Fatty-acylation of proteins in eukaryotes is associated with many fundamental cellular processes but has been challenging to study due to limited tools for rapid and robust detection of protein fatty-acylation in cells. The development of azido-fatty acids enabled the nonradioactive detection of fatty-acylated proteins in mammalian cells using the Staudinger ligation and biotinylated phosphine reagents. However, the visualization of protein fatty-acylation with streptavidin blotting is highly variable and not ideal for robust detection of fatty-acylated proteins. Here we report the development of alkynyl-fatty acid chemical reporters and improved bioorthogonal labeling conditions using the Cu(I)-catalyzed Huisgen [3 + 2] cycloaddition that enables specific and sensitive fluorescence detection of fatty-acylated proteins in mammalian cells. These improvements allow the rapid and robust biochemical analysis of fatty-acylated proteins expressed at endogenous levels in mammalian cells by in-gel fluorescence scanning. In addition, alkynyl-fatty acid chemical reporters enable the visualization of fatty-acylated proteins in cells by fluorescence microscopy and flow cytometry. The ability to rapidly visualize protein fatty-acylation in cells using fluorescence detection methods therefore provides new opportunities to interrogate the functions and regulatory mechanisms of fatty-acylated proteins in physiology and disease.

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Here we report an efficient CRISPR-Cas9 knock-in strategy to activate silent biosynthetic gene clusters (BGCs) in streptomycetes. We applied this one-step strategy to activate multiple BGCs of different classes in five Streptomyces species and triggered the production of unique metabolites, including a novel pentangular type II polyketide in Streptomyces viridochromogenes. This potentially scalable strategy complements existing activation approaches and facilitates discovery efforts to uncover new compounds with interesting bioactivities.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains the leading cause of mortality from a single infectious agent. Each year around 9 million individuals newly develop active TB disease, and over 2 billion individuals are latently infected with M.tb worldwide, thus being at risk of developing TB reactivation disease later in life. The underlying mechanisms and pathways of protection against TB in humans, as well as the dynamics of the host response to M.tb infection, are incompletely understood. We carried out whole-genome expression profiling on a cohort of TB patients longitudinally sampled along 3 time-points: during active infection, during treatment, and after completion of curative treatment. We identified molecular signatures involving the upregulation of type-1 interferon (α/β) mediated signaling and chronic inflammation during active TB disease in an Indonesian population, in line with results from two recent studies in ethnically and epidemiologically different populations in Europe and South Africa. Expression profiles were captured in neutrophil-depleted blood samples, indicating a major contribution of lymphocytes and myeloid cells. Expression of type-1 interferon (α/β) genes mediated was also upregulated in the lungs of M.tb infected mice and in infected human macrophages. In patients, the regulated gene expression-signature normalized during treatment, including the type-1 interferon mediated signaling and a concurrent opposite regulation of interferon-gamma. Further analysis revealed IL15RA, UBE2L6 and GBP4 as molecules involved in the type-I interferon response in all three experimental models. Our data is highly suggestive that the innate immune type-I interferon signaling cascade could be used as a quantitative tool for monitoring active TB disease, and provide evidence that components of the patient’s blood gene expression signature bear similarities to the pulmonary and macrophage response to mycobacterial infection.

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Microbial fermentation provides as an attractive alternative to chemical synthesis for the production of structurally complex natural products. In most cases, however, production titers are low and need to be improved for compound characterization and/or commercial production. Owing to advances in functional genomics and genetic engineering technologies, microbial hosts can be engineered to overproduce a desired natural product, greatly accelerating the traditionally time-consuming strain improvement process. This review covers recent developments and challenges in the engineering of native and heterologous microbial hosts for production of bacterial natural products, focusing on the genetic tools and strategies for strain improvement. Special emphasis is placed on bioactive secondary metabolites from actinomycetes. The considerations for the choice of host systems will also be discussed in this review.

The functional significance and regulation of reversible S-acylation on diverse proteins remain unclear because of limited methods for efficient quantitative analysis of palmitate turnover. Here, we describe a tandem labeling and detection method to simultaneously monitor dynamic S-palmitoylation and protein turnover. By combining S-acylation and cotranslational fatty acid chemical reporters with orthogonal clickable fluorophores, dual pulse-chase analysis of Lck revealed accelerated palmitate cycling upon T-cell activation. Subsequent pharmacological perturbation of Lck palmitate turnover suggests yet uncharacterized serine hydrolases contribute to dynamic S-acylation in cells. In addition to dually fatty-acylated proteins, this tandem fluorescence imaging method can be generalized to other S-acylated proteins using azidohomoalanine as a methonine surrogate. The sensitivity and efficiency of this approach should facilitate the functional characterization of cellular factors and drugs that modulate protein S-acylation. Furthermore, diverse protein modifications could be analyzed with this tandem imaging method using other chemical reporters to investigate dynamic regulation of protein function.bioorthogonal ligation | chemical reporters | click chemistry | S-palmitoylation | fatty acylation P rotein S-palmitoylation (S-acylation) targets proteins to discrete intracellular membrane compartments, controls protein stability, and mediates protein-protein interactions (1, 2). Furthermore, the reversibility of S-acylation offers spatial and temporal control of protein function akin to protein phosphorylation (Fig. 1A). Notably, the differential S-palmitoylation and membrane targeting of H-and N-Ras isoforms have been shown to activate discrete signaling pathways for cellular growth and differentiation (3-6). Accelerated deacylation of G-protein subunits and G-protein coupled receptors upon receptor stimulation (7,8) suggest active mechanisms regulating S-acylation/deacylation cycles. In addition, receptor activity-regulated palmitate turnover of PSD-95 is proposed to modulate synaptic strength and plasticity in postsynaptic neurons by mediating receptor clustering (9). Development of the acyl-biotinyl exchange protocol (10-12) and fatty acid chemical reporters (13-16) enabled improved detection and identification of many S-palmitoylated proteins from diverse biological pathways in eukaryotes (17). Nonetheless, it remains to be determined if S-acylation is dynamic and regulated for identified fatty-acylated proteins.Quantitative analysis of S-acylation/deacylation cycle on proteins would provide further insight into the biological significance and function of dynamic S-palmitoylation, but tools to efficiently visualize palmitate turnover on proteins are limited. While photoactivation/bleaching of S-palmitoylated proteins fused to fluorescent reporters allow visualization of protein dynamics in cells (4,5,18), these methods yield indirect readouts of protein S-acylation. Acyl-biotinyl exchange enables nonradioactive det...

Symbiont choice has been proposed to play an important role in shaping many symbiotic relationships, including the fungus-growing ant-microbe mutualism. Over millions of years, fungusgrowing ants have defended their fungus gardens from specialized parasites with antibiotics produced by an actinomycete bacterial mutualist (genus Pseudonocardia). Despite the potential of being infected by phylogenetically diverse strains of parasites, each ant colony maintains only a single Pseudonocardia symbiont strain, which is primarily vertically transmitted between colonies by the founding queens. In this study, we show that Acromyrmex leaf-cutter ants are able to differentiate between their native actinomycete strain and a variety of foreign strains isolated from sympatric and allopatric Acromyrmex species, in addition to strains originating from other fungusgrowing ant genera. The recognition mechanism is sufficiently sensitive for the ants to discriminate between closely related symbiont strains. Our findings suggest that symbiont recognition may play a crucial role in the fungus-growing ant-bacterium mutualism, likely allowing the ants to retain ecological flexibility necessary for defending their garden from diverse parasites and, at the same time, resolve potential conflict that can arise from rearing competing symbiont strains.