The genome of antibiotic-producing colonies of the Pelagophyte alga Chrysophaeum taylorii reveals a diverse and non-canonical capacity for secondary metabolism.

Chrysophaeum taylorii is a member of an understudied clade of marine algae that can be responsible for harmful coastal blooms and is known to accumulate bioactive natural products including antibiotics of the chrysophaentin class. Whole genome sequencing of laboratory-cultivated samples revealed an extensive and diverse complement of secondary metabolite biosynthetic genes in C. taylorii, alongside a small microbiome with a more limited biosynthetic potential. 16S microbiome analysis of laboratory cultured alongside wild-collected samples revealed several common taxa; however, analysis of biosynthetic genes suggested an algal origin for the chrysophaentins, possibly via one of several non-canonical polyketide synthase genes encoded within the genome.

Lentzeacins A-E, New Bacterial-Derived 2,5- and 2,6-Disubstituted Pyrazines from a BGC-Rich Soil Bacterium Lentzea sp. GA3-008.

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.

Vertirhodins A-F, C-Linked Pyrrolidine-Iminosugar-Containing Pyranonaphthoquinones from Streptomyces sp. B15-008.

Six novel pyranonaphthoquinones, vertirhodins A-F (1-6), were discovered from a soil-derived Streptomyces sp. B15-008. Their chemical structures and absolute configurations were determined using nuclear magnetic resonance and comparison of experimental and theoretical electronic circular dichroism spectra. The vertirhodins feature an unusual C-8 N-methyl-2-pyrrolidinemethanol moiety, a 5,14-epoxide rarely seen in streptomyces-derived natural products, and a C-13 hydroxyl group that forms the semiquinone. A plausible ver biosynthetic gene cluster was identified through whole genome sequencing and provides insights into these features.

Antimicrobial Chrysophaentin Analogs Identified from Laboratory Cultures of the Marine Microalga Chrysophaeum taylorii.

A laboratory culture of the colonial marine alga Chrysophaeum taylorii NIES-1699 yielded a set of new bioactive chrysophaentin analogs, and their structures were determined by HRESIMS and NMR spectroscopy. Differences in the metabolites identified between cultured C. taylorii NIES-1699 and field-collected strains from the U.S. Virgin Islands revealed additional structure-activity relationships for the Gram-positive antibiotic activity of the chrysophaentins. The presence of new hemichrysophaentins and a C-C linked biphenyl analog suggest novel features of their biosynthetic pathway. Bayesian analysis of the alignment of the 18S rRNA gene places the microalga C. taylorii in the pelagophyte clade.

Chemical and Biophysical Approaches for Complete Characterization of Lectin-Carbohydrate Interactions.

Lectins are carbohydrate-binding proteins unrelated to antibodies or enzymes. While carbohydrates are present on all cells and pathogens, lectins are also ubiquitous in nature and their interactions with glycans mediate countless biological and physical interactions. Due to the multivalency found in both lectins and their glycan-binding partners, complete characterization of these interactions can be complex and typically requires the use of multiple complimentary techniques. In this chapter, we provide a general strategy and protocols for chemical and biophysical approaches that can be used to characterize carbohydrate-mediated interactions in the context of individual oligosaccharides, as part of a glycoprotein, and ending with visualization of interactions with whole virions.

A New Natural Product Analog of Blasticidin S Reveals Cellular Uptake Facilitated by the NorA Multidrug Transporter.

The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics.

Insights from NMR Spectroscopy into the Conformational Properties of Man-9 and Its Recognition by Two HIV Binding Proteins.

Man9 GlcNAc2 (Man-9) present at the surface of HIV makes up the binding sites of several HIV-neutralizing agents and the mammalian lectin DC-SIGN, which is involved in cellular immunity and trans-infections. We describe the conformational properties of Man-9 in its free state and when bound by the HIV entry-inhibitor protein microvirin (MVN), and define the minimum epitopes of both MVN and DC-SIGN by using NMR spectroscopy. To facilitate the implementation of 3D 13 C-edited spectra to deconvolute spectral overlap and to determine the solution structure of Man-9, we developed a robust expression system for the production of 13 C,15 N-labeled glycans in mammalian cells. The studies reveal that Man-9 interacts with HIV-binding proteins through distinct epitopes and adopts diverse conformations in the bound state. In combination with molecular dynamics simulations we observed receptor-bound conformations to be sampled by Man-9 in the free state, thus suggesting a conformational selection mechanism for diverse recognition.

Trimeric HIV-1-Env Structures Define Glycan Shields from Clades A, B, and G.

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ∼90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B, and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, that encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed, and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans among known broadly neutralizing antibodies that target the glycan-shielded trimer.