The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity.

Type III CRISPR systems provide immunity against genetic invaders through the production of cyclic oligo-adenylate (cA) molecules that activate effector proteins that contain CRISPR-associated Rossman fold (CARF) domains. Here, we characterized the function and structure of an effector in which the CARF domain is fused to an adenosine deaminase domain, CRISPR-associated adenosine deaminase 1 (Cad1). We show that upon binding of cA or cA to its CARF domain, Cad1 converts ATP to ITP, both in vivo and in vitro.

Oxydifficidin, a potent antibiotic due to DedA assisted uptake and ribosomal protein RplL sensitivity.

Gonorrhea, which is caused by , is the second most reported sexually transmitted infection worldwide. The increasing appearance of isolates that are resistant to approved therapeutics raises the concern that gonorrhea may become untreatable. Here, we serendipitously identified oxydifficidin as a potent antibiotic through the observation of a contaminant in a lawn of .

Cinnamosyn, a Cinnamoylated Synthetic-Bioinformatic Natural Product with Cytotoxic Activity.

Most biosynthetic gene clusters (BGCs) are functionally inaccessible by using fermentation methods. Bioinformatic-coupled total synthesis provides an alternative approach for accessing BGC-encoded bioactivities. To date, synthetic bioinformatic natural product (synBNP) methods have focused on lipopeptides containing simple lipids.

DNA glycosylases provide antiviral defence in prokaryotes.

Bacteria have adapted to phage predation by evolving a vast assortment of defence systems. Although anti-phage immunity genes can be identified using bioinformatic tools, the discovery of novel systems is restricted to the available prokaryotic sequence data. Here, to overcome this limitation, we infected Escherichia coli carrying a soil metagenomic DNA library with the lytic coliphage T4 to isolate clones carrying protective genes.

Identification of an Optimized Clinical Development Candidate from Cilagicin, an Antibiotic That Evades Resistance by Dual Polyprenyl Phosphate Binding.

Cilagicin is a dual polyprenyl phosphate binding lipodepsipeptide antibiotic with strong activity against clinically relevant Gram-positive pathogens while evading antibiotic resistance. Cilagicin showed high serum binding that reduced its in vivo efficacy. Cilagicin-BP, which contains a biphenyl moiety in place of the N-terminal myristic acid found on cilagicin, showed reduced serum binding and increased in vivo efficacy but decreased potency against some pathogens.

Tapcin, an In Vivo Active Dual Topoisomerase I/II Inhibitor Discovered by Synthetic Bioinformatic Natural Product (Syn-BNP)-Coupled Metagenomics.

DNA topoisomerases are attractive targets for anticancer agents. Dual topoisomerase I/II inhibitors are particularly appealing due to their reduced rates of resistance. A number of therapeutically relevant topoisomerase inhibitors are bacterial natural products.

Bacterial cGAS senses a viral RNA to initiate immunity.

Cyclic oligonucleotide-based antiphage signalling systems (CBASS) protect prokaryotes from viral (phage) attack through the production of cyclic oligonucleotides, which activate effector proteins that trigger the death of the infected host. How bacterial cyclases recognize phage infection is not known. Here we show that staphylococcal phages produce a structured RNA transcribed from the terminase subunit genes, termed CBASS-activating bacteriophage RNA (cabRNA), which binds to a positively charged surface of the CdnE03 cyclase and promotes the synthesis of the cyclic dinucleotide cGAMP to activate the CBASS immune response.

A Family of Antibiotics That Evades Resistance by Binding Polyprenyl Phosphates.

Cilagicin is a Gram-positive active antibiotic that has a dual polyprenyl phosphate binding mechanism that impedes resistance development. Here we bioinformatically screened predicted non-ribosomal polypeptide synthetase encoded structures to search for antibiotics that might similarly avoid resistance development. Synthesis and bioactivity screening of the predicted structures that we identified led to three antibiotics that are active against multidrug-resistant Gram-positive pathogens, two of which, paenilagicin and virgilagicin, did not lead to resistance even after prolonged antibiotic exposure.

Present and future outlooks on environmental DNA-based methods for antibiotic discovery.

Novel antibiotics are in constant demand to combat a global increase in antibiotic-resistant infections. Bacterial natural products have been a long-standing source of antibiotic compounds, and metagenomic mining of environmental DNA (eDNA) has increasingly provided new antibiotic leads. The metagenomic small-molecule discovery pipeline can be divided into three main steps: surveying eDNA, retrieving a sequence of interest, and accessing the encoded natural product.

High-throughput retrieval of target sequences from complex clone libraries using CRISPRi.

The capture of metagenomic DNA in large clone libraries provides the opportunity to study microbial diversity that is inaccessible using culture-dependent methods. In this study, we harnessed nuclease-deficient Cas9 to establish a CRISPR counter-selection interruption circuit (CCIC) that can be used to retrieve target clones from complex libraries. Combining modern sequencing methods with CCIC cloning allows for rapid physical access to the genetic diversity present in natural ecosystems.

Multiplexed mobilization and expression of biosynthetic gene clusters.

Bacterial genomes contain large reservoirs of biosynthetic gene clusters (BGCs) that are predicted to encode unexplored natural products. Heterologous expression of previously unstudied BGCs should facilitate the discovery of additional therapeutically relevant bioactive molecules from bacterial culture collections, but the large-scale manipulation of BGCs remains cumbersome. Here, we describe a method to parallelize the identification, mobilization and heterologous expression of BGCs.

Discovery of Paenibacillaceae Family Gram-Negative-Active Cationic Lipopeptide Antibiotics Using Evolution-Guided Chemical Synthesis.

Cationic nonribosomal lipopeptides (CNRLPs) from spp. have been a rewarding source of Gram-negative-active antibiotics. Here we systematically screened sequenced bacterial genomes for CNRLP biosynthetic gene clusters (BGCs) that we predicted might encode additional Gram-negative-active antibiotics.

Unraveling function and diversity of bacterial lectins in the human microbiome.

The mechanisms by which commensal organisms affect human physiology remain poorly understood. Lectins are non-enzymatic carbohydrate binding proteins that all organisms employ as part of establishing a niche, evading host-defenses and protecting against pathogens. Although lectins have been extensively studied in plants, bacterial pathogens and human immune cells for their role in disease pathophysiology and as therapeutics, the role of bacterial lectins in the human microbiome is largely unexplored.

Bioinformatic prospecting and synthesis of a bifunctional lipopeptide antibiotic that evades resistance.

Emerging resistance to currently used antibiotics is a global public health crisis. Because most of the biosynthetic capacity within the bacterial kingdom has remained silent in previous antibiotic discovery efforts, uncharacterized biosynthetic gene clusters found in bacterial genome-sequencing studies remain an appealing source of antibiotics with distinctive modes of action. Here, we report the discovery of a naturally inspired lipopeptide antibiotic called cilagicin, which we chemically synthesized on the basis of a detailed bioinformatic analysis of the biosynthetic gene cluster.

Lapcin, a potent dual topoisomerase I/II inhibitor discovered by soil metagenome guided total chemical synthesis.

In natural product discovery programs, the power of synthetic chemistry is often leveraged for the total synthesis and diversification of characterized metabolites. The synthesis of structures that are bioinformatically predicted to arise from uncharacterized biosynthetic gene clusters (BGCs) provides a means for synthetic chemistry to enter this process at an early stage. The recent identification of non-ribosomal peptides (NRPs) containing multiple ρ-aminobenzoic acids (PABAs) led us to search soil metagenomes for BGCs that polymerize PABA.

A naturally inspired antibiotic to target multidrug-resistant pathogens.

Gram-negative bacteria are responsible for an increasing number of deaths caused by antibiotic-resistant infections. The bacterial natural product colistin is considered the last line of defence against a number of Gram-negative pathogens. The recent global spread of the plasmid-borne mobilized colistin-resistance gene mcr-1 (phosphoethanolamine transferase) threatens the usefulness of colistin.

Identification of structurally diverse menaquinone-binding antibiotics with in vivo activity against multidrug-resistant pathogens.

The emergence of multidrug-resistant bacteria poses a threat to global health and necessitates the development of additional in vivo active antibiotics with diverse modes of action. Directly targeting menaquinone (MK), which plays an important role in bacterial electron transport, is an appealing, yet underexplored, mode of action due to a dearth of MK-binding molecules. Here we combine sequence-based metagenomic mining with a motif search of bioinformatically predicted natural product structures to identify six biosynthetic gene clusters that we predicted encode MK-binding antibiotics (MBAs).

Synthesis and evaluation of dual-action kanglemycin-fluoroquinolone hybrid antibiotics.

Bacterial resistance threatens the utility of currently available antibiotics. Rifampicin, a cornerstone in the treatment of persistent Gram-positive infections, is prone to the development of resistance resulting from single point mutations in the antibiotic's target, RNA polymerase. One strategy to circumvent resistance is the use of 'hybrid' antibiotics consisting of two covalently linked antibiotic entities.

Metabolites with SARS-CoV-2 Inhibitory Activity Identified from Human Microbiome Commensals.

The COVID-19 pandemic has highlighted the need to identify additional antiviral small molecules to complement existing therapies. Although increasing evidence suggests that metabolites produced by the human microbiome have diverse biological activities, their antiviral properties remain poorly explored. Using a cell-based SARS-CoV-2 infection assay, we screened culture broth extracts from a collection of phylogenetically diverse human-associated bacteria for the production of small molecules with antiviral activity.

Multiplexed functional metagenomic analysis of the infant microbiome identifies effectors of NF-κB, autophagy, and cellular redox state.

The human microbiota plays a critical role in host health. Proper development of the infant microbiome is particularly important. Its dysbiosis leads to both short-term health issues and long-term disorders lasting into adulthood.

Metagenome-Guided Analogue Synthesis Yields Improved Gram-Negative-Active Albicidin- and Cystobactamid-Type Antibiotics.

Natural products are a major source of new antibiotics. Here we utilize biosynthetic instructions contained within metagenome-derived congener biosynthetic gene clusters (BGCs) to guide the synthesis of improved antibiotic analogues. Albicidin and cystobactamid are the first members of a new class of broad-spectrum ρ-aminobenzoic acid (PABA)-based antibiotics.

Biosynthetic Interrogation of Soil Metagenomes Reveals Metamarin, an Uncommon Cyclomarin Congener with Activity against .

Tuberculosis (TB) remains one of the deadliest infectious diseases. Unfortunately, the development of antibiotic resistance threatens our current therapeutic arsenal, which has necessitated the discovery and development of novel antibiotics against drug-resistant (). Cyclomarin A and rufomycin I are structurally related cyclic heptapeptides assembled by nonribosomal peptide synthetases (NRPSs), which show potent anti- activity with a new cellular target, the caseinolytic protein ClpC1.

Refactoring biosynthetic gene clusters for heterologous production of microbial natural products.

Microbial natural products (NPs) are of paramount importance in human medicine, animal health and plant crop protection. Large-scale microbial genome and metagenomic mining has revealed tremendous biosynthetic potential to produce new NPs. However a majority of NP biosynthetic gene clusters (BGCs) are functionally inaccessible under standard laboratory conditions.