Milk has a rich microbiota that engages in complex interactions. These interactions can be mutualistic or antagonistic, shaping the microbial composition of dairy products and affecting fermentation, preservation, and product quality. In this study, we investigate the interaction between Pseudomonas bacteria and the yeast Candida zeylanoides in the context of traditional dairy fermentation. We identified a collection of cyclic lipopeptides (CLPs) from Pseudomonas sp. SM4, isolated from the traditional Mongolian dairy product öröm and elucidated the structure of six CLPs belonging to the amphisin family using a combination of bioinformatic predictions, mass spectrometry, Marfey's analysis, nuclear magnetic resonance (NMR) spectroscopy, and X-ray crystallography. Milk-based bioassays and 16S rRNA gene amplicon sequencing analyses revealed changes in the microbial composition of milk due to the addition of the CLP tensin A and allowed us to identify its function in promoting the growth of C. zeylanoides, a yeast commonly found in dairy environments. Through transcriptomic analysis, we obtained preliminary insights into the potential metabolic pathways that may contribute to the growth-promoting effect, which indicates a role for the glyoxylate metabolism. This represents the first report of a CLP directly stimulating yeast growth. Understanding how Pseudomonas-derived lipopeptides modulate microbial communities, particularly by supporting yeasts relevant to dairy fermentations, could inform strategies for optimizing dairy fermentation processes like kefir production and improving the stability and quality of fermented dairy products.

Chemically induced proximity of biomacromolecules is a powerful strategy to regulate cellular processes using small molecule ligands that act as “molecular glues” to influence complex lifetime, localization and function. However,...

Peptidases are indispensable tools in biotechnology and chemical biology. However, the enzyme repertoire for the selective hydrolysis of d l-amide bonds in peptides is small. Here, we describe novel dl-peptidases that mediate complex microbial interactions. These enzymes, Lip3 and Lip7, convert lipopeptides into potent amoebicidal agents via selective d l-peptide bond cleavage. Using structural analyses and mutagenesis, we identified an unusual Ser-Lys-Lys-Tyr catalytic tetrad required for dl-specificity. Despite their high structural similarity, both enzymes show distinct substrate preferences: Lip3 acts primarily as a carboxypeptidase, removing a single C-terminal residue, while Lip7 excises a tripeptide. Although their substrate scopes are broad, they are highly specific with regard to their respective cutting sites. These features make these dl-peptidases powerful tools for elucidating the structure of complex peptide-based natural products, including tensin and WLIP. Overall, this work elucidates the molecular mechanisms of cooperative microbial defense and provides a new enzymatic toolbox for biocatalysis and natural product discovery.

Amoebal predation exerts a strong evolutionary selection pressure on bacteria, thus driving the development of effective predator-defense strategies. However, little is known about the molecular interplay between bacteria and predators, particularly how bacteria can sense and kill their microbial predators. We show how the ubiquitous bacterium Pseudomonas syringae detects and kills the social amoeba Polysphondylium pallidum. Combining comparative genomics, molecular biology, and chemical analyses, we identified a chemical radar system. The system relies on P. syringae secreting the lipopeptide syringafactin, which is deacylated by the amoeba. The resulting peptides are sensed via the bacterial sensor protein chemical radar regulator (CraR) that activates genes for converting the predator-derived signal into the amoebicide pyrofactin. This system is widespread in P. syringae and enables bacteria to infect A. thaliana in the presence of amoebae. Our study advances the understanding of microbial sensing and opens new avenues for the discovery of natural products.

Bacteria are social organisms, which are constantly exposed to predation by nematodes or amoebae. To counteract these predation pressures, bacteria have evolved a variety of potent antipredator strategies. Bacteria of the genus Pseudomonas, for instance, evade amoebal predation by the secretion of amoebicidal natural products. The soil bacterium Pseudomonas fluorescens HKI0770 produces pyreudione alkaloids that can kill amoebae. Even though the mode of action of the pyreudiones has been elucidated, the spatiotemporal dynamics underlying this predator-prey interaction remain unknown. Using a combination of microscopy and analytical techniques, we elucidated the intricate relationship of this predator-prey association. We used the chromatic bacteria toolbox for intraspecific differentiation of the amoebicide-producing wildtype and the non-producing mutant within microcosms. These allow for variations in nutrient availability and the emergence of predation-evasion strategies of interacting microorganisms. Imaging of the co-cultures revealed that the amoebae initially ingest both the non-producer as well as the toxin-producer cells. The outcomes of predator-prey interactions are governed by the population size and fitness of the interacting partners. We identified that changes in the cell density coupled with alterations in nutrient availability led to a strong Allee effect resulting in the diminished production of pyreudione A. The loss of defense capabilities renders P. fluorescens HKI0770 palatable to amoebae. Such a multifaceted regulation provides the basis for a model by which predator-prey populations are being regulated in specific niches. Our results demonstrate how the spatiotemporal regulation of bacterial toxin production alters the feeding behavior of amoeba.

Covering: 2000 up to the first half of 2024Milk and its derived dairy products have long been integral to the human diet, with evidence of consumption dating back over 9000 years. Milk's high nutritional value renders dairy products an important element of human diet while also offering a fertile environment for microbial growth. Beneficial microorganisms in dairy products are often associated with biogenic and probiotic effects, whereas spoilage or pathogenic microorganisms can pose health risks. Fermentation is a key method to preserve milk. Whereas dairying practices in most parts of the world have been highly altered by industrialization over the past century, nomadic pastoralists in Mongolia notably retain a rich tradition of household-level dairy fermentation that has been practiced since 3000 BC. Milk-associated microorganisms produce a vast number of low molecular weight natural products that can mediate beneficial and detrimental interactions. Bacteria of the genus Pseudomonas are found in traditional Mongolian dairy products and are common contaminants in commercial dairy products, and they can strongly impact the quality and shelf-life of dairy products. These bacteria are well known for their ability to produce a variety of secondary metabolites, including nonribosomal (lipo)peptides, which are both structurally and functionally diverse. Lipopeptides can have antimicrobial properties, act as quorum sensing molecules, and contribute to biofilm formation due to their amphiphilic nature. Although often associated with spoilage, some of these natural products can also exhibit positive effects with potential beneficial applications in the dairy industry. This review aims to provide a comprehensive overview of the interplay between culinary fermentation and the production and activities of microbial-derived natural products.

Bacterially produced antimicrobial peptides (AMPs), or bacteriocins, play key roles in shaping microbial communities via interspecies competition. Unlike the more temporally dynamic gut microbiome, the oral microbiome exhibits long-term stability and is preserved into deep time in dental calculus, enabling evolutionary analysis across time. Here, we combine metagenomics, structural modeling, and experimental validation to investigate AMP diversity in ancient and modern dental biofilms from humans, Neanderthals, and nonhuman primates spanning 100,000 years. Using our newly developed platform, AMPcombi, we uncover evolutionary trajectories of bacteriocins and elucidate their ecological functions. Among these, we identify a conserved family of Actinomyces-derived defensin-like peptides, termed actifensins, present across all time periods. Phylogenetic, structural, and functional analyses revealed shared ancestry and adaptive diversification between ancient (paleo-) and modern actifensins, with evidence of positive selection and maintained antimicrobial activity. Our findings position the oral microbiome as a valuable reservoir for natural product discovery. In the face of rising antimicrobial resistance, evolutionary insights into AMP function open a door to next-generation therapeutics. AMPcombi streamlines this process, linking ancient biomolecules with biotechnology.

Chemically induced dimerization of proteins is a powerful approach to regulate biomolecular functions through small molecule ligands acting as "molecular glues". Here, we demonstrate that simple, thienopyrimidinone scaffold-based inhibitors efficiently promote homodimerization of an essential oxidoreductase from the human pathogenic parasite Trypanosoma brucei through selective covalent attachment and self-assembly. A fluorine walk strategy, commonly used to optimize small molecule properties, resulted in tuning induced dimer affinity across two orders of magnitude. NMR spectroscopy, MD simulations, chromatography, multi-angle light scattering, mass spectrometry, calorimetry, and functional assays reveal how the inhibitor fluorination pattern alters the dynamics and interactions of the enzyme-bound inhibitor and surface-exposed aromatic protein side chains, affecting both enzyme inhibition kinetics and induced dimerization. This work highlights how site-specific fluorination can modulate protein interactions and offers a framework for the design of novel molecular glues with broad applications in chemical biology and drug development.

Microbial polyketides represent a structurally diverse class of secondary metabolites with medicinally relevant properties. Aromatic polyketides are produced by type II polyketide synthase (PKS) systems, each minimally composed of a ketosynthase-chain length factor (KS-CLF) and a phosphopantetheinylated acyl carrier protein (holo-ACP). Although type II PKSs are found throughout the bacterial kingdom, and despite their importance to strategic bioengineering, type II PKSs have not been well-studied in vitro. In cases where the KS-CLF can be accessed via E. coli heterologous expression, often the cognate ACPs are not activatable by the broad specificity Bacillus subtilis surfactin-producing phosphopantetheinyl transferase (PPTase) Sfp and, conversely, in systems where the ACP can be activated by Sfp, the corresponding KS-CLF is typically not readily obtained. Here, we report the high-yield heterologous expression of both cyanobacterial Gloeocapsa sp. PCC 7428 minimal type II PKS (gloPKS) components in E. coli, which allowed us to study this minimal type II PKS in vitro. Initially, neither the cognate PPTase nor Sfp converted gloACP to its active holo state. However, by examining sequence differences between Sfp-compatible and -incompatible ACPs, we identified two conserved residues in gloACP that, when mutated, enabled high-yield phosphopantetheinylation of gloACP by Sfp. Using analogous mutations, other previously Sfp-incompatible type II PKS ACPs from different bacterial phyla were also rendered activatable by Sfp. This demonstrates the generalizability of our approach and breaks down a longstanding barrier to type II PKS studies and the exploration of complex biosynthetic pathways.

Pseudomonas syringae is a soil-dwelling bacterium that exhibits remarkable niche adaptability, and it is known for its devastating impact as a plant pathogen. This bacterium has an outstanding capability to produce a wide array of biologically active natural products. P. syringae coexists with amoebal predators and fungal strains, which drives the production of secondary metabolites for predator evasion in addition to niche adaptation. In this study, we conducted a broad pangenomic analysis of 18 taxonomically distinct P. syringae strains, leading to the identification of 231 biosynthetic gene clusters (BGCs). Among these, nonribosomal peptide synthetases (NRPSs) were particularly abundant, indicating their potential significance within this ecological context. We discovered and elucidated the structures of two novel classes of bioactive compounds, the syrilipamides and chlorosecimides. Furthermore, a bioinformatic analysis enabled the identification of an undescribed halogenase, SecA, essential for the chlorination of secimide A. We observed that syrilipamides and secimides and in particular mixtures thereof, exhibit amoebicidal activities. Additionally, secimides showed selective antifungal activity. These findings provide valuable insights into the ecological roles of P. syringae natural products and highlight their potential for biotechnological and therapeutic applications.

The total synthesis of Pseudomonas-derived cyclic lipodepsipeptide anikasin was achieved. Using a depsipeptide building block and balanced protecting groups on the branching d-allo-Thr residue, the synthesis was established semiautomatically on a synthesizer. Buffered deprotections minimized side reactions and afforded synthetic anikasin and its enantiomer. Biological activity studies indicated that anikasin's mode of action is directly resulting from its physicochemical properties.

Polyketides represent a structurally diverse class of natural products with a wide range of biological functions, including antimicrobial activity, defense responses, developmental regulation, pigmentation, and intercellular and intracellular communication signals. The social amoeba Dictyostelium discoideum harbors 40 polyketide synthase (PKS) genes, yet the specific and collective functions remain poorly understood. PKSs require activation by the phosphopantetheinyl transferase DiSfp, which converts inactive apoenzymes into functional holo forms. Disruption of the DiSfp gene abolished the production of PKS-derived metabolites across all developmental stages. Integrated phenotypic, transcriptomic, and metabolomic analyses revealed impaired growth in liquid culture, defects in macropinocytosis, aberrant chemotaxis, and diminished spore formation, associated with altered expression of genes regulating these processes. Comparative metabolomic profiling of the mutant identified candidate polyketide metabolites across different developmental stages, providing a valuable resource for targeted identification and isolation of previously undescribed compounds. This study establishes a functional link between the PKS machinery and the metabolic and developmental networks of D. discoideum , highlighting the essential roles of polyketides in cellular physiology and offering a framework for future polyketide discovery.

A recent study by Granton et al.in Cell investigates the role of exopolysaccharide(EPS)in Pseudomonas aeruginosa's biofilm formation,immune evasion,and lung-brain signaling during pneumonia.1 This research significantly advances our under-standing of how P.aeruginosa's biofilms influence disease symptoms and suggests potential therapeutic strategies targeting the lung-brain axis to improve clinical outcomes in pneumonia patients.

Microbial natural products-low molecular weight compounds biosynthesized by microorganisms-form the foundation of important modern therapeutics, including antibiotics, immunomodulators, and anti-cancer agents. This perspective discusses and contrasts two emerging approaches for uncovering natural products of the past. On the one hand, ancestral sequence reconstruction allows recreating biosynthetic pathways that date back hundreds of millions of years. On the other hand, sequencing and de novo assembly of ancient DNA reveals the biosynthetic potential of ancient microbial communities up to 100 000 years. Together, these approaches unveil an otherwise hidden reservoir of functional and structural molecular diversity. They also offer new opportunities to study the biological function and evolution of these molecules within an archaeological context.

Photosynthetic protists, named microalgae, are key players in global primary production. The green microalga Chlamydomonas reinhardtii is a well-studied model organism. In nature, it dwells in acetate-rich paddy rice soil, which is not mimicked by standard liquid laboratory conditions. Here, we maintained the algae in a liquid environment with spatially structured 3-D components (S3-D) and acetate recreating natural conditions. We perform transcriptome sequencing, immunoblotting, fluorescence and electron microscopy, and Raman microspectroscopy to characterize the algae in S3-D vs homogeneous conditions. The algae undergo a metamorphosis-like process when transitioned from homogeneous aquatic to a lifestyle simulating acetate-rich rice soil. These conditions result in reduced cell size and cilia length, an enlarged eyespot and many cells with double-layered cell walls. RNA-Seq reveals alterations in c. 2400 transcripts. Four key photoreceptors including CRY-DASH1 and phototropin governing plastid metabolism along with its eyespot are altered in their protein expression. Consequently, photosynthetic pigments, lipids and starch levels vary as do starch distribution patterns. Fitness against antagonistic bacteria is enhanced concurrently with the downregulation of an involved Ca2+ channel transcript. This study highlights the profound impact of S3-D initiating processes inaccessible under homogeneous laboratory conditions. Thus, overexpression lines for certain photoreceptors and starch are naturally created.

Low molecular weight compounds biosynthesized by microorganisms, commonly known as natural products, are indispensable in modern medicine. They serve as the basis for many antibiotics, immunomodulators, and anti cancer agents. The new research field of paleobiotechnology allows access to natural products of the past. Based on ancient DNA from dental calculus, it was possible to generate bacterial natural products dating back over 100,000 years.

Correction for 'Tyrosine bioconjugation with hypervalent iodine' by Nina Declas et al., Chem. Sci., 2022, 13, 12808-12817, https://doi.org/10.1039/D2SC04558C.

Investigating the ecological context of microbial predator-prey interactions enables the identification of micro-organisms, which produce multiple secondary metabolites to evade predation or to kill the predator. In addition, genome mining combined with molecular biology methods can be used to identify further biosynthetic gene clusters that yield new antimicrobials to fight the antimicrobial crisis. In contrast, classical screening-based approaches have limitations since they do not aim to unlock the entire biosynthetic potential of a given organism. Here, we describe the genomics-based identification of keanumycins A-C. These nonribosomal peptides enable bacteria of the genus Pseudomonas to evade amoebal predation. While being amoebicidal at a nanomolar level, these compounds also exhibit a strong antimycotic activity in particular against the devastating plant pathogen Botrytis cinerea and they drastically inhibit the infection of Hydrangea macrophylla leaves using only supernatants of Pseudomonas cultures. The structures of the keanumycins were fully elucidated through a combination of nuclear magnetic resonance, tandem mass spectrometry, and degradation experiments revealing an unprecedented terminal imine motif in keanumycin C extending the family of nonribosomal amino acids by a highly reactive building block. In addition, chemical synthesis unveiled the absolute configuration of the unusual dihydroxylated fatty acid of keanumycin A, which has not yet been reported for this lipodepsipeptide class. Finally, a detailed genome-wide microarray analysis of Candida albicans exposed to keanumycin A shed light on the mode-of-action of this potential natural product lead, which will aid the development of new pharmaceutical and agrochemical antifungals.

The rhizosphere is a highly competitive environment forcing bacteria to evolve strategies to oppose their enemies. The production of toxic secondary metabolites allows bacteria to counteract predators. In this study, we describe the anti-predator armamentarium of the soil-derived bacterium Pseudomonas nunensis 4A2e. Based on a genome mining approach, we identified several biosynthetic gene clusters coding for nonribosomal peptide synthetases. Generation of gene deletion mutants of the respective clusters shows a loss of defense capabilities. We isolated the novel lipopeptides keanumycin D and nunapeptins B and C, and fully elucidated their structures by a combination of in-depth mass spectrometry experiments, stable isotope labelling, and chemical synthesis. Additionally, investigation of the quorum sensing-dependent biosynthesis allowed us to elucidate parts of the underlying regulation of the biosynthetic machinery. Ecology-inspired bioassays highlight the role of these peptides as a defence strategy against protozoans and led us to find a previously unknown function against the bacterivorous nematode Oscheius myriophilus.

Interactions between microorganisms are often mediated by specialized metabo-lites. Although the structures and biosynthesis of these compounds may have been elucidated, microbes exist within complex microbiomes and chemical signals can thus also be subject to community-dependent modifications. Increasingly powerful chemical and biological tools allow to shed light on this poorly understood aspect of chemical ecology. We provide an overview of loss-of-function and gain-of-function chemical mediator (CM) modifications within mi-crobial multipartner relationships. Although loss-of-function modifications are abundant in the literature, few gain-of-function modifications have been described despite their important role in microbial interactions. Research in this field holds great potential for our understanding of microbial interactions and may also provide novel tools for targeted interference with microbial signaling.