David T. Fox

Exploring new strategies for cellulosic biofuels production

A research program has been initiated to formulate new strategies for efficient low-cost lignocellulosic biomass processing technologies for the production of biofuels. This article reviews results from initial research into lignocellulosic biomass structure, recalcitrance, and pretreatment. In addition to contributing towards a comprehensive understanding of lignocellulosic biomass, this work has contributed towards demonstrated optimizations of existing pretreatment methods, and the emergence of new possible pretreatment strategies that remain to be fully developed.

Ionic liquids as a class of materials for transdermal delivery and pathogen neutralization

Significance Effective treatment of skin-based bacterial biofilms has been identified as a serious and unmet medical need. Biofilm-protected bacteria account for ∼80% of bacterial infections in humans and are 50–1,000 times more resistant to antibiotics than their planktonic counterparts. Biofilms in skin are further protected by the outermost layer of skin, the stratum corneum, which serves as a natural barrier to most therapeutics. Here, we present compelling evidence for exploiting ionic liquids (ILs) as an arsenal of materials both in a concerted effort to combat antibiotic-resistant bacterial biofilms in skin as well as for topical transdermal drug delivery. Our comprehensive strategy resulted in the identification of ILs that are effective at disrupting biofilms, neutralizing pathogens, and enhancing delivery of antibiotic into skin. Moreover, ILs did not show skin irritation that is typically associated with topical formulations. Biofilm-protected microbial infections in skin are a serious health risk that remains to be adequately addressed. The lack of progress in developing effective treatment strategies is largely due to the transport barriers posed by the stratum corneum of the skin and the biofilm. In this work, we report on the use of Ionic Liquids (ILs) for biofilm disruption and enhanced antibiotic delivery across skin layers. We outline the syntheses of ILs, analysis of relevant physicochemical properties, and subsequent neutralization effects on two biofilm-forming pathogens: Pseudomonas aeruginosa and Salmonella enterica. Further, the ILs were also examined for cytotoxicity, skin irritation, delivery of antibiotics through the skin, and treatment of biofilms in a wound model. Of the materials examined, choline-geranate emerged as a multipurpose IL with excellent antimicrobial activity, minimal toxicity to epithelial cells as well as skin, and effective permeation enhancement for drug delivery. Specifically, choline-geranate was comparable with, or more effective than, bleach treatment against established biofilms of S. enterica and P. aeruginosa, respectively. In addition, choline-geranate increased delivery of cefadroxil, an antibiotic, by >16-fold into the deep tissue layers of the skin without inducing skin irritation. The in vivo efficacy of choline-geranate was validated using a biofilm-infected wound model (>95% bacterial death after 2-h treatment). This work establishes the use of ILs for simultaneous enhancement of topical drug delivery and antibiotic activity.

Siderophore-mediated iron acquisition in Bacillus anthracis and related strains.

Recent observations have shed light on some of the endogenous iron-acquisition mechanisms of members of the Bacillus cereus sensu lato group. In particular, pathogens in the B. cereus group use siderophores with both unique chemical structures and biological roles. This review will focus on recent discoveries in siderophore biosynthesis and biology in this group, which contains numerous human pathogens, most notably the causative agent of anthrax, Bacillus anthracis.

The missing link in petrobactin biosynthesis: asbF encodes a (-)-3-dehydroshikimate dehydratase.

The siderophore petrobactin harbors unique 3,4-dihydroxybenzoyl iron-liganding groups. These moieties are known to be synthesized from shikimate pathway precursors, but no reports of the biosynthetic enzymes responsible for this conversion have been published. The gene encoding AsbF from Bacillus thuringiensis 97-27 was overexpressed in an Escherichia coli host. AsbF rapidly and efficiently transforms (-)-3-dehydroshikimate (DHS) into 3,4-dihydroxybenzoate (k(cat)(DHS) = 217 +/- 10 min(-1); K(m)(DHS) = 125 +/- 14 microM) at 37 degrees C and has an absolute requirement for divalent metal. Finally, the pH versus k(cat)(DHS) profile revealed two ionizable groups (pK(a1) = 7.9 +/- 0.1, and pK(a2) = 9.3 +/- 0.1).

Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry

We created a single cell sorting system to screen for enzyme activity in Escherichia coli producing 3,4 dihydroxy benzoate (34DHB). To do so, we engineered a transcription factor regulon controlling the expression of green fluorescent protein (GFP) for induction by 34DHB. An autoregulated transcription factor, pcaU, was borrowed from Acinetobacter sp ADP1 to E. coli and its promoter region adapted for activity in E. Coli. The engineered pcaU regulon was inducible at >5 μM exogenous 34DHB, making it a sensitive biosensor for this industrially significant nylon precursor. Addition of a second plasmid provided IPTG inducible expression of dehydroshikimate dehydratase enzyme (AsbF), which converts endogenous dehydroshikimate to 34DHB. This system produced GFP fluorescence in an IPTG dose-dependent manner, and was easily detected in single cell on flow cytometer despite a moderate catalytic efficiency of AsbF. Using fluorescence-activated cell sorting (FACS), individual cells carrying the active AsbF could be isolated even when diluted into a decoy population of cells carrying a mutant (inactivated) AsbF variant at one part in a million. The same biosensor was also effective for further optimization of itself. FACS on E. coli carrying randomized loci in the promoter showed several variants with enhanced response to 34DHB.

Evaluation of ionic liquids on phototrophic microbes and their use in biofuel extraction and isolation

Multiple ionic liquids (ILs) were assessed for their ability to extract branched, unsaturated hydrocarbons from an aqueous medium. In addition, IL cytotoxicity studies were performed on two phototrophic microbes, Synechocystis sp. PCC6803 and Botryococcus braunii var Showa. The optimum IL for use in an isoprenoid hydrocarbon extraction may vary based on the biological source of the isoprenoids. Our results suggest that ionic liquids have the potential to serve as novel biocompatible milking agents for extracting high-value chemicals from the microbes, with toxicity to both species minimized by considerations of ionic liquid structure and hydrophobicity.

Rosetta comparative modeling for library design: Engineering alternative inducer specificity in a transcription factor

Structure‐based rational mutagenesis for engineering protein functionality has been limited by the scarcity and difficulty of obtaining crystal structures of desired proteins. On the other hand, when high‐throughput selection is possible, directed evolution‐based approaches for gaining protein functionalities have been random and fortuitous with limited rationalization. We combine comparative modeling of dimer structures, ab initio loop reconstruction, and ligand docking to select positions for mutagenesis to create a library focused on the ligand‐contacting residues. The rationally reduced library requirement enabled conservative control of the substitutions by oligonucleotide synthesis and bounding its size within practical transformation efficiencies (∼107 variants). This rational approach was successfully applied on an inducer‐binding domain of an Acinetobacter transcription factor (TF), pobR, which shows high specificity for natural effector molecule, 4‐hydroxy benzoate (4HB), but no native response to 3,4‐dihydroxy benzoate (34DHB). Selection for mutants with high transcriptional induction by 34DHB was carried out at the single‐cell level under flow cytometry (via green fluorescent protein expression under the control of pobR promoter). Critically, this selection protocol allows both selection for induction and rejection of constitutively active mutants. In addition to gain‐of‐function for 34DHB induction, the selected mutants also showed enhanced sensitivity and response for 4HB (native inducer) while no sensitivity was observed for a non‐targeted but chemically similar molecule, 2‐hydroxy benzoate (2HB). This is unique application of the Rosetta modeling protocols for library design to engineer a TF. Our approach extends applicability of the Rosetta redesign protocol into regimes without a priori precision structural information. Proteins 2015; 83:1327–1340.

Biosynthesis of the 3,4-Dihydroxybenzoate Moieties of Petrobactin by Bacillus anthracis

The biosynthesis of the 3,4-dihydroxybenzoate moieties of the siderophore petrobactin, produced by B. anthracis str. Sterne, was probed by isotopic feeding experiments in iron-deficient media with a mixture of unlabeled and D-[(13)C6]glucose at a ratio of 5:1 (w/w). After isolation of the labeled siderophore, analysis of the isotopomers was conducted via one-dimensional (1)H and (13)C NMR spectroscopy, as well as (13)C-(13)C DQFCOSY spectroscopy. Isotopic enrichment and (13)C-(13)C coupling constants in the aromatic ring of the isolated siderophore suggested the predominant route for the construction of the carbon backbone of 3,4-DHB (1) involved phosphoenol pyruvate and erythrose-4-phosphate as ultimate precursors. This observation is consistent with that expected if the shikimate pathway is involved in the biosynthesis of these moieties. Enrichment attributable to phosphoenol pyruvate precursors was observed at C1 and C6 of the aromatic ring, as well as into the carboxylate group, while scrambling of the label into C2 was not. This pattern suggests 1 was biosynthesized from early intermediates of the shikimate pathway and not through later shikimate intermediates or aromatic amino acid precursors.

Single-pot extraction-analysis of dyed wool fibers with ionic liquids.

Analytical capabilities to identify dyes associated with structurally robust wool fibers would critically assist crime-scene and explosion-scene forensics. Nondestructive separation of dyes from wool, removal of contaminants, and dye analysis by MALDI- or ESI-MS, were achieved in a single-pot, ionic liquid-based method. Ionic liquids (ILs) that readily denature the wool α-keratin structure have been identified and are conducive to small volume, high-throughput analysis for accelerated threat-response times. Wool dyed with commercial or natural, plant-based dyes have unique signatures that allow classification and matching of samples and identification of dyestuffs. Wool released 0.005 mg of dye per mg of dyed wool into the IL, allowing for analysis of single-thread sample sizes. The IL + dye mixture promotes sufficient ionization in MALDI-MS: addition of common MALDI matrices does not improve analysis of anionic wool dyes. An inexpensive, commercially available tetrabutylphosponium chloride IL was discovered to be capable of denaturing wool and was determined to be the most effective for this readily fieldable method.

Choline and Geranate Deep Eutectic Solvent as a Broad-Spectrum Antiseptic Agent for Preventive and Therapeutic Applications

Antiseptic agents are the primary arsenal to disinfect skin and prevent pathogens spreading within the host as well as into the surroundings; however the Food and Drug Administration published a report in 2015 requiring additional validation of nearly all current antiseptic agents before their continued use can be allowed. This vulnerable position calls for urgent identification of novel antiseptic agents. Recently, the ability of a deep eutectic, Choline And Geranate (CAGE), to treat biofilms of Pseudomonas aeruginosa and Salmonella enterica was demonstrated. Here it is reported that CAGE exhibits broad-spectrum antimicrobial activity against a number of drug-resistant bacteria, fungi, and viruses including clinical isolates of Mycobacterium tuberculosis, Staphylococcus aureus, and Candida albicans as well as laboratory strains of Herpes Simplex Virus. Studies in human keratinocytes and mice show that CAGE affords negligible local or systemic toxicity, and an ≈180-14 000-fold improved efficacy/toxicity ratio over currently used antiseptic agents. Further, CAGE penetrates deep into the dermis and treats pathogens located in deep skin layers as confirmed by the ability of CAGE in vivo to treat Propionibacterium acnes infection. In combination, the results clearly demonstrate CAGE holds promise as a transformative platform antiseptic agent for preventive as well as therapeutic applications.