Functional metagenomic analysis of the human gut microbiome to identify novel salt tolerance genes

The ability to adapt to and respond to increases in external osmolarity is an important characteristic that enables bacteria to survive and proliferate in different environmental niches. When challenged with increased osmolarity, due to sodium chloride (NaCl) for example, bacteria elicit a phased response; firstly via uptake of potassium (K+), which is known as the primary response. This primary response is followed by the secondary response which is characterised by the synthesis or uptake of compatible solutes (osmoprotectants). The overall osmotic stress response is much broader however, involving many diverse cellular systems and processes. These ancillary mechanisms are arguably more interesting and give a more complete view of the osmotic stress response. The aim of this thesis was to identify novel genetic loci from the human gut microbiota that confer increased tolerance to osmotic stress using a functional metagenomic approach. Functional metagenomics is a powerful tool that enables the identification of novel genes from as yet uncultured bacteria from diverse environments through cloning, heterologous expression and phenotypic identification of a desired trait. Functional metagenomics does not rely on any previous sequence information to known genes and can therefore enable the discovery of completely novel genes and assign functions to new or known genes. Using a functional metagenomic approach, we have assigned a novel function to previously annotated genes; murB, mazG and galE, as well as a putative brp/blh family beta-carotene 15,15’-monooxygenase. Finally, we report the identification of a completely novel salt tolerance determinant with no current known homologues in the databases. Overall the genes identified originate from diverse taxonomic and phylogenetic groups commonly found in the human gastrointestinal (GI) tract, such as Collinsella and Eggerthella, Akkermansia and Bacteroides from the phyla Actinobacteria, Verrucomicrobia and Bacteroidetes, respectively. In addition, a number of the genes appear to have been acquired via lateral gene transfer and/or encoded on a prophage. To our knowledge, this thesis represents the first investigation to identify novel genes from the human gut microbiota involved in the bacterial osmotic stress response.

Hidden land and changing landscape: Narratives about Mount Khangchendzonga among the Lepcha and the Lhopo

This article explores contemporary ‘hidden land’ narrative constructs of Máyel Lyáng and Beyul Dremojong in Sikkim, India, as conceived by the Lepcha and the Lhopo, two ‘scheduled tribes’. Lepcha and Lhopo narratives about these hidden lands in Mount Khangchendzonga inform us about their contemporary and historical, indigenous and Buddhist contexts and the interactions between these contexts. Lhopo perspectives on the hidden Beyul Dremojong echo classical Tibetan Buddhist ‘revealed treasure’ guidebooks and exist within the complex and reciprocal relationship between the Lhopo and the land they inhabit; development initiatives are understood to have caused illness and death in the Lhopo community of Tashiding, often referred to as the geographical ‘center’ of Beyul Dremojong. Contemporary Lepcha comprehensions of Máyel Lyáng, described in oral narratives within an ethnic community whose cosmology is intimately connected with Mount Khangchendzonga, today show some influence of Lhopo interpretations of Beyul Dremojong and the treasure texts; they also reflect Lepcha fears about cultural dispersion. Present-day narratives about both hidden lands reference notable political events in modern Sikkimese history (encounters with the British; the Chinese occupation of Tibet).