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Among the ribosomally synthesized and post-translationally modified peptide (RiPP) natural products, “graspetides” (formerly known as microviridins) contain macrocyclic esters and amides that are formed by ATP-grasp ligase tailoring enzymes using the side chains of Asp/Glu as acceptors and Thr/Ser/Lys as donors. Graspetides exhibit diverse patterns of macrocylization and connectivities exemplified by microviridins, that have a caged tricyclic core, and thuringin and plesiocin that feature a “hairpin topology” with cross-strand ω-ester bonds. Here, we characterize chryseoviridin, a new type of multicore RiPP encoded by Chryseobacterium gregarium DS19109 (Phylum Bacteroidetes) and solve a 2.44 Å resolution crystal structure of a quaternary complex consisting of the ATP-grasp ligase CdnC bound to ADP, a conserved leader peptide and a peptide substrate. HRMS/MS analyses show that chryseoviridin contains four consecutive five- or six-residue macrocycles ending with a microviridin-like core. The crystal structure captures respective subunits of the CdnC homodimer in the apo or substrate-bound state revealing a large conformational change in the B-domain upon substrate binding. A docked model of ATP places the γ-phosphate group within 2.8 Å of the Asp acceptor residue. The orientation of the bound substrate is consistent with a model in which macrocyclization occurs in the N- to C-terminal direction for core peptides containing multiple Thr/Ser-to-Asp macrocycles. Using systematically varied sequences, we validate this model and identify two- or three-amino acid templating elements that flank the macrolactone and are required for enzyme activity in vitro. This work reveals the structural basis for ω-ester bond formation in RiPP biosynthesis.

Edited by Ruma BanerjeeClinical isolates of Yersinia, Klebsiella, and Escherichia coli frequently secrete the small molecule metallophore yersiniabactin (Ybt), which passivates and scavenges transition metals during human infections. YbtT is encoded within the Ybt biosynthetic operon and is critical for full Ybt production in bacteria. However, its biosynthetic function has been unclear because it is not essential for Ybt production by the in vitro reconstituted nonribosomal peptide synthetase/polyketide synthase (NRPS/ PKS) pathway. Here, we report the structural and biochemical characterization of YbtT. YbtT structures at 1.4 -1.9 Å resolution possess a serine hydrolase catalytic triad and an associated substrate chamber with features similar to those previously reported for low-specificity type II thioesterases (TEIIs). We found that YbtT interacts with the two major Ybt biosynthetic proteins, HMWP1 (high-molecular-weight protein 1) and HMWP2 (high-molecular-weight protein 2), and hydrolyzes a variety of aromatic and acyl groups from their phosphopantetheinylated carrier protein domains. In vivo YbtT titration in uropathogenic E. coli revealed a distinct optimum for Ybt production consistent with a tradeoff between clearing both stalled inhibitory intermediates and productive Ybt precursors from HMWP1 and HMWP2. These results are consistent with a 6 The abbreviations used are:

Short-read sequencing of GC-rich genomes such as those from actinomycetes results in a fragmented genome assembly and truncated biosynthetic gene clusters (often 10 to >100 kb long), which hinders our ability to understand the biosynthetic potential of a given strain and predict the molecules that can be produced. The current study demonstrates that contiguous DNA assemblies, suitable for analysis of BGCs, can be obtained through low-coverage, multiplexed sequencing on Flongle, which provides a new low-cost workflow ($30 to 40 per strain) for sequencing actinomycete strain libraries.

Genome-mining is an important tool for discovery of new natural products; however, the number of publicly available genomes for natural product-rich microbes such as Actinomycetes, relative to human pathogens with smaller genomes, is small. To obtain contiguous DNA assemblies and identify large (ca. 10 to greater than 100 Kb) biosynthetic gene clusters (BGCs) with high-GC (>70%) and -repeat content, it is necessary to use long-read sequencing methods when sequencing Actinomycete genomes. One of the hurdles to long-read sequencing is the higher cost. In the current study, we assessed Flongle, a recently launched platform by Oxford Nanopore Technologies, as a low-cost DNA sequencing option to obtain contiguous DNA assemblies and analyze BGCs. To make the workflow more cost-effective, we multiplexed up to four samples in a single Flongle sequencing experiment while expecting low-sequencing coverage per sample. We hypothesized that contiguous DNA assemblies might enable analysis of BGCs even at low sequencing depth. To assess the value of these assemblies, we collected high-resolution mass-spectrometry data and conducted a multi-omics analysis to connect BGCs to secondary metabolites. In total, we assembled genomes for 20 distinct strains across seven sequencing experiments. In each experiment, 50% of the bases were in reads longer than 10 Kb, which facilitated the assembly of reads into contigs with an average N50 value of 3.5 Mb. The programs antiSMASH and PRISM predicted 629 and 295 BGCs, respectively. We connected BGCs to metabolites for N,N-dimethyl cyclic-ditryptophan, a novel lassopeptide and three known Actinomycete-associated siderophores, namely mirubactin, heterobactin and salinichelin.

Pyrazines (1,4-diazirines) are an important group of natural products that have tremendous monetary value in the food and fragrance industries and can exhibit a wide range of biological effects including antineoplastic, antidiabetic and antibiotic activities. As part of a project investigating the secondary metabolites present in understudied and chemically rich Actinomycetes, we isolated a series of six pyrazines from a soil-derived Lentzea sp. GA3-008, four of which are new. Here we describe the structures of lentzeacins A-E (1, 3, 5 and 6) along with two known analogues (2 and 4) and the porphyrin zincphyrin. The structures were determined by NMR spectroscopy and HR-ESI-MS. The suite of compounds present in Lentzea sp. includes 2,5-disubstituted pyrazines (compounds 2, 4, and 6) together with the new 2,6-disubstituted isomers (compounds 1, 3 and 5), a chemical class that is uncommon. We used long-read Nanopore sequencing to assemble a draft genome sequence of Lentzea sp. which revealed the presence of 40 biosynthetic gene clusters. Analysis of classical di-modular and single module non-ribosomal peptide synthase genes, and cyclic dipeptide synthases narrows down the possibilities for the biosynthesis of the pyrazines present in this strain.

E. coli urinary tract infections (UTIs) are among the most common infectious diseases and account for a substantial proportion of antibiotic prescriptions. The continuous increase of multi‐drug resistance (MDR) among E. coli motivates efforts to identify new therapeutic approaches to UTIs. One strategy is to diminish the pathogenic potential of E. coli by pharmacologically inactivating key virulence factors. The Yersinia High Pathogenicity Island (HPI), which encodes a distinctive metallophore system implicated in passivation and trafficking of Fe (III), Cu (II) and Ni (II) ions, is one such candidate drug target in uropathogenic E. coli (UPEC). Key to this system is yersiniabactin (Ybt), a small molecule chelator which is biosynthesized from salicylic acid and other precursor molecules by a hybrid nonribosomal peptide synthetase and polyketide synthase (NRPS/PKS). With the knowledge that exogenous salicylic acid can be taken up and utilized by the model uropathogen UTI89, we hypothesized that some chemically modified forms of salicylic acid may be incorporated into the Ybt biosynthetic pathway and inhibit native Ybt biosynthesis. We created a mass spectrometric screen to monitor Ybt production by UPEC in the presence of salicylate analogs. Counter‐screens monitored bacterial growth and biosynthesis of enterobactin, a chemically distinct siderophore produced by E. coli. For inhibitory compounds, production of chemically altered (“mutasynthesized”) forms of Ybt pathway were identified using LC‐MS constant neutral loss (CNL) scans based upon detection of the putatively conserved, carboxy terminal fragment of Ybt. Among over 40 substrate analogs tested, we identified four that inhibited Ybt production without affecting bacterial growth or enterobactin biosynthesis. Each of these substrates was associated with production of varying levels of mutasynthetic Ybt. These results demonstrate that a mutasynthetic strategy may identify useful modulators of Ybt in growing bacteria. These compounds will serve as distinctive biochemical probes of the Ybt metallophore system in intact bacteria. Moreover, these suggest a mechanistically distinctive therapeutic strategy to diminish the pathogenic potential of uropathogenic E. coli and related Enterobacteriaceae.