Microbial methane utilization (also known as methanotrophy) stands out as an exceptional form of chemoorganotrophy, which requires a set of unique cellular machineries (membrane compartments and metabolic pathways) fully dedicated for energy recovery from the simplest organic compounds (methane or methanol) and reconstruction of all cellular parts from a single carbon unit. Despite significant progress in understanding fundamentals of methanotrophy, several critical elements remain unresolved. The most critical knowledge gaps include the lack of mechanistic understanding of methane oxidation, such as the source of electrons for the copper dependent methane monooxygenase (pMMO), the plasticity of metabolic pathways, and their dynamic interplay modulated by metal availability. These elements need to be thoroughly understood before they can be established as a chassis organism for biomanufacturing. To solve these mysteries, the research in this thesis is presented in three chapters, each dedicated to different cellular and molecular aspects of the methane metabolism using Methylotuvimicrobium alcaliphilum 20ZR as a model methanotrophic organism. The first chapter of my thesis is focused on characterization of methane oxidation machinery in the strain 20ZR at a cellular level. Exploration into this machinery was performed by a series of biochemical studies with strains that lack different elements of the methane oxidation network, such as methanol dehydrogenase (MDH), formate dehydrogenase (FDH), and downregulation/ expression of particulate methane monooxygenase (pMMO) proteins. This is followed by the second chapter of the thesis, which investigates cellular function of the surface layer proteins (SLPs). I performed comparative genetic analysis, knockout studies and a series of phenotyping tests complemented by -omics studies to gain insights into the SLP function. The data revealed that the surface layer protein matrix not only stabilizes the cell envelope at suboptimal conditions, but also contributes to metal scavenging. The last chapter of my thesis is dedicated to evaluating the feasibility of utilizing SLPs from methanotrophic bacteria for biomanufacturing. I developed an array of genetic tools to functionalize the surface layer proteins and use them as a platform to display heterologous proteins and peptides. The identification and utilization of surface layer proteins in methanotrophic bacteria introduces a novel approach for biomining.

Methane levels have rapidly increased in concentration in the atmosphere due to anthropogenic inputs making it a crucial abatement target against global climate change. The aerobic methanotrophs are bacteria capable of growth solely on methane as substrate and are genetically tractable and amenable to large scale fermentation making them ideal model system for the engineering of C1- biocatalysts. In this dissertation I examine the effects of trace minerals and substrate on the cell performance of Methylotuvimicrobium alcaliphilum 20ZR by contextualizing multi-omics with structural characterization through electron microscopy. This reveals the development of the intracytoplasmic membrane network within the cell which is affected by metals and leads to the increased gas consumption. Occupying the cell volume with membranes leads to an overflow metabolic state in conditions with sufficient substrate and oxygen in which fermentation products such as formate are excreted.. The nature of structural features revealed by microscopy is explored to determine the genetic elements for expression and transport of the surface layer protein. Genes responsible for the large and small proteins of the surface layer protein matrix are identified and a Type 1 Secretion System is explored as the potential mechanism of export for this cell surface feature. Concluding with an exploration of a novel methane chemotactic phenotype, which was identified by structural modelling and confirmed through a methane-based chemotaxis assay. This work demonstrates the importance of tying systems-level data such as proteomics with structural information gathered by electron microscopy to explain and explore phenotypes of performance such as growth rate and substrate consumption to cell biology of a model organism used as a methane-based biotechnology strain.

This work takes the first steps towards linking symbiotic associations of microbes to desert plants with global methane cycling. Drylands occupy 37.1% of the Earth’s terrestrial surface and are increasingly being converted into agricultural fields. Sustainable and productive use of arid land requires a better understanding of the functional traits of microbes inhabiting native arid environments. To improve our knowledge of native arid ecosystem functioning, we performed preliminary studies with soil samples collected from Anza-Borrego Desert State Park. The following discoveries were made: 1) Metagenomic data identified the methanotrophic bacteria Methylocaldum sp. as the predominant genus within the methanotrophic community in vegetated and unvegetated soil of the Anza-Borrego desert. 2) The consumption rate of atmospheric methane positively correlated with the presence of vegetation; 3) Genome annotations of methanotrophs from the rhizosphere of desert plants highlight several functions that can contribute to interactions with plants; 4) Methanotrophs have the potential to improve plant survival during drought stress. Based on these data we propose that in arid environments plants facilitate methane transport to the ground and cooperate with methane-consuming microbes to capture methane efficiently evolved in deeper soil layers and atmospheric methane (i.e. reverse chimney), which is then metabolized to provide supplemental resources for survival in water-limiting conditions. The results suggest that the plant-methanotroph associations represent an overlooked sink of atmospheric methane in dryland ecosystems. We show that supplementation with native Methylocaldum isolates from the Anza-Borrego Desert can potentially improve crop drought tolerance (e.g. Sorghum). Furthermore, this work provides essential evidence that targeted engineering of association between crops and methanotrophs in dryland soils can enhance atmospheric methane sink, ultimately influencing the global climate.

The recently generated database of microbial genes from an oligotrophic environment populated by a calculated 1,800 of major phylotypes (the Sargasso Sea metagenome) presents a great source for expanding local databases of genes indicative of a specific function. In this paper we analyze the Sargasso Sea metagenome in terms of the presence of methanopterin-linked C1 transfer genes that are signature for methylotrophy. We conclude that more than 10 phylotypes possessing genes of interest are present in this environment, and a few of these are relatively abundant species. The sequences representative of the major phylotypes do not appear to belong to any known microbial group capable of methanopterin-linked C1 transfer. Instead, they separate from all known sequences on phylogenetic trees, pointing towards their affiliation with a novel microbial phylum. These data imply a broader distribution of methanopterin-linked functions in the microbial world than previously known.