Jason M. Crawford

Detection of preinvasive cancer cells

More than 85% of all cancers originate in the epithelium that lines the internal surfaces of organs throughout the body. Although these are readily treatable provided they are diagnosed in one of the preinvasive stages, early lesions are often almost impossible to detect. Here we present a new optical-probe technique based on light-scattering spectroscopy that is able to detect precancerous and early cancerous changes in cell-rich epithelia.

Siderophores from Neighboring Organisms Promote the Growth of Uncultured Bacteria

The majority of bacterial species do not grow on synthetic media. Many non-growers require growth factors from other bacteria, but the nature of these compounds is largely unknown. We show here that previously uncultured isolates from marine sediment biofilm grow on a Petri dish in the presence of cultured organisms from the same environment. The growth factors produced by one cultured helper strain were identified as new acyl-desferrioxamine siderophores. A panel of previously uncultured isolates exhibited a range of siderophore promiscuity for growth promotion. This siderophore-based approach has enabled the culturing of organisms only distantly related to previously cultured microbes. The lack of growth in the laboratory for many strains from this habitat stems from an inability to autonomously produce siderophores, and the resulting chemical dependence on other microorganisms regulates community establishment in the environment.

Deconstruction of Iterative Multidomain Polyketide Synthase Function

PksA, which initiates biosynthesis of the environmental carcinogen aflatoxin B1, is one of the multidomain iterative polyketide synthases (IPKSs), a large, poorly understood family of biosynthetic enzymes. We found that dissection of PksA and its reconstitution from selected sets of domains allows the accumulation and characterization of advanced octaketide intermediates bound to the enzyme, permitting the reactions controlled by individual catalytic domains to be identified. A product template (PT) domain unites with the ketosynthase and thioesterase in this IPKS system to assemble precisely seven malonyl-derived building blocks to a hexanoyl starter unit and mediate a specific cyclization cascade. Because the PT domain is common among nonreducing IPKSs, these mechanistic features should prove to be general for IPKS-catalyzed production of aromatic polyketides.

Identification of a starter unit acyl-carrier protein transacylase domain in an iterative type I polyketide synthase

Polyketides are a class of natural products that exhibit a wide range of functional and structural diversity. They include antibiotics, immunosuppressants, antifungals, antihypercholesterolemics, and cytotoxins. Polyketide synthases (PKSs) use chemistry similar to fatty acid synthases (FASs), although building block variation and differing extents of reduction of the growing polyketide chain underlie their biosynthetic versatility. In contrast to the well studied sequential modular type I PKSs, less is known about how the iterative type I PKSs carry out and control chain initiation, elongation, folding, and cyclization during polyketide processing. Domain structure analysis of a group of related fungal, nonreducing PKSs has revealed well defined N-terminal domains longer than commonly seen for FASs and modular PKSs. Predicted structure of this domain disclosed a region similar to malonyl-CoA:acyl-carrier protein (ACP) transacylases (MATs). MATs play a key role transferring precursor CoA thioesters from solution onto FASs and PKSs for chain elongation. On the basis of site-directed mutagenesis, radiolabeling, and kinetics experiments carried out with individual domains of the norsolorinic acid PKS, we propose that the N-terminal domain is a starter unit:ACP transacylase (SAT domain) that selects a C6 fatty acid from a dedicated yeast-like FAS and transfers this unit onto the PKS ACP, leading to the production of the aflatoxin precursor, norsolorinic acid. These findings could indicate a much broader role for SAT domains in starter unit selection among nonreducing iterative, fungal PKSs, and they provide a biochemical rationale for the classical acetyl “starter unit effect.”

Structural basis for biosynthetic programming of fungal aromatic polyketide cyclization

Polyketides are a class of natural products with diverse structures and biological activities. The structural variability of aromatic products of fungal nonreducing, multidomain iterative polyketide synthases (NR-PKS group of IPKSs) results from regiospecific cyclizations of reactive poly-β-keto intermediates. How poly-β-keto species are synthesized and stabilized, how their chain lengths are determined, and, in particular, how specific cyclization patterns are controlled have been largely inaccessible and functionally unknown until recently. A product template (PT) domain is responsible for controlling specific aldol cyclization and aromatization of these mature polyketide precursors, but the mechanistic basis is unknown. Here we present the 1.8 Å crystal structure and mutational studies of a dissected PT monodomain from PksA, the NR-PKS that initiates the biosynthesis of the potent hepatocarcinogen aflatoxin B1 in Aspergillus parasiticus. Despite having minimal sequence similarity to known enzymes, the structure displays a distinct ‘double hot dog’ (DHD) fold. Co-crystal structures with palmitate or a bicyclic substrate mimic illustrate that PT can bind both linear and bicyclic polyketides. Docking and mutagenesis studies reveal residues important for substrate binding and catalysis, and identify a phosphopantetheine localization channel and a deep two-part interior binding pocket and reaction chamber. Sequence similarity and extensive conservation of active site residues in PT domains suggest that the mechanistic insights gleaned from these studies will prove general for this class of IPKSs, and lay a foundation for defining the molecular rules controlling NR-PKS cyclization specificity.

New insights into the formation of fungal aromatic polyketides.

Fungal aromatic polyketides constitute a large family of bioactive natural products and are synthesized by the non-reducing group of iterative polyketide synthases (PKSs). Their diverse structures arise from selective enzymatic modifications of reactive, enzyme-bound poly-β-keto intermediates. How iterative PKSs control starter unit selection, polyketide chain initiation and elongation, intermediate folding and cyclization, selective redox or modification reactions during assembly, and product release are central mechanistic questions underlying iterative catalysis. This Review highlights recent insights into these questions, with a particular focus on the biosynthetic programming of fungal aromatic polyketides, and draws comparisons with the allied biosynthetic processes in bacteria.

The colibactin warhead crosslinks DNA

Members of the human microbiota are increasingly being correlated to human health and disease states, but the majority of the underlying microbial metabolites that regulate host-microbe interactions remain largely unexplored. Select strains of E. coli present in the human colon have been linked to initiating inflammation-induced colorectal cancer through an unknown small molecule-mediated process. The responsible nonribosomal peptide-polyketide hybrid pathway encodes “colibactin,” a largely uncharacterized family of small molecules. Genotoxic small molecules from this pathway capable of initiating cancer formation have remained elusive due to their high instability. Guided by metabolomic analyses, here we employ a combination of NMR spectroscopy and bioinformatics-guided isotopic labeling studies to characterize the colibactin warhead, an unprecedented substituted spirobicyclic structure. The warhead crosslinks duplex DNA in vitro, providing direct experimental evidence for colibactin’s DNA-damaging activity. The data support unexpected models for both colibactin biosynthesis and its mode of action.

Bacterial symbionts and natural products

The study of bacterial symbionts of eukaryotic hosts has become a powerful discovery engine for chemistry. This highlight looks at four case studies that exemplify the range of chemistry and biology involved in these symbioses: a bacterial symbiont of a fungus and a marine invertebrate that produce compounds with significant anticancer activity, and bacterial symbionts of insects and nematodes that produce compounds that regulate multilateral symbioses.

Small molecule perimeter defense in entomopathogenic bacteria

Two Gram-negative insect pathogens, Xenorhabdus nematophila and Photorhabdus luminescens, produce rhabduscin, an amidoglycosyl- and vinyl-isonitrile-functionalized tyrosine derivative. Heterologous expression of the rhabduscin pathway in Escherichia coli, precursor-directed biosynthesis of rhabduscin analogs, biochemical assays, and visualization using both stimulated Raman scattering and confocal fluorescence microscopy established rhabduscin’s role as a potent nanomolar-level inhibitor of phenoloxidase, a key component of the insect’s innate immune system, as well as rhabduscin’s localization at the bacterial cell surface. Stimulated Raman scattering microscopy visualized rhabduscin at the periphery of wild-type X. nematophila cells and E. coli cells heterologously expressing the rhabduscin pathway. Precursor-directed biosynthesis created rhabduscin mimics in X. nematophila pathway mutants that could be accessed at the bacterial cell surface by an extracellular bioorthogonal probe, as judged by confocal fluorescence microscopy. Biochemical assays using both wild-type and mutant X. nematophila cells showed that rhabduscin was necessary and sufficient for potent inhibition (low nM) of phenoloxidases, the enzymes responsible for producing melanin (the hard black polymer insects generate to seal off microbial pathogens). These observations suggest a model in which rhabduscin’s physical association at the bacterial cell surface provides a highly effective inhibitor concentration directly at the site of phenoloxidase contact. This class of molecules is not limited to insect pathogens, as the human pathogen Vibrio cholerae also encodes rhabduscin’s aglycone, and bacterial cell-coated immunosuppressants could be a general strategy to combat host defenses.

A Single Promoter Inversion Switches Photorhabdus Between Pathogenic and Mutualistic States

Twin Tales of Two Toxins The luminescent bacterium, Photorhabdus luminescens, is carried in the gut of an insect-parasitic nematode as a stealth weapon. By using an allele swapping technique, Somvanshi et al. (p. 88) investigated the promoter-switching mechanism that flips the bacterium from the almost dormant M forms, which stick to the adult nematodes posterior gut, into the motile, luminous P forms, which are armed with the toxic virulence factors needed to overcome the insect prey of the nematode. Similar switches may operate in bacteria that flip between harmless commensals and lethal pathogens. The bio-control agent Bacillus thuringiensis also kills insects by means of a crystal toxin, which allows the bacteria to penetrate the host gut and access nutrients. Release of nutrients also allows bacterial cheats that do not make toxin, to grow and outcompete the toxin-producing colonizers. In field experiments, Raymond et al. (p. 85) found that, consequently, the bacterial population becomes less virulent. Because these type of virulence factors are secreted from the cell and are widespread in pathogens, such social interactions may affect the fitness and constrain the virulence of many toxin-producing bacteria. A stochastic switch allows a bacterium to toggle between virulent and symbiotic forms in its hosts. Microbial populations stochastically generate variants with strikingly different properties, such as virulence or avirulence and antibiotic tolerance or sensitivity. Photorhabdus luminescens bacteria have a variable life history in which they alternate between pathogens to a wide variety of insects and mutualists to their specific host nematodes. Here, we show that the P. luminescens pathogenic variant (P form) switches to a smaller-cell variant (M form) to initiate mutualism in host nematode intestines. A stochastic promoter inversion causes the switch between the two distinct forms. M-form cells are much smaller (one-seventh the volume), slower growing, and less bioluminescent than P-form cells; they are also avirulent and produce fewer secondary metabolites. Observations of form switching by individual cells in nematodes revealed that the M form persisted in maternal nematode intestines, were the first cells to colonize infective juvenile (IJ) offspring, and then switched to P form in the IJ intestine, which armed these nematodes for the next cycle of insect infection.