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Motivation The quest for efficient drug discovery processes necessitates a comprehensive approach that integrates molecular feature analysis with accurate compound-protein interaction (CPI) prediction. This study introduces models that combine deep learning (DL) techniques for intricate molecular feature engineering and innovative CPI prediction methods. This integration responds to the need for detailed molecular dataset analysis and the prediction of interactions between novel compounds and proteins, thereby enhancing drug discovery. Methods and Results Chapter 3 - Molecular Clustering and Feature Analysis: The framework implements a feature engineering scheme focusing on molecule-specific atomic and bonding information. It utilizes principal component analysis (PCA) for encoding this information and a variational autoencoder (VAE)-based method for embedding both global chemical properties and local features. This approach facilitated the clustering of a large dataset containing over 47,000 molecules. Using the K-means method with 32 embedding`s size based on the VAE method, 50 distinct molecular clusters were identified. These clusters were visualized through t-distributed Stochastic Neighbor Embedding (t-SNE), showcasing the framework's capability in effectively grouping molecules based on their complex features. Chapter 4 - CPI Prediction with GraphBAN: For CPI prediction, the study introduces GraphBAN, a novel inductive-based approach using graph knowledge distillation (KD). This component incorporates a deep bilinear attention network (BAN) and a KD module for graph analysis, enabling the alignment of interaction features across different distributions. GraphBAN's functionality extends to both transductive and inductive link predictions in a bi-partite graph of CPIs. Tested against three benchmark datasets, GraphBAN demonstrated superior performance, outperforming six baseline models. It shows that it is able to predict interactions between unseen compounds a

Human adenovirus (HAdV) is a critical tool and essential model for studying virology, cell biology and molecular biology. Research into HAdV has led to a comprehensive understanding of the virus and its interaction with the host cell. These properties have been used to exploit HAdV to develop therapeutics and vaccines, and to understand the cell’s inner workings. This study first explores the use of HAdV as a molecular biology tool to develop and create a set of recombinant HAdV (rHAdV) to be used as potential vaccines or research reagents for the coronavirus disease 2019 (COVID-19) pandemic. Secondly, it utilizes the critical viral protein Early 1A (E1A) to identify the cellular E1A interaction hub protein, arginine and glutamate rich 1 (ARGLU1).

Motivation: With the advent of large-scale compound screening facilitated by the high-throughput technologies, a variety of machine learning methods have been integrated into the pipelines of antibiotic discovery. Feature engineering, a type of data mining technique, is often used as one of the first steps to mine patterns from big data and optimize predictive models for the goal. Clustering analysis, on the other hand, is a critical approach to get insights into the underlying biological relationships between the gene products in the high-dimensional chemical-genetic data. Converting molecules into computer-interpretable features with rich molecular information is a core problem of data-driven machine learning applications in chemical and drug-related tasks. As small molecules can be considered as non-structural data, graph convolutional neural networks (GCNs), which can learn and aggregate the local information of molecules, have been used to predict molecular properties with great success. Furthermore, given the merits and various successful practices of transformers in multiple artificial intelligence (AI) domains, it is desirable to integrate the self-attention mechanism into GCNs for better molecular representation construction. Methods and Results: This thesis begins with a review of different types of molecular representations and deep learning architectures to which they are applicable in Chapter 1 and the research objectives were described in Chapter 2. Next, Chapter 3 first applied statistical and machine learning approaches to evaluate the important features for predicting bacterial growth inhibitory activity , then applied a directed-message passing neural network to analyze a large-scale compound screen against Burkholderia cenocepacia to predict the bacterial growth inhibition of drugs. In Chapter 4, a directed-message passing neural network-based analytic framework was developed to model the large-scale chemical-genetic interaction profiles against M

Motivation: With the advent of large-scale compound screening facilitated by the high-throughput technologies, a variety of machine learning methods have been integrated into the pipelines of antibiotic discovery. Feature engineering, a type of data mining technique, is often used as one of the first steps to mine patterns from big data and optimize predictive models for the goal. Clustering analysis, on the other hand, is a critical approach to get insights into the underlying biological relationships between the gene products in the high-dimensional chemical-genetic data. Converting molecules into computer-interpretable features with rich molecular information is a core problem of data-driven machine learning applications in chemical and drug-related tasks. As small molecules can be considered as non-structural data, graph convolutional neural networks (GCNs), which can learn and aggregate the local information of molecules, have been used to predict molecular properties with great success. Furthermore, given the merits and various successful practices of transformers in multiple artificial intelligence (AI) domains, it is desirable to integrate the self-attention mechanism into GCNs for better molecular representation construction. Methods and Results: This thesis begins with a review of different types of molecular representations and deep learning architectures to which they are applicable in Chapter 1 and the research objectives were described in Chapter 2. Next, Chapter 3 first applied statistical and machine learning approaches to evaluate the important features for predicting bacterial growth inhibitory activity , then applied a directed-message passing neural network to analyze a large-scale compound screen against Burkholderia cenocepacia to predict the bacterial growth inhibition of drugs. In Chapter 4, a directed-message passing neural network-based analytic framework was developed to model the large-scale chemical-genetic interaction profiles against M

Human adenovirus (HAdV) is a critical tool and essential model for studying virology, cell biology and molecular biology. Research into HAdV has led to a comprehensive understanding of the virus and its interaction with the host cell. These properties have been used to exploit HAdV to develop therapeutics and vaccines, and to understand the cell’s inner workings. This study first explores the use of HAdV as a molecular biology tool to develop and create a set of recombinant HAdV (rHAdV) to be used as potential vaccines or research reagents for the coronavirus disease 2019 (COVID-19) pandemic. Secondly, it utilizes the critical viral protein Early 1A (E1A) to identify the cellular E1A interaction hub protein, arginine and glutamate rich 1 (ARGLU1).

Degradation of extracellular Polyhydroxyalkanoates (PHAs) in the environment depends on the secretion of specific extracellular PHA depolymerases and lipases. Compared to extracellular scl-PHA degrading enzymes, extracellular enzymes that can degrade mcl-PHAs are poorly investigated. In this project, Proteobacteria members that produce extracellular mcl-PHAs depolymerases/ lipases were screened on mcl-PHA agar plates. Then, genes involved in the production and regulation of mcl-PHA depolymerases/lipases were identified using transposon mutagenesis, insertional mutagenesis, and bioinformatics prediction tools. Once the genes were identified, the mcl-PHA depolymerase/lipase encoding genes were cloned and characterized. Burkholderia thailandensis E264, Stenotrophomonas maltophilia K279a, S. maltophilia D457, and S. maltophilia NCPPB1683 were found to produce extracellular mcl-PHA depolymerases/lipases. In Pseudomonas chlororaphis PA23, transposon mutagenesis showed that disruption of certain metabolic pathways positively affects mcl-PHA depolymerase/lipase production. Transposon mutagenesis of B. cepacia ATCC 25416 indicated that disruption of the APZ15_13320 gene negatively affected the production of pigmented metabolites and positively affected mcl-PHA depolymerase/lipase production. A novel S. maltophilia strain that can produce mcl-PHA depolymerase/lipase was discovered during the screening process as a contaminant strain of the T. caryophylli DSMZ16666. Using whole-genome sequencing and bioinformatics, a putative extracellular mcl-PHA depolymerase/lipase known as fig|40323.255.peg.2780 was identified in the novel S. maltophilia HT01 strain. The in-silico prediction of a putative extracellular mcl-PHA depolymerase/lipase in S. maltophilia K279a resulted in twenty-five protein-coding sequences. Five of the twenty-five putative depolymerase/lipases were cloned in E. coli. The E. coli clones expressing these genes were screened for degradation of polyhydroxyoctanaoate

Degradation of extracellular Polyhydroxyalkanoates (PHAs) in the environment depends on the secretion of specific extracellular PHA depolymerases and lipases. Compared to extracellular scl-PHA degrading enzymes, extracellular enzymes that can degrade mcl-PHAs are poorly investigated. In this project, Proteobacteria members that produce extracellular mcl-PHAs depolymerases/ lipases were screened on mcl-PHA agar plates. Then, genes involved in the production and regulation of mcl-PHA depolymerases/lipases were identified using transposon mutagenesis, insertional mutagenesis, and bioinformatics prediction tools. Once the genes were identified, the mcl-PHA depolymerase/lipase encoding genes were cloned and characterized. Burkholderia thailandensis E264, Stenotrophomonas maltophilia K279a, S. maltophilia D457, and S. maltophilia NCPPB1683 were found to produce extracellular mcl-PHA depolymerases/lipases. In Pseudomonas chlororaphis PA23, transposon mutagenesis showed that disruption of certain metabolic pathways positively affects mcl-PHA depolymerase/lipase production. Transposon mutagenesis of B. cepacia ATCC 25416 indicated that disruption of the APZ15_13320 gene negatively affected the production of pigmented metabolites and positively affected mcl-PHA depolymerase/lipase production. A novel S. maltophilia strain that can produce mcl-PHA depolymerase/lipase was discovered during the screening process as a contaminant strain of the T. caryophylli DSMZ16666. Using whole-genome sequencing and bioinformatics, a putative extracellular mcl-PHA depolymerase/lipase known as fig|40323.255.peg.2780 was identified in the novel S. maltophilia HT01 strain. The in-silico prediction of a putative extracellular mcl-PHA depolymerase/lipase in S. maltophilia K279a resulted in twenty-five protein-coding sequences. Five of the twenty-five putative depolymerase/lipases were cloned in E. coli. The E. coli clones expressing these genes were screened for degradation of polyhydroxyoctanaoate

For successful bacterial infection, bacteria need to sense and respond to their environment. Quorum sensing (QS) allows bacteria to regulate their virulence in a cell-density dependent manner. Polymicrobial infections are prevalent in cystic fibrosis (CF) infections but the interactions between these microbes are not well understood. The phenylacetic acid (PAA) pathway is upregulated in CF-like conditions and PAA was linked to the attenuation of virulence in two major CF pathogens, Burkholderia cenocepacia and Pseudomonas aeruginosa. However, the mechanism of attenuation remains unknown. The goals of my thesis were to investigate the role of PAA release in CF pathogen interactions, identify the PAA pathway metabolite responsible for the attenuation of virulence, and determine if the mechanism of inhibition is due to an effect on the CepIR QS system (either directly or indirectly). I investigated the accumulation of PAA in CF pathogens. I determined that B. cenocepacia was the most likely candidate for the accumulation of PAA but that PAA did not affect the pathogen interactions. I then characterized knockout mutants of the first steps of the PAA pathway (PaaK or PaaABCDE) to determine which PAA metabolite(s) are responsible for the attenuation of virulence. While loss of the paaABCDE operon resulted in decreased virulence, a paaK deficient strain had increased virulence compared to wild type despite the fact that both mutations resulted in higher levels of external PAA. Although we found no evidence of direct cepI or cepR downregulation by PAA or PAA-CoA, a low-virulence cepR mutant reverted to a virulent phenotype upon removal of the paaK genes. Whereas removal of the paaABCDE operon in the cepR mutant had no bearing on its attenuated phenotype. These results demonstrate that B. cenocepacia can elicit a pathogenic response if PAA-CoA is not produced. Conversely, the accumulation of PAA-CoA appears to attenuate pathogenicity. In summary, this thesis demonstrates tha

Pseudomonas chlororaphis PA23 is a biocontrol agent (BCA) that is able to protect canola against the pathogenic fungus Sclerotinia sclerotiorum. PA23 secretes a number of metabolites that contribute to fungal antagonism, including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN) and degradative enzymes. Beyond pathogen suppression, the success of a BCA is dependent upon its ability to persist in the environment and avoid the threat of grazing predators, including protozoa. The first part of this thesis investigated whether PA23 is able to resist predation by Acanthamoeba castellanii (Ac) and defined the role of antifungal (AF) compounds in the bacterial-protozoan interaction. We discovered that PRN, PHZ and HCN contribute to PA23 toxicity towards Ac trophozoites. Ac preferentially migrated towards regulatory mutants devoid of AF metabolites as well as a PRN biosynthetic mutant, indicating that AF metabolites act to repel Ac. We also discovered that toxin-producing strains were able to survive inside trophozoites for up to 24 h. Collectively, our findings indicate that PRN, PHZ and HCN are involved in amoebicidal activity, and through the production of these molecules, PA23 is able to avoid the threat of predation. PA23 accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds. The second part of this thesis aimed to elucidate the role of PHAs in PA23 stress resistance and interaction with Ac. Three PHA biosynthesis mutants were created, PA23phaC, PA23phaC1ZC2 and PA23phaC1ZC2D, which no longer accumulated PHA. We observed that PA23phaC1ZC2D produced less PHZ compared to wild type (WT). All three mutants exhibited enhanced sensitivity to UV radiation, starvation, heat and cold stress, and exposure to hydrogen peroxide. Moreover, a lack of PHA production resulted in increased motility, biofilm formation, exopolysaccharide production and root attachment. Interaction studies with the protozoan predator Ac revealed that the WT, PA2

For successful bacterial infection, bacteria need to sense and respond to their environment. Quorum sensing (QS) allows bacteria to regulate their virulence in a cell-density dependent manner. Polymicrobial infections are prevalent in cystic fibrosis (CF) infections but the interactions between these microbes are not well understood. The phenylacetic acid (PAA) pathway is upregulated in CF-like conditions and PAA was linked to the attenuation of virulence in two major CF pathogens, Burkholderia cenocepacia and Pseudomonas aeruginosa. However, the mechanism of attenuation remains unknown. The goals of my thesis were to investigate the role of PAA release in CF pathogen interactions, identify the PAA pathway metabolite responsible for the attenuation of virulence, and determine if the mechanism of inhibition is due to an effect on the CepIR QS system (either directly or indirectly). I investigated the accumulation of PAA in CF pathogens. I determined that B. cenocepacia was the most likely candidate for the accumulation of PAA but that PAA did not affect the pathogen interactions. I then characterized knockout mutants of the first steps of the PAA pathway (PaaK or PaaABCDE) to determine which PAA metabolite(s) are responsible for the attenuation of virulence. While loss of the paaABCDE operon resulted in decreased virulence, a paaK deficient strain had increased virulence compared to wild type despite the fact that both mutations resulted in higher levels of external PAA. Although we found no evidence of direct cepI or cepR downregulation by PAA or PAA-CoA, a low-virulence cepR mutant reverted to a virulent phenotype upon removal of the paaK genes. Whereas removal of the paaABCDE operon in the cepR mutant had no bearing on its attenuated phenotype. These results demonstrate that B. cenocepacia can elicit a pathogenic response if PAA-CoA is not produced. Conversely, the accumulation of PAA-CoA appears to attenuate pathogenicity. In summary, this thesis demonstrates tha

Antibiotic resistance is a fast-growing crisis worldwide and has been estimated to cause 10 million deaths by the year 2050, which is more than the present number of deaths caused by cancer and road accidents combined. Recently, WHO has published a priority list of pathogens against which new antibiotics are urgently needed. In this list, Pseudomonas aeruginosa has been listed among the Priority-1 (critical) pathogens. Among the various resistance mechanisms used by P. aeruginosa, the use of Resistance-Nodulation-Division (RND) efflux pumps is an intrinsic resistance mechanism. RND efflux pumps are the tripartite complex consisting of outer membrane protein (OMP), membrane-fusion protein (MFP), and RND antiporter. Among 12 well-characterized RND efflux pumps found in the genome of P. aeruginosa, the MexXY efflux system has been reported to be overexpressed in the clinical isolates, especially isolates obtained from Cystic Fibrosis (CF) patients. Overexpression of the MexXY efflux pump leads to reduced susceptibility against aminoglycosides, a routinely used antibiotic for the treatment of CF patients. Understanding the process of antibiotic resistance through RND efflux pumps requires the knowledge of protein-protein interaction among RND complexes and their mechanism and determinants of substrate selectivity. This study is focused on understanding the role of RND efflux systems in multidrug resistance among CF isolates and determining the substrate determinants of an RND complex. Determinants of substrate selectivity were studied using MexXY-OprA/OprM efflux system as a model due to its clinical significance among CF isolates of P. aeruginosa, and its ability to form a functional complex with two different OMPs. Since OprA is only found in P. aeruginosa PA7 strain (and PA7-type genomes), we also compared the genome of PA7 with PAO1 (type-strain) to find any other clinically-relevant antibiotic resistance and virulence genes. Our study shows MexXY to be the most ove

Lichens are traditionally described as symbionts of fungi and algae and are renowned for their diverse secondary metabolites. How lichens produce these natural products, such as their biosynthetic pathways and the genes that are required to produce these molecules, remains unknown. The genome of the fungal partner of the lichen Cladonia uncialis was de novo sequenced and its genetic secondary metabolome was annotated. This work revealed 48 secondary metabolite biosynthetic gene clusters, providing a first glimpse into the genetic programming of lichen polyketides, terpenes, and non-ribosomal peptides. A ‘deductive approach’ employing retro-biosynthetic reasoning was applied to find the genes responsible for the biosynthesis of usnic acid, the most extensively studied lichen secondary metabolite. A ‘homology mapping’ approach, combining phylogenetics and rational pathway deductions, provided putative assignments of biosynthetic function to nine gene clusters in C. uncialis, including what appears to be gene clusters encoding patulin, the betaenones, 6-hydroxymellein, and grayanic acid. The development of a reliable heterologous expression protocol is a prerequisite to an advanced understanding of lichen secondary metabolite biosynthesis. The heterologous expression of seven C. uncialis polyketide synthases was investigated using the NSAR1 A. oryzae platform. These experiments revealed that A. oryzae can transcribe lichen genes and remove introns to produce translation-coherent mRNA. Though de novo biosynthesis of lichen metabolites was not observed in A. oryzae, this work propels lichen natural product studies into the genomic era and provide a foundation upon which future studies may strive to uncover the rich biosynthetic potential of these enigmatic organisms.

This study mainly focuses on the rational design, synthesis and bioassay testing of netrin-1/UNC5 and netrin-1/DCC inhibitors. This study began with the development of a virtual screening method to select out candidates from virtual libraries. These candidates were then synthesized and the binding affinity between the synthesized molecules and netrin-1 was evaluated by microscale thermophoresis.

Antibiotic resistance is a fast-growing crisis worldwide and has been estimated to cause 10 million deaths by the year 2050, which is more than the present number of deaths caused by cancer and road accidents combined. Recently, WHO has published a priority list of pathogens against which new antibiotics are urgently needed. In this list, Pseudomonas aeruginosa has been listed among the Priority-1 (critical) pathogens. Among the various resistance mechanisms used by P. aeruginosa, the use of Resistance-Nodulation-Division (RND) efflux pumps is an intrinsic resistance mechanism. RND efflux pumps are the tripartite complex consisting of outer membrane protein (OMP), membrane-fusion protein (MFP), and RND antiporter. Among 12 well-characterized RND efflux pumps found in the genome of P. aeruginosa, the MexXY efflux system has been reported to be overexpressed in the clinical isolates, especially isolates obtained from Cystic Fibrosis (CF) patients. Overexpression of the MexXY efflux pump leads to reduced susceptibility against aminoglycosides, a routinely used antibiotic for the treatment of CF patients. Understanding the process of antibiotic resistance through RND efflux pumps requires the knowledge of protein-protein interaction among RND complexes and their mechanism and determinants of substrate selectivity. This study is focused on understanding the role of RND efflux systems in multidrug resistance among CF isolates and determining the substrate determinants of an RND complex. Determinants of substrate selectivity were studied using MexXY-OprA/OprM efflux system as a model due to its clinical significance among CF isolates of P. aeruginosa, and its ability to form a functional complex with two different OMPs. Since OprA is only found in P. aeruginosa PA7 strain (and PA7-type genomes), we also compared the genome of PA7 with PAO1 (type-strain) to find any other clinically-relevant antibiotic resistance and virulence genes. Our study shows MexXY to be the most ove

Lichens are traditionally described as symbionts of fungi and algae and are renowned for their diverse secondary metabolites. How lichens produce these natural products, such as their biosynthetic pathways and the genes that are required to produce these molecules, remains unknown. The genome of the fungal partner of the lichen Cladonia uncialis was de novo sequenced and its genetic secondary metabolome was annotated. This work revealed 48 secondary metabolite biosynthetic gene clusters, providing a first glimpse into the genetic programming of lichen polyketides, terpenes, and non-ribosomal peptides. A ‘deductive approach’ employing retro-biosynthetic reasoning was applied to find the genes responsible for the biosynthesis of usnic acid, the most extensively studied lichen secondary metabolite. A ‘homology mapping’ approach, combining phylogenetics and rational pathway deductions, provided putative assignments of biosynthetic function to nine gene clusters in C. uncialis, including what appears to be gene clusters encoding patulin, the betaenones, 6-hydroxymellein, and grayanic acid. The development of a reliable heterologous expression protocol is a prerequisite to an advanced understanding of lichen secondary metabolite biosynthesis. The heterologous expression of seven C. uncialis polyketide synthases was investigated using the NSAR1 A. oryzae platform. These experiments revealed that A. oryzae can transcribe lichen genes and remove introns to produce translation-coherent mRNA. Though de novo biosynthesis of lichen metabolites was not observed in A. oryzae, this work propels lichen natural product studies into the genomic era and provide a foundation upon which future studies may strive to uncover the rich biosynthetic potential of these enigmatic organisms.

This study mainly focuses on the rational design, synthesis and bioassay testing of netrin-1/UNC5 and netrin-1/DCC inhibitors. This study began with the development of a virtual screening method to select out candidates from virtual libraries. These candidates were then synthesized and the binding affinity between the synthesized molecules and netrin-1 was evaluated by microscale thermophoresis.

Immunoglobulins (IgGs) play a central role in the immune system of living organisms. Human IgG comprises of 4 subclasses, ranging from IgG1 to IgG4, of which IgG1 and IgG2 are the most abundant in healthy individuals. In an effort to develop an absolute MALDI-ToF-MS quantitation method for these subclasses and their Fc N-glycoforms, peptides and (glyco)peptides were synthesized using a solid phase approach and used as internal standards for enriched fractions from human IgG tryptic digests. Mass spectrometry was used to analyze biomolecules quantitatively throughout the experiment. Tryptic digest glycopeptides from monoclonal IgG1 and IgG2 samples were first quantified using EEQYN(GlcNAc)STYR and EEQFN(GlcNAc)STFR, respectively. It was observed that simultaneous quantitation of IgG1 and IgG2 yielded non-quantitative results, and more success was obtained when subclasses were quantified one by one. The study also explored the relative sensitivities of two synthetic Fc tryptic peptides of IgG1 and IgG2 under MALDI conditions.

Immunoglobulins (IgGs) play a central role in the immune system of living organisms. Human IgG comprises of 4 subclasses, ranging from IgG1 to IgG4, of which IgG1 and IgG2 are the most abundant in healthy individuals. In an effort to develop an absolute MALDI-ToF-MS quantitation method for these subclasses and their Fc N-glycoforms, peptides and (glyco)peptides were synthesized using a solid phase approach and used as internal standards for enriched fractions from human IgG tryptic digests. Mass spectrometry was used to analyze biomolecules quantitatively throughout the experiment. Tryptic digest glycopeptides from monoclonal IgG1 and IgG2 samples were first quantified using EEQYN(GlcNAc)STYR and EEQFN(GlcNAc)STFR, respectively. It was observed that simultaneous quantitation of IgG1 and IgG2 yielded non-quantitative results, and more success was obtained when subclasses were quantified one by one. The study also explored the relative sensitivities of two synthetic Fc tryptic peptides of IgG1 and IgG2 under MALDI conditions.

The metabolism and physiology of Pseudomonas putida strain LS46 was investigated using biodiesel-derived waste streams as potential low cost substrates for production of medium chain length polyhydroxyalkanoates (mcl-PHA). Proteomic and trranscriptomic analyses were used to correlate specific gene and gene product expression patterns with differences in phenotypes of mcl-PHA biosynthesis by P. putida LS46. Growth and mcl-PHA content of P. putida LS46 were similar in cultures containing biodiesel-derived waste glycerol versus pure glycerol, and mcl-PHA synthesis occurred during stationary phase after nitrogen concentrations in the medium were exhausted. Waste glycerol cultures contained elevated concentrations of heavy metal ions, such as copper, which induced significant changes in gene expression levels related to heavy metal resistance. Several membrane-bound proteins, such as CusABC efflux and CopAB were identified and putatively play a role in regulating cellular copper concentrations. Cultures containing waste free fatty acids synthesized mcl-PHA throughout the exponential growth phase. Protein expression levels of two mcl-PHA synthases were suppressed during exponential phase growth in waste glycerol cultures, putatively via post-transcriptional regulation. Culture specific expression of monomer supplying proteins (PhaJ1 and PhaG), and sets of fatty acid oxidation enzymes were observed, and may have contributed to differences in the composition of polymers synthesized by P. putida LS46 cultured on the two substrates. Expression levels of the majority of mcl-PHA biosynthesis pathway genes were stable during active polymer synthesis in waste glycerol cultures. However, variations in protein expression levels, and in some cases their corresponding mRNAs, were observed in a number of other metabolic patheays, such as glycerol transportation, partial glycolysis, pyruvate metabolism, the TCA cycle, and fatty acid biosynthesis. These data suggest potential regulatory

Pseudomonas aeruginosa causes various infections in humans, animals and plants. Type III secretion system (T3SS) is one of the essential virulence factors used by P. aeruginosa. In this study, a previously uncharacterized gene PA0466 and its role in T3SS regulation have been examined. The results indicate that PA0466 is a novel T3SS regulator. It regulates T3SS directly through an unknown pathway and has a minor effect on the GacA-RsmA pathway. Besides the role in the interaction between the pathogen and the host, T3SS may also play a role in the interspecies interaction. A real-time PCR based Competitive Index (CI) assay was used to compare the wild type and T3SS mutant with and without the presence of Staphylococcus spp.. The results indicate that PAO1 was more competitive than exsA mutant and the difference was even bigger in the presence of Staphylococcus, suggesting T3SS may play a significant role in bacterium-bacterium interaction.

Nidoviruses are an order of positive-sense RNA viruses, which include the families Arteriviridae and Coronaviridae. Nidoviruses express their complement of non-structural proteins (nsps) as a single polyprotein, which is cleaved into functional domains by proteases encoded within. The equine arterivirus (EAV) and Middle East respiratory syndrome coronavirus (MERS-CoV) encode for papain-like protease domains within nsp2 and nsp3, respectively, which serve a replicative role through their polyprotein processing activities. During infection, the host innate immune response is triggered by incoming pathogens, and engages multiple signalling pathways which are in-part dependent on the post-translational modification by ubiquitin (Ub). These signalling processes are tightly regulated by deubiquitinases (DUBs), which remove Ub from their cellular targets in order to reverse their effects. The EAV papain-like protease 2 (PLP2) and the MERS-CoV papain-like protease (PLpro), in addition to their replicative functions, were proposed to interfere with the induction of the cellular innate immune response to infection by acting as deubiquitinating enzymes. The fact that these enzymes rely on a single active site in order to carry out both DUB and polyprotein processing activities complicates our ability to assess the role of these functions independently. Here, the crystal structures of EAV PLP2, and the MERS-CoV PLpro in covalent complex with their Ub substrates were determined, and structure-guided mutagenesis was used to selectively remove DUB activity permitting the independent study of their DUB activities. Specific mutations targeting the Ub-binding interface of these enzymes inhibited DUB activity while permitting replicative polyprotein processing. Studies using these DUB-deficient enzymes demonstrated directly their role in the down regulation of cellular innate immune responses. In an effort to further define the molecular basis for substrate recognition by these viral

The metabolism and physiology of Pseudomonas putida strain LS46 was investigated using biodiesel-derived waste streams as potential low cost substrates for production of medium chain length polyhydroxyalkanoates (mcl-PHA). Proteomic and trranscriptomic analyses were used to correlate specific gene and gene product expression patterns with differences in phenotypes of mcl-PHA biosynthesis by P. putida LS46. Growth and mcl-PHA content of P. putida LS46 were similar in cultures containing biodiesel-derived waste glycerol versus pure glycerol, and mcl-PHA synthesis occurred during stationary phase after nitrogen concentrations in the medium were exhausted. Waste glycerol cultures contained elevated concentrations of heavy metal ions, such as copper, which induced significant changes in gene expression levels related to heavy metal resistance. Several membrane-bound proteins, such as CusABC efflux and CopAB were identified and putatively play a role in regulating cellular copper concentrations. Cultures containing waste free fatty acids synthesized mcl-PHA throughout the exponential growth phase. Protein expression levels of two mcl-PHA synthases were suppressed during exponential phase growth in waste glycerol cultures, putatively via post-transcriptional regulation. Culture specific expression of monomer supplying proteins (PhaJ1 and PhaG), and sets of fatty acid oxidation enzymes were observed, and may have contributed to differences in the composition of polymers synthesized by P. putida LS46 cultured on the two substrates. Expression levels of the majority of mcl-PHA biosynthesis pathway genes were stable during active polymer synthesis in waste glycerol cultures. However, variations in protein expression levels, and in some cases their corresponding mRNAs, were observed in a number of other metabolic patheays, such as glycerol transportation, partial glycolysis, pyruvate metabolism, the TCA cycle, and fatty acid biosynthesis. These data suggest potential regulatory

Pseudomonas aeruginosa causes various infections in humans, animals and plants. Type III secretion system (T3SS) is one of the essential virulence factors used by P. aeruginosa. In this study, a previously uncharacterized gene PA0466 and its role in T3SS regulation have been examined. The results indicate that PA0466 is a novel T3SS regulator. It regulates T3SS directly through an unknown pathway and has a minor effect on the GacA-RsmA pathway. Besides the role in the interaction between the pathogen and the host, T3SS may also play a role in the interspecies interaction. A real-time PCR based Competitive Index (CI) assay was used to compare the wild type and T3SS mutant with and without the presence of Staphylococcus spp.. The results indicate that PAO1 was more competitive than exsA mutant and the difference was even bigger in the presence of Staphylococcus, suggesting T3SS may play a significant role in bacterium-bacterium interaction.

The Burkholderia cepacia complex (Bcc) are a group of closely related species known for their intrinsic multidrug resistance, large multipart genomes and ability to infect people with cystic fibrosis. The clinical relevance of the Bcc and their large multipart genomes make the study of their essential genes of broad interest. Essential genes are those required for survival in standard laboratory conditions this makes them potential targets for novel antibiotics against a group of species where few existing antibiotics are effective. Furthermore, while essential gene studies have been carried out in a number of bacterial species, only one of these species had multiple chromosomes and none had a genome as large as the Bcc. In my research I identified essential genes in B. cenocepacia K56-2, a member of the Bcc, by using transposon mutagenesis to deliver a rhamnose inducible promoter randomly into the genome and screening for a conditional growth (CG) phenotype. The utility of the CG mutant library was confirmed by showing that, when grown in suboptimal concentrations of rhamnose, only mutants that under-expressed the target of the antibiotic were hypersensitive. The CG mutant library included transposon insertions upstream from widely conserved, well-characterized essential genes suggesting that the system is capable of recovering essential gene mutants. A number of genes with either no or mixed records of essentiality in other microorganisms were also recovered. Among these was one of the three electron transfer flavoproteins (ETFs) in B. cenocepacia. The ETFs are a family of proteins found in a large number of eukaryotic, archaeal and bacterial species, which are required for the metabolism of specific substrates or for symbiotic nitrogen fixation in some bacteria. Despite these non-essential functions, high throughput screens have identified ETFs as putatively essential in several species. I showed that ETF expression is required for both viability and growth both

Burkholderia cenocepacia K56-2 is a clinical isolate of the Burkholderia cepacia complex, a group of intrinsically antibiotic resistant opportunistic pathogens, for which few treatment options are available. The identification of essential genes is an important step for exploring new antibiotic targets. Previously, a conditional growth mutant library was created using transposon mutagenesis to insert a rhamnose-inducible promoter into the genome of B. cenocepacia. It was then demonstrated that conditional growth mutants under-expressing an antibiotic target were sensitized to sub-lethal concentrations of that antibiotic. This thesis describes the use of next generation sequencing to measure the enhanced sensitivity of conditional growth mutants after being grown together competitively. Evidence is provided to show that the sensitivity of a GyrB conditional growth mutant to its cognate antibiotic, novobiocin, was enhanced due to competitive growth. Screening eight antibiotics with known mechanisms of action against a pilot conditional growth mutant library revealed a previously uncharacterized two-component system involved antibiotic resistance in B. cenocepacia. With the goal of exploring new Burkholderia antibacterial targets, the essential genome of B. cenocepacia K56-2 was identified by sequencing a high density transposon mutant library. Comparison of the B. cenocepacia K56-2 essential gene set with the essential genomes predicted for B. thailandensis, B. pseudomallei, and B. cenocepacia J2315 revealed strain-specific essential genes and 158 essential genes that are common to species in the Burkholderia genus. Three genes were identified that are essential in Burkholderia but not in other bacterial essential genomes identified so far, suggesting that specific cell envelope functions are critical to survival of Burkholderia species. To increase the diversity of the conditional growth mutant library, a method to enrich conditional growth mutants from the high dens

Methicillin resistant Staphylococcus aureus presents clinical challenges. The ability of S. aureus to cause human disease is correlated with the nematocidal activity of that isolate. This thesis investigates the relationship between SCCmec type, colony spreading, and the presence of the fudoh/psm-mec region of MRSA isolates to their ability to kill Caenorhabditis elegans. PCR was used to test for the fudoh/psm-mec region in 420 CMRSA2 isolates and 155 CMRSA1 isolates. Spread plate assays were performed on 149 CMRSA2 isolates to determine correlation between SCCmec type and colony spreading. Fudoh/psm-mec was correlated with the infection/colonization status of a patient. SCCmec type IV was correlated with increased spreading ability. SCCmec type, PFGE type, spa type, and colony spreading ability of 40 MRSA isolates were determined; these isolates were characterized using a nematode killing assay. It was found that the colony spreading ability of an isolate correlated with the nematocidal ability of that isolate.

In Rhizobium leguminosarum the ABC transporter responsible for rhamnose transport is dependent on RhaK, a sugar kinase that is necessary for the catabolism of rhamnose. It was hypothesized that RhaK has two separate functions; phosphorylation of rhamnose, and an unknown interaction with the rhamnose ABC transporter. To address this hypothesis a linker-scanning mutagenesis of rhaK was carried out. Two generated variants (RhaK72 and RhaK73) were found to maintain kinase activity, but were severely impaired in rhamnose transport function. Structural modelling suggested that both RhaK72 and RhaK73 affect surface exposed residues in two distinct regions localized to one face of the protein. This suggests that this proteins face may play a role in a protein-protein interaction that affects rhamnose transport. Using a two-hybrid system, an N-terminal and a C-teminal fragment of RhaK were both shown to interact with the N-terminal fragment of RhaT. These fragments span the regions that contain the rhaK73 and rhaK72 inserts respectively. When the rhaK72 and rhaK73 insert alleles were cloned and assayed using the two-hybrid system, these they were unable to interact with the RhaT fragment, suggesting these inserts abolish transport by interfering with a physical interaction between RhaT and RhaK. A phylogeny was generated based on the amino acid sequence of RhaK like proteins found in syntenous opereons. To gain insight into what residues may constitute a binding domain a PRALINE alignment of the orthologous kinases was combined with secondary structure analysis, known informative mutations, and functional residue predictions. A putative 12 amino acid binding site was identified using this method. An alanine scanning mutagenesis and subsequent two-hybrid analysis was carried out on this region. The substitution of any of these residues greatly affected the interaction between RhaT and RhaK. Although heterologous complementation of RhaK is possible, cosmid complementation anom

The Burkholderia cepacia complex (Bcc) are a group of closely related species known for their intrinsic multidrug resistance, large multipart genomes and ability to infect people with cystic fibrosis. The clinical relevance of the Bcc and their large multipart genomes make the study of their essential genes of broad interest. Essential genes are those required for survival in standard laboratory conditions this makes them potential targets for novel antibiotics against a group of species where few existing antibiotics are effective. Furthermore, while essential gene studies have been carried out in a number of bacterial species, only one of these species had multiple chromosomes and none had a genome as large as the Bcc. In my research I identified essential genes in B. cenocepacia K56-2, a member of the Bcc, by using transposon mutagenesis to deliver a rhamnose inducible promoter randomly into the genome and screening for a conditional growth (CG) phenotype. The utility of the CG mutant library was confirmed by showing that, when grown in suboptimal concentrations of rhamnose, only mutants that under-expressed the target of the antibiotic were hypersensitive. The CG mutant library included transposon insertions upstream from widely conserved, well-characterized essential genes suggesting that the system is capable of recovering essential gene mutants. A number of genes with either no or mixed records of essentiality in other microorganisms were also recovered. Among these was one of the three electron transfer flavoproteins (ETFs) in B. cenocepacia. The ETFs are a family of proteins found in a large number of eukaryotic, archaeal and bacterial species, which are required for the metabolism of specific substrates or for symbiotic nitrogen fixation in some bacteria. Despite these non-essential functions, high throughput screens have identified ETFs as putatively essential in several species. I showed that ETF expression is required for both viability and growth both

Burkholderia cenocepacia K56-2 is a clinical isolate of the Burkholderia cepacia complex, a group of intrinsically antibiotic resistant opportunistic pathogens, for which few treatment options are available. The identification of essential genes is an important step for exploring new antibiotic targets. Previously, a conditional growth mutant library was created using transposon mutagenesis to insert a rhamnose-inducible promoter into the genome of B. cenocepacia. It was then demonstrated that conditional growth mutants under-expressing an antibiotic target were sensitized to sub-lethal concentrations of that antibiotic. This thesis describes the use of next generation sequencing to measure the enhanced sensitivity of conditional growth mutants after being grown together competitively. Evidence is provided to show that the sensitivity of a GyrB conditional growth mutant to its cognate antibiotic, novobiocin, was enhanced due to competitive growth. Screening eight antibiotics with known mechanisms of action against a pilot conditional growth mutant library revealed a previously uncharacterized two-component system involved antibiotic resistance in B. cenocepacia. With the goal of exploring new Burkholderia antibacterial targets, the essential genome of B. cenocepacia K56-2 was identified by sequencing a high density transposon mutant library. Comparison of the B. cenocepacia K56-2 essential gene set with the essential genomes predicted for B. thailandensis, B. pseudomallei, and B. cenocepacia J2315 revealed strain-specific essential genes and 158 essential genes that are common to species in the Burkholderia genus. Three genes were identified that are essential in Burkholderia but not in other bacterial essential genomes identified so far, suggesting that specific cell envelope functions are critical to survival of Burkholderia species. To increase the diversity of the conditional growth mutant library, a method to enrich conditional growth mutants from the high dens

Methicillin resistant Staphylococcus aureus presents clinical challenges. The ability of S. aureus to cause human disease is correlated with the nematocidal activity of that isolate. This thesis investigates the relationship between SCCmec type, colony spreading, and the presence of the fudoh/psm-mec region of MRSA isolates to their ability to kill Caenorhabditis elegans. PCR was used to test for the fudoh/psm-mec region in 420 CMRSA2 isolates and 155 CMRSA1 isolates. Spread plate assays were performed on 149 CMRSA2 isolates to determine correlation between SCCmec type and colony spreading. Fudoh/psm-mec was correlated with the infection/colonization status of a patient. SCCmec type IV was correlated with increased spreading ability. SCCmec type, PFGE type, spa type, and colony spreading ability of 40 MRSA isolates were determined; these isolates were characterized using a nematode killing assay. It was found that the colony spreading ability of an isolate correlated with the nematocidal ability of that isolate.

In Rhizobium leguminosarum the ABC transporter responsible for rhamnose transport is dependent on RhaK, a sugar kinase that is necessary for the catabolism of rhamnose. It was hypothesized that RhaK has two separate functions; phosphorylation of rhamnose, and an unknown interaction with the rhamnose ABC transporter. To address this hypothesis a linker-scanning mutagenesis of rhaK was carried out. Two generated variants (RhaK72 and RhaK73) were found to maintain kinase activity, but were severely impaired in rhamnose transport function. Structural modelling suggested that both RhaK72 and RhaK73 affect surface exposed residues in two distinct regions localized to one face of the protein. This suggests that this proteins face may play a role in a protein-protein interaction that affects rhamnose transport. Using a two-hybrid system, an N-terminal and a C-teminal fragment of RhaK were both shown to interact with the N-terminal fragment of RhaT. These fragments span the regions that contain the rhaK73 and rhaK72 inserts respectively. When the rhaK72 and rhaK73 insert alleles were cloned and assayed using the two-hybrid system, these they were unable to interact with the RhaT fragment, suggesting these inserts abolish transport by interfering with a physical interaction between RhaT and RhaK. A phylogeny was generated based on the amino acid sequence of RhaK like proteins found in syntenous opereons. To gain insight into what residues may constitute a binding domain a PRALINE alignment of the orthologous kinases was combined with secondary structure analysis, known informative mutations, and functional residue predictions. A putative 12 amino acid binding site was identified using this method. An alanine scanning mutagenesis and subsequent two-hybrid analysis was carried out on this region. The substitution of any of these residues greatly affected the interaction between RhaT and RhaK. Although heterologous complementation of RhaK is possible, cosmid complementation anom

Burkholderia cenocepacia causes Cepacia syndrome, a fatal pneumonia that affects Cystic Fibrosis patients. Aromatic degradation has been linked to virulence in B. cenocepacia by insertional mutagenesis of genes involved in phenylacetic acid catabolism. B. cenocepacia has two paralogous copies of PaaK, the phenylacetyl-CoA ligase, which produces PA-CoA, the inducer of the pathway. Our objective was to assess a role for PaaK1 and PaaK2 in PA metabolism and virulence by constructing clean deletion mutants for each gene, and a double paaK mutant, as well as to quantify virulence using the nematode host model. Deletion and complementation of paaK1 revealed no change in killing phenotype. Reporter activity assays revealed PaaK1-dependent induction of the PA pathway 3-hydroxyphenylacetic acid but not 4-hydroxyphenylacetic acid. Altogether, these results demonstrate that 3-OHPA induces the PA degradation pathway in a paaK1 dependent manner and that PaaK1 is not involved in pathogenicity.

Burkholderia cenocepacia causes Cepacia syndrome, a fatal pneumonia that affects Cystic Fibrosis patients. Aromatic degradation has been linked to virulence in B. cenocepacia by insertional mutagenesis of genes involved in phenylacetic acid catabolism. B. cenocepacia has two paralogous copies of PaaK, the phenylacetyl-CoA ligase, which produces PA-CoA, the inducer of the pathway. Our objective was to assess a role for PaaK1 and PaaK2 in PA metabolism and virulence by constructing clean deletion mutants for each gene, and a double paaK mutant, as well as to quantify virulence using the nematode host model. Deletion and complementation of paaK1 revealed no change in killing phenotype. Reporter activity assays revealed PaaK1-dependent induction of the PA pathway 3-hydroxyphenylacetic acid but not 4-hydroxyphenylacetic acid. Altogether, these results demonstrate that 3-OHPA induces the PA degradation pathway in a paaK1 dependent manner and that PaaK1 is not involved in pathogenicity.

Serratia marcescens, a member of Enterobacteriaceae family, is a causative agent of nosocomial and opportunistic infections. Numerous reports show that the multidrug resistance among S. marcescens is growing. This organism has high-level intrinsic resistance to a variety of antimicrobial agents, which makes the treatment of infections caused by this bacterium very difficult. The major mechanism for antibiotic resistance, especially to fluoroquinolones, in Gram-negative organisms is the active efflux of the antibiotic molecule mediated by efflux pumps belonging to the Resistance-Nodulation-Cell Division (RND) family. It was previously shown that the SdeAB and SdeXY multidrug efflux pumps are important for conferring the intrinsic drug resistance in S.marcescens. In Escherichia coli, the up-regulation of transcriptional activators, such as MarA, SoxS and Rob, affect transcription of acrAB, tolC and micF. Over-expression of Rob results in increased expression of the E. coli AcrAB-TolC efflux pump and decreases outer membrane permeability through up-regulation of micF, resulting in multidrug, organic solvent and heavy metal resistance. In the present study, we report the identification of a rob gene in S. marcescens which has a 70% identity at the DNA level and 71% identity at the amino acid level to that of E. coli. Moreover, the S. marcescens rob demonstrated similar properties to the E. coli rob including having an effect on expression of outer membrane protein F (OmpF) and over-expression of SdeAB and SdeXY, conferring antibiotic resistance to divergent antibacterial agents and tolerance to organic solvents. We performed rob promoter evaluations using transcriptional fusions to the Green Fluorescence Protein (GFP) in the vector pGlow-TOPO and constructed a rob knock-out using the TargeTron Gene Knockout System. Promoter activity assessment, using the pGlow-TOPO reporter plasmid, showed that rob had higher promoter activity at 37°C than 30°C. In the presence of 2,2’

Serratia marcescens, a member of Enterobacteriaceae family, is a causative agent of nosocomial and opportunistic infections. Numerous reports show that the multidrug resistance among S. marcescens is growing. This organism has high-level intrinsic resistance to a variety of antimicrobial agents, which makes the treatment of infections caused by this bacterium very difficult. The major mechanism for antibiotic resistance, especially to fluoroquinolones, in Gram-negative organisms is the active efflux of the antibiotic molecule mediated by efflux pumps belonging to the Resistance-Nodulation-Cell Division (RND) family. It was previously shown that the SdeAB and SdeXY multidrug efflux pumps are important for conferring the intrinsic drug resistance in S.marcescens. In Escherichia coli, the up-regulation of transcriptional activators, such as MarA, SoxS and Rob, affect transcription of acrAB, tolC and micF. Over-expression of Rob results in increased expression of the E. coli AcrAB-TolC efflux pump and decreases outer membrane permeability through up-regulation of micF, resulting in multidrug, organic solvent and heavy metal resistance. In the present study, we report the identification of a rob gene in S. marcescens which has a 70% identity at the DNA level and 71% identity at the amino acid level to that of E. coli. Moreover, the S. marcescens rob demonstrated similar properties to the E. coli rob including having an effect on expression of outer membrane protein F (OmpF) and over-expression of SdeAB and SdeXY, conferring antibiotic resistance to divergent antibacterial agents and tolerance to organic solvents. We performed rob promoter evaluations using transcriptional fusions to the Green Fluorescence Protein (GFP) in the vector pGlow-TOPO and constructed a rob knock-out using the TargeTron Gene Knockout System. Promoter activity assessment, using the pGlow-TOPO reporter plasmid, showed that rob had higher promoter activity at 37°C than 30°C. In the presence of 2,2’

Synthetic cystic fibrosis sputum medium (SCFM) is rich in amino acids and supports robust growth of Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc). Previous work demonstrated that B. cenocepacia phenylacetic acid (PA) catabolic genes are up-regulated during growth in SCFM and are required for full virulence in a Caenorhabditis elegans host model. In this work, we investigated the role of phenylalanine, one of the aromatic amino acids present in SCFM, as an inducer of the PA catabolic pathway. Phenylalanine degradation intermediates were used as sole carbon sources for growth and gene reporter experiments. In addition to phenylalanine and PA, phenylethylamine, and phenylpyruvate could be used as sole carbon sources by wild type B. cenocepacia K56-2 but not by a PA catabolism defective mutant. These intermediates also induced a PA-inducible reporter system. Furthermore, proteomic analysis utilizing iTRAQ were used to study the protein expression of B. cenocepacia K56-2 grown in the amino acid-rich SCFM. Our results showed the over-expression of several proteins involved in amino acid and carbohydrate transport and metabolism. Interestingly, our results also showed the over-expression of flagellin and membrane efflux protein which are involved in the virulence of B. cenocepacia.

Immune cytokines tumor necrosis factor alpha and type I interferons provide front-line defense against viral infection and are regulated in part by ubiquitin (Ub) and Ub-like molecules. Ubiquitin and Ub-like molecule ISG15 share a conserved C-terminal motif where a terminal glycine residue becomes attached to cellular target proteins. Nairoviruses and arteriviruses contain an ovarian tumor domain-containing protease (OTU protease) that was found to corrupt pathways by removing Ub or ISG15 from target proteins. This broad substrate specificity is unlike mammalian deubiquitinating enzymes, which cannot recognize both substrates. To understand how viral OTU domain-containing proteases remove Ub and ISG15, the crystal structure of the Crimean-Congo Heamorhaggic Fever nairovirus (CCHFV) was determined with Ub to 2.5 Å resolution. A computational model was built of the CCHFV Otu protease bound to ISG15 as well. The CCHFV Otu protease has several structural differences from known OTU proteases, manifesting in its broad substrate recognition capability.

Immune cytokines tumor necrosis factor alpha and type I interferons provide front-line defense against viral infection and are regulated in part by ubiquitin (Ub) and Ub-like molecules. Ubiquitin and Ub-like molecule ISG15 share a conserved C-terminal motif where a terminal glycine residue becomes attached to cellular target proteins. Nairoviruses and arteriviruses contain an ovarian tumor domain-containing protease (OTU protease) that was found to corrupt pathways by removing Ub or ISG15 from target proteins. This broad substrate specificity is unlike mammalian deubiquitinating enzymes, which cannot recognize both substrates. To understand how viral OTU domain-containing proteases remove Ub and ISG15, the crystal structure of the Crimean-Congo Heamorhaggic Fever nairovirus (CCHFV) was determined with Ub to 2.5 Å resolution. A computational model was built of the CCHFV Otu protease bound to ISG15 as well. The CCHFV Otu protease has several structural differences from known OTU proteases, manifesting in its broad substrate recognition capability.

Synthetic cystic fibrosis sputum medium (SCFM) is rich in amino acids and supports robust growth of Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc). Previous work demonstrated that B. cenocepacia phenylacetic acid (PA) catabolic genes are up-regulated during growth in SCFM and are required for full virulence in a Caenorhabditis elegans host model. In this work, we investigated the role of phenylalanine, one of the aromatic amino acids present in SCFM, as an inducer of the PA catabolic pathway. Phenylalanine degradation intermediates were used as sole carbon sources for growth and gene reporter experiments. In addition to phenylalanine and PA, phenylethylamine, and phenylpyruvate could be used as sole carbon sources by wild type B. cenocepacia K56-2 but not by a PA catabolism defective mutant. These intermediates also induced a PA-inducible reporter system. Furthermore, proteomic analysis utilizing iTRAQ were used to study the protein expression of B. cenocepacia K56-2 grown in the amino acid-rich SCFM. Our results showed the over-expression of several proteins involved in amino acid and carbohydrate transport and metabolism. Interestingly, our results also showed the over-expression of flagellin and membrane efflux protein which are involved in the virulence of B. cenocepacia.