A pyrA mutation in Pseudomonas putida was isolated using transposon mutagenesis for the first time. Transposon Tn5 was used to inactivate the pyrA gene for carbamoylphosphate synthetase in these mutants. Accordingly, these mutants were defective in pyrimidine and arginine biosynthesis. The suicide vector, pM075, from Pseudomonas aeruginosa, was used to introduce the transposon into the cells. Tn5 was subsequently used to supply homology so that the plasmid pM075 could be introduced in its entirety into the Pseudomonas putida chromosome at the locus of the Tn5 insertion in the pyrA gene. Consequently, these strains exhibited high frequency of recombination and were capable of chromosome mobilization.

Directing groups (DGs) are moieties installed onto organic molecules to confer regioselectivity in subsequent reactions. DGs have found utility in selective CH activations catalyzed by transition metal (TM) catalysis on starting materials with multiple CH bonds. Despite their utility, DGs are scarcely used in industrial applications due to the generally wasteful nature of conventional DG strategies and their associated increase in step-count. Transient directing groups (TDGs) have been developed to overcome these limitations, with additives reversibly forming adducts with compounds of interest prior to the DG-mediated CH activation, in one-pot processes. However, the use of TDGs still requires harsh conditions to achieve significant yields, hindering broad applications. Chemoenzymatic catalytic cascades have attracted attention due to the mild and environmentally friendly nature of biocatalysis, with the greatest challenge being compatibility issues between biocatalytic and traditional chemical transformations. Here we propose a concurrent chemoenzymatic catalytic cascade that would enable TM-catalyzed DG chemistry via flanking biocatalytic reductive amination to install, and oxidative deamination to remove, a TDG. Preliminary efforts have identified some incompatibilities arising from the biocatalytic portion of the cascade, namely substrate specificity and organic co-solvent tolerance, that need to be addressed to achieve the proposed chemoenzymatic cascade in a one-pot concurrent protocol.

Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-resistant Staphylococcus aureus (MRSA) in vitro. To randomly generate A. oryzae mutant strains, spores were treated with ethyl methanesulfonate (EMS). Over 3000 EMS-treated A. oryzae cultures were tested in the screen, and one isolate, CAL220, exhibited altered morphology and antibacterial activity. Culture supernatant from this isolate showed antibacterial activity against Methicillin-sensitive Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa, but not Klebsiella pneumonia or Proteus vulgaris. The results of this study support our hypothesis and suggest that the screen used is sufficient and appropriate to detect secreted antibacterial fungal compounds resulting from mutagenesis of A. oryzae. Because the genome of A. oryzae has been sequenced and systems are available for genetic transformation of this organism, targeted as well as random mutations may be introduced to facilitate the discovery of novel antibacterial compounds using this system.

京都大学 / 新制・課程博士 / 博士(人間・環境学) / 甲第23392号 / 人博第1005号 / 新制||人||237(附属図書館) / 2021||人博||1005(吉田南総合図書館) / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)准教授 三浦 智行, 教授 川本 卓男, 准教授 西川 完途 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

Mammals receive more germline mutations from fathers than mothers. While the paternal bias in mutation has historically been attributed to errors in DNA replication during spermatogenesis, evidence suggests that in humans mutational mechanisms independent from cell division may play a more prominent role. Understanding how the ratio of paternal-to-maternal mutations, 𝛼, varies across animals differing in their gametogenic development, physiologies, and habitats can provide unique insights into the processes by which mutation arises in male and female germlines. To these ends, this thesis examines features of paternal mutation bias in dozens of amniote species using a combination of sequencing and evolutionary approaches. A direct way of measuring the strength of paternal mutation bias involves sequencing pedigrees of related individuals and detecting mutations arising in a single generation. In Chapter 2, we applied this approach to measure 𝛼 in olive baboons (Papio anubis) and humans. Strikingly, we estimated that in baboons 𝛼 = 4.5, similar to humans, despite baboons experiencing far fewer spermatogenic cell divisions than humans. A model of mutation based on cell division differences in the two species failed to explain this observation. Our results provide added evidence for non-replicative processes driving paternal bias in mutation and suggest that these causes are likely shared across mammals. In Chapter 3, we expanded our analysis to survey 𝛼 across 42 amniote species. We estimated 𝛼 from putatively neutral substitution rates of sex chromosomes and autosomes and found that in mammals, 𝛼 ranges up to 4 and correlates with generation times. In contrast, birds and snakes harbor a stable 𝛼 of roughly 2. These results are well predicted by modeling sex bias in mutation as a product of an early developmental phase when mutation occurs equally in both parents and a late phase after sexual differentiation when the male germline is more mutagenic. That the paternal mutation bias is widespread and occupies a narrow range of values suggests that it is caused by endogenous damage sources that are similar across species. Through a combination of pedigree sequencing and evolutionary techniques, this work demonstrates how a comparative approach across diverse taxa can shed light on the origins of sex-bias in germline mutation.

Microtubule motor proteins play fundamental roles in transporting a broad range of cellular cargoes in most eukaryotic cells. While there are over 40 kinesins helping to accommodate these diverse cellular demands, there is only one major form of cytoplasmic dynein (dynein, hereafter) carrying out almost all aspects of microtubule (MT) minus end-directed cargo transport. Dynein achieves this versatility by utilizing a wide range of adaptor proteins. Bicaudal D2 (BicD2) is a dynein adaptor protein responsible for linking cytoplasmic dynein to multiple forms of subcellular cargo. These include Rab6A, which contributes to Golgi function (Grigoriev et al., 2007; Matanis et al., 2002); the nucleoporin RanBP2 (Splinter et al., 2010); and the nucleus-cytoplasmic linker LINC complex protein Nesprin-2 (Goncalves et al., 2020). The latter two proteins were found to play important specific roles in the developing brain, respectively in the oscillatory interkinetic nuclear migration (INM) behavior characteristic of Radial Glial Progenitor (RGP) cells and in the directed nuclear migration in postmitotic neurons traveling to the expanding cortical plate. The BICD2 gene was implicated in the autosomal dominant forms of neuromuscular diseases such as Spinal Muscular Atrophy with Lower Extremity Dominance 2 (SMA-LED2) (Neveling et al., 2013; Oates et al., 2013; Peeters et al., 2013; Synofzik et al., 2014) and, more recently in at least three developmental brain pathologies: polymicrogyria (Ravenscroft et al., 2016), cerebellar hypoplasia (Fiorillo et al., 2016), and Lissencephaly (Tsai et al., 2020) expanding the clinical spectrum from neuromuscular only to potentially the entire central nervous system. It is largely unknown how these diverse clinical presentations have any relations to the BicD2 mutational sites. To investigate the genotype-phenotype relationship, it is inevitable to study each point mutation molecularly and characterize the mutational effects in in vivo setting. To investigate the mutational effects of BicD2, I used five different BicD2 fragments and nine missense mutants and characterized their behavior using biochemical and cellular approaches. Four of the missense mutations were further tested in the rat embryonic brain system, in which our lab has previously elucidated BicD2's roles during neurogenesis and post-mitotic neuronal migration. The chapters are organized by mutational effects on the BicD2-cargo interactions (Chapter 2) and the BicD2-dynein interactions (Chapter 3). In Chapter 4, I include supplemental materials to Chapters 2 and 3. In Chapter 5, I summarize, discuss, and provide my perspectives on all the mutational phenotypes found in the previous chapters. Lastly, all experimental procedures and reagents are described in Chapter 6. Here, I describe the first identification of novel gain-of-function (GoF) defects in BicD2- nuclear cargo interaction associated with two pathogenic mutations, R690C and E770G (Chapter 2). Furthermore, I characterize the GoF defects in the embryonic rat brain by in utero electroporation. The cell-type specific expression of R690C or E770G constructs revealed mutation-specific impairment of nuclear migrations in the developing cerebral cortex. In addition, I found GoF defects in BicD2-dynein interaction associated with three pathogenic mutations, L679R, R690C, and T699, surprisingly, in the C-terminal cargo binding domain of BicD2 (Chapter 3). I demonstrate that these missense mutations cause a defect in BicD2 autoinhibition control, which in turn results in abnormally enhanced dynein association. I provide evidence for hyper-activation of BicD2 for dynein binding contributes to Golgi fragmentation, which has been associated with many neuromuscular diseases (Martinez-Menarguez et al., 2019), including SMA-LED2 (Martinez-Carrera and Wirth, 2015). The entirety of this work describes molecular defects in 9 representative BicD2 mutations in vitro and demonstrates the mutational effects in vivo. I propose that the differential mutational effects associated with the BicD2 mutations might contribute to the broad spectrum of clinical phenotypes seen in patients with BicD2 diseases.

In the fission yeast S. pombe, Mto1p and Mto2p are involved in MT nucleation from cytoplasmic MTOCs and recruit components of the γ-TuC. We wished to discover whether Ben1p plays a similar role. ben1+ was tagged at its normal chromosomal location and the intracellular localization was analyzed. It showed that Ben1p localized to cytoplasmic microtubules. We generated ben1Δ. cells by replacing it with a copy of ura4+. We observed that 20% of the cells were undergoing division. Next we observed the effects that deleting ben1+ would have on the functions of Mto1p and Mto2p. We setup crosses between these strains and observed their localization. We did not observe any specific Mto2-GFP localization in ben1Δ. cells. This suggests that Ben1p plays a role in the proper localization of a γ-TuC associated protein.

The CB1 cannabinoid receptor is a G-protein coupled receptor highly expressed throughout the central nervous system, that has been suggested as a target for the treatment of various disorders, including anxiety, pain and neurodegeneration. Despite the wide therapeutic potential of CB1, development of potential drug candidates has long been hindered by concerns about adverse effects, rapid tolerance development and abuse potential. Ligands that produce biased signaling have been proposed as a strategy to dissociate therapeutic and adverse effects for a variety of G-protein coupled receptors. Biased signaling involves selective activation of a signaling transducer in detriment of another, mainly involving selective activation of G-protein signaling or b-arrestin signaling. However, biased signaling at the CB1 receptor is poorly understood due to the lack of strongly biased agonists. The development of biased agonists would be aided by understanding the molecular mechanism that leads to biased signaling. Although the structure of CB1 has been resolved in the inactive state and in the canonical active state, which allows G-protein signaling, little is known about the alternative active state that allows b-arrestin biased signaling. Therefore, we set out to investigate molecular and pharmacological tools that could shed light on the mechanism of CB1 biased signaling and to characterize novel allosteric ligands with a biased signaling profile. Using molecular dynamics stimulation of CB1 bound to a ORG27569, an allosteric ligand that stimulates b-arrestin signaling and inhibits G-protein signaling, we proposed single amino acid mutations that were predicted to impact b-arrestin signaling, and expressed wild-type and mutated CB1 receptor in HEK293 cells to measure signaling through different signaling transducers. We found that N7.49 and Y7.53, two amino acids in the highly conserved NPXXY motif, were essential for b-arrestin recruitment and signaling, but mutating them to Ala and Phe, respectively, did not impact G-protein signaling. We also found that I2.43, a functionally conserved amino acid on transmembrane helix 2, negatively regulates a switch in the rotameric position of Y7.53, as mutating I2.43 to Ala reduced steric hindrance upon Y7.53 and enhanced b-arrestin1 recruitment and signaling, while mutating it to Thr, a polar residue that would further hinder Y7.53, partially inhibited b-arrestin recruitment. Therefore, we concluded that N7.49 and Y7.53 form a hydrogen bond network along with D2.50 that is essential for the alternative active state that stimulates b-arrestin biased signaling. N7.49 acts as a fulcrum on which transmembrane helix 7 can bend, and Y7.53 acts as a rotamer toggle switch, stabilizing conformational changes on the intracellular end of transmembrane helix 7. This is the first record of a molecular mechanism for CB1 b-arrestin biased signaling involving the NPXXY motif. Due to the highly conserved character of these residues, it is possible that this mechanism can also be applied to other class A G-protein coupled receptors. In addition, we characterized novel biased allosteric ligands that stimulate or inhibit b-arrestin1 signaling. Two ORG27569 analogs were found to enhance orthosteric agonist induced b-arrestin1 recruitment and extracellular-signal regulated kinase 1/2 phosphorylation (pERK), with no effect on G-protein signaling. Two pregnenolone analogs absent of the steroid scaffold were found to inhibit pERK signaling independent of Gprotein signaling, indicating that they hinder b-arrestin dependent signaling. Since these analogs are believed to mediate their effects via stimulation or inhibition of conformational changes on transmembrane helix 7, our findings support a role for this domain on the alternative active state of CB1. In contrast, a GAT211 analog, GAT1601, had no effect on recruitment of b-arrestin1, but stimulated G-protein signaling and slightly enhanced barrestin2 recruitment. This compound binds to an allosteric site, where it stimulates the canonical active state of CB1 by facilitating the outward movement of transmembrane helix 6. Altogether, the results presented in this dissertation suggest that CB1 b-arrestin biased signaling is regulated by the NPXXY motif, which stimulates conformational changes on the transmembrane helix 7/helix 8 elbow, and that stimulating or hindering these conformational changes can enhance or disrupt CB1 b-arrestin biased signaling. However, facilitating the movement of transmembrane helix 6 favors G-protein biased signaling. Our findings provide molecular and pharmacological tools that will be of great importance to structure guided drug design and to future studies on the functional consequences of biased signaling at the CB1 receptor.

No description available.

Salmonella enterica serovar Typhimurium is one of the most prevalent food-borne pathogens, affecting millions around the world every year, making it a threat to global health. Salmonella possesses the ability to survive the normally lethal condition of desiccation, however, discovery of the genes and mechanisms behind this phenomenon are still ongoing. Using a transposon mutagenesis approach to construct a broad transposon library, this study aimed to uncover genes that may be contributing to changes in Salmonella’s survivability under desiccation, particularly when exposed to the antimicrobial molecule salicylic acid. Building on previous findings showing salicylic acid can alter cell viability through differential gene regulation, transposon mutants were exposed to salicylic acid and subsequently desiccated to screen for mutants that displayed an alteration in survival phenotypes. This work identified a transposon mutant with an interruption of the porin-coding gene ompC that displayed an augmented survivability phenotype under these conditions, leading to further exploration into the origin of this phenomenon.