Biology subjects

Bailey, C. B.

Publications and source records attributed to Bailey, C. B..

4 recordsLinked to original sources

bioRxiv2026

Mechanism of phospholipid transport to the bacterial outer membrane by TAM.

Gram-negative bacteria transport phospholipids from the inner membrane (IM) to the outer membrane (OM) via poorly understood processes. These processes are essential for cell growth and the establishment of an antibiotic-resistant barrier. Here, we conducted single-particle cryo-electron microscopy, in vivo functional assays, and lipidomics to investigate the role of the "translocation and assembly module" (TAM) in lipid transport. We found that the OM-embedded subunit TamA anchors the IM-embedded bridge-like subunit TamB to the OM by forming a functional stable hybrid-barrel structure with the highly conserved C-terminal domain of unknown function 490 (DUF490). Using in vivo disulfide-tethering experiments we found that a highly conserved amphipathic helix within TamB DUF490 is important for TAM to function in OM maintenance. We also found that TamB DUF490 forms a {beta}-taco channel containing lipid-like densities and that the lipophilic property of the channel is important for TAM to maintain the levels of cardiolipin in the OM. Not only do our data support a novel model in which TAM acts to direct specific lipid classes into the OM, but it also supports the notion that TamB is a bacterial evolutionary prototype of a structurally homologous superfamily of eukaryotic bridge-like lipid transfer proteins.

biochemistry↗

bioRxiv2025

Establishing Conserved Biosynthetic Gene Clusters of the Phylum Myxococcota

A surge in sequenced myxobacteria catalyzed by advancements in long read genome and metagenome sequencing has provided sufficient data to scrutinize the conserved biosynthetic gene clusters (BGCs) within the phylum Myxococcota. Provided the utility of myxobacteria in environmental nutrient cycles and discovery of novel therapeutic leads, we sought to determine any conserved specialized metabolism in the phylum. Using a pan-genome approach to analyze eleven genera and 195 sequenced genomes including ten newly reported myxobacterial isolate, we observed five conserved BGCs. All five clusters encode for characterized metabolites with established ecological roles for four of the metabolites, and none of the metabolites are known toxins. Validation of our approach was done by analyzing Myxococcota genera without sufficient, sequenced representatives for pan-genome analysis to observe the presence/absence of these five clusters. This approach enabled observation of genus-level conservation of BGCs with varying degrees of confidence due to diversity of sequenced species within each genus. The indigoidine BGC typically found in Streptomyces spp. was notably conserved in Melittangium; heterologous expression of the core biosynthetic gene bspA in Escherichia coli and subsequent detection of indigoidine confirmed the identity of the indigoidine cluster. Conserved BGCs in myxobacteria reveal maintenance of biosynthetic pathways and cognate metabolites with ecological roles as chemical signals and stress response; these observations suggest competitive specialization of secondary metabolism and toxin production in myxobacteria.

microbiology↗

bioRxiv2023

Profiling Expression Strategies for a Type III Polyketide Synthase in a Lysate-Based, Cell-free System

Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli. Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli frequently results in a myriad of unpredictable issues with protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts, such as BGC refactoring, can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as 1) a valuable expression platform for enzymes that are challenging to synthesize in vivo, and as 2) a testbed for rapid prototyping that can improve cellular expression. Here, we use a type III polyketide synthase from Streptomyces griseus, RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We synergistically tune promoter and codon usage to improve flaviolin production from cell-free expressed RppA. We then assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs. By showing the coordinators between CFE versus in vivo expression, this work advances CFE as a tool for natural product research. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/569483v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1aebfceorg.highwire.dtl.DTLVardef@1b289f1org.highwire.dtl.DTLVardef@7e46b4org.highwire.dtl.DTLVardef@53f480_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2019

Chemoinformatic-guided engineering of polyketide synthases

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge in PKS design is engineering a partially reductive module to produce a saturated {beta}-carbon through a reductive loop exchange. In this work, we sought to establish that chemoinformatics, a field traditionally used in drug discovery, could provide a viable strategy to reductive loop exchanges. We first introduced a set of donor reductive loops of diverse genetic origin and chemical substrate structures into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with atom pair chemical similarity between the substrate of the donor reductive loops and recipient LipPKS1, reaching a titer of 165 mg/L of short chain fatty acids produced by Streptomyces albus J1074 harboring these engineered PKSs. Expanding this method to larger intermediates requiring bimodular communication, we introduced reductive loops of divergent chemosimilarity into LipPKS2 and determined triketide lactone production. Collectively, we observed a statistically significant correlation between atom pair chemosimilarity and production, establishing a new chemoinformatic method that may aid in the engineering of PKSs to produce desired, unnatural products.

biochemistry↗