Academic literature on the topic 'Microbiology Bioinformatics'

Ebolavirus (EBOV) is a negative sense, single stranded RNA virus that causes Ebola hemorrhagic fever. This disease causes substantial morbidity and mortality in humans, with death occurring in 50-90% of cases. Despite years of intensive research, much of the molecular mechanism underlying the entry of EBOV remains unknown. We performed a bioinformatics screen to identify novel entry cofactors by correlating mRNA expression in a panel of human cancer cell lines with permissivity to the EBOV entry glycoprotein. This assay identified several known EBOV entry cofactors such as actin and the tyrosine kinase Axl. In addition, several genes involved in macropinocytosis and endosomal maturation were also correlated with EBOV permissivity. Subsequent evaluation of plasma membrane proteins correlated by this screen showed T-cell immunoglobulin and mucin domain-1 (TIM-1) mRNA expression correlated extremely well with EBOV pseudovirion transduction. Depletion of TIM-1 from highly-permissive cells inhibits EBOV pseudovirion transduction. Conversely, expression of TIM-1 in poorly-permissive cells significantly and specifically enhances EBOV pseudovirion transduction and infection. TIM-1 binds to EBOV GP and this binding is important in the initial interaction between the virus and the host cell. ARD5, a TIM-1 mAb, significantly inhibits EBOV GP-mediated entry into several cell lines and primary human airway epithelia in a dose and time-dependent manner. Therefore, TIM-1 is the first receptor identified for EBOV. Additionally, AMP-activated protein kinase (AMPK) mRNA correlated strongly with EBOV pseudovirion transduction. Compound C, a specific AMPK inhibitor, inhibited EBOV pseudovirion transduction and infection in a time and dose-dependent manner into several cell lines and primary human monocyte derived macrophages. Mouse embryonic fibroblasts (MEFs) lacking functional AMPK were significantly less permissive to EBOV GP-mediated infection that WT MEFs. Visualization of virus entry into these cells revealed that EBOV causes actin polymerization independently of AMPK, but AMPK-/- cells do not form lamellipodia in the presence of EBOV and, consequently, cannot internalize virus into cells by macropinocytosis.

3-Methylindole and 4-methylphenol are cytotoxic and malodorant compounds derived from tryptophan and tyrosine, respectively. Each is present in swine waste lagoons and contributes to malodorous emissions from agricultural facilities. Clostridium scatologenes ATCC 25775 produces both compounds and serves as a model organism to study their metabolism and function. Through the repeated assembly and annotation of the Clostridium scatologenes genome, we propose a novel pathway for tryptophan degradation and 3-methylindole production by this organism. The genome of Clostridium scatologenes was sequenced, and re-assembled into contigs. Key elements of the tryptophan and shikimate pathways were identified. Contigs containing these elements were extracted from assemblies and matched to the reference genome of Clostridium carboxidivorans. Sequence for both pathways was then extended and defined using these joined sequence fragments. This sequence could serve as a starting point for the isolation of genes related to 3-methylindole synthesis using biochemical and enzyme analysis

<p> <i>Vibrio parahaemolyticus,</i> a human pathogenic bacterium, is a naturally occurring member of the microbiome of the Eastern oyster. As the nature of this symbiosis in unknown, the oyster presents the opportunity to investigate how microbial communities interact with a host as part of the ecology of an emergent pathogen of importance. To define how members of the oyster bacterial microbiome correlate with <i>V. parahaemolyticus,</i> I performed marker-based metagenetic sequencing analyses to identify and quantify the bacterial community in individual oysters after culturally-quantifying <i> V. parahaemolyticus</i> abundance. I concluded that despite shared environmental exposures, individual oysters from the same collection site varied both in microbiome community and <i>V. parahaemolyticus</i> abundance, and there may be an interaction with <i>V. parahaemolyticus</i> and <i> Bacillus</i> species. In addition, to elucidate the ecological origins of pathogenic New England ST36 populations, I performed whole genome sequencing and phylogenetic analyses. I concluded ST36 strains formed distinct subpopulations that correlated both with geographic region and unique phage content that can be used as a biomarker for more refined strain traceback. Furthermore, these subpopulations indicated there may have been multiple invasions of this non-native pathogen into the Atlantic coast.</p>

Bacteria interact with their eukaryotic hosts using a variety of mechanisms that range from being beneficial to detrimental. This dissertation focuses on Pantoea stewartii subspecies stewartii (P. stewartii), an endosymbiont in the corn flea beetle gut that causes Stewart's wilt disease in corn. Gaining insights into the interactions occurring between this bacterial pathogen and its plant host may lead to informed intervention strategies. This phytopathogen uses quorum sensing (QS) to coordinate cell density-dependent gene expression and successfully colonize corn leading to wilt disease. Prior to the research presented in this dissertation, the QS master regulator EsaR was shown to regulate two major virulence factors of P. stewartii, capsule production and surface motility. However, the function and integration of EsaR downstream targets in P. stewartii were still largely undefined. Moreover, only a draft genome of a reference strain of P. stewartii was publicly available for researchers, limiting bioinformatics and genome-scale genetic approaches with the organism. The work described in this dissertation has now addressed these important issues. The function of two EsaR direct targets, LrhA and RcsA, was explored (Chapter Two) and the existence of integration in the regulation between them was discovered (Chapters Two and Four). RcsA and LrhA are transcription factors controlling capsule production and surface motility in P. stewartii, respectively. In Chapter Two, the RcsA and LrhA regulons were investigated using RNA-Seq. This led to the discovery of a potential regulatory interaction between them that was confirmed by qRT-PCR and transcriptional gene fusion assays. The involvement of LrhA in surface motility and virulence was also established in this project. A direct interaction between LrhA and promoter of rcsA was defined in Chapter Four. Additional direct regulatory targets of LrhA were also identified. A project to generate a complete assembly of the P. stewartii genome (Chapter Three) enabled more thorough genome-wide analysis and revealed the existence of a previous unknown 66-kb region in the P. stewartii genome believed to contain genes important for motility and virulence. In addition, completion of the genome sequence permitted genes for two distinctive Type III secretion systems, used for interactions with corn or the corn flea beetle, to be placed on two mega-plasmids. Furthermore, the complete genome sequence facilitated a Tn-Seq approach (Chapter Five). Tn-Seq is a potent tool used to identify bacterial genes required for certain environmental test conditions. This project is a pioneering utilization of a Tn-Seq analysis in planta to investigate genes important for colonization and survival of P. stewartii within its corn host. It was discovered that OmpC and Lon are important to in planta growth and OmpA plays a role in plant virulence. In conclusion, these studies have broadened our understanding about the role of the QS regulon and other genes important for the pathogenesis of this phytopathogen. This knowledge may now be applied toward the development of future disease intervention strategies against P. stewartii and other wilt-disease causing plant pathogens.<br>PHD

The study of microbial evolution has been recently accelerated by the advent of comparative genomics, an approach enabling investigation of organisms at the whole-genome level. Tools of comparative genomics, including the DNA microarray, have been applied in bacterial genomes towards studying heterogeneity in DNA content, and to monitor global gene expression. When focused upon the study of microbial pathogens, genome analysis has provided unprecedented insight into their evolution, virulence, and host adaptation. Contributing towards this, I herein explore the evolutionary change affecting genomes of the Mycobacterium tuberculosis complex (MTC), a group of closely related bacterial organisms responsible for causing tuberculosis (TB) across a diverse range of mammals. Despite the introduction nearly a century ago of BCG, a family of live attenuated vaccines intentioned on preventing human TB, the uncertainty surrounding its usefulness is punctuated by the reality that TB continues to be responsible for claiming over 2 million lives per year. As pursued throughout this thesis, a precise understanding of the differences in genomic content among the MTC, and its impact on gene expression and biological function, promises to expose underlying mechanisms of TB pathogenesis, and suggest rational approaches towards the design of improved diagnostics and vaccines to prevent disease.<br>With the availability of whole-genome sequence data and tools of comparative genomics, our publications have advanced the recognition that large sequence polymorphisms (LSPs) deleted from Mycobacterium tuberculosis, the causative agent of TB in humans, serve as accurate markers for molecular epidemiologic assessment and phylogenetic analysis. These LSPs have proven informative both for the types of genes that vary between strains, and for the molecular signatures that characterize different MTC members. Genomic analysis of atypical MTC has revealed their diversity and adaptability, illuminating previously unexpected directions of MTC evolution. As demonstrated from parallel analysis of BCG vaccines, a phylogenetic stratification of genotypes offers a predictive framework upon which to base future genetic and phenotypic studies of the MTC. Overall, the work presented in this thesis has provided unique insights and lessons having direct clinical relevance towards understanding TB pathogenesis and BCG vaccination.

<p> The <i>C. acetobutylicum</i> genome annotation has been markedly improved by integrating bioinformatic predictions with RNA sequencing(RNA-seq) data. Samples were acquired under butanol, butyrate, and unstressed treatments across various growth stages to sample the transcriptome from a range of physiologically relevant conditions. Analysis of an initial assembly revealed errors due to technical and biological background signals, challenges with few solutions. Hurdles for RNA-seq transcriptome mapping research include optimizing library complexity and sequencing depth, yet most studies in bacteria report low depth and ignore the effect of ribosomal RNA abundance and other sources on the effective sequencing depth. </p><p> In this work, workflows were established to address type I and II errors associated with these challenges. An integrative analysis method was developed to combine motif predictions, single-nucleotide resolution sequencing depth, and library complexity to resolve these errors during assembly curation. This contextualization minimized false positive error and determined gene boundaries, in some cases, to the exact basepair of prior studies. Curation of the pSOL1 megaplasmid reconciled transcriptome assembly statistics with findings from <i>E. coli</i>. </p><p> The resulting annotation can be readily explored and downloaded through a customized genome browser, enabling future genomic and transcriptomic research in this organism. This work demonstrates the first strand-specific transcriptome assembly in a <i>Clostridium</i> organism. This method can improve the precision of transcript boundary estimates in bacterial transcriptome mapping studies.</p>

<p> Lyme disease is the most commonly reported vector-borne disease in the United States. The causative agent of Lyme disease, can alter gene expression to enable survival in a diverse set of conditions, including the tick midgut and the mammalian host. External environmental changes can trigger gene expression in <i>B. burgdorferi,</i> and the data demonstrate that <i> B. burgdorferi</i> can similarly alter gene expression as a stress-response when it is treated with the antibiotic doxycycine. After treatment with the minimum bactericidal concentration (MBC) of doxycycline, a subpopulation can alter its phenotype to survive antibiotic treatment, and to host adapt and successfully infect a mammalian host. Furthermore, our data demonstrate that if a population is treated with the MBC of doxycycline, a subpopulation may alter its phenotype to adopt a state of dormancy until the removal of the antibiotic, whereupon the subpopulation can regrow. We demonstrate that the chance of regrowth occurring increases as a population reaches stationary phase, and present a mathematical model for predicting the probability of a persister subpopulation within a larger population, and ascertain the quantity of a persister subpopulation. To determine which genes are expressed as stress-response genes, RNA Sequencing analysis, or RNASeq, was performed on treated, untreated, and treated and regrown <i>B. burgdorferi</i> samples. The results suggest several genes were significantly different in the treated group, compared to the untreated group, and in the untreated and regrown group compared to the untreated group, including a 50S ribosomal stress-response protein, coded from BB_0786. The appendices discuss the theory and methods that were used in RNA Sequencing (RNASeq) analysis, and provide an overview of the database that was created for the <i>B. burgdorferi</i> transcriptome. Additional studies may demonstrate further how persister subpopulations form, and which genes can trigger a persister state in <i>B. burgdorferi.</i></p>