Functional and comparative studies of members of the genus Methanosphaera, and their adaptations to the gut environment

Hoedt, Emily Cornelia (2017). Functional and comparative studies of members of the genus Methanosphaera, and their adaptations to the gut environment PhD Thesis, School of Chemistry and Molecular Biosciences, The University of Queensland. doi:10.14264/uql.2017.366

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Author Hoedt, Emily Cornelia
Thesis Title Functional and comparative studies of members of the genus Methanosphaera, and their adaptations to the gut environment
Formatted title
Functional and comparative studies of members of the genus Methanosphaera, and their adaptations to the gut environment
School, Centre or Institute School of Chemistry and Molecular Biosciences
Institution The University of Queensland
DOI 10.14264/uql.2017.366
Publication date 2017-02-28
Thesis type PhD Thesis
Supervisor Mark Morrison
Phil Hugenholtz
Gene Tyson
Total pages 173
Language eng
Subjects 0604 Genetics
0605 Microbiology
0603 Evolutionary Biology
Formatted abstract
The methanogenic archaea are responsible for maintaining an efficient scheme of fermentation in many environments and habitats, including the gastrointestinal tracts of animals and humans. The principal members of gut methanogenic communities are members of the Methanobrevibacter genus, with lesser numbers of Methanosphaera and Methanomassiliicoccus spp. Much of our understanding of gut methanogens has been produced using axenic cultures and genomic data for ~30 Methanobrevibacter spp. but the functional relevance of these other archaeal lineages to digestive function remains poorly understood. With this background, the goals of my PhD research are: i) to increase the biotic representation of the Methanosphaera genus through the recovery of axenic isolates from different environments; ii) characterise the metabolic properties of these isolates in terms of their methanogenic pathways and; iii) expand our functional understanding of this genus via reconstruction of “population genomes” from existing metagenomic datasets and comparative genome analyses. During the latter stages of my PhD, I chose to make a transition in my research activities to include some biomedical focus and take advantage of my relocation to the University of Queensland Diamantina Institute. Here, I have examined variations in methanogenic archaeal populations in some clinical studies, as well as evaluated the immunostimulatory properties of some gut archaea.

Chapter Two describes the enrichment, isolation, and pathways of methane formation by a strain of Methanosphaera sp. (WGK6) recovered from the Western Grey kangaroo (Macropus fulginosus). In contrast with the human isolate, strain WGK6 was found to utilize ethanol to support growth, but principally as a source of reducing power. Both the WGK6 and Msp. stadtmanae DSMZ 3091T genomes are very similar in terms of their size (~1.7 Mbp), synteny, and G:C content. However, the WGK6 genome was found to encode contiguous genes encoding putative alcohol- and aldehyde- dehydrogenases, which are absent from the Msp. stadtmanae genome. These two genes provide a plausible explanation for the ability of WGK6 to utilize ethanol for methanol reduction to methane. Furthermore, my in vitro studies suggest that ethanol supports a greater cell yield per mol of methane formed compared to hydrogen-dependent growth. Taken together, this expansion in metabolic versatility can explain the persistence of these archaea in the kangaroo foregut, and their abundance in these “low methane emitting” herbivores.

Chapter Three describes the isolation of a hydrogen-dependent methylotrophic archaeon assigned to the Methanosphaera genus from bovine animals in northern Australia (strain BMS). The BMS genome was sequenced to closure and surprisingly, found to be substantially larger (2.9 Mbp) than the WGK6 and Msp. stadtmanae genomes (~1.8 Mbp). I then interrogated metagenomic datasets produced from human stool and ruminant digesta samples, to recover 7 “population genomes” assigned to the Methanosphaera genus, with 5 of these genomes also predicted to be large (> 2.1 Mbp). The Methanosphaera pan-genome consists of 5321 genes and 305 of these were assigned to the core-genome, which principally consists of the genes coordinating methanogenesis, anaerobic metabolism, and related functions. The whole genome phylogeny analysis of all genomes suggests a monophyletic origin for the genus Methanosphaera, with those isolates possessing smaller genomes being the most recently evolved lineages.

Chapter Four investigates whether and how the composition of the methanogenic archaea in patients with chronic kidney disease is altered in response to synbiotic administration, as an intervention designed to mitigate uremic toxin production. The synbiotic intervention was found to have no measurable impact on the methanogen profiles of the patients, with Methanobrevibacter smithii the predominant archaeon and an infrequent occurrence of Methanomassiliicoccus, but no detectable populations of Methanosphaera spp. I also have assessed the immunogenic properties of Mbb. smithii DSMZ 861T, Msp. stadtmanae DSMZ 3091T, Mbb. ruminantium DSMZ 1093T, and my new Methanosphaera isolates, using human peripheral blood mononuclear cells (PBMC) and a mouse macrophage cell line. I first showed that Msp. stadtmanae DSMZ 3091T cell preparations produce a stronger TNF-α response from PBMC than Mbb. smithii DSMZ 861T, as well as the other Methanosphaera isolates WGK6 and BMS, but Mbb. ruminantium DSMZ 1093T cells produced the strongest TNF-α response. In subsequent studies using methanogen cell preparations and different sources of PBMC (also from healthy subjects) I measured the release of a broader range of cytokines, using a multi-bead array and flow cytometry techniques. In these studies, Mbb. smithii DSMZ 861T was more immunogenic compared to Msp. stadtmanae DSMZ 3091T, but both types of cells induce a similar cytokine profile from the PBMC preparations. Last, I used a murine macrophage cell line bearing a reporter gene activated by the NF-κB transcription system, which further confirmed that cell preparations of all these archaea induce reporter gene activation via this important pathway of stress response. Collectively, these results suggest all these methanogens possess immunostimulatory properties that remain cell associated, and the findings are discussed with respect to gastrointestinal inflammation and also the development of anti-methanogen vaccines.

Chapter 5 provides an integrative overview of the findings arising from my research, which provide a deeper understanding into the genus Methanosphaera, in terms of its evolutionary development, nutritional ecology, and host adaptation. I believe my findings are novel and provide new insights into the role of methanogenic archaea in gut function; and this knowledge will be useful to address key challenges facing agriculture (e.g. methane abatement strategies) and human health (e.g. as triggers of inflammation and immune response in gut and respiratory disease).
Keyword Methanogen
Methane
Macropodid
Ruminant
Human
Methanosphaera
Population genome
Pan-genome
Immunostimulatory

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Created: Wed, 08 Feb 2017, 11:16:40 EST by Emily Hoedt on behalf of Learning and Research Services (UQ Library)