Ehud Herbst

To defend against bacteriophage (phage) infection, bacteria have developed various defense systems, dozens of which were discovered and mechanistically studied recently. To date, almost all defense systems whose mechanisms were deciphered were shown to operate within the bacterial cell. Here we describe a secreted protease from the Actinobacterium Salinispora mooreana which, when expressed heterologously in Streptomyces coelicolor , reduces titers of two taxonomically related Siphoviridae phages. Antiphage effects were maintained when concentrated supernatant from S. coelicolor expressing the Salinispora protease was added externally to phage-containing medium, in either the presence or absence of bacterial cells, supporting an extracellular mechanism. We further show that phages can escape the antiphage effect of the Salinispora protease by mutating a tail-associated protein. The antiphage effect is associated with an increased proportion of phage particles devoid of DNA. Our data suggest antiphage activity of a secreted bacterial protease.

Despite decades of study, large parts of the mammalian metabolome remain unexplored¹. Mass spectrometry-based metabolomics routinely detects thousands of small molecule-associated peaks in human tissues and biofluids, but typically only a small fraction of these can be identified, and structure elucidation of novel metabolites remains challenging^2,3,4. Biochemical language models have transformed the interpretation of DNA, RNA and protein sequences, but have not yet had a comparable impact on understanding small molecule metabolism. Here we present an approach that leverages chemical language models^5,6,7 to anticipate the existence of previously uncharacterized metabolites. We introduce DeepMet, a chemical language model that learns from the structures of known metabolites to anticipate the existence of previously unrecognized metabolites. Integration of DeepMet with mass spectrometry-based metabolomics data facilitates metabolite discovery. We harness DeepMet to reveal several dozen structurally diverse mammalian metabolites. Our work demonstrates the potential for language models to advance the mapping of the mammalian metabolome.

To defend against bacteriophage (phage) infection, bacteria have developed various defense systems, dozens of which were discovered and mechanistically studied recently. To date, almost all defense systems whose mechanisms were deciphered were shown to operate within the bacterial cell. Here we describe a secreted protease from the Actinobacterium Salinispora mooreana which, when expressed heterologously in Streptomyces coelicolor , reduces titers of two taxonomically related Siphoviridae phages. Antiphage effects were maintained when concentrated supernatant from S. coelicolor expressing the Salinispora protease was added externally to phage-containing medium, even in the absence of bacterial cells, supporting an extracellular mechanism. We further show that phages can escape the antiphage effect of the Salinispora protease by mutating a tail-associated protein. The antiphage effect is associated with an increased proportion of phage particles devoid of DNA. Our data suggest antiphage activity of a secreted bacterial protease.

Toll/interleukin-1 receptor (TIR) domains are central components of pattern recognition immune proteins across all domains of life^1,2. In bacteria and plants, TIR-domain proteins recognize pathogen invasion and then produce immune signalling molecules exclusively comprising nucleotide moieties^2,3,4,5. Here we show that the TIR-domain protein of the type II Thoeris defence system in bacteria produces a unique signalling molecule comprising the amino acid histidine conjugated to ADP-ribose (His-ADPR). His-ADPR is generated in response to phage infection and activates the cognate Thoeris effector by binding a Macro domain located at the C terminus of the effector protein. By determining the crystal structure of a ligand-bound Macro domain, we describe the structural basis for His-ADPR and its recognition and show its role by biochemical and mutational analyses. Our analyses furthermore reveal a family of phage proteins that bind and sequester His-ADPR signalling molecules, enabling phages to evade TIR-mediated immunity. These data demonstrate diversity in bacterial TIR signalling and reveal a new class of TIR-derived immune signalling molecules that combine nucleotide and amino acid moieties.

Developing treatments for antibiotic resistant bacterial infections is among the highest priority public health challenges worldwide. Tetracyclines, one of the most important classes of antibiotics, have fallen prey to antibiotic resistance, necessitating the generation of new analogs. Many tetracycline analogs have been accessed through both total synthesis and semisynthesis, but key C-ring tetracycline analogs remain inaccessible. New methods are needed to unlock access to these analogs, and heterologous biosynthesis in a tractable host such as Saccharomyces cerevisiae is a candidate method. C-ring analog biosynthesis can mimic nature’s biosynthesis of tetracyclines from anhydrotetracyclines, but challenges exist, including the absence of the unique cofactor F₄₂₀ in common heterologous hosts. Toward this goal, this paper describes the biosynthesis of tetracycline from anhydrotetracycline in S. cerevisiae heterologously expressing three enzymes from three bacterial hosts: the anhydrotetracycline hydroxylase OxyS, the dehydrotetracycline reductase CtcM, and the F₄₂₀ reductase FNO. This biosynthesis of tetracycline is enabled by OxyS performing just one hydroxylation step in S. cerevisiae despite its previous characterization as a double hydroxylase. This single hydroxylation enabled us to purify and structurally characterize a hypothetical intermediate in oxytetracycline biosynthesis that can explain structural differences between oxytetracycline and chlortetracycline. We show that F_o, a synthetically accessible derivative of cofactor F₄₂₀, can replace F₄₂₀ in tetracycline biosynthesis. Critically, the use of S. cerevisiae for the final steps of tetracycline biosynthesis described herein sets the stage to achieve a total biosynthesis of tetracycline as well as novel tetracycline analogs in S. cerevisiae with the potential to combat antibiotic-resistant bacteria.

The Toll/interleukin-1 receptor (TIR) domain is a canonical component of animal and plant immune systems. In plants, intracellular pathogen sensing by immune receptors triggers their TIR domains to generate a molecule which is a variant of cyclic ADP-ribose (v-cADPR). This molecule is hypothesized to activate plant cell death via a yet unresolved pathway. TIR domains were recently also shown to be involved in a bacterial anti-phage defense system called Thoeris, but the mechanism of Thoeris defense remained unknown. In this study we report that phage infection triggers Thoeris TIR-domain proteins to produce an isomer of cyclic ADP-ribose. This molecular signal activates a second protein, ThsA, which then depletes the cell of the essential molecule nicotinamide adenine dinucleotide (NAD) and leads to abortive infection and cell death. We further show that similar to eukaryotic innate immune systems, bacterial TIR-domain proteins determine the immunological specificity to the invading pathogen. Our results describe a new antiviral signaling pathway in bacteria, and suggest that generation of intracellular signaling molecules is an ancient immunological function of TIR domains conserved in both plant and bacterial immunity.