Nearly all life requires iron; however, under aerobic conditions, Fe(III) is nearly insoluble. To meet their metabolic needs, many bacteria synthesize siderophores: small-molecule, high-affinity Fe(III) chelators. The discovery of new siderophores has been streamlined by genome mining, where bacterial genomes of interest are scanned for siderophore biosynthetic gene clusters (BGCs). This dissertation is focused on the development of new bioinformatics tools to improve siderophore structural predictions, as well as the application of those tools towards traditional natural product isolation.

Some peptidic siderophores contain the chelating ligand β-hydroxyaspartate (β-OHAsp), which provides bidentate OO′ coordination to Fe(III). Current genome mining methods cannot reliably predict which aspartate residues will be hydroxylated, nor the resulting stereochemistry, leaving structural ambiguities which must be rectified experimentally. Through coupling BGCs with verified structures, the origin of the β-OHAsp diastereomers in siderophores is reported. Two functional subtypes of nonheme Fe(II)/α-ketoglutarate–dependent aspartyl β-hydroxylases were identified in siderophore BGCs. Each aspartyl β-hydroxylase subtype effects distinct diastereoselectivity. A previously undescribed, noncanonical member of the nonribosomal peptide synthetase (NRPS) condensation domain superfamily is identified, named the interface domain, which is proposed to position the β-hydroxylase and the NRPS-bound amino acid prior to hydroxylation. Through mapping characterized β-OHAsp diastereomers to the phylogenetic tree of siderophore β-hydroxylases, methods to predict β-OHAsp stereochemistry in silico are realized.

The DHB-CAA-Ser family of triscatechol siderophores each contain a different cationic amino acid (CAA = D-Arg, D-Lys, or L-Orn), hinting at the existence of their diastereomers with L-Arg, L-Lys, and D-Orn. With no knowledge of which bacteria, if any, are capable of their production, a novel high-throughput genome mining workflow was developed, the Catechol Siderophore Cluster Analysis (CatSCAn). Over 11,000 bacterial genomes were scanned for siderophore BGCs, revealing about 1% potentially capable of the biosynthesis of the combinatoric suite of DHB-CAA-Ser siderophores with L- and D-CAA. The NRPS from Marinomonas sp. TW1 was found to be unusually promiscuous, producing a relatively rare example of a suite of siderophores with D-Arg, D-Orn, and L-Orn amino acids.

CatSCAn produced a rich dataset of putative BGCs that may be coupled with structurally- characterized siderophores to study the chemistry and biology of triscatechol siderophores. A detailed analysis of BGCs highlighted key features within siderophore pathways. Mapping known and predicted siderophores onto a phylogenetic tree of NRPS proteins reveals repeated changes in CAA stereochemistry and identity within the DHB-CAA-Ser family. Parsimony suggests that the ancestor of all DHB-CAA-Ser siderophores was trivanchrobactin, (DHB-DArg-Ser)3. A model of DHB-CAA-Ser siderophore evolution is presented where enzyme promiscuity allows for the biosynthesis of new siderophores.

Genome mining for VibH homologs reveals several species of Acinetobacter with a gene cluster that putatively encodes the biosynthesis of catechol siderophores with an amine core. A. bouvetii DSM 14964 produces three novel biscatechol siderophores: propanochelin, butanochelin, and pentanochelin. This strain has a relaxed specificity for the amine substrate, allowing for the biosynthesis of a variety of non-natural siderophore analogs by precursor directed biosynthesis. Of potential synthetic utility, A. bouvetii DSM 14964 condenses 2,3-dihydroxybenzoic acid (2,3-DHB) to allylamine and propargylamine, producing catecholic compounds which bind iron(III) and may be further modified via thiol–ene or azide–alkyne click chemistry.

Cells are known to sense and respond to their mechanical microenvironment in profound ways. Various evidence has implicated the adhesome, a body of adhesion associated proteins, in several cell fate decisions, including differentiation and proliferation. Furthermore, recent work has demonstrated that substrate topography can modulate the epigenetic state of fibroblasts, facilitate cell reprogramming, and temper inflammatory activation. However, the effectors responsible for such phenomena remain poorly understood. Here we explore the effect of biomaterial design, adhesion, and intracellular forces of the epigenetic and transcriptional regulation of cell state during macrophage activation and somatic cell reprogramming.

Regarding macrophage activation, we relied entirely on murine bone marrow-derived macrophages (BMDMs). To study the effect of adhesion on BMDM behavior, we utilized microcontact printing to produce fibronectin patterned substrates ontop of which we seeded BMDMs. Next, we induced macrophage activation using lipopolysaccharide (LPS) and interferon-γ (INFγ) and found patterned substrates reduce inflammatory gene expression and histone-3 acetylation (H3Ac) while also increasing cellular elongation. We incorporated these results into a bioinformatic reanalysis of transcriptional and epigenetic data derived from a study of H3Ac protein binding during macrophage polarization. This analysis allowed us to identify epigenetic mechanisms linking the adhesome and inflammatory gene expression.

In our investigation of somatic cell reprogramming, we found extracellular matrix binding (ECM) and mechanosensitive ion channel activation reduce reprogramming efficiency. In addition to these results, we discovered that 104 adhesome genes are dynamically regulated during the reprogramming process. Subsequently, we performed an shRNA knockdown of each of these genes and found over 90% of our knockdowns increased the number of TRA-1-60 positive pluripotent colonies. Our knockdown of SHROOM3, a gene associated with apical cellular constriction, increased reprogramming efficiency by 27-fold. Further investigation into SHROOM3 identified a mechano-sensitive critical state transition, which may be necessary for successful reprogramming.

These observations establish adhesome gene expression and mechanical signaling as influential regulators of cell fate during macrophage activation and reprogramming. Moreover, our findings may guide future attempts to regulate cell state and fate using biophysical stimuli. They may also help shed light on cell behavior in various disease states involving cancer and inflammation.