CAGO: A Software Tool for Dynamic Visual Comparison and Correlation Measurement of Genome Organization

CAGO (Comparative Analysis of Genome Organization) is developed to address two critical shortcomings of conventional genome atlas plotters: lack of dynamic exploratory functions and absence of signal analysis for genomic properties. With dynamic exploratory functions, users can directly manipulate chromosome tracks of a genome atlas and intuitively identify distinct genomic signals by visual comparison. Signal analysis of genomic properties can further detect inconspicuous patterns from noisy genomic properties and calculate correlations between genomic properties across various genomes. To implement dynamic exploratory functions, CAGO presents each genome atlas in Scalable Vector Graphics (SVG) format and allows users to interact with it using a SVG viewer through JavaScript. Signal analysis functions are implemented using R statistical software and a discrete wavelet transformation package waveslim. CAGO is not only a plotter for generating complex genome atlases, but also a platform for exploring genome atlases with dynamic exploratory functions for visual comparison and with signal analysis for comparing genomic properties across multiple organisms. The web-based application of CAGO, its source code, user guides, video demos, and live examples are publicly available and can be accessed at http://cbs.ym.edu.tw/cago

BACTERIAL BIOTRANSFORMATION OF LIGNIN IN ANOXIC ENVIRONMENTS

There is a growing need to reduce reliance on non-renewable fuels, especially fossil fuels that contribute to the climate crisis. Plant lignocellulose is an abundant and undervalued source of energy, but its use is hindered due to the recalcitrance of the lignin-component. Current methods to remove lignin have sustainability concerns and are costly for industrial applications such as paper mill pulping. An alternative and greener approach is biopulping, which uses microbes and their enzymes to break down lignin. However, there are limitations to biopulping that prevent it from outcompeting other pulping processes, such as requiring constant aeration and mixing. The work presented in this dissertation investigates anaerobic bacteria as a promising alternative source for consolidated depolymerization of lignin and its conversion to valuable byproducts. We first ask if anaerobic aromatic metabolism is vertically inherited or horizontally transferred across bacteria. We analyzed seven out of the nine known central intermediate pathways. Of the seven, benzoyl-CoA metabolism had the strongest phylogenetic signal, suggesting vertical inheritance is the driver of its phylogenetic distribution. This information can be used in future studies to test if predictions can be made for uncharacterized taxa and anaerobic benzoyl-CoA related metabolism. We also investigated the mechanisms of two uncharacterized isolates, Sodalis sp. strain 159R and Tolumonas lignolytica BRL6-1. Strain 159R contains many genes related to both aerobic and anaerobic aromatic metabolism but lacks extracellular enzymes for anaerobic lignin depolymerization. Conversely, strain BRL6-1 did not demonstrate lignin metabolism but instead relies on iron redox and organic radicals to potentially modify lignin structure under anoxic conditions. The electron exchange between iron, lignin, and BRL6-1 suggests a protein that acts as a chelator and redox molecule is the intermediate between the bacteria and substrate. The two isolates demonstrate the importance that lignin depolymerization and metabolism may be found separately in organisms and should be considered in future designs for anaerobic biopulping and lignin valorization to be a competitive process on the market

<i>Staphylococcus aureus </i>Transcriptome Architecture:From Laboratory to Infection-Mimicking Conditions

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria

Conserved Genome Organization and Core Transcriptome of the Lactobacillus acidophilus Complex

The Lactobacillus genus encompasses a genetically and functionally diverse group of species, and contains many strains widely formulated in the human food supply chain as probiotics and starter cultures. Within this genetically expansive group, there are several distinct clades that have high levels of homology, one of which is the Lactobacillus acidophilus group. Of the uniting features, small genomes, low GC content, adaptation to dairy environments, and fastidious growth requirements, are some of the most defining characteristics of this group. To better understand what truly links and defines this clade, we sought to characterize the genomic organization and content of the genomes of several members of this group. Through core genome analysis we explored the synteny and intrinsic genetic underpinnings of the L. acidophilus clade, and observed key features related to the evolution and adaptation of these organisms. While genetic content is able to provide a large map of the potential of each organism, it does not always reflect their functionality. Through transcriptomic data we inferred the core transcriptome of the L. acidophilus complex to better define the true metabolic capabilities that unite this clade. Using this approach we have identified seven small ORFs that are both highly conserved and transcribed in diverse members of this clade and could be potential novel small peptide or untranslated RNA regulators. Overall, our results reveal the core features of the L. acidophilus complex and open new avenues for the enhancement and formulation and of next generation probiotics and starter cultures

An Extended Network of Genomic Maintenance in the Archaeon Pyrococcus abyssi Highlights Unexpected Associations between Eucaryotic Homologs.

In Archaea, the proteins involved in the genetic information processing pathways, including DNA replication, transcription, and translation, share strong similarities with those of eukaryotes. Characterizations of components of the eukaryotic-type replication machinery complex provided many interesting insights into DNA replication in both domains. In contrast, DNA repair processes of hyperthermophilic archaea are less well understood and very little is known about the intertwining between DNA synthesis, repair and recombination pathways. The development of genetic system in hyperthermophilic archaea is still at a modest stage hampering the use of complementary approaches of reverse genetics and biochemistry to elucidate the function of new candidate DNA repair gene. To gain insights into genomic maintenance processes in hyperthermophilic archaea, a protein-interaction network centred on informational processes of Pyrococcus abyssi was generated by affinity purification coupled with mass spectrometry. The network consists of 132 interactions linking 87 proteins. These interactions give insights into the connections of DNA replication with recombination and repair, leading to the discovery of new archaeal components and of associations between eucaryotic homologs. Although this approach did not allow us to clearly delineate new DNA pathways, it provided numerous clues towards the function of new molecular complexes with the potential to better understand genomic maintenance processes in hyperthermophilic archaea. Among others, we found new potential partners of the replication clamp and demonstrated that the single strand DNA binding protein, Replication Protein A, enhances the transcription rate, in vitro, of RNA polymerase. This interaction map provides a valuable tool to explore new aspects of genome integrity in Archaea and also potentially in Eucaryotes

3D Organization of Eukaryotic and Prokaryotic Genomes

There is a complex mutual interplay between three-dimensional (3D) genome organization and cellular activities in bacteria and eukaryotes. The aim of this thesis is to investigate such structure-function relationships. A main part of this thesis deals with the study of the three-dimensional genome organization using novel techniques for detecting genome-wide contacts using next-generation sequencing. These so called chromatin conformation capture-based methods, such as 5C and Hi-C, give deep insights into the architecture of the genome inside the nucleus, even on a small scale. We shed light on the question how the vastly increasing Hi-C data can generate new insights about the way the genome is organized in 3D. To this end, we first present the typical Hi-C data processing workflow to obtain Hi-C contact maps and show potential pitfalls in the interpretation of such contact maps using our own data pipeline and publicly available Hi-C data sets. Subsequently, we focus on approaches to modeling 3D genome organization based on contact maps. In this context, a computational tool was developed which interactively visualizes contact maps alongside complementary genomic data tracks. Inspired by machine learning with the help of probabilistic graphical models, we developed a tool that detects the compartmentalization structure within contact maps on multiple scales. In a further project, we propose and test one possible mechanism for the observed compartmentalization within contact maps of genomes across multiple species: Dynamic formation of loops within domains. In the context of 3D organization of bacterial chromosomes, we present the first direct evidence for global restructuring by long-range interactions of a DNA binding protein. Using Hi-C and live cell imaging of DNA loci, we show that the DNA binding protein Rok forms insulator-like complexes looping the B. subtilis genome over large distances. This biological mechanism agrees with our model based on dynamic formation of loops affecting domain formation in eukaryotic genomes. We further investigate the spatial segregation of the E. coli chromosome during cell division. In particular, we are interested in the positioning of the chromosomal replication origin region based on its interaction with the protein complex MukBEF. We tackle the problem using a combined approach of stochastic and polymer simulations. Last but not least, we develop a completely new methodology to analyze single molecule localization microscopy images based on topological data analysis. By using this new approach in the analysis of irradiated cells, we are able to show that the topology of repair foci can be categorized depending the distance to heterochromatin