Salty Genetics:a genetic toolbox for the study of haloarchaea and their viruses

Viruses have a profound impact on members of all three domains of life, eukarya, bacteria and archaea. Archaea are single celled microorganisms which are most well known for thriving in extreme environments such as hot springs or salt lakes. They are however ubiquitous and can be found even in the human microbiome. While viruses of bacteria and eukaryotes have been studied in detail, archaeal viruses remain understudied. Therefore, this thesis aims to establish a new model system for the detailed study of archaea and their associated viruses. A halophilic (salt-loving) archaeon, Haloferax gibbonsii LR2-5, and its associated virus, HFTV1, were used for this purpose. First, both the archaeon and the virus are described in detail, granting first insights into their individual features and their interaction. Next, the archaeal host organism was genetically modified to allow for the use of more sophisticated molecular tools. Experiments showed that the modification was successful and can be used for the intended biomolecular tools.Lastly, similar approaches to genetically modify viruses are described and first experiments are used to explore which of these could be used with the virus HFTV1. Taken together, this thesis introduces a new model system for the study of archaeal virus-host interactions

The architecture of polyketide synthases

Since the discovery of penicillin over a century ago, secondary metabolites from all kingdoms of life have proven to be of high medical value. One class of proteins prevalent in the production of secondary metabolites are polyketide synthases (PKSs). Their polyketide products are complex organic compounds based on carbon chains assembled from carboxylic acid precursors. Many polyketides are produced by their hosts with the primary purpose of gaining an advantage in their ecological niche. To contribute to such an advantage, a significant proportion of polyketides are active against pro- and eukaryotic microorganisms. Type I PKSs are giant multienzyme proteins employing an assembly line logic for the synthesis of the most complex polyketides. They are composed of one or more functional and structural modules, each capable of carrying out one step of precursor elongation during the formation of an extended polyketide product. In this thesis, I address two fundamental and open questions in the biosynthesis of polyketides: First, what is the unique architecture underlying the assembly line logic of multimodular PKS assembly lines; and second, how is atomic accuracy achieved in cyclization and aromatic ring formation in the final step of PKS action. The first aim is addressed in chapter two, which provides for the first time detailed structural insights into the organization of type I PKS multimodules. This is achieved by cryo-electron microscopic analysis of filamentous and non-filamentous forms of K3DAK4, a bimodular trans-acyltransferase (AT) PKS fragment from Brevibacillus brevis. Overall reconstructions are provided at an intermediate resolution of 7 Å, with detailed insights into individual domains at sub-3Å resolution from cryo-electron microscopy and X-ray crystallography. The bimodule core displays a vertical stacking of its two modules along the central dimer axis of all three enzymatic domains involved. Additionally, K3DAK4 oligomerizes into filaments horizontally via small scaffolding domains in a trans-AT PKS-specific manner. In chapter three the second aim is tackled, as I visualize an intermediate of the enigmatic targeted cyclization and aromatic ring formation in the product template domain (PT) of the aflatoxin-producing PksA at 2.7 Å resolution using X-ray crystallography. To this end a substrate-analogue mimicking the transient intermediate after the first of two cyclization steps facilitated by the enzyme is covalently crosslinked to the active site. The positioning of the ligand relative to previously known ligands representing the pre-and post-cyclization states indicate an outward movement of the substrate throughout the process and a substantial effect of progressing cyclization on the meticulous positioning of the intermediates. The work provides detailed insights into core aspects of PKS biology from the atomistic picture of guided product modification to the giant overall assembly line architecture. In chapter four, both of these levels are put into context with current advances in the analysis of modular structure and dynamics of PKSs, such as recent structural models of cis-AT PKS modules and iterative PKSs. Furthermore, it addresses currently open questions, such as the interaction of trans-AT PKS with their cognate trans-acting enzymes. Altogether, the current progress in mechanistic understanding of PKS systems makes systematic and structure-guided efforts to unleash the full potential of PKS bioengineering ever more achievable

Flea Genetic Diversity in Gunnison's Prairie Dog Colonies and Its Implications for Flea Transmitted Diseases

Understanding disease-causing organisms from a broader ecological perspective has proven a valuable tool for understanding the causes of disease outbreaks in various organisms. Several insect species act as both parasites and pathogen carriers, making them important players in the spread of diseases in human and wildlife communities. This study aimed to determine what could be used to predict the distribution of flea genetic diversity parasitizing Gunnison’s prairie dogs (Cynomys gunnisoni) as a foundation for understanding the potential influence and implications this may have for transmission of disease causing microbes such as Rickettsia, Bartonella, and Yersinia pestis. A much higher level of flea genetic diversity was found in the colonies compared to what has been observed for fleas parasitizing black-tailed prairie dogs (Cynomys ludovicanus). Although none of the factors tested (location of colony relative to others, prairie dog genetic diversity, or number of mammals species) were able to predict the genetic diversity of fleas observed across colonies, potential implications for the spread of disease causing microbes are still considered, with recommendations for further research. The present study emphasizes the need to collect further data on mammals that frequently interact with Gunnison’s prairie dogs, as well as abiotic factors such as climate and temperature, both of which could be used to further investigate the survival and transmission of pathogens in this system

Predicting Evolution and Inferring Its Consequences

This dissertation concerns the roles of genetic and environmental factors in producing trait variation in evolving populations, with an emphasis on the creation and use of statistical tools that facilitate predictions. The research concerns evolution across a variety of spatial and temporal scales and environmental conditions. In each study I employ statistical approaches to make predictions about how observed trait variation is derived from variation due to the environment, or genetics, or the interaction between the two. The first chapter investigates the evolution of species' performance curves through the construction of a Bayesian model that facilitates comparisons among groups. The model is used to investigate how performance curves have evolved among taxa in the genus Lasthenia, and how variation in their performance curves predict where they occur in nature with respect to fine-scale hydrological gradients. I find evidence that the microhabitats taxa occupy along fine-scale hydrological gradients is best predicted by their overall productivity rather than the conditions that optimize their performance. The second chapter concerns predictability of evolution during colonization. Using flour beetle microcosm experiments, this work demonstrates that the genetic and phenotypic predictability of evolution during colonization decays over time, highlighting the relative contributions of stochastic and deterministic forces that shape variation in dispersal, fecundity, and body size. The last chapter addresses key challenges in predicting phenotypes from genetic sequence data. I develop a novel approach to representing DNA sequences, and demonstrate its value to capture multiple types of genetic variation, which I then show can be effectively used as input for models predicting phenotype. Collectively, the three studies provide insights into the complex and interacting roles of genetic and environmental variation in generating traits, and the development and use of statistical methods to make predictions that are important to our understanding of evolutionary processes

Versatility of the BID Domain: Conserved Function as Type-IV-Secretion-Signal and Secondarily Evolved Effector Functions Within Bartonella-Infected Host Cells

Bartonella spp. are facultative intracellular pathogens that infect a wide range of mammalian hosts including humans. In order to subvert cellular functions and the innate immune response of their hosts, these pathogens utilize a VirB/VirD4 type-IV-secretion (T4S) system to translocate Bartonella effector proteins (Beps) into host cells. Crucial for this process is the Bep intracellular delivery (BID) domain that together with a C-terminal stretch of positively charged residues constitutes a bipartite T4S signal. This function in T4S is evolutionarily conserved with BID domains present in bacterial toxins and relaxases. Strikingly, some BID domains of Beps have evolved secondary functions to modulate host cell and innate immune pathways in favor of Bartonella infection. For instance, BID domains mediate F-actin-dependent bacterial internalization, inhibition of apoptosis, or modulate cell migration. Recently, crystal structures of three BID domains from different Beps have been solved, revealing a conserved fold formed by a four-helix bundle topped with a hook. While the conserved BID domain fold might preserve its genuine role in T4S, the highly variable surfaces characteristic for BID domains may facilitate secondary functions. In this review, we summarize our current knowledge on evolutionary and structural traits as well as functional aspects of the BID domain with regard to T4S and pathogenesis

Genetic diversity and thermal performance in invasive and native populations of African fig flies

During biological invasions, invasive populations can suffer losses of genetic diversity that are predicted to negatively impact their fitness/performance. Despite examples of invasive populations harboring lower diversity than conspecific populations in their native range, few studies have linked this lower diversity to a decrease in fitness. Using genome sequences, we show that invasive populations of the African fig fly, Zaprionus indianus, have less genetic diversity than conspecific populations in their native range and that diversity is proportionally lower in regions of the genome experiencing low recombination rates. This result suggests that selection may have played a role in lowering diversity in the invasive populations. We next use interspecific comparisons to show that genetic diversity remains relatively high in invasive populations of Z. indianus when compared to other closely related species. By comparing genetic diversity in orthologous gene regions, we also show that the genome-wide landscape of genetic diversity differs between invasive and native populations of Z. indianus, indicating that invasion not only affects amounts of genetic diversity, but also how that diversity is distributed across the genome. Finally, we use parameter estimates from thermal performance curves measured for 13 species of Zaprionus to show that Z. indianus has the broadest thermal niche of measured species, and that performance does not differ between invasive and native populations. These results illustrate how aspects of genetic diversity in invasive species can be decoupled from measures of fitness, and that a broad thermal niche may have helped facilitate Z. indianus's range expansion.Funding provided by: National Science FoundationCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000001Award Number: Dimensions of Biodiversity award number 1737752Data used to generate genome annotations was generated by extracting whole RNA from groups of ~5 adult flies (24 hours after eclosion). Transcripts were assembed using Trinity (Grabherr et al. 2011; Hass et al. 2013) and annotations were generated using the MAKER pipeline (v3.01.02; Holt and Yandell 2011; Campbell et al. 2014). Data on thermal performance we generated in the lab under controlled conditions. All scripts used to fit thermal performance curves are given in this Dryad deposit. Software available for the method used are available at github.com/silastittes/performr

Cellular and Genomic Properties of Haloferax gibbonsii LR2-5, the Host of Euryarchaeal Virus HFTV1

Hypersaline environments are the source of many viruses infecting different species of halophilic euryarchaea. Information on infection mechanisms of archaeal viruses is scarce, due to the lack of genetically accessible virus-host models. Recently, a new archaeal siphovirus, Haloferax tailed virus 1 (HFTV1), was isolated together with its host belonging to the genus Haloferax, but it is not infectious on the widely used model euryarcheon Haloferax volcanii. To gain more insight into the biology of HFTV1 host strain LR2-5, we studied characteristics that might play a role in its virus susceptibility: growth-dependent motility, surface layer, filamentous surface structures, and cell shape. Its genome sequence showed that LR2-5 is a new strain of Haloferax gibbonsii. LR2-5 lacks obvious viral defense systems, such as CRISPR-Cas, and the composition of its cell surface is different from Hfx. volcanii, which might explain the different viral host range. This work provides first deep insights into the relationship between the host of halovirus HFTV1 and other members of the genus Haloferax. Given the close relationship to the genetically accessible Hfx. volcanii, LR2-5 has high potential as a new model for virus-host studies in euryarchaea

The structure of a polyketide synthase bimodule core

Polyketide synthases (PKSs) are predominantly microbial biosynthetic enzymes. They assemble highly potent bioactive natural products from simple carboxylic acid precursors. The most versatile families of PKSs are organized as assembly lines of functional modules. Each module performs one round of precursor extension and optional modification, followed by directed transfer of the intermediate to the next module. While enzymatic domains and even modules of PKSs are well understood, the higher-order modular architecture of PKS assembly lines remains elusive. Here, we visualize a PKS bimodule core using cryo-electron microscopy and resolve a two-dimensional meshwork of the bimodule core formed by homotypic interactions between modules. The sheet-like organization provides the framework for efficient substrate transfer and for sequestration of trans-acting enzymes required for polyketide production

Auswirkungen des Ausstiegs aus der Kohleverstromung in Deutschland – Auswertung einer Umfrage

Der Beitrag analysiert die Ergebnisse einer Umfrage von Dezember 2018 - Januar 2019 zu den erwarteten Auswirkungen eines Kohleausstiegs in Deutschlan