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Glycosyltransferases (GTs) are important proteins that are widely distributed in both prokaryotes and eukaryotes and play a crucial role in the biosynthesis of carbohydrates and glycoconjugates. They catalyze the formation of specific glycosidic linkages by transferring sugar moieties from activated sugars to a variety of biomolecules such as carbohydrates, lipids, proteins, or water. Progress in functional genomics technologies, such as DNA sequencing and proteomics, has allowed the identification of a large number of GT genes in several species. In general, functional genomics approaches, which include genomics, genetics, proteomics, and biochemistry, are used to determine the function of a gene (i.e., GTs). In biochemical approaches, the most direct way to assign a function to a gene is through direct testing of the enzyme activity of its product (carbohydrate) in vitro. However, in contrast to genomics/proteomics approaches, the biochemical approaches are the most difficult to adapt to high-throughput screening. The reason is that biochemical approaches require the use of isolated/purified proteins, which is labor-intensive and prone to the formation of undesired products resulting from background enzyme activity. Therefore, there is a need for the development of protein-based in vitro high-throughput platforms for determination of biochemical functions of proteins, including GTs, and their interactions with other proteins. To be advantageous, a protein-based high-throughput platform should have the following characteristics: i) the platform can be adapted to all GTs and synthases, ii) the detection method should be sensitive enough to demonstrate the formation of GT products, and iii) the platform should be simple and easy to implement or accessible to any laboratory. This work describes the development of a novel platform for screening of enzyme activities of GTs in vitro. It is called the in vitro GT-array (i-GTray) platform. This platform uses an in vitro cell-free expression system to produce tagged proteins directly from plasmid DNA and capture the tagged proteins on a solid surface, such as microplate wells, using a capture antibody (anti-tag antibody). Thus, i-GTray combines protein synthesis and purification with post-assay desalting paper spray-mass spectrometry (DPS-MS) analysis. DPS-MS has the capability to detect as low as 50 fmol amounts of transferase products.As a proof-of-concept, the i-GTray platform was used to investigate the activity of 21 putative fucosyltransferases (FUTs) members of the GT37 family (CAZy database) using five different acceptor substrates representing fucosylated plant CW polymers, such as xyloglucan (XyG), arabinogalactan-proteins (AGPs), and pectin (rhamnogalacturonan I and II (RG-I and RG-II)). This screening consisted in 288 assays (four microplates) and resulted in the identification of five rice XyG-FUTs, four rice AGP-FUTs, three Arabidopsis RG-I-FUTs, and one Arabidopsis RG-II-FUT. However, none of the 15-rice putative FUTs acted on RG-I and RG-II acceptors used in this study. The i-GTray platform was also used to investigate the specificity of protein interactions among rice GTs belonging to the GT43 and GT47 families, and the results obtained corroborated BiFC data. Data from both i-GTray and BiFC are in agreement and demonstrated the assembly of at least six OsGT43/OsGT47 complexes that represent the central core complexes of three xylan synthase complexes (OsXSCs).

Poplar (Populus sp.) is a promising biofuel feedstock due to advantageous features such as fast growth, the ability to grow on marginal land, and relatively low lignin content. However, there is tremendous variability associated with the composition of biomass. Understanding this variability, especially in lignin, is crucial to developing and implementing financially viable, integrated biorefineries. Although lignin is typically described as being comprised of three primary monolignols (syringyl, guaiacyl, p-hydroxyphenyl), it is a highly irregular biopolymer that can incorporate non-canonical monolignols. It is also connected by a variety of interunit linkages, adding to its complexity. Secondary cell wall formation requires the coordination of many metabolic pathways. Additionally, complex traits such as lignin are highly polygenic. While there are several methods to analyze lignin structure, 2D HSQC NMR is a powerful analytical tool that elucidates many structural traits of lignin simultaneously. This work examined the lignin structure of 409 unique Populus trichocarpa genotypes via HSQC NMR. Twelve lignin phenotypes were subsequently utilized for a genome-wide association study (GWAS) – a powerful approach for identifying loci contributing to natural phenotypic diversity. This deep phenotyping enabled GWAS to identify 756 candidate genes associated with at least one lignin phenotype. Many of these candidate genes have not been previously reported to be associated with lignin or cell wall biosynthesis. These results provide a valuable resource for gaining insights into the molecular mechanisms of lignin biosynthesis and offer new targets for future genetic improvement in poplar.

This dissertation outlines research quantifying the entering and exiting the quinone reaction chamber in NADH dehydrogenase or respiratory complex I. Respiratory complex I, the first complex in the respiratory electron transport chain. The respiratory electron transport is essential for all aerobic life. The methods used to quantify the entrance and exit process are geometric modeling, steered molecular dynamics, and singular value decomposition of the process. Five structures were analyzed: bacterial, yeast, ovine mt, mice mt, and human complex I. The structures reveal an almost 30 angstrom tunnel-like chamber for quinone binding in the core part of the enzyme, at the joint between the membrane and hydrophilic arms of the enzyme. The entrance of this quinone chamber located in ND1 subunit and has an apparent bottleneck of quinone/quinol passage. The first chapter introduces complex I and how transition state theory using diffusion kinetics gives an approximate maximum for the energy of crossing the bottleneck for quinone/quinol passage. Chapter 2 introduces the techniques used to quantify the difficulty of passage as well as methods to identify modes for collective confirmational changes for bottleneck opening. Chapters 3 and 4 are reproductions of the published papers based on this work. The appendices are reproductions of the supplemental information for those two papers.

The coronavirus disease 2019 (COVID-19) pandemic caused millions of deaths and serious economic disruptions, also boosted the unprecedented development of novel nucleic acid detection methods. Although polymerase chain reaction (PCR) is the golden-standard and has been widely used for practical nucleic acid detection, isothermal amplification strategies capable of rapid, inexpensive, and accurate nucleic acid detection also provide new options for large-scale pathogen detection, disease diagnosis, and genotyping. Here we are going to describe an assay development journey from a simple COVID-19 detection assay to a genotyping strategy and eventually to a droplet-based amplification-free assay.First, we described a highly sensitive multicomponent XNA-based nucleic acid detection platform that combines analyte preamplification with X10–23 mediated catalysis to detect the viral pathogen responsible for COVID-19. It is termed RNA-Encoded Viral Nucleic Acid Analyte Reporter (REVEALR), functions with a detection limit of ≤20 aM (∼10 copies/μL) using conventional fluorescence and paper-based lateral flow readout modalities. With a total assay time of 1 h, REVEALR provides a convenient nucleic acid alternative to equivalent CRISPR-based approaches, which have become popular methods for SARS-CoV-2 detection. The assay shows no cross-reactivity for other in vitro transcribed respiratory viral RNAs and functions with perfect accuracy against COVID-19 patient-derived clinical samples.Second, we explained how we design REVEALR into a novel genotyping assay that detects single-base mismatches corresponding to each of the major SARS-CoV-2 strains found in the United States. Of 34 sequence-verified patient samples collected in early, mid, and late 2021 at the UCI Medical Center in Orange, California, REVEALR accurately identified the correct variant. The assay, which is programmable and amenable to multiplexing, offers an important new approach to personalized diagnostics.Third, we talked about an improved REVEALR platform, termed digital droplet REVEALR (ddREVEALR), that can achieve direct viral detection and absolute quantitation utilizing a signal amplification strategy that relies on DNAzyme multiplexing and volume compression. Using an AI-assisted image-based readout, ddREVEALR was found to achieve 95% positive predictive agreement from a set of 20 nasopharyngeal swabs collected at UCI Medical Center in Orange, California. We suggest that the combination of amplification-free and protein-free analysis makes ddREVEALR a promising approach for direct viral RNA detection of clinical samples.Finally, we summarized the developed DNAzyme-based nucleic acid detection methods, offered some alternatives, compared the DNAzyme-based platforms with CRISPR based platforms, and gave insight on potential future directions to further elevate the REVEALR system.

Synthetic biology holds promise for adapting and modifying molecular systems found in nature toward improving human life. This has sparked a modern bioindustry with applications ranging from biomanufacturing pharmaceutical products to engineered microbes for supporting space exploration. In the last decade two biotechnological advances have revolutionized the field of synthetic biology: CRISPR/Cas-mediated genome editing and directed protein evolution. As these principles have matured, advances in automation and artificial intelligence have been quickly adopted as major tools toward the generation of new molecular biotechnologies.This thesis incorporates the use of specific genetic systems and broad principles of evolution toward engineering new biotechnologies. To adopt novel genetic systems in synthetic biology applications they must first be fully understood. Thus, an important interest underlying the applied aspect of synthetic biology is solving genetic and proteomic mechanisms of action through basic science. One way of doing so is to understand the effects that these genetic systems of interest have had on the natural evolution of the organisms that utilize them.Toward this interest, my first chapter aims to better understand the impact that diversity generating retroelements (DGRs), may have had on evolving vast morphological, physiological, or adaptive complexity observed within the cyanobacterial phylum. This first chapter develops the hypothesis that, in cyanobacteria, DGRs have evolved to accelerate the evolution of proteins of a distinct functional class, compared to the DGRs of all other prokaryotic organisms currently known. This further sets apart cyanobacteria as an incredibly unique and diverse taxonomic clade within bacteria and sets DGRs apart as an important player in the evolutionary success of cyanobacteria. A paper has been communicated on the work presented in my first chapter, detailed below. Aside from the potential evolutionary and ecological impact DGRs may have had within the cyanobacterial phylum they are also of interest for biotechnological applications. DGRs are a potential tool for continuous in-vivo, targeted hyper-diversification within a small region of a gene. This is a desirable tool for directed evolution efforts, as the field currently lacks a means for generating highly diverse random mutations in-vivo in targeted genomic loci. As such, in-vivo directed evolution schemes are generally not attempted, though the efficiency of in-vivo natural selection schemes may be much greater when compared to in-vitro screening of individual protein variants as a selection scheme.The remaining two chapters of my thesis deal with mining the marine environment for other molecular systems that hold significant promise for developing new biotechnologies including products that may aid in space exploration. Marine isolates of Bacillus subtilis have been shown to display a unique propensity for producing highly complex and highly bioactive secondary metabolites. Furthermore, their ability to form endospores that are highly resistant to extreme environmental conditions make them an ideal candidate for engineering toward bioremediation and drug production for space conditions.The majority of pharmaceuticals are set, conservatively, to expire within two years of the manufacturing date, after which the reported potency and pharmacokinetics are no longer guaranteed. Though there are efforts to study and extend pharmaceutical expiration dates, viability of a product will vary based on manufacturing lot, storage conditions, and packaging. The environs of spaceflight may also affect the decay of pharmaceuticals, though the extent remains unclear. As such, for longer crewed missions, the pharmaceutical supply at launch is unlikely to remain viable for the duration of the mission. However, replenishment becomes complicated on any space mission, and particularly on longer- duration missions, when resupply is difficult, and it is impossible to predict the specific medication needs of the crew. Thus, a platform for on-demand drug synthesis is necessary to ensure the success of long-term space exploration and colonization missions to Mars and beyond.Thus, my second chapter presents the first step in a three-step process known as an “Astropharmacy”, a peptide drug production platform that uses genetically engineered bacterial spores or a cell-free system to produce drugs on-demand. Not only would this platform allow astronauts to produce drugs according to their needs, but it would also have utility on Earth, with applications including bioterror prevention, orphan drug production, and drug production in remote areas where refrigeration is impossible. Whereas my second chapter deals with mining the marine environment for microbial hosts which have unique and favorable evolutionary characteristics for space, my third chapter seeks to isolate a microalgal marine protein and study its ability to be engineered further to meet different needs we may have on earth.Microalgae have been considered the most promising biological chemists for the mass production of biologically sourced fuels. Algal organisms may have adapted this ability as a way of regulating cell membrane curvature and hydrophobicity. One goal in biofuel-focused synthetic biology is to reverse-engineer pathways that lead to the production of different hydrocarbon molecules to increase the efficiencies, yields, and culture survivability. One of the newest discoveries that apply to this effort is fatty acid photodecarboxylase (FAP) – an enzyme which uses light to make fuel from acidic fats.My third chapter details the need for confirming homologs of the originally discovered enzyme. Can mutations that work in the original enzyme be successfully transferred to its homologs? If so, it would allow us to consider homologs for directed evolution strategies including chimeric gene splicing. It would also allow us to add molecular tools found in other organisms to the biocatalytic toolbox of enzymes that enable light-driven decarboxylation of fatty acid substrates.

Cyanobacteria are photosynthetic microbes that have shaped the very environment of the world we live in over several billion of years. This is, in large part, because of their ability to perform the chemically exclusive processes of photosynthesis and nitrogen fixation. To segregate these processes temporally, they have evolved an elegant and complex circadian clock that aligns their physiology with the solar day to maximize biological fitness. This clock keeps time through the action of a biochemical oscillator comprising just three proteins: KaiA, KaiB and KaiC, that, along with ATP, can recapitulate a 24-h pacemaking activity in vitro. This process is achieved by a negative feedback loop akin to a biochemical game of Rock, Paper, Scissors, whereby the phosphorylation state of the hexameric ATPase KaiC is controlled by the nucleotide exchange factor KaiA, the ability of KaiA to stimulate KaiC repression is controlled by the metamorphic protein KaiB, and the ability of KaiB to inactivate KaiA is controlled by KaiC phosphorylation state. This creates a repetitive biochemical cycle that takes just bout 24 hours to complete. Additionally, the output proteins SasA and CikA interact with the clock in various phases of the biochemical oscillation that influence their ability to activate the master circadian transcription factor RpaA and orchestrate circdain gene expression throughout the cyanobacterial cell.This thesis focuses on the relationship between KaiC and KaiB. In particular, on positive cooperativity that increases KaiB’s affinity for KaiC, or in other words, the process by which initial binding of KaiB to the KaiC hexamer enables more efficient recruitment of KaiB to the remaining 5 binding sites. While this effect is well documented when considering KaiB and KaiC on their own, in Chapter 2 we expand this concept in the in the context of the entire reconstituted clock system including output pathways that link biochemical oscillation to DNA binding by the transcription factor RpaA. In doing so, we uncovered the unexpected result that SasA expands the range of permissive KaiB concentrations for biochemical oscillation to occur. I showed that SasA does this by binding to KaiC analogously to how KaiB does, and then recruiting additional KaiB molecules through positive heterotropic cooperativity. Integrating a novel crystal structure of the interacting domains of KaiC and SasA with existing crystal structures of the KaiC hexamer, I identified SasA mutations that abrogate heterotropic cooperativity but have only minor effects on the output signaling function of SasA. Remarkably, cyanobacteria bearing these mutations have defective circadian rhythms, demonstrating that SasA’s ability to bolster KaiB recruitment is an evolved aspect of biochemical oscillation.Chapter 3 describes our structural analysis of phosphomimetic variants of KaiC that differ in their ability to bind KaiB. Because crystallography has failed to identify the structural basis of this discrimination previously, we employed cryo-electron microscopy to analyze KaiC particles as a structural ensemble frozen in ice. Each KaiC protomer is composed of two ATPase domains, termed CI and CII. The phase-determining phosphosites are located on CII, and KaiB binds to KaiC over 70 Å away to the ADP-bound form of CI. Our data corroborated previous studies that daytime KaiC, which does not bind KaiB, loses interactions amongst the CII domain protomers within the hexamer, while maintaining a hexameric CI domain. Additionally, we obtained a relatively high resolution (3.2 Å) structure of a compressed form of KaiC where the CII domain breaks into a split washer with 2-fold symmetry, causing two of the CII domain subunits to interact more tightly with their respective CI domains. Using mutagenesis and various functional assays, I identified an allosteric conduit that connects the ATPase domains of the CI and CII domains of KaiC in both intra-protomer and inter-protomer contexts. Importantly, I trace these interactions back to cooperativity in KaiB association, with mutants along the pathway disrupting both KaiB affinity and cooperativity. Furthermore, I link KaiB cooperativity to ATP hydrolysis in the CI domain by identifying a key residue that senses CI nucleotide state. This residue is dispensable for both ATP hydrolysis as well as KaiB association, but is critical for both KaiB cooperativity and in vivo circadian rhythms. This, along with additional nucleotide dependence studies reported in Chapter 4, suggests that allosteric control of cooperativity through the CI active site is the structural basis for restriction of KaiB association to the nighttime KaiC phosphostates.Finally, in the latter half of Chapter 4 I summarize experiments that used the solvatocromatic dye Sypro Orange to detect changes in KaiC structure as a function of temperature and mutagenesis. I describe our preliminary efforts to build on these results by identifying additional solvatochromatic dyes that can leverage this effect to report continuously on the phase of biochemical oscillation by discriminate binding to different KaiC phosphostates at constant temperature.

Wildlife trafficking is a global issue with devastating ecological effects and synergistic relationships with other forms of international illicit networks. Furs and pelts are a major component of this trade and are more practical to transport. Proper identification of such items is important to be able to prosecute and punish poachers. Wildlife forensic investigators depend on reliable tools and methods to either include or exclude illegal versus legal species. However, due to the chemically and physically harsh production process, DNA-based methods often fail because the sample DNA is degraded. Morphological-based methods have poor species resolution, are time-consuming, and are dependent on a limited number of highly trained individuals. However, these challenges may be by-passed by focusing on the phylogenetic information in protein. DNA changes between closely related species are reflected at the protein level, allowing the phylogenetic information between species to lead to a species-level identification. Proteins are chemically more stable than DNA. Therefore, proteomic analysis of heavily processed items may provide an alternative, and robust, method of species identification. However, determining the amino acid sequences in mass spectrometry data requires the precise sequences to be present in a reference protein database. The hypothesis is that the protein sequences, or reference proteome, of a given species would match more than proteomes from other species and be more efficient at identifying peptide sequences in fur digests from the same species. This research focuses on the phylogenetic relationship between six cat species, lion (Panthera leo), leopard (Panthera pardus), tiger (Panthera tigris), cheetah (Acinonyx jubatus), Canada lynx (Lynx canadensis), and domestic cat (Felis catus); of which the first four had good-quality, morphologically-identified materials that were selected for analysis. The remaining two species, Canada lynx and domestic cat, were included in the phylogenetic comparison using their reference proteome databases. The selected fur or pelt reference material items were digested based on a previously-published method for protein extraction from human hair and analyzed by LC-MS/MS and PEAKS DB software. Species identification was achieved at three different levels: total peptide yield, protein coverage patterns, and presence of peptides containing species-specific markers. For each item sampled, the maximum number of total peptides identified using the PEAKS scoring algorithm was obtained using the corresponding theoretical database. For example, the raw peptides generated from the lion paw pelt yielded 13928 total identified peptides using the lion database. As evolutionary distance between the lion and related felid species increases, the total peptide yield decreases. Using the leopard, tiger, Canada lynx, domestic cat, and cheetah databases, the total peptide yields were less - 13766, 13758, 12604, 12307, and 11774, respectively. Similar results were obtained with the leopard pelt, tiger pelt, and cheetah coat sampled, demonstrating how species identification can be achieved through comparison of total peptide yields. Protein coverage patterns also indicated species of origin. Many keratin and keratin-associated proteins were identified from the hair and skin samples, including, but not limited to, Keratin 14, Keratin 16, Keratin 32, Keratin 35, Keratin 38, Keratin 75, Keratin 82, Keratin 85, Desmoplakin, and Corneodesmosin. Coverage patterns demonstrated the highest percent coverage when analyzing the raw peptides from a sample with its corresponding database. For example, the raw peptides generated from the lion sample that matched with Keratin 35 resulted in 84% coverage using the lion database, while the tiger, leopard, domestic cat, Canada lynx, and cheetah databases resulted in 77%, 75%, 77%, 75%, and 65%, respectively. Species-specific marker locations were identified by protein alignments between the sequences of all six species. Peptides containing these species-specific markers were then identified in the raw data and found to be present only in the samples that correlated with the species of origin. This paper demonstrates four individual examples of species-specific markers within Keratin 35 corresponding to the respective species of origin for all four sampled items: lion, leopard, tiger, and cheetah. In summary, the three approaches used in this project – the total peptide approach, the protein coverage pattern approach, and the species-specific peptide approach – individually demonstrated that species identification is plausible using phylogenetic proteomics. This data demonstrates the robustness and reliability of proteomic approaches to species identification from fur and lays the foundation for future research, such as the development of targeted proteomic assays, for broader application to wildlife forensic casework.

Pathogenic multidrug resistant bacteria cause a range of serious infections in humans. These bacteria have developed mechanisms to counteract the lethal effects of currently used antibiotics, creating a need for novel therapeutics. Gram-positive bacteria display a wide assortment of cell surface proteins that are important for bacterial survival and host-pathogen interactions. These key surface structures included pili, proteinaceous fibers that assist in microbial survival by mediating adhesion to host tissues and aid in the formation of biofilm. A large number of gram-positive bacterial species assemble pili and append surface proteins to the cell wall using sortase cysteine transpeptidase enzymes. These enzymes link the components of the pilus together via covalent lysine isopeptide bonds which confer enormous tensile strength. This dissertation describes my investigation of the assembly mechanism of the archetypal SpaA-pilus from Corynebacterium diphtheriae. It also describes my contributions to develop small molecule sortase inhibitors that could function as anti-infective agents and my work towards exploiting the activity of sortase enzymes as a protein engineering tool. This thesis focuses on sortase enzymes and can be divided into two major sections: studies to determine how sortases construct pili (Chapters 2-4) and work designed to discover a small molecule inhibitor for the Staphylococcus aureus Sortase A enzyme (Chapter 5). All of the studies have been published in peer reviewed papers, with the exception of work detailed in Chapter 4. Chapter 2 describes the NMR solution structure of the lysine isopeptide bond interface that connects the pilin components of the pilus. This structural information combined with biophysical and cellular analyses led to the formulation of the “latch” mechanism of pilus assembly. Chapter 3 describes an enzyme kinetic study of the sortase enzyme from C. diphtheriae (CdSrtA) which catalyzes the formation of lysine isopeptide bonds between components of the SpaA pilus. In this study, the rate-limiting step of catalysis was determined and variants of CdSrtA with improved activity were discovered. Chapter 4 describes research that employed biochemical and structural approaches to investigate how the incorporation of the SpaB pilin subunit terminates pilus assembly. Key differences were observed between the reaction that terminates assembly and the process of polymerization that builds the shaft of the pilus. Chapter 5 describes efforts to discover a small molecule inhibitor of the Staphylococcus aureus Sortase A enzyme that has the potential to be a therapeutically useful anti-infective agent. The first half of Chapter 5 describes the work done to improve the activity of previously discovered pyridazinone-based molecules using computational and synthetic chemistry methods. The second half describes the implementation of a novel cell-based screen to identify sortase inhibitors. This work leveraged the unique sortase-dependent growth phenotype of Actinomyces oris to screen for sortase inhibitors. Over 200,000 small molecules were screened for their ability to impair A.oris’s growth, which led to the identification of three molecular scaffolds that inhibit sortase activity in vitro. In totality, my thesis research has shed light on how sortase enzymes assemble pili in gram-positive bacteria, led to improved variants of CdSrtA sortase that can be used in protein engineering, and helped identify new small molecule sortase inhibitors with potential therapeutic applications.

HP1(Heterochromatin Protein 1) is a major component of heterochromatin, which is a highly condensed form of DNA playing an important role in multiple cellular activities including gene silencing. New research proposes that HP1 proteins could compartmentalize DNA into compacted chromatin by phase separation, which could be promoted by diverse HP1-binding proteins. STAT is a promising candidate. Besides its canonical role in JAK-STAT signaling pathway, previous research in our lab indicated that STAT contains HP1-binding PXVXI motif. Unphosphorylated STAT could bind to HP1 and maintain the stability of heterochromatin while phosphorylated STAT disperses from HP1, resulting in heterochromatin disruption. Thus, I hypothesized that phosphorylation induced conformational change on STAT, switching it from an HP1-binding state to a DNA-binding state. In this paper, I constructed computational models among HP1α, STAT3, and DNA to examine the influence of phosphorylation on STAT3’s binding affinity to both HP1α and DNA. During the preparation stage, I modified and constructed biomolecular structures for protein docking by Pymol and SWISS-MODEL. I imported the prepared biomolecular structures into HADDOCK and the web server provided potential binding complexes as output. I used the PRODIGY program to measure the Gibbs free energy and equilibrium constant of binding among unphosphorylated STAT3, HP1α and DNA as well as phosphorylated STAT3, HP1α and DNA. Compared to phosphorylated STAT3 homodimers, unphosphorylated STATA3 homodimers have higher binding affinity to HP1α and lower binding affinity to DNA. Although computational model has limitations and needs confirmation by further experiments in vitro, my results support the conclusion that phosphorylation drives STAT from HP1-binding to DNA-binding.

For much of human history, microbial infections have been a problem. The discovery of antibiotics has led to the prevention of many deaths and allowed for surgeries that would be too risky otherwise to be performed. In the post-antibiotic era antibiotic resistance has become an ever-growing crisis. Overuse and misuse of antibiotics have allowed for the development of resistance to antibiotics by many strains of bacteria. There have also been very few new antibiotics that have been developed in the past three decades. Antibiotics act on single biological pathways of bacteria and can be deactivated with relative ease, particularly when bacteria are exposed to a subinhibitory concentration. Pseudomonas aeruginosa (PA) is a gram-negative bacterium that has been able to develop resistance to many antibiotics. Nanoparticulate matter has properties that differ from their bulk counterparts. Many metallic nanoparticles have been examined as antimicrobial agents. These nanoparticles tend to harm bacterial growth by many different pathways simultaneously, making the development of resistance by bacteria much more difficult. Silver has been used as an antimicrobial agent for much of human history. Metallic silver nanoparticles have been explored as antimicrobial agents, but systematic evaluation of other forms of silver nanoparticles has been rare. Silver halides are photoreactive materials, most used in paper photography. In this work, I systematically evaluate the antimicrobial activity of silver halide nanoparticles against PA. The preliminary results indicate that silver halide nanoparticles exhibit higher antimicrobial activity and better silver resistance profiles when compared with the metal silver nanoparticles.