Global Genomic Arrangement of Bacterial Genes Is Closely Tied with the Total Transcriptional Efficiency

AbstractThe availability of a large number of sequenced bacterial genomes allows researchers not only to derive functional and regulation information about specific organisms but also to study the fundamental properties of the organization of a genome. Here we address an important and challenging question regarding the global arrangement of operons in a bacterial genome: why operons in a bacterial genome are arranged in the way they are. We have previously studied this question and found that operons of more frequently activated pathways tend to be more clustered together in a genome. Specifically, we have developed a simple sequential distance-based pseudo energy function and found that the arrangement of operons in a bacterial genome tend to minimize the clusteredness function (C value) in comparison with artificially-generated alternatives, for a variety of bacterial genomes. Here we extend our previous work, and report a number of new observations: (a) operons of the same pathways tend to group into a few clusters rather than one; and (b) the global arrangement of these operon clusters tend to minimize a new “energy” function (C+ value) that reflects the efficiency of the transcriptional activation of the encoded pathways. These observations provide insights into further study of the genomic organization of genes in bacteria

From Genes to Ecosystems: Resource Availability and DNA Methylation Drive the Diversity and Abundance of Restriction Modification Systems in Prokaryotes

Together, prokaryotic hosts and their viruses numerically dominate the planet and are engaged in an eternal struggle of hosts evading viral predation and viruses overcoming defensive mechanisms employed by their hosts. Prokaryotic hosts have been found to carry several viral defense systems in recent years with Restriction Modification systems (RMs) were the first discovered in the 1950s. While we have biochemically elucidated many of these systems in the last 70 years, we still struggle to understand what drives their gain and loss in prokaryotic genomes. In this work, we take a computational approach to understand the underlying evolutionary drivers of RMs by assessing ‘big data’ signals of RMs in prokaryotic genomes and incorporating molecular data in trait-based mathematical models. Focusing on the Cyanobacteria, we found a large discrepancy in the frequency of RMs per genome in different environmental contexts, where Cyanobacteria that live in oligotrophic nutrient conditions have few to no RMs and those in nutrient-rich conditions consistently have many RMs. While our models agree with the observation that increased nutrient inputs make the selective pressure of RMs more intense, they were unable to reconcile the high numbers of RMs per genome with their potent defensive properties- a situation of apparent overkill. By incorporating viral methylation, an unavoidable effect of RMs, we were able to explain how organisms could carry over 15 RMs. With this discovery, we then tried and reassess the distribution of methyltransferases, an essential component of RMs that can also have alternate physiological rolls in the cell. We expand on conventional wisdom, that methyltransferases that are widely phylogenetically conserved are associated with global cellular regulation. However, we also find that organisms with high numbers of RMs also have a surprising amount of conservation in the methyltransferases that they carry. This data suggests caution should be used in associating phylogenic signals with functional rolls in methyltransferases as different functional rolls seem to overlap in their phylogenetic signal. Indeed, we suggest trait-based modeling may be the best tool in elucidating why organisms with a high selective pressure to maintain RMs appear to have conserved methyltransferase

Prediction of Host-Microbe Interactions from Community High-Throughput Sequencing Data

Microbial ecology is a diverse field, with a broad range of taxa, habitats, and trophic structures studied. Many of the major areas of research were developed independently, each with their own unique methods and standards, and their own questions and focus. This has changed in recent decades with the widespread implementation of culture-independent techniques, which exploit mechanisms shared by all life, regardless of habitat. In particular, high-throughput sequencing of environmentally isolated DNA and RNA has done much to expand our knowledge of the planet’s microbial diversity and has allowed us to explore the complex interplay between community members. Additionally, metatranscriptomic data can be used to parse relationships between individual members of the community, allowing researchers to propose hypotheses that can be tested in a laboratory or field setting. However, use of this technology is still relatively young, and there is a considerable need for broader consideration of its pitfalls, as well as the development of novel approaches that allow those without a computational background or with fewer resources to navigate its challenges and reap its rewards. To address these needs, we have developed targeted computational approaches that simplify next-generation sequencing datasets to a more manageable size, and we have used these techniques to address specific questions in environmental ecosystems. In a dataset sequenced for the purpose of identifying ecological factors that drive Microcystis aeruginosa to dominate cyanobacterial harmful algal blooms worldwide, we used a targeted approach to predict replication and lysogenic dormancy in bacteriophage. We used RNA-seq data to characterize viral diversity in the Sphagnum peat bog microbiome, identifying a wealth of novel viruses and proposing several host-virus pairs. We were able to assemble and describe the genome of a freshwater giant virus as well as that of a virophage that may infect it, and we used our techniques to describe its activity in publicly available datasets. Lastly, we have extended our efforts into the realm of medicine where we showed the influence exerted by the mouse gut microbiome on the host immune response to malaria, identifying several genes that may play a key role in reducing disease severity

Inactivation of pathogens on food and contact surfaces using ozone as a biocidal agent

This study focuses on the inactivation of a range of food borne pathogens using ozone as a biocidal agent. Experiments were carried out using Campylobacter jejuni, E. coli and Salmonella enteritidis in which population size effects and different treatment temperatures were investigate

Molecular techniques and their limitations shape our view of the holobiont

It is now recognised that the biology of almost any organism cannot be fully understood without recognising the existence and potential functional importance of associated microbes. Arguably, the emergence of this holistic viewpoint may never have occurred without the development of a crucial molecular technique, 16S rDNA amplicon sequencing, which allowed microbial communities to be easily profiled across a broad range of contexts. A diverse array of molecular techniques are now used to profile microbial communities, infer their evolutionary histories, visualise them in host tissues, and measure their molecular activity. In this review, we examine each of these categories of measurement and inference with a focus on the questions they make tractable, and the degree to which their capabilities and limitations shape our view of the holobiont

Role of the Nla6S and Nla28S histidine kinases in fruiting body development of \u3ci\u3eMyxococcus xanthus\u3c/i\u3e

The complex life cycle of Myxococcus xanthus makes it a model organism for studying multicellular developmental processes in bacteria. In response to adverse environmental conditions, M. xanthus aggregates and forms multicellular structures known as fruiting bodies. Two component signal transduction systems (TCS) are widely used by bacteria to detect and respond to environmental cues by regulating large-scale changes in gene expression. They contain a histidine kinase sensor that detects environmental cues and a response regulator that modulates cellular processes. Two key regulators of the early stages of fruiting body development are the Nla6S/Nla6 and Nla28S/Nla28 TCSs. The response regulators of these TCSs, Nla6 and Nla28, are important for the successful completion of fruiting body formation. However, the histidine kinase sensors that modulate the activity of these key response regulators were previously unknown. Here we report the identification and characterization of the Nla6S and Nla28S histidine kinases. Analysis of Nla6S reveals that it represents a new family of bacterial histidine kinases. Furthermore, it plays an important role in the M. xanthus life cycle. Analysis of Nla28S shows that this is an important sensor of early developmental events. Our data suggests that Nla28S is involved in sensing the two important events of early development, nutrient depletion and cell density. In response to these signals Nla28S regulates sporulation of M. xanthus. Characterization of Nla6S and Nla28S expands our knowledge of the signaling networks that regulate initiation of the multicellular developmental process of M. xanthus

IDENTIFICATION AND CHARACTERIZATION OF GATase1-LIKE AraC-FAMILY TRANSCRIPTIONAL REGULATORS IN BURKHOLDERIA THAILANDENSIS.

The ability of bacteria to detect their surroundings and enact an appropriate response is critical for survival. Translation of external signals into a coherent response requires specific control over the transcription of DNA into RNA. Much of the regulation at this step is accomplished by transcriptional regulators, proteins that bind to DNA and alter gene expression. A wide-spread variety of regulators in bacteria is the AraC-family. These regulators are divided into two conserved domains and respond to a variety of compounds owing to different N-terminal domains. A subfamily of these regulators, GATase1-like AraC-family transcriptional regulators (GATRs), is described. These proteins contain an N-terminal domain with structural characteristics similar to enzymes that synthesize amine-containing compounds. Members of this subfamily of transcriptional regulators are found in a wide range of bacteria, however, few are characterized. A relatively high number of GATRs are encoded in the Burkholderia thailandensis genome. Therefore, we utilized this bacterium as a model to explore the function and diversity of these regulators. GATRs in B. thailandensis divided into two groups based on bioinformatics analysis. The first group includes three members which we identified that contribute to the positive regulation of glycine betaine (GB) catabolism. GB can be utilized as a nutrient source or as a potent osmoprotectant. The regulation of this pathway in B. thailandensis differs from previously established models due to the interplay of these regulators. Homologs of two other GATRs in this group were identified that regulate carnitine and arginine catabolism. The second group of GATRs contains uncharacterized members with no known functions. A genetic strategy for engineering constitutive GATRs was developed and employed to investigate the transcriptional regulons of these GATRs. This approach yielded the identification of a novel GATR that represses expression of an operon producing a formaldehyde detoxification system, and is the first example of a GATR that functions as a repressor

Impact of Chromosomal Architecture on the Function and Evolution of Bacterial Genomes

The bacterial nucleoid is highly condensed and forms compartment-like structures within the cell. Much attention has been devoted to investigating the dynamic topology and organization of the nucleoid. In contrast, the specific nucleoid organization, and the relationship between nucleoid structure and function is often neglected with regard to importance for adaption to changing environments and horizontal gene acquisition. In this review, we focus on the structure-function relationship in the bacterial nucleoid. We provide an overview of the fundamental properties that shape the chromosome as a structured yet dynamic macromolecule. These fundamental properties are then considered in the context of the living cell, with focus on how the informational flow affects the nucleoid structure, which in turn impacts on the genetic output. Subsequently, the dynamic living nucleoid will be discussed in the context of evolution. We will address how the acquisition of foreign DNA impacts nucleoid structure, and conversely, how nucleoid structure constrains the successful and sustainable chromosomal integration of novel DNA. Finally, we will discuss current challenges and directions of research in understanding the role of chromosomal architecture in bacterial survival and adaptation

Beyond protein factories : expanding the synthetic biology toolkit for engineering mammalian hosts

The incredible clinical and commercial successes of recombinant protein therapeutics cemented the use of mammalian cells as the premier production hosts for these products. However, we can further exploit these cells to harness their potential for addressing current and future medical needs through metabolic and advanced engineering of these cells. To do so, we need a deeper understanding of the intricate gene regulation network that governs these cells and the ability to attain precise control of gene expression levels. In addition, some of these applications, such as gene therapy and immunotherapy, could benefit greatly by refraining from using viral-derived genetic elements. Therefore, this work seeks to establish additional transcriptional control elements to improve our ability to regulate expression with generalizable approaches and methods, facilitating the adaptation of these techniques for any mammalian cell type of interest. Here, we successfully demonstrated three key genetic elements can be utilized to tune gene expression in a rational manner. First, we conducted a genome-wide screen to survey genomic integration sites that support high transcriptional activity. We showed that CRISPR/Cas9-mediated de novo integration into one of these transcriptional hot-spots at the GRIK1 locus resulted in a 2.4-fold increase in heterologous gene expression over random integration. Subsequently, we set the groundwork necessary to evaluate a cell line development strategy that aims to increase the frequency of successful de novo targeted integrations. Second, we utilized two approaches for rational promoter engineering. We established a transcriptomics-guided workflow for de novo synthetic promoter design based on the Design-Build-Test paradigm. By using this workflow, we generated two synthetic designs that were comparable to a strong viral promoter and a strong endogenous promoter. We also employed an alternative approach by creating hybrid promoters, which resulted in a hybrid promoter variant that was also comparable to the same viral and endogenous promoters. Third, we exploited the general mammalian terminator structure and created a synthetic terminator that was comparable to a strong viral terminator. We evaluated 12 endogenous and 30 synthetic terminators for heterologous gene expression and revealed interactions between several key components of the terminator. Critically, we showed that transgene expression was 1.9x higher with endogenous and synthetic elements when compared with strong viral-derived elements. Ultimately, we showed that transgene expression can be finely adjusted by the approaches and methods described in this dissertation, and that viral-derived elements can be readily substituted by our synthetic designs.Chemical Engineerin

Nitrogen metabolism and butanol production by South African clostridium beijerinckii and clostridium saccharobutylicum strains

Includes bibliographical references.The acetone- butanol-ethanol (ABE) fermentation was one of the first fermentation processes to be industrialized on a large scale, and the dominant product, butanol is particularly significant due to its potential as a modern day fuel additive or fuel extender in the petrochemical industry. A collection of 19 solventogenic Clostridium beijerinckii and 11 Clostridium saccharobutylicum strains isolated from the National Chemical Products (NCP) ABE fermentation plant in Germiston, South Africa, were classed according to species by a quick species-specific colony PCR and by rifampicin screening methods respectively. The speciesspecific PCR aims to provide a rapid means of assessing any contamination of an ABE batch fermentation by differentiating between C. saccharobutylicum and C. beijerinckii species. Random Amplification of Polymorphic DNA (RAPD) analysis generated four C. beijerinckii and two C. saccharobutylicum strain groups respectively. Multilocus Sequence Typing (MLST) was developed for a smaller selection of strains and showed a further two strain groups within the NCP C. beijerinckii strains and three groups within the C. saccharobutylicum strains