanisms of chemical reaction and regulation of nonheme diiron cluster-containing oxygenases. The categories are: (a) mass properties that allow substrate-to-product conversions to be tracked, (b) atomic properties that allow specialized spectroscopies, (c) mass properties that impact primarily vibrational spectroscopies, and (d) bond dissociation energy shifts that permit dynamic isotope effect studies of many types. The application of these categories of isotope effects is illustrated using the soluble methane monooxygenase system and CmlI, which catalyzes the multistep arylamine to arylnitro conversion in the biosynthetic pathway for chloramphenicol.

CmlI N-Oxygenase Catalyzes the Final Three Steps in Chloramphenicol Biosynthesis without Dissociation of Intermediates.

CmlI catalyzes the six-electron oxidation of an aryl-amine precursor (NH2-CAM) to the aryl-nitro group of chloramphenicol (CAM). The active site of CmlI contains a (hydr)oxo- and carboxylate-bridged dinuclear iron cluster. During catalysis, a novel diferric-peroxo intermediate P is formed and is thought to directly effect oxygenase chemistry. Peroxo intermediates can facilitate at most two-electron oxidations, so the biosynthetic pathway of CmlI must involve at least three steps. Here, kinetic techniques are used to characterize the rate and/or dissociation constants for each step by taking advantage of the remarkable stability of P in the absence of substrates (decay t1/2 = 3 h at 4 °C) and the visible chromophore of the diiron cluster. It is found that diferrous CmlI (CmlIred) can react with NH2-CAM and O2 in either order to form a P-NH2-CAM intermediate. P-NH2-CAM undergoes rapid oxygen transfer to form a diferric CmlI (CmlIox) complex with the aryl-hydroxylamine [NH(OH)-CAM] pathway intermediate. CmlIox-NH(OH)-CAM undergoes a rapid internal redox reaction to form a CmlIred-nitroso-CAM (NO-CAM) complex. O2 binding results in formation of P-NO-CAM that converts to CmlIox-CAM by enzyme-mediated oxygen atom transfer. The kinetic analysis indicates that there is little dissociation of pathway intermediates as the reaction progresses. Reactions initiated by adding pathway intermediates from solution occur much more slowly than those in which the intermediate is generated in the active site as part of the catalytic process. Thus, CmlI is able to preserve efficiency and specificity while avoiding adventitious chemistry by performing the entire six-electron oxidation in one active site.

Unprecedented (µ-1,1-Peroxo)diferric Structure for the Ambiphilic Orange Peroxo Intermediate of the Nonheme N-Oxygenase CmlI.

The final step in the biosynthesis of the antibiotic chloramphenicol is the oxidation of an aryl-amine substrate to an aryl-nitro product catalyzed by the N-oxygenase CmlI in three two-electron steps. The CmlI active site contains a diiron cluster ligated by three histidine and four glutamate residues and activates dioxygen to perform its role in the biosynthetic pathway. It was previously shown that the active oxidant used by CmlI to facilitate this chemistry is a peroxo-diferric intermediate (CmlIP). Spectroscopic characterization demonstrated that the peroxo binding geometry of CmlIP is not consistent with the µ-1,2 mode commonly observed in nonheme diiron systems. Its geometry was tentatively assigned as µ-η2:η1 based on comparison with resonance Raman (rR) features of mixed-metal model complexes in the absence of appropriate diiron models. Here, X-ray absorption spectroscopy (XAS) and rR studies have been used to establish a refined structure for the diferric cluster of CmlIP. The rR experiments carried out with isotopically labeled water identified the symmetric and asymmetric vibrations of an Fe-O-Fe unit in the active site at 485 and 780 cm-1, respectively, which was confirmed by the 1.83 Å Fe-O bond observed by XAS. In addition, a unique Fe···O scatterer at 2.82 Å observed from XAS analysis is assigned as arising from the distal O atom of a µ-1,1-peroxo ligand that is bound symmetrically between the irons. The (µ-oxo)(µ-1,1-peroxo)diferric core structure associated with CmlIP is unprecedented among diiron cluster-containing enzymes and corresponding biomimetic complexes. Importantly, it allows the peroxo-diferric intermediate to be ambiphilic, acting as an electrophilic oxidant in the initial N-hydroxylation of an arylamine and then becoming a nucleophilic oxidant in the final oxidation of an aryl-nitroso intermediate to the aryl-nitro product.

Mechanism for Six-Electron Aryl-N-Oxygenation by the Non-Heme Diiron Enzyme CmlI.

The ultimate step in chloramphenicol (CAM) biosynthesis is a six-electron oxidation of an aryl-amine precursor (NH2-CAM) to the aryl-nitro group of CAM catalyzed by the non-heme diiron cluster-containing oxygenase CmlI. Upon exposure of the diferrous cluster to O2, CmlI forms a long-lived peroxo intermediate, P, which reacts with NH2-CAM to form CAM. Since P is capable of at most a two-electron oxidation, the overall reaction must occur in several steps. It is unknown whether P is the oxidant in each step or whether another oxidizing species participates in the reaction. Mass spectrometry product analysis of reactions under (18)O2 show that both oxygen atoms in the nitro function of CAM derive from O2. However, when the single-turnover reaction between (18)O2-P and NH2-CAM is carried out in an (16)O2 atmosphere, CAM nitro groups contain both (18)O and (16)O, suggesting that P can be reformed during the reaction sequence. Such reformation would require reduction by a pathway intermediate, shown here to be NH(OH)-CAM. Accordingly, the aerobic reaction of NH(OH)-CAM with diferric CmlI yields P and then CAM without an external reductant. A catalytic cycle is proposed in which NH2-CAM reacts with P to form NH(OH)-CAM and diferric CmlI. Then the NH(OH)-CAM rereduces the enzyme diiron cluster, allowing P to reform upon O2 binding, while itself being oxidized to NO-CAM. Finally, the reformed P oxidizes NO-CAM to CAM with incorporation of a second O2-derived oxygen atom. The complete six-electron oxidation requires only two exogenous electrons and could occur in one active site.

An unusual peroxo intermediate of the arylamine oxygenase of the chloramphenicol biosynthetic pathway.

Streptomyces venezuelae CmlI catalyzes the six-electron oxygenation of the arylamine precursor of chloramphenicol in a nonribosomal peptide synthetase (NRPS)-based pathway to yield the nitroaryl group of the antibiotic. Optical, EPR, and Mössbauer studies show that the enzyme contains a nonheme dinuclear iron cluster. Addition of O(2) to the diferrous state of the cluster results in an exceptionally long-lived intermediate (t(1/2) = 3 h at 4 °C) that is assigned as a peroxodiferric species (CmlI-peroxo) based upon the observation of an (18)O(2)-sensitive resonance Raman (rR) vibration. CmlI-peroxo is spectroscopically distinct from the well characterized and commonly observed cis-µ-1,2-peroxo (µ-η(1):η(1)) intermediates of nonheme diiron enzymes. Specifically, it exhibits a blue-shifted broad absorption band around 500 nm and a rR spectrum with a ν(O-O) that is at least 60 cm(-1) lower in energy. Mössbauer studies of the peroxo state reveal a diferric cluster having iron sites with small quadrupole splittings and distinct isomer shifts (0.54 and 0.62 mm/s). Taken together, the spectroscopic comparisons clearly indicate that CmlI-peroxo does not have a µ-η(1):η(1)-peroxo ligand; we propose that a µ-η(1):η(2)-peroxo ligand accounts for its distinct spectroscopic properties. CmlI-peroxo reacts with a range of arylamine substrates by an apparent second-order process, indicating that CmlI-peroxo is the reactive species of the catalytic cycle. Efficient production of chloramphenicol from the free arylamine precursor suggests that CmlI catalyzes the ultimate step in the biosynthetic pathway and that the precursor is not bound to the NRPS during this step.