Microbiology

Fungi are a diverse group of organisms that play crucial roles in various ecological processes and have immense economic importance. Understanding the intricate mechanisms underlying fungal growth and development is fundamental to harnessing their potential and exploring their applications in different fields. Signalling molecules, such as hormones, have been identified as key regulators in fungal physiology, orchestrating intricate processes and modulating biological responses to the environment. Phytohormones, commonly associated with plants, have been proposed as potential myco-hormones due to their production in a wide variety of fungi. Metabolomic analyses were performed via LC-MS/MS to investigate the role of phytohormones, specifically cytokinins (CKs) and indole-3-acetic acid (IAA), along with lipids and energetic metabolites such as organic acids during the growth and development of the model fungus Sordaria macrospora. The results revealed a clear switch between CK ribosides and CK free bases during the ascosporegenesis stage, with increased levels of cZ and iP and decreased levels of iPR and cZR. A similar pattern was observed in the mutant strain smgpi1 but demonstrating higher levels of CK free bases and increased fruiting body formation compared to the wild type. These findings provide insights into the regulation of phytohormones especially during fungal fruiting body development. In terms of IAA, the levels increased during the transition to sexual development in all strains, with the per5 mutant, unable to produce lipids via the cytosol, demonstrating a higher concentration than the wild type. The interplay between energetic metabolites and IAA suggests a potential role in the transition to sexual development. Additionally, the dose-dependent effects of exogenous CK application were investigated, showing the potential of low concentrations, from 1 to 10 μM, of CKs in promoting biomass accumulation or sexual development. Furthermore, gene editing in S. macrospora was proposed as a future direction to explore the functions of CKs and other metabolites during fungal development. Overall, this research contributes to our understanding of phytohormone-mediated processes in fungi and opens avenues for future investigations in fungal biology.

Author Keywords: fungi, LC-MS, metabolomics, myco-hormone, phytohormone, sexual development

Euglena gracilis is an enigmatic and adaptable organism that has great bioremediationpotential and is best known for its metabolic flexibility. The research done in this dissertation addresses (1) how growth conditions impact cellular composition, and (2) how chemometric approaches (such as statistical design of experiments and artificial neural networks) are viable alternatives to the conventional biosorption models for process optimization. Using high-resolution mass spectrometry for biosorbent characterization is a powerful way to assess the chemical characteristics of lyophilized and fractionated cells with high precision, especially to screen for compound classes that may have potentiality for rare earth element removal. Growth conditions impacted cellular composition and separated size fractions of cells yielded different molecular/chemical properties as described by compositional abundances, thus different biosorptive potential. Untargeted analysis demonstrated that exponential dark-grown cells with glucose supplementation were abundant in polyphenolic- and carbohydrate-like compounds, molecular species highly involved in rare earth element binding. Light grown cells had more heterogeneity and the highest molecular weighted fractions from light grown cells (fraction D) had the most abundances of polyphenolic- and protein-like structures. Chemometric modeling used identified the best and worst conditions for iii dysprosium sorption and showed that pH had the most significant influence on bioremoval. Bioremoval ranged from 37% at pH 8 to 91% at pH 3 at Dy concentration ranging from 1 to 100 μg L-1. The work presented in the PhD dissertation will aid in understanding the chemical characteristics of biosorbents by using a Van krevelen analysis of elemental ratios whether algal cells are grown in different environmental growth conditions, or when algal cell are size fractionated. This is especially applied for the screening for metal binding potentiality to Dysprosium. Chemometric methods provide an alternative method for the investigating factors for bioremoval, and applications for process optimization and for real-world applications. This dissertation will aid in understanding chemical characteristics when a biosorbent is grown in a given condition and which factors are important for rare earth element (REE) bioremoval. The significance of this work aims to look for alternate ways to screen biosorbents and using a more efficient experimental design for REE bioremoval.

Author Keywords: bioremoval, biosorption, chemometrics, dysprosium, euglena, mass spectrometry

Population density regulation is a fundamental principle in ecology, however there remain several unknowns regarding the functional expression of density dependence. One prominent view is that the patterns by which density dependence is expressed are largely fixed across a species, irrespective of environmental conditions. Our study investigated the expression of density dependence in Chlamydomonas reinhartti grown under a gradient of nutrient densities, and hypothesized that the relationship between per capita growth rate (pgr) and population density would vary from concave-up to concave-down as nutrients became less limiting. Contrary to prediction, we found that the relationship between a population's pgr and density became increasingly concave-up as nutrient levels increased. Our results suggest that density dependence is strongly variable depending on exogenous and endogenous processes acting on the population, implying that expression of density regulation depends extensively on local conditions. Population growth suppression may be attributable to environments with high intraspecific competition. Additional work should reveal the mechanisms influencing how the expression of density dependence varies across populations through space and time.

Author Keywords: Chlamydomonas reinhartti, density dependence, logistic model, population dynamics, single species growth, theta-logistic equation

Ustilago maydis is a basidiomycete smut fungus and the causal agent of common smut of corn. Disease progression and fungal development in this pathogen occur in planta, terminating in the production of dormant teliospores. Dormant spores of many fungi are characterized by reduced metabolic activity, which is restored during spore germination. The transition out of dormancy requires the rapid translation of stored mRNAs, which may be stabilized through natural antisense transcript (NAT)-mediated mechanisms. Transcript analysis revealed that as-ssm1, a NAT to the mitochondrial seryl-tRNA synthetase (ssm1), is detected in the dormant teliospore and absent in haploid cells. Disruption of ssm1 leads to cell lysis, indicating it is essential for cellular viability. Presented data supports the hypothesis that as-ssm1 has a role in facilitating teliospore dormancy through stabilizing ssm1 transcripts, which reduces mitochondrial function. as-ssm1 expression during in planta development begins 10 days post-infection, coinciding with the first appearance of dormant teliospores. To assess the impact of as-ssm1 expression on cell division, virulence and mitochondrial function, as-ssm1 was ectopically expressed in haploid cells, leading to increased ssm1 transcript levels and the formation of double-stranded RNA. These expression mutants are characterized by attenuated growth rate, virulence, mitochondrial membrane potential and oxygen consumption. Together, these findings support a role for NATs in moderating mitochondrial function during the onset of teliospore dormancy.

Author Keywords: Dormant teliospore, Mitochondria, mRNA stability, Natural antisense transcripts, Non-coding RNA, Ustilago maydis